Thesis LK TCD Neuroscience

8/11/2019 Thesis LK TCD Neuroscience

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UNIVERSITY OF DUBLIN,

TRINITY COLLEGE DUBLIN

The Role of Circadian Rhythms in Object

Recognition and Displacement Memory

Student Name:Lua Koenig

Student Number: 10701183

Supervisor:Daniel Ulrich

Date of Submission: 3rdApril 2014

Thesis submitted as partial fulfilment of requirement for the award of

B.A. (Honours) Moderatorship in Neuroscience, by the School of

Natural Sciences, University of Dublin, Trinity College Dublin.


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Table of Contents

1. Introduction .................................................................................................................................................. 5

1.1 Circadian Rhythms ................................................................................................................................ 5

1.1.1 Time-of-day effects on behaviour ................................................................................................... 6

1.1.2 Time-of-day effects on long-term potentiation and synaptic plasticity ............................................ 7

1.1.3 Time-of-day effects on pleiotropic molecules ................................................................................. 8

1.2 Sleep and Memory Formation ............................................................................................................. 10

1.2.1 Behavioural studies ...................................................................................................................... 11

1.2.2 The contribution of different sleep stages to memory consolidation ............................................ 12

1.2.3 Theories on the mechanisms of memory consolidation in sleep .................................................. 14

1.2.4 Alternative views on the role of sleep ........................................................................................... 15

1.3 Integration of time-of-day and sleep effects on memory ..................................................................... 16

2. Material and Methods ................................................................................................................................ 18

2.1 Subjects ............................................................................................................................................... 18

2.2 Apparatus ............................................................................................................................................ 18

2.3 Experimental procedure ...................................................................................................................... 18

2.3.1 Reversal of LD cycle for the DL group ......................................................................................... 19

2.3.2 Novel-object recognition task ....................................................................................................... 19

2.3.3 Novel-location recognition task .................................................................................................... 19

2.3.4 Increased sleep and increased wake ........................................................................................... 20

2.4 Data Analysis and Statistics ................................................................................................................ 20

3. Results ....................................................................................................................................................... 21

3.1 Training Times ..................................................................................................................................... 21

3.2 Influence of sleep/wake on memory performance ............................................................................... 21

3.2.1 NOR task ...................................................................................................................................... 21

3.2.2 NLR task ....................................................................................................................................... 22

3.3 Influence of time-of-day (TOD) on memory performance .................................................................... 23

3.3.1 NOR task ...................................................................................................................................... 23

3.3.2 NLR task ....................................................................................................................................... 24

3.4 Joint effects of TOD and sleep ............................................................................................................ 24

3.4.1 NOR task ...................................................................................................................................... 24

3.4.2 NLR task ....................................................................................................................................... 25

3.5 Influence of increased sleep/wake on memory performance .............................................................. 26

3.5.1 Effect of increased sleep on NOR ................................................................................................ 26

3.5.2 Effect of increased wake on NOR ................................................................................................ 27

3.5.3 Effect of increased sleep on NLR ................................................................................................. 27

3.5.4 Effect of increased wake on NLR ................................................................................................. 28

4. Discussion ................................................................................................................................................. 29

BIBLIOGRAPHY ............................................................................................................................................ 37


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Acknowledgements

I would like to sincerely thank my supervisor, Prof. Daniel Ulrich, for being available and

helpful throughout the duration of the project, especially in such a busy and exciting time for

him, during which the correction of drafts, I can imagine, is not ones priority

Thank you to Ruth Hennessy, for being so patient when I was lost with statistics, and for

communicating to me her love for behavioural animal research. Thank you to Dr. Aine Kelly

too, for her help explaining the set-up of the experiments.

Most of all, thank you to my lab partner, Hannah Coogan Murphy, for living through this

research with me for endless weeks and late nights, and for keeping me sane in the

confinement of the solitary red-lit lab. Candy Crush and Desert Island Discs kept us going

during the long hours, as well as a common distaste for statistics. We will miss Gonzales and

Spoon dearly!


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Abstract

Memory is a complex phenomenon that has been shown to be affected by both circadian

rhythms and sleeping patterns. The extent to which both of these factors influence memory is

uncertain and many theories hypothesize how the molecular mechanisms of sleep, as wellas circadian pacemakers of the central nervous system, affect memory centres. However,

the fields of sleep and of circadian rhythms are largely separated, and few studies have

attempted to dissociate these effects on memory. The present study made use of an

experimental design which aimed to avoid the use of sleep deprivation, a method used in

sleep studies which has the drawback of producing stress, which is presumably harmful to

memory formation. Two groups of rats were used: the LD group (n=14) was housed in a

12h:12h light-dark cycle, and the DL group (n=14) was housed in a 12h:12h dark-light cycle.

Novel object recognition and Novel location recognition tasks were used to assess memoryperformance 2h, 5h and 24h after training. Two experimental sessions were conducted for

each task: one with training at 9am and the other at 1pm. This allowed for the consideration

of the influence of sleep on memory, and the influence of time-of-day. Joint effects of the two

factors were also assessed. In an additional experiment, one group of rats (n=10) was put on

a 24h dark cycle, and another group (n=10) was put on a 24h light cycle to assess the effects

of constant dark and light, respectively, on memory. The results showed that sleep was

overall more beneficial to the hippocampus-dependent NLR task. There was an effect of

time-of-day which was dependent on time of training rather than time of testing, and the

results also suggested the existence of a peak in memory performance between ZT17

(Zeitgeber Time) and ZT18. The joint effects of time-of-day and sleep showed a fluctuation in

memory performance consistent with the sequential hypothesis for NOR intermediate-term

memory and NLR long-term memory. Constant light was detrimental to memory formation for

the NOR task but not the NLR task. Constant dark, intriguingly, was overall beneficial to

memory performance in both tasks. These results have important implications in the

understanding of the pathophysiology that results from the disruption of circadian rhythms

and sleeping patterns.


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1. Introduction

Circadian rhythms are generated by a collection of neural networks that regulate many

physiological processes in the organism. These include temperature, hormonal cycling, cell

cycle, metabolism, appetite, mood and sleep-wake cycles. These processes all display, to acertain extent, a 24-hour endogenous oscillation. It is thought that this internal clock may

function to coordinate biological processes with the external environment, with its daily light-

dark cycle. Evidence has converged to demonstrate a role for circadian rhythms in memory

formation. The role of sleep, in particular, has been shown to greatly impact on memory

formation, in behavioural, neurophysiological and in vitro studies. Research has been

conducted to determine which type of sleep benefits memory the most, and which types of

memory are affected in each case. However, the sleep-wake cycle is not the only product of

the circadian rhythm on memory. Indeed, memory consolidation has been shown to follow atime-of-day variation (Valentinuzzi et al., 2008), and disruption of signalling pathways that are

subject to diurnal oscillation has been shown to negatively impact cognitive performance and

learning (Rajaratnam and Arendt, 2001). Evidence points to the existence of a central

pacemaker in the brain, in the form of the suprachiasmatic nucleus (SCN), a region of the

hypothalamus (Kononenko et al., 2008). Research in the fields of sleep and time-of-day

effects on memory are important for the understanding of pathophysiology resulting from the

dysfunction of these processes, especially in a society that tends to gradually depend less on

24h light/dark cycles.

