THE EFFECTS OF SLEEP DEPRIVATION ON SPATIAL MEMORY, DREAM AND
HYPERPHOSPHORYLATED TAU PROTEINS EXPRESSION IN RATS
NORHIDA BINTI RAMLI
UNIVERSITI SAINS MALAYSIA
2021
THE EFFECTS OF SLEEP DEPRIVATION ON SPATIAL MEMORY, DREAM AND
HYPERPHOSPHORYLATED TAU PROTEINS EXPRESSION IN RATS
by
NORHIDA BINTI RAMLI
Thesis submitted in fulfilment of the requirements for the Degree of
Doctor of Philosophy
MARCH 2021
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ACKNOWLEDGEMENTS
In the name of ALLAH the Most Merciful and the Most Compassionate.
First, I would like to thank ALLAH for everything, without which, I won’t be able to breathe on my own, let alone completing this research and writing.
For the most honourable supervisors, Dr Idris Long and Prof Dr Zalina Ismail, I would like to express the deepest gratitude for their patience, guidance, encouragement and lifelong guidance in this never ending endeavour, learning and researching. I pray to ALLAH the greatest blessing for them in this world and hereafter.
The largest appreciation to my late father, Mr Ramli Zain, my supportive mother, Mrs Aisah Kudsi, and my dear husband, Dr Mohd Anuar Ramdhan bin Ibrahim, my children, Wafaa, Fawwaz, Ziyaad, Adeena and the whole family for their continuous support and care.
Not to forget my thankfulness to my colleagues in Brainetwork Center PPSK, U.S.M., whom I believed are now successful person of their own organisation, for their help and guidance during my research works. Thank you too to all staffs in PPSK USM and Animal House U.S.M. for all their helps.
Finally, the biggest thanks to Universiti Sains Malaysia for the change given to me as a student in this degree. This research project was financially supported by USM Research grant. [1001.PSKBP.8630021].
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TABLE OF CONTENTS
ACKNOWLEDGEMENTS ii
TABLE OF CONTENTS iii
LIST OF TABLES viii
LIST OF FIGURES ix
LIST OF APPENDICES xii
LIST OF ABBREVIATIONS xiii
ABSTRAK xvii
ABSTRACT xx
INTRODUCTION 1
Rationale of The Study 2
General Objective 2
Specific Objectives 2
LITERATURE REVIEW 3
Sleep 3
2.1.1 Stages of Sleep, REM Sleep Versus NREM Sleep 3
2.1.2 Physiological Benefits of Sleep 9
2.1.3 Sleep-Wake – Regulating System 10
2.1.4 Neuromodulator for Wake-Sleep Cycle 11
2.1.5 Sleep Initiation Process 12
Circadian rhythm 13
2.2.1 The molecular event under the circadian rhythm regulation 13
2.2.2 Zeitgeber time 14
Memory 14
2.3.1 Memory classification 15
2.3.2 Memory Stages 17
2.3.3 Cellular and Molecular Level of Memory Formation 17
Sleep and Memory 21
2.4.1 Roles of Sleep on Memory Consolidation 21
2.4.2 Molecular Modulator for LTP in Memory Consolidation 22
Circadian Rhythm and Memory 24
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Hippocampus and Memory 24
2.6.1 Hippocampal formation 26
2.6.2 Hippocampal Neuronal Circuit 28
2.6.3 Hippocampal Neural Complex and Memory 30
DREAM Protein 32
2.7.1 Role of DREAM Protein 33
2.7.2 DREAM Protein in Learning and Memory 36
Tau Protein 36
2.8.1 Tau Structure 39
2.8.2 Neurodegenerative Disease and Tau Protein Properties. 41
2.8.3 Paired Helical Filament (PHF) 41
2.8.4 Roles of Tau Protein in The Pathogenesis of Neurodegenerative
Diseases 44
2.8.5 Tau Phosphorylation and It's Regulation. 44
2.8.6 Clinical Manifestation of Hyperphosphorylation of Tau Protein. 46
Neurobehavioral Analysis Involving Animal 47
2.9.1 Morris Water Maze Analysis of Spatial Memory 47
MATERIALS AND METHODS 50
Materials 50
Preparation of Solutions 50
3.2.1 Ammonium Persulphate (APS) (20%) 50
3.2.2 Diaminobenzidine (DAB) 50
3.2.3 Loading Buffer 51
3.2.4 Ponceau S 51
3.2.5 Phosphate-buffered Saline (PBS) pH 7.4 51
3.2.6 Phosphate Buffer (PB) Stock (0.2 M) 51
3.2.7 Paraformaldehyde Stock (8%) 52
3.2.8 Paraformaldehyde (PFA) (4%) in 0.1 M PB pH 7.4 52
3.2.9 Resolving Buffer 52
3.2.10 Running Buffer 52
3.2.11 Stacking Buffer 52
3.2.12 Sample Buffer 53
3.2.13 Sucrose (20%) in 0.1 M PB pH 7.4 53
3.2.14 Trizma Base Stock Buffer 53
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3.2.15 Tris-buffered Saline (TBS) pH 7.4 53
3.2.16 Tris-triton (TBS/TX) pH 7.4 53
3.2.17 Transfer Buffer 53
3.2.18 Tris Buffer Saline (TBST) 54
3.2.19 Tris Buffer Saline-Tween 20 (TBS-T20) (0.05%) 54
Animals Preparation 54
3.3.1 Sample Size for Animal Study. 54
3.3.2 Experimental Groups 55
3.3.3 Circadian disruption experiment 58
3.3.4 REM Sleep Experiment 61
Morris Water Maze 65
3.4.1 Equipment for Morris Water Maze 65
3.4.2 Morris Water Maze Procedure 65
Immunohistochemistry for DREAM, Tau, and Hyperhosphorylated Tau
Proteins. 69
3.5.1 Brain sampling for Immunohistochemistry 69
3.5.2 Brain Dissection and Cryostat Sectioning 69
3.5.3 Preparation of DREAM, Tau, and Hyperphosphorylated Tau
Immunopositive Neurons 70
3.5.4 Positive DREAM, Tau, and Hyperphosphorylated Tau Protein
Counting. 71
Western Blot Analysis 74
3.6.1 Brain Sampling for Western Blot Analysis 74
3.6.2 Protein Extraction for Western Blot Analysis 74
3.6.3 Measurement of Protein Concentration 75
3.6.4 Preparation of Sodium Dodecyl Sulphate Polyacrylamide gel (SDS-
PAGE). 76
3.6.5 Electrophoresis of SDS-PAGE gel 79
3.6.6 Electrophoretic Transfer and Immunoblotting 79 3.6.7 Measurement of Mean Relative Intensity (fold changes) 82
Statistical Analysis 83
RESULTS 85
Effects of REM Sleep Deprivation on Food Consumption and Body Weight
Gain. 85
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4.1.1 Food Consumption 85
4.1.2 Body Weight Gain 87
Serum Melatonin Level for Circadian Disruption Experiment. 89 Neurobehavioral Analysis Using Morris Water Maze (MWM) Test. 91
4.3.1 Spatial Learning and Memory. 91
4.3.2 Probe test. 94
4.3.3 Reverse Spatial. 96
Immunohistochemical Analysis 98
4.4.1 Results of DREAM Positive Neuronal Cells (DPN) 98 4.4.2 Hyperphosphorylated Tau Protein Expression. 102
Western Blot Analysis 109
4.5.1 Mean Relative DREAM Protein Level in Hippocampal Nucleus. 109 4.5.2 Mean Relative Phosphorylated Tau Protein Level in Hippocampal
Nucleus. 112
4.5.3 Mean Relative of Total Tau Protein in Hippocampal Nucleus. 115 4.5.4 Mean Relative PPN / Total Tau Proteins Ratio. 118 4.5.5 Mean Relative DREAM Protein Level in Hippocampal Cytoplasm.
