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Modulators of synaptic plasticity in the hippocampus Robillard, Julie 2010

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Modulators of synaptic plasticity in the hippocampus by Julie Robillard  B.Sc., Université de Montréal, 2003  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF Doctor of Philosophy in The Faculty of Graduate Studies (Ncuroscicncc)  The University Of British Columbia May, 2010  ©  Julie Robillard 2010  Abstract The two main forms of hippocampal synaptic plasticity, long-term poten tiation (LTP) and long-term depression (LTD) represent a cellular model for learning and memory. While synaptic plasticity has been studied exten sively, questions still remain on how exogenous and endogenous modulators can impact hippocampal LTP and LTD. Here, we use electrophysiology and imaging to investigate the effects of two types of modulators on synaptic plasticity. First, we look at the effects of an antagonist of the 5-HT6 re ceptor on LTP and LTD in two regions of the hippocampus, the CAl and the dentate gyrus (DG). We find that our 5-HT6 antagonist differentially affects LTP in each region and blocks hippocampal LTD. These findings are the first report of an involvement of the 5-HT6 receptor in synaptic plastic ity and are particularly relevant in light of evidence showing a key role of the 5-HT6 receptor in cognition and memory. Second, we look at the effects of glutathione (GSH) supplementation on LTP in aged animals. We show that supplementing aged mice with a precursor for GSH formation reverses the mechanisms underlying hippocampal LTP from L-type calcium channel dependence back to NMDA receptor-dependence. These results suggest an important role for GSH as a modulator of synaptic plasticity in aging.  11  Table of Contents Abstract  ii  Table of Contents  iii  List of Tables  vi  List of Figures  vii  List of Abbreviations  x  Acknowledgements  xiii  Dedication  xiv  Statement of Collaboration 1  xv  Introduction  1  1.1  Research hypothesis and objectives  1  1.2  Introduction to the hippocampus  3  1.3  Synaptic plasticity: changes in synaptic transmission  4  1.4  Synaptic plasticity in the mammalian hippocampus  5  1.4.1  5  Long-term potentiation  111  1.4.2  Molecular mechanisms of LTP induction and mainte nance  6  1.4.3  Long-term depression  9  1.4.4  Molecular mechanisms of LTD induction and mainte nance  1.4.5 1.5  Short-term forms of plasticity  9 11  Induction protocols  12  1.5.1  Induction protocols used to induce LTP  12  1.5.2  Induction protocols used to induce LTD  13  1.5.3  Spike-timing protocols  14  1.6  Particularities of plasticity in hippocampal dentate gyrus  14  1.7  Modulation of LTP by ndurotransmittcr systems  16  1.7.1  Serotonergic system and receptors in the hippocampus  17  1.7.2  Modulation of hippocampal synaptic plasticity by sero  1.8  1.9  toniii  18  1.7.3  The 5-HT6 receptor  21  1.7.4  The 5-HT6 receptor and memory  22  LTP in the aged mammalian hippocampus  24  1.8.1  Impact of aging on the hippocampus  24  1.8.2  Impact of aging on hippocampal LTP  27  Modulation of LTP by oxidative stress  29  1.9.1  Introduction to oxidative stress in the brain  29  1.9.2  Glutathione  31  1.9.3  Oxidative stress and GSH in the aged brain  37  1.9.4  Impact of oxidative stress on LTP  39 iv  2  3  4  The Role of the 5-HT6 Receptor in Hippocampal Synaptic Plasticity in the rat  47  2.1  Introduction  47  2.2  Experimental procedures  49  2.3  Results  53  2.4  Discussion  57  Glutathione Alters the Mechanism of Synaptic Memory in Aged Mice  77  3.1  Introduction  77  3.2  Experimental procedures  80  3.3  Results  86  3.4  Discussion  93  General Discussion  128  4.1  Summary of findings  128  4.2  Impact of oxidative stress on the L-type calcium channel pathway  130  4.3  Clinical relevance of work  133  4.4  Future directions  139  References  143  Appendices A Research Ethics Approval  181  V  List of Tables 1.1  Relevant studies looking at the effects of aging on synaptic plasticity in vitro in wild-type animals  2.1  46  In vitro binding affinities for R9237 at various 5-HT receptors. 