1.1 Circadian Rhythm s

The suprachiasmatic nucleus (SCN), located in the hypothalamus, has been defined as the

central pacemaker of circadian rhythms in the human brain (Antle and Silver, 2005). This

nucleus generates neuronal and hormonal activity and regulates other local pacemakers

within the central nervous system. Notably, it has been shown that pathways exist that link

the SCN to limbic structures essential to memory and learning, such as other parts of the

hypothalamus and the amygdala (Sheeba et al., 2008). This structure is sensitive to photic

input and has endogenous oscillations of approximately 24 hours. Central pacemakers

regulate many neurophysiological and behavioural processes like body temperature, mood,

appetite, sleep, hormone secretion and gene expression. Some of these processes affect

learning and memory performance and thus provide an indirect link between circadian

rhythms and memory formation. Many studies have also shown that circadian rhythms affect

synaptic plasticity and long-term potentiation, two processes that have been directly related

to memory consolidation. Research on circadian rhythms and their effects on cognition have

implications in conditions like jet lag, shift work sleep disorder or chronic sleep pattern


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disorganization, which significantly decrease cognitive and memory-related performances

(Cho et al., 2000,Medeiros et al., 2001, Rajaratnam and Arendt, 2001). Learning deficits

were reported in humans with internal desynchronisation conditions, which cause the sleep-

wake cycle to fall out of phase with internal biological time (Wright et al., 2006). Insight into

the mechanisms of circadian modulation of cognition and intellect therefore has great

implications in public health.

1.1.1 Time-of-day effects on behaviour

Different behavioural experiments have been developed to evaluate time-of-day effects on

memory performance. These models have shown that different forms of memory and

learning are affected by circadian cycling, such as social recognition memory (Moura et al.,

2009), spatial memory (Valentinuzzi et al., 2004), acoustic memory (Valentinuzzi and Ferrari,

1997), odour discrimination memory (Decker et al., 2007)and reward memory (Valentinuzzi

et al., 2008). Chaudhury and Colwell conducted a study in which they fear-conditioned mice

and tested them for possible circadian regulation of three stages of memory: acquisition,

recall and extinction (Chaudhury and Colwell, 2002). The mice acquired the fear conditioning

faster during the day and showed a greater extinction of memories formed during the night.

This pattern was maintained after the reversal of the light-dark cycle and in constant dark

conditions, showing that the rhythm was endogenous and most likely circadian.

These studies outline the need to determine which component of memory is affected by

circadian cycling: acquisition, recall or retention. Researchers have developed various tasks

using different animals to distinguish between these distinct processes. In an experiment

usingAplysia Californica, long-term sensitization (LTS) training was used as a measure of

memory performance (Fernandez et al., 2003). A circadian effect on LTS memory was found,

which depended on the time of training rather than on the time of testing. In a landmark study

conducted with mice, a fear condition paradigm was used (Chaudhury and Colwell, 2002). It

investigated the influence of time-of-day effect on memory acquisition and found that thiseffect was stronger during the light period, especially at ZT3 (zeitgeber time 3, the notation

used for circadian cycles with ZT0 being the beginning of the light phase). Mice were trained

at ZT3 under light/dark conditions and maintained peak memory performance at ZT3 for 3

days. This was maintained in constant dark conditions, suggesting that the peak time of

recall could coincide with the peak time of acquisition. However, when rats were trained at

ZT15, they still maintained a peak performance at ZT3. In view of this result, it would seem

that the peak time of memory recall is under time-of-day-dependent control and is in this

sense independent of training time. Another study conducted with rats suggested that animal

memory performance is best if they are tested at the circadian phase at which they were


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trained (Garren et al., 2013). This would make circadian rhythm a contextual cue for

memory retrieval.

Certain studies have focused on particular aspects of memory formation, such as the type of

task used for animal training. Garren et al. conducted a study to consider the effects of

circadian rhythm on memory in two distinct types of tasks: classical and operant conditioning

(2013). The former provokes an association between two stimuli and focuses on involuntary

behaviours. The latter, in contrast, constructs a relationship between a stimulus and an

animals voluntary reaction to it. It generally depends onpunishment or reward to increase or

decrease a behaviour. It has been demonstrated that circadian cycling has a significant

effect on animal memory with classical conditioning paradigms (Chaudhury and Colwell,

2002,Rawashdeh et al., 2007). An operant conditioning setup was used in an odour

discrimination task, using identical sensory cues as in a previous classical conditioning task.

Whereas circadian rhythm had been shown to influence short- and long-term memories in

classical conditioning, no such circadian impact was observed in the operant conditioning

task. This data suggests that circadian regulation of learning may be different depending on

what type of conditioning is used for training.

Furthermore, Takahashi et al studied time-of-day effects on different memory tasks like

object-recognition and novel location recognition in rats (Takahashi et al., 2013). They found

there was memory improvement during the day and impairment at night for the novel location

task, but found no such variation in performance for the novel object recognition task. They

suggested that circadian modulation is task-dependent and hypothesized that only

hippocampus-dependent tasks like novel location recognition are subject to this variation.

However, more studies are needed to confirm this hypothesis.

This array of behavioural studies illustrates the existing consensus that memory performance

in different tasks is differentially influenced by circadian rhythms.

1.1.2 Time-of-day effects on long-term potentiation and synaptic plasticity

An aspect of circadian systems that researchers have found interesting to investigate is their

anatomical basis and their influence on processes like long-term potentiation (LTP), a form of

synaptic plasticity. LTP occurs as a consequence of simultaneous stimulation of two

neurons, resulting in the enhancement of synaptic transmission between them. This is the

basis of synaptic plasticity, the process by which synapses are able to become stronger or

weaker depending on their stimulation. Synaptic connections within the hippocampus are

subject to such activity-dependent changes in strength, and it is thought that there is a


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functional relationship between synaptic plasticity and memory consolidation (Martin et al.,

2000).

Direct and indirect pathways connecting this circadian system to limbic structures associated

with memory have been shown (Moga et al., 1995, Peng and Bentivoglio, 2004). One of

these involves hypocretin-expressing cells of the lateral hypothalamus (Peyron et al., 1998),

although it has not been demonstrated that these pathways are the instigators of circadian

influence on memory. Central pacemaker cells of several species have been shown to have

circadian oscillations in physiological properties, like their spontaneous firing rates and

resting membrane potential (de Jeu et al., 1998). This would suggest the existence of a

conserved mechanism across phylogeny for time-of-day-dependent oscillation of

physiological processes, which presumably impact on cellular mechanisms of memory.

Time-of-day-dependent changes have also been observed in the changes in hippocampal

LTP and synaptic plasticity (Harris and Teyler, 1983). It remains to be demonstrated that

circadian modulation of hippocampal cells and the time-of-day-dependent firing of

pacemaker cells are in phase with each other, before a functional connectivity between these

two circadian rhythms can be confirmed.

1.1.3 Time-of-day effects on pleiotropic molecules

Many different theories outlining how the circadian system affects memory focus on

molecules that impact on both systems. These molecules include hormones that display a

circadian modulation of expression like melatonin or stress hormones, signalling pathways

like the cAMP-MAPK-CREB pathway, and genetic modulators that modify epigenetic

mechanisms. The research on these pleiotropic molecules is extensive. These molecular and

cellular events have been shown to affect many aspects of memory.

Melatonin is a signalling molecule that follows a circadian rhythm of expression (Pandi-Perumal et al., 2006)and is present throughout phylogeny (Cahill, 1996,Goto et al., 1989).