120 4.5.6 Mean Relative Phosphorylated Tau Protein Level in Hippocampal
Cytoplasm 123
4.5.7 Mean Relative of Total Tau Protein in Hippocampal Cytoplasm. 126 4.5.8 Mean Relative PPN Tau / Total Tau Proteins Ratio. 129 Summary on Semi-Quantitative Analysis of Dream Protein Level in
Hippocampal Neurons and Cytoplasm. 131
Summary on Semi-Quantitative Analysis of Tau Protein Level, PPN Protein Level, and PPN/Tau Ratio in Hippocampal Neurons and Cytoplasm. 132
DISCUSSION 134
Assessment of REMsd 134
Circadian Disruption Measurement. 136
Effects of REMsd and CD on MWM test. 137
5.3.1 Effects of REMsd and CD on spatial learning MWM paradigm. 137 5.3.2 Effects of REMsd and CD in probe test MWM paradigm. 139 5.3.3 Effects of REMsd and CD on reversal spatial learning MWM
paradigm. 141
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Effects of Remsd and CD on DREAM Protein Expression and Tau Protein
Hyperphosphorylation. 144
5.4.1 Effect of REMsd and CD on DREAM protein expression. 144 5.4.2 Effects of REMsd and CD on Tau protein expression (TPN) and
hyperphosphorylated Tau positive neuron (PPN). 147
Summarization of Findings from This Study 150
CONCLUSION, LIMITATION AND RECOMMENDATION 152
Conclusion 152
Limitation 152
Recommendation 153
REFERENCES 154
APPENDICES
LIST OF PRESENTATION AND PUBLICATION
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LIST OF TABLES
Page Table 2.1 Brainwaves in different states of mind and stages of sleep. 8
Table 3.1 A cycle of phase-shifting stage table. 60
Table 4.1 The average of food intake during dry tank (FID) and weight gain after dry tank(WGD) and food intake during wet tank (FIW) and weight gain after wet tank (WGW) between PC and
REMsd groups. 88
Table 4.2 Overall EL time within all groups during MWM test for 5
days’ acquisition for both experimental groups. 93 Table 4.3 Mean relative value of DREAM protein measured in nucleus
and cytoplasm in REMsd and CD experiments. 131 Table 4.4 Mean relative level of Tau and PHF proteins and the PHF/Tau
ratio. 133
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LIST OF FIGURES
Page
Figure 2.1 Stages of sleep. 5
Figure 2.2 Memory classification. 16
Figure 2.3 Mechanisms of LTP initiation 20
Figure 2.4 The limbic lobe. 25
Figure 2.5 The hippocampal formation. 27
Figure 2.6 The hippocampal circuit of trisynaptics loop. 29 Figure 2.7 Nuclear Magnetic Resonance structure of DREAM protein. 35
Figure 2.8 Tau protein. 38
Figure 2.9 The isoforms of Tau in human neurons. 40
Figure 2.10 Formation of PHF and NFT. 43
Figure 2.11 Water tank and equipment used in MWM test analysis. 49 Figure 3.1 Study flow chart for REM sleep deprivation experiment. 56 Figure 3.2 Study flow chart for circadian disruption experiment. 57
Figure 3.3 Rats in circadian disruption group. 59
Figure 3.4 Inverted flower pot technique for REM sleep induction. 64 Figure 3.5 Morris water maze tank (view from recording camera). 68 Figure 3.6 Steps in immunohistochemistry – Free-floating technique. 73
Figure 3.7 Gel for sample loading. 78
Figure 3.8 Semidry trans-blotting equipment. 81
Figure 3.9 Protein confirmation using Ponceau S solution. 81
Figure 4.1 Food consumption results dry tank. 86
Figure 4.2 Food consumption results wet tank. 86
Figure 4.3 Body weight (g) gain during 72 hours of adaptation period. 88 Figure 4.4 Body weight gain (g) during experimental period. 88
Figure 4.5 Serum melatonin level. 90
Figure 4.6 Daily escape latency (EL) for all groups in REM. 92
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Figure 4.7 Daily escape latency (EL) for all groups in CD. 92
Figure 4.8 Probe test for REM. 95
Figure 4.9 Probe test for CD. 95
Figure 4.10 EL for reverse spatial for REM. 97
Figure 4.11 EL for reverse spatial for CD. 97
Figure 4.12 The hippocampal at 4x magnification. 99
Figure 4.13 DPN expression in hippocampus. 100
Figure 4.14 Mean DPN in REM sleep deprivation experiment. 101
Figure 4.15 Mean DPN in CD experiment. 101
Figure 4.16 PHF Tau staining in CA region of hippocampus. 104 Figure 4.17 Tau protein staining in CA region of hippocampus. 105 Figure 4.18 Mean TPN in REM sleep deprivation experiment. 106
Figure 4.19 Mean TPN in CD experiment. 106
Figure 4.20 Mean PPN in REM sleep deprivation experimen. 107 Figure 4.21 Mean rank score for PPN in CD experiment. 107 Figure 4.22 Mean rank score for PPN/TPN ratio in REM sleep deprivation. 108 Figure 4.23 Mean PPN/TPN ratio in CD experiment groups. 108 Figure 4.24 Mean relative DREAM protein levels of hippocampal nucleus
REM sleep deprivation experiment group. 110
Figure 4.25 Mean relative DREAM protein levels in hippocampal nucleus
of CD experiment group. 111
Figure 4.26 Mean relative PHF protein levels in hippocampal nucleus of
REM sleep deprivation experiment groups. 113
Figure 4.27 Mean relative PHF protein levels in hippocampal nucleus of
CD experiment groups. 114
Figure 4.28 Mean relative total Tau protein levels in hippocampal nucleus
in REM sleep deprivation experiment groups. 116 Figure 4.29 Mean relative Tau protein levels in hippocampal nucleus of
CD experiment group. 117
Figure 4.30 Mean relative PHF/Tau ratio in hippocampal nucleus of REM
sleep deprivation experiment groups. 119
Figure 4.31 Mean relative PHF/Tau ratio in hippocampal nucleus of CD
experiment groups. 119
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Figure 4.32 Mean relative DREAM protein levels in hippocampal