76  vi  List of Figures 1.1  Metabolism of glutathione  1.2  Impact of N-acetylcysteine on glutathione metabolism  2.1  Structure of R9237  62  2.2  Dose-response for the effect of R9237 on CAl LTP  63  2.3  R9237 increases the magnitude of LTP in the CAl region  64  2.4  Input-output curves for CAl LTP  65  2.5  R9237 completely blocks LTD in the CAl region  66  2.6  Input-output curves for CAl LTD  67  2.7  Paired-pulse analysis from the CAl region  68  2.8  R9237 does not affect LTP in the dentate gyrus  69  2.9  Input-output curves for DG LTP  70  .  .  .  .  .  43 44  2.10 R9237 reverses LTD in the dentate gyrus  71  2.11 Input-output curves for DC LTD  72  2.12 R9237 does not significantly affect LTP induced using a weak stimulus in the dentate gyrus  73  2.13 Input-output curves for DC LTP induced using a weak stimulus 74 3.1  Effect of stimulation protocol on LTP in adult mice  102  vii  3.2  Effect of stimulation protocol on LTP in aged mice  103  3.3  LTD in adult and aged mice  104  3.4  LTD in adult and aged mice using an altered calcium/magnesium ratio in the aCSF  105  3.5  APV blocks LTP in the CAl region of adult mice  106  3.6  LTP is unaffected by nimodipine in the CAl region of adult mice  107  3.7  LTP is NMDA receptor-dependent in adult mice  108  3.8  LTP is unaffected by APV in the CAl region of aged mice.  109  3.9  LTP is significantly decreased in the presence of nimodipinc in the CAl region of aged mice  3.10 LTP is L-typc calcium channel-dependent in aged mice.  110 .  .  .  111  3.11 NAC supplementation in aged mice leads to increased levels of hippocampal GSH 3.12 Example of MCB labeling within a single stack of images  112 113  .  3.13 The intensity of MCB labeling is increased in NAC-fed aged mice  114  3.14 NAC supplementation in aged mice leads to increased levels of GSH in hippocampal neurons 3.15 APV blocks LTP in the CAl region of NAG-fed aged mice  115 .  .  116  3.16 LTP is unaffected by the presence of nimodipine in the CAl region of NAC-fed aged mice 3.17 LTP is NMDA receptor-dependent in NAC-fed aged mice  117 .  .  118  3.18 LTP is not significantly blocked by APV in the CAl region of control-fed aged mice  119  viii  3.19 LTP is significantly decreased in the presence of nimodipine in the CAl region of control-fed aged mice  120  3.20 LTP is L-type calcium channel-dependent in control-fed aged mice  121  3.21 NMDA receptor blockade reduces HFS-induced calcium sig nals in NAC-fed mice  122  3.22 NAC supplementation restores NMDA receptor-mediated cal cium signals in aged mice  123  3.23 L-type calcium channel blockade reduces HFS-induced cal cilim signals in control-fcd mice 3.24 Control-fed mice exhibit nimodipine-sensitive calcium signals  124 125  3.25 GSH in the intrapipette solution leads to significant LTP in whole-cell conditions 3.26 GSH restores whole-cell LTP  126 127  ix  List of Abbreviations 5-HT  5-Hydroxytryptamine, serotonin  AHP  Afterhyperpolarization  AMPA  -amino-3-hydroxyl-5-methyl-4-isoxazole-propionate  ApoE4  Apolipoprotein E4  APV  (2R)-amino-5-phosphonovaleric acid  ATP  Adenosine triphosphate  BSO  Buthionine sulfoxide  CA  Cornu Ammonis  CaMKII  Calcium/calmodulin-dependent protein kinase  cAMP  Cyclic adenosine monophosphate  C0P39653  DL- (E)-2-amino-4-propyl-5-phosphono-3-pentenoic acid  CICR  Calcium-induced calcium release  CNS  Central nervous system  CRE  Cyclic AMP-response element  CREB  Cyclic AMP-response element-binding protein  DAG  Diacyiglycerol  DC  Dentate gyrus  DNA  Deoxyribonucleic acid  DTNB  5,5-Dithiohis(2-nitrohenzoic acid) x  E-LTD  Early long-term depression  E-LTP  Early long-term potentiation  EPSC  Excitatory postsynaptic current  EPSP  Excitatory postsynaptic potential  fEPSC  Field excitatory postsynaptic current  fEPSP  Field excitatory postsynaptic potential  GABA  Gainma-Amiiiobutyric acid  CCL  7-glutamylcysteine ligase  CPCR  G-protein-coupled receptor  GPx  Glutathionc peroxidase  CR  Glutathioiie reductase  GS  Glutathione synthetase  GSH  Glutathione  GSSG  Oxidized glutathione  HFS  High-frequency stimulation  I/O  Input-output  lEG  Immediate early genes  1P3  Inositol triphosphate  JNK  c-Jun N-terminal kinase  L-LTD  Late long-term depression  L-LTP  Late long-term potentiation  LTD  Long-term