There is also evidence that melatonin influences memory. That is, low levels of melatonin

correspond with high levels of memory performance (Gerstner and Yin, 2010). In an

experiment conducted with rats, their memory performance was lower in the dark period,

when large amounts of melatonin are endogenously secreted. Conversely, when little

melatonin was secreted in the light period, their memory performance was enhanced

(Takahashi et al., 2013). Rawashdeh et al conducted an experiment in zebrafish in which

they found that melatonin secretion from the pineal gland provoked suppression of memory

consolidation at night; removal of the pineal gland or treatment with melatonin receptor


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to day in these rats, and hypothesized that there exists a signalling pathway between the

SCN and the hippocampus via the adrenal gland.

Epigenetic mechanisms refer to the processes through which chromatin structure is modified

through acetlytranferase activity or DNA modification, and consequently alters gene

expression. It is possible that these mechanisms affect synaptic plasticity and circadian

oscillations, therefore providing a causal relationship between circadian rhythms and

memory. It has been found that light provokes chromatin remodelling in the nucleus of the

SCN and of memory-related hippocampal cells (Crosio et al., 2000). A second breakthrough

was the observation that chromatin undergoes rhythmic modifications (Etchegaray et al.,

2003). The circadian gene CLOCK has also been shown to have a role in long-term memory

formation (Kondratova et al., 2010); and its protein product has acetlytransferase activity (Doi

et al., 2006). It can be hypothesized that specific signalling pathways perpetrate these

epigenetic changes and help the formation of long-term memory through the expression of

circadian genes.

Therefore, research has shown that circadian rhythms influence behaviour, excitability,

synaptic plasticity, and memory-related molecules. The central pacemaker of the central

nervous system, the SCN, seems to hold a crucial role in the relationship between circadian

rhythms and memory consolidation. However, some studies have implied the existence of

local pacemakers too, with the SCN coordinating their activities. That is, many tissues have

cell-autonomous clocks that potentially control the circadian modulation of physiology and

processes like synaptic plasticity, independently of the SCN (Yamazaki et al., 2000). As an

example, the central pacemakers ofAplysia californicawere excised and time-of-day effects

on long-term sensitization were nevertheless observed (Lyons et al., 2006b). These findings

illustrate the necessity to investigate to what extent local pacemakers are coupled with the

SCN and how they specifically influence memory. Further research on the importance of

time-of-day effects on memory has wide implications in pathologies that disrupt circadianrhythms. What is more, circadian rhythms regulate sleep/wake cycles and therefore have a

role in the widely studied influence of sleep on memory. The extent to which both processes

are linked is yet to be determined, because although sleep is regulated in a circadian

fashion, it is a unique phenomenon from a neurophysiological perspective.

1.2 Sleep and Memory Form ation

It has never been determined with certainty what the function of sleep is, making it the

subject of substantial research. It is a state characterized by a reduction or absence of


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consciousness, inactivity of voluntary muscles (especially during rapid eye movement sleep)

and reduced sensory responsiveness. There is a consensus that performance of newly

acquired declarative and procedural skills is actively improved by sleep, although the

evidence is more scarce for declarative memory. Declarative memory refers to the encoding

of explicit information like facts and knowledge, whereas procedural memory encompasses

implicit skills like learning motor sequences, to read or how to play an instrument. The

process which sleep presumably improves is consolidation, which transforms newly encoded

information into stable representations that can be integrated into pre-existing networks of

long-term memories (Diekelmann and Born, 2010). However, there is a school of thought

that argues that sleep has no effect on cognitive performance or memory.

1.2.1 Behavioural studies

Studies detailing the significant increase in memory recall after sleep are numerous for

procedural memories (Plihal and Born, 1997,Fischer et al., 2002)and more scant for

declarative memory (Rasch et al., 2007,Barrett and Ekstrand, 1972). One particular study

provides evidence of declarative memory improvement after sleep: Gais et al investigated

human subjects and their retention of vocabulary and found that their performance was

enhanced after sleep and that the improvement was persistent for at least two days (Gais et

al., 2006). Fatigue is usually a confounding factor in studies that use sleep-deprived subjects

as a control for memory performance. Gais et als study design was interesting in that it

showed that memory improvement in this task was independent of time-of-day and acute

fatigue. Research in the field of sleep and memory has evolved to more specific aspects of

sleep like its effects on emotional memory (Payne et al., 2008), episodic memory (Inostroza

et al., 2013)or the optimal timing of sleep for memory improvement. For example, Inostroza

et al investigated the sleep-dependency of episodic-like memory in rats (Inostroza et al.,

2013); this refers to the memory that binds a specific event to its spatial and temporal

contexts. Novel object recognition and novel location recognition tests were used, as well as

experimental models to test episodic memory as a whole. The results suggested that sleepwas beneficial to hippocampus-dependent memories; that is, memories with associative

learning components. It also inferred that sleep was most beneficial when it occurred soon

after memory encoding.

Several studies investigated the optimal timing of sleep and one of them found that in the

case of procedural memory, longer durations of sleep proportionally increase memory recall

(Walker et al., 2003). In a study on declarative memory, an interval of 3h after learning

rendered sleep more effective than immediately after the learning event (Gais et al., 2006),


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suggesting that a short delay was necessary for optimization of the effect. However, this

study did not allow for the effects of forgetting during the interval.

Another focus of behavioural studies in this field is the difference between explicit and implicit

memories. The former refers to conscious recollection and the former refers to unconscious,

unintentional memory. A serial reaction time task was developed (Robertson et al., 2004)to

account for the benefits of sleep on both types, which involves learning of an identical

sequence either by explicit or implicit means. The explicitly learnt task was improved by

sleep more so than its implicit counterpart, suggesting that encoding of explicit memories is

more prone to sleep-dependent consolidation.

Behavioural research has also demonstrated that memories with a motivational component

are preferentially enhanced by sleeping (Fischer and Born, 2009)that is, memories for

which a better performance of recall will earn subjects a reward. Another interesting result

was produced from a study in which subjects trained for a procedural skill and underwent

declarative learning of words immediately afterwards (Brown and Robertson, 2007). Subjects

who did not sleep were unable to reproduce the procedural skill whereas those who did

recalled the task without difficulty. This would suggest that competing memory systems

disconnect during sleep and independently consolidate memories, and that this is impossible

in the waking state.

These results demonstrate the consensus that sleep actively enhances memory

performance, but that this effect can only be observed under specific conditions.

1.2.2 The contribution of different sleep stages to memory consolidation

The characteristics of sleep stages have been well defined, by electrophysiological means

and brain imaging techniques. Two distinctive phases of sleep have been identified and

characterized: REM (rapid eye movement) sleep and non-REM sleep, which includes SWS(slow wave sleep) and lighter stages 1 and 2. The cycling of the phases differs among

species but in humans SWS occurs in the first half of the night and REM sleep is more

widespread. Each phase has specific neuromodulator activity and electric field potential

oscillations.

A core feature of memory consolidation is the reactivation of the encoding circuits to create

long-term memory. It was shown that this occurred in the first hours after learning in SWS

sleep. These reactivations nearly always happen in the same order in which they were

experienced (Foster and Wilson, 2006). During an experiment in which subjects were re-


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exposed to an odour in SWS that they had smelt for the first time during a spatial learning

task, their hippocampus was strongly activated and their spatial memory enhanced (Rasch et

al., 2007). Therefore, reactivation is a process essential to consolidation, and it is

hypothesized that it executes the transfer of hippocampal memories to the neocortex for

long-term storage.

It was found that SWS-rich sleep benefited declarative memory, whereas REM-rich sleep

was beneficial for non-declarative memories (Plihal and Born, 1997). Tying in with this,

studies found that REM sleep particularly promoted emotional aspects of memory.