cytoplasm of REM sleep deprivation experiment group 121 Figure 4.33 Mean relative DREAM protein in hippocampal cytoplasm of
CD experiment group. 122
Figure 4.34 Mean relative PHF Tau protein levels in hippocampal
cytoplasm of REM sleep deprivation experiment group 124 Figure 4.35 Mean relative PHF protein in hippocampal cytoplasm of CD
experiment group. 125
Figure 4.36 Mean relative Tau protein levels in hippocampal cytoplasm of
REM sleep deprivation experiment group 127
Figure 4.37 Mean relative Tau protein in hippocampal cytoplasm of CD
experiment group. 128
Figure 4.38 Mean relative PHF / total Tau protein ratio in hippocampal
cytoplasm of REM sleep deprivation experiment group. 130 Figure 4.39 Mean relative PHF/Tau ratio in hippocampal cytoplasm of CD
experiment group. 130
Figure 5.1 The summary of findings in the current study. 151
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LIST OF APPENDICES
Appendix A : Animal ethics approval
Appendix B : List of materials used in this study Appendix C : ELISA Steps
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LIST OF ABBREVIATIONS
ACD : Acute Circadian Disruption AChE : acetylcholinesterase
Aβ : amyloid β peptide
ANOVA : Analysis of variance APS : Ammonium persulphate
ARASC : Animal research and service centre BCA : bicinchoninic acid
BDNF : brain-derived neurotrophic factor
BF : basal forebrain
BSA : bovine serum albumin BWg : body weight gain
CA : connus ammonis
CA1 : connus ammonis region 1 CA2 : connus ammonis region 2 CA3 : connus ammonis region 3
Ca2+ : calcium
CaM : calmodulin
cAMP : cyclic adenosine monophosphate
Cavβ3 : a beta intracellular subunit of the voltage-gated calcium channel
CC : Cage control
CCD : Control Circadian Disruption CD : Circadian disruption
Cdk5 : cyclin-dependent kinas CHCD : Chronic circadian disruption CNS : central nervous system CRE : cAMP-responsive element
CREB : cyclic AMP response element binding protein
DAB : diaminobenzidine
DG : dentate gyrus
dH2O : deionised water
xiv DNA : deoxyribonucleic acid DPN : DREAM positive neuron DRE : downstream response element
DREAM : downstream regulatory element antagonist modulator DT : distance travelled
EC : entorhinal cortex EEG : electroencephalogram EF1 : first EF hand
EF2 : second EF hand
EF3 : third EF hand
EF4 : fourth EF hand
EL : escape latency
ELISA : enzyme linked immunosorbent assay
EMG : electromyogram
ER : endoplasmic reticulum ETC : electron transport chain
Fc : food consumption
GA : Golgi apparatus
GABA : gamma-aminobutyric acid GlcNAc : β‐N‐acetylglucosamine
Glu : glutamate
GLUT : glucose transport system H2O2 : hydrogen peroxide HCl : hydrochloric acid HRP : horseradish peroxidase IDV : integrated density values
IgG : immunoglobulin G
IHC : immunohistochemistry
K+ : potassium
KChIPs : voltage-gated potassium (Kv) channels-interacting proteins Kv : voltage-gated potassium
LD : Light: Day
xv LTD : long term depression LTP : long term potentiation
MAPK : mitogen- activated protein kinase mPRF : medial pontine reticular formation
mRNA : messenger RNA
MS : medial septum
MWM : Morris water maze
n : number
Na2+ : sodium
Na2HPO4 : disodium hydrogen phosphate Na2HPO4.7H2O : sodium phosphate heptahydrate NaCl : sodium chloride
NaH2PO4.H2O : sodium dihydrogen phosphate dehydrate NaHCO3 : sodium bicarbonate
NaOH : sodium hydroxide
NCS : Neuronal Calcium Sensor NCX2 : sodium-calcium exchange 2 NEPER : nuclear protein extraction NGS : normal goat serum NMDA : N-methyl-D-aspartate NPY : neuropeptide Y
NREM : non-rapid eye movement
NT : neurotrophin
OD : optical density
PAGE : polyacrylamide gel electrophoresis
PB : phosphate buffer
PBS : phosphate buffered saline PC : Pedestal control
PFA : paraformaldehyde
PHF : Paired helical filament PKA : protein kinase A PKC : protein kinase C
xvi POMC : pro-opiomelanocortin PPN : Positive PHF Neuron PS : paradoxical sleep
PSD-95 : post synaptic dendrite -95
PT : probe test
R : rapid eye movement group
REM : Rapid Eye Movement
REMsd : REM sleep deprivation RER : rough endoplasmic reticulum
RN : rapid eye movement and nicotine treated group RNA : ribonucleic acid
rpm : revolutions per minutes S.E.M : standard error mean SDS : sodium dodecyl sulphate
SDS-PAGE : sodium dodecyl sulphate polyacrylamide gel SPSS : statistical package of social sciences software
SWS : slow wave sleep
TBS : tris buffer saline
TBS-T20 : tris buffer saline-Tween 20 TBS-TX : tris buffer saline- Triton X-100 TEM : transmission electron microscopy TEMED : N,N,N’N’-tetramethylenediamine TF : Transcription factor
TPN : Tau Positive Neuron
Zt : Zeitgeber
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KESAN KEKURANGAN TIDUR PADA MEMORI, PENGHASILAN PROTEIN DREAM DAN FOSFORILASI BERLEBIHAN PROTEIN TAU KE
ATAS TIKUS
ABSTRAK
Ritma sirkadian dikawal selia oleh biologi dalaman yang mengawal aktiviti fisiologi yang berbeza termasuk kitaran sel, suhu badan dan metabolisma badan.
Pemantapan memori berlaku semasa fasa tidur yang dinamakan ‘rapid eye movement’
(REM), jadi apabila ritma sirkadian terganggu dan juga fasa REM ini terganggu, memori akan turut terjejas. Protein yang dinamakan Downstream regulatory element antagonist modulator (DREAM) didapati memberi kesan kepada pembelajaran dan memori. Proses fosforilasi Tau protein yang berlebihan akan menyebabkan ketidakstabilan pada fungsi sel neuron dan akan mempengaruhi proses pembelajaran.
