Pharmacological Manipulation of Long-term Potentiation and Cortical Inhibition from the Dorsolateral Prefrontal Cortex, a Model to Understand Cognitive Deficits of Schizophrenia
by
Bahar Salavati
A thesis submitted in conformity with the requirements for the degree of doctor of philosophy
Institute of Medical Science University of Toronto
© Copyright by Bahar Salavati 2017
ii
Pharmacological Manipulation of Long-term Potentiation and
Cortical Inhibition from the Dorsolateral Prefrontal, a Model to
Understand Cognitive Deficits of Schizophrenia
Bahar Salavati
Doctor of Philosophy
Institute of Medical Science University of Toronto
2017
Abstract
Several studies have assessed the pharmacological modulation of cortical inhibition (CI) and
long-term potentiation (LTP) using transcranial magnetic stimulation (TMS) from the motor
cortex and recorded these effects through surface electromyography (EMG). However, recording
CI and LTP from the dorsolateral prefrontal cortex (DLPFC), a cortical region that is more
closely associated with the pathophysiology of severe psychiatric disorders including
schizophrenia were previously not done. Objectives: This study, therefore, was designed to
investigate whether CI, indexed by long interval cortical inhibition (LICI), and LTP measured
using paired associative stimulation (PAS) could be modulated and measured from the DLPFC
in healthy participants through the use of TMS combined with electroencephalography ( EEG )
using a placebo-controlled, randomized double-blinded crossover study design. Methods: 12
healthy participants were given a single oral dose of baclofen, a GABAB agonist,
dextromethorphan, a NMDA antagonist, L-DOPA, a dopamine precursor, rivastigmine, an
acetylcholine esterase inhibitor for each arm of the study. Results: LICI was enhanced and
suppressed by increasing GABAergic tone and cholinergic tone, respectively. LTP was increased
by enhancing cholinergic tone and dopaminergic tone and abolished when NMDA receptors
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were blocked. Conclusion: These results are important and have the potential for therapeutic
application in manipulating neural plasticity and CI in the DLPFC to treat a variety of conditions,
including depression, Parkinson's disease, and schizophrenia.
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Acknowledgments
I would like to take this opportunity to express my sincere gratitude to everyone who helped me
with this endeavor. Foremost, I would like to thank my supervisors and mentors, Dr. Jeff.,
Zafiris, Daskalakis and Dr. Tarek Rajji. Jeff and Tarek, thank you for challenging me
intellectually for the past four years. I am truly grateful for this opportunity and words cannot
express my appreciation. The completion of this thesis would have been possible without both
of your guidance and support. Next, I would like to thank my two phenomenal committee
members, Dr. Chen and Dr. Pollock for always finding time to provide timely feedback and
guidance. You are both an inspiration and I truly appreciate your support and patience. I would
also like to extend my gratitude to Reza Zoomordi and Yinming Sun and my other CAMH
friends for all their support. Also a special thank you to the volunteers who participated and
made this study possible, as well as, to the Ontario Metal Health Foundation and the Ontario
Graduate Scholarship for partially funding my studentship. I would also like to thank my
wonderful family for all for their support and for letting me believe I could do anything I set my
mind to. Last but not least, I would like to thank my wonderful husband Bruce, for the
encouragement he has given to me throughout my academic career, without him, my dream
would still be a dream.
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Contributions
Chapter 1. Introduction This chapter was written solely by Bahar Salavati to serve as the introduction of this dissertation
Chapter 2. Imaging-based Neurochemistry in Schizophrenia: A Systematic Review and Implications for Dysfunctional Long-Term Potentiation. Bahar Salavati fully wrote this chapter. Bahar Salavati conducted the systematic literature search and data interpretation. Yinming Sun designed figure 2. Neurochemicals and Receptors in Patients with Schizophrenia Relative to Healthy Controls in Different Brain Regions for this paper. All the authors critically reviewed, edited and approved the final version for publication.
Chapter 3. Pharmacological Modulation of Long-term Potentiation in the Dorsolateral Prefrontal Cortex. Tarek Rajji and Zafiris Daskalakis designed the study. Bahar Salavati conducted the recruitment and screened all research participants to ensure eligibility. Bahar Salavati collected and analyzed all the data. Reza Zoomordi provided consultation and expertise with the EEG analysis and approved the final EEG analysis. Bahar Salavati wrote the paper but all authors reviewed and edited this paper and approved the final version for publication.
Chapter 4. Pharmacological Manipulation of Cortical Inhibition in the Dorsolateral Prefrontal Cortex Bahar Salavati, Tarek Rajji, and Zafiris Daskalakis designed the study. Bahar Salavati conducted the recruitment and screened all research participants to ensure eligibility. Bahar Salavati collected and analyzed all the data. Reza Zoomordi provided consultation and expertise with the EEG analysis and approved the final EEG analysis. Bahar Salavati wrote the paper and all authors reviewed and edited this paper and approved the final version for publication.
Chapter 5 Discussion. This chapter was written solely by Bahar Salavati to serve as the general discussion of this dissertation
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Table of Contents Abstract .......................................................................................................................................... ii
Acknowledgments ........................................................................................................................ iv
Contributions................................................................................................................................. v
List of Abbreviations .................................................................................................................... x
List of Tables .............................................................................................................................. xiii
List of Figures ............................................................................................................................. xiv
Chapter 1 ....................................................................................................................................... 1 1. Introduction ........................................................................................................................... 1 1. 1 Schizophrenia and Cognitive Deficits ............................................................................... 1
1.1.1 Schizophrenia Symptoms ............................................................................................... 1 1.2. What is Long-term Potentiation? ..................................................................................... 2
1.2.1 NMDA-Dependent LTP ................................................................................................. 3 1.2.2 Properties of Long-term Potentiation ............................................................................. 4 1.2.3 NMDA-Dependent Long-term Potentiation is Associated with Learning and Memory 5
1.3 Abnormal Plasticity and Long-term Potentiation in Schizophrenia .............................. 5 1.3.1 In vivo LTP Studies in Schizophrenia ............................................................................ 6
1.4 Dorsolateral Prefrontal Cortex and Schizophrenia: ........................................................ 7 1.4.1 Functional Abnormalities ............................................................................................... 7 1.4.2 Anatomical Abnormalities .............................................................................................. 7
1.5 Long-term Potentiation of the Dorsolateral Prefrontal Cortex ...................................... 8 1.6 Glutamate ............................................................................................................................. 8
1.6.1 Glutamate and Glutamate Receptors .............................................................................. 8 1.7 Glutamatergic Activity on Long-term Potentiation ......................................................... 9 1.8 Glutamatergic Activity Associated Schizophrenia ......................................................... 10
1.9.1 Dopamine...................................................................................................................... 11 1.9.2 Dopaminergic Pathways ............................................................................................... 11 1.9.3 Dopamine Receptors ..................................................................................................... 12 1.9.4 Dopamine Release ........................................................................................................ 13
1.10 Dopaminergic Activity on Long-term Potentiation ...................................................... 13 1.10.1 D1 Receptor Activation ............................................................................................... 13 1.10.2 D2 Receptor Activation ............................................................................................... 14 1.10.3 D1 and D2 Receptor Activation ................................................................................... 14 1.10.4 Dose-dependent Effects of Dopamine ........................................................................ 15
1.11 Dopaminergic Activity Associated with Schizophrenia ............................................... 16 1.12 Acetylcholine .................................................................................................................... 17
1.12.1 Cholinergic Pathways ................................................................................................. 17 1.12.2 Nicotinic Receptors .................................................................................................... 18
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1.12.3 Muscarinic Receptors ................................................................................................. 19 1.13 Cholinergic Activity in Modulating Long-term Potentiation ...................................... 20 1.14 Cholinergic Activity in Schizophrenia........................................................................... 22 1.15 Gamma-aminobutyric Acid (GABA) ............................................................................ 23
1.15.1 GABAergic Interneurons ............................................................................................ 23 1.15.2 GABAA Receptors ...................................................................................................... 24 1.15.3 GABAB Receptors ...................................................................................................... 25
1.16 GABAergic Activity in Modulating Long-term Potentiation ...................................... 25 1.17 GABAergic Activity in Schizophrenia........................................................................... 26 1.18 Transcranial Magnetic Stimulation ............................................................................... 28
1.18.1 TMS Coil .................................................................................................................... 29 1.18.2 TMS Activation .......................................................................................................... 31
1.19 Transcranial Magnetic Stimulation and Electromyography ...................................... 31 1.20 Paired Associative Stimulation ...................................................................................... 32 1.21 Electroencephalogram .................................................................................................... 33
1.21.1 Cortical Oscillations ................................................................................................... 34 1.22 EEG Artifacts and Independent Component Analysis (ICA) ..................................... 35 1.23 Cortical Inhibition (CI) ................................................................................................... 36
1.23.1 Long Interval cortical Inhibition................................................................................. 36 1.23.2 Cortical Silent Period.................................................................................................. 37 1.23.3 Short Interval Cortical Inhibition ............................................................................... 38
1.24 Cortical Excitation .......................................................................................................... 39 1.24.1 Resting Motor Threshold ............................................................................................ 39 1.24.2 Intracortical Facilitation ............................................................................................. 40
1.25 Pharmaco-TMS Experiments ......................................................................................... 40 1.25.1 GABAergic Activity ................................................................................................... 41 1.25. 2 Glutamatergic Activity .............................................................................................. 42 1.25.3 Dopaminergic Activity ............................................................................................... 42 1.25.4 Cholinergic Activity ................................................................................................... 43
1.26. Abnormal Cortical Inhibition in Schizophrenia ......................................................... 43 1.27 Pharmacology of the Drugs Used in this Dissertation ................................................. 45
1.27.1 Baclofen ...................................................................................................................... 45 1.27. 2 Dextromethorphan ..................................................................................................... 45 1.27.3 Levodopa .................................................................................................................... 46 1.27.4 Rivastigmine ............................................................................................................... 46
Chapter 2 ..................................................................................................................................... 52
2. Imaging-based Neurochemistry in Schizophrenia: A Systematic Review and Implications for Dysfunctional Long-Term Potentiation .............................................................................. 52
2.1 Abstract .............................................................................................................................. 53 2.2 Introduction ....................................................................................................................... 54
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2.3 Methods .............................................................................................................................. 55 2.3 Results ................................................................................................................................ 56
2.3.1 Glutamatergic System................................................................................................... 56 2.3.2 Dopaminergic System................................................................................................... 63 2.3.3 GABAergic System ...................................................................................................... 72
2.4 Discussion ........................................................................................................................... 73 2.5 Limitations ......................................................................................................................... 76 2.6 Conclusion .......................................................................................................................... 77
Chapter 3 ..................................................................................................................................... 80
Pharmacological Modulation of Long-term Potentiation in the Dorsolateral Prefrontal Cortex ........................................................................................................................................... 80
3.1 Abstract .............................................................................................................................. 81 3.2 Introduction ....................................................................................................................... 82 3.3 Participants and Methods ................................................................................................. 83
3.3.1 Overall Study Design.................................................................................................... 83 3.3.2 Participants ................................................................................................................... 84 3.3.3 Locating and Co-Registering the DLPFC .................................................................... 84 3.3.4 Electromyography (EMG) recordings from the Motor Cortex and TMS-EEG in the DLPFC ................................................................................................................................... 85 3.3.5 PAS to the DLFPC ....................................................................................................... 87 3.3.6 EEG Data Processing.................................................................................................... 87 3.3.7 Statistical Analysis ....................................................................................................... 88
3.4 Results ................................................................................................................................ 89 3.5 Discussion ........................................................................................................................... 96 3.6 Conclusion .......................................................................................................................... 98
Chapter 4 .......................................................................................... Error! Bookmark not defined.
Pharmacological Manipulation of Cortical Inhibition in the ............................................... 100
Dorsolateral Prefrontal Cortex ................................................................................................ 100 Abstract 4.1. ........................................................................................................................... 101 4.2 Introduction ..................................................................................................................... 102 4.3 Methods and Participants ............................................................................................... 105
4.3.1 Overall Study Design.................................................................................................. 105 4.3.2 Participants ................................................................................................................. 107 4.3.3 Locating and Co-Registering the DLPFC .................................................................. 107 4.3.4 TMS-EMG in the Motor Cortex and TMS-EEG in the DLPFC ................................ 108 4.3.5 EEG Data Processing.................................................................................................. 109 4.3.6 LICI Quantification .................................................................................................... 109 4.3.7 Statistical Analysis ..................................................................................................... 111
4.4 Results .............................................................................................................................. 111
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4.5 Discussion ......................................................................................................................... 116 4.6 Conclusion ........................................................................................................................ 120
Chapter 5 ................................................................................................................................... 121
5. Discussion............................................................................................................................... 121 5.1 Summary of the Dissertation ......................................................................................... 121 5.1.1 Summary of the First Paper ........................................................................................ 122 5.1.2 Summary of the Second Paper .................................................................................... 122 5.1.3 Summary of the Third Paper ...................................................................................... 123
5.2 General Discussion .......................................................................................................... 123 5.2.1 Glutamate.................................................................................................................... 123 5.2.2 Dopamine.................................................................................................................... 125 5.2.3 GABA ......................................................................................................................... 128 5.2.4 Nicotine ...................................................................................................................... 130 5.2.5 PAS as Therapeutic Tool ............................................................................................ 131
5.3 Significance of this Work ................................................................................................ 133 5.4 Limitations ....................................................................................................................... 133 5.5 Conclusion ........................................................................................................................ 134 5.6 Future Direction .............................................................................................................. 136
x
List of Abbreviations
APV 2-amino-5-phosphonopentanoic acid
L-DOPA (3, 4-dihydroxyphenylalanine)
Acetyl CoA acetyl coenzyme A
AD adenylyl cyclase
APB abductor pollicis brevis
Ca2+ calcium
CaMKII calmodulin dependent protein kinase II
cAMP cyclic adenoside monophosphate
ChAT choline acetyltransferase
CI cortical inhibition
COMT catechol-O-methyl transferase
CNS central nervous system
CS conditioning stimulus
CSP cortical silent period
D-AP5 D-(-) 2-amino-5-phosphonopentanoic acid
DLPFC dorsolateral prefrontal cortex
EEG electroencephalography
EMG electromyography
EPSP excitatory post synaptic potential
GABA gamma-aminobutyric acid
GAD glutamic acid decarboxylase
GAT1 GABA transporters
GDP guanosine diphosphate
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GTP guanosine triphosphate
ICA independent component analysis
ICF intracortical facilitation
IPSP inhibitory post synaptic potential
ISI interstimulus interval
L-DOPA levodopa
LICI long interval cortical inhibition
LTP long-term potentiation
Mg2+ magnesium
MAO monoamine oxidase
MEP motor evoked potential
MPFC medial prefrontal cortex
MRI magnetic resonance image
MRS magnetic resonance spectroscopy
mV millivolt
Na+ sodium
NMDA N-methyl D-aspartate
PAS paired associative stimulation
PET position emission tomography
PCP phencyclidine
PFC prefrontal cortex
PKC protein kinase C
PNS peripheral nerve stimulation
RMT resting motor threshold
SCZ schizophrenia
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SICF short interval cortical facilitation
SICI short interval cortical inhibition
tDCs transcranial direct current stimulation
TMS transcranial magnetic stimulation
TS test stimulus
VAChT vesicular acetylcholine transporter
VMAT vesicular monoamine transporter
VTA ventral tegmental area
WCST Wisconsin Card Sorting Test
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List of Tables
Table 1. Image Studies Assessing the Glutamatergic Systems in Antipsychotic-
Naïve/Antipsychotic-Free Patients with Schizophrenia………………………... Appendix 140
Table 2. Image Studies Assessing Dopaminergic Systems in Antipsychotic-Naïve or Antipsychotic-
Free Patients with Schizophrenia.…………………………………….…………….. Appendix 145
Table. 3 Demographic and Basic Neurophysiologic Characteristics………………………..91
Table 4. Potentiation over the Dorsolateral Prefrontal Cortex under each Drug Condition………………………………………………………………………………………..96
Table 5. Properties of Drugs ………………………………………………………..………..108
Table 6. Demographic and Neurophysiologic Characteristics……………………………..113
Table 7. Pre-Drug vs Post-Drug LICI from stimulation to Dorsolateral Prefrontal Cortex under each Drug Condition……………………………………………………….……….…116
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List of Figures
Figure 1. PAS in the motor Vs PAS in the DLPFC…………………………………………..33
Figure 2. Cortical Inhibition Measures………………………………………………………..40
Figure 3. Neurochemical Model …………….............................................................................79
Figure 4. Neurochemicals and Receptors in Patients with Schizophrenia Relative to Healthy Controls in Different Brain Regions…………………………………………………80
Figure 5. Experimental design…………………………………………………………...…… 87
Figure 6. Effects of Drugs on DLPFC Neuroplasticity……………………………………….93
Figure 7. Event-Related Potentials (ERPs) Across All Conditions…………………………..94
Figure 8. LICI Protocol………………………………………………………………………..107
Figure 9. Effects of Drugs on DLPFC LICI……………………………………………….….114
Figure 10. Topographical plots for LICI………………………………………………….….115
1
Chapter 1
1. Introduction
1.1 Schizophrenia and Cognitive Deficits
Schizophrenia is a psychiatric disorder that affects 1 % of the world’s population, (Torrey 1987;
Whiteford, Degenhardt et al. 2013) and is among the top 10 causes of life-long functional
disability worldwide, occupying 10% of all hospital beds, and exacting enormous personal,
social and economic costs. Worse of all, 15% of those diagnosed eventually commit suicide
(Kaplan HI 1994).
1.1.1 Schizophrenia Symptoms
Symptoms of schizophrenia are separated into two categories, positive and negative symptoms.
Positive symptoms include hallucinations, delusions, and disorganized speech (Schultz and
Andreasen 1999). These symptoms reflect excesses from normal experience, whereas, negative
symptoms are deficits in normal emotional responses or thought processes. These symptoms
include poverty of speech, affective flattening, and avolition (Andreasen and Olsen 1982;
Schultz and Andreasen 1999). Severe cognitive deficits involving learning, memory, and
executive function are also central to the disorder (Heinrichs and Zakzanis 1998; van Os and
Kapur 2009). In fact, first episode patients typically score half a standard deviation below the
mean on tasks requiring executive function and memory (Bilder, Goldman et al. 2000). These
results are independent of medication, as drug-naïve first episode patients also demonstrate
impaired executive function (Krieger, Lis et al. 2005). Furthermore, these patients also show
2
greater difficulties on alertness, information maintenance, sustained and selective attention, as
well as, explicit and implicit recall (Lussier and Stip 2001) functions associated with cognition.
These findings highlight that cognitive impairment in this population is independent of
medication and is truly illness-related.
Despite some success for positive symptoms, treatment has not been promising for cognitive
symptoms (i.e. memory and learning impairments) which are the strongest predictors of
functional disability (Green 1996). Although the etiology of these symptoms are unknown,
several researchers have proposed that impaired cortical inhibition (CI) and long-term
potentiation (LTP), two widely accepted cellular mechanism that govern learning and memory,
(Malenka 2003; Lynch 2004) may contribute and promote cognitive deficits in schizophrenia
(Daskalakis, Christensen et al. 2002; Hasan, Nitsche et al. 2011).
1.2. What is Long-term Potentiation?
Neuroplasticity represents the ability of the brain to reorganize its anatomical and functional
properties in response to a changing environment (Pascual-Leone, Amedi et al. 2005). LTP is a
synaptic form of neuroplasticity that involves a persistent strengthening of synapses between
neurons. This can result from the upregulation of receptors located on presynaptic and/or
postsynaptic neurons, changes in the quantity of neurotransmitters released into the synaptic
cleft, or how effectively neurons respond to those neurotransmitters (Collingridge and Bliss
1995). Given that memory and learning are associated with the modification of synaptic strength,
LTP is widely considered to be one of the cellular mechanisms that underlies cognitive deficits
(Bliss and Lomo 1973; Malenka 2003; Malenka and Bear 2004; Cooke and Bliss 2006; Citri and
Malenka 2008).
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1.2.1 NMDA-Dependent LTP
Although there are different types of LTP, the most commonly studied is mediated by N-methyl-
D-aspartate (NMDA) receptors and involves -amino-3-hydroxy-5-methyl-4-isoxazolepropionic
acid (AMPA) glutamate receptors (Morris 1989; Collingridge and Bliss 1995), and therefore, the
focus of this thesis. At resting membrane potential, NMDA receptors are blocked by magnesium
(Mg2+) ions and cannot be activated. When presynaptic glutamate release coincides with the
postsynaptic membrane depolarization, via AMPA receptors, NMDA receptors become available
due to the expulsion of Mg2+ions by electrostatic repulsion of sodium (Na+) ions. (Ascher and
Nowak 1988). Dislodge of Mg2+ ions allow calcium (Ca2+) and Na+ ions to flow freely through
NMDA receptors. Intracellular calcium is a major determinant of LTP plasticity (Malenka 2003).
High calcium concentrations result in LTP, while small concentrations result in long-term
depression (LTD), the opposing phenomenon, characterized by a decrease in synaptic
transmission (Mulkey and Malenka 1992).
This influx of calcium subsequently activates calmodulin, which in turn activates both
calmodulin-dependent protein kinase II (CaMKII) and protein kinase C (PKC) (Malinow,
Schulman et al. 1989; Lisman, Schulman et al. 2002). Calmodulin also activates adenylyl cyclase
(AD) which increases the signaling of cyclic adenosine monophosphate (cAMP) and activates
protein kinase A (Wu, Thomas et al. 1995; Wong, Athos et al. 1999). The activation of protein
kinases leads to the phosphorylation and upregulation of postsynaptic AMPA receptors (Hayashi,
Shi et al. 2000). This upregulation induces LTP by strengthening synaptic connections. This
effect is long-lasting and can last up to several months (Malenka and Bear 2004).
4
1.2.2 Properties of Long-term Potentiation
NMDA-dependent LTP exhibits four main properties, these include input specificity,
cooperativity, associativity, and persistence. First, input specificity means that the induction of
LTP is restricted to only the inputs that received stimulation and does not spread to adjacent
synapses that were not activated. That is, there is a spatially restricted increase in intracellular
Ca2+ in only the relevant neuron. Second, cooperativity means that LTP can be induced either by
a strong stimulation of one presynaptic neuron that synapses onto a postsynaptic neuron,
or cooperatively by several weaker stimulations of many presynaptic neurons that converge onto
a postsynaptic neuron. Third, associativity means a weak stimulus from a single pathway is
insufficient to induce LTP, but when simultaneously paired with a strong stimulus from another
pathway, will be sufficient enough to induce LTP at the postsynaptic neuron (Bliss and Lomo
1973; Malenka 2003; Malenka and Bear 2004). That is, a weak input is potentiated when
activated in “association” with a strong input. This is the basis of paired associative stimulation
(PAS), a noninvasive neurostimulation technique that combines TMS with peripheral nerve
stimulation (PNS) to artificially induce LTP-like plasticity in the human cortex (Stefan, Kunesch
et al. 2000). This technique is described in greater detail below. Finally, persistence means that
the induced LTP is long-lasting, which lasts from several minutes to many months, it is this long-
lasting effect that separates LTP from other forms of synaptic plasticity.
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1.2.3 NMDA-Dependent Long-term Potentiation is Associated with Learning and Memory
LTP is required for cognitive functions including learning and memory. This was first proposed
in 1940’s by Donald Hebb who claimed that when presynaptic and postsynaptic activity is
coupled, associative memories are formed through the strengthening of synaptic connections.
This concept is better known as “neurons that fire together wire together” (Larry. R Squire
2008). Subsequently, in the early 1980’s Collingridge discovered that NMDA activation is
required for the induction of LTP (Collingridge, Kehl et al. 1983). Later, in 1986 Morris et al.
demonstrated that memory is NMDA receptor dependent. He bathed the hippocampi of one
group of rodents in the NMDA receptor blocker, 2-amino-5-phosphonopentanoic acid (APV) and
noticed this group failed to perform the Morris swim task, a spatial learning, and memory task
(Morris 1989). Similar results were later shown through gene targeting, where spatial
impairments were evident in rodents who had their NMDA receptors knocked from the
hippocampus (Tsien, Huerta et al. 1996). Therefore, the cumulative efforts of these studies
suggest that impairments in NMDA-dependent LTP may underlie cognitive deficits as seen in
schizophrenia.
1.3 Abnormal Plasticity and Long-term Potentiation in Schizophrenia
Abnormal LTP weakens synaptic connections, which in turn impairs connections between
populations of neurons, and ultimately impairs highly plastic regions such as the hippocampus,
amygdala, and neocortex. These brain regions are commonly the most affected and implicated in
schizophrenia (Friston 2002). Abnormal LTP also impairs connections between different brain
6
regions. In fact, patients demonstrate a reduction in connectivity between the PFC and temporal
cortex, and between the motor and sensory cortex (Pettersson-Yeo, Allen et al. 2011). This
abnormal connection is linked to cognitive and self-monitoring impairments and hallucinations
(Friston 2002; Ford, Roach et al. 2008).
1.3.1 In vivo LTP Studies in Schizophrenia
In vivo LTP can be induced and measured using several neurostimulation techniques. Through
the use of these techniques, it has consistently been shown that patients with schizophrenia have
impaired in vivo LTP in the motor cortex. For example, by applying transcranial direct current
stimulation (TDCS) on multi-episode patients, results showed a reduction in LTP-like plasticity
compared with recent-onset patients and healthy controls (Hasan, Nitsche et al. 2011). This
finding illustrates that disease severity or duration may be a contributing factor in LTP
dysfunction. In another study, patients showed impaired use-dependent plasticity, a transcranial
magnetic stimulation (TMS) paradigm that assesses the brain's adaptation to the direction of
trained movement.(Daskalakis, Christensen et al. 2008) This deficit was independent of
medication, as patients who were on and off medication exhibited impairments in LTP to the
same degree (Daskalakis, Christensen et al. 2008). Impairments have also been demonstrated
using PAS. Following PAS-induced LTP (PAS-LTP) was present in healthy controls but absent
in patients with schizophrenia (Frantseva, Fitzgerald et al. 2008). These patients also showed
impaired motor learning, an outcome of abnormal LTP facilitation, (Frantseva, Fitzgerald et al.
2008) which lasted one-week post-PAS (Rajji, Liu et al. 2011). Most importantly, PAS-LTP was
also impaired in patients when assessed from the dorsolateral prefrontal cortex (DLPFC) using
the combination of PAS and electroencephalography (Rajji 2014). This finding is important
7
given that the DLPFC is directly involved in cognitive functions including learning and memory,
and abnormalities in function may underlie cognitive impairments in this population.
1.4 Dorsolateral Prefrontal Cortex and Schizophrenia
1.4.1 Functional Abnormalities
The DLPFC is a part of the prefrontal cortex (PFC) and connected to the orbital frontal cortex,
thalamus, basal ganglia and the primary and secondary area of the neocortex (Fuster 1995). It is
involved in higher order cognitive processes, including executive function, learning and working
memory, (Fuster 1995; Curtis and D'Esposito 2003) and damage to this region causes severe
deficits (Barbey, Koenigs et al. 2013). As such, compromised DLPFC function is thought to
underlie cognitive disturbances in schizophrenia. (Goto, Yang et al. 2010). In fact, abnormalities
have consistently been shown in the left DLFC, as both hypo-activity (Weinberger, Berman et al.
1986) and hyperactivity (Manoach, Press et al. 1999) which have been reported in both
medicated and unmedicated patients during working memory tasks.
1.4.2 Anatomical Abnormalities
Anatomically, the DLPFC of patients with schizophrenia is also different. For example, the right
DLPFC in patients is denser compared to the left, while the left is denser in healthy controls.
Also, in healthy controls, pyramidal neurons are larger and rounder in the left DLPFC compared
to the right, while no significant difference has been seen between the right and left DLPFC in
patients with schizophrenia (Cullen, Walker et al. 2006). As such, these findings highlight that
abnormal DLPFC function and anatomy in schizophrenia may be a contributing factor for
cognitive impairments.
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1.5 Long-term Potentiation of the Dorsolateral Prefrontal Cortex
The PFC has been proposed to be involved in memory because LTP has been induced in the
DLPFC, (Wang and Arnsten 2015), medial prefrontal cortex (MPFC), (Otani, Daniel et al. 2003)
and its afferents from the hippocampus (Laroche, Jay et al. 1990; Gurden, Tassin et al. 1999),
amygdala (Maroun and Richter-Levin 2003), thalamus (Herry, Vouimba et al. 1999),
and sensory cortex (Kim, Chun et al. 2003). LTP in the PFC is mediated by glutamate (Jay,
Burette et al. 1995) and gamma-aminobutyric acid (GABA)(Vickery, Morris et al. 1997), and
modulated by dopamine (Gurden, Tassin et al. 1999; Otani, Daniel et al. 2003; Huang, Simpson
et al. 2004; Goto and Grace 2006; Matsuda, Marzo et al. 2006) and acetylcholine (Couey,
Meredith et al. 2007; Lopes Aguiar, Romcy-Pereira et al. 2008). These neurochemicals are also
abnormal in schizophrenia. As such, these neurochemicals and their effect on LTP and impact on
schizophrenia will be discussed in the next section.
1.6 Glutamate
1.6.1 Glutamate and Glutamate Receptors
Glutamate is the most abundant excitatory neurotransmitter and the metabolic precursor of
GABA, the major inhibitory neurotransmitter (Petroff 2002) in the brain. Glutamate binds to
three classes of ionotropic receptors, AMPA, kainate, and NMDA receptors (Nakanishi 1992).
These receptors are ion channels that when activated cause an influx of Na+ and/or Ca2+ ions and
an efflux of K+ ions (Honore 1989).
Glutamate also activates eight metabotropic guanosine triphosphate (GTP) (G)-protein coupled
receptors labeled from mGluR1 to mGluR8 (Nakanishi 1992). In general, when a G-protein
9
receptor is activated, guanosine diphosphate (GDP) is converted to GTP causing the α and βγ
subunits to dissociate from each other, which allows both subunits to activate downstream
signaling pathways. Due to the scope of this thesis, the effects of these receptors will be not be
explored further.
1.7 Glutamatergic Activity on Long-term Potentiation
As the main glutamatergic receptors, AMPA and NMDA receptors play an essential role in
mediating the induction of LTP (McEntee and Crook 1993). Normally, a weak stimulation
activates only AMPA receptors, allowing a small amount of Na+ ions to enter the postsynaptic
neuron (Malenka 2003). This influx slightly depolarizes the neuron but not sufficiently to
activate NMDA receptors. In contrast, a strong stimulation causes a large amount of Na+ ions to
enter. This influx expels Mg2+ ions that are blocking the pore of NMDA receptors. Once
unblocked Ca2+ and Na+ ions can rush in and depolarize the neuron (Malenka and Bear 2004).
Increased concentration of Ca2+ sets off a cascade of biochemical reactions, which ultimately
upregulate AMPA receptors and makes the synapse more efficient (McEntee and Crook 1993).
Several manipulation studies have also shown that NMDA receptors are vital for the induction of
LTP. For example, in animal studies, the non-competitive NMDA receptor antagonists,
dextromethorphan (Church, Lodge et al. 1985) abolished LTP in the hippocampus (Krug 1993).