Substantial research has been conducted on the effects of REM sleep deprivation:

pharmacological suppression by anti-depressant drugs resulted in procedural memories

remaining intact (Rasch et al., 2009), although these drugs have been found to affect

synaptic plasticity, potentially compensating for the beneficial effects of REMS on these

memories. Evidence was found for the existence of an REM sleep window during which

REM sleep is increased after learning depending on the type of task, and disruptions of REM

in these windows has severe consequences on memory (Smith, 2003). The sequential

hypothesis seems to offer a solution for the controversial roles of sleep stages on different

types of memory. It states that the close succession of SWS and REM sleep is optimal for

the consolidation of declarative and non-declarative memories (Ambrosini et al., 1988). This

was apparent when SWS-REM cycling was disrupted, and the time spent in the different

phases was unaltered (Ficca and Salzarulo, 2004). Memory performance was significantly

poorer than pre-sleep testing. This shows that the order in which the phases occur is the

determining factor for memory consolidation. This hypothesis is further backed up by

electrophysiological studies.

The intermediate sleep stage corresponds to non-REM sleep stage 2, and has been shown

to impact on consolidation too. Stage 2 sleep was disrupted pharmacologically and resulted

in an immediate enhancement of procedural skills (Rasch et al., 2009). This result wasunexpected and suggests that sleep stages themselves are not responsible for specific types

of memory consolidation, but most likely the molecular events they encompass, which are

sometimes shared by different sleep stages.

SWS is characterized by neocortical slow oscillations, thalamocortical spindles and

hippocampal ripples. These field potential rhythms have been shown to contribute to memory

consolidation. For example, an increase in spindle activity after learning correlated with post-

sleep memory enhancement and was local to areas dedicated to encoding information duringlearning (Clemens et al., 2006). In line with this, disruption of hippocampal ripples led to


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suppression of long-term memory formation in rats (Girardeau et al., 2009). REM sleep is

characterized by PGO waves and EEG theta rhythms. An increase in PGO waves after an

active avoidance task was proportional to an increase in post-sleep performance (Datta,

2000).

1.2.3 Theories on the mechanisms of memory consolidation in sleep

There are currently two competing theories on the mechanisms of memory consolidation in

sleep. The first one, which is older and more widely accepted, is the system consolidation

hypothesis. It suggests that learning may potentiate specific synapses by strengthening them

during sleep. The second theory is the synaptic homeostasis hypothesis, which proposes

that consolidation occurs as a consequence of weakening of neural connections; in other

words, a global downscaling of brain activity. These seemingly antagonistic hypotheses may

not be mutually exclusive, but more research is necessary in order to produce a model

whereby both systems work together to produce long-lasting memory consolidation.

The active system consolidation hypothesis assumes that information is encoded in different

parts of the neocortex and the hippocampus during waking and that re-activation of the

encoded pathways during SWS provokes the redistribution of memory traces so that

connections gradually become strengthened. This acts to integrate the new memories into

existing networks of long-term memories (Marshall and Born, 2007). There is evidence that

the depolarizing up-states of SWS could generate the reactivation of memory traces in the

hippocampus, and it is thought that this may allow for feedback from the hippocampus to the

neocortex. It has been demonstrated that reactivation occurs during these slow oscillations.

This mechanism of redistribution of memory traces is consistent with brain imaging studies

(Gais et al., 2007,Takashima et al., 2006). This imaging also revealed that hippocampus-

dependent memories were mostly redistributed to the medial prefrontal cortex, which is the

generator of slow oscillations, suggesting a reciprocal relationship between redistribution and

SWS (Murphy et al., 2009).

The synaptic homeostasis hypothesis suggests that sleep offers the opportunity for the brain

to downscale synaptic strength after the net increase that occurs in wakefulness (Vyazovskiy

et al., 2008). This would allow for synapses to be available for re-use for future memory

consolidation. A reason this theory came about is that genes involved in synaptic LTP that

are very active in wakefulness, are not expressed during sleep stages responsible for

reactivation of memory traces (Cirelli and Tononi, 2004). Slow oscillations are characteristic

of downscaling and have been shown to decrease in amplitude with SWS cycles as

synapses are depotentiated. This downscaling presumably weakens all synapses by the


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same amount, thus cancelling weak potentiation and improving the ratio of significant traces

to irrelevant information by leaving these memory traces potentiated compared to other

synapses. Research is on-going regarding how the brain identifies which information

constitutes noise and which corresponds to signals.Three main pieces of evidence exist

to support this hypothesis. Firstly, a study found that in rodents, the number of AMPA

glutamate receptors is decreased during sleep (Kemp and Bashir, 2001), meaning there is a

lower level of excitatory neurotransmission during SWS. Secondly, the number of synaptic

spines on neuronswhich are responsible for detecting neural signalsis decreased during

sleep. Finally, studies in rats and humans found that neurons have stronger reactions to

electrical stimulation in subjects that are sleep-deprived than those who have slept

(Eschenko and Sara, 2008). This shows that synaptic strength is reduced during sleep. This

theory implies that weak memories are erased with sleep, which is in contrast with

behavioural results. However, it does account for the finding that sleep facilitates the

encoding of new memories during subsequent waking through downscaling of previously

saturated synapses.

These two models of consolidation each have their limitations, but also suggest that SWS

and REM sleep have complementary roles in the mechanism of memory consolidation, in

line with the aforementioned sequential hypothesis.That is, active system consolidation

integrates new memories into long-term memories during the slow oscillations of SWS. This

process most likely works in tandem with synaptic downscaling to avoid synaptic saturation.

Subsequent REM sleep would then act to stabilize new memories by desynchronizing neural

networks and allowing for the process of consolidation to occur uninterrupted.

1.2.4 Alternative views on the role of sleep

Some researchers argue that the role of sleep in memory consolidation is overestimated.

The main argument is that sleep could simply be a state of rest and metabolic recovery, that

predisposes the body to an indirect improvement of post-sleep memory performance (Siegel,2001). Subjects who underwent an auditory learning task followed by either a period of sleep

or a period of restful waking had the same degree of improvement (Gottselig et al., 2004).

Furthermore, different species have very different sleep patterns. For example, many

maritime animals sleep unilaterally with one hemisphere awake (LM, 1984), and some

undergo total sleep deprivation like dolphins who have just given birth and stay awake for

very long periods (Lyamin et al., 2005). These differences are a main counter-argument for a

generalized role of sleep in memory. Secondly, Vertes argues that improvements in post-

sleep memory performance could simply be due to the passage of time (Vertes, 2004). It was

found that subjects deprived of rapid-eye moment (REM) sleep had no difference in memory


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and learning, and that exposing them to learning situations did not provoke increases in

subsequent REM sleep (Smith, 2001). Furthermore, patients with brainstem lesions that

completely disrupted REM sleep were found to show no cognitive deficits (Vertes, 2004).

Evidence that sleep promotes forgetting is very scarce (Crick and Mitchison, 1983).

However, this data lacks empirical evidenceand existing experimental studies strongly

argue that sleep does indeed have significant impact on memory.

1.3 Integrat ion of t im e-of-day and sleep effects on memory

Although circadian rhythms and sleep/wake cycles are presumably products of similar neural

systems, they are two distinct fields of research that are rarely considered to be overlapping.

