Marisela Morales
NIDA Intramural Research ProgramCellular Neurobiology Branch
Cellular Neurophysiology Section
Interactions Between Reward and Stress Systems
The Science of Drug Abuse & Addiction
National Advisory Council on Drug Abuse
Identification of neuronal pathways , neurons and molecules that may be affected or participate in the biology of drugs of abuse
Diversity. Brain is made of neurons with different phenotypes
Connectivity. Different phenotypes of neurons establish functional interactions (synapses) that determine specific neuronal pathways (specific behaviors)
Information. Exchange of information among different neurons in a neuronal pathway is mediated by molecules
Drugs of abuse affect the structure and function of the brain
Interactions between the stress and reward systems
Stressors increase drug self-administration
Different models of stress have shown that it increases vulnerability to addictive drugs
Single or repeated exposure to stressful stimuli can augment the motor stimulant action of amphetamine, cocaine, or morphine
Prenatal stress increases amphetamine self-administration in the adult rat
Stressors reinstate drug seeking (model of relapse). Recent findings
Foot shock reinstates cocaine seeking and induces release of CRF, glutamate and DA in VTA of cocaine-experienced rats (Wang, et al., 2005)
Foot shock reinstates cocaine seeking, however, transient inhibition of the VTA blocks drug seeking (McFarland, 2004)
Investigate neuronal pathways , type of neurons and molecules that might mediate functional interactions between stress and reward systems
Stress responses are mediated by corticotrophin-releasing factor (CRF) originated from different cell types located in several brain areas
Reward responses are mediated by dopamine (DA) produced by neurons located in the ventral tegmental area (VTA)
Ventral TegmentalArea (VTA)
Amygdala
Olfactory tubercle
Nucleus accumbens
Prefrontal cortexHippocampus
Mesocorticolimbic DA system
Dopamine neurons
Interactions between stress and reward systems. Brain area?
CRF cell
Do CRF target VTA cells?
(1) Application of CRF into VTA increases locomotor activity(Kalivas et al., 1987
(2) Footshock induces CRF release in VTA (Wang et al., 2005)
VTA
(3) In vivo administration of drugs of abuse or acute stress increase strength at excitatory synapses on DA neurons (Saal et al., 2003)
GABAergic or DAergic neurons?
Do CRF cells establish functional interactions (synapses) with cells located in VTA?
(1) Rat brain sections were incubated with specific antibodies to label neurons containing CRF
(2) VTA ultra thin sections (70 nm in thickness) were obtained from labeled brain tissue
(3) Material was analyzed under the electron microscope
Do CRF cells establish functional interactions (synapses) with cells located in VTA? Yes
CRF (+) axonal terminalsCRF (-) axonal terminal
CRF (-) dendrite
PresynapticCRF
Postsynapticdopamine?
Synapse
Do CRF cells establish synapses with dopaminergic neurons in VTA?
(1) Rat brain sections were incubated with antibodies against CRF and tyrosine hydroxylase (TH, marker of dopamine neurons in VTA)
(2) VTA ultra thin sections (70 nm in thickness) were obtained from double labeled brain tissue
(3) Material was analyzed under the electron microscope
Do CRF cells establish synapses with dopaminergic neurons in VTA? Yes
CRF (+) axonal terminals
TH (+) dendritesAsymmetrical
synapses
SymmetricalSymmetricalsynapsesynapse
17 %
EXCITATORY INHIBITORY
83 %
At the molecular level, CRF mediates its biological effects by interacting with three different proteins
CRF receptor 1 (CRF-R1) CRF receptor 2 (CRF-R2) CRF binding protein (CRF-BP)
Are these proteins present in DAergic neurons in VTA?
Which of these molecules mediate the functional interactions between CRF and VTA dopaminergic neurons?