Similarly, the NMDA antagonist D-(-) 2-amino-5-phosphonopentanoic acid (D-AP5) blocked
LTP at the hippocampal-prefrontal cortex pathway (Jay, Burette et al. 1995). This effect was also
shown in humans as dextromethorphan abolished PAS-LTP in the motor cortex (Stefan, Kunesch
et al. 2002). This finding is important as it demonstrates that PAS-LTP is NMDA-dependent and
shares similar mechanisms as LTP induced in animals.
10
Therefore, since glutamate plays an important role in LTP facilitation, then abnormal
glutamatergic activity may lead to aberrant synaptic plasticity, which in turn can promote
cognitive deficits in schizophrenia. For this reason, abnormal glutamatergic activity has been the
focus of several hypotheses attempting to explain cognitive symptoms of schizophrenia.
1.8 Glutamatergic Activity Associated Schizophrenia
The glutamate hypothesis of schizophrenia posits that cognitive symptoms are associated with
abnormal glutamatergic signaling, in particular, hypofunction of NMDA receptors (Kim,
Kornhuber et al. 1980; Olney, Newcomer et al. 1999; Coyle and Tsai 2004). Support for this
hypothesis comes from several lines of evidence. First, recreational drugs, such as phencyclidine
(PCP) and ketamine, which are NMDA receptor antagonists induce both positive and negative
symptoms similar to those observed in schizophrenia (Luby, Cohen et al. 1959; Javitt 2007).
Second, low levels of glutamate have been reported in the cerebrospinal fluid of patients with
schizophrenia (Kim, Kornhuber et al. 1980). Third, cognitively impaired patients display a 30%
reduction in the expression of NMDA receptors in the temporal cortex compared to healthy
controls, whereas patients without cognitive impairment show no such reduction (Humphries,
Mortimer et al. 1996). Fourth, NMDA receptors are reduced in the superior frontal region of
drug-naïve patients but not in antipsychotic-treated patients, suggesting that antipsychotics may
have upregulated NMDA receptors back to normal (Sokolov 1998). Lastly, the administration of
ketamine to healthy participants promotes abnormal hyper-connectivity and activation, and this
hyper-connectivity is related to positive and negative symptoms (Driesen, McCarthy et al. 2013).
Thus, overall these studies provide support for abnormal glutamatergic activity, which may
contribute to cognitive symptoms of schizophrenia. Nevertheless, glutamatergic activity is
11
regulated by complex systems in the brain, which involves other neurotransmitters including
dopamine, acetylcholine, and GABA. Thus, although there is strong support for the
glutamatergic hypothesis, these alterations are unlikely to exist in isolation.
1.9. Dopamine
Dopamine (3, 4-dihydroxyphenethylamine) is the catecholamine neuromodulator. The metabolic
pathway for the synthesis of dopamine starts with tyrosine. Tyrosine is converted into L-DOPA
(3, 4-dihydroxyphenylalanine) by tyrosine hydroxylase, and L-DOPA is decarboxylated into
dopamine by aromatic L-amino acid decarboxylase. After synthesis, dopamine is transported
into synaptic vesicles known as vesicular monoamine transporter (VMAT) that are released into
the synaptic cleft via an action potential. After release dopamine is degraded into homovanillic
acid by three main enzymes know as, monoamine oxidase (MAO), catechol-O-methyl
transferase (COMT), and aldehyde dehydrogenase. There are two isoforms of monoamine
oxidase, MAO-A and MAO-B and both metabolize dopamine (Larry. R Squire 2008). The
metabolites are then removed out of the bloodstream and filtered out by the kidneys to be
excreted in the urine.
1.9.1 Dopaminergic Pathways
Dopamine is the most abundant catecholamine neuromodulator and as such plays a vital role in
voluntary movement, motivation, sleep, mood, and working memory (Larry. R Squire 2008).
These actions are mediated by four main dopaminergic pathways within the brain, these are
known as the neostriatal, mesolimbic, tuberoinfundibular and mesocortical pathways. The first
pathway, the nigrostriatal pathway originates from the substania nigra and innervates the dorsal
12
striatum, which is composed of the caudate and putamen. This pathway plays a significant role in
the motor control and learning new motor skills. The second pathway, the mesolimbic pathway
starts from the ventral tegmental area (VTA) as well as, the retrorubral area and projects to
several regions of the limbic system, including the nucleus accumbens (or ventral striatum), the
septum, olfactory tubercle, amygdala, and piriform cortex. These pathways play a central role in
reward, memory and other aspects of motivation. The third pathway is the tuberoinfundibular
pathway, it starts from the periventricular and arcuate nuclei of the hypothalamus and innervates
the median eminence of the hypothalamus. This pathway modulates the secretion of prolactin
from the anterior pituitary. The last pathway, the mesocortical pathway originates from the VTA
and innervates the cortex, including the prefrontal, cingulate, and entorhinal cortex.
Dopaminergic neurons in this pathway are vital as they regulate information flow from other
areas of the brain and modulate cognitive function (Larry. R Squire 2008). There is also a source
of dopaminergic neurons in the hypothalamus which innervates the central nucleus of the
amygdala, Broca’s area, and paraventricular nucleus of the hypothalamus, which are suggested
to play a key role in learning, motivation and memory.
1.9.2 Dopamine Receptors
Dopamine activates 5 types of metabotropic G-protein-coupled receptors referred to as, D1, D2,
D3, D4 and D5 (Kebabian and Calne 1979; Neve, Seamans et al. 2004). D1 and D5 receptors are
generally categorized as excitatory, while D2, D3 and D4 receptors are classified as inhibitory
(Neve, Seamans et al. 2004). D1 and D5 are grouped together because their G-protein (Go)
stimulates adenylate cyclase, which in turn increases Ca2+ concentrations. These receptors are
mainly present in the striatum, nucleus accumbens, olfactory tubucle, limbic system,
13
hypothalamus, thalamus, and cortex. In contrast, D2, D3, and D4 receptors are grouped together
because their G-protein (Gi) inhibits adenylate cyclase (Neve, Seamans et al. 2004). These
receptors are found in the striatum, olfactory tubercle, nucleus accumbens, substania nigra pars
compacta and ventral tegmental area, on both presynaptic and postsynaptic dopaminergic
neurons (Larry. R Squire 2008). For simplicity, throughout the rest of the thesis D1/D5 receptors
will be referred to as D1 receptors and D2/D3/D4 as D2, unless otherwise specified.
1.9.3 Dopamine Release
Dopamine release is regulated by two states known as phasic and tonic release. Tonic release
maintains a steady “pacemaker “ background level of dopaminergic tone, while phasic release
consists of spontaneous bursts of firing, which is in response to behaviorally relevant stimuli. D2
receptors are more sensitive to changes in the tonic release, while D1 receptors are sensitive to
changes in the phasic release. Although dopamine activates both types of receptors, it has a
greater affinity for D2 receptors compared to D1 receptors. Thus, D1 receptors may play a greater
role in triggering plasticity, while D2 receptors modulate the direction plasticity.
1.10 Dopaminergic Activity on Long-term Potentiation
1.10.1 D1 Receptor Activation
Several studies have investigated the role of D1 and D2 receptors in the induction of LTP.
Numerous studies have been shown that D1 activation facilitates the induction of LTP. For
instance, this has been demonstrated in pyramidal neurons in the hippocampus (Roggenhofer,
Fidzinski et al. 2010, striatum {Hu, 1997 #6182), and PFC (Tseng and O'Donnell 2004). This has
also been shown in the hippocampal-prefrontal synapse (Gurden, Takita et al. 2000). This study
14
showed that D1 antagonism causes a dose-dependent impairment in LTP induction, illustrating
the importance of D1 receptors in transferring information from the hippocampus to the PFC
(Gurden, Takita et al. 2000).
1.10.2 D2 Receptor Activation
The activation of D2 receptors on presynaptic GABAergic interneurons also plays a role in LTP
facilitation. For instance, in the striatum D2 receptors on presynaptic GABAergic interneurons
reduced GABA release, which in turn increased postsynaptic glutamatergic transmission (Cooper
and Stanford 2001). Similar results were shown in the amygdala, as dopamine promoted LTP by
suppressing feed-forward inhibition from local interneurons (Bissiere, Humeau et al. 2003).
1.10.3 D1 and D2 Receptor Activation
The cooperative activation of D1 and D2 receptors have also been shown to facilitate LTP. For
example, in the striatum, the co-application of D1 receptor agonist SKF 38393 combined with the
D2 receptor agonist quinpirole (Hu and White 1997) enhanced LTP. Similar results were shown
using dopamine. When primed with dopamine a weak stimulation which would lead to LTD
instead, induced LTP (Matsuda, Marzo et al. 2006). This study highlights the importance of tonic
dopamine activity in the modulation of LTP. Further, in the monkey PFC, the application of
dopamine was shown to enhance neuronal activity associated with memory. This enhancement
was hindered by D2 antagonists, fluphenazine, and haloperidol, but a weaker D2 antagonist,
sulpiride had no significant effect (Sawaguchi, Matsumura et al. 1988).
15
1.10.4 Dose-dependent Effects of Dopamine
Furthermore, it has been shown that the facilitating effects of dopamine follow an inverted-U
shaped dose response curve, whereas concentrations that are too low or too high impair LTP.
(Seamans and Yang 2004; Kolomiets, Marzo et al. 2009). This is because when dopaminergic
neurons are stimulated, dopamine is phasically released, which when combined with low tonic
background dopamine, induces LTP via D1 receptor activation. However, excessive amounts of
tonic or phasic dopamine can activate D2 receptors and impair the induction of LTP.
Several human studies have also assessed the effects of dopamine on in vivo LTP from the motor
cortex. First, it has been shown that L-DOPA, a dopamine precursor increases the magnitude and
duration of PAS-LTP (Kuo, Paulus et al. 2008), implying, as noted previously, that a balanced co-
activation of D1 and D2 receptors is necessary for optimal LTP induction (Hu and White 1997).
Second, this effect by L-DOPA was not affected by the D2 receptor antagonist sulpiride (Nitsche,
Kuo et al. 2009) or the D2 agonist cabergoline (Korchounov and Ziemann 2011) suggesting that
D1 receptors play a slightly greater role in enhancing PAS-LTP. Third, haloperidol a stronger D2
antagonist impaired PAS-LTP, indicating that D2 receptors, although to a lesser degree than D1
receptors, play a vital role in facilitating LTP (Nitsche, Kuo et al. 2009; Korchounov and Ziemann
2011)(Nitsche, Kuo et al. 2009; Korchounov and Ziemann 2011). Lastly, it has been shown that
the facilitating effects of L-DOPA, similar to dopamine, follows an inverted U-shaped dose-
response curve, where concentrations that are too high or too low abolish PAS-LTP
(Thirugnanasambandam, Grundey et al. 2011). Therefore, these studies suggest that LTP and
memory are promoted by increasing dopaminergic tone through both D1 and D2 receptor
activation.
16
1.11 Dopaminergic Activity Associated with Schizophrenia
Abnormal dopaminergic activity has long been hypothesized to be associated with
schizophrenia. The first version of the dopamine hypothesis suggested that increased dopamine
transmission was responsible for schizophrenic symptoms. This association was postulated based
on the observation that drugs such as cocaine and amphetamine, which increase dopaminergic
activity induce psychotic symptoms similar to those observed in schizophrenia (Snyder 1972;
Lieberman, Kane et al. 1987). This hypothesis was later strengthened by the finding that D2
antagonists such chlorpromazine effectively reduce positive symptoms, and were subsequently
used as antipsychotics (Seeman and Lee 1975).
While the first version of the dopamine hypothesis accounted for positive symptoms, it did not
explain negative symptoms. As such a second version was developed that included an
explanation for negative symptoms and regional specificity. This theory proposed that
dopaminergic signaling is increased in the striatum, producing positive symptoms and reduced in
the PFC, producing negative symptoms (Davis, Kahn et al. 1991; Howes and Kapur 2009). This
reconceptualization supported the observation that drug-naïve and drug-free patients displayed
increased D1 receptors in the DLPFC, possibly due to a compensatory effect of
hypodopaminergic activity (Abi-Dargham, Mawlawi et al. 2002). They also have increased
baseline and amphetamine-induced dopamine release in the striatum, suggesting
hyperdopaminergic activity (Abi-Dargham, van de Giessen et al. 2009).
17
1.12 Acetylcholine
Acetylcholine is a neuromodulator that is synthesized from two precursors, acetyl coenzyme A
(acetyl-CoA) and choline by the enzyme choline acetyltransferase (ChAT). It is packaged into
synaptic vesicles by the vesicular acetylcholine transporter (VAChT), which is subsequently
released into the synaptic cleft. After release, acetylcholine is hydrolyzed by the enzyme
acetylcholine esterase (Dani and Bertrand 2007). This enzyme breaks down acetylcholine to
acetyl and choline, which is then taken up by the neuron and recycled (Dani and Bertrand 2007).
1.12.1 Cholinergic Pathways
Acetylcholine is produced by cholinergic neurons in the basal forebrain, a brain area composed
of several cholinergic nuclei, including the nucleus basalis of magnocellularis , medial septum
nucleus, substantia innominata and diagonal band of Broca (Mesulam 1995; Woolf and Butcher
2011). It is also produced by the pedunculopontine nucleus, medial habenula and laterodorsal
tegmental area in the brain stem (Mesulam, Mufson et al. 1983), and in sparsely distributed
cholinergic interneurons (Eckenstein and Baughman 1984; von Engelhardt, Eliava et al. 2007).
Cholinergic neurons in the medial septum and the diagonal band of Broca project to the
hippocampus, while neurons in the nucleus basalis of magnocellularis project to the neocortex
and to the amygdala. The PFC receives cholinergic innervation from the basal forebrain, basal
nucleus, parts of the diagonal band of Broca, magnocellular preoptic nucleus and substantia
innominate. Innervations to the PFC have been shown to regulate and modulate cognitive
functions (Ragozzino 2000).
18
1.12.2 Nicotinic Receptors
Acetylcholine binds to two classes of receptors, referred to as muscarinic and nicotinic,
originally named for their specific activation by nicotine and muscarine, respectively (Dani and
Bertrand 2007).Nicotinic acetylcholine receptors are widespread and found on all neurons,
including GABAergic, glutamatergic, and dopaminergic. These receptors are ligand-gated ion
channels composed of five subunits (Gotti and Clementi 2004), which are further divided into
two main subfamilies, the homopentameric, and heteropentameric. The homopentameric
receptors are composed of five α7 subunits, while, the heteropentameric receptors are composed
of two α4 subunits, two β2 subunits and a fifth subunit, which can be α4, β2 or α5 (Albuquerque,
Pereira et al. 2009). Further, there are seven isoforms of the α subunit (α2 – α7), and three
isoforms of the β subunit (β2 – β4), which allow for multiple arrangements that create for various
biological effects (Mineur and Picciotto 2008).
Nicotinic receptors can modify neuronal state depending on their location. Receptors that are
located on postsynaptic neurons depolarize the neuron through an influx of Na+, and efflux of
K+, while nicotinic receptors that are on presynaptic neurons regulate neurotransmitter
release(Dani and Bertrand 2007). Further, it should also be noted that some homopentameric, α7
nicotinic receptors are permeable to calcium, and these receptors play an important role in
facilitating LTP by influencing both neurotransmitter release and neuronal depolarization {Shen,
2009 #10844
19
1.12.3 Muscarinic Receptors
Muscarinic acetylcholine receptors are seven transmembrane G-protein coupled receptors.
There are five types of muscarinic receptors, numbered M1-M5 {Bubser, 2012 #8803}. These
receptors are sub-grouped based on the type of G-protein that binds to the α subunit. The first
group includes M1, M3 and M5 receptors, which interact with Gq/11 proteins, whereas the second
group consists of M2 and M4 receptors, and these interact with Gi proteins (Brown 2010).
M1, M3 and M5 Receptors
The first group, the M1, M3 and M5 receptors are mainly excitatory and postsynaptic (Levey,
Kitt et al. 1991). The G-protein of these receptors activates phospholipase C, which initiates the
phosphatidylinositol triphosphate signaling cascade leading to an increase in intracellular Ca2+
concentration and activation of protein kinase C while reducing K+ conductance. M1 receptors
are mainly found in cerebral cortex, hippocampus, and corpus striatum (Oki, Takagi et al. 2005),
while M3 and M5 receptors are expressed consistently throughout the central nervous system
(CNS), but are highly expressed on smooth muscle and glandular tissues (Levey 1993).
M2 and M4 Receptors
The second group, the M2, and M4 receptors are usually inhibitory and decrease intracellular
levels of cAMP by inhibiting adenylate cyclase (Gulledge and Stuart 2005). Generally, M2
receptors mediate postsynaptic inhibition, whereas M4 receptors mediate presynaptic inhibition
(Zhang and Warren 2002). M4 receptors are predominately found in the striatum, whereas M2
receptors are most abundant in the thalamus-hypothalamus and the pons-medulla region, and
20
some are found in the cortex, hippocampus and striatum where they control acetylcholine release
(Raiteri, Marchi et al. 1990; Wei, Walton et al. 1994).
1.13 Cholinergic Activity in Modulating Long-term Potentiation
Research has shown that cholinergic nicotinic and muscarinic receptors are essential for
attention, learning and working memory. As such these receptors play a vital role in regulating
plasticity (Tracy, Monaco et al. 2001; Sarter, Bruno et al. 2003; Pepeu and Giovannini 2004).
Nicotinic receptors modulate plasticity through both presynaptic and postsynaptic activity.
Presynaptic nicotinic activation leads to an influx of Ca2+, which increases the probability of
neurotransmitter release (Albuquerque, Pereira et al. 1997; Mansvelder and McGehee 2000),
while, postsynaptic activation, increases the probability of neuronal depolarization (Blitzer, Gil
et al. 1990; Jones, Sudweeks et al. 1999).
Several animal studies have assessed the effects of nicotinic receptor activation on LTP
plasticity. For example, in the hippocampus and piriform cortex nicotinic activation enhanced
LTP in vivo (Blitzer, Gil et al. 1990; Auerbach and Segal 1994; Patil, Linster et al. 1998;
Matsuyama, Matsumoto et al. 2000). Similar results were demonstrated in the PFC,
glutamatergic neurons from the medial dorsal thalamus that project to the PFC were potentiated
by nicotinic agonists (Vidal and Changeux 1993).
Muscarinic receptors also play a role in modulating LTP. For instance, postsynaptic activation of
M1 receptors enhances LTP in the hippocampus (Natsume and Kometani 1997) and cortex
21
(Gulledge, Bucci et al. 2009) by facilitating pyramidal neuron firing and increasing Ca2+
concentration from intracellular stores. This enhancement in the hippocampus may be regionally
sensitive as in the CA3 region, muscarine decreased LTP (Williams and Johnston 1988), whereas
in the dentate gyrus LTP was facilitated by muscarine (Burgard and Sarvey 1990). In contrast,
scopolamine, an M1 muscarinic receptor antagonist blocked LTP (Hirotsu, Hori et al. 1989),
illustrating the importance of M1 receptors in facilitating LTP.
Like M1 receptors, M2 receptors also play a key role in modulating LTP. For example, M2 knockout
rodents exhibit significant problems in working memory, hippocampal plasticity, and behavioral
flexibility (Seeger, Fedorova et al. 2004). In contrast, M3 knockout rodents do not present
behavioral or cognitive problems (Yamada, Miyakawa et al. 2001), illustrating that these receptors
play less of a role in cognitive processes. Furthermore, several studies have shown that signaling
through M4 receptors facilitates plasticity. For example, studies using M4 genetic knock-out
rodents or the M4- antagonist, MT3, demonstrate impaired plasticity, indicating that M4 receptors
also contribute to synaptic plasticity (Bonsi, Martella et al. 2008; Dasari and Gulledge 2011).
Further, the application of MT3 impairs memory performance in rodents (Ferreira, Furstenau et
al. 2003), highlighting the vital role of M4 receptors in plasticity and memory.
Cholinergic activity on LTP has also been assessed in vivo in the human motor cortex. For
example, use-dependent plasticity was blocked by the non-selective muscarinic antagonist
scopolamine (Sawaki, Boroojerdi et al. 2002; Meintzschel and Ziemann 2006). The muscarinic
M1 receptor antagonist, biperiden, also suppressed both use-dependent plasticity (Meintzschel
and Ziemann 2006) and PAS-LTP (Korchounov and Ziemann 2011). In contrast, tacrine, another
22
cholinesterase inhibitor, had no significant effect on PAS-LTP but enhanced use-dependent
plasticity (Meintzschel and Ziemann 2006). On the other hand, the cholinesterase inhibitor,
rivastigmine enhanced PAS-LTP by inactivating acetylcholine esterase, which allows for longer
cholinergic activation (Kuo, Grosch et al. 2007). Similar results were also seen with nicotine
(Thirugnanasambandam, Grundey et al. 2011) and varenicline, a nicotinic agonist (Batsikadze,
Paulus et al. 2015), suggesting that enhancement of PAS-LTP may be mediated through nicotinic
receptors. (Metherate and Ashe 1993; Letzkus, Wolff et al. 2011)
1.14 Cholinergic Activity in Schizophrenia
Considering cholinergic activity is pivotal for cognition, dysregulation may be partly responsible
for cognitive impairments seen in schizophrenia (Smucny, Olincy et al. 2013; Smucny and
Tregellas 2013; Ahlers, Hahn et al. 2014). In fact, nicotinic receptors in the frontal cortex,
hippocampus, and striatum are downregulated and reduced in patients (Freedman, Hall et al.
1995; Guan, Zhang et al. 1999; Breese, Lee et al. 2000; Durany, Zochling et al. 2000; Esterlis,
Ranganathan et al. 2014). Patients also demonstrate reduced expression and function of the α7
nicotinic receptors, which play an important role in cognition and plasticity (Leonard, Adams et
al. 1996; Freedman, Adams et al. 2000). Similarly, muscarinic receptors are downregulated in
the frontal cortex, (Crook, Tomaskovic-Crook et al. 2001), hippocampus (Crook, Tomaskovic-
Crook et al. 2000) and striatum (Dean, Crook et al. 1996). This reduction is also evident in
unmedicated patients with schizophrenia, suggesting that these abnormalities are illness related
and independent of medication use (Raedler, Knable et al. 2003). Furthermore, the M1/M4
agonist, xanomeline has been shown to have antipsychotic effects, in addition to enhancing
cognition (Shannon, Rasmussen et al. 2000; Felder, Porter et al. 2001). As such, based on these
23
findings impaired nicotinic and muscarinic receptors in patients with schizophrenia may play a
role in the abnormal plasticity observed in this population.
1.15 Gamma-aminobutyric Acid (GABA)
Gamma-aminobutyric acid (GABA) is the most abundant inhibitory neurotransmitter, as 15-30
% of all neurons are GABAergic (DeFelipe 2002). GABA is synthesized from glutamate by the
enzyme glutamic acid decarboxylase (GAD) and pyridoxal phosphate (an active form of vitamin
B6), which acts as a co-factor. There are two forms of GAD that are named by their molecular
weight, GAD65, and GAD67. Synaptic GABA acts on two main classes of GABA receptors, the
GABAA and, GABAB type, each which mediates inhibition through a distinct mechanism. After
release, GABA is taken up via presynaptic GABA transporters (GAT1) (DeFelipe 2002).
1.15.1 GABAergic Interneurons
A subgroup of GABAergic neurons are interneurons, which are classified by their morphology,
or their synaptic connections with pyramidal neurons (DeFelipe 2002). The three most common
types include basket, chandelier, and double bouquet cells. Basket cells act at the soma or
proximal dendrite of pyramidal neurons and as result have powerful inhibitory effects (Amitai
2001). These cells play an essential role in controlling sensory information from the thalamus via
feed-forward inhibition (Amitai 2001). Chandelier cells, on the other hand, act at the axon
hillock or initial segment of pyramidal neurons. Due to their location, these cells can prevent the
propagation of an action potential and as such play an important role in regulating the output of
pyramidal activity (DeFelipe 2002). These cells are abundant in the cortex and hippocampus and
play an important role in learning and memory (Benes and Berretta 2001). In contrast, double
24
bouquet cells synapse at the dendrite of pyramidal neurons and as a result have less of an
influence on pyramidal firing (DeFelipe 2002).
Interneurons are further sub-grouped by their expression of neuropeptides (somatostatin and
cholecystokinin) and calcium-binding proteins (calbindin and parvalbumin). Calcium binding
proteins include calbindin, which is abundant and expressed in double bouquet cells, than
parvalbumin, which is expressed in only 25% of interneurons, predominantly in basket and
chandelier cells (DeFelipe 2002). Parvalbumin-expressing cells are fast-spiking and
consequently have high temporal control over pyramidal neuron firing, playing an essential role
in the modulation of cortical oscillations that mediate cognition (DeFelipe 2002).
1.15.2 GABAA Receptors
GABAA receptors are heterooligomeric and made up from a combination of five subunits from
a possible sixteen (α1-6, β1-3, γ1-3 (with two splice variants, γ2Long and γ2Short), δ, ε, and θ)
(Macdonald and Olsen 1994). This diversity in GABAA subunits suggests that there are many
different subtypes of GABAA receptors in the brain. GABAA receptors are mainly found in the
frontal cortex, granule cell layers of the cerebellum, olfactory bulb and thalamic medial
geniculate (Bowery, Hudson et al. 1987). These receptors are ligand-gated chloride channels and
when activated cause an influx of chloride (Cl−) ions that hyperpolarize the neuron (Bowery and
Smart 2006). These receptors are fast acting and contribute to the early part of the inhibitory
inhibitory post synaptic potential (IPSP) with a short synaptic time constant between 10 to 25ms
(McCormick 1989).
25
1.15.3 GABAB Receptors
Unlike GABAA receptors, GABAB receptors are metabotropic receptors. These receptors are a
heterodimer with two subunits, GABAB1 and GABAB2. GABAB receptors are coupled to second
messenger system that activates a G-protein that inhibits adenylyl cyclase, reducing cAMP
production and phosphorylation (Bowery 1993). GABAB receptors also activate nearby K+
channels that hyperpolarize the neuron by inhibiting Ca2+ channels (Bowery 1993) (McCormick
1989). These receptors are abundant in the thalamic nuclei, cerebellum, and dorsal horn of the
spinal cord (Bowery, Hudson et al. 1987). In contrast to GABAA receptors which are mainly
postsynaptic, GABAB receptors are located on both presynaptic and postsynaptic neurons.
Presynaptic GABAB receptors are autoreceptors and regulate GABA release by preventing Ca2+
entry. Postsynaptic GABAB receptors hyperpolarize the neuron and mediate the late part of the
IPSP with a long synaptic time constant between 50 to 200ms (Karlsson and Olpe 1989;
McCormick 1989).
1.16 GABAergic Activity in Modulating Long-term Potentiation
GABA neurotransmission plays a pivotal role in modulating LTP. For example, it can facilitate
LTP by decreasing the release of GABA by inhibiting presynaptic voltage-gated calcium channels
via GABAB receptor-mediated auto-inhibition. (Mott, Lewis et al. 1990; Davies, Starkey et al.
1991). In fact, in rodents, the deletion of these auto-receptors impaired the induction of LTP (Vigot,
Barbieri et al. 2006). GABA can also impair LTP through activation of postsynaptic GABAB
receptors, by hyperpolarizing the neuron and making LTP induction more difficult (Olpe and
Karlsson 1990; Olpe, Worner et al. 1993). This hyperpolarizing effect is also demonstrated through
GABAA receptor activation. For instance, in rat hippocampal slices, midazolam, a positive
26
allosteric modulator of the GABAA receptor, prevented the induction of LTP following theta burst
stimulation. This effect was reversed, by the GABAA receptor antagonist, bicuculline (Evans and
Viola-McCabe 1996), suggesting that GABAA action is critical for the regulation of LTP. In
contrast, the GABAB agonist, baclofen, reduced the population spike amplitude, but had no
significant effect on the induction of LTP, while the GABAB antagonist, phaclofen facilitated the
induction of LTP, suggesting that GABAB has a modulatory role on LTP (Olpe and Karlsson 1990).
Several human studies have assessed GABAergic activity on in vivo PAS-LTP from the motor
cortex. For example, the GABAB agonist, baclofen, impaired PAS-LTP (McDonnell, Orekhov et
al. 2007), potentially by hyperpolarizing the neuron. In contrast, diazepam and topiramate,
tiagabine, all had no significant effect on PAS-LTP (Heidegger, Krakow et al. 2010). However,
the activation of GABAA receptors prior to PAS using a TMS paradigm known as short interval
cortical inhibition (SICI) impaired PAS-LTP (Elahi, Gunraj et al. 2012). This suggests that SICI
may have blocked the induction of PAS-LTP via depotentiation mechanisms. As such these studies
indicate that GABAergic activity plays an important role in the regulation of cortical plasticity in
the human motor cortex.
1.17 GABAergic Activity in Schizophrenia
It has been proposed that altered GABAergic neurotransmission, particularly in the frontal
lobes, is associated with cognitive symptoms of schizophrenia (Coyle 2004) (Hashimoto, Arion
et al. 2008; Tse, Piantadosi et al. 2015). This hypothesis was first postulated by Roberts based on
the inhibitory role that GABA has in the central nervous system and how a reduction can lead to
excessive neuronal excitation (Roberts and Frankel 1950). Several post-mortem studies have
since provided support for this hypothesis. For instance, reduced density of GABAergic
27
interneurons has been shown in the PFC and anterior cingulate (Benes, McSparren et al. 1991). It
may be that only a subset of these neurons is affected, as the density of GABA cells that express
only the NMDA NR2A subunit have been shown to be reduced in anterior cingulate of patients,
demonstrating impaired glutamate-GABA interaction (Woo, Walsh et al. 2004). Further, reduced
levels of GABA have also been shown in the thalamus, nucleus accumbens and amygdala (Perry,
Kish et al. 1979; Blum and Mann 2002). This reduction may reflect altered GAD expression, the
enzyme responsible for the production of GABA, which has also been reported in the DLPFC
(Akbarian and Huang 2006; Curley, Arion et al. 2011), amygdala, hippocampus, nucleus
acumens and putamen (Bird, Spokes et al. 1977). This reduction is predominately in fast-spiking
parvalbumin GABAergic interneurons (Hashimoto, Volk et al. 2003) and shown to be
independent of antipsychotic use, suggesting that this effect is inherent to the disorder (Volk,
Austin et al. 2000). Given that these fast-spiking neurons play an essential role in the generation
of gamma oscillations, which are vital for cognitive function, then abnormalities may underlie
cognitive deficits seen in this population (Sohal, Zhang et al. 2009; Carlen, Meletis et al. 2012).
Furthermore, position emission tomography (PET) studies have revealed abnormal GABAA and
GABAB activity in patients. GABAA activity is elevated in both the PFC and hippocampus, two
regions important for cognitive function, in which abnormalities may lead to impairments
(Rudolph and Mohler 2014). Such alteration may reflect a compensatory upregulation of
GABAA receptors as a result of impaired inhibitory input onto pyramidal cells due to reduced
inhibitory interneurons, or GABA release (Nestler and Hyman 2010). On the other hand,
GABAB expression has been shown to be reduced in the hippocampus and temporal regions
(Mizukami, Sasaki et al. 2000; Mizukami, Ishikawa et al. 2002). Similar abnormalities have also
28
been reported in patients with the use of TMS paradigms known as long-interval cortical
inhibition (LICI) and cortical silent period (CSP), which measure GABAB neurotransmission,
and SICI, a measure for GABAA neurotransmission (these findings are discussed in detail below)
(Daskalakis, Christensen et al. 2002; Farzan, Barr et al. 2010). Intriguingly, it should be
mentioned that benzodiazepines have not been successful for cognitive or negative symptoms
but, baclofen, a GABAB agonist, improves cognition in animal models of schizophrenia,
suggesting that GABAB receptors may play a bigger role in cognitive deficits (Carpenter,
Buchanan et al. 1999; Hashimoto, Arion et al. 2008).Together, the evidence suggests that
GABAergic activity including, interneuron density, GAD and GABA levels are impaired in
schizophrenia. Thus, cognitive dysfunction in schizophrenia may be related to abnormal
GABAergic neurotransmission in the DLPFC, with GABAB receptors having a prominent role.