Sleep is regulated in a circadian manner, and it is a unique phenomenon in many respects,

which explains why it is considered as distinct from circadian rhythm research. However,

there is a lack of research integrating time-of-day effects versus sleep effects on memory. A

problem that results from this is in judging if a memory enhancement observed at a specific

time is due to circadian modulation or to a post-sleep improvement. Studies that have tried to

address this issue are rare. Many studies that investigate the roles of circadian rhythms

would be extremely informative for this problem if they took into account sleeping patterns in

the analysis of their results. A problem often encountered in sleep studies is in finding

appropriate control groups for memory performance. Sleep deprivation is often used, but this

method has drawbacks in that it provokes acute fatigue which presumably alters cognitive

performance. Sleep experiments designed to control for time-of-day effects (Binder et al.,

2012, Cirelli et al., 2004)often use sleep deprivation as a control, making it difficult to

extrapolate their results. Huber at al designed a study in which a motor learning task was

performed by subjects, and their memory was tested after a night of sleep, as well as after 8h

of spontaneous wakefulness (Huber et al., 2004). The group that slept displayed an increase

in performance, whilst the awake group did not. It was concluded that the improvement was

specifically due to sleep. However, the study was not specifically designed to integrate time-

of-day versus sleep effects, and only aimed to control for confounding circadian factors.

Another method that has potential in analysing this dichotomy in the field is the investigation

of circadian rhythm disruption. An initial study found that long-term memory performance was

decreased for a fear-conditioning paradigm in mice after they were exposed to constant light

(Eckel-Mahan et al., 2008). Similarly, rats in constant light conditions had impaired

performance in the Morris water maze (Ma et al., 2007). These findings seem to agree on a

direct effect of circadian rhythm on memory performance in different tasks. Although these

studies had not aimed to directly account for joint effects of sleep and circadian rhythms on

memory, it has previously been demonstrated that exposure to constant light for nocturnal


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animals like mice induces increased sleep (Yasuda et al., 2005), as well as circadian rhythm

disruption. It would be very interesting to repeat these studies with the aim of investigating

both effects simultaneously.

In order to address the lack of research that integrates time-of-day effects and sleep/wake

effects on memory, our study is specifically designed to understand to what extent these

factors are influential in novel object recognition (NOR) and novel location recognition (NLR).

These one-trial object recognition/location tasks were originally developed for rats (Ennaceur

and Delacour, 1988)and constitute a behavioural paradigm for the investigation of sleep and

TOD effects on memory consolidation and retrieval. The appeal of these tasks is that the

former (NOR) is hippocampus-independent and the latter (NLR) is hippocampus-dependent.

Moreover, they are one-trial tasks which can be repeated on the same animals, and they

reflect learning. They are also completely stress-free. Animals are taught to recognize

objects based on their shape and size or location, and tested for learning in three memory

tests aimed to represent short-term, intermediate-term and long-term memory. The

distinctive feature of this study is that these tasks are conducted on one group of rats on a

normal light/dark cycle, as well as on a group of rats on a reversed dark/light cycle. Stress

factors will thus be ruled out by the lack of sleep deprivation. The first aim of the present

study is to confirm the influence of sleep in memory consolidation. Secondly, it is to

investigate the effects of time-of-day on memory performance, by conducting testing

sessions (which include training and three tests) at two different times. The main aim is then

to consolidate these two streams of research in order to determine how these effects interact

in their impact on memory. A final purpose for the study will be to analyse the effects of

increased sleep and increased wake, by the use of constant light and constant dark

conditions, respectively. This will help to understand to what extent sleep and wake are

influential in the amelioration of memory performance. This is to our knowledge the first

experiment to specifically target the distinction of these two effects on memory. Although it

will hopefully shed light on memory consolidation by these two processes, it will only partiallyresolve the issue because the phenomena of circadian rhythms and sleep/wake cycles are

so closely interlinked. Studies with similar aims are necessary at the cellular level in order to

better define the molecular mechanisms relating to these phenomena.


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2. Materials and Methods

2.1 Subj ects

Twenty-eight eight-week-old male, Wistar rats were purchased from Harlan, UK. Upon arrival

the rats weighed on average 280g. The rats were divided into 2 groups of 14 rats. The first

group was named the LD group and was housed under a daily light/dark cycle, consisting

of 12 h of light conditions (lights on at 9:00 am, defined as ZT0) and 12 h of dark conditions

(lights off at 9:00 pm, defined as ZT12). The second group was named the DL group and

was housed under a daily dark/light cycle, consisting of 12 h of dark conditions (lights off at

9:00 am, defined as ZT12) and 12 h of light conditions (lights on at 9:00 pm, defined as ZT0).

Prior to the start of behavioural testing, the rats were habituated to the empty open field for 6

consecutive days, by exploring it for 5min each day between 1pm and 3pm. They had free

access to food and water. All experimental procedures were approved by the Bioresources

Unit committee.

2.2 Ap paratus

Novel-object recognition and novel-location recognition testing took place in a circular,

painted wood, black open field (diameter: 120cm, height: 51cm), under red light, in a dark

room. The open field had a blue triangle on one side, and a green cross on the other, as

spatial markings. Only the triangle was apparent for novel-object recognition, whereas both

markings were visible for novel-location recognition. The objects to be explored were

constructed from Lego bricks. The objects had different sizes, shapes and colours with a

bottom diameter of 8-16cm and a height of 11-21cm. Blu-Tack was attached to the bottom

of the objects to ensure the rats would not push them over during testing. Objects and open

field were cleaned with water between each rat, and cleaned with Dettol between each set

of experiments and each group of rats.

2.3 Experimental pro cedure

Each session consisted of a training trial of 5min, a 5min test for short-term memory 2 hours

after training, a 5min test for intermediate-term memory 5 hours after training, and a 5min

test for long-term memory 24 hours after training. All training and testing was conducted in a

dark room, under red light. Depending on which memory test, the Lego objects were placed

at different locations in the open-field. Exploration of the objects was monitored with two

stopwatches, one for each objects. The rats were placed on the perimeter of the open-field,

face to the wall, and cumulated times for exploration behaviour were timed. Exploration

behaviour was considered as sniffing the objects or touching with forelegs. Close proximity to


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an object or contact while passing were not counted. Preference for the novel object or

displaced object in the testing phase indicated memory for the object in the training phase.

Two different memory tests were conducted on the rats, at two different times of the day.

Therefore, special care was taken so that each rat had a minimum of 7 days between eachof the tests. In this way it was considered that interference of memory for the different tests

would be negligible.

2.3.1 Reversal of LD cycle for the DL group

14 rats that were originally on a LD cycle were progressively switched to a DL cycle over the

course of 4 days, a week prior to the beginning of experiments. That is, the rats were put on

a 12:12 LD cycle delayed by 6h with lights on 3pm-3am for 4 days until their cycle was fully

reversed, such that the group was eventually on a 12:12 dark/light cycle.

2.3.2 Novel-object recognition task

In this task, two different Lego objects, object A and object B, were placed equidistantly

from the open-field walls, on the same line, during the training phase. In the first test, 2 hours

after training, object B was replaced with a different object, object C. Similarly, in the two

following tests, object C was replaced with object D and object E, respectively. Objects B, C,

D and E were placed at exactly the same location (see Fig. 1). Object A remained at the

same location, but was washed between each session and each rat. The NOR task was

conducted in four sessions: the first session was conducted at 9am (training phase) for the

LD group. The second session was conducted at 9am for the DL group. The third session

was conducted at 1pm for the LD group. The fourth session was conducted at 1pm for the

DL group.