Kajian ini bertujuan untuk mengkaji kesan gangguan tidur dan sirkadian (circadian disruption - CD) pada memori. Seratus empat puluh empat ekor tikus Sprague Dawley telah digunakan dalam kajian ini. Dalam eksperimen CD, gangguan sirkadian akut (acute circadian disruption - ACD) telah dilakukan dengan mengawal ritma sirkadian pada tikus ACD tiga jam awal dari hari sebelumnya. Gangguan ini telah dilakukan selama 6 hari diikuti oleh 10 hari rehat (re-entrainment). Proses yang sama telah diulang selama empat kali dalam kajian gangguan sirkadian kronik (chronic circadian disruption - CHCD). Pada kumpulan lain, pengurangan tidur fasa REM telah laksanakan dengan meletakkan mereka pada platfom kecil di dalam tangki berisi air (kumpulan REMsd). Kumpulan kawalan REMsd (PC) telah dimasukkan ke dalam keadaan yang sama seperti kumpulan REMsd kecuali mereka telah diletakkan di atas
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platform yang lebih luas di mana mereka dapat tidur dan berehat dengan baik.
Kumpulan kawalan dalam sangkar (CC) telah dimasukkan ke dalam sangkar biasa. Ia diikuti oleh ujian Morris water maze (MWM) untuk menilai kebolehan memori berkenaan ruang (spatial), platform tersembunyi (probe) dan ruang secara berbalik (reverse spatial). Kemudian, otak tikus bahagian hipokampus telah dikeluarkan dan nukleus serta sitoplasma neuron diekstrak untuk protein DREAM, Tau dan Tau terfosforilasi dianalisa dengan kaedah pewarnaan immunohistochemistry (IHC) dan Western Blot (WB). Ujian MWM mengukur tempoh masa untuk tikus berenang untuk mencari platform (EL) dari setiap kumpulan. Tempoh perbezaan EL selama lima hari didapati tidak berbeza secara signifikan (p> 0.05) di antara semua kumpulan dalam eksperimen REMsd tetapi didapati signifikan lebih lama pada kumpulan ACD dalam eksperimen CD. Ujian probe untuk MWM menunjukkan perbezaan yang tidak signifikan (p> 0.05) di antara kumpulan dalam eksperimen REMsd dan CD. Ujian mengukur memori secara berbalik juga menunjukkan perbezaan yang tidak signifikan di antara semua kumpulan (p> 0.05) dalam eksperimen REMsd dan CD. Ekspresi protein DREAM menunjukkan tiada perbezaan secara statistik direkodkan di antara kumpulan dalam eksperimen REMsd dan CD. Protein Tau dan Tau terfosfolirasi didapati berbeza secara statistik di antara kumpulan REMsd dan CC (p <0.05) dan kumpulan ACD dan CCD (p<0.01) dalam eksperimen REMsd dan CD, tetapi tidak signifikan berbeza terhadap ekspresi protein Tau yang terfosfolirasi berlebihan.
Manakala untuk analisis WB menunjukkan tahap protein DREAM secara relatifnya tidak signifikan berbeza di antara kumpulan dalam eksperimen REMsd dan CD. Purata relatif Tau terfosforilasi berlebihan adalah tidak mempunyai perbezaan statistik yang signifikan di antara kumpulan dalam eksperimen REMsd dan CD. Kesimpulannya, protokol yang digunakan dalam kajian ini tidak berjaya menghasilkan perubahan dari
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segi tingkah-laku otak dan tahap ekspresi protein DREAM dan fosforilasi berlebihan protein Tau.
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THE EFFECTS OF SLEEP DEPRIVATION ON SPATIAL MEMORY, DREAM AND HYPERPHOSPHORYLATED TAU PROTEINS EXPRESSION
IN RATS ABSTRACT
Circadian rhythm is regulated by an endogenous biological regulator controlling various physiological activities, including cell cycle, body temperature, and metabolism. Some consolidation of memory occurs during Rapid Eye Movement (REM) sleep, so when the circadian system breaks down and REM sleep depressed, the memory will be affected. Downstream regulatory modulator antagonist (DREAM) proteins were discovered to affects learning and memory, while Tau protein hyperphosphorylation indicates some degree of neuronal instability influencing learning processes. This study aimed to examine the effects of sleep and circadian disruption (CD) on memory. One hundred and fourty four Sprague Dawley rats were used in this study. In the CD experiment, acute circadian disruption (ACD) was induced by putting the rats on a three-hour phase advance method for 6 days followed by 10 days of re-entrainment. This cycle was induced four times in a chronic circadian disruption (CHCD) study. On the other hand, REM sleep deprivation was induced by putting the rats on a small platform in a water tank (REMsd group). The REMsd control group (PC) was placed in the same condition as the REMsd group except that they were put in a broader platform to sleep and relax (PC group). The free-moving control group (CC) was placed in normal propylene cages (CC group). It was followed by the Morris water maze (MWM) task to assess spatial, probe test, and reverse spatial test. Then, the rat’s brain from the hippocampal region was removed. The neuron's nucleus and cytoplasm were extracted for DREAM, normal Tau, and phosphorylated
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Tau protein expression analyses by Immunohistochemistry and Western Blot (WB) analysis. In MWM test, the mean daily escape latency was measured for all rats from each group. The differences of EL between the groups were not statistically significant (p > 0.05) when compared between all groups in REM and CD experimental groups.
The probe test showed no statistically significant (p>0.05) difference between the groups in the REMsd and CD experiment. The reverse spatial trial showed no significant difference between all groups (p>0.05) in the REMsd and CD experiment.
DREAM protein expression was not statistically significant difference between groups in the REMsd and CD experiment. Tau and phosphorylated Tau protein was found to be significantly different when compared between REMsd and CC groups (p<0.05) and ACD and CCD group in the REMsd and CD experiment, but not significant difference for hyperphosphorylated Tau protein expression. WB analysis showed that the mean relative DREAM protein level was not statistically significant between all groups in the REMsd and CD experiment groups. The mean relative hyperphosphorylated Tau difference also was found not statistically significant between groups in the REMsd and CD experiments. In conclusion, the current study protocol failed to adequately elicited significant changes in neurobehavioral and level of the DREAM and hyperphosphorylated Tau protein expression.
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INTRODUCTION
Learning and memory consolidation and sleep-dependent are one of the areas of interest in neuroscience. Many studies have shown the importance of sleep in memory consolidation, which is a crucial part of the learning process (Stickgold and Walker, 2005, Maquet, 2001, Smith, 1995). Rapid Eye Movement sleep stage (REM) is related to non-declarative memory consolidation, which is essential in learning a new skill related to the procedure (Rasch and Born, 2013).
Lifestyle has now been increasingly stressful; failure to manage it may be affecting the quality and quantity of sleep needed. Furthermore, the arrival of portable touchscreen devices which are widely used in online social interaction and essential in fitting the needs of excellent work performance might worsen the situation of sleep quality. Studies have shown the sleep duration has become less, and the onset of sleep has become longer due to difficulties of falling asleep, as observed among high school and college students. These are the stages where learning is an essential part of their life (Peach et al., 2015).
Active learning induces neuronal excitation in the brain cortex. During sleep, neuronal excitation will be transferred to the inner part of the brain. This process will firmly consolidate the memory (Ngo and Staresina, 2017, Marshall and Born, 2007, Stickgold and Walker, 2005; BuzsÁk, 1998). This explains briefly and superficially how sleep in learning and memory are interrelated.