In conclusion, these diverse findings highlight that not one neurotransmitter alone can account
for cognitive symptoms in schizophrenia. Cognitive symptoms associated with schizophrenia
likely arise from dysfunctions within each of these neurotransmitter systems. It is clear however
that many unknowns still exist regarding the pathophysiology and etiology of this disorder and
continued research studies are necessary.
1.18 Transcranial Magnetic Stimulation
In 1985 Anthony Barker and his team developed a brain stimulation device known today as TMS.
It is a noninvasive tool used to examine the functioning and interconnections of the brain
(Kobayashi and Pascual-Leone 2003). The circuitry of the TMS machine is composed of four main
parts: the power source, the capacitor, a thyristor switch that closes the circuit and a coil. TMS
functions by periodic discharges of electrical energy that travels from the TMS machine to the coil
29
(Hallett 2000). When the switch is closed the capacitor stores the electrical energy, and when
sufficient energy is stored the switch opens and the current flows to the coil. Essentially, when the
current is zero, all the energy is in the capacitor, and when the current is at a maximum, the energy
is in the coil. When the coil is placed on the scalp, the changing electrical current induces a
transient magnetic field of about 2.5 Tesla, which is perpendicular to the plane of the TMS coil.
This magnetic field penetrates the scalp painlessly and unimpeded to induce a secondary electric
field in the brain, via the principle of electromagnetic induction (Barker, Jalinous et al. 1985). The
human brain being a conductive substance allows for a flow of electric current. The effect on the
brain is depolarization of neurons, not due to the magnetic field, but rather by the secondary electric
field produced in the brain. (Thielscher and Kammer 2004).
1.18.1 TMS Coils
Different coil shapes can be utilized depending on the degree of precision and stimulation depth.
There are primarily two types, a circular or figure-8 configuration.(Hallett 2000) The circular
shape is typically 8 to 15 cm in diameter and produces an antiparallel circular flow of opposite
direction in the brain underneath. The outer edges of the coil have the greatest induced current,
but the magnetic field produced is concentrated directly at the center of the coil. The circular coil
lacks the ability to focus on a single place in the brain, as the radius of the field applied is quite
large.
On the other hand, the figure 8 coil, resembling the number “8”, is more focal as it consists of
two coils placed side by side to create a junction. This concept is based on the mechanism of
electromagnetic induction, which states that when two loops of wire are in close proximity, the
changing primary current in one of the coils, and the resultant changing magnetic field generates
30
an electric field and consequently a secondary current of the opposite direction in the other coil.
Due to the opposite direction of flow, where they meet creates a concentration of current.
1.18.2 Potential TMS Risks
TMS has no significant health risk to healthy participants. Studies designed to systematically
evaluate health effects have not reported changes in blood pressure, heart rate, serum cortisol, serum
prolactin, cerebral blood flow (Rossi, Hallett et al. 2009). The FDA has concluded that stimulation
at <1 Hz carries only a slight risk of in inducing a seizure and is therefore classified as a device
with no significant risk.
Seizures in Patients with Neurological Abnormalities
To date no seizures have been reported in healthy participants receiving single-pulse TMS, nor
has an evaluation in serum prolactin levels associated with limbic after discharges been found in
normal controls receiving single-pulse TMS. Nonetheless, seizures have been reported in recent
stroke patients who were receiving single-pulse TMS for clinical evaluation purposes. To ensure
the participant’s safety, safety guidelines for TMS that minimize this possible seizure risk
including careful assessments of seizure vulnerability and history are assessed during the
eligibility assessments.
Headache and Scalp Pain
The most commonly reported side effect of TMS is a headache (~5%). Participants may also
experience some mild discomfort under the coil due to the contraction of facial muscles and
stimulation of nerves on the scalp. If the subject is discomforted by a headache, it is usually
managed with acetaminophen (Tylenol, 500 mg).
31
Magnetic Effects
Human tissue is virtually non-conductive to magnetic fields. The peak magnetic field strength of
the stimulator is approximately 2T. Exposure to static magnetic fields is considered safe up to 2T
in the context of a clinical MRI. The total time of exposure to the magnetic field in a TMS study
is usually brief compared to a clinical MRI.
1.18.3 TMS Activation
The current through the coil being parallel to the surface of the scalp strongly activates
horizontally-oriented interneurons and transynaptically activates pyramidal neurons rather than
directly. This activation area is several square centimeters (ie.15 mA/cm2) with a limited depth
(ie. 2.5cm), which depends on the stimulation intensity. When an above threshold intensity, a
TMS pulse that is delivered to the motor cortex depolarizes the neurons underneath the coil,
which in turn activates spinal neurons, and ultimately activates the peripheral muscle of interest.
This muscle activation is referred to as motor evoked potentials (MEPs), which can be measured
using electromyography (EMG) (Day, Dressler et al. 1989).
1.19 Transcranial Magnetic Stimulation and Electromyography
TMS combined with surface EMG allows for the exploration of the corticospinal tract in vivo.
Stimulation of the scalp over the motor cortex by a single TMS pulse produces a corresponding
contralateral MEP, which is non-invasively measured by EMG. Muscles at rest do not produce
detectable EMG signals, however, when activated EMG can detect the summation of several
superimposed motor unit action potentials over the muscle of interest. Several important factors,
32
including the shape of the magnetic coil, the intensity of the pulse, number of pulses and the
interstimulus interval separating the pulses influence the effect that the TMS pulse has on MEP
activity.
1.20 Paired Associative Stimulation
PAS is a non-invasive TMS paradigm used to assess LTP-like activity from the human cortex.
PAS involves the pairing of a weak electrical PNS to the median nerve with a strong TMS pulse
to the contralateral cortex (Stefan, Kunesch et al. 2000). Conventionally, PAS is administered
over the motor cortex (M1) with a 21.5 to 25ms time interval between the peripheral and cortical
stimulation and is referred to as PAS-25. This ~25 ms delay is important as it allows for the two
stimuli to arrive simultaneously in the cortex (Stefan, Kunesch et al. 2000). This combination
reflects associative plasticity and spike-timing dependent plasticity (Dan and Poo 2006).
Repetitive PAS stimulations result in increased cortical excitability known as LTP-like plasticity
(Stefan, Kunesch et al. 2000). This increase in cortical excitability from the motor cortex is
captured as an increase in MEP amplitude (Stefan, Kunesch et al. 2000). The ratio of change in
MEP amplitude before and after PAS-25 indicates the degree of potentiation. Potentiation of
MEP is greatest when ~25ms interval is used between the peripheral and cortical stimulation,
and with longer time intervals between the two being ineffective (Stefan, Kunesch et al. 2000).
Although PAS-25 is conventionally performed in the motor cortex, it can also be used to induce
plasticity in other regions of the cortex, with changes in cortical excitability measured through
electroencephalogram (EEG) (Rajji, Sun et al. 2013) (Figure 1.) . The magnitude of cortical
excitability is a neurological index that is similar to MEP amplitude, which is known as cortical
evoked activity (CEA). Recently, PAS has been induced and measured from the DLPFC. CEA
33
was greatly increased Post-PAS when compared to Pre-PAS. This effect was focal and localized
to the left frontal brain region and greatest in electrodes overlying the DLPFC (Rajji, Sun et al.
2013).
Figure 1. PAS in the motor Vs PAS in the DLPFC. This figure shows how PAS is performed
in the motor and in the DLPFC using TM-EEG
1.21 Electroencephalogram
EEG is a non-invasive device that records in vivo cortical electrical activity from the surface of
the scalp over a period of time (Swartz and Goldensohn 1998). It uses several metal-based
electrodes that are placed on the scalp that are filled with a conductive saline solution (Buzsáki
2006). This conductive gel reduces impedance, as it has a salt content of 3-10%. EEG readings
are derived from a summation of the electrical activity mainly from the pyramidal neurons
directly below the electrode as their axons run parallel to one another while their dendrites run
perpendicular to the cortical surface. Other neurons such as interneurons and glial cells
34
minimally contribute to the EEG signal because these neurons are not perpendicular to the
cortical surface.
Collectively these pyramidal neurons create an ionic current that the EEG picks up as a voltage
difference across low resistance extracellular space, namely, the distance between the brain and
the scalp. This current results from the movement of ions (Na+, K+, Ca2+ and Cl-) through various
kinds of channels (i.e. voltage-gated, ligand-gated, ion-dependent gated, and second-messenger
gated)(Buzsáki 2006). This electrical signal is processed through the use of three different types
of electrodes, known as the active, reference and ground electrodes. The active electrode is
compared to the reference electrode and the difference in voltage between these two electrodes is
transmitted over time. The ground electrode is needed for obtaining differential voltage by
subtracting the recorded measures from the active and reference points. This detected weak
signal is then amplified typically by a factor of 10,000 which is transmitted to a computer that
displays traces of electrical activities, showing EEG waves. When combined with TMS, EEG
can assess cortical activity known CEA. This technique is reliable and has been used to assess
activity from the motor (Ilmoniemi, Virtanen et al. 1997; Paus, Sipila et al. 2001; Bonato,
Miniussi et al. 2006) and prefrontal cortices (Daskalakis, Farzan et al. 2008; Sun, Farzan et al.
2016).
1.21.1 Cortical Oscillations
An EEG signal is composed of several brain frequencies known as cortical oscillations. These
oscillations are suggested to be generated by the movement of ions through various kinds of
channels (Buzsáki 2006). As such, a change in the voltage, release of neurotransmitters, or a
35
change in ion concentration can all change the state of these channels (i.e. open versus closed)
and give rise to oscillatory activity (Buzsáki 2006).
Cortical oscillations are categorized into five conventional groups based on their oscillating
frequency: alpha, beta, theta, delta and gamma (Buzsáki 2006). Alpha waves have a frequency
between 8 to 12Hz, with a large amplitude of approximately 50V peak to peak, and
predominately observed in the occipital region of the brain. These waves are evident when the
eyes are closed and in a purely relaxed state. Beta waves have a frequency range between 12 to
30Hz and generally associated with movement. In particular, beta waves increase when
movement is resisted or voluntary suppressed, but also occur when one is alert, actively
concentrating or anxious thinking. These waves are also evident during REM or deep sleep.
Delta waves have a frequency between 0 to 4Hz and are characterized by a slow wave and occur
in deep sleep. Theta waves have a frequency range of 4 to7Hz and are present during the light
stages of sleep, typically the earliest stage of sleep. Finally, gamma waves have the highest
frequency range of 30 to 80Hz and present during times of high concentration and cognitive
function. The precise mechanism involved in the generation of gamma oscillations are not fully
understood. Although GABAA receptors have been proposed to contribute to their generation,
while GABAB receptors play a role in the modulation of these oscillations (Whittington, Traub et
al. 1995).
1.22 EEG Artifacts and Independent Component Analysis (ICA)
EEG signals that are detected but that do not originate from cortical activity are called artifacts.
These artifacts can be separated into either biological or environmental noise. Some
environmental artifacts come from lights and electronic devices, and can be avoided. On the
36
other hand, biological artifacts are more difficult to avoid, which include eye movement,
blinking, muscle twitches and heart beats (Yuval-Greenberg, Tomer et al. 2008; Rogasch,
Thomson et al. 2014). These artifacts, however, can be teased out using independent component
analysis (ICA), a common technique used to remove artifacts from EEG recordings (Rogasch,
Thomson et al. 2014). Essentially, ICA assumes that each of the several components teased out
from the signal is independent of one another and that removing one component has no effect on
the others. This technique allows for accurate and precise removal of artifacts without affecting
brain related components (Rogasch, Thomson et al. 2014).
1.23 Cortical Inhibition (CI)
Synaptic plasticity may also be a corollary of CI since mechanisms mediating plasticity include
both cortical excitation and inhibition (Schieber and Hibbard 1993). CI has been defined as a
neurophysiological process by which inhibitory interneurons selectively suppress the activity of
pyramidal neurons, modulating and regulating excitatory activity (Daskalakis, Fitzgerald et al.
2007). TMS can be used to measure and index both cortical inhibition and excitation. CI consists
of two phases, a fast IPSP followed by a slow IPSP (Davies, Davies et al. 1990). Fast IPSPs are
mediated by GABAA receptor activation, whereas slow IPSPs are mediated by GABAB receptor
activation. (Sanger, Garg et al. 2001). Three TMS paradigms are commonly used to measure CI,
these are known as LICI, CSP, and SICI.
1.23.1 Long Interval cortical Inhibition
LICI is a paired-pulse TMS paradigm that consists of a suprathreshold conditioning pulse (i.e.
120% of the RMT) followed by a suprathreshold unconditioned testing pulse (i.e. 120% of the
RMT) at a long interstimulus interval (e.g. 50 to 100 ms) (Valls-Sole, Pascual-Leone et al. 1992).
37
The conditioning stimulus primes the test pulse and leads to inhibition of test MEP (Figure 2.)
This effect is usually expressed by a ratio of conditioned MEP amplitude divided by
unconditioned test MEP amplitude. Further, LICI can be assessed directly from the cortex
through the combination of TMS with EEG (Daskalakis, Farzan et al. 2008; Premoli, Rivolta et
al. 2014). This method has been shown to be reliable with high test-retest and intraclass
correlation coefficient > 0.38 (Farzan, Barr et al. 2010).
Several pharmacological evidence proposes that LICI reflects GABAB receptor activation.
First, the GABAB agonist, baclofen potentiates LICI (McDonnell, Orekhov et al. 2006). Second,
LICI is optimal when the conditioning stimulus precedes the TS by 100 to 150ms (Sanger, Garg
et al. 2001), which is comparable to the time course of the GABAB receptor activation, which
has been shown to typically peak around 150 to 200ms post-stimulus(McCormick 1989). Third,
LICI is evoked by a high-intensity suprathreshold conditioning pulse, which is consistent with
the finding that GABAB receptor activation has a high activation threshold (Deisz 1999; Sanger,
Garg et al. 2001).
1.23.2 Cortical Silent Period
Another TMS measure that indexes GABAB receptor-mediated inhibition is CSP (Cantello,
Gianelli et al. 1992). CSP is measured during voluntary muscle contraction (e.g., 20% of
maximum contraction). A suprathreshold intensity TMS pulse (110% to 160% of the RMT) is
delivered to the contralateral motor cortex causing a temporary interruption of voluntary muscle
activity (Figure 2.). CSP is determined by the duration from the onset of muscle activity to the
return of voluntary muscle activity (Day, Dressler et al. 1989). It is this duration of “silent
period” that provides a measure of GABAB inhibition (Cantello, Gianelli et al. 1992). Although
38
spinal inhibition contributes to the early part of CSP (50 to75ms), such as Renshaw inhibition,
the latter part is of supraspinal mechanism and mediated by cortical inhibitory interneurons
(Fuhr, Agostino et al. 1991; Chen, Lozano et al. 1999).
Converging lines of evidence suggest that CSP is mediated through GABAB activity. First, the
intrathecal administration of the GABAB agonist, baclofen, increases CSP duration(Siebner,
Dressnandt et al. 1998). Second, there is a strong correlation (Pearson’s r=0.90, p<0.001)
between LICI and CSP in the motor cortex, suggesting a common mechanism (Farzan, Barr et al.
2010). Third, similar to LICI, CSP is evoked by a high-intensity pulse, which is consistent with
the finding that GABAB receptor activation has a high activation threshold. Lastly, the time
course of CSP duration is similar to the duration of GABAB receptor activation, approximately
150 to 200ms post-stimulus (McCormick 1989; Siebner, Dressnandt et al. 1998).
1.23.3 Short Interval Cortical Inhibition
SICI is a paired-pulse TMS paradigm. It consists of a subthreshold conditioning pulse (i.e. 80%
of RMT) followed by a suprathreshold test pulse with a short interstimulus interval of 1 to 5ms
(Kujirai, Caramia et al. 1993; Ziemann 1999). The conditioning pulse leads to an inhibition of
the test MEP amplitude. This effect is expressed by a ratio of conditioned divided by
unconditioned test MEP amplitude (Kujirai, Caramia et al. 1993; Ziemann 1999). A lack of
change in spinal reflexes suggests that SICI is due to synaptic interactions occurring cortically
rather than spinally (Kujirai, Caramia et al. 1993). Studies propose that SICI is mediated by short
lasting IPSPs (Kujirai, Caramia et al. 1993). Further, SICI has been pharmacologically enhanced
using GABAA acting agents , such as lorazepam, suggesting that this type of inhibition is
mediated through GABAA activation (Ziemann, Lonnecker et al. 1996). Also, SICI activity
39
displays a similar time course to GABAA receptor activation, with a synaptic time constant of
approximately 10 to 25ms (Wang and Buzsaki 1996).
Figure 2. Cortical Inhibition Measures. This image illustrates SICI, CSP and LICI, three common measures used to assess CI in the motor cortex.
1.24 Cortical Excitation
1.24.1 Resting Motor Threshold
TMS can also be used to examine cortical excitability through paradigms that include, resting
motor threshold and intracortical facilitation (ICF). Resting motor threshold is defined as the
minimum stimulus intensity that elicits an MEP of >50mV in at least 5 out of 10 trials in a
relaxed target muscle (Kujirai, Caramia et al. 1993). It is a global measure of corticospinal
excitability and depends on glutamatergic synaptic excitability mediated mainly through fast
40
acting AMPA receptors (Paulus, Classen et al. 2008). This measure is also dependent on voltage-
gated sodium channels, as drugs that block these channels such as carbamazepine, lamotrigine,
and losigamone, increase RMT (Ziemann, Lonnecker et al. 1996).
1.24.2 Intracortical Facilitation
ICF is a paired-pulse TMS paradigm that assesses cortical excitability. It consists of a
conditioning stimuli followed by a test stimulus with an interstimulus interval of 7ms to 20ms
(Nakamura, Kitagawa et al. 1997). It has been proposed that ICF is mediated by NMDA
neurotransmission based on several findings (Nakamura, Kitagawa et al. 1997). For example, the
latency of onset of NMDA-mediated EPSP is approximately 10ms, which is consistent with the
time course of ICF (Kujirai, Caramia et al. 1993; Ziemann, Lonnecker et al. 1996). Also,
pharmacological studies have shown that NMDA receptor antagonists such as dextromethorphan
and memantine decrease ICF (Ziemann, Lonnecker et al. 1996; Schwenkreis, Witscher et al.
1999).
1.25 Pharmaco-TMS Experiments
Several pharmaco-TMS experiments have shown that cortical inhibition and excitation can be
manipulated after a single dose of a central nervous system (CNS) active drug that influence
neuromodulators including, acetylcholine and dopamine, and neurotransmitters including GABA
and glutamate. As such, in the next section, these findings will be discussed.
41
1.25.1 GABAergic Activity
GABAA Activity
Several TMS studies have looked at the effects of GABA on CI. With regards to GABAA
activity, GABAA activating agents, benzodiazepines, lorazepam (Ziemann, Lonnecker et al.
1996; Teo, Terranova et al. 2009) and diazepam increased SICI(Di Lazzaro, Pilato et al. 2005;
Di Lazzaro, Pilato et al. 2007), and CSP (Inghilleri, Berardelli et al. 1996; Ziemann, Lonnecker
et al. 1996; Kimiskidis, Papagiannopoulos et al. 2006), but reduced ICF (Ziemann, Lonnecker et
al. 1996; Mohammadi, Krampfl et al. 2006). In contrast, zolpidem, a benzodiazepine with low
affinity for the GABAA receptor, because it is selective for the GABAA-alpha1 receptor,
enhanced LICI (Mohammadi, Krampfl et al. 2006) but did not affect SICI (Di Lazzaro, Pilato et
al. 2006; Di Lazzaro, Pilato et al. 2007). Similarly, ethanol, which binds to the α6
GABAA receptor subunit increased SICI, enhanced CSP, and reduced ICF (Olsen, Hanchar et al.
2007). In contrast, baclofen, a GABAB receptor agonist decreased SICI (McDonnell, Orekhov et
al. 2006), presumably through presynaptic GABAB autoreceptors (Daskalakis, Christensen et al.
2002).
GABAB Activity
With regards to GABAB activity, the GABAB receptor agonist, baclofen has inconsistent effects
on SICI (Ziemann, Lonnecker et al. 1996; Ziemann, Tergau et al. 1998; McDonnell, Orekhov et
al. 2006), but increases LICI (McDonnell, Orekhov et al. 2006) and CSP duration (Siebner,
Dressnandt et al. 1998; Stetkarova and Kofler 2013). These findings suggest that the effects of
baclofen are mediated through GABAB neurotransmission. Further, increasing GABA levels by
blocking GABA reuptake with tiagabine, resulted in a dose-dependent increase in CSP duration,
42
predominately through the activation of GABAB receptors (Thompson and Gahwiler 1992) and
an increase in SICI (Werhahn, Kunesch et al. 1999). Additionally, inhibiting GABA
transaminase with vigabatrin (Pierantozzi, Marciani et al. 2004), or possibly enhancing GABA
levels using gabapentin (Ziemann, Lonnecker et al. 1996) both enhanced inhibition assessed by
CSP, SICI, LICI and/or decreased ICF.
1.25. 2 Glutamatergic Activity
Several studies have looked at the effects of NMDA antagonists on CI using TMS.
Dextromethorphan (Ziemann, Chen et al. 1998) and memantine (Schwenkreis, Witscher et al.
1999), which are NMDA antagonists, both increased CI assessed through SICI and decreased
excitation assessed through ICF. Riluzole and amantadine, which are also anti-glutamatergic
drugs, showed similar results of suppressing cortical excitation (Schwenkreis, Liepert et al. 2000;
Reis, John et al. 2006).
1.25.3 Dopaminergic Activity
Several studies have looked at the effects of dopamine on CI using TMS. Dopamine can either
directly increase inhibition by acting on cortical pyramidal neurons (Gao, Wang et al. 2003) or
indirectly through GABAergic interneurons (Tseng and O'Donnell 2004; Floyer-Lea,
Wylezinska et al. 2006). As such, dopamine agonists, bromocriptine (Ziemann, Tergau et al.
1997), cabergoline (Korchounov, Ilic et al. 2007) and pergolide (Ziemann, Bruns et al. 1996)
have been shown to increase SICI and CSP (Ziemann, Bruns et al. 1996) and decrease ICF
(Korchounov, Ilic et al. 2007), while dopamine antagonists, such as haloperidol has been shown
to decrease inhibition (Ziemann, Tergau et al. 1997) or have no significant(Daskalakis,
Christensen et al. 2003). Similarly, L-DOPA, a dopamine precursor, prolonged CSP duration
43
(Priori, Berardelli et al. 1994). Also, methylphenidate and amphetamine, agents which increase
dopamine by blocking dopamine transporters have been shown to decrease SICI (Ilic,
Korchounov et al. 2003; Khoshbouei, Sen et al. 2004) and increase ICF (Kirschner, Moll et al.
2003; Moll, Heinrich et al. 2003; Gilbert, Ridel et al. 2006).
1.25.4 Cholinergic Activity
Very few studies have assessed the effects of cholinergic activity. One study found that the
acetylcholine esterase inhibitor tacrine decreases SICI and increases ICF (Korchounov, Ilic et al.
2005). While another study that assessed the effects of rivastigmine on cortical excitability
reported an enhancement (Langguth, Bauer et al. 2007). Finally, a different study found that the
M1/M2 antagonist atropine decreases SICI and increases ICF (Liepert, Schardt et al. 2001), while
another study found that scopolamine had no significant effect (Di Lazzaro, Oliviero et al. 2000).
In conclusion, TMS provides a quantitative way to indirectly measure CI from the cortex. These
responses have been shown to be altered by CNS drugs that affect neurotransmitters and
neuromodulators. While there has been considerable work exploring the effects of these drugs on
TMS responses, most of it has been done by using only TMS without EEG and from the motor
cortex with results demonstrated from MEP activity. By combining TMS with EEG, the influence
of these drugs can be measured directly from the cortex with potentially greater clinical relevance.
1.26. Abnormal Cortical Inhibition in Schizophrenia
Several lines of evidence suggest that CI is impaired in patients with schizophrenia and
implicated in the pathophysiology of the disorder (Lewis, Pierri et al. 1999; Radhu, Garcia
Dominguez et al. 2015) (Daskalakis, Christensen et al. 2002; Wobrock, Kadovic et al. 2007).
First, a reduction in SICI amplitude and CSP duration has been shown in first-episode patients
44
with limited exposure to antipsychotics, which may be due to compensatory mechanisms
(Wobrock, Schneider-Axmann et al. 2009; Hasan, Wobrock et al. 2012). In contrast, in patients
diagnosed with schizophrenia both SICI and CSP were shown to be reduced (Fitzgerald, Brown
et al. 2002; Pascual-Leone, Manoach et al. 2002). Second, medication may alleviate such
impairments as only unmedicated patients showed a reduction in SICI amplitude and in CSP
duration, while medicated patients did not (Daskalakis, Christensen et al. 2002). Third,
clozapine-treated patients showed a longer CSP duration compared to healthy participants and
unmedicated patients with schizophrenia, which may be related to the potentiation of GABAB
receptor neurotransmission (Daskalakis, Farzan et al. 2008; Liu, Fitzgerald et al. 2009). Fourth,
reduced SICI has been shown to be correlated with the severity of psychotic symptoms
(Daskalakis, Farzan et al. 2008). Further investigations have shown that SICI is inversely
correlated with positive symptoms, while CSP is inversely associated with negative symptoms,
suggesting the involvement of GABAA and GABAB neurotransmission in positive and negative
symptoms, respectively (Liu, Fitzgerald et al. 2009). Lastly, LICI is reduced in the DLPFC of
patients and this reduction was seen only in schizophrenia, and not in obsessive-compulsive
disorder (OCD), a psychiatric disorder with similar characteristics (Radhu, Garcia Dominguez et
al. 2015) Thus, the evidence illustrates that CI deficits are specific to patients with schizophrenia
and are not a generalized to similar disorders of severe psychopathology.
45
1.27 Pharmacology of the Drugs Used in this Dissertation
1.27.1 Baclofen
Baclofen, a GABAB agonist is primarily used for the treatment of spasticity including cerebral
palsy and multiple sclerosis. Once baclofen binds to GABAB receptors, it opens nearby
potassium channels, which slightly hyperpolarizes the neuron. When a 10mg dose of baclofen is
administered, the smallest dose available on the market, the bioavailability recorded is 74% and a
plasma protein binding of 30%. The median peak time for baclofen is one hour (McDonnell,
Orekhov et al. 2007), while its half-life is 4 to 6.54 hours, with complete elimination within 72
hours (Faigle et al. 1972). The drug is predominately (85%) eliminated in urine/feces unchanged
and 15% metabolized by deamination.
1.27. 2 Dextromethorphan
Dextromethorphan is primarily a cough suppressant of the morphine class and found in some
pain medications. It is a noncompetitive NMDA antagonist that works by blocking Ca2+ ion
channels that would normally depolarize the neuron (Church, Lodge et al. 1985; Linn, Long et
al. 2014). When orally administered the drug is quickly absorbed by the GI tract and permeable
to the blood-brain barrier with a bioavailability of 11%. Maximal plasma concentration is
reached within 3 hours with a half-life of approximately 2 to 4 hours (Ziemann, Chen et al.
1998). Dextromethorphan is converted into dextrorphan, its metabolite, by O-demethylation and
eliminated through the hepatic system with CYP2D6 playing a major role, while CYP3A4 and
CYP3A5 have a minor role and excretion is mainly renal.
46
1.27.3 Levodopa
Levodopa (L-DOPA) is a dopaminergic agent used to increase dopamine concentrations in the
treatment of Parkinson's disease and dopamine-responsive dystonia. Dopamine is unable to pass
through the blood-brain barrier due to size restrictions. L-DOPA, a smaller amino acid, and
dopamine precursor, however, is able to pass through the blood-brain barrier. For this reason, L-
DOPA is administered instead of dopamine. L-DOPA is then converted into dopamine by
the enzyme aromatic L-amino acid decarboxylase and pyridoxal phosphate (vitamin B6), which
acts as a cofactor for this reaction. Often, L-DOPA is combined with carbidopa, an inhibitor of
the enzyme that would normally decarboxylase L-DOPA into dopamine in the peripheral system.
This inhibition enhances the amount of L-DOPA that reaches the brain. Upon oral
administration, L-DOPA is absorbed by the GI tract via an active transport system and reaches
peak concentration after 0.5 to 2 hours (Crevoisier, Hoevels et al. 1987; Kuo, Paulus et al. 2008).
Its half-life is approximately one hour, but in combination with a DOPA-decarboxylase inhibitor
it can reach 1.5 to 2 hours. In the young, the combination of 100 mg L-DOPA and 25 mg
carbidopa reaches a bioavailability of 41%, while in the elderly it reaches 86%. (Okereke 2002).
The consumption of food also plays a factor as it affects its movement through the system, with
low protein diets contributing to greater rates of absorption (Eriksson, Granerus et al. 1988).
Elimination of L-DOPA and its metabolites are mainly renal (70-80%).
1.27.4 Rivastigmine
Rivastigmine is a cholinergic agent used in the treatment of mild to moderate dementia of
Alzheimer's or Parkinson's disease. It is an acetylcholinesterase inhibitor that renders the enzyme
inactive, so it is unable to break down acetylcholine to choline and acetate, increasing its
47
availability for neurotransmission (Polinsky 1998). The drug can be administered orally or by a
transdermal patch. When a 3 mg dose is given orally the drug is quickly absorbed and permeable
to the blood-brain barrier with a bioavailability of about 40%, and plasma protein binding of
40% (Polinsky 1998; Jann, Shirley et al. 2002). Maximal plasma concentration is reached within
2 hours (Kuo, Grosch et al. 2007) with a half-life of approximately one hour, but effects on the
brain can last up to 10 hours. Elimination bypasses the hepatic system and cytochrome P450, and
fully out of the system after 24 hours of intake primarily through the urine, with less than 1%
found in feces (Polinsky 1998).
1.28 Potential Risks Associated with Experimental Drugs
a) Carbidopa and Levodopa:
2-10%- dyskinesia, nausea, hallucinations, confusions, dizziness
b) Baclofen
>10%: drowsiness, headache, vertigo, dizziness, trouble sleeping, slurred speech, ataxia-
lack of coordination of muscles, hypotonia- low muscle tone, neuromuscular and skeletal
weakness
1%-10%: low blood pressure, fatigue, confusions, headache, rash, nausea, constipation,
polyuria-excessive urination
Currently, there are no reported cases of severe side effects following a single oral dose
of baclofen within the daily dosage range (40mg to 80 mg). Previous studies using single
oral dose of baclofen (50 mg) (McDonnell, Orekhov et al. 2006; McDonnell, Orekhov et
48
al. 2007) did report some sedation and fatigue among the participants but without any
consequence
c) Rivastigmine
2-10%: nausea, vomiting, loss of appetite, dizziness, abdominal pain, fatigue
d) Dextromethorphan
>10%: diarrhea
1-5%: cough, vomiting, peripheral edema, asthenia- lack of muscle strength.