2.3.3 Novel-location recognition task

In this task, two identical Lego objects, object A and object B, were placed equidistantly from

the open-field walls, on the same line, during the training phase. In the first test, 2 hours after


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training, object B was moved to a different location in the open-field (see Fig. 2). Similarly,

object B was moved to a new location for the second test, and another location for the third

test. The two objects were washed between each session and each rat. The NLR task was

conducted in four sessions: the first session was conducted at 9am (training phase) for the

LD group. The second session was conducted at 9am for the DL group. The third session

was conducted at 1pm for the LD group. The fourth session was conducted at 1pm for the

DL group.

2.3.4 Increased sleep and increased wake

To examine the effect of increased sleep on performance in the NOR and NLR tasks, 10 rats

from the LD group were put under 24 hour dark conditions, starting at 9am when lights

should have been turned on. The NOR or the NLR task was immediately carried out, and the

rats remained in the dark until the 24 hour test had been carried out. Similarly, to examine

the effect of increased wake on performance in the NOR and NLR tasks, 10 rats from the DL

group were put under 24 hour light conditions, starting at 9am when lights should have been

turned off. The NOR or the NLR task was immediately carried out, and the rats remained in

the light until the 24 hour test had been carried out. The procedures for the NOR and NLR

tasks were identical to the previous descriptions.

2.4 Data An alysis and Statist ics

The times spent exploring each object were converted into a discrimination ratio (D.R.),

which was the percentage of total exploration time spent at the novel/displaced object. That

is: D.R. = [(time at novel or displaced object)/(time at object A + time at novel or displaced

object)] * 100. A value of D.R. significantly higher than random exploration (50%) indicated

that the rats had discriminated between the two objects or two locations in the test phase.

Difference between the D.R. and random exploration was tested by Students one-sample t

test. To determine the joint effects of time-of-day and sleep on memory performance, a two-

way analysis of variance (ANOVA) was conducted with sleep/wake group as between-

subjects variable and time-of-day as within-subjects variable (with 2 levels, 9am and 1pm).


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Significance was established for P-values < 0.05. D.R.s are shown as mean standard error

mean (S.E.M.). Statistical analyses were performed using GraphPad Prism 5 for Windows.

3. Results

3.1 Training Tim es

NOR was performed on both

groups of rats, the LD and the DL

group, at two times of day: 9am

and 1pm. Similarly, NLR was

performed on the LD group and

the DL group, at these two times.

The training phases were graphedwith percentages of total

exploration for each object (Fig.3).

As expected, during the training

phases, the object exploration

against random exploration (50%)

was not significant.

3.2 Influenc e of sleep/wake onmemory performance

Figure 4 shows the influence of

sleep on memory performance in

both the NOR and the NLR tasks.

This is done by comparing the LD

group, in which the rats sleep

between experiments, with the DL

group, in which the rats are awake

between experiments.

3.2.1 NOR task

For the LD group, D.R. was significantly higher than chance level for all three memory tests

(2h test, ZT2: p=0.0132; 5h test, ZT5: p=0.0013; 24h test, ZT0: p


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were able to encode novel object information under both sleep and wake conditions, except

the awake rats (DL group) which were unable to form a short-term memory (2h test). At the

2h test, the LD group had a significantly higher memory performance than the DL group,

which did not exceed chance level. At the 5h test, the DL group had a significantly higher

memory performance than the LD group. At the 24h test, the LD group had a significantly

higher memory performance than the DL group. Taken together, these data suggest that the

sleep group (LD) has a higher memory performance than the wake group (DL) at the short-

term (2h) and long-term (24h) memory tests in the NOR task. The wake group (DL) displays

a better memory performance than the sleeping rats for the intermediate-term memory test

(5h).

3.2.2 NLR task

For the LD group, the D.R. was significantly higher than chance level for the short- and

intermediate-term memory tests (2h test, ZT2: p=0.0233; 5h test, ZT5: p


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3.3.1 NOR task

For the early tasks, D.R. was significantly higher than chance level for the 5h test and the

24h test (5h test, ZT17: p=0.001; 24h test, ZT12: p=0.0028) but there was no difference

between D.R. and chance level for the 2h test (2h test, ZT14: p=0.9824). For the late tasks,

D.R. was significantly higher than chance level for the 2h test and the 24h test (2h test,

ZT18: p=0.0219; 24h test, ZT16: p


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3.3.2 NLR task

For the early tasks, D.R. was significantly higher than chance level for the three memory

tests (2h test, ZT14: p=0.0122; 5h test, ZT17: p=0.0493; 24h test, ZT12: p


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reached significance (TOD: F(1,26) = 0.2462, p=0.6239; sleep/wake group: F(1,26) =

0.04537, p=0.833). It can be concluded that the effect of sleep/wake is dependent on the

TOD of testing. However, post-hoc testing did not reveal at which level sleep/wake was most

influential.

3.4.2 NLR task

Results of the 2-way ANOVA reveal that for the short-term memory test, neither the

sleep/wake factor, the TOD factor or the interaction reached significance (Sleep/Wake group:

F(1,26) =0.2317,p=0.6343; TOD: F(1,26) = 0.235, p=0.631; Sleep/wake group x TOD:

F(1,26) = 0.8483, p=0.3655). Therefore, it cannot be concluded from the P-values which of

the factors was most influential on memory performance in this test.


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In the intermediate-term memory test, there is a significant interaction between TOD and

sleep/wake group (F(1,26) = 5.479, p=0.0272). There is also a significant effect of TOD

(F(1,26) = 4.309, p=0.0479). These results indicate that there is a TOD effect, which is

dependent on which sleep/wake group is considered. A post-hoc Bonferonni test revealed

that memory performance was higher in the sleep group than in the wake group (p


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sleep are beneficial on memory, in the short-term memory test, but that the memory trace

obtained during LL training does not persist for intermediate- and long-term memory tests.

It can be hypothesized that the effects of 24h light are a source of stress, potentially

explaining why the increased wake and sleep groups were worse than their controls atforming memories past the 2h test.

3.5.2 Effect of increased wake on NOR

For the DL group, D.R. was significantly higher than chance level for the 5h test and the 24h

test (5h test, ZT14: p=0.0008; 24h test, ZT12: p=0.0042) but there was no difference

between D.R. and chance level for the 2h test (2h test, ZT14: p=0.0708). For the DD group,

D.R. was significantly higher than chance level for the 2h test and the 24h test (2h test, ZT2:

p=0.0018; 24h test, ZT0: p=0.0181) but there was no difference between D.R. and chance

level for the 5h test (5h test, ZT5: p=0.2572). At the 2h test, the increased wake group

performed significantly better than the wake group at the memory test. At the 5h test, the

wake group had a significantly better memory performance than the increased wake group,

which did not differ from chance level. At the 24h test, both the increased wake and the wake

groups reached significance compared to chance level, but it cannot be concluded which

group had a better memory retention at this test from the P-values. These results suggest

that the immediate effects of increased wake are beneficial to memory, in the short-term

memory test. These beneficial effects do not extend to the intermediate-term memory test, in

which the normal wake group has a better performance.

3.5.3 Effect of increased sleep on NLR

For the LD group, the D.R. was significantly higher than chance level for the short- and

intermediate-term memory tests (2h test, ZT2: p= 0.0395; 5h test, ZT5: p


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3.5.4 Effect of increased wake on NLR

For the DL group, the D.R. was significantly higher than chance level only for the 24h test

(24h test, ZT0: p


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4. Discussion

The fields of circadian rhythms and of sleep are both extremely well studied as a result of

experimental designs ranging from behavioural and psychological paradigms to

electrophysiological and biochemical studies. The separation of the two fields, however, isincreasingly unjustified, as several studies are showing how sleep follows circadian

modulation, and that many neural systems of these two processes work in tandem, with

common signalling pathways. As it has become apparent that memory is affected in different

ways both by sleeping patterns and circadian rhythms, a few studies have tried to address

the issue of dissociating these simultaneous effects. However, these studies are scant, and

are limited by their use of sleep deprivation, or in some cases by the failure to address

certain issues that arise from procedures designed to control for one effect but not the other.