2 Rationale of The Study
Therefore, the current study aimed to explore the effects of REM sleep deprivation and circadian rhythm disruption on learning and memory by investigating the behavioural analysis and brain proteins measurement.
General Objective
The general objective is to measure the expression and level of DREAM and hyperphosphorylated Tau protein expression in REM sleep deprivation and circadian rhythm disruption in Sprague Dawley rats.
Specific Objectives
1.3.1 To evaluate the effects of REM sleep deprivation and circadian rhythm disruption on spatial memory in Sprague Dawley rats.
1.3.2 To measure DREAM and Hyperphosphorylated Tau proteins expression in Sprague Dawley rat’s hippocampus after REM sleep deprivation and circadian rhythm disruption experiment.
1.3.3 To measure the level of DREAM/calsenilin and Hyperphosphorylated Tau protein expression in Sprague Dawley rat’s hippocampus after REM sleep deprivation and circadian rhythm disruption experiment.
3
LITERATURE REVIEW
Throughout all living organisms, sleep is a part of the most crucial cycle of their daily life. It carries a long list of physiological functions, and some of them are still elusive. Sleep deprivation affects many aspects of all living organisms. Sleep is not merely just a state of unconsciousness. It is defined as the behaviour of temporary perceptual disengagement from the external environment. However, from the inside, it involves a very complex physiological process and changes which carry all the essential function for human body revitalization before the next wake up cycle (Carskadon, 2011).
Sleep
A few physiological changes are associated with sleep. Physically, sleep dissociates a person from the environment, and it is associated with loss of consciousness. During sleep, the muscular hypotonic leads the postural changes to a recumbent position. Eye closure inhibits light entrance to the retina, thus cascading the sensory insensitivity in which its threshold gets higher as the sleep moves from arousal to deeper sleep stages (Datta, 2010).
2.1.1 Stages of Sleep, REM Sleep Versus NREM Sleep
Sleep can be divided into two stages: non-rapid eye movement (NREM, pronounced Non-REM) and rapid eye movement (REM). The classifications of various sleep stages are made based on the physiological parameters and brain wave activity recorded using an electroencephalogram (EEG). More than one cycle of NREM and REM sleep stages may occur throughout the night of a healthy individual.
The mentioned two sleep cycles alternate cyclically. In a sleep disorder, the irregularity
4
of cycling and/or devoid of any cycle stage may occur (Altevogt and Colten, 2006).
(Figure 2.1)
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Figure 2.1: States of mind in awake and various sleep stages. There are four NREM stages, which move from light sleep to deeper sleep stage from one to four. After stage 4 is the REM sleep stage, where the states of mind resemble the awake and active stage. Adapted from Altevogt and Colten, 2006
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A single sleep cycle consists of NREM, the initial stage, which is then followed by REM. The first cycle lasts for an average of 70 to 100 minutes, and the subsequent cycles are longer, with an average of 90 to 120 minutes per cycle. NREM sleep stage accounts for about 75 – 80% of the cycle, and 20 – 25 % is the REM sleep stage (Altevogt and Colten, 2006).
NREM sleep is further subdivided into stages 1, 2, 3, and 4. This subdivision is based on the EEG pattern observed (Table 2.1 – NREM sleep stages brain wave).
From the active brain wave during wakefulness to the alpha wave, an active but clam or resting stage of the brain in Stage 1 of NREM. Stage 1, which lasts for about seven minutes, is easily interrupted by noise. The second stage of NREM has a lower voltage of brain wave and the main difference from Stage 1 is the presence of the K wave complex and sleep spindle (Table 2.1). Stage 3 and 4 are collectively called slow-wave sleep (SWS). They have high voltage and slow activity brain waves. Stage 4 has a higher voltage wave, and it lasts longer than Stage 3. Brain waves in NREM sleep are well synchronised (Altevogt and Colten, 2006).
REM sleep cycle started when the brain waves desynchronised, with low- voltage waves with mixed frequencies. The brain waves in a REM sleep cycle is described as having a sawtooth appearance. Along with the sawtooth brain waves activity, the muscles atonia and rapid eye movement occur in the REM sleep cycle.
This stage lasts only for about one to five minutes during the initial cycle but getting longer in subsequent cycles. Unlike NREM sleep, REM sleep is not divided into any substages.
Dreaming is mostly associated with the REM sleep stage, during which the muscles atonia are important to avoid muscle actions during dreaming (Altevogt and Colten, 2006, Carskadon, 2011). Sleepwalking disorder is related to dreaming in the
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latter two stages of NREM sleep, where the muscle tone is present (Guilleminault et al., 2006).
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Table 2.1: Brainwaves in various states of mind and stages of sleep (Adapted from Altevogt and Colten, 2006).
State of mind
Brainwave Features
Awake and active
Beta brain wave.
Awake and drowsy
Alpha brain wave.
Stage 1 of NREM
A mixture of alpha and theta waves.
Stage 2 of NREM
The appearance of sleep spindle and K- complex in theta wave.
Stage 3 of NREM
A mixture of theta and delta wave.
Stage 4 of NREM
Delta wave.
REM Fast active brain
wave. Resembles brainwave in active awake brain.
9 2.1.2 Physiological Benefits of Sleep
Although the actual functions of sleep are elusive, there are a few theories and ongoing studies that have been done regarding the functions of sleep for the human body. Researchers are still working on a few hypotheses about the actual functions of sleep. They mentioned the hypothesis about energy conservation in sleep. Energy conservation is related to the release of specific genes regulated based on the circadian cycle that controls the food or energy released during sleeping (Sen et al., 2017, Schmidt et al., 2017). Meanwhile, brain metabolic down-regulation is related to the NREM sleep cycle, which is postulated to preserve and restore the brain glucose level during sleep. However, brain metabolism is higher during the wake-up stage of the REM sleep cycle; this indicates more neuronal excitation occurs during the REM sleep stage (Krueger et al., 2016). This explains how a daytime power nap that involves only Stage 1 and 2 of NREM sleep stages has a rejuvenating body effect (Hayashi et al., 2005).
Sleep helps in body recuperation after being affected by certain diseases by enhancing the immune systems (Krueger et al., 2016). One of the most interesting functions of sleep is how it is related to many aspects of brain wellness. For instance, cognitive impairment is related to poor sleep quality and sleep insufficiency. A study showed that sleep insufficiency in adolescents is related to more significant risk-taking behaviour due to poor cognitive judgment (Telzer et al., 2013). Good quality of sleep is crucial for emotional stability, proved by the amygdala's negative stimulation in the limbic system of a sleep-deprived person (Yoo et al., 2007). And for learning and memory benefits, sleep-associated neuroplasticity is currently being one of the major neuroscience fields. Neuroscientists are working out at the various levels of how sleep affects learning and memory.