Aim:To investigate neuronal pathways , type of neurons and molecules that might mediate functional interactions between stress and reward
Method (Double in situ hybridization)
Brain sections were hybridized with a non-radioactive anti-sense TH riboprobe to label DAergic neurons
CRF-R2 mRNA was not detected in VTA neurons
CRF-R1 and CRF-BP mRNA were detected in VTA neurons
Results
Same sections were hybridized with a radioactive anti-sense CRF-R1, CRF-R2 and CRF-BP riboprobes to determine expression of any of these molecules within DAergic neurons
DNA mRNA Protein
Expression of CRF Receptor 1 (CRF-R1) mRNA in the Ventral Tegmental Area
VTA
SNC
VTA
SNC
TH mRNA CRF-R1 mRNA
VTA = Ventral Tegmental Area
SNC = Substantia Nigra Compacta
Regional Distribution
Hybridization with radioactive antisense RNA probes to detect CRF-R1 mRNA
Hybridization with non radioactive antisense RNA probes to detect TH mRNA
Expression of CRF receptor 1 (CRF-R1) mRNA in dopaminergic neurons in the VTA
Hybridization with non radioactive antisense
RNA probes to detect TH mRNA
Hybridization with radioactive antisense RNA probes to detect
CRF-R1 mRNA
TH mRNA CRF-R1 mRNA
Arrows indicate cellular co-expression of TH (dark color) and
CRF-R1 (green grains) in VTA
71.46% of all CRF-R1 expressing neurons are dopaminergic in VTA
CRF binding protein (CRF-BP)
At the molecular level, CRF mediates its biological effects by interacting with three different proteins
CRF receptor 1 (CRF-R1)
CRF receptor 2 (CRF-R2)
Peripheral CRF-BP plays a role in lowering free circulating CRF levels
CRF binding protein
CRF-BP is expressed in different type of cells in many brain regions(What is the role of CRF-BP in the brain?)
Studies with mouse midbrain slices indicates that CRF-BP is required for CRF to potentiate synaptic transmission by N-MDA (N-methyl-D-aspartate) receptors in VTA dopaminergic neurons
Expression of CRF Binding protein (CRF-BP) mRNA in the Ventral Tegmental Area
VTA = Ventral Tegmental Area
SNC = Substantia Nigra Compacta
Regional Distribution
Hybridization with radioactive antisense RNA probes to detect CRF-BP mRNA
Hybridization with non radioactive antisense RNA probes to detect TH mRNA
SNCSNC SNCSNC
VTAVTA VTAVTA
TH mRNA CRF-BP mRNA
Expression of CRF Binding Protein (CRF-BP) mRNA in VTA Dopaminergic Neurons
Hybridization with non radioactive antisense RNA probes to detect TH mRNA
Hybridization with radioactive antisense RNA probes to detect CRF-BP mRNA
TH mRNA CRF-BP mRNA
Expression of CRF Binding Protein (CRF-BP) mRNA in VTA Dopaminergic Neurons
TH mRNA CRF-BP mRNA
Summary Within the VTA, CRF axonal terminals establish mainly asymmetrical
(presumably excitatory) synapses with dopaminergic dendrites
We suggest CRF excitatory synapses on dopaminergic dendrites as a locus for the known interaction of stress mechanisms and the mesocorticolimbic dopamine system (a system implicated in addiction, a number of stress-related psychiatric syndromes) and co-morbidity between the two
Implications: Following stress, synaptical release of CRF in VTA may directly activate dopaminergic neurons, inducing release of dopamine within the mesocorticolimbic system
Within the VTA, CRF-R1 and CRF-BP are preferentially expressed in dopaminergic neurons
Implications: At the cellular level, CRF may affect dopaminergic neurotransmition by interacting with CRF-R1 and CRF-BP located with VTA dopaminergic cell bodies
Why is this important?
We provide evidences indicating that stress system may directly activate the reward system through CRF-R1 and CRF-BP
New targets for medication development
CRF-BP is a molecule that interacts with CRF and is selectively present in DAergic neurons involved in the rewarding effects of drugs of abuse
Current and future studies
(2) Evaluation of effects of drugs of abuse on the CRF, CRF-R1 and CR-BP system
(1) Brain circuitry involved in the direct interaction between stress and reward systems
Identification of CRF neurons that synapse on VTA dopaminergic neurons [brain distribution, cellular phenotype (receptors, neurotransmitters, etc.), afferents, etc.]
(3) Evaluation of the participation of CRF, CRF-R1 and CRF-BP system in cocaine and methamphetamine induced behaviors (collaboration with Dr. Roy Wise)
(4) Set up in vitro studies to determine functional molecular interactions among CRF, CRF-R1 and CRF-BP
Determination the neurotransmitters (glutamate, GABA) present in CRF axonal terminals, and establish at the ultratructural level the distribution of CRF-R1 and CRF-BP
Acknowledgement
Patricia Tagliaferro Ph.D.(Ultrastructural studies)
Emma Roach(In situ hybridization studies)
Support: NIDA IRP