49
2. Objectives, Hypotheses and Participants
2.1 Objectives
The objective of the first paper was to assess studies that used proton magnetic resonance
spectroscopy (1H MRS), positron emission tomography (PET) and single-photon emission
computed tomography (SPECT) imaging techniques to measure glutamate, dopamine and
GABA levels in drug-naïve and drug-free patients with schizophreia. The main aim of this paper
was to provide evidence for abnormal dopaminergic, GABAergic and glutamatergic
neurotransmission in antipsychotic-naïve/free patients with schizophrenia compared with healthy
controls, and to build a model illustrating how these abnormalities could lead to impaired LTP in
patients with schizophrenia and consequently cognitive deficits.
The objective of the second paper was to assess the effects of rivastigmine, an acetyl
cholinesterase inhibitor, L-DOPA, a dopamine precursor, baclofen, a GABAB receptor agonist,
and dextromethorphan, a NMDA receptor antagonist on PAS-induced LTP in the DLPFC using a
double-blind randomized controlled design. This was achieved by assessing the effects of each
drug to a placebo agent. LTP was assessed by comparing pre-PAS and post-PAS (0, 15, 30, and
60 mins post PAS) CEA using TMS-EEG.
The objective of the third study was to assess the effects of the aforementioned agents on LICI
from DLPFC stimulation. This was done on a separate day from the PAS experiment but in the
same participants who took part in the first study using a double-blind randomized controlled
design. LICI was measured at pre-drug and post-drug and then the change was compared to
placebo.
50
2.2. Hypothesis
1) 50 mg baclofen will impair LTP and enhance LICI in the DLFPC when compared to a placebo
agent.
2) 100 mg L-DOPA will enhance LTP and enhance LICI in the DLFPC when compared to a
placebo agent.
3) 3 mg rivastigmine will enhance LTP and reduce LICI in the DLFPC when compared to a
placebo agent.
4) 150 mg dextromethorphan will block LTP and enhance LICI in the DLFPC when compared to
a placebo agent.
2.3 Participants
Participants were recruited through advertisements posted at the Center for Addiction and Mental
Health, University of Toronto, from our recruitment database, and Craigslist, a classified
website.
Inclusion Criteria
1. Age of 18-55 years
2. Non-smoker
51
3. Males and females, females with potential childbearing must have a negative urine
pregnancy test.
4. Speak English
5. Willingness to provide informed consent and is competent.
6. Free of psychopathology based on the Personality Assessment Screener (PAS)
7. Right handedness
Exclusion Criteria
1. Past or current history of drug abuse disorder, illicit drug use was determined by a
positive urine drug screen
2. History of a medical or neurological disorder that affects CNS (such as, traumatic brain
injury, stroke, Parkinson).
3. Current or past history of seizures
4. Any metal implant or dentures
5. Electroconvulsive Therapy (ECT) within 6 months prior to study participation
6. Any of the following; breastfeeding, immediate post-myocardial infarction, life-
threatening arrhythmias, angina pectoris
7. Psychotropic medication
8. no acute/chronic medication during or up to 2 weeks before participating in the study
52
Chapter 3
3. Imaging-based Neurochemistry in Schizophrenia: A Systematic Review and Implications for Dysfunctional
Long-Term Potentiation
Contents of this chapter have been reprinted by permission from Schizophrenia Bulletin
Bahar Salavati, Tarek K. Rajji, Rae Price, Yinming Sun, Ariel Graff-Guerrero, Zafiris J. Daskalakis. Imaging-based Neurochemistry in Schizophrenia: A Systematic Review and Implications for Dysfunctional Long-Term Potentiation. Schizophrenia Bulletin (2015) 41 (1): 44-56.doi: 10.1093/schbul/sbu132
53
3.1 Abstract
Cognitive deficits are commonly observed in patients with schizophrenia. Converging lines of
evidence suggest that these deficits are associated with impaired long-term potentiation (LTP). In
our systematic review, this hypothesis is evaluated using neuroimaging literature focused on
proton magnetic resonance spectroscopy (1H MRS), positron emission tomography (PET) and
single-photon emission computed tomography (SPECT). The review provides evidence for
abnormal dopaminergic, GABAergic and glutamatergic neurotransmission in antipsychotic-
naïve/free patients with schizophrenia compared with healthy controls. The review concludes
with a model illustrating how these abnormalities could lead to impaired LTP in patients with
schizophrenia and consequently cognitive deficits.
54
3.2 Introduction
Schizophrenia is a psychiatric disorder that affects 1% of the world population (Ross, Margolis
et al. 2006) (Freedman 2003). Cognitive deficits such as learning and memory impairments are
considered core features of the illness. (Rajji, Ismail et al. 2009; van Os and Kapur 2009). LTP is
a key determinant of learning and memory function (Lynch 2004) and may be a key
neurophysiological mechanism underlying cognitive impairment in schizophrenia.
LTP is defined as an activity dependent long-lasting enhancement in synaptic efficacy (Citri and
Malenka 2008). LTP is typically dependent on the glutamatergic NMDA receptor (Coan and
Collingridge 1987) (Errington, Lynch et al. 1987). Glutamate activates NMDA receptors
allowing Ca+2 entry, which in turn acts on calmodulin-dependent protein kinases (CaM Kinases)
II and IV and leads to the upregulation of AMPA receptors (Miyamoto 2006).
LTP is modulated by the dopaminergic (Frey, Schroeder et al. 1990) and GABAergic systems
(Lopez-Gil, Babot et al. 2007) (Lewis and Moghaddam 2006). Dopaminergic modulation of LTP
depends on the type of receptors. Dopamine D1 receptor activation enhances LTP (Bailey,
Andrews et al. 2000) (Huang and Kandel 1995), while dopamine D2/3 receptor activation
suppresses NMDA activity and GABA activity (Chen, Ito et al. 1996) (Tseng and O'Donnell
2004) GABAergic modulation of LTP also depends on the subtype of GABA receptor.
Antagonism of GABAA receptor facilitates LTP (Ruiz, Campanac et al. 2010). Activation of
GABAB receptor modulates GABAA receptor through presynaptic auto-inhibition of interneurons
which facilitates LTP (Davies, Starkey et al. 1991) (Deisz 1999).
A number of imaging studies using proton magnetic resonance spectroscopy (1H MRS), positron
emission tomography (PET) and single-photon emission computed tomography (SPECT)
55
assessed these systems (glutamatergic, dopaminergic and GABAergic) in patients. To date, there
has been one meta-analysis, and one review paper on glutamate 1H MRS studies (Marsman, van
den Heuvel et al. 2013; Poels, Kegeles et al. 2014) two meta-analyses on dopamine PET and
SPECT studies (Laruelle 1998; Howes, Kambeitz et al. 2012) and one narrative review on
imaging studies assessing dopamine, serotonin, GABA and glutamate systems in schizophrenia
(Soares and Innis 1999). This last review was performed more than a decade ago and included
patient with- and without exposure to antipsychotic treatment. Thus, our aim was to perform a
systematic review of imaging studies assessing these three neurotransmitter systems, focusing
only on antipsychotic-naïve or antipsychotic-free patients with schizophrenia. Assessing this
subgroup helps to disentangle changes in neurochemistry related to illness compared to changes
related to medications. Differences between medicated and unmedicated patients are also
highlighted throughout the review only for comparison purposes. Lastly, we present a model
linking these systems to abnormal LTP and cognitive deficits associated with schizophrenia.
3.3 Methods
A literature search was performed on November 18, 2013, using PUBMED with no date limits
and the following terms were used: schizo* AND drug naiv* OR antipsychotic naiv* OR
untreat* OR unmedicat* OR never treat* OR neuroleptic free OR antipsychotic free OR first
episod* AND glutamate OR GABA OR dopamine. The inclusion criteria were determined a
priori and were (1) in-vivo human studies, (2) imaging studies and (3) studies including
antipsychotic-free and/or antipsychotic-naïve patients with schizophrenia or schizoaffective
disorder. In total 2,383 publications were identified. Articles were excluded after reviewing titles
and abstracts, leaving 63 studies. Considering there was only a few studies for GABA we
56
summarized the finding and findings for dopamine and glutamate were separated into two tables,
one for dopamine, and glutamate (see Table 1, and 2 in the appendix).
3.4 Results
Our search identified 16 studies on the glutamatergic system, 44 studies on the dopaminergic
system and three studies on the GABAergic system.
3.4.1 Glutamatergic System
Several 1H MRS studies and one SPECT study demonstrated altered glutamatergic activity in
antipsychotic-naïve or antipsychotic-free patients. Changes were reported in the concentrations
of glutamate, glutamine, a precursor of glutamate (Bradford and Thomas 1969) and/or GLX, a
combination of both. We summarize the findings below and have chosen to divide these findings
based on various regions of the brain due to intrinsic variations that exist in the healthy brain
(Sailasuta, Ernst et al. 2008).
Medial Prefrontal Cortex (MPFC)
Two studies assessed glutamatergic activity in the MPFC of antipsychotic-free/naïve patients
with schizophrenia compared with healthy controls. One of these studies reported a 30%
increase in GLX levels of nine antipsychotic-naïve and seven antipsychotic-free patients (M=11,
F=5) (mean age 32 years) compared with 22 healthy controls (M=14, F=8) and 16 medicated
patients (M=11, F=5) (Kegeles, Mao et al. 2012). The authors proposed that antipsychotics may
have normalized GLX levels in the MPFC of medicated patients. Elevated GLX levels were also
evident in the right MPFC of 20 adolescents (M=7, F=13) (mean age 16.4 years), those who are
at high-risk for developing schizophrenia by having a parent with schizophrenia (Tibbo,
57
Hanstock et al. 2004). Since glutamine concentration is 40-60% lower than that of glutamate
(Govindaraju, Young et al. 2000) (Jensen, Licata et al. 2009), it can be inferred that elevated
GLX levels mostly reflect elevated glutamate concentrations (Bradford, Ward et al. 1978)
(Kaiser, Schuff et al. 2005). These findings suggest that high-risk adolescents and young
patients with schizophrenia have elevated levels of glutamate in the MPFC early in the illness.
However, a study assessing both glutamine and glutamate levels independently reported an
increase in only glutamine levels in the MPFC of ten antipsychotic-naïve patients (M=8, F=2)
(Bartha, Williamson et al. 1997). The authors concluded that schizophrenia may be associated
with an abnormal conversion of glutamine to glutamate, resulting in elevated glutamine levels
(Bartha, Williamson et al. 1997). Alternatively, this finding may be explained by experimental
limitations. To accurately measure glutamate and glutamine levels separately, specialized 1H
MRS techniques (e.g. high magnetic field (>3T) with short echo and long acquisition time) or
editing techniques (e.g. J-editing) are necessary due to glutamine and glutamate’s analogous
signals (Magistretti and Pellerin 1999; Hurd, Sailasuta et al. 2004; Mullins, Chen et al. 2008). In
this study, a 1.5 T magnetic field without editing techniques was used, which could be unreliable
in distinguishing peaks arising from glutamine and glutamate independently, potentially
confounding the results. While glutamine level in antipsychotic-free/naive patients might be still
elusive, a meta-analysis including medicated and unmedicated patients indicated that glutamine
is higher in patients than healthy controls (Marsman, van den Heuvel et al. 2013).
In contrast, a study comparing glutamate levels in the MPFC of older 12 patients with
schizophrenia (M=7, F=5) (medication status unknown; mean age 49.5 years) and their
unaffected twin with healthy controls (M=12, F=9) found that both patients and their unaffected
twins had decreased glutamate levels (Lutkenhoff, van Erp et al. 2010). Taken together, these
58
studies suggest that patients have elevated glutamate levels in the MPFC early in their illness but
then experience a decline in glutamate concentrations as they age. This age-related change in
glutamate levels in schizophrenia was shown by a recent meta-analysis describing a drop below
healthy controls after the age of 35. Since some of the studies included in this meta-analysis
include medicated patients, medication effects cannot be ruled out and therefore little is known
about glutamate changes over the course of the illness in unmedicated patients(Marsman, van
den Heuvel et al. 2013).
Dorsolateral Prefrontal Cortex (DLPFC)
Four studies assessed the DLPFC of antipsychotic-free/naïve patients compared with healthy
controls. A study using a 3T MRS found no difference in GLX levels in the DLPFC of
antipsychotic-free patients (M=11, F=5) (Kegeles, Mao et al. 2012). This finding is in line with
other studies that reported similar results in antipsychotic-naïve patients (Ohrmann, Siegmund et
al. 2005) (Stanley, Williamson et al. 1996) (Ohrmann, Siegmund et al. 2007), high-risk
individuals (Yoo, Yeon et al. 2009) and childhood-onset patients (Seese, O'Neill et al. 2011).
However, a study that assessed 23 chronic antipsychotic-free patients using 1.5 T MRS found
significantly greater combination of glutamate and GABA levels in patients than healthy controls
(Choe, Kim et al. 1994). Inconsistent results may be explained by the differences in acquisition
and analysis techniques employed in these studies. In contrast, a decrease in GLX levels were
noted when 20 chronic medicated patients (M=14, F=6) were compared with 20 healthy controls
(M=13, F=7) (Ohrmann, Siegmund et al. 2007), suggesting either an aging or chronicity
(including chronic exposure to antipsychotics) effect. As such, further studies using more
specific 1H MRS acquisition and quantification techniques are required.
59
Thalamus
Three different studies comparing antipsychotic-naïve patients with healthy controls reported
elevated glutamine levels in the thalamus of patients (Theberge, Bartha et al. 2002; Theberge,
Williamson et al. 2007; Aoyama, Theberge et al. 2011). The first study assessed 21
antipsychotic-naïve patients (M=14, F=7) and reported elevated glutamine levels in the left
thalamus (Theberge, Bartha et al. 2002). In contrast, a follow-up study conducted in 21 chronic
medicated patients with schizophrenia (M=20, F=1) detected reduced glutamine levels in the left
thalamus of patients (Theberge, Al-Semaan et al. 2003). This finding was replicated and
extended in a cohort of 16 antipsychotic-naïve patients (M=14, F=2), which included 12 patients
from the earlier study (Theberge, Williamson et al. 2007). Baseline glutamine levels in the left
thalamus remained elevated until 30 months of antipsychotic treatment (Theberge, Williamson et
al. 2007). Another study also found high glutamine levels in antipsychotic-naïve patients
(M=14, F=3), which decreased over 80 months (Aoyama, Theberge et al. 2011). These findings
may suggest an aging or treatment effect. In contrast, another study detected no difference in
glutamine/glutamate (Gln/Glu) ratio between 14 (M=12, F=2) minimally treated patients and 10
healthy controls (M=12, F=2) (Bustillo, Rowland et al. 2010). Medication effects could have
played a role in this inconsistent finding, since these patients had some, albeit minimal exposure
to antipsychotics, lasting less than three weeks. On the other hand, glutamate levels were found
to be decreased in the thalamus of 27 high-risk adolescents (M=14, F=13) (Stone, Day et al.
2009). However, recently Tandon et al. (2013) reported increased GLX in the thalamus of 23
high-risk adolescents (M=10, F=13) (Tandon, Bolo et al. 2013).
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These findings support a dysfunctional glutamate-glutamine cycle in the brains of patients. It is
postulated that an abnormal conversion of glutamine to glutamate would result in high glutamine
and low glutamate levels, consistent with the majority of the above-mentioned findings.
Basal Ganglia (BG)
Two studies assessed the BG in antipsychotic-naïve/free patients compared to healthy controls.
A study looking at the precommissural dorsal caudate (PCDC) of 14 antipsychotic-free patients
detected elevated glutamate/creatine (Glu/Cr) ratio, suggesting elevated glutamate levels (de la
Fuente-Sandoval, Favila et al. 2009). Another study that assessed the PCDC of first episodes
antipsychotic-free (N=18) (M=10, F=8) and ultra-high risk for psychosis patients (N=18) (M=14,
F=4) detected elevated glutamate levels in both groups (de la Fuente-Sandoval, Leon-Ortiz et al.
2011). A longitudinal study of 24 antipsychotic-naïve patients (M=13, F=11) reported elevated
glutamate in the PCDC of patients (de la Fuente-Sandoval, Leon-Ortiz et al. 2013). This study
also showed that after 4 weeks of exposure to antipsychotics, glutamate levels in PCDC
decreased to similar levels as controls. The same group followed 19 ultra-high-risk subjects for
two years and showed that transition to psychosis was associated with higher glutamate levels in
the PCDC. Another study including 23 ultra-high-risk subjects (M=13, F=10) reported increases
in GLX in the caudate nucleus (Tandon, Bolo et al. 2013). When 40 high-risk adolescents were
assessed, a gender effect was noted, that is, elevated glutamate and GLX levels were detected in
the BG of only male adolescents (N=18)(Keshavan, Dick et al. 2009). Overall, these results
suggest that high glutamate and GLX levels in the BG precede the onset of schizophrenia,
predict the onset of the first episode of psychosis and remain elevated until patients are treated
with antipsychotics.
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Anterior Cingulate
Three publications reported increased glutamine levels in the anterior cingulate of high-risk
adolescents (mean age 16) or antipsychotic-naïve first-episode patients (mean age 21) (Theberge,
Bartha et al. 2002; Tibbo, Hanstock et al. 2004; Stone, Day et al. 2009). In contrast, a study
assessing the anterior cingulate of 17 antipsychotic-naïve patients found no difference in
glutamate or glutamine levels (Aoyama, Theberge et al. 2011). Another group reported
increased Gln/Glu ratio but did not find elevated glutamine levels in the anterior cingulate of 14
minimally treated patients (M=12, F=2) (mean age 27) (Bustillo, Rowland et al. 2010). A study
assessing chronic medicated patients found decreased glutamate and glutamine levels in the
anterior cingulate of patients (Theberge, Williamson et al. 2007). Overall, these findings suggest
that the levels of glutamine and glutamate are abnormal in the anterior cingulate of high-risk
adolescents and first-episode antipsychotic-naïve patients. Such findings suggest that the
glutamate-glutamine cycle may be dysfunctional in anterior cingulate, resulting in excessive
glutamine levels that decline as the disease progresses. The reason for the decline in glutamine
level is still elusive.
Occipital, Parietal and Hippocampal Regions
Several imaging studies have focused on glutamatergic activity in the occipital, parietal and
temporal regions of antipsychotic-naïve or antipsychotic-free patients. A study looking at the
hippocampus of 10 male patients (7 antipsychotic-free and 3 antipsychotic medicated) found
elevated GLX/Cho levels in patients (Kegeles, Shungu et al. 2000). It is important to note that
although in this study GLX is a combination of glutamate, glutamine, and GABA, the
contribution of GABA and glutamine are almost negligible. A recent study assessing 27 patients
(M=20, F=7) (11 antipsychotic -naïve and 16 antipsychotic -free) found elevated GLX in the
62
hippocampus of patients compared with healthy controls (Kraguljac, White et al. 2013). In
contrast, no differences in glutamate or glutamine were found in studies that assessed the medial
temporal lobes of 11 antipsychotic-naïve patients (M=9, F=2) (Bartha, al-Semaan et al. 1999), or
glutamate in 14 twins discordant for schizophrenia. (Lutkenhoff, van Erp et al. 2010). It is
important to note that the first study used a higher MRS field strength ( 3T) and a larger sample
size compared to the second study. No difference in GLX levels were reported when assessing
the temporal gyri of 28 youths with childhood-onset schizophrenia (M=15, F=13) (Seese, O'Neill
et al. 2011). Also, one study found elevated GLX levels in the inferior parietal lobe of only high-
risk male adolescents (M=18, F=22) (Keshavan, Dick et al. 2009). In keeping with the
glutamatergic dysfunction hypothesis, one SPECT study found reduced NMDA binding in the
medial temporal lobe of antipsychotic-free patients, but not in antipsychotic medicated patients
compared to healthy controls, suggesting that antipsychotic medication may have a normalizing
effect (Pilowsky, Bressan et al. 2006). Taken together, these studies suggest increased
glutamatergic activity in the occipital and parietal region and in the medial temporal lobes of
antipsychotic-naïve or antipsychotic-free patients when compared with healthy controls.
Cerebellum
When assessing the cerebellum, two studies did not find a difference in glutamatergic levels and
one reported increased glutamate and GLX. The first negative study included first episode
antipsychotic-free patient and looked at the Glu/Cr ratio (de la Fuente-Sandoval, Favila et al.
2009). The second negative study included 18 antipsychotic-naïve patients (M=14, F=4) and 18
patients with ultra-high risk for psychosis (M=14, F=4) (de la Fuente-Sandoval, Leon-Ortiz et al.
2011). In contrast, a third study, which included only 24 antipsychotic-naïve first episode
patients (M=13, F=11), reported increased glutamate and GLX levels. Interestingly, glutamate
63
levels normalized after 4 weeks of antipsychotic treatment and GLX remained increased (de la
Fuente-Sandoval, Leon-Ortiz et al. 2013). Glutamine could not be quantified to understand its
contribution to the GLX signal.
Summary of Glutamatergic System Findings
The above sections evaluated studies that assessed glutamate, glutamine or GLX levels in the
brains of antipsychotic-free or antipsychotic-naïve patients with schizophrenia and patients at
high-risk of psychosis compared with healthy controls. Overall, our findings revealed the
following: elevated GLX levels in the MPFC, parietal, anterior cingulate, thalamus, basal
ganglia, and occipital region; elevated glutamine levels in the MPFC, thalamus, and anterior
cingulate; elevated glutamate levels in the basal ganglia; decreased glutamate levels in the
thalamus; and no differences or uncertainty in glutamatergic metabolites in the DLPFC, temporal
and cerebellum regions. These results support the notion that the pathophysiology of
schizophrenia may stem from dysfunctional glutamate and glutamine neurotransmission.
3.4.2 Dopaminergic System
Several PET and SPECT imaging studies assessed dopamine levels and receptors in different
regions of the brains of antipsychotic-naïve and antipsychotic-free patients with schizophrenia.
This section will review the literature pertaining to abnormalities in the dopamine D1 and D2/3
receptors because these dopamine receptors are highly relevant to LTP modulation(Gurden,
Takita et al. 2000; Granado, Ortiz et al. 2008; Xu and Yao 2010) as well as, dopamine synthesis,
and release.
64
Dopamine D1 Receptor Studies
Four studies assessed dopamine D1 receptor binding in the prefrontal cortex of patients. One of
these studies using the PET radiotracer [11C]-NNC112 reported greater dopamine D1 receptor
binding in seven antipsychotic-naïve and nine antipsychotic-free patients (M=13, F=3) (Abi-
Dargham 2002). In a follow-up study, an elevation in dopamine D1 receptor binding was
detected in the prefrontal cortex of only antipsychotic-naïve patients (N=12) (M=5, F=7), and not
in antipsychotic-free patients (N=13) (M=11, F=2) when compared with healthy controls (N=24)
(Abi-Dargham, Xu et al. 2012). On the contrary, studies using the radiotracer [11C]-SCH23390
reported decreased (Okubo, Suhara et al. 1997) or no change (Karlsson, Farde et al. 2002) in the
dopamine D1 receptor binding in the prefrontal cortex of antipsychotic-naïve or antipsychotic-
free patients. Discrepancies between studies might be accounted for by differences in
demographic, clinical characteristics, previous antipsychotic exposure and PET radiotracers
([11C]-NNC112versus [11C]-SCH23390). Dopamine depletion studies in rodents showed
increased [11C]-NNC112 binding and decreased [11C]-SCH23390 binding (Guo, Hwang et al.
2003), indicating opposite sensitivity for dopamine levels. In addition, 5-HT2A binding was
shown to contribute to the cortical binding of both radiotracers in non-human primates (Ekelund,
Slifstein et al. 2007) and in humans for [11C]-NNC112 only (Catafau, Searle et al. 2010). As
such, these limitations should be taken into consideration when evaluating the aforementioned
studies. Regarding other brain regions, no difference in dopamine D1 binding was found in the
striatal, limbic and thalamic regions when patients were compared with healthy controls.(Okubo,
Suhara et al. 1997; Abi-Dargham 2002; Abi-Dargham, Xu et al. 2012). Taken together, these
results illustrate inconsistent differences in dopamine D1 receptor binding in the DLPFC and no
65
difference in D1 receptor binding in the striatum, limbic and thalamic regions of the
antipsychotic-naïve/antipsychotic-free patient.
Dopamine D2/3 Receptor Studies
Striatum and Substantia Nigra
i) Studies without Pharmacological Challenges
Fifteen publications assessing dopamine D2/3 receptor binding reported no difference between
patients and healthy controls in the striatum (Farde, Wiesel et al. 1990; Martinot, Paillere-
Martinot et al. 1991; Hietala, Syvalahti et al. 1994; Pilowsky, Costa et al. 1994; Breier, Su et al.
1997; Knable, Egan et al. 1997; Okubo, Suhara et al. 1997; Abi-Dargham, Gil et al. 1998; Abi-
Dargham, Rodenhiser et al. 2000; Yang, Yu et al. 2004; Talvik, Nordstrom et al. 2006; Graff-
Guerrero, Mizrahi et al. 2009; Kessler, Woodward et al. 2009; Kegeles, Slifstein et al. 2010;
Schmitt, Dresel et al. 2012). In contrast, one study reported reduced D2/3 binding in 23 acutely ill
patients (M=19; F=4) compared with healthy controls (Schmitt, Meisenzahl et al. 2009). In the
above-mentioned studies, patients had in general mild to moderate symptoms. The mean scores
on the positive and negative symptom scale (PANSS) positive subscale ranged from 18 to 21.9
and on the brief psychiatric rating scale (BPRS) ranged from 28.8 to 60. One exception was a
study in which patients had a mean PANSS positive subscale score of 30.92 (Schmitt, Dresel et
al. 2012). In contrast, the publication showing reduced D2/3 binding in patients included patients
with severe symptoms (PANNS positive score=21.9; PANSS general score = 60.4; BPRS score=
73.6) (Schmitt, Meisenzahl et al. 2009). As such, lower dopamine D2/3 receptor binding may be a
result of greater endogenous dopamine concentrations which compete with the D2/3 receptor
ligand, resulting in reduced D2/3 binding (Abi-Dargham 2004). Thus, given that there is an
66
inverse correlation between severity of psychosis and D2/3 binding potential (Tuppurainen,
Kuikka et al. 2003), the differences in severity of patients’ symptoms may account for the
differences detected in D2/3 binding among these studies.
On the contrary, four studies reported increased D2/3 receptors binding in the striatal region.
(Tune, Wong et al. 1993; Pearlson, Wong et al. 1995; Corripio, Perez et al. 2006; Corripio,
Escarti et al. 2011). Corripio et al. (2011) found that D2/3 striatal/frontal binding ratio was
increased in 25 first-episode antipsychotic-naïve patients (compared with 12 healthy controls and
12 patients with a psychotic disorder different to schizophrenia using 123 I-IBZM SPECT
(Corripio, Escarti et al. 2011). Increased D2/3 receptor binding was also reported in 11 patients
(M=6, F=5) compared with 18 healthy controls (M=10, F=8) using 123 I-IBZM SPECT (Corripio,
Perez et al. 2006). This finding is in line with an earlier study that reported increased D2/3
receptor striatal binding in 25 antipsychotic-naïve and antipsychotic-free chronic patients (M=17,
F=8) (Tune, Wong et al. 1993). Notwithstanding, a meta-analysis by Laruelle reported
approximately 12% elevation in D2/3 receptor binding in antipsychotic-free patients with
schizophrenia compared to healthy controls (Laruelle 1998).
Studies that assessed the caudate or putamen independently reported inconsistent findings that
seemed to be influenced by the radiotracer. For instance, when [11C]-raclopride was used to
separately assess the caudate and putamen of 18 antipsychotic-naïve patients (M=10, F=8),
elevation in D2/3 receptor binding was not detected (Farde, Wiesel et al. 1990). In contrast, two
other studies that used [11C]-methylspiperone reported greater D2/3 receptor binding in the
caudate nucleus of ten antipsychotic-naïve and antipsychotic-free patients (M=8, F=2) (Wong,
Wagner et al. 1986) and 22 antipsychotic-naïve patients (M=13, F=9) (Wong, Singer et al. 1997).
The radiotracer[11C]-methylspiperone has been shown to be less sensitive to endogenous
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dopamine and binds to dopamine D4 receptors unlike [11C]-raclopride (Seeman, Guan et al.
1993; Ishiwata, Hayakawa et al. 1999). Therefore, considering that [11C]-raclopride and [11C]-
methylspiperone have different pharmacological properties, it may be difficult to compare results
obtained with these two radiotracers. Nevertheless, a study comparing antipsychotic-free (N=16)
(M=13, F=3) and antipsychotic-naïve patients (N=12) (M=5, F=7) detected no difference in D2/3
receptor binding in the striatum between the two groups of patients (Lomena, Catafau et al.
2004).
Lastly, three studies employing the dopamine D2/3 receptor high-affinity radiotracers
[123I]epidepride (SPECT) (Tuppurainen, Kuikka et al. 2006) and [18F]fallypride (PET) (Kessler,
Woodward et al. 2009) and the agonist [11C]-(+)-PHNO (Graff-Guerrero, Mizrahi et al. 2009)
assessed the substantia nigra and reported inconsistent results. The study employing [123I]
epidepride detected decreased D2/3 receptor binding, the study employing [18F] fallypride
detected greater D2/3 receptor binding and the study employing [11C]-(+)-PHNO did not find any
difference in antipsychotic-free patients with schizophrenia in comparison with controls. The
reason for the discrepancy in results is still elusive and could be due to differences in the
radiotracers employed and/or differences in the characteristics of the clinical population. Thus,
the majority of the present results reveal no difference in D2/3 receptor binding in the striatum,
however, a meta-analysis reported an elevation in D2 receptors (Laruelle 1998) and the results in
the substantia nigra require further exploration.
ii) Studies assessing dopamine synthesis capacity
In addition to changes in the D2/3 receptor, several PET studies performed on antipsychotic-naïve
and antipsychotic-free patients reported increased dopamine synthesis capacity in the striatum.
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Three studies found greater dopamine synthesis in the caudate nucleus and putamen of patients.
(Hietala, Nagren et al. 1999; Lindstrom, Gefvert et al. 1999; Nozaki, Kato et al. 2009).
Specifically, Nozaki et al. found significantly greater dopamine synthesis in only the left caudate
of 14 antipsychotic-naïve and four antipsychotic-free patients who were 3-months antipsychotic-
free (M=10, F=3) (Nozaki, Kato et al. 2009). Another study revealed increased dopamine
synthesis in the striatum of eight male antipsychotic-free/antipsychotic-naïve patients (N= 3
antipsychotic-naïve and N= 5 antipsychotic-free for at least 6 months) (Kumakura, Cumming et
al. 2007). This difference was nearly twofold, the greatest biochemical difference reported to
date. In contrast, one study found no difference between six untreated male patients (2
antipsychotic-naïve) and seven male healthy controls (Dao-Castellana, Paillere-Martinot et al.
1997). Contradictory findings may be explained by age, type of schizophrenia and gender, as
patients in this study were generally younger (mean age 26 years), more catatonic compared with
the other studies (30+ years), and consisted exclusively of male patients. Comparable results
were also evident in the high-risk individuals (N=30) (M=17, F=13) (Howes, Bose et al. 2011)
and dopamine synthesis in these individuals determined their clinical outcome three years later.