For example, Chaudhury and Colwell studied fear-conditioned mice and found there was alarger extinction of memories during the night than in the day; this pattern was maintained

after reversal of the LD cycle and under constant dark (2002). This shows a definite

existence of an endogenous circadian rhythmbut it would have been very informative to

consider the effects of sleep in conjunction with these observations. In another study aiming

to challenge the accepted view that sleep is beneficial to memory (Gottselig et al., 2004), a

group of subjects that slept and one that engaged in restful wake were found to have

identical performances in the memory tests. This study does not control for effects of time-of-

day and it could be that the times of testing corresponded to an existing peak in memory

performance which is not affected by sleep. Binder et al had the aim of disentangling sleep

effects from circadian effects in an experiment using sleep deprivation, and found that

memories for object location were only formed in the morning (Binder et al., 2012). These

results are interesting but are difficult to extrapolate because they disregard effects of acute

fatigue and stress resulting from sleep deprivation.

As a result, the present study has several aims. The first aim is to confirm the established

beneficial influence of sleep on memory, in a hippocampus-dependent and a hippocampus-independent task. The aim is then to investigate the effects of time-of-day on memory

performance, in order to then consolidate these results and try to understand how circadian

rhythms and sleep effects interact. Additionally, the effects of constant dark and constant

light were investigated, to explore how they affect sleep and memory performance in the two

chosen memory tasks.

The chosen experimental protocol made use of a normal LD cycle and a reversed DL cycle

in order to eliminate the limitations resulting from sleep deprivation. The use of the NOR and

NLR tasks allowed for emotional- and stress-free learning processes that rely on


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Results can be interpreted accordingly if these confounding factors are taken into account.

Future studies should perhaps make use of the present study design, conducting one

training session for each memory test, instead of increasing animals habituation to one

object when testing is conducted three times for short-, intermediate- and long-term tests.

This would also help to avoid the limitations resulting from long-term memory tested after 12h

spent in the opposite phase. The experiments would also largely benefit from the monitoring

of sleep.

The consideration of the influence of sleep is interesting in view of the current literature.

Novel object recognition is considered to be a hippocampus-independent task (Bussey et al.,

2000), whereas novel location recognition relies on the hippocampus (Inostroza et al., 2013).

The short-term memory test revealed that sleep was beneficial in both NOR and NLR tasks,

although the effect of wake was just as beneficial in the NLR task. In the intermediate-term

test, the hippocampus-dependent NLR task was more prone to the effect of sleep, which is

consistent with theories that sleep is critical only for the learning of associative components

(Binder et al., 2012,Inostroza et al., 2013, Lau et al., 2010). Intriguingly, memory

performance was better in the awake group during the NOR task. This is in line with the fact

that evidence is scant for a beneficial effect of sleep on novel object recognition. The long-

term memory tests yielded surprising results. That is, the LD group had better memory

performance in the NOR task, and the DL group had better performance in the NLR task.

These results are contradictory with the previous memory tests, and it is possible to consider

that these results can be inverted, because of the 12 hour phase of sleep (for the wake

group) and wake (for the sleep group) that the rats underwent just before testing. These

factors were not planned for in the design of the study, and future studies should control for

this element. It is known that in rats, sleep occurs within the first two hours of the light period.

Similarly, rats awake nearly immediately with the onset of the dark phase (Trachsel et al.,

1986). It is therefore justifiable to assume that following a 12h light phase, the rats have

slept, and the opposite is also true. In this case, the LD group would in fact be a wake

group, and the DL group would be a sleep group. If this is an accurate presumption, then

sleep is overall beneficial in the NLR task, and not in the NOR task. These results provide

further support for such a role for sleep selectively supporting the consolidation of

hippocampus-dependent memory like the spatial memory tested in the NLR task.

A time-of-day effect was undoubtedly observed in both the NOR and the NLR tasks.

Interestingly, for both the short- and intermediate-term memory tests, similar effects were

observed. All tests were conducted on the DL group, in which rats were awake between

tests, so as to observe only effects of time-of-day. In the short-term memory test, the rats

performed better when they were trained at ZT16 than at ZT12, in both NOR and NLR tasks.


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Subjective time is the name given to the time which is experienced by rats on a reversed

cycle, if they had been on a normal LD cycle. Therefore, the rats had better memory

retention when trained at subjective time 1am than 9pm. Conversely, in the intermediate-

term memory test the opposite is true, namely that rats trained at ZT12 (subjective time 9pm)

performed significantly better than those trained at ZT16 (subjective time 1am), in both tasks.

It is interesting that the two memory tests have the same pattern for memory performance in

both tasks, which presumably model different types of memory consolidation. An important

aspect of these results is that in the short-term memory test, memory performance was

highest when training occurred at ZT16, meaning that the test occurred at ZT18. In the

intermediate-term memory test, memory performance was highest when training occurred at

ZT12, meaning that the test occurred at ZT17. This is true both NOR and NLR tasks. It

seems reasonable to suggest that there is a peak in memory performance between ZT17

and ZT18, as suggested by the results. These results are also in agreement with previous

studies that show that circadian effects are indeed dependent on time of training and not on

time of testing (Fernandez et al., 2003). It would be interesting to conduct a similar study as

ours with more than two training and testing times, and verify that TOD effects were indeed

dependent on training rather than testing, and that such a memory peak exists between

ZT17 and ZT18. This would help the understanding of how circadian cycling affects cognitive

performance, and perhaps shed light on what molecular and systematic investigations could

be conducted to fully understand this effect.

The long-term memory test results are more difficult to extrapolate, because these tests

intend to investigate TOD effects only, and rats, being tested at the L/D transition (for the

early test) or 4h after it (for the late test), have undergone a 12h light period, during which the

nocturnal animals supposedly slept. In the NOR task, the later trained rats yielded better

performances than the earlier trained; the opposite is true in the NLR task. Garren et al.

suggested that there is a peak in memory performance when rats are tested at the ZT at

which they were trained (2013). This is the case in both tasks, in that the rats best

performance compared to training occurred at their 24h test. However, as previously

mentioned, it is possible that interest for object A is increasingly reduced with each test,

proportionally increasing exploration time for the novel or displaced object. A more detailed

analysis of this hypothesis is required to gain further insight into the mechanism behind time-

of-day fluctuation of memory retention. It would be very informative to train animals at

enough time points during the light and dark phases to subsequently verify that there is a

strong relationship between training and testing times, with peak performance at these times.

One study proposes a joint role of sleep and time-of-day by proposing that an interval of 3

hours after learning renders sleep more beneficial than immediately after learning (Gais et


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al., 2006). Inostroza et al confirm that an interval of wake before sleep renders its beneficial

effects better (2013). The intermediate-term memory tests conducted in the later tasks are

trained for at 1pm and occur at 6pm. Three hours later, the DL group rats enter a light phase

during which they spend the best of 12 hours asleep, and they are subsequently tested in the

24h test. As the intermediate-term test constitutes a training phase for the next memory test,

it is very interesting to note that our results are in agreement with this supposition, because

memory performance in the NOR task was considerably better for those later trained rats,

which slept 3 hours after learning, than the earlier ones.