10 2.1.3 Sleep-Wake – Regulating System
Some essential brain areas are involved in sleep-wake regulation. Four regions are located in the hypothalamus, one in pineal gland, and one region is in the brainstem.
In the hypothalamus, there are suprachiasmatic nucleus (SCN), lateral hypothalamus, ventral preoptic nucleus and tuberomammillary nucleus. SCN is the region involved in sleep-wake homeostasis and is related to the circadian rhythm. Sleep homeostasis determines the amount of sleep needed. It is explained when a person has been awake for a longer duration, that person will have a stronger sleeping drive. In turn, the sleeping drive decreases after a period of sleep (Krystal et al., 2013). In the lateral hypothalamus, orexin, also known as hypocretin, is the neuropeptide involved in wakefulness. They start firing during the sleep-wake transition. Narcolepsy, a chronic sleep disorder is characterised by extreme daytime sleepiness, is associated with orexin deficiency (Krystal et al., 2013, Kalia, 2006, Krebs, 2012).
The ventrolateral preoptic (VLPO) nucleus of the hypothalamus is important for the slow-wave sleep oscillation, the NREM stages. Neurons in the VLPO nucleus are activated by adenosine and prostaglandin D2. The tuberomammillary nucleus of the hypothalamus, mediated by the histamine neurotransmitter, is essential in promoting wakefulness (Kalia, 2006). Hence this is explained when drowsiness is the effect of anti-histamine medication.
The suprachiasmatic nucleus (SCN) of the hypothalamus acts as a regulator for the endogenous biological clock by controlling the wake-sleep cycle known as the circadian rhythm. SCN is located at the anterior hypothalamus. It serves as a ‘master clock’ that controls both physiological and behavioural circadian rhythm. SCN is a neural substrate for day and night cycles in many physiological regulations, including motor activity, body temperature secretion of many hormones, sleep-waking, and
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many more (Gray, 2005, Krebs, 2012). SCN operated as a luminance detector when they received light input from the retina through retinothalamic fibres that terminate in SCN of the hypothalamus and regulate all physiological regulation based on it. The retinal ganglion cells' activation upon receiving light in the retina stimulates the melanopsin in the SCN. This triggers the sympathetic nervous system's activation in the thoracic region, leading to a negative feedback loop mechanism towards inhibition of melatonin released by the pineal gland (Kalia, 2006).
A region known as reticular formation has an ascending reticular activation system (ARAS) in the brainstem. This system has two pathways known to induce wakefulness. The deactivation of these pathways leading to sleep generating process.
The pathways are the dorsal ascending pathway and the ventral ascending pathway.
Fibres in the dorsal ascending pathway arise from the nuclei in the pons and medulla of the brainstem. They ascend towards the thalamic nuclei, and then they are projected to several parts of the brain cortex. Acetylcholine is the neuropeptide that mediates neuronal firing in this region during wakefulness and during the REM sleep stage.
Fibres in the ventral ascending pathway arise from locus coeruleus, known as a noradrenergic neuron, while dorsal and median raphe nuclei are called serotoninergic neurons. They project toward the basal forebrain by passing by the lateral hypothalamus. Neurons of these fibres actively fire during wakefulness and not in REM sleep or REM sleep stages (Kalia, 2016). Lesions involving ARAS causes unconsciousness.
2.1.4 Neuromodulator for Wake-Sleep Cycle
Having known the regions involved in the sleep-wake regulating system, the neurotransmitter released by neurons in these regions is in charge of maintaining wakefulness or actively being released during sleeping. So, the neurons involved in
12
maintaining wakefulness are called the wake-active neurons, and neurons that are released during sleep are called sleep-active neurons (Szymusiak and McGinty, 1989, Morairty et al., 2004). Noradrenaline, histamine, and orexin are the neurotransmitters released by the neurons categorized in the wake-active neurons. Their release is abundant during awake, reduced during SWS, and ceased during REM sleep.
Acetylcholine in the same group as neurotransmitters above, being actively released during the wake and REM sleep stage but decreases during SWS. The neurons in the sleep-active category release GABAergic neurotransmitter. They are mainly found in the basal forebrain and preoptic (VLPO) of the hypothalamus and reciprocal to the wake-active neurons (Jones, 2005).
2.1.5 Sleep Initiation Process
A passive closure of the cerebral sensory gate leads to deafferentation. This quiet initiation of sleep shuts up all the excitatory event of all wake-active neurons in the ARAS and all regions involved in arousal. The neurons in the preoptic or anterior hypothalamus that contain inhibitory neurotransmitters influence the wake-active neurons. The deactivation of ARAS stopping the sensory impulse firing towards the cerebral cortex (Datta, 2010).
Another theory mentioned the accumulation of endogenous metabolites that facilitates the movement from wakefulness to sleep. This theory is based on the homeostatic regulation of sleep, which means the longer the awake period, the higher the metabolites accumulate. The known metabolites that initiate the SWS are adenosine, prostaglandin D2, neuroinhibitory amino acid GABA, cytokine (interleukin I beta), and tumour necrosis factor α (TNFα) (Datta, 2010, Datta and Maclean, 2007).
13 Circadian rhythm
2.2.1 The molecular event under the circadian rhythm regulation
An endogenous circadian clock controls the circadian rhythm. The study of genetic factors that influence the circadian rhythm was done a few decades ago. In 1988, they finally found the first clue to how our body controls the biological clock through a fruit fly study when they found a protein named period (PER). This protein was scattered throughout the fruit fly cells' nucleus in a profound amount during the night time than in the day time, and it shuttles between cytoplasm and nucleus. The PER mRNA was also circulating high at night, indicating PER level is high in the presence of increased mRNA PER and PER oscillation follow the day and night cycle (Saez and Young, 1988).
In 1995, they discovered another gene, timeless (TIM). TIM and TIM mRNA circulate in a circadian manner like PER. TIM directly reacts in PER by preventing degradation of PER in the day time. In humans, the oscillator of circadian rhythm is regulated by a transcription-translation feedback loop (TTFL) (Sehgal et al., 1994, Price et al., 1995).
PER and TIM proteins transcription are activated by another gene call clock (CLK) and cycle (CYC) genes. These two genes interact with each other in the PER gene and produce mRNA for PER. On the other hand, PER and TIM is the negative regulator for CLK and CYC genes, thus promoting a circadian feedback loop cycle (Jin et al., 1999).
Besides the CYC and CLK that activate PER, the degradation of PER is done by a protein kinase known as doubletime (DBT). DBT phosphorylates PER and degrades them. Meanwhile, the TIM protein degradation occurs by the interaction of TIM with cryptochrome (CRY) protein. CRY genes that interact with the TIM in the
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process of degradation of TIM are activated by light. That results in a gradual degradation of TIM as the morning arrives. On the other hand, leaving PER protein awaiting the time to be phosphorylated. The transcription and degradation of PER and TIM cycling followed the circadian rhythm and controlled the internal biological events of the organisms (Price et al., 1998, Jin et al., 1999).