The psychotic transition group (N=9) had greater dopamine synthesis in the striatum (effect
size=1.18) compared with the healthy control (N=29) (M=20, F=9) and the non-transition group
(N=15). This finding is consistent with another study that reported elevated dopamine levels in
the striatum of high-risk individuals (Fusar-Poli, Howes et al. 2011). One study reported
significantly higher dopamine synthesis in only the putamen, with no difference found in the
caudate (Hietala, Syvalahti et al. 1995). Overall the evidence shows that patients with
schizophrenia and individuals at high-risk for psychosis have increased dopamine release in the
striatum and may be related to the illness severity.
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iii) Studies under dopamine release conditions
To study dopamine release, investigators used the amphetamine challenge method, as
amphetamine has been shown to be linked to psychosis (Ellinwood, Sudilovsky et al. 1973).
These studies reported elevated dopamine release in the striatum of antipsychotic-free patients
(Abi-Dargham, Gil et al. 1998) (Laruelle, Abi-Dargham et al. 1996) (Laruelle, Abi-Dargham et
al. 1999) and a sample of antipsychotic-naïve and antipsychotic-free patients (Breier, Su et al.
1997). Overall, these results illustrate increased dopamine release in patients with schizophrenia.
iv) Studies under dopamine depletion conditions
To investigate indirectly the dopamine levels at the synaptic cleft, a few studies have used alpha-
methyl-para-tyrosine (AMPT) to inhibit transiently the synthesis of dopamine. The first study
compared 18 antipsychotic-naïve and antipsychotic-free patients (M=11, F=7) to 18 healthy
controls (M=11, F=7) (Abi-Dargham, Rodenhiser et al. 2000). They demonstrated that patients
have greater amounts of dopamine occupying the D2/3 receptors in the striatum. In a follow-up
study, the same group assessed only six antipsychotic-naïve patients (M=2, F=4) with
schizophrenia and demonstrated a greater increase in dopamine D2/3 binding in the striatum,
suggesting greater dopamine levels at the synaptic cleft in the striatum compared to 8 healthy
controls (M=6, F=2) (Abi-Dargham, van de Giessen et al. 2009).
Furthermore, another study that used [11C]-raclopride after dopamine depletion with AMPT
found greater D2/3 receptor binding in the PCDC of 18 antipsychotic-naïve and antipsychotic-free
patients (M=13, F=5) (Kegeles, Slifstein et al. 2010). It is important to note that among the 18
patients assessed in this study, twelve were chronically ill and previously medicated.
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In summary, based on the presented evidence, antipsychotic-naive and antipsychotic-free
patients with schizophrenia present increased dopamine synthesis capacity, release after
amphetamine challenge and baseline dopamine levels in the striatum after dopamine depletion.
Thalamus
Nine studies assessed the thalamus, one of these studies using [18F] fallypride PET found
increased binding in six antipsychotic-naïve and 12 antipsychotic-free (M=14, F=7) (Kegeles,
Slifstein et al. 2010). Another study using the same technique assessing 15 antipsychotic-naïve
patients (M=10, F=5), however, reported reduced D2/3 receptor binding, with the greatest
difference in the left medial dorsal nucleus and left pulvinar (Buchsbaum, Christian et al. 2006).
Several other studies also reported decreased D2/3 receptor binding in the thalamus (Kessler,
Woodward et al. 2009) (Talvik, Nordstrom et al. 2006) (Abi-Dargham, van de Giessen et al.
2009) (Kegeles, Abi-Dargham et al. 2010). Talvik et al. demonstrated decreased D2/3 receptor
binding in the right medial thalamus (Talvik, Nordstrom et al. 2003); Yasuno et al., in the central
medial (Yasuno, Suhara et al. 2004) and posterior sub-region of the thalamus; and Kessler et al,
in the left medial thalamus (Kessler, Woodward et al. 2009). A later study by Talvik and
colleagues confirmed their earlier findings by demonstrating lower D2/3 receptor binding in the
right thalamus of patients compared with healthy controls, but they detected no difference in the
left thalamus (Talvik, Nordstrom et al. 2006). In contrast, four studies found no overall
difference in D2/3 receptor binding in the thalamus (Graff-Guerrero, Mizrahi et al. 2009)
(Tuppurainen, Kuikka et al. 2006) (Suhara, Okubo et al. 2002) (Glenthoj, Mackeprang et al.
2006). One assessed only 11 antipsychotic-naïve male patients using [11C] FLB 457 PET
(Suhara, Okubo et al. 2002)and the other assessed 25 antipsychotic-naïve patients (M=2, F=4)
using 123I-epidepride SPECT, the largest sample to date (Glenthoj, Mackeprang et al. 2006).
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Although most of the evidence suggests reduced D2/3 receptor binding in the thalamus of patients
with schizophrenia, the only study that performed partial volume correction, found increased
D2/3 binding in this region (Kegeles, Slifstein et al. 2010). As such, inconsistency in findings
for the thalamus warrants further studies.
Temporal, Limbic and Frontal Regions
Studies comparing D2/3 receptor binding between patients and healthy controls found patients had
equal amounts of D2/3 receptors in the limbic, sensorimotor, temporal, and frontal regions
(Talvik, Nordstrom et al. 2003). In contrast, a study specifically assessing the amygdala,
cingulate gyrus, and temporal cortices reported reduced D2/3 receptor binding in these regions
(Buchsbaum, Christian et al. 2006). Furthermore, a study that assessed the anterior cingulate of
11 antipsychotic-naïve male patients reported a 12.5 % reduction in D2/3 binding in patients
(Suhara, Okubo et al. 2002). As such, discrepancies may be attributed to sample and sex
differences.
A study that assessed dopaminergic synthesis capacity in the limbic and temporal regions
reported elevated dopamine levels (Kumakura, Cumming et al. 2007). In this study, a 50%
increase in F-DOPA clearance was detected in the amygdala of eight male patients (Kumakura,
Cumming et al. 2007). Greater dopamine synthesis capacity was also detected in the MPFC of 12
patients (M=12, F=2) (Lindstrom, Gefvert et al. 1999). Thus, although further investigations are
needed, preliminary results demonstrate reduced D2/3 receptor binding and potentially elevated
dopaminergic synthesis capacity in temporal and limbic regions of patients with schizophrenia
compared with healthy controls.
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In conclusion, evidence revealed no difference in D2/3 receptor binding, increased dopamine
synthesis capacity, increase dopamine release, increase dopamine occupying the D2/3 receptors in
the striatum, reduced D2/3 receptor binding in the thalamus and potentially increased dopamine
synthesis capacity in the temporal and limbic regions. Inconsistent results were reported in the
anterior cingulate and substantia nigra. The findings pertaining to D1 receptor binding were
inconsistent and further studies are needed to clarify inconclusive results.
3.4.3 GABAergic System
Presently, only one study has compared GABA levels independently between antipsychotic-free
patients and healthy controls. The study reported elevated GABA concentrations in MPC of 32
patients (M=11, F=5) (Kegeles, Mao et al. 2012). This study, albeit preliminary, suggests the
involvement of the GABAergic anomalies in schizophrenia. MRS studies assessing medicated
patients compared with healthy controls reported increased GABA/Cr in the medial frontal and
parietooccipital regions (Benes, Todtenkopf et al. 2000), reduced GABA/Cr concentrations in the
left basal ganglia but no difference in the frontal or occipital-parietal regions of early-stage
patients with schizophrenia (Goto, Yoshimura et al. 2009), lower GABA/Cr levels in the
occipital region of patients (Yoon, Maddock et al. 2010), but no difference from the medial
prefrontal and left basal ganglia (Tayoshi, Nakataki et al. 2010), and increased GABA/Cr in
medial frontal and parietooccipital regions (Ongur, Prescot et al. 2010). Furthermore, as
suggested by a recent study, GABA levels were elevated in younger patients compared with
older patients with schizophrenia, suggesting an association between the stage of illness and
GABA levels (Rowland, Kontson et al. 2013).
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3.5 Discussion
A number of studies revealed abnormalities in the glutamatergic system in antipsychotic-naïve or
antipsychotic-free patients with schizophrenia. In brief, studies focusing on the glutamatergic
system demonstrated that among individuals at high-risk for psychosis or during the first episode
of schizophrenia, GLX, glutamine and glutamate levels are elevated in most regions of the brain.
In contrast, studies looking at patients who were older than 35 years of age or labeled as chronic
showed low GLX levels, which may be a medication effect. In addition, one study reported
decreased NMDA binding in the hippocampus of antipsychotic-free patients. (Pilowsky, Bressan
et al. 2006).
Studies focusing on the dopaminergic system demonstrated a decrease in the dopamine D2/3
receptor binding in the thalamus, an increase in dopamine synthesis capacity in the striatum,
enhanced dopamine release and increased dopamine at baseline. Lastly, one study reported
elevation of GABA levels in MPFC of antipsychotic-free patients. Below we describe a model
that could explain these various findings.
It has been proposed that at the onset of the disorder, hyperfunctioning NMDA receptors on
GABAergic interneurons lead to excessive release of glutamate from pyramidal neurons (Olney
and Farber 1995). Excessive glutamate levels lead to excitotoxicity-mediated neuronal death
(Rothstein 1995). As a precursor for glutamine, some of the glutamate is converted to glutamine
within astrocytes (Daikhin and Yudkoff 2000) and result in high levels glutamine as
demonstrated by in vivo imaging studies. Elevated glutamate levels may also overstimulate
dopaminergic neurons resulting in high levels of dopamine, as suggested by striatal studies and
yet to be confirmed in the cortex (Exposito, Del Arco et al. 1999) (Segovia, Del Arco et al.
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1997). Further, given that glutamate is a precursor to GABA and that the current literature
suggests high levels of GABA early in the course of the illness, our model proposes that high
levels of GABA are driven by high levels of glutamate and that glutamate-to-GABA conversion
is intact. Another possible scenario is that excessive glutamatergic activity stimulates
interneurons to release more GABA. Finally, elevated GABA levels could be independent of
high glutamate levels as they could reflect abnormal GABA re-uptake by transporters. This
finding is supported by postmortem studies that reported reduced presynaptic GAT1 transporters
in patients lead to increased GABAergic transmission at the synapse due to diminished reuptake
(Menzies, Ooi et al. 2007). As a compensatory measure, post-synaptic GABAA receptors are up-
regulated, followed by the down-regulation of GAD67 and parvalbumin-positive interneurons
(Benes, Todtenkopf et al. 2000) (Lewis and Moghaddam 2006) (Benes, Vincent et al. 1992)
(Lisman, Coyle et al. 2008), eventually leading to reduced GABAergic activity. Irrespective of
the underlying mechanism, high levels of GABA could be contributing to the relative stability of
the excitation-inhibition system.
Dopamine effect on LTP facilitation depends on the concentration and activated sub-receptors.
Dopaminergic receptors are in close proximity to glutamatergic receptors and appear to have a
major role in synaptic modulation, by affecting the phosphorylation of glutamatergic NMDA and
AMPA receptors (Chase and Oh 2000). The relationship between dopamine levels and LTP
facilitation is reported as an inverted “U” shape dose response curve (Seamans and Yang 2004;
Monte-Silva, Liebetanz et al. 2010). Low levels of dopamine preferentially activate presynaptic
D2/3 receptors, which reduces the release of dopamine and essentially LTP facilitation. On the
other hand, high levels equally activate postsynaptic D1 and D2/3 receptors, counteracting each
other’s effect. However, at optimal dopamine levels, D1 postsynaptic receptors are stimulated
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and LTP is facilitated. That is, insufficient or excessive dopamine levels impair LTP facilitation
and optimal facilitation is achieved at moderate concentrations. Thus, high levels of dopamine in
the striatum and potentially in the cortex of patients with schizophrenia likely result in impaired
LTP because excessive dopamine may lead to the up-regulation of the D2/3 receptors (Wang, Pei
et al. 2010) or the functional sensitisation of the D2/3 receptors (Seeman, Weinshenker et al.
2005). Presynaptic D2/3 receptors on interneurons enable LTP facilitation by suppressing
GABAergic inhibition on pyramidal neurons (Xu and Yao 2010). Low levels of dopamine in the
cortex can also result in impaired LTP. When D2/3 receptors are hyperfunctioning,
understimulated pyramidal neurons are not sufficiently suppressed, thereby leading to excessive
excitation. When D1 receptors are stimulated, LTP activity is facilitated and resting
glutamatergic neurons increase their production of neurotransmitters and receptors by
stimulating the CAMP/ protein-kinase-A (PKA) pathway (Gurden, Takita et al. 2000; Matsuda,
Marzo et al. 2006). As such, dopamine regulates both glutamatergic excitatory and GABAergic
inhibitory circuits (Wigstrom and Gustafsson 1983) and the balanced concentration of dopamine
and interplay between excitation and inhibition facilitates the induction of LTP (Homayoun and
Moghaddam 2007). Several studies have demonstrated in vivo evidence for impaired LTP in
patients with schizophrenia. Using transcranial direct current stimulation, Hasan et al (2011)
showed that multi-episode patients had reduced LTP-like plasticity compared to healthy controls
and recent-onset patients. (Hasan, Nitsche et al. 2011). LTP impairments have also been
revealed in the motor cortex and dorsolateral prefrontal cortex of patients using paired
associative stimulation.(Frantseva, Fitzgerald et al. 2008; Rajji 2014) LTP plasticity was also
shown to be impaired in both medicated and unmedicated patients using transcranial magnetic
stimulation (Fitzgerald, Brown et al. 2004; Daskalakis, Christensen et al. 2008). Lastly,
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impaired LTP has been demonstrated in the visual cortex using high-frequency stimulation
(Cavus, Reinhart et al. 2012).
3.6 Limitations
First, discrepancies in findings may be accounted for by the difference in patient population,
such as sex. Not all the studies included in this review assessed antipsychotic-naïve patients, as
some assessed antipsychotic-free; therefore, the effects of antipsychotics cannot be completely
discounted, as studies in animals suggest that even minimal exposure to antipsychotics can
modulate glutamatergic activity (Lopez-Gil, Babot et al. 2007). Second, the interpretations of
GABA and GLX measurements present another limitation. The validity of early 1H-MRS studies
may be less compared with recent studies, which employed better 1H-MRS technology including
acquisition and quantification that allows the separation of overlapping resonance signals arising
from glutamate, glutamine, and GABA. Third, MRS is capable of detecting the total
concentration of a neurochemical and currently cannot distinguish between intracellular and
extracellular glutamate, glutamine or GABA (Stagg, Bachtiar et al. 2011). However, one study
showed a relationship between MRS-derived measures of GABA and glutamate and behavior,
suggesting that what is measured by MRS is associated with neurotransmission (Stagg, Bachtiar
et al. 2011). Fourth, discrepancies among PET studies may have resulted from the differences in
the selectivity and affinity of the radiotracers used. For instance, [11C]-N-methylspiperone
(NMSP) binds to D2/3 receptors and 5-HT2 serotonin receptors in vivo and has an affinity for
dopamine D4 receptors in vitro (Seeman, Guan et al. 1993). The increase in D2/3 binding
detected with this tracer may include the binding of serotonin receptors, which are not detected
using other ligands (Frost, Smith et al. 1987). Also, not all radioligands have the same affinity
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for D2/3 receptors, presenting a major limitation when comparing one study to another. Lastly,
since the sample size in most of the studies was small and heterogeneous, larger homogenous
samples are needed to verify such findings. Therefore, future studies using better 1H-MRS
technology, more selective PET ligands and large homogenous samples are necessary in order to
verify these observations.
3.7 Conclusion
LTP is a neuronal mechanism mediating learning and memory. This review presented evidence
highlighting abnormal glutamatergic, dopaminergic and GABAergic systems in antipsychotic-
naïve and antipsychotic-free patients with schizophrenia. As these systems are essential for LTP
facilitation, cognitive impairments associated with schizophrenia may be explained by impaired
LTP formation. This proposed model does not negate that these same systems could be
mediating other dimensions of schizophrenia, e.g. positive and negative symptoms, and not
necessarily through LTP impairments. Lastly, it is important to note that medicated patients also
experience cognitive deficits and that understanding the neurochemical abnormalities underlying
these deficits among these patients could lead to better remediation interventions.
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Figure 3. Neurochemical Model. Hypoactive NMDA receptor causes downstream hyper
glutamatergic activity, which leads to the conversion of glutamate to glutamine by the enzyme
glutaminase, as such increasing glutamine levels (Lisman, Coyle et al. 2008). Glutamine is a
molecule which cannot exert neurotoxic effects (Rowland, Bustillo et al. 2005). To balance out
excitatory activity with inhibitory activity, glutamate is converted into GABA, the main
inhibitory neurotransmitter. Extracellular dopamine is regulated by NMDA receptors located on
the dopaminergic neuron. Hypoactive NMDA receptors on cortico-brainstem pathway reduce
inhibition of tonic dopamine neurons of the mesocortical pathway, which leads to increase in DA
release.(Javitt 2007; Lisman, Coyle et al. 2008). To attenuate the dopamine release, D2/3 receptor
density is upregulated.
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Figure 4. Neurochemicals and Receptors in Patients with Schizophrenia Relative to
Healthy Controls in Different Brain Regions. * Evidence is based on one study
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Chapter 4.
4. Pharmacological Modulation of Long-term Potentiation in the Dorsolateral Prefrontal Cortex
Bahar Salavati, Zafiris J. Daskalakis, Reza Zomorrodi, Daniel M. Blumberger, Robert Chen,Bruce G. Pollock, Tarek K. Rajji,
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4.1 Abstract
Synaptic long-term potentiation (LTP) depends on glutamatergic neurotransmission and is
modulated by cholinergic, dopaminergic, and GABAergic inputs. Paired Associative Stimulation
(PAS) is a neurostimulation paradigm that assesses LTP-like activity (PAS-induced LTP) in the
dorsolateral prefrontal cortex (DLPFC) in vivo. We conducted a hypothesis-driven
pharmacological study to assess the role of cholinergic, dopaminergic, GABAergic, and
glutamatergic neurotransmission on PAS-induced LTP in the DLPFC in vivo. We hypothesized
that increasing dopaminergic tone with L-DOPA and cholinergic tone with rivastigmine will
enhance PAS-induced LTP while increasing GABAergic tone with baclofen and inhibiting
glutamatergic neurotransmission with dextromethorphan will reduce it. In this randomized
controlled, double-blind cross-over within-subject study, 12 healthy participants received five
sessions of PAS to the DLPFC in a random order, each preceded by the administration of placebo
or one of the four active drugs. As predicted, L-DOPA and rivastigmine enhanced PAS-induced
LTP in the DLPFC and dextromethorphan inhibited it compared to placebo. In contrast, baclofen
did not have a significant effect. This study demonstrates for the first time the role of the
dopaminergic, cholinergic, and glutamatergic neurotransmission in DLPFC neuroplasticity. It also
provides a novel approach to study DLPFC neuroplasticity and its modulation in patients with
DLPFC dysfunction.
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4.2 Introduction
Neuroplasticity refers to the ability of the brain to change and adapt in response to experiences
(Pascual-Leone, Amedi et al. 2005). Long-term potentiation (LTP) is a synaptic form of
neuroplasticity that is considered to be fundamental for learning and memory (Collingridge and
Bliss 1995). The dorsolateral prefrontal cortex (DLPFC) is critical to several cognitive functions
including learning and memory (Fuster 2008). Further, abnormalities in the DLPFC structure and
function are observed in various brain disorders including Alzheimer’s disease (Kaufman, Pratt et
al. 2010), depression (Koenigs and Grafman 2009), and schizophrenia (Callicott, Bertolino et al.
2000). Thus, studying LTP and its modulation in the DLPFC could advance knowledge of DLPFC
function and lead to the development of effective cognitive interventions for these brain disorders.
Paired associative stimulation (PAS) is a neurostimulation paradigm that induces in vivo LTP-like
activity in the human cortex (Stefan, Kunesch et al. 2000; Rajji, Sun et al. 2013). PAS simulates a
spike-timing dependent plasticity protocol, resulting in the potentiation of cortical output in
response to single-pulse transcranial magnetic stimulation (TMS). Using well-established methods
of combining TMS with electroencephalography (EEG), PAS has been shown to result in LTP-
like activity in the human DLPFC as captured by the potentiation of TMS-induced cortical evoked
activity over the DLPFC (Rajji, Sun et al. 2013). PAS-induced LTP has also been shown to be
impaired in several brain disorders, e.g. Alzheimer’s disease (Battaglia, Wang et al. 2007),
depression (Player, Taylor et al. 2013), and schizophrenia (Frantseva, Fitzgerald et al. 2008).
Synaptic LTP depends on glutamatergic neurotransmission (Luscher and Malenka 2012) and is
modulated by cholinergic (Picciotto, Higley et al. 2012), dopaminergic (Tritsch and Sabatini 2012)
and GABAergic (Nugent and Kauer 2008) neurotransmission. A few studies assessed the
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pharmacological modulation of PAS in humans, with PAS applied to the motor cortex. In one
study, baclofen (50 mg), which increases GABAergic tone, decreased PAS-induced LTP
(McDonnell, Orekhov et al. 2007). In a second study, dextromethorphan (150 mg), which blocks
NMDA glutamatergic receptors, decreased PAS-induced LTP (Stefan, Kunesch et al. 2002). In a
third study, L-DOPA (100 mg), which increases dopaminergic tone, increased LTP (Kuo, Paulus
et al. 2008; Thirugnanasambandam, Grundey et al. 2011). In a fourth study, rivastigmine (3 mg),
which increases cholinergic tone, enhanced PAS-induced LTP (Kuo, Grosch et al. 2007).
To date, no study has assessed the pharmacological modulation of PAS-induced plasticity in the
DLPFC. Further, no study assessed all of these drugs in the same participants and not all of the
above studies were double-blind or randomized. Thus, we conducted the first pharmacological
modulation study of DLPFC plasticity in vivo using PAS-EEG and a double-blind randomized
controlled within-subject design that included all of the above four drugs. We hypothesized that,
compared to placebo, L-DOPA and rivastigmine would increase PAS-induced LTP, while
baclofen and dextromethorphan would decrease it.
4.3 Participants and Methods
4.3.1 Overall Study Design
This was a double-blind randomized controlled within-subject crossover study. Each participant
received five sessions of PAS in a random order, each preceded by the administration of placebo
or one of the four active drugs, and separated by at least one week to minimize drug interference
and carryover effects. The time of each drug administration before PAS was based on the time of
its plasma peak level, i.e. 1 hour for baclofen, 3 hours for dextromethorphan, 1 hour for L-DOPA,
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and 2 hours for rivastigmine. Placebo was randomly given to each participant at 1, 2 or 3 hours
prior to the administration of PAS. The doses of the drugs (Baclofen 50 mg, dextromethorphan
150 mg, L-DOPA 100 mg, and rivastigmine 3 mg) were based on the previous studies
demonstrating effects at similar doses on PAS-induced LTP in the motor cortex. Across the
participants, the sequences of drug administration were counterbalanced. The administrator of the
experiments and participants were blind to drug assignment. All data processing and analyses were
also completed under blind condition.
4.3.2 Participants
Participants were females and males; aged 18 to 55 years because cortical neuroplasticity as
measured using neurophysiologic methods starts to decline around age 50 (Muller-Dahlhaus,
Orekhov et al. 2008); non-smokers, not diagnosed with any neurologic or psychiatric disorder;
right-handed to ensure homogeneity in hemisphere dominance; had no contraindication to TMS
(Rossi, Hallett et al. 2009) or MRI; and provided written informed consent. The study was
approved by the Centre for Addiction and Mental Health Research Ethics Board.
4.3.3 Locating and Co-Registering the DLPFC
The left DLPFC is located at the junction of the middle and anterior third of the middle frontal
gyrus (Talairach Coordinates (x, y, z) = (-50, 30, 36)), which corresponds to the posterior region
of Brodmann area 9 and the superior section of area 46. Following previously published methods
localization of the DLPFC was achieved through neuronavigation techniques using the MINIBIRD
system (Ascension Technologies) and each participant’s T1- weighted MRI with fiducial markers
placed on the nasion, inion, left and right tragus and vertex (Rajji, Sun et al. 2013).
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4.3.4 Electromyography (EMG) Recordings from the Motor Cortex and TMS-EEG in the DLPFC
Following established methods, we used a 7-cm figure-eight coil and a Magstim 200 stimulator
(The Magstim Company, Whitland, UK) to determine the participant’s resting motor threshold
(RMT) (defined as the minimum stimulus intensity that elicits a motor evoked potential (MEP) of
more than 50mV in 5 of 10 trials) (Rajji, Sun et al. 2013). MEP activity was measured through
EMG recordings from the right abductor pollicis brevis muscle. The RMT was then adjusted to a
suprathreshold intensity with mean peak-to-peak MEP amplitude of ~1 mV over 20 trials, which
corresponded to approximately 120% of the RMT. This intensity referred to as SI1mV was then
used to deliver 100 single TMS pulses at 0.1 Hz to the scalp over the DLPFC throughout the PAS
experiment. We acquired EEG through a 64-channel Synamps 2 (Neuroscan Inc.) EEG
system. All electrodes (Ag/AgCl ring electrodes) impedance were ≤5 kΩ and referenced to an
electrode positioned posterior to Cz electrode. In addition, EEG signals were recorded using DC
and a low pass filter, anti-aliasing filter, of 200 Hz, at 20 kHz sampling rate, which was shown to
avoid saturation of amplifiers and minimize TMS-related artifact (Figure 4).
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Figure 5. Experimental design. This image illustrates one session of the PAS protocol
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4.3.5 PAS to the DLFPC
PAS consisted of repetitive simultaneous pairing of peripheral nerve stimulation (PNS) to the
median nerve followed 25 ms later by a transracial magnetic stimulation (TMS) pulse to the scalp
over the DLPFC. There were 180 paired pulses delivered over a 30 min period at 0.1 Hz. This
paradigm has been shown to induce LTP-like activity over the DLPFC in healthy individuals
(Rajji, Sun et al. 2013). PNS was delivered at 300% of the sensory threshold, defined as the
minimum intensity that the participant perceives sensation. Given that attention has been shown
to affect the level of potentiation following PAS (Stefan, Wycislo et al. 2004), participants were
asked to maintain attention by looking at their wrist and continuously counting the total number
of PAS pulses delivered. Before PAS, 100 single pulses TMS was delivered to the left DLPFC
while recording EEG using Scan 4.1 (Compumedics, USA) to generate cortical evoked activity
(CEA) pre-PAS. Then the drug was given and at 1-3 hours post-drug administration PAS was
delivered. The time interval between drug administration and PAS was equal to the time of plasma
peak for each drug. After PAS, 100 single pulse TMS combined with EEG were delivered to the
left DLPFC to generate CEA at time 0, 15, 30, and 60 min post-PAS.
4.3.6 EEG Data Processing
Using MATLAB (The MathWorks Inc. Natick, MA, USA), raw EEG recordings were first
downsampled from 20 to 1 kHz and then segmented from -1000 ms to + 2000 ms relative to the
onset of the TMS pulse. Epochs were then baseline corrected -500 ms to -110 ms with respect to
the pre-stimulus interval. To minimize TMS artifacts, the data was re-segmented from 25ms to
2000ms. Thereafter, the EEG data was digitally filtered using a second-order, Butterworth, zero-
phase shift 1-55 Hz band pass filter (24dB/Oct). EEG recordings from all five time points of the
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study (pre and at time 0, 15, 30, and 60 min post-PAS) were then concatenated in order to apply
the same objective criteria for de-noising the data (Rajji, 2014). Then, an electrodes-by-trials
matrix of ones and zeros was created and assigned a value of zero if an epoch had the following:
1) amplitude larger than +/- 150 μV; 2) power spectrum that violated 1/f power law; or 3) standard
deviation 3 times larger than the average of all trials. An electrode was rejected if its corresponding
row had more than 60% of columns (trials) coded as zeros. An epoch was removed if its
corresponding column had more than 20% of rows (electrodes) coded as zeros. Next, independent
component analysis (ICA) (EEGLAB toolbox; Infomax algorithm) was performed to remove
remaining artifacts such as eye blink traces, muscle artifacts and other noise from the EEG data.
Finally, the data was re-referenced to the average, generating a clean signal devoid of noise for
each participant.
At each time point before and after PAS, CEA was calculated as the mean of the 100 rectified
areas under the curve (AUC) generated from the 100 TMS pulses and using the electrode under
the site of stimulation, i.e. over the left DLPFC. Each AUC was calculated using the interval
between 50 and 275 ms post-TMS pulse. The first interval cutoff (i.e. 50 ms) was chosen as it
represents the earliest artifact-free data, while the second interval cutoff (i.e., 275 ms) was chosen
because it represents the end of the window during which potentiation of CEA is still significant
post-PAS (Rajji, Sun et al. 2013).
4.3.7 Statistical Analysis
To measure LTP-like activity over the DLPFC, mean CEA at each time point after PAS was
divided by mean CEA before PAS. This ratio represented potentiation of CEA at each of the time
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points after PAS. Since the timing of maximum potentiation post-PAS varies among participants,
the maximum CEA ratio at any of these time points was selected for each participant. This
maximum CEA ratio for each drug condition represented PAS-induced LTP for each participant
under the influence of each drug.
All data was first checked for normality using the Kolmogorov–Smirnov test. To test our primary
hypotheses and assess whether there is a drug effect on PAS-induced LTP, a repeated measures
analysis of variance (rmANOVA) was conducted with the drug condition (placebo vs. baclofen
vs. dextromethorphan vs. L-DOPA vs. rivastigmine) as the repeated measure. This was followed
by a series of posthoc analyses, with Bonferroni correction, to compare PAS-induced LTP under
each of the active drug conditions to PAS-induced LTP under placebo.
To assess whether there is PAS-induced LTP under each drug condition, we ran a series of one-
sample t-tests to compare PAS-induced LTP under each drug condition to a test value of 1
representing no LTP. Bonferroni correction was also applied in this analysis.
4.4 Results
Thirteen participants (4 females and 9 males) took part in this study. All participants completed all
sessions except for one participant who dropped out after only one of the five sessions and data
for this participant was not used. Participants’ demographics and basic neurophysiologic
characteristics are described in Table 3.
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Table 3. Demographic and Basic Neurophysiologic Characteristics
Characteristic Mean (SD)
Age (years) 31.3 (10.5)
Gender (Female, %) 4 (25)
Education (years) 15.3 (2.3)
Resting Motor Threshold 49.0 (4.9)
SI1mV 61.5 (8.3)
Peripheral Nerve Stimulation Count*
Placebo
Baclofen
Dextromethorphan
L-DOPA
Rivastigmine
175.5 (9.6)
171.6 (11.2)
183.3 (22.3)
176.7 (6.0)
174.3(7.1)
*There was no significant drug effect on peripheral nerve stimulation count (F (1.65, 18.15) =
1.28, p = 0.30) and under each drug condition, the count did not differ significantly from the actual
number of peripheral nerve stimulations (i.e. 180) (p’s > 0.05).
SI1mV = Stimulation intensity with a mean peak-to-peak motor evoked potential amplitude of 1
mV over 20 trials; SD = standard deviation.
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All outcome data was normally distributed. RmANOVA revealed that there was a drug effect on
PAS-induced LTP as measured from the electrode over the site of stimulation (F (4, 44) = 10.08,
p <0.001). Further, posthoc pairwise comparisons against placebo, with Bonferroni correction,
revealed that LTP was increased after the intake of L-DOPA (p =0.004) or rivastigmine (p =0.009),
and decreased after the intake of dextromethorphan (p =0.007). In contrast, there was no change
after the intake of baclofen (p =0.54) (Figures 6 and 7).