Our study, designed to investigate the simultaneous effects of sleep and TOD, was, to our

knowledge, the first of its kind. The two-way analysis of variance was inconclusive for the

short-term memory tests in the NOR and the NLR task. It is therefore impossible to say

which of sleep or TOD was the most influential factor in this test. In the intermediate-term test

there was an interaction of sleep and TOD factors for both the NOR and the NLR tasks. In

the NOR task, there was also a main effect of sleep, with post-hoc test revealing that sleep

was more beneficial in the late testing. In the NLR task, there was a main effect of TOD, with

post-hoc testing revealing that this effect was most important for the sleep group. Taken

together these data reveal that recognition memory and spatial memory are both prone to the

joint effects of sleep and TOD on intermediate-term memory. Similarly, in the long-term

memory test, both NOR and NLR tests showed a significant interaction between sleep and

TOD effects on memory performance. In the case of the NOR task, post-hoc testing did not

reveal where this interaction was most significant, but in the NLR task it showed that the

effect of the interaction was most significant on the later testing. That is, the effect of sleep is

stronger in the later tasks. A study on human adults put forward the idea that the circadian

system has a modulatory effect on cognitive impairment resulting from sleep restriction

(Mollicone et al., 2010). This suggests that the systems regulating circadian rhythms and

sleep have interacting effects on cognition, which is confirmed by our results. These findings

confirm the need for further studies which would use simultaneous sleeping pattern and

circadian rhythm disruptions to inspect possible consequences on cognitive capacities and

learning.

It is also interesting to note that in the NOR intermediate-term memory task, and the NLR

long-term memory task, sleep was more beneficial to later testing. This suggests that sleep

in the second part of the night, as opposed to early sleep at the beginning of the light period,

is more efficient at consolidating recognition memories. The sequential hypothesis,

interpreted in light of the active system consolidation and synaptic homeostasis hypotheses,

states that the first part of the night, during SWS, integrates new memory traces into longer

lasting memories with a general synaptic downscaling. The second part of the night is


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dedicated to the desynchronisation of neural networks, when new memories are stabilised.

This process allows for memories that have been temporarily encoded in hippocampal

networks to be redistributed into long-term storage sites (Marshall and Born, 2007). This

suggests that it is in the second part of the night that memories are fully formed. Moreover,

many studies have shown the importance of REM sleep in the consolidation of memory in

rats (Smith, 1996), and this sleep stage is known to occur especially in the later stages of the

night. In order to confirm that REM was in fact more abundant during the later sleep testing,

an EEG study would need to be conducted in conjunction with the present study design.

These theories provide insight into why our results showed higher performance after sleep

occurring in the second part of the night. This finding is important because it ties together

molecular theories of the influence of sleep, with behavioural observations.

An enquiry into why this effect was observed in the intermediate-term NLR task and the long-

term NOR task would shed light on how the sequential hypothesis may only apply to certain

memory systems in the central nervous system (CNS). Perhaps the time-scale of memory

consolidation for spatial memory tasks is longer, which would explain why this effect was not

observed in the intermediate-term memory task.

The last aim of the study was to observe the effects of increased sleep and increased wake

on memory performance. The time scale of the study did not permit the intended second goal

of investigating the effects of circadian rhythm disruption by measuring memory performance

at later stages (training at 1pm). The extrapolation of these is limited, however, as they show

an immediate effect of constant light or constant dark exposure, because training for the

tasks occurred at the very beginning of 24-hour exposure. Many studies that have

investigated constant light or dark exposure use a longer time-scale.

The assumption that 24h light leads to increased sleep, and that inversely 24h dark leads to

increased wake, is due to Yasuda et als study(2005). However, this stress effect kicks in

with time, but the minimum time for this factor is unknown. In the case of constant light, the

immediate effects on both NOR and NLR tasks were in fact beneficial. However, the rats

performed significantly worse than their controls in the NOR task in the subsequent memory

tests. Several studies have shown that exposure of animals to constant light greatly

increases stress, both behaviourally and physiologically (Giannetto et al., 2014,Milosevic et

al., 2005). Stress hormones are known to interfere with memory acquisition and recall

(Kuhlmann et al., 2005)and to follow a circadian rhythm which directly impacts LTP

(Pavlides et al., 2002). More specifically, Eckel-Mahan et al. showed that exposure to

constant light led to a decrease in long-term memory formation (2008). Ma et al also showed

that performance by rats in the Morris Water Maze was greatly impaired by constant light


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exposure (2007). Therefore, the results of the NOR task are in line with previous findings,

and explain why performance in the intermediate- and long-term memory tests was poor

compared to the normal sleep group. The results in the NLR task are very intriguing,

however, because the rats exposed to constant light performed just as well as the sleep

group, and even better in the long-term memory test. This discrepancy with previous

research may be due to different effects of increased sleep on different types of memory

tasks. Perhaps the effect of stress hormones such as cortisol and corticosterone impact quite

differently on hippocampus-dependent memories like those encoded during the NLR task. A

more detailed analysis of exposure to constant light is required to gain further insight into the

mechanism behind the surprising increase in memory performance in this task, as well as the

temporary beneficial effect of increased light on short-term memory in the NOR task.

The exposure to constant dark had comparable results in the NOR and the NLR task. In the

NOR task memory was improved by exposure to constant dark in the short-term memory

test, then performance was similar to the wake group in the long-term memory test. For the

NLR task, the short- and intermediate-term tests showed a better performance for the

constant dark group, but in the long-term this memory was not retained. To some extent, the

effects of constant dark or not dissimilar to those of normal wake performance patterns. It is

interesting to note that the stress apparently associated to constant light exposure does not

seem to impact negatively on memory in the case of constant dark. The literature is

considerably lacking on the effects of constant dark exposure, and this is therefore the first

study to investigate the effects of such conditions on memory performance. It would appear

that the effects of increased dark are negligible, but it would nonetheless be revealing to

measure cortisol and corticosterone levels during constant dark exposure, to understand if

this condition is also a source of stress. These results seem to refute the theory of how

melatonin affects memory. That is, melatonin is known to have a specific pattern of

expression, with consequences on memory formation (Gerstner and Yin, 2010). Its

concentration increases in dark phases, and this is correlated with low memory performance;

whereas it decreases in light phases, leading to high memory performance. It is essential to

conduct further studies to understand the mechanisms of this memory increase due to

constant dark, as this is a poorly understood area.

The results of these studies confirm the well-established notion that sleep is beneficial to

memory, and in particular to the binding of hippocampus-dependent memories with

associative components. It also confirms the hypothesis that circadian effects on memory are

dependent on time of training, rather than on time of testing, and that there is a peak in

memory performance when memory testing is conducted at the Zeitgeber time of training.

The results also suggested the existence of a peak in memory performance between ZT17


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and ZT18, for NOR and NLR tasks. Memory performance, in this way, follows a circadian

modulation as well as an influence of sleep. It is suggested in view of the results that memory

performance is affected by sleep in such a way that is consistent with the sequential

hypothesis of sleep, in the cases of intermediate-term memory in the NOR task, and long-

term memory in the NLR task. Finally, exposure to constant light had primarily adverse

effects on the encoding of NOR memories, but had little effects on NLR memories, even

yielding better performance in the long-term. Exposure to constant dark was overall

beneficial to memory in both NOR and NLR tasks, bringing into question how the absence of

light affects memory centres, and possibly refuting the theory of how melatonin influences

memory consolidation.


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