2.2.2 Zeitgeber time
The exogenous factor that synchronises the organism’s endogenous circadian rhythm is known as Zeitgeber. The factor can be a form of light, social and chemical.
Zeitgeber time is a system that refers to the time in the period of Zeitgeber, for instance, the 12:12 light: dark cycle used in light factor Zeitgeber. The Zeitgeber time 0 (Zt 0) refers to the beginning of the light on, and ZT 12 is the beginning of the light off.
(Daan and Merrow, 2002) Memory
Memory is a part of learning where its quality is evaluated based on the performance of a person after a series of training. Memory involves the processes of data acquisition, consolidation, retention, retrieval, and performance at various stages and occurs at the cellular and molecular level of the human brain complex (Abel and Lattal, 2001). Memory acquisition happens during training, followed by up to a few days of memory consolidation and memory retrieval during re-training. Good memory consolidation is indicated by improvement in the last training session.
External sensory signals gain from learning or training will be received and transcribed in the brain cortex. The transcribed signal will then be transmitted to the hippocampus, where new memory formation occurs. After a strong or repeated signal transmission at the hippocampus, the recent memory is established. This memory is
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then wired back to the brain cortex for storage. Disorder of hippocampus structure and function lead to the inability to produce recent memory while they still have the storage of the old one.
2.3.1 Memory classification
Memory is subjected to different ways of classification. One of the most common ways is dividing it into two main prongs: declarative and non-declarative memory (Figure 2.2). Neuropsychologists defined this classification based on the involvement of the medial temporal lobe’s region during memory acquisition.
Declarative memory critically involves encoding at the brain's medial temporal lobe region during the memory acquisition process (Rasch and Born, 2013). Hippocampus located at the medial temporal lobe is where the new memory formation occurs.
Meanwhile, the entorhinal, perirhinal, and parahippocampus are the areas of memory storage. Thus, declarative memory is also known as hippocampal-dependent memory (Squire et al., 2004). Declarative memory is further divided into episodic and semantic memory. Episodic memory is the memory of events, for instance, the memory of our first day in university, and semantic memory is the general knowledge or fact that we learn in school, like the capital city of certain countries. Non-declarative memory is regarded as an unconscious type. It is more for procedural kinds like how to ride a bicycle (Squire, 1992).
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Figure 2.2: This figure depicted the classification of memory. Memory is classified as long and short-term memory. Long-term memory is further divided into declarative in the form of data or theory, while non- declarative is best explained as acquired skills.
Long term
Declarative memory (knowledge or data that are explainable – hippocampal
dependant memory)
Non-Declarative memory (knowledge about doing or performing, i.e. cooking), non- hippocampal dependant memory)
Episodic memory:
Remembering your first experience.
Semantic memory:
Knowledg e about facts or idea.
Skill:
Learning how to drive a
car.
Priming:
Words you heard
recently.
Conditioning:
Salivating when you see your favourite
foods.
17 2.3.2 Memory Stages
Memory processing involves three main stages, which are memory acquisition, memory consolidation, and memory retrieval. Memory acquisition is the stage where we gain new information or data from learning or training. Through a strong or effective way of the information or data collected, it will stay in our memory once it stabilised in storage, which is the brain cortex. The process of stabilisation is called the memory consolidation stage. Next is when we want to retrieve the information or data that we stored, we will recall the data from our memory. The latter is known as a memory retrieval stage.
2.3.3 Cellular and Molecular Level of Memory Formation
Neurons in the nervous system that connected at a small cleft are called a synapse. A typical neuron can have thousands of synapses connecting to other neurons.
The connections work in an intricate manner that allows the brain to function normally.
The synaptic connection can change over time. Following repeated stimulations, the connection can become more substantial, and it even may produce a new pair of synaptic connections. In the case of ignorance or without further stimulus, the synapse connection may weaken and eventually be lost. The ability of neurons to change the structure of their synaptic ends is known as neuronal plasticity. The established, recent memory is formed after a robust synaptic connection. The strength of the connection is measured by the level of excitability of the postsynaptic neurons. A strong through repeated and high-frequency stimulus strengthens the synaptic connection after some time. This is known as long term potentiation (LTP), a cellular basis of learning and memory. (Abel and Lattal, 2001)
LTP can occur at various locations in the brain. One of them being the hippocampus, where LTP in this site involves glutamate as their neurotransmitter.
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Glutamate is the most common excitatory neurotransmitter in the central nervous system. Neurons with glutamate neurotransmitters are known as glutamatergic neurons. Stimulations of glutamatergic neurons lead to the action’s potential travels down through its axon and glutamate releases at its synaptic end. Glutamates bind to the α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptor and N- methyl-D-aspartate (NMDA) receptor at the postsynaptic neurons (Abel and Lattal, 2001).
When a weak signal is transmitted in the presynaptic neuron, it releases a small number of glutamates. Those glutamates will bind to both AMPA and NMDA receptors and allow AMPA receptors to influx cations, sodium (Na+), and no cations influx through NMDA receptors due to magnesium (Mg2+) blockage in the NMDA receptor channel. These amounts of Na+ produce weak depolarisation of the postsynaptic neuron. However, a strong signal will release many glutamates and allow more Na+ influx through the AMPA receptor. An adequate amount of Na+ allowed greater depolarisation of postsynaptic neurons, which removes the Mg2+ blockage of the NMDA receptor. When the NMDA channel opened, it will enable the influx of Na+ and calcium (Ca2+). Calcium is the mediator of LTP induction by initiating the signaling pathway that activating several protein kinases (Figure 2.3).
Calcium/calmodulin-dependent protein kinase II (CaMKII) and protein kinase C (PCK) are two types of protein kinases in early LTP. In early LTP, those protein kinases phosphorylate the AMPA receptor increases the AMPA receptor conductions to sodium. Protein kinases help in bringing out more AMPA receptors from the intracellular storage to the postsynaptic membrane. This signal of early LTP is the basis of short-term memory, which lasts for several hours. In the late LTP phase, it involves new protein synthesis, which enhances the connection between pre and
19
postsynaptic neurons. It includes the synthesis of new AMPA receptors and proteins involved in the formation of new synaptic dendrites and synaptic connections. The late phase of LTP is the basis of long-term memory formation (Abel and Lattal, 2001).
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Figure 2.3: Diagram of the molecular mechanism of Long Term Potentiation (LTP) initiation in neuronal plasticity. It is started with the strong or repeated impulse from the presynaptic neuron that releases the glutamate neurotransmitter. Those glutamates are then attached to the α- amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPA) receptor, which results in the influx of sodium ions through the receptor.
The increased concentration of sodium ion causes depolarisation and, at the same time, magnesium blockage at N-methyl-D-aspartate (NMDA) receptor was removed resulting in the influx of calcium ion and eventually initiate the LTP (Adapted from Abel and Lattal, 2001).