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Figure 6. Effects of Drugs on DLPFC Neuroplasticity. Figure 6A: This figure illustrates the
effects of drugs (L-DOPA, baclofen, rivastigmine, dextromethorphan, and placebo on PAS-
induced LTP-like activity (PAS-induced LTP) expressed as a ratio of post-PAS CEA / Pre-PAS
CEA over the DLPFC (Ratio). The p-values refer to the comparisons between each active drug
and placebo. Error bars: +/-1 SE. Figure 6B: These topographical plots illustrate the effects of
drugs (L-DOPA, baclofen, rivastigmine, dextromethorphan, and placebo) on PAS-induced LTP-
like activity in the DLPFC. The value of 1 represents no LTP-like activity.
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Figure 7. Event-Related Potentials (ERPs) Across All Conditions. This figure illustrates the
ERPs as captured from the electrode over the DLPFC before drug administration and following
placebo or one of the four active drugs. Each ERP represents the average across all participants,
and “Pre” represents the average across all time points of Pre-PAS conditions.
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Finally, compared to a test value of 1 which represents no LTP, participants experienced PAS-
induced LTP under placebo, L-DOPA, and rivastigmine, but not under baclofen or
dextromethorphan condition, after Bonferroni corrections (Table 4).
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Table 4: Potentiation over the Dorsolateral Prefrontal Cortex under each Drug Condition
Drug PAS-induced LTP
(SD)
t (df) p-value
Placebo 1.25 (0.14) 4.31 (11) 0.001
Baclofen 1.15 (0.52) 1.0 (11)
0.34
Dextromethorphan 0.95 (0.19)
-0.95 (11)
0.36
L-DOPA 1.64 (0.37) 6.0 (11) <0.001
Rivastigmine 1.63 (0.40)
5.36 (11)
<0.001
PAS-induced LTP = Paired Associative Stimulation (PAS)-induced Long-Term Potentiation
(LTP)-like activity as measured by Cortical Evoked Activity (CEA) post-drug/CEA pre-drug at
maximum LTP-like activity; SD = standard deviation; t(df) = one sample t-test (degrees of
freedom) with a test value of 1 which is equivalent of no LTP.
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4.5 Discussion
This study confirmed our hypotheses that L-DOPA and rivastigmine enhance neuroplasticity in
the DLPFC in vivo and that dextromethorphan suppresses it. It did not confirm the fourth
hypothesis that baclofen reduces DLPFC neuroplasticity compared to placebo although under
baclofen exposure participants did not experience significant DLPFC neuroplasticity compared to
baseline. To our knowledge, this is the first study to assess the pharmacological modulation of
DLPFC neuroplasticity in humans.
Our finding that L-DOPA enhanced DLPFC neuroplasticity is consistent with animal studies that
reported enhanced LTP in the prefrontal cortex following dopaminergic intervention (Otani 2003).
Dopaminergic neurons project from the ventral tegmental area to the prefrontal cortex. These
projections activate dopamine D1 receptors on prefrontal pyramidal neurons and facilitate NMDA
receptor activity (Seamans, Durstewitz et al. 2001; Wang and O'Donnell 2001). L-DOPA is a
dopamine precursor that is converted to dopamine, which activates these dopaminergic receptors
(Okereke 2002), resulting in enhanced LTP.
Our finding is also consistent with human studies that assessed dopaminergic modulation of PAS-
induced LTP in the motor cortex (Kuo, Paulus et al. 2008; Nitsche, Kuo et al. 2009; Korchounov
and Ziemann 2011; Thirugnanasambandam, Grundey et al. 2011; Kishore, Popa et al. 2014). In
the motor cortex, L-DOPA increases the magnitude and duration of PAS-induced LTP (Kuo,
Paulus et al. 2008). This effect was not affected by sulpiride (Ross, Heinlein et al. 2006; Nitsche,
Kuo et al. 2009), a D2 receptor antagonist, underlining the role of D1 receptors in L-DOPA
enhancement of PAS-induced LTP. Of note, ropinirole, a dopamine D3 receptor agonist also
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enhanced PAS-induced LTP following an inverted U-shaped dose-response curve (Monte-Silva,
Kuo et al. 2009).
We also found that rivastigmine enhances DLPFC neuroplasticity. Rivastigmine increases
synaptic levels of acetylcholine by inhibiting acetylcholine esterase, allowing for longer
cholinergic receptors activation (Polinsky 1998). In animal and slice studies cholinergic activity
plays a pivotal role in LTP facilitation in the prefrontal cortex (Vidal and Changeux 1993). It has
been shown that cholinergic agonists enhance LTP (Blitzer, Gil et al. 1990; Brocher, Artola et al.
1992). This effect on LTP is thought to be mediated by a transient reduction in inhibitory
transmission, which in turn, lowers the threshold for NMDA receptor-dependent LTP (Metherate
and Ashe 1993; Letzkus, Wolff et al. 2011). In the human motor cortex, biperiden, a muscarinic
M1 receptor cholinergic antagonist suppressed (Korchounov and Ziemann 2011), while
rivastigmine enhanced PAS-induced LTP (Kuo, Grosch et al. 2007).
Our third and confirmed hypothesis was that dextromethorphan suppresses DLPFC
neuroplasticity. This finding is consistent with previous studies assessing the effects of
dextromethorphan on LTP in the slice, animal and human studies (Krug 1993; Stefan, Kunesch et
al. 2002). Dextromethorphan is a non-competitive NMDA receptor antagonist (Church, Lodge et
al. 1985). Thus, it is expected to suppress NMDA-receptor dependent LTP. Only one study
assessed the effects of dextromethorphan on PAS-induced LTP, which reported abolishment of
PAS-induced LTP plasticity in the motor cortex compared to placebo (Stefan, Kunesch et al.
2002). Our finding with dextromethorphan also provides evidence that PAS-induced LTP in the
DLPFC represents synaptic LTP by being dependent on functional NMDA receptors.
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Contrary to our fourth hypothesis, we did not find a difference in PAS-induced LTP under baclofen
compared to placebo. However, we still found that under baclofen exposure, participants did not
experience significant PAS-induced LTP compared to baseline. Our hypothesis was based on a
study that assessed baclofen effects in the motor cortex and that included only five participants
(McDonnell, Orekhov et al. 2007). Thus, the discrepancy could be due to the different site of
plasticity and to the difference in power. Baclofen is a GABAB receptor agonist that, through the
promotion of inhibitory neurotransmission, could suppress PAS-induced LTP. However, through
presynaptic GABAB receptor, it could also lead to decreased release of GABA via GABAB
receptor-mediated autoinhibition (Jablensky 1997), thus, facilitating LTP. In mice, deletion of
GABAB autoreceptors leads to a failure in LTP expression (Vigot, Barbieri et al. 2006).
This study is limited by a relatively small sample size. However, the sample size was calculated
based on previously published literature in the motor cortex. Another limitation is that we did not
measure blood levels of the drugs to time the delivery of PAS. However, this limitation is mitigated
by our delivery of PAS based on published plasma levels peak values. Finally, this study assessed
the impact of a single dose on PAS-induced LTP. These medications are used chronically in
clinical settings. Thus, future studies should assess the effects of chronic exposure to these
medications in healthy individuals as well as patients with brain disorders associated with
abnormalities in these neurochemical systems.
4.6 Conclusion
In conclusion, this is the first study investigating the pharmacological modulation of DLPFC
neuroplasticity in humans. The study confirmed our hypotheses that dopaminergic and cholinergic
neurotransmission enhance DLPFC neuroplasticity while suppressing glutamatergic
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neurotransmission reduces it. Future studies should assess this modulation in clinical conditions to
better understand the pathophysiology underlying these conditions as well the mechanisms that
these drugs target in various brain disorders.
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Chapter 5
5. Pharmacological Manipulation of Cortical Inhibition in the Dorsolateral Prefrontal Cortex
Bahar Salavati, Tarek K. Rajji, Reza Zomorrodi, Daniel M. Blumberger, Robert Chen, Bruce G. Pollock, Zafiris J. Daskalakis
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5.1 Abstract
Cortical inhibition (CI) occurs largely through GABA receptor mediated inhibitory
neurotransmission, which can be modulated by cholinergic, dopaminergic, and glutamatergic
inputs. Transcranial magnetic stimulation (TMS) can be used to index CI through a paradigm
known as long interval cortical inhibition (LICI). When TMS is combined with
electroencephalography (EEG), LICI can index GABA receptor mediated inhibitory
neurotransmission in the dorsolateral prefrontal cortex (DLPFC). We conducted a hypothesis-
driven pharmacological study to assess the role of cholinergic, dopaminergic, GABAergic, and
glutamatergic neurotransmission on LICI from the DLPFC using TMS-EEG. In this randomized
controlled, double-blind crossover within-subject study, 12 healthy participants received five
sessions of LICI to the DLPFC in a random order, each preceded by the administration of placebo
or one of the four active drugs. LICI was assessed after each drug administration and compared to
LICI after placebo. Relative to placebo, baclofen resulted in a significant increase in LICI, while
rivastigmine resulted in a significant decrease in LICI. Dextromethorphan and L-DOPA did not
result in a significant change in LICI relative to placebo. Our study confirms that LICI in the
DLPFC is largely mediated by GABAB receptor mediated inhibitory neurotransmission and also
suggests that cholinergic modulation decreases LICI in the DLPFC. Such findings may help guide
future work examining the neurophysiological impact of these neurotransmitters in healthy and
diseased states.
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5.2 Introduction
The dorsolateral prefrontal cortex (DLPFC) is a critical brain region that is involved in several
important domains of cognition including learning and memory (Fuster 2008). Abnormalities in
DLPFC structure and function are observed in various brain disorders including addiction (Naim-
Feil, Bradshaw et al. 2015), Alzheimer’s disease (Kaufman, Pratt et al. 2010), depression (Koenigs
and Grafman 2009), Parkinson’s disease (Ko, Antonelli et al. 2013), and schizophrenia (Goto,
Yang et al. 2010). GABA plays an important role in DLPFC function as it synchronizes the activity
of pyramidal neurons (Sederberg, Schulze-Bonhage et al. 2007). This synchronization is closely
related to GABA receptor function and shown to play a role in learning and memory (Heaney and
Kinney 2016). Thus, studying the mechanisms involved in GABA receptor mediated inhibitory
neurotransmission from the DLPFC could advance our knowledge of the mechanisms involved in
cognition while also helping to identify treatment for disorders in which the DLPFC has been
shown to be dysfunctional (e.g., depression, schizophrenia).
Transcranial magnetic stimulation (TMS) combined with electroencephalography (EEG) can be
used to assess in vivo GABA neurotransmission from the DLFPC through a paradigm known as
long-interval cortical inhibition (LICI) with high test-retest reliability (Farzan, Barr et al. 2010).
LICI is a paired-pulse inhibitory paradigm that consists of a suprathreshold conditioning stimulus
(CS), followed by a suprathreshold test stimulus at a long interstimulus intervals (e.g. 50 - 200 ms)
(Valls-Sole, Pascual-Leone et al. 1992).
There are several lines of evidence that suggest that LICI reflects GABAB receptor mediated
inhibitory neurotransmission. First, LICI reduces short interval cortical inhibition (SICI), a
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GABAA receptor mediated inhibitory paradigm (Sanger, Garg et al. 2001). This is consistent with
the finding that presynaptic GABAB activation inhibits GABA release with a concomitant
reduction in GABAA receptor mediated inhibition (Werhahn, Kunesch et al. 1999; Werhahn,
Kunesch et al. 1999). Second, LICI is evoked with a superthreshold intensity CS, which produces
a long lasting inhibition (Valls-Sole, Pascual-Leone et al. 1992) supporting the finding that
GABAB receptor mediated inhibition has a greater activation threshold and longer inhibitory effect
(Sanger, Garg et al. 2001). Third, the administration of GABAB receptor agonist baclofen has been
shown to enhance LICI (McDonnell, Orekhov et al. 2006). Furthermore, LICI has been linked to
DLPFC function, as prefrontal LICI strength correlates with individual performance on a working
memory task (Rogasch, Daskalakis et al. 2015) and was found to be dysfunctional in disorders
including schizophrenia (Radhu, Garcia Dominguez et al. 2015) Parkinson’s (Chu, Wagle-Shukla
et al. 2009) and depression (Croarkin, Nakonezny et al. 2014).
Although LICI is closely linked to GABAB receptor mediated inhibitory neurotransmission, the
influence of other neurotransmitters cannot be excluded. The interaction between GABAergic with
dopaminergic, cholinergic and glutamatergic neurotransmission is complex. Dopamine facilitates
GABA release via dopamine D1 receptors and inhibits release via dopamine D2 receptors (Harsing
and Zigmond 1997). GABAergic activity is also enhanced through cholinergic nicotinic receptors
or muscarinic M3 receptors, but inhibited through muscarinic M4 receptors (Zhang and Warren
2002). Lastly, NMDA activation on GABAergic neurons enhances GABAergic activity, while
NMDA antagonism on glutamatergic neurons reduces excitatory drive on GABAergic neurons
resulting in decreased inhibition in the cortex (Olney, Newcomer et al. 1999)
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The application of a single dose of a central nervous system (CNS) drug that acts on a specific
neurotransmitter or neuromodulator system have been used to understand TMS measures of
cortical inhibition and excitation. For instance CNS drugs, such as, baclofen, and
dextromethorphan have been used to increase and decrease GABAergic and glutamatergic tone,
respectively, while rivastigmine and L-DOPA have been used to increase cholinergic and
dopaminergic tone, respectively.
Several studies suggest that in vivo LICI from the motor cortex in healthy controls is enhanced by
increasing GABAergic tone, as GABAergic drugs such as, baclofen (McDonnell, Orekhov et al.
2006; Premoli, Rivolta et al. 2014) vigabatrin (Pierantozzi, Marciani et al. 2004) and tiagabine
(Werhahn, Kunesch et al. 1999) increased LICI, tiagabine possibly through GABAB activation due
to the increased availability of GABA in the synaptic cleft (Ziemann, Reis et al. 2015).
Nonetheless, the contribution of other neurotransmitters on LICI is unknown (Paulus, Classen et
al. 2008). A few studies have assessed the pharmacological modulation of these neurotransmitters
on in vivo cortical excitability in the motor cortex. Both dextromethorphan and L-DOPA decreased
cortical excitability (Priori, Berardelli et al. 1994; Ziemann, Chen et al. 1998), while rivastigmine
had no significant effect (Langguth, Bauer et al. 2007). One limitation of these findings is that
TMS was applied to the motor cortex as opposed to the DLPFC, the latter being a cortical region
whose physiological function is of considerable significance in attempting to understand
pathophysiology of severe psychiatric disorders.
To date, no study has assessed the pharmacological modulation of LICI from DLPFC stimulation.
Further, no study has assessed all of these drugs in the same participants using a double-blind
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randomized controlled design. Thus, we conducted the first pharmacological modulation of
DLPFC LICI in vivo using TMS-EEG and a double-blind, randomized controlled within-subject
design that included all of the above four drugs. We hypothesized that, compared to placebo,
baclofen, L-DOPA and dextromethorphan and would increase LICI, while rivastigmine would
decrease it.
5.3 Methods and Participants
5.3.1 Overall Study Design
This was a double-blinded randomized controlled within-subject crossover study. Each participant
received five sessions of LICI in a random order, each preceded by the administration of a placebo
or one of the four active drugs, and separated by at least one week to minimize drug interference
and carryover effects (Korchounov and Ziemann 2011). LICI was measured Pre and Post-Drug,
and Post-LICI was administered after the drug had reached plasma peak level (Table 5). The doses
of the drugs were based on the previous studies demonstrating effects at similar doses on LICI in
the motor cortex. Across the subjects, the sequences of drug administration were counterbalanced.
The administrator of the experiments and participants were blind to drug assignment. All data
processing and analyses were also completed under blind condition.
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Figure 8. LICI Protocol. This figure illustrates one session of LICI
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Table 5. Properties of Drugs
Drugs Main mechanism of action
Dose (mg) Plasma Peak (hr)
Baclofen GABA-B agonist 50 1
Dextromethorphan NMDA antagonist 150 3
L-DOPA Dopamine precursor 100 1
Rivastigmine Acetylcholine esterase inhibitor
3 2
Placebo -- -- 1, 2, or 3*
*Placebo was randomly given to each participant at 1, 2 or 3 hours prior to the administration of
5.3.2 Participants
Participants were 4 females and 9 males; average age 31.3 (10.5) years; not diagnosed with any
medical problems; non-smokers, negative for urine toxicology screen for drugs of abuse; right-
handed to ensure homogeneity in hemisphere dominance; had no contraindication to TMS (Rossi,
Hallett et al. 2009) or MRI; and provided written informed consent. The study was conducted in
accordance with ethical standards of the responsible committee on human experimentation and
approved by the Centre for Addiction and Mental Health Research Ethics Board.
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5.3.3 Locating and Co-Registering the DLPFC
The left DLPFC is located at the junction of the middle and anterior third of the middle frontal
gyrus (Talairach Co-ordinates (x,y,z)= (-50, 30, 36)), which corresponds to the posterior region of
Brodmann area 9 and superior section of area 46 (Rusjan, Barr et al. 2010). Following previous
published methods the localization of the DLPFC was achieved through neuronavigation
techniques using the MINIBIRD system (Ascension Technologies) and each participant’s T1-
weighted MRI with fiducial markers placed on the nasion, inion, left and right tragus and vertex
(Daskalakis, Farzan et al. 2008).
5.3.4 TMS-EMG in the Motor Cortex and TMS-EEG in the DLPFC
Following established methods, we used a 7-cm figure-eight coil and a Magstim 200 stimulator
(The Magstim Company, Whitland, UK) to determine the participant’s resting motor threshold
(RMT) (defined as the minimum stimulus intensity that elicits a motor evoked potential (MEP) of
more than 50mV in 5 of 10 trials) (Sun, Farzan et al. 2016). MEP activity was measured through
EMG recordings from the right abductor pollicis brevis muscle. The stimulus intensity was then
adjusted to a suprathreshold intensity with mean peak-to-peak MEP amplitude of ~1 mV over 20
trials, which corresponded to approximately 120% of the RMT. This intensity referred to as SI1mV
was then used to deliver 100 TMS pulses pre-drug (paired-pulse and single-pulse) with an
interstimulus of 5 seconds to the scalp over the DLPFC, and then again post-drug, to assess change.
LICI was delivered at the optimal interstimulus of 100 ms.
To evaluate TMS-induced cortical evoked potentials, we acquired EEG through a 64-channel
Synamps 2 EEG system. All electrodes (Ag/AgCl ring electrodes) impedance were ≤5 kΩ and
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referenced to an electrode positioned posterior to Cz electrode. In addition, EEG signals were
recorded using DC and a low pass filter, anti-aliasing filter, of 200 Hz, at 20 kHz sampling rate,
which was shown to avoid saturation of amplifiers and minimize TMS-related artifact.
5.3.5 EEG Data Processing
EEG data was analyzed using MATLAB (The MathWorks Inc. Natick, MA, USA) and a custom
script that was developed based on previous work (Sun, Farzan et al. 2016). The recorded EEG
data for both single-pulse and paired-pulse were first down sampled from 20 to 1 kHz and then
segmented from -1000 ms to + 2000 ms after the test TMS stimulus. Epochs were then baseline
corrected -500 ms to -110 ms with respect to the pre-stimulus interval. To minimize TMS artifacts,
the data was re-segmented from 25ms to 2000ms. Thereafter, the EEG data was digitally filtered
using a second order, Butterworth, zero-phase shift 1-55 Hz band pass filter (24dB/Oct). EEG
recordings from pre-LICI and post-LICI were then concatenated in order to apply the same
objective criteria for de-noising the data. Then, an electrodes-by-trials matrix of ones and zeros
was created and assigned a value of zero if an epoch had the following: 1) amplitude larger than
+/- 150 μV; 2) power spectrum that violated 1/f power law; or 3) standard deviation 3 times larger
than the average of all trials. An electrode was rejected if its corresponding row had more than
60% of columns (trials) coded as zeros. An epoch was removed if its corresponding column had
more than 20% of rows (electrodes) coded as zeros. Next, independent component analysis (ICA)
(EEGLAB toolbox; Infomax algorithm) was performed to remove remaining artifacts such as
eyeblink traces, muscle artifacts and other noise from the EEG data (Sun, Farzan et al. 2016).
Finally, the data was re-referenced to the average, generating a clean signal devoid of noise for
each participant.
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5.3.6 LICI Quantification
The modulating effects of the drugs on LICI were calculated by the following steps: (I)
determining the cortical evoked activity (CEA) by averaging 100 single-pulses, and 100 paired-
pulses. (II) Subtracting single-pulse CEA waveform from paired-pulse CEA (Sun, Farzan et al.
2016) (Premoli, Rivolta et al. 2014). (III) Calculating the area under the rectified waveform from
50-250ms post test stimulus for both single-pulse CEA and paired-pulse CEA. The first interval
(50ms) was chosen as it represents the earliest artifact-free data, while the second interval (250ms)
was chosen because it represents the end of GABAB inhibitory postsynaptic potential (Sun, Farzan
et al. 2016). To quantify LICI as a ratio, paired-pulse CEA was divided by single-pulse CEA.
Lastly, to capture the effects of LICI from the frontal brain region, the average value from the
following frontal electrodes were used: FP1, FPZ, FP2, AF3, AF4, F7, F5, F3, F1, FZ, F2, F4, F6,
and F8. These frontal electrodes were selected for two main reasons. First, these electrodes are
the least influenced by muscle activity and TMS-related artifacts. Second, frontal electrodes show
the greatest and most consistent inhibitory response from DLPFC stimulation (Sun, Farzan et al.
2016)
Calculation for LICI:
Area under rectified curve (paired‐pulse)
Area under rectified curve (single‐pulse) LICI =
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5.3.7 Statistical Analysis
All data was first checked for normality using the Kolmogorov–Smirnov test. To test our primary
hypotheses and assess whether there is a drug effect on LICI, a repeated measures analysis of
variance (rmANOVA) was conducted with the drug condition (placebo vs. baclofen vs.
dextromethorphan vs. L-DOPA vs. rivastigmine) as the repeated measure. This was followed by a
series of post-hoc analyses, to compare LICI under each of the active drug condition to LICI under
placebo.
5.4 Results
Thirteen participants (4 females and 9 males) took part in this study. All participants completed all
sessions except for one participant who dropped out after only one of the five sessions and data
for this participant was not used. Participants’ demographics and basic neurophysiologic
characteristics are described in Table 6.
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Table 6. Demographic and Neurophysiologic Characteristics
Characteristic Mean (SD)
Age (years) 31.3 (10.5)
Gender (Female, %) 4 (25)
Education (years) 15.3 (2.3)
Resting Motor Threshold (% stimulator
output)
49.0 (0.74)
SI1mV (% stimulator output) 61.7 (1.5)
* SI1mV = Stimulation intensity with a mean peak-to-peak motor evoked potential amplitude of
1 mV over 20 trials; SD = standard deviation.
All outcome data were normally distributed. rmANOVA revealed that there was a drug effect on
LICI (F (4,44)= 6.34, p <0.001). Further, post-hoc pairwise comparisons against placebo, revealed
that LICI was decreased after the intake of rivastigmine (p =0.009) and increased after baclofen (p
=0.038). In contrast, there was no significant change after the intake of L-DOPA (p =0.17) or
dextromethorphan (p =0.79) when compared with placebo. (Figures 9). The topography of all
DLPFC LICI values across all electrodes is shown in Figure 10.
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Figure 9. Effects of Drugs on DLPFC LICI. This figure illustrates the effects of drugs (L-DOPA,
baclofen, rivastigmine, dextromethorphan, and placebo) on DLPFC LICI expressed as a change in
the ratio of Post-LICI from Pre-LICI CEA. The p-values refer to the comparisons between each
active drug and placebo. Error bars: +/-1SE.
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Figure 10: Topographical plots of LICI. These topographical plots illustrate the effects of drugs
(L-DOPA, baclofen, rivastigmine, dextromethorphan (DMO), and placebo) on inhibition from
DLPFC stimulation. Rivastigmine significantly decreased and baclofen increased inhibition
compared to placebo, while L-DOPA and dextromethorphan did not. Increased inhibition is shown
as more red, while decreased inhibition is shown as more blue. LICI from DLPFC stimulation is
most prominent at frontal locations when plotted topographically across all electrodes.
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Finally, compared Pre-LICI to Post-LICI, participants showed a significant difference under L-
DOPA, rivastigmine, and baclofen but not for dextromethorphan condition (Table 7).
Table 7. Pre-Drug vs Post-Drug LICI from stimulation to Dorsolateral Prefrontal Cortex
under each Drug Condition
Drug Pre-Drug
LICI
Post-Drug
LICI
t (df) p-value
Placebo 0.52 (0.14) 0.51(1.0) 0.48 (11) 0.64
Baclofen 0.63 (0.19) 0.53 (0.17) 3.14 (11)
0.009*
Dextromethorphan 0.62 (0.13)
0.62 (0.23) 0.85 (11)
0.93
L-DOPA 0.56 (0.14) 0.47 (0.12) 2.79 (11) 0.017*
Rivastigmine 0.47 (0.09)
0.61 (0.15) -2.79 (11)
0.018*
LICI= Long-Interval Cortical inhibition activity as measured by Cortical Evoked Activity (CEA)
Pre-Drug and Post-drug; SD = Standard Deviation; t (df) = Paired T-test (degrees of freedom)
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5.5 Discussion
This study confirmed our hypotheses that baclofen enhances and rivastigmine decreases LICI from
the DLPFC in vivo. It did not confirm our hypotheses that dextromethorphan and L-DOPA
decrease LICI compared to placebo. To our knowledge, this is the first study to assess the
pharmacological modulation of LICI from DLPFC stimulation in humans.
As hypothesized we found that baclofen enhanced LICI compared to placebo. Baclofen is a
GABAB receptor agonist (Faigle and Keberle 1972) that increases inhibition through the allosteric
modulation of GABAB receptor-mediated neurotransmission (Mann-Metzer and Yarom 2002).
This finding is consistent with animal studies that showed baclofen enhances inhibition in the
cortex (Porter and Nieves 2004). Our result also replicate and extend to TMS human studies that
assessed the effect of baclofen on LICI in the motor cortex (McDonnell, Orekhov et al. 2006;
Premoli, Rivolta et al. 2014).
Furthermore, in disorders where LICI has been shown to be dysfunctional (e.g., schizophrenia,
(Radhu, Garcia Dominguez et al. 2015), Parkinson’s (Chu, Wagle-Shukla et al. 2009) and
depression (Croarkin, Nakonezny et al. 2014), these findings suggest that drugs targeting the
GABAB receptor may reverse these deficits and even have a therapeutic role. As an example,
clozapine, which is one of the most effective treatments for schizophrenia, has been shown to
increase GABAB receptor mediated neurotransmission (Kaster, de Jesus et al. 2015). These results,
therefore, also suggest that measuring LICI in the DLPFC may be a possible treatment or
biomarker for schizophrenia.
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We also found that rivastigmine reduces LICI from DLPFC stimulation. To the best of our
knowledge no study has examined the effects of rivastigmine on LICI. One study, however,
assessed the effects of rivastigmine on cortical excitability from the human motor cortex and
reported an enhancement of MEP amplitude after a single dose (Langguth, Bauer et al. 2007),
which supports our finding given that enhanced MEP indicates reduced cortical inhibition
(Bestmann and Krakauer 2015). These findings are also consistent with animal studies that
reported increased cortical excitation in the prefrontal cortex following cholinergic intervention
(Vidal and Changeux 1993) Lastly, Rivastigmine is known to enhance short afferent inhibition
(SAI), which is partly cholinergic mediated and SAI decreases LICI (Udupa, Ni et al. 2009),
further supporting our finding.
The prefrontal cortex receives glutamatergic inputs from the medial dorsal thalamus
(Groenewegen and Uylings 2000). These thalamo-cortical glutamatergic projections are
modulated by highly expressed presynaptic and postsynaptic cholinergic α7- nicotinic receptors
(Yang, Paspalas et al. 2013). Activation of these receptors increases glutamate release, which
results in reduced cortical inhibition (Parikh, Ji et al. 2010). Rivastigmine increases synaptic levels
of acetylcholine by inhibiting acetylcholine-esterase, allowing for longer cholinergic receptor
activation (Polinsky 1998). This subtype of the nicotinic receptors are also permeable to calcium,
which are important in facilitating NMDA activity and mediating LTP plasticity (Yang, Paspalas
et al. 2013). In fact, LTP in the motor cortex of healthy participants was enhanced following the
administration of rivastigmine (Kuo, Grosch et al. 2007). Furthermore, activation of α7 nicotinic
receptor has been shown to be essential for cognitive circuits in the DLPFC (Yang, Paspalas et al.
2013). Therefore, based on our observation and prior studies, rivastigmine may have pro-cognitive
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effects by reducing cortical inhibition and, as a corollary, increasing neural plasticity (Ziemann,
Hallett et al. 1998).
Contrary to our hypothesis, we did not find a difference in LICI for L-DOPA compared to placebo.
However, we found that L-DOPA exposure enhanced LICI Pre-drug to Post-drug, but this was
only a trend when corrected for multiple comparisons, which may due to a limited sample size.
These findings are in line with animal studies that reported enhanced inhibition in the prefrontal
cortex following dopaminergic intervention (Kroner, Krimer et al. 2007; Towers and Hestrin
2008). In the prefrontal cortex, dopaminergic axons connect with fast spiking GABAergic neurons
(Sesack, Hawrylak et al. 1998). Dopamine increases the firing of these neurons through activation
of D1 receptors (Gorelova, Seamans et al. 2002). Given that D1 receptors are highly expressed in
the prefrontal cortex (Gaspar, Bloch et al. 1995), L-DOPA could have enhanced inhibition through
these receptors, however, considering that these dopaminergic effects are downstream and not
direct then smaller effects and effect sizes may be due to these indirect influences. Also, these
findings support TMS studies that reported increased cortical silent period (CSP) following L-
DOPA administration from the motor cortex (Ziemann, Tergau et al. 1997). These studies are
relevant given that CSP similar to LICI is mediated through GABAB neurotransmission.
We did not confirm our hypothesis that dextromethorphan would enhance LICI. No previous study
assessed the effect of dextromethorphan on LICI. As such our hypothesis was based on a study
that reported that dextromethorphan enhanced cortical inhibition and decreased excitation in the
motor cortex (Ziemann, Chen et al. 1998). This study, however, examined the effects of
dextromethorphan on SICI and not LICI. Although both SICI and LICI are cortical inhibitory
circuits, several studies have shown that LICI reduces SICI, suggesting that these two inhibitory
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paradigms are mediated by different mechanisms (Sanger, Garg et al. 2001). Also,
dextromethorphan being a non-competitive NMDA receptor antagonist (Church, Lodge et al.
1985) can reduce cortical inhibition through activation of NMDA receptors on GABAergic
neurons or, enhance inhibition by acting on NMDA receptors on glutamatergic neurons (Olney,
Newcomer et al. 1999). Further, dextromethorphan also binds to other non-NMDA sites including,
opioid sigma binding sites (Musacchio, Klein et al. 1989), nicotinic receptors (Hernandez,
Bertolino et al. 2000) and calcium channels (Netzer, Pflimlin et al. 1993) and, therefore, is not a
direct NMDA antagonist, potentially explaining our observed effects on LICI. As such further
studies are needed to determine the effects of dextromethorphan on LICI.