AMPA receptor
NMDA receptor
Mg
= Glutamate Na+
Na+
Na+
Na+ Na+ Na+ Na+
↑ Na+ = depolarisation
Depolarisation expel Mg++ blockage in NMDA receptor
Ca++
Ca++
Ca++
Ca++
Strong impulse or repeated impulse in the presynaptic neuron releases of
glutamate neurotransmitter
1
Glutamates attach to AMPA receptor.
2
Activated AMPA receptor result
in Na+ influx.
3
4
More Na+ influx with additional Ca++
influx causes initiation of LTP 5
21 Sleep and Memory
Memory acquisition is an initial step in-memory processing. Acquisition of memory occurs during learning or training, and it relies on the integrity of the prefrontal cortex functions (PFC). Memory encoding is a process that occurs during the wake-up state.
The deprivations of the pre-training sleep affecting the PFC metabolic rate, thus causing the memory acquisition difficulty (Walker and Stickgold, 2006). The cerebral metabolic rate is measured based on the oxygen level in the cerebral blood flow (Clarke, 1999). During SWS, the rate recorded slightly decreased than in the state of wakefulness but increased during the REM sleep stage (Madsen and Vorstrup, 1991, DiNuzzo and Nedergaard, 2017). While other researchers support that consolidation of memory is supported by sleep through various experiments (Ahrberg et al., 2012, Genzel et al., 2014). Post-training sleep stabilises newly obtained memory by increasing its resistance to interference (Ellenbogen et al., 2006). This allows this memory to stay longer before consolidation takes place. Sleeping after memory acquisition has better memory retrieval than wakefulness (Korman et al., 2007).
2.4.1 Roles of Sleep on Memory Consolidation
For more than a hundred years, scientists have established the importance of sleep in memory consolidation through thousands of research. Among those, the most acceptable finding is the ability to sleep to stabilize memory by increasing memory interference from external stimuli. From the results of REM sleep's involvement, they revealed the importance of SWS for the consolidation of memory. Memory acquisition allows the memory encoding process in the brain. During the SWS stage, optimisation of memory encoding occurs, and consolidation occurs during the REM sleep stage (Rasch and Born, 2013).
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It is described that memory storage in declarative memory involved two standard storages: fast learning and slow learning storage. Fast learning store refers to the hippocampal that is engaged in new memory formation. A slow learning store is long-term memory storage located at the neocortex. Fast learning store receives new data and stores it temporarily in it. In other recent data, it encoded the previous data that may sometimes be unstable and easily disrupted by the new ones. So, because of sleep adequacy, pre-learning sleep is found to strengthen the fast learning storage center by making it more stable and resistant to deletion upon other data encoding.
On the other hand, going to sleep after learning makes the memory storage more stable before its projection to the slow learning storage after a certain learning period (Rasch and Born, 2013). To prove the importance of REM sleep in stabilising the newly stored memory, Davis et al., (2006) have studied REM sleep deprivation's effects on the up-regulation of cortactin, an important cytoskeleton in neuronal plasticity. Their study confirmed the incidence of inhibition of up-regulation of the cortactin, which leads to interference of LTP in rats and the whole process in memory consolidation (Davis et al., 2006).
2.4.2 Molecular Modulator for LTP in Memory Consolidation
A few molecules and proteins are critically crucial for the modulation of LTP in hippocampal-dependent memory consolidation processes. They are the brain- derived neurotrophic factor (BDNF), cAMP response-element-binding (CREB), and calcium–calmodulin-dependent protein kinase II (CAMKII). These molecules involve in many directions of strengthening synaptic plasticity.
BDNF is a growth factor for neurons in the nervous system. Thus, it is known as a neurotrophic factor. BDNF, translated from the chromosome in the human brain, binds to tyrosine kinase B (TrkB) and low-affinity nerve growth factor receptor
23
(LANGF) before eliciting their action, neuronal growth, and maturity. BDNF is crucial for the growth and survival of dopaminergic, GABAergic, serotoninergic, and cholinergic neurons, which are important for neuronal excitatory (Pillai, 2008). In the LTP signaling process, BDNF in the presynaptic neurons enhances the release of neurotransmitters. Meanwhile, in postsynaptic neurons, they increase the influx of calcium and potassium ion through enhancing the ion channels in NMDA receptors and eventually helps in the initiation of LTP through the early and late phase of LTP (Poo, 2001, Lu et al., 2008) cited by (Autry and Monteggia, 2012).
CREB is a cellular transcription factor that binds to DNA and influences the up-regulation or down-regulation of certain genes. BDNF gene is one of the genes that CREB regulates. A previous study that involved the disruption of the CREB gene leads to impairment of long-term memory formation (Bourtchuladze et al., 1994). CREB involve in molecular cascades mediating the transformation of short-term memory to long-term memory. They influence synaptic plasticity during long term memory consolidation. Besides, it is also found that they increase neuronal excitability (Benito and Barco, 2010). As mentioned in LTP cellular mechanism, CREB is a factor that is opening the gate for protein phosphatases that is important in maintaining the influx of sodium through AMPA receptor for induction of powerful depolarisation, cascading for the LTP initiation (Blitzer et al., 1995).
CAMKII is a protein kinase that regulates the calcium/calmodulin complex. It is involved in many cellular signaling cascades, and one of them is in learning and memory. Regulation of this protein kinase induces protein phosphorylation is a catalyst for various intracellular LTP induction changes in a neuron. The intracellular function includes neurotransmitter synthesis, cytoskeletal functions, gene expression, synaptic plasticity and all process involved in LTP (Yamauchi, 2005).
24 Circadian Rhythm and Memory
The circadian rhythm is controlled by a central pacemaker, the SCN of the anterior hypothalamus, is associated with the hippocampal formation. Hippocampus is critical in learning and memory, integrating the circadian information through direct or indirect input from SCN or other oscillators input, modulating the hippocampal neurogenesis through clock genes expression and genetic modification. (Smarr et al., 2014, Ruby et al., 2008). A study concluded that the memory impairment in circadian rhythm disruption is associated with inhibition of GABAergic communication to the hippocampus (Ruby et al., 2008). Clock gene expression in the hippocampus, which is rhythmically oscillated postulated to be synchronised by SCN (Smarr et al., 2014).
These clock genes then regulate the hippocampus's neurogenesis (Gery et al., 2006, Matsuo et al., 2003). Melatonin is an endogenous hormone released based on the circadian clock, where its levels are higher during the dark phase, promoting neuron maturation and survival (Ganguly et al., 2002).
Hippocampus and Memory
The cerebral cortex is the largest part of the brain, which contains lobes that include the limbic lobe. The limbic lobe, located at deep brain area is also known as the medial temporal lobe in the cerebral hemisphere's medial wall. The limbic lobe is made of subcallosal (corpus callosum), cingulate and para-hippocampal gyri (Figure 2.4).