This study is limited by a relatively small sample size but sample size was calculated based on
previous published studies. This small sample may have obscured finding smaller effects through
agents such as L-DOPA. Another limitation is that we did not measure blood levels of the drugs
to time the delivery of Post-LICI. However, this limitation was mitigated by delivering Post-LICI
based on published peak times of plasma levels. Finally, this study assessed the impact of a single
dose on LICI. These medications are used chronically in clinical settings. Thus, future studies
should assess the effects of longer exposure to these medications in healthy individuals as well as
patients with neuropsychiatric disorders associated with abnormalities in LICI.
In conclusion, this study confirmed our hypotheses that baclofen – a GABAergic agent- enhanced
LICI in the DLPFC while rivastigmine – a cholinergic agent - reduced it. Future studies should
assess this modulation in clinical conditions to better understand the pathophysiology underlying
these conditions as well the mechanisms that these drugs target in various brain disorders.
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5.6 Conclusion
In conclusion, this study confirmed our hypotheses that baclofen – a GABAergic agent- enhanced
LICI in the DLPFC while rivastigmine – a cholinergic agent - reduced it. Future studies should
assess this modulation in clinical conditions to better understand the pathophysiology underlying
these conditions as well the mechanisms that these drugs target in various brain disorders.
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Chapter 6
6. Discussion
6.1 Summary of the Dissertation
The introduction of this dissertation starts off by stating that there are currently no effective
treatments for cognitive deficits, which are the most debilitating symptoms of schizophrenia.
Next, it introduces the mechanisms behind two important cellular processes that mediate
cognition, LTP and CI, and how the main neurotransmitters (GABA and glutamate) and
neuromodulators (dopamine and acetylcholine) modulate these mechanisms. It further goes on to
explain how these mechanisms are dysfunctional in patients with schizophrenia and links this
abnormality to dysfunctional neurotransmitter and neuromodulator activity while looping it back
to aberrant cognition in schizophrenia.
The original research component of this dissertation consists of three components. The first
component is a systematic review that provides evidence for abnormal dopaminergic,
glutamatergic and GABAergic neurotransmission in drug-naïve and drug-free patients with
schizophrenia. The second component is a study that evaluates the impact of four CNS drugs that
affect the main neurotransmitters (dextromethorphan and baclofen) and neuromodulators (L-
DOPA and rivastigmine), on PAS-LTP from the DLPFC in healthy participants using a within-
subject, double-blinded, placebo-controlled design. The third and final component is a study that
assesses the impact of the aforementioned drugs on LICI from the DLPFC in the same healthy
participants who took part in the previous study using a within-subject, double-blinded, placebo-
controlled design
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6.1.1 Summary of the First Paper
The first paper provided the rationale for the second and third papers, linking abnormal
neurotransmission to dysfunctional plasticity measured by LTP in patients with schizophrenia.
This publication summarized all data pertaining to drug-naïve and drug-free patients focusing on
abnormal glutamatergic, dopaminergic and GABAergic neurotransmission assessed through
neuroimaging studies that used MRS, PET, and SPECT. The analysis pertaining to the
glutamatergic system showed elevated glutamatergic levels during the initial period of diagnosis,
which decreased as time progressed, suggesting that high levels of glutamatergic activity may be
responsible for the initial symptoms. Results pertaining to the dopaminergic system showed a
decrease in the dopamine D2/3 receptor binding in the thalamus, as well as, an increase in
dopamine synthesis, dopamine release, and dopamine at baseline in the striatum. Findings
pertaining to GABAergic activity were inconsistent, with one study reporting an elevation of
GABA levels in MPFC of antipsychotic-free patients. Finally, this paper concluded with a model
explaining and summarizing the various findings.
6.1.2 Summary of the Second Paper
The second study evaluated the effects of four CNS drugs on PAS-LTP from the DLPFC of 12
healthy subjects, which was captured through the use of TMS-EEG. The main objective of this
study was to evaluate how these four CNS drugs compare to placebo and whether they potentiate
or inhibit PAS-LTP in the DLPFC. Our results showed that L-DOPA, and rivastigmine enhanced
LTP, and dextromethorphan blocked LTP when compared to placebo. No significant differences
were found between baclofen and placebo. Thus, our findings suggest that LTP in the DLPFC
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can be facilitated by enhancing cholinergic and dopaminergic tone and abolished by blocking
NMDA receptors.
6.1.3 Summary of the Third Paper
The last study included in this thesis assessed the effects of the same four CNS drugs used in the
earlier study on frontal LICI measured using TMS-EEG in the same 12 healthy subjects who
took part in the previous study. Although the same subjects took part in the study, the subjects
were tested on a different day than the first study. We found that baclofen and rivastigmine
significantly enhanced and reduced frontal LICI respectively, while dextromethorphan and L-
DOPA had no significant effect when compared to placebo. Also, under the influence of L-
DOPA, there was a trend effect of an increase in LICI after correcting for multiple comparisons
when Pre-LICI was compared to Post-LICI, which may be due to a small sample size, and
therefore warranting further investigation.
6.2 General Discussion
6.2.1 Glutamate
The glutamate hypothesis of schizophrenia is based on the deficiency or defect of NMDA
receptors. These receptors have been shown to be reduced in the frontal cortex of patients
through both in vivo neuroimaging (Pilowsky, Bressan et al. 2006) and in vitro postmortem
studies (Akbarian, Sucher et al. 1996). It has been speculated that increased pyramidal activity
overwhelms the neural circuits with excessive glutamate, which leads to neurotoxicity and the
downregulation of NMDA receptors. In fact, several studies have reported enhanced GLX, a
measure of glutamine and glutamate levels in first episode drug-naïve patients during the initial
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period of diagnosis, indicating an over-activity of the glutamatergic system (Salavati, Rajji et al.
2015).
Additionally, the blockade of NMDA receptors by antagonists such as PCP and ketamine have
been shown to have neurotoxic and psychotomimetic effects in healthy participants (Lahti,
Weiler et al. 2001; Javitt 2010). These drugs induce not only psychotic symptoms but also
cognitive symptoms, including memory and learning impairment. For this reason, in this study,
we evaluated the effects of dextromethorphan, an NMDA antagonist, on LTP in the DLPFC. We
demonstrated that dextromethorphan blocks the induction of LTP. This finding is expected given
that dextromethorphan prevents Ca2+ and Na+ from passing through NMDA channels, which
would abolish LTP. Our finding is also consistent with a study that assessed the effects of
dextromethorphan in the motor cortex using PAS (Stefan, Kunesch et al. 2002). It also supports
animal studies that showed that dextromethorphan (Krug, Matthies et al. 1993) and other NMDA
antagonists such as D-(-)2-amino-5-phosphonopentanoic acid (Davis, Butcher et al. 1992; Jay,
Burette et al. 1995) and MK 80l (Frankiewicz, Potier et al. 1996), abolish the induction of LTP.
Furthermore, our finding confirms the idea that PAS-LTP from the DLPFC is an NMDA
receptor-dependent process similar to cellular LTP.
Overall, these observations suggest that improper activation of NMDA receptors through
deficiency or defect may be involved in the manifestation of dysfunctional LTP seen in
schizophrenia. Based on these observations, conceivably, NMDA agonists may facilitate the
induction of LTP and have therapeutic implications. In fact, agents that directly or indirectly
activate NMDA receptors, such as glycine and D-cycloserine have been used as an adjunct or
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antipsychotic replacement with promising results (Strzelecki and Rabe-Jablonska 2011;
McCullumsmith, Hammond et al. 2012). In one placebo-controlled study glycine administration
significantly improved negative symptoms (Heresco-Levy, Javitt et al. 1999). The effect of D-
cycloserine, a partial agonist of the glycine site, however, has been inconclusive, with
improvement in negative symptoms reported only in one study (Goff, Tsai et al. 1995).
Therefore, the approach of using an NMDA agonist although promising still remains
controversial and warrants further studies.
6.2.2 Dopamine
Another widely considered neurochemical hypothesis of schizophrenia is the dopamine
hypothesis. This hypothesis postulates that positive symptoms of schizophrenia arise from hyper-
dopaminergic neurotransmission in the striatum, and hypodopaminergic activity in the PFC
contributes to negative and cognitive symptoms (Stahl 2007). Dopaminergic inputs to the PFC
play a major role in regulating working memory, planning, and attention, and for this reason,
dysfunctions have been postulated to underlie cognitive deficits associated with schizophrenia.
Also, as mentioned previously patients show a reduction in the expression of NMDA receptors in
the frontal cortex, and hypo-functioning of NMDA receptors reduces downstream dopamine
activity. Thus, in this case, LTP facilitation is not limited to the glutamatergic system, but also
the dopaminergic system.
A critical component of the effect of dopamine is its modulation of glutamatergic activity, which
can be different depending on which receptors are activated. Dopamine increases the excitation
of glutamatergic neurons via D1 receptors and inhibition via D2 receptors. To explore this
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interaction in the DLPFC, we used PAS, an NMDA-mediated paradigm, in combination with L-
DOPA, a dopamine precursor. As expected, L-DOPA significantly enhanced PAS-LTP
compared to placebo. This finding is consistent with an animal study that assessed the effects of
L-DOPA on LTP induction in the frontal cortex (Otani, Daniel et al. 2003). Similar results were
also observed when PAS was applied to the motor cortex; 100 mg dose of L-DOPA significantly
enhanced the magnitude and duration of PAS-LTP (Kuo, Paulus et al. 2008). Also, in a different
study, PAS alone was ineffective in potentiating LTP in the elderly, but not when combined with
L-DOPA (Kishore, Popa et al. 2014), illustrating the potent effect that L-DOPA has on PAS-
LTP.
The modulatory effect by L-DOPA, however, follows an inverted-U shape concentration curve
and only a moderate dose (100mg) facilitates LTP, while concentrations that are too high
(200mg) or too low (25mg) hinder it (Thirugnanasambandam, Grundey et al. 2011). This
facilitation is presumed to be through pyramidal cell excitability via D1 receptors, given that 2mg
of cabergoline, a D2 agonist, had no significant effect on PAS- LTP (Korchounov and Ziemann
2011). Also, ropinirole, a D2 agonist, was found to hinder PAS-LTP and this effect was stronger
at a high (1.0mg) and a low (0.125mg) dose compared to a medium dose (0.5mg), suggesting an
inverted-U shape concentration curve (Monte-Silva, Kuo et al. 2009). Lastly, sulpiride (400mg),
a weak D2/D3 receptor antagonist, had no significant effect on PAS- LTP but the administration
of haloperidol (2.5mg), a strong D2 antagonist blocked PAS- LTP in the motor cortex
(Korchounov and Ziemann 2011). For these reasons, the enhancement in PAS-LTP through the
use of L-DOPA is assumed to be mainly through D1 receptor activation.
When D1 receptors are activated LTP is initiated and resting glutamatergic neurons increase their
activity (Gurden, Takita et al. 2000). As such, D1 receptors play a vital role in the facilitation of
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LTP, specifically in the PFC, where there is a greater concentration of D1 receptors than D2
receptors (Hall, Sedvall et al. 1994). Interestingly, there is a reduction of D1 receptors in the PFC
of drug-naive and drug-free patients with schizophrenia, and this reduction has been associated
with poor performance on the Wisconsin Card Sorting Test(WCST), a cognitive test used to test
executive function (Okubo, Suhara et al. 1997). Thus, based on the aforementioned studies,
abnormal D1 receptor activation may contribute to dysfunctional LTP, which in turn may lead to
cognitive deficits seen in schizophrenia.
Furthermore, it is important to note, that this endogenous release of dopamine only serves as a
trigger for LTP in the presence of tonic/background dopamine and triggers LTD in its absence
(Matsuda, Marzo et al. 2006). In fact, there is a strong correlation between cortical dopamine
levels and cortical LTP amplitude (r = 0.8; P < 0.001), and a depletion of more than 50%
corresponds to a dramatic decrease in LTP induction (Gurden, Tassin et al. 1999). Also, it has
been shown that the pairing of dopamine with weak tetanic stimulation that would normally
induce LTD, instead induces LTP in the PFC slice (Matsuda, Marzo et al. 2006). These results
indicate that an appropriate level of tonic/background dopamine needs to present in order to
facilitate the induction of LTP. Several studies have shown that patients with schizophrenia have
low levels of dopamine in the frontal cortex (Brisch, Saniotis et al. 2014).Therefore this work in
combination with previous works demonstrates the importance of mesocortical dopamine for the
induction of LTP, which plays a vital role in cognition and abnormal levels may contribute to
impaired LTP reported in schizophrenia.
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6.2.3 GABA
The glutamatergic hypothesis pertaining to schizophrenia proposes that hypo-functioning of
NMDA receptors on GABAergic neurons leads to a reduction in activities of GABAergic
neurons, and in turn CI (Moghaddam and Javitt 2012). In fact, several studies have demonstrated
reduced CI in patients with schizophrenia (Rogasch, Daskalakis et al. 2014). This reduction has
also been observed in drug-naïve patients with schizophrenia (Hasan, Wobrock et al. 2012),
suggesting that this impairment is inherent to the illness rather than induced through
antipsychotic medication. Post-mortem studies suggest a subset of GABAergic interneurons,
known as parvalbumin (PV) interneurons in the PFC and hippocampus are the most affected
(Wulff, Ponomarenko et al. 2009). These interneurons are fast-spiking and as such play a critical
role in the generation of gamma oscillations via GABAA neurotransmission, and the modulation
of gamma oscillations via GABAB neurotransmission (Wulff, Ponomarenko et al. 2009; Buzsaki
and Wang 2012).
As mentioned in the introduction, there are several ways of measuring CI; however, LICI is the
only measure that has been used outside of the motor cortex. Recent studies have shown that
LICI is reduced in the DLPFC of patients with schizophrenia, the brain region greatly associated
with this disorder (Farzan, Barr et al. 2010; Radhu, Garcia Dominguez et al. 2015). For this
reason, we examined the pharmacological modulation of LICI from this region.
As expected we showed that baclofen increases LICI compared to placebo. LICI is a putative
measure of GABAB receptor activity as it is measured at ISIs between 50 and 200ms (Werhahn,
Kunesch et al. 1999; McDonnell, Orekhov et al. 2006). Baclofen is a GABAB receptor agonist,
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which would explain the increase in LICI expression as it hyperpolarizes the neuron through K+
efflux and Ca+ blockage. Our result is consistent with a study that evaluated the effects of
baclofen on LICI using TMS-EEG from the motor cortex (Premoli, Rivolta et al. 2014). Our
finding also supports studies that evaluated the effect of baclofen from the animal PFC, and the
human motor cortex using TMS (McDonnell, Orekhov et al. 2006).
We also found that the acetylcholine esterase inhibitor, rivastigmine, reduces LICI. This effect
may be through the activation of nicotinic receptors on glutamatergic neurons, given that these
receptors are abundant in the PFC (Wallace and Bertrand 2013). To our knowledge, only one
study looked at the effect of rivastigmine on cortical activity using TMS. This study used the
same dose of 3mg on 16 subjects and found that MEP activity, a measure of brain excitability
increased for 7 days but stabilized after consistent intake (Langguth, Bauer et al. 2007). This
finding indirectly supports our result since increased excitation leads to reduced inhibition.
Lastly, we found a trending increase in LICI from Pre-LICI to Post-LICI under the influence L-
DOPA, which may be due to our small sample size.
We also assessed the influence of baclofen on PAS-LTP and found no significant effect. A
previous study found that PAS-LTP was blunted in the motor cortex by 50mg of baclofen
(McDonnell, Orekhov et al. 2007). This inconsistency in results may be due to our sample size,
which we do not assume is the case as the previous study only enrolled seven subjects, or due to
different brain regions being analyzed.
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6.2.4 Nicotine
Cholinergic inputs to the DLPFC have been shown to enhance attention and working memory.
The DLPFC is critical for these cognitive functions and also rich in the expression of
acetylcholine receptors (Wallace and Bertrand 2013). The specific effects of these receptors on
LTP remain elusive. Therefore, the present study was conducted to clarify the role of
acetylcholine receptors on LTP in vivo in the DLPFC using rivastigmine, which allows
acetylcholine to activate both the nicotinic and muscarinic receptors. Under rivastigmine
exposure LTP significantly increased when compared to placebo. Similar results were also
shown in animal studies (Blitzer, Gil et al. 1990; Vidal and Changeux 1993). Our finding is also
in line with a study that administered PAS to the motor cortex, whereby 3mg of rivastigmine
enhanced PAS-LTP relative to a placebo (Kuo, Grosch et al. 2007). Rivastigmine may facilitate
LTP by enhancing glutamatergic transmission. This is achieved either through the activation of
presynaptic alpha7 nicotinic receptors, enhancing Ca2+ influx leading to enhanced
neurotransmitter release or by postsynaptic alpha7 nicotinic receptors which enhance Ca2+ and
Na+ influx leading to neuronal depolarization and the enhancement of intracellular Ca2+.
Intracellular Ca2+ is a key determinant of plasticity and under conditions where glutamate can
activate NMDA receptors; LTP is induced.
Although our results are likely mediated by nicotinic receptor activity, it is important to note that
muscarinic activity also plays a role. While little is known about the muscarinic cholinergic
effects on prefrontal synapses, one study found that the muscarinic agonist, pilocarpine
potentiates the late phase of LTP in the PFC without affecting the early portion of LTP (20min)
(Lopes Aguiar, Romcy-Pereira et al. 2008). In contrast, 8mg of biperiden, an M1 muscarinic
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receptor antagonist significantly reduces PAS-LTP in the motor cortex, suggesting that
muscarinic receptors play a greater role in inhibiting LTP rather than facilitating LTP
(Korchounov and Ziemann 2011). Thus, the cumulative findings of previous studies in
combination with our finding suggest that nicotinic receptor activation exerts a boosting effect
on LTP plasticity. Therefore, given that nicotinic receptors are pivotal for synaptic plasticity, and
important for learning, memory, and attention, then a loss or disruption of these receptors may
contribute to cognitive dysfunctions like those manifested in schizophrenia and other disorders
like Alzheimer's disease.
6.2.5 PAS as Therapeutic Tool
In the recent years, there has been heightened interest in electrophysiological techniques to study
and induce plasticity. One of those techniques is PAS, which as mentioned in the introduction, is
a TMS paradigm that consists of a low frequency peripheral electrical nerve stimulation,
generally to the median nerve paired with TMS over the area of interest, conventionally the
motor cortex (Stefan, Kunesch et al. 2000). Repeated pairing, about 180 pulses of these two
associative stimuli over an extended period using an interstimulus of 25ms induces a plastic
change of increased excitability in the human cortex. This 25ms interval allows for both stimuli
to arrive synchronously at the cortex (Stefan, Kunesch et al. 2000).
The principle behind PAS is based on associative LTP and resembles spike timing dependent
plasticity, as the effective inter-stimulus interval lies within a restricted (milliseconds) range
(Stefan, Kunesch et al. 2000; Rajji, Sun et al. 2013). Both associative and spike timing dependent
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plasticity are important cellular mechanisms that govern the induction of LTP, thus making PAS
an optimal tool for the exploration of in vivo LTP from the cortex of an intact brain as a whole,
in an awake state. This is a major advantage of PAS as opposed to cellular in vitro recordings
which focus on a small group of cells. This is important considering that functions associated
with LTP, such as learning and memory are hierarchical and distributed mechanisms linked with
different brain regions. Initially, in 2000, when Stefan et al first introduced PAS as a tool for
assessing LTP, explorations were assessed from the motor cortex, due to recordable muscle
contractions (Stefan, Kunesch et al. 2000). Since then PAS has been combined with EEG which
extends this exploration to other cortical regions including the frontal brain, which is important
for psychiatric research (Rajji, Sun et al. 2013).
There are several reasons as to why PAS is an optimal non-invasive LTP-inducing
neurostimulation technique when compared to other techniques (TBS, TDCS, and rTMS). First,
PAS has the ability to induce, instead of augmenting, focal, not widespread LTP (Stefan,
Kunesch et al. 2000). Second, PAS-LTP shares a number of physiological properties with
cellular LTP, which include, rapidly evolving (within 30min), persistent (minimum duration 30-
60min) beyond the period of stimulation, yet reversible, and topographically specific (Stefan,
Kunesch et al. 2000). Moreover, NMDA antagonist abolishes PAS-LTP, which further supports
the concept that PAS-LTP behaves in accordance with cellular LTP (Stefan, Kunesch et al.
2002). As such, PAS has tremendous potential as a therapeutic tool for restoring aberrant LTP in
motor and non-motor regions implicated in several neurological and psychiatric disorders.
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6.3 Significance of this Work
Studies exploring LTP and CI are conventionally limited to the hippocampus and to animals. To
the best of our knowledge, this is the only study that assessed the pharmacological manipulation
of LTP and CI from the DLPFC in humans, a critical brain region underlying higher order
cognition and which abnormalities are implicated in several disorders including schizophrenia.
The results from this project will not only help to clarify the functional neurobiology of LTP and
CI from the DLPFC but may also pave the way for the development of new treatments to help
treat complex cognitive impairments seen in schizophrenia, as well as, other psychiatric and
neurological disorders.
6.4 Limitations
There are several limitations to this work which need to be discussed. The first limitation
pertains to EEG recordings as there are various sources of biological and environmental artifacts
that can contaminate brain signals captured through this method. These include face and scalp
muscle activity, eye activity such as movement and blink, as well as, TMS-related artifacts.
These artifacts, however, can be removed with minimal impact on brain activity using ICA.
Secondly, although we controlled for the most important confounding variables, being attention
and age, there are other variables that may influence the PAS response, such as, cortisol level
(Yuval-Greenberg, Tomer et al. 2008), and circadian cycle (Sale, Ridding et al. 2007), which
were not directly controlled. These variables, however, should not influence the data too much,
as all testing sessions started in the morning and ended in the evening. Third, although the
evidence shows that an interstimulus interval of 25ms used for PAS in the DLPFC is optimal, it
134
may be better to use an individualized PAS interstimulus interval by using the subject’s negative
potential occurring at 20ms, known as N20 (Yamada, Kayamori et al. 1984; Valeriani, Restuccia
et al. 2000). Previous studies have demonstrated that the N20 latency or N20+2ms used as the
PAS interstimulus interval results in better potentiation of LTP compared to the standardized
interval of 25ms (Ziemann, Ilic et al. 2004; Muller-Dahlhaus, Orekhov et al. 2008; Jung and
Ziemann 2009). This approach would also take into account an individual’s arm length, as
shorter arm length has been correlated with shorter sensory evoked potential (SEP) latencies in
response to median nerve stimulation (Poornima, Ali et al. 2013). This is important given men
typically have longer arms compared to women, as they are generally taller (Stetson, Albers et al.
1992). This individualized method would also control for age, as older individuals have a slower
conduction velocity compared to younger and generally have a longer SEP latency (Dorfman and
Bosley 1979; Stetson, Albers et al. 1992; Poornima, Ali et al. 2013). Fourth, plasma levels were
not obtained in this study, thus the bioavailability of the drugs could not be accurately accounted.
Also, the amount given was standardized and not based on the weight of the participant,
however, this should not affect the data too much, as none of the subjects were overweight.
Lastly, although we used a within-subject design, which allows for a heterogeneous sample, our
sample size may be too small to draw accurate conclusions.
6.5 Conclusion
In conclusion, it has been proposed that cognitive deficits seen in schizophrenia are associated
with impaired LTP and CI. These mechanisms are modulated by various neurochemical systems,
particularly the cholinergic, dopaminergic, GABAergic, and glutamatergic systems. Over the
135
past few decades, several neurostimulation protocols have been developed to assess LTP and CI
in vivo. PAS and LICI are TMS based protocols that assess LTP and CI activity, respectively
from the human cortex. Combined with EEG, it can assess these activities in the DLPFC in
which LTP and CI are disrupted in several neurological and psychiatric disorders including
schizophrenia.
Using PAS-EEG and LICI-EEG we conducted the first pharmacological modulation study from
the DLPFC in vivo in a double-blind randomized controlled within-subject design. We used four
drugs that affect the above mentioned neurochemical systems: rivastigmine (cholinergic), L-
DOPA (dopaminergic), baclofen (GABAergic), and dextromethorphan (glutamatergic). In the
first arm of the study we tested four hypotheses, we hypothesized that, compared to placebo,
rivastigmine and L-DOPA would enhance PAS-LTP, while baclofen and dextromethorphan
would inhibit it. Our study confirmed three out of the four hypotheses (baclofen being the
exception). In the second arm of the study, we hypothesized that, compared to placebo;
rivastigmine would reduce LICI, while baclofen, L-DOPA, and dextromethorphan would
enhance it. Our study confirmed two out of the four hypotheses (rivastigmine and baclofen).
These results, therefore, provide compelling evidence for the role of these modulatory agents in
affecting LTP and CI in the DLPFC and support the pursuit of combining pharmacological and
neurostimulation interventions to enhance LTP and CI in several brain disorders including
schizophrenia.
136
6.6 Future Direction
There are several future studies that could be conducted as an extension of this work. First,
although PAS-LTP is associated with learning in the motor and somatosensory cortex, it is
unclear as to whether PAS-LTP in the DLPFC is correlated with working memory performance,
as to date no study has been conducted to assess this correlation. One would, however, expect
that there is a strong correlation, given that LTP in the DLPFC and afferent pathways from the
hippocampus and amygdala have been suggested to play a prominent role in learning and
memory formation (Laroche, Jay et al. 1990; Laroche, Davis et al. 2000; Maroun and Richter-
Levin 2003).
Second, a study should assess the functional significance of the effect of these drugs on cognitive
function, such as working memory assessed through a cognitive test, such as the N-Back. This
would allow for a better link between neurophysiological findings and cognitive symptoms of
schizophrenia.
Third, structural differences in the DLPFC, i.e. cortical thickness and/or surface area, have been
shown to be associated with cognitive function. Two studies have examined the relationship
between potentiation and cortical thickness/surface, in healthy subjects. A correlation between
PAS-LTP and cortical thickness has been shown in the primary sensorimotor cortex of young
(Conde, Vollmann et al. 2012)and in older healthy individuals when assessing the motor
cortex(List, Kubke et al. 2013). However, to date, no studies have been conducted to evaluate the
relationship between cortical thickness or surface area and PAS-LTP in the DLPFC of healthy
participants.
137
Fourth, this study due to recruitment challenges included a limited sample of 12 healthy subjects;
potentially a replication study may recruit a larger sample to further assess if these effects are
reproducible. Lastly, future studies may attempt to use PAS as a therapeutic tool in restoring
impaired plasticity in neurologic and psychiatric disorders characterized by this dysfunction.
138
Appendix
Table 1. Image Studies assessing the Glutamatergic Systems in Antipsychotic-Naïve/Antipsychotic-Free Patients with Schizophrenia
Publication Subjects (n): Age mean ±
(SD)
Method
Findings
Kragulijac, et
al.,2013(Kraguljac,
White et al. 2013)
n=27 patients: 11 antipsychotic‐naïve, 16 antipsychotic‐free for at least two weeks (M=20, F=7) (32.63± 9.28)
n=27 healthy controls (M=20, F=7) (32.89 ± 9.39)
3‐T (1H) MRS was used for assess GLX levels from the hippocampus
Elevation in GLX in the hippocampus of antipsychotic‐free patients compared to healthy controls
Kegeles, et. al.,
2012(Kegeles,
Mao et al. 2012)
n=32 patients: n=16 unmedicated patients, for a minimum of 14 days: (M=11, F=5) (32 ± 11 years)
n=16 medicated patients: (M=11, F=5) (32 ±10 years)
n= 22 healthy controls: (M=14, F=8) (33 ± 8 years)
3‐T (1H) MRS and the J‐edited spin‐echo difference method was used to assess GLX and GABA levels in the medial‐ and dorsolateral prefrontal cortex of unmedicated patients, medicated patients, and healthy controls
30% elevations in GLX and GABA levels in MPFC of unmedicated patients compared with healthy controls, and medicated patients
No difference was detected in dorsolateral prefrontal cortex among the three groups
There was a correlations between GABA and GLX levels in both the medial‐ and dorsolateral prefrontal cortex of patients and healthy controls
139
Seese, et al., 2011
(Seese, O'Neill et
al. 2011)
n=28 youth with childhood‐onset schizophrenia: (M=15, F=13) (14.1 ± 3.0 years)
n=34 healthy controls: (M=15, F=19) (11.5 ± 2.9 years)
1.5 T (1H) MRS and short echo time was used to assess glutamatergic metabolites bilaterally in the inferior frontal, middle frontal, and superior temporal gyri of patients and healthy controls
No difference was detected in the regions specified between healthy controls and patients
Aoyama, et al.,
2011(Aoyama,
Theberge et al.
2011)
n=17 antipsychotic‐naïve patients: (M=14, F=3) (27 ± 8 years)
n=17 healthy controls: (M=13, F=4) (30 ±10 years)
3 T (1H) MRS was used to assess glutamatergic metabolites in the left anterior cingulate and thalamus before medication, 10 and 80 months after treatment
Thalamic glutamate and glutamine levels decreased over 80 months
No difference was detected in the anterior cingulate
De la Fuente‐
Sandoval, et al.,
2011(de la
Fuente‐Sandoval,
Leon‐Ortiz et al.
2011)
n=18 antipsychotic‐naïve patients with prodromal symptoms ‐ considered to be at high‐risk for schizophrenia (M=14, F=4) (19.56 ± 3.46 years)
n=18 antipsychotic‐naïve first‐episode patients: (M=10, F=8) (23.44 ± 4.93
years)
n=40 healthy controls: (M=28, F=12) (21.83 ± 4.47 years)
3 T (1H) MRS was used to assess glutamate level in the precommissural dorsal‐caudate and the cerebellar cortex in the three groups
Greater glutamate levels were detected in the precommissural dorsal‐caudate in both the high‐risk and first‐episode groups compared with healthy controls
No differences were detected between the three groups in the cerebellum
Lutkenhoff, et al.,
2010(Lutkenhoff,
van Erp et al.
2010)
n=12 co‐twins discordant for schizophrenia : (M=7, F=5) (49.5 ± 10 years)
3T (1H) MRS was used to assess the medial prefrontal gray matter, left prefrontal white matter and left hippocampus
Glutamate levels were significantly lower in the medial prefrontal of patients with schizophrenia and unaffected co‐twin in
140
n= 21 healthy twins: (M=12, F=9) (55.7 ± 3.8 years)
n= 9 proband twin: (M=5, F=4) (48.8 ± 11.5 years)
the cortex comparedwith healthy twins.
No differences in glutamate levels were detected in the left hippocampus or the left prefrontal white matter
Bustillo, et al.,
2010(Bustillo,
Rowland et al.
2010)
n= 14 minimally treated (less than 3 weeks) patients with schizophrenia: (M=12, F=2) (27.2 ± 8.9 years)
n=10 healthy controls: (M=8, F=2) (28.8± 9.7 years)
4 T(1H) MRS proton echo planar spectroscopic imaging was used to assess Gln/Glu ratio, N‐ acetylaspartic acid (NAA) and Inositol levels in anterior cingulate and thalamus of patients and healthy controls
No difference in Gln/Glu ratio was found between healthy controls and patients with schizophrenia in the thalamus, but an elevation in Gln/Glu was detected in the anterior cingulate before treatment
De la Fuente‐
Sandoval, et al.,
2009(de la
Fuente‐Sandoval,
Favila et al. 2009)
n=14 antipsychotic‐free patients (N/A)
n=14 healthy controls (N/A)
(1H) MRS was assessed twice, once before treatment and another 6 weeks after treatment and were compared with and healthy controls
Glu/Cr levels were higher in the dorsal caudate nucleus in patients compared with healthy controls during the antipsychotic‐free period and after treatment
No difference found in the cerebellum
Theberge, et al.
2007 (Theberge,
Williamson et al.
2007)
n=16 antipsychotic‐naïve patients before and after 10 months and 30 months of antipsychotic treatment: (M=14, F=2) (25 ± 8 years)
n=16 controls on two occasions: 30 months apart: (M=14, F=2) (29 ±12 years) and (32 ±12 years)
4T (1H) MRS and MRI images were used to assess the anterior cingulate and thalamic glutamatergic metabolite levels as well as grey‐matter volumes of patients and controls
Greater glutamine levels were detected in the anterior cingulate and thalamus of antipsychotic‐naïve patients.
Thalamic glutamine levels were reduced after 30 months of antipsychotic treatment
No correlation was found between medication levels and glutamine levels
141
Ohrmann, et al.,
2007(Ohrmann,
Siegmund et al.
2007)
n =15 first‐episode antipsychotic‐naïve patients: (M=10, F=5) (27 ± 6.9 years
n=20 chronic patients: (M=14, F=6) (30.3 ± 7.3 years)
n=20 healthy controls (M=13, F=7) (28.1 ± 6.5 years
1.5 T (1H) MRS was used to assess glutamatergic metabolites from the dorsolateral prefrontal cortex of patients and healthy controls
Patients with chronic schizophrenia had reduced levels of glutamate/glutamine in the dorsolateral prefrontal cortex compared with first‐episode patients and healthy controls, while no difference in GLX was detected in the dorsolateral prefrontal cortex of first‐episode patients compared with healthy controls
Ohrmann, et al.,
2005 (Ohrmann et
al,. 2005)
n=18 first‐episode antipsychotic‐naïve patients: (M=13, F=6) (29.3 ± 7.3 years)
n=21 chronic patients: (M=15, F=6) (29.7 ±7.4 years)
n=21 healthy controls: (M=13, F=8) (28.0± 6.8 years)
1.5 T (1H) MRS was used to assess glutamatergic metabolites from the dorsolateral prefrontal cortex of patients and healthy controls
Chronic patients had significantly reduced levels of GLX, and N‐ acetylaspartic acid (NAA) compared with healthy controls and first‐episode patients.
Theberge, et al.,
2002(Theberge,
Bartha et al.
2002)
n=21 antipsychotic‐naïve patients: (M=14, F=7) (26 ± 7 years)
n=21 healthy controls: (M=14, F=7) (26
± 7 years)
4T (1H) MRS was used to assess the left anterior cingulate and thalamus of patients and healthy controls
Greater glutamine levels were detected in both the left anterior cingulate and the left thalamus of patients compared with healthy controls
Kegeles, et al.,
2000(Kegeles,
Shungu et al.
2000)
n=10 male patients: ( 3 medicated and 7 antipsychotic‐free) (28 ± 7 years)
1.5 T (1H)MRS was used to assess GLX and other glutamatergic metabolites from the medial temporal lobe of patients and healthy controls
Greater GLX/Cho levels were detected in the right medial temporal lobe of patients compared with healthy controls
142
n=10 male healthy controls (29 ± 5 years)
Bartha, et al.,
1999(Bartha, al‐
Semaan et al.
1999)
n=11 antipsychotic‐naïve first‐episode patients: (M=9, F=2) (27.4 ± 6 7.7)
n=11 healthy controls: (M=9, F=2) (25.9 ± 6 5.9 years)
1.5T (1H)MRS was used to assess glutamate, glutamine and N‐ acetylaspartic acid (NAA) levels from the left (hippocampus) medial temporal lobe of patients and healthy controls
No difference was detected in the hippocampus when patients were compared with healthy controls.
Bartha, et al.,
1997(Bartha,
Williamson et al.
1997)
n=10 antipsychotic‐naïve patients: (M=8, F=2) (24±5 years )
n=10 healthy controls: (M=8, F=2) (26.3±6.4 years )
1.5 T (1H)MRS was used to assess glutamate and glutamine from the medial prefrontal cortex of patients and healthy controls
Greater glutamine levels were detected in the medial prefrontal cortex of patients compared with healthy controls
Stanley et al.,
1996(Stanley,
Williamson et al.
1996)
n=29 patients with schizophrenia : n= 13 antipsychotic‐naïve first‐episode patients (M=11, F=2) ; n=12 acute medicated patients (M=10, F=2); n=10 chronic medicated patients (M=11, F=1)
n=24 male healthy controls
1.5 T short echo (1H)MRS was used to assess glutamate and glutamine from the left dorsolateral prefrontal cortex of patients and healthy controls
No difference in glutamate or glutamine levels were detected in the dorsolateral prefrontal cortex of antipsychotic‐naïve patients compared to healthy controls, however, glutamine levels were elevated in chronic patients compared with healthy controls
143
Table 2: Image Studies Assessing Dopaminergic Systems in Antipsychotic-Naïve or Antipsychotic-Free Patients with Schizophrenia
Publication Subjects (n): Sex: Age mean
± (SD)
Method
Findings
Schmitt, et al.,
2012 (Schmitt,
Dresel et al.
2012)
N=12 antipsychotic-naïve patients: (M=10, F=2) (27.57 ± 5.34 years)
N= 12 treated patients: (M=10, F=2) (26.41 ± 5.29 years)
N= 12 healthy controls: (M=9, F=3) (N/A)
123I‐IBZM SPECT was used
to assess D2/3 receptor binding in the striatum of patients and healthy controls
No difference in D2/3 binding was detected in striatum of antipsychotic‐naive patients compared with healthy controls, but a reduction was detected in treated patients
Corripio, et
al., 2011
(Corripio,
Escarti et al.
2011)
N=37 Antipsychotic-naïve first-episode patients: (28.3 ± 8.4 years): N=12 Non-schizophrenia: (Schizophreniform
disorder N=3;
Schizoaffective
disorder N=2:
Delusional
Disorder: N=2;
Brief Psychotic
Disorder
N= 2; Bipolar
Disorder n=2;
Psychotic disorder
N =1 ) : (M=26,
F=11) (29.3± 9.2
years)
N=18 healthy controls: (M=10, F=8) (24.3 ± 5.3 years)
123I‐IBZM SPECT was used to assess D2/3 receptor binding in striatum, caudate and putamen of patients and healthy controls
D2/3 striatal/frontal binding ratio was greater in patients with schizophrenia compared with healthy controls ,but there was no difference between patients with schizophrenia and non‐schizophrenia
144
Abi‐Dargham,
et al.,
2011(Abi‐
Dargham, Xu
et al. 2012)
N=25 patients (12 antipsychotic-naïve: (M=5, F=7) (24.42 ± 4.81 years) and 13 antipsychotic-free: (M=11, F=2) (30.56 ± 10.21 years)
N=24 healthy controls antipsychotic-naive: (M=12, F=12) (25.42 ± 4.81 years)
N=24 healthy controls antipsychotic-free: (M=21, F=4) (30.34 ± 9.81 years)
[11C]NNC112 PET was used to assess D1 receptor binding in the striatal subregions : associative striatum: dorsal and pre‐post commissural caudate and pre‐ post commissural putamen, the limbic striatum: the ventral striatum, the sensorimotor striatum: post‐commissural putamen, the cortical regions: the dorso‐ medial prefrontal cortex and the orbito‐frontal cortex of patients and healthy controls
D1 receptor binding was greater in dorso‐, medial prefrontal cortex and the orbito‐frontal cortex of antipsychotic‐naïve patients compared with healthy controls , but no difference when antipsychotic‐free patients were compared with healthy controls
Kegeles, et al.,
2010(Kegeles,
Abi‐Dargham
et al. 2010)
N=18 untreated patients: 6 antipsychotic-naïve and 12 antipsychotic-free(minimum, 20 days; maximum, 300 days : (M=13, F=5) (29± 8 years)
N=18 healthy controls: (M=13, F=5) (29 ± 7 years)
[11C] raclopride PET was used to measure D2/3
receptor binding in the ventral striatum ,precommissural dorsal caudate, precommissural dorsal
putamen,
postcommissural
caudate,and
postcommissural putamen
of patients and healthy
controls
Greater D2/3 receptor binding in the associative striatum of patients: most pronounced in the precommissural
No difference were detected in the other regions when patients were compared with healthy controls
Kegeles et al.,
2010(Kegeles,
Slifstein et al.
2010)
N=21 patients with schizophrenia: 5 antipsychotic-naïve, 15 antipsychotic-free (M=14, F=7) (31 ± 12)
N=22 healthy controls (M=17, F= 5) (26 ± 6)
[18F]fallypride PET was used to measure D2/3 receptor binding in the striatal: postcommissural putamen, precommissural putamen, ventral striatum, precommissural dorsal caudate and extrastriatal regions :thalamus, amygdala, insula, midbrain, incus, hippocampus, temporal cortex and entorhinal cortex
Greater D2/3 receptor binding in the postcommisural caudate and thalamus, and decrease D2/3 binding in the uncus
145
Abi‐Dargham,
et al.,
2009(Abi‐
Dargham, van
de Giessen et
al. 2009)
N=6 first‐episode antipsychotic‐naïve patients: (M=2, F=4) (29 ± 6 years)
N=8 healthy controls: (M=6; F=2) (28 ± 8 years)
123I‐IBZM SPECT was used under three conditions: baseline, after amphetamine administration and after dopamine depletion to assess striatal D2/3 receptor binding and dopamine release
Greater D2/3 receptor binding and dopamine transmission were detected in the striatum of patients compared with healthy controls
Graff, et al.,
2009(Graff‐
Guerrero,
Mizrahi et al.
2009)
N=13 first episode patients, antipsychotic‐ free for at least 2 weeks (M=9, F=4) (26 ± 6 years)
N=13 healthy controls (M=9, F=4) (27 ± 6 years)
[11C]‐[+][PHNO] was used to assess D2/3 high receptor binding in the caudate, putamen, ventral striatum, globus pallidus, substantia niagra and anterior thalamus of patients and healthy controls
No difference in D2/3 high receptor binding was found in any of the regions when patients were compared to healthy controls
Kessler, et al.,
2009(Kessler,
Woodward et
al. 2009)
N=11 patients: 4 ‐naïve, 7 antipsychotic‐free: (M=6, F=5) (30+ 8 years)
N=11 healthy controls: (M=5, F=6) ( 31.6 + 9.2 years)
[18F] fallypride PET was used to assess D2/3 receptor binding in the caudate, putamen, ventral striatum, medial thalamus, posterior thalamus, substantia nigra, amygdale, temporal cortex, anterior cingulate, and hippocampus of patients and healthy controls
Greater D2/3 receptor binding was detected bilaterally in the substantia nigra of patients
Decreased D2/3 receptor binding was detected in the left medial thalamus of patients
Nozaki, et al.,
2009(Nozaki,
Kato et al.
2009)
N=18 patients:
14 antipsychotic ‐naive and 4 antipsychotic‐free(3 months) patients: (M=10,
F=8) ( 35.1± 9.5 years)
N=20 healthy controls: (M=10, F=10) (35.6± 7.4 years)
L‐[beta‐11C] DOPA PET was used to assess
presynaptic dopamine synthesis in the prefrontal
cortex, temporal cortex,
anterior cingulate,
parahippocampus,
thalamus, caudate
nucleus, and putamen of
patients and healthy
controls
Greater
dopamine synthesis was detected in the left caudate nucleus and thalamus, which positively correlated with overall symptom severity.
Schmitt, et al.,
2009 (Schmitt,
Meisenzahl et
al. 2009)
N=23 acutely ill first‐episode antipsychotic‐naïve patients: (M=19, F=4) (28.18± 6.23 years)
123I–IBZM SPECT was used to assess D2/3 receptor binding in the striatum of patients and healthy controls
Reduced D2/3 receptor binding was detected in patients compared with healthy controls
146
N= 10 healthy controls: (M=5, F=5) (32.4± 12.73 years)
Kumakura, et
al.
2007(Kumakur
a, Cumming et
al. 2007)
N=8 male patients: 3 antipsychotic‐naïve and 5 antipsychotic‐free for at least 6 months : (37.3 ± 6.3 years)
N=15 healthy male controls: (37.3 ± 6.4 years)
[18F]fluorodopa [FDOPA] PET was used to assess dopamine synthesis in the striatum of patients and healthy controls
Greater synthesis and turnover of radiolabeled dopamine was detected in patients
[18F]fluorodopa was greater nearly twofold in striatum of patients
FDOPA clearance was increased by 20% in caudate and putamen and by 50% in amygdala and midbrain of the patients
FDOPA and its decarboxylated metabolites were reduced by one-third in the caudate nucleus and amygdala of patients compared to healthy controls
147
Buchsbaum, et al., 2006(Buchsbaum, Christian et al. 2006)
N=15 antipsychotic‐naïve patients: (M=10, F=5) (28.5 ± 8.9 years)
N=15 healthy controls: (M=9, F=6) (27.4 ± 7.9 years)
[18F] fallypride PET and MRI images were used to detect D2/3/D3 receptor binding in the thalamus, amygdala region, cingulate gyrus, and temporal cortices of patients and healthy controls
Reduced D2/3/D3
receptor binding was detected in the thalamus; mostly in left medial dorsal nucleus and left pulvinar ,but also in amygdale, cingulated gyrus and temporal cortices of patients compared with healthy controls
Corripio, et al., 2006 (Corripio, Perez et al. 2006) .
N = 11 first-episode antipsychotic-naïve patients with schizophrenia: (M=6, F=5) (25.6 ± 4.5 years)
N=7 patients with non-schizophrenia (Schizophreniform, schizoaffective and bipolar): (M=4, F=3) (22.6 ± 3.4 years)
N = 18 control: (M=10, F=8) (24.2 ± 4.4 years)
123I‐IBZM SPECT was used to assess striatal D2/3 receptor binding of patients and controls
Patients with schizophrenia showed greater D2/3 receptor binding-(striatal/occipital) then non-schizophrenia patients and healthy controls; D2/3
receptor binding at diagnosis predicted a high probability for developing schizophrenia after a 2-year follow-up
Talvik, et al.,
2006(Talvik,
Nordstrom et
al. 2006)
N=18 antipsychotic‐naïve patients: (M=9, F=9) (16–50 years)
N= 17 controls: (M=13, F=4) (17–50 years)
[11C] raclopride PET was used to assess D2/3 receptor binding in the putamen, caudate and thalamus of patients and healthy controls
No group differences were detected for D2/3 binding in the putamen or caudate, and there was no hemispheric difference for any region.
D2/3 receptor binding was reduced in the right thalamus of patients compared with healthy controls, however did not reach statistical significance for the left thalamus
Glenthoj, et
al.,
N=25 antipsychotic‐
123I‐epidepride SPECT was used to measure D2/3/D3
No difference was found in D2/3
148
2006(Glenthoj
, Mackeprang
et al. 2006)
naïve patients: (M=17, F=8) (26.8 years)
N=20 healthy controls: (M=13, F=7) ( 26.5 years)
receptor binding in the frontal, temporal, and thalamic region of patients and controls
receptor binding between patients and healthy controls in the any of the regions
Patients, however, had greater D2/3 binding in the right compared to the left thalamus, whereas no hemispheric imbalance was detected in healthy controls
Binding values were greater in male (n=17) compared to female patients (n=8)
Tuppurainen,
et al.,
2006(Tuppurai
nen, Kuikka et
al. 2006)
N= 6 antipsychotic ‐naive patients: (M=2, F=4) (33 ± 14 years)
N= 7 healthy controls: (M=4, F=3) (31 ± 9 years)
123I‐epidepride SPECT was used to assesses D2/3/D3
receptor binding in the thalamus and midbrain of patients and healthy controls
Reduced D2/3/D3
receptor binding was found in the midbrain, substantia niagra, of patients compared with healthy controls
No difference was detected in the thalamus between patients and healthy controls
Lomena, et
al.,
2004(Lomena,
Catafau et al.
2004)
N=12 antipsychotic ‐naïve: (M=5, F=7) (26 ± 6 years)
N= 16 antipsychotic‐free after 7 days: (M=13, F=3) (30 ± 9 years)
123I‐IBZM SPECT was used to assess basal ganglia/ frontal cortex D2/3 receptor binding ratio of antipsychotic‐naïve and antipsychotic‐free patients
No difference in basal ganglia/frontal cortex D2/3 receptor binding was detected between antipsychotic‐naïve and antipsychotic‐free patients
Yang, et al.,
2004(Yang, Yu
et al. 2004)
N=11 antipsychotic-naïve patients: (M=6, F=5) (25.4 ± 10.2 years)
N= 12 healthy controls: (M=9, F=3) (33.3 ± 12.9 years)
123I‐IBZM SPECT was used to assess
striatal dopamine D2/3/D3 receptor binding
No difference in striatal D2/3/D3
receptor binding was detected between healthy controls and patients
149
Yasuno, et al.,
2004(Yasuno,
Suhara et al.
2004)
N=10 antipsychotic -naive male patients: (29. ± 5 7.8 years)
N=19 healthy male controls : (29.6 ± 7.5 years)
[11C]FLB457 PET was used to assess D2/3 receptor binding in subregions of the thalamus of patients and healthy controls
D2/3 receptor binding was reduced in the central medial and posterior subregions of the thalamus in patients compared with healthy controls.
Talvik, et al.,
2003 (Talvik,
Nordstrom et
al. 2003)
N=9 antipsychotic-naïve patients: (M=3, F=6) (36 ± 12 years)
N=8 healthy controls: (M=4, F=4) (31 ± 12 years)
[11C]FLB 457 PET was used to assess D2/3/D3 receptor binding in the thalamus, anterior cingulate, frontal and temporal cortices of patients and healthy controls
D2/3/D3 receptor binding was reduced in the medial thalamus of patients compared with healthy controls
No significance was detected in the anterior cingulated, frontal or temporal regions when patients were compared with healthy controls
Abi‐Dargham,
et al.,
2002(Abi‐
Dargham
2002)
N=16 patients: 7 antipsychotic-naïve and 9 antipsychotic-free for at least 21 days: (M=13, F=3) (33 ± 12 years)
N=16 healthy controls: (M=11, F=5) (34 ± 10 years)
[11C ]NNC 112 PET was used to assess D1 receptor binding in the dorso‐ medial prefrontal cortex subcortical regions: dorsal caudate, dorsal putamen, ventral striatum, thalamus, amygdale, and hippocampus of patients and healthy controls
Greater D1 receptor binding was detected in the dorsolateral prefrontal cortex of patients (in both antipsychotic‐naïve and antipsychotic‐free patients ) compared with healthy controls
Suhara, et al.,
2002(Suhara,
Okubo et al.
2002)
N=11 antipsychotic‐naïve male patients: (28.1 ±7.9 years)
N=8 healthy controls: (27.3 ±6.2 years)
[11C] FLB 457 PET was used to assess D2/3 receptor binding in the anterior cingulate, prefrontal cortex, temporal cortex, occipital cortex, hippocampus, and thalamus of patients and healthy controls
Reduced D2/3 receptor binding was detected in only the anterior cingulate of patients when compared with healthy controls
Abi‐Dargham,
et al.,
2000(Abi‐
Dargham,
Rodenhiser et
al. 2000)
N=18 patients :8 antipsychotic‐naïve and 10 antipsychotic‐free: (M=11, F=7) (31 ± 8 years)
SPECT was used to assess D2/3 receptor binding in the striatum of patients and healthy controls before and during AMPT dopamine depletion
Greater D2/3 receptor binding and dopamine levels were found in patients compared with healthy controls
150
N= 18 healthy controls: (M=11, F=7) (31 ±8 years)
Hietala, et al.,
1999(Hietala,
Nagren et al.
1999)
N= 10 patients all antipsychotic‐naïve, but 6 had received occasional doses of benzodiazepine for sedation 2 weeks before the PET scan (M=4, F=6) (29.8 ± 8.8 years)
N= 13 healthy controls: (M=8, F=5) (30.4 ± 9.4 years)
PET with fluorodopa [FDOPA] was used to assess dopamine synthesis in the striatum of patients and healthy controls
Greater pre‐synaptic dopaminergic synthesis was detected in patients compared with healthy controls, the increase was greater in the putamen than caudate and was predominately greater in the left caudate
Laruelle, et al.,
1999 (Laruelle
et al. 1999)
N=34 patients: 7 antipsychotic‐
naïve
and 27
antipsychotic‐ free
for at least 21
days: (M=32, F=4)
(40 ±9 years )
N=36 healthy controls (M=28, F=6) (40 ±9 years)
1231‐IBZM SPECT and amphetamine were used to assess dopamine transmission in the striatum of patients and healthy controls
Greater dopamine transmission was detected in patients at the onset of illness and during periods of exacerbation, but not during periods of remission
Lindström, et
al.,
1999(Lindstro
m, Gefvert et
al. 1999)
N= 14 patients: 12 antipsychotic-naïve (1 antipsychotic-free for at least 10 years and 1 antipsychotic-free for at least 2 years): (M=12, F=2) (31 years)
N=10 healthy controls (M=8, F=2) (N/A)
[11C] L‐DOPA was used to assess L‐DOPA influx rate in the striatum of patients and healthy controls
Greater L-DOPA influx rate was detected in the caudate nucleus, putamen and in parts of medial prefrontal cortex (Brodmann 24) of patients compared with healthy controls
Abi‐Dargham,
et al.,
1998(Abi‐
Dargham, Gil
et al. 1998)
N=15 antipsychotic‐free patients (for a least 21 days): (M=12, F=3) (41 ± 9 years)
123IBZM PET and amphetamine were used to assess D2/3 receptor binding in the striatum of patients and healthy controls
No difference in D2/3 receptor binding was detected in the striatum of patients when compared with healthy controls ; but increased dopamine
151
N=15 healthy controls: (M=12, F=3) (40 ± 11 years)
transmission was detected in patients when compared with healthy controls
Breier, et al.,
1997(Breier,
Su et al. 1997)
N=11 patients: 6 antipsychotic-naïve and 5 antipsychotic -free, mean 7.2 days: (M=8, F=3) (32.4 6 ± 3.0 years)
N=12 healthy controls: (M=9, F=3) (29.2 6 ± 2.6 years)
[11C) raclopride PET and amphetamine were used to assess amphetamine-induced dopamine release in the striatum of patients and healthy controls
Greater dopamine synthesis, but no difference in striatal D2/3 receptor binding was detected in patients compared with healthy controls
Dao‐
Castellana, et
al., 1997(Dao‐
Castellana,
Paillere‐
Martinot et al.
1997)
N=6 male untreated patients: 2 antipsychotic-naïve (26 ± 9 years)
N=7 male healthy controls (25 ± 5 years)
[18F ]-DOPA PET was used to assess dopamine synthesis in the caudate and putamen of patients and healthy controls
No difference between patients and controls was found for Ki mean values
[18F]-DOPA uptake variability was higher in the caudate and putamen of patients compared with healthy controls
Knable, et al.,
1997(Knable,
Egan et al.
1997)
N=21 patients (M=18, F=3) (38.5 ± 9 years)
N=16 healthy controls (M=11, F=5) ( 28.8 ± 7.8 years)
123I-idobenzamide SPECT was used to assess D2/3 receptor binding
No difference was detected between the groups
Okubo, et al.,
1997 (Okubo
et al. 1997)
N=10 antipsychotic-naive male patients: (26.1 ± 3.8 years)
N=7 antipsychotic-free male patients: (29.2 ± 8.1years)
N=18 male healthy controls: (27.7 ± 5.6 years)
PET with [11C] SCH23390 and [ 11C]N-methylspiperone was used to assess D1 and D2/3 receptor binding ,respectively in prefrontal cortex of antipsychotic-naïve patients, antipsychotic-free patients and healthy controls
No difference was detected in both patient groups compared with healthy controls in the striatum, however D1 receptor binding was reduced in the prefrontal cortex
152
Wong, et
al.,1997(Wong
, Singer et al.
1997)
N=22 patients with schizophrenia: antipsychotic-naïve patients: (M=13, F=9) 24 years)
N= 14 patients with bipolar: N= 7 with psychotic symptoms: (M=4, F=3) (41 ± 13 years); N=7 with non-psychotic symptoms: (M=5, F=2) (41 ± 14 years)
N=24 healthy controls: (M=19, F=5) (40 ± 22 years)
PET with [11C]N‐methylspiperone was used to assess D2/3
receptor binding in the caudate nucleus of patients and healthy controls
D2/3 receptor binding was greater in the caudate nucleus of patients with psychotic symptoms
D2/3 receptor binding decreased with age and there was no difference between patients and controls
Laruelle, et al.,
1996(Laruelle,
Abi‐Dargham
et al. 1996)
N=15 antipsychotic-free patients: (M=14, F=1) (42± 2)
N=15 healthy controls: (M=14, F=1) (41 ±2)
SPECT with 123I-IBZM and amphetamine were used to assess dopamine transmission of patients and healthy controls
Greater dopamine transmission in the striatum of patients compared with healthy controls
Hietala, et al.,
1995(Hietala,
Syvalahti et al.
1995)
N= 7 patients: all antipsychotic‐naïve but 5 had received occasional doses of benzodiazepine for sedation 2 weeks before PET: (M=4, F=3) (26± 7 years)
N=8 healthy controls: (M=6, F=2) (26± 7 years)
PET with FDOPA was used to assess dopamine synthesis in the striatum of patients and healthy controls
Greater pre‐synaptic dopamine synthesis was detected in the striatum of patients compared with healthy controls
The increase was greater in putamen than the caudate and was predominately greater in the left caudate
Nordstorm, et
al.,
1995(Nordstro
m, Farde et al.
1995)
N= 7 antipsychotic‐naïve patients: (M=5, F=2) (28.4 ± 6.8 years)
N=7 male healthy controls: (27.7 ± 6.8 years)
PET with [11C]N‐methylspiperone was used to assess D2/3 receptor binding in the basal ganglia of patients and healthy controls
No difference in D2/3
receptor binding was detected in the basal ganglia of patients compared with healthy controls
Pearlson, et
al.,
N= 10 antipsychotic ‐naïve patients with
PET was used to assess D2/3 receptor binding in patients with
Greater D2/3 receptor binding was detected in psychotic patients
153
1995(Pearlson
, Wong et al.
1995)
schizophrenia :( M=6, F=4) (31 ± 11.4 years )
N= 12 healthy controls: (M=9, F=4) (28 ± 12.6 years )
N=14 patients with bipolar ( antipsychotic ‐ free for more than 6 months or antipsychotic‐naïve (N=11) :N= 7 bipolar with non‐psychosis: (M=4, F=3) (41 ± 13.4 years ); N=7 bipolar with psychosis: (M=5, F=2) (39.4 ± 13.9 years)
schizophrenia, bipolar and healthy controls
with bipolar disorder and
schizophrenic
patients
compared with
healthy controls
Greater D2/3 receptor binding was detected in schizophrenic patients and psychotic patients with bipolar disorder when compared with non‐psychotic patients
.
Hietala, et al.,
1994(Hietala,
West et al.
1994)
N=13 antipsychotic‐free patients: (M=9, F=4) (26.8± 7.3 years)
N=10 healthy controls: (M=6, F=4) (25.2± 6.8 years)
Raclopride PET was used to assess D2/3 receptor binding in the striatum of patients and healthy controls
No difference in D2/3
receptor binding or affinity was detected in the striatum of patient compared with healthy controls.
Martinot, et
al., 1994
(Martinot,
Paillere‐
Martinot et al.
1994)
N=10 young patients presenting with negative symptoms (8 antipsychotic‐naïve and 2 antipsychotic‐free for at least 4 months): (M=7, F=3) (26.8± 7.3 years)
N=10 male healthy controls: (25.2± 6.8 years)
[76BR) Bromolisuride PET was used to assess striatal D2/3 receptor binding in the striatum of patients and healthy controls
No difference in D2/3
receptor binding was detected in the striatum when patients were compared with healthy controls
Pilowsky, et
al.,1994(Pilow
N=20 patients : 17 never‐medicated and 3 antipsychotic‐
SPECT with 123I‐IBMZ was used to assess striatal D2/3 receptor binding in
No difference in D2/3 receptor binding was detected in the striatum when
154
sky, Costa et
al. 1994)
free(>5 years): (M=11, F=9) (29 ±2.2 years)
N=20 healthy controls: (M=11, F=9) ( 29 ± 3.3 years)
the striatum of patients and healthy controls
patients were compared with healthy controls
Pearlson, et
al.,
1993(Pearlson
, Tune et al.
1993)
N=13 late-onset antipsychotic-naïve patients: (M=3, F=10) (74 ± 13 years)
N= 17 healthy controls: (M=12, F=5) (39 ± 25years)
PET was used to assess D2/3 receptor binding in patients and healthy controls
Late onset patients had greater D2/3 receptor binding compared with age and gender matched healthy controls.
Tune, et al.,
1993(Tune,
Wong et al.
1993)
N=25 chronic patients: 18 antipsychotic-naïve and 7 antipsychotic-free: (M=17, F=8) (34.88 ± 7.08 years)
N=17 healthy controls (M=13, F=4) (39 ± 5.93 years
PET with [11 C]-N-methylspiperone was used to assess D2/3 receptor binding in striatum of patients and healthy controls
D2/3 receptor binding was greater in the striatum of patients compared with healthy controls; showed to decline with age
.
Martinot, et
al.,
1991(Martinot
, Paillere‐
Martinot et al.
1991)
N=19 untreated patient: 10 antipsychotic-naïve and 9 antipsychotic-free for at least 6 months: (M=12, F=7) (23 ± 5 years)
N=14 male healthy controls: (23 ± 4 years)
[76BR] Bromospiperone PET was used to measure striatal to cerebellar radioactivity as an index of D2/3 receptor binding in patients and healthy controls
No difference in D2/3 receptor binding was detected in the striatum of patients when compared with healthy controls
Farde, et
al.,1990(Farde
, Wiesel et al.
1990)
N=18 first-episode antipsychotic-naïve patients: (M=10, F=8) (24.2 ± 3.3 years)
[11C]raclopride PET was used to assess D2/3 receptor binding
No difference in were detected in D2/3 receptor binding in the putamen or caudate nucleus
Greater binding was found in the left than
155
N=20 healthy controls: (M=10, F=10) (27.5 ± 4.9 years)
in the right putamen in patients but not in healthy controls
Martinot, et
al.,
1990(Martinot
, Peron‐
Magnan et al.
1990)
N= 19 patients: 10 antipsychotic‐naïve; 9 antipsychotic‐free: (M=12) (22 ± 4 years) and (F=7) (24 ± 6 years)
N=14 male healthy controls: (23 ± 4 years).
[76BR] Bromolisuride PET was used to measure striatal to cerebellar radioactivity as an index of D2/3 receptor binding in patients and healthy controls
No difference was found between patients and healthy controls
Wong, et al.,
1986(Wong,
Wagner et al.
1986)
N=10 antipsychotic-naïve patients: (M=8, F=2) (31.2 ± 3.6 years)
N=5 male antipsychotic-free patients: (26.8± 2.6 years)
N=11 healthy controls: (M=8, F=3) (24.3 ± 2 years)
[11 C]-N-methylspiperone PET was used to assess D2/3 receptor binding in caudate nucleus of patients and healthy controls
D2/3 receptor binding was greater in nucleus caudate of both the patient groups compared healthy controls
156
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