depression  LTP  Long-term potentiation  MAPK  Mitogen-activated protein kinase  xi  mGluR  Metabotropic glutamate receptor  mtDNA  Mitochondrial deoxyribonucleic acid  NAC  N-acetylcysteine  NMDA  N-methyl-D-aspartate  nNOS  Neuronal nitric oxide synthase  NO  Nitric oxide  NOS  Nitric oxide synthase  PKA  Protein kinase A  PKC  Protein kinase C  PP1  Protein phosphatase 1  PSD-95  Postsynaptic density 95  ROS  Reactive oxygen species  RyR  Ryanodine receptor  SNOC  S-nitrosocysteine  SOD  Superoxide dismutase  SSRI  Selective serotonin reuptake inhibitor  STDP  Spike-timing dependent plasticity  STP  Short-term potentiation  TBS  Theta-burst stimulation  wTBS  Weak theta-burst stimulation  ‘yGluCys  ‘y-glutamylcysteine  X11  Acknowledgements First, I would like to extend my sincere appreciation to  my supervisor,  Dr.  Brian MacVicar, for his support, guidance and time. Under Dr MacVicar’s supervision, I experienced a fantastic research environment, and I enjoyed every  minute of my time in the lab.  Many thanks to Dr. Brian Christie for his support and assistance. I  am very grateful that Dr. Christie welcomed me in his lab and gave me an opportunity to pursue quality research in Neuroscience. Thank you to my committee members, Dr. Shernaz Bamji, Dr. Robert Douglas, and Dr. Yu—Tian Wang, for their advice and support of my work. I extend my appreciation to my colleagues and friends at the Brain Re search Centre. Their friendship, support and advice helped me overcome many challenges.  Special thanks to Andrea Titterness, Denise Feighan,  Hyun Beom Choi and Grant Gordon for their help with experiments. I am truly grateful for the financial support I have received from the Natural Science and Engineering Research Council and the University of British Columbia. Thank you to my parents for their unconditional support in this great adventure.  Finally, thanks to Craig Hennessey. I couldn’t have done it without you. xiii  Dedication Dedicated to my parents, Madeleine and Pierre, and to craig. Thank you for your encouragcmcnts, your support, and for constantly inspiring me to be the best I can be.  xiv  Statement of Collaboration For the work described in Chapter 2, 1 conceived and designed the exper iments, analyzed the data, prepared the figures and wrote the manuscript on which the chapter is based. While the majority of the experimental work was performed by myself, Kyle Russell assisted with electrophysiological  recordings in the CAl region. The data in Table 2.1 was provided by Roche (Palo Alto LLC, CA). Dr. Brian Christie provided advice on the overall experimental design and helped edit the manuscript. For the work described in Chapter 3, I conceived and designed the exper iments, analyzed the data, prepared the figures and wrote the manuscript on which the chapter is based. While the majority of the experimental work was performed by myself, Dr. Hyun Beom Choi assisted with the molecular  biology work, and Dr. Grant Gordon provided assistance with the imag ing and whole-cell recordings. Dr. Brian MacVicar provided advice on the overall experimental design and helped edit the manuscript.  xv  Chapter 1  Intro 1.1  ion  Research hypothesis and objectives  Synaptic plasticity represents the ability of a synapse to change in strength. Long-term potentiation (LTP) and long-term depression (LTD), the two main forms of long-term synaptic plasticity, are thought to be means by which the brain adapts to the external environment, and represent a cel lular model for learning and memory. This work addresses the clinically important question of how exogenous and endogenous modulators can im pact long-term synaptic plasticity in the hippocampus. While there is an extensive body of literature pertaining to hippocampal synaptic plasticity, many questions remain about the potential for clinically relevant modula tion of LTP. Our goals were to establish new modulators of LTP in both the adult and aged brain. The first major aim of this work (described in Chapter 2) was to assess the modulation of hippocampal LTP and LTD by the serotonergic system through the 5-HT6 receptor. Serotonin has been implicated in the processes underlying memory formation in the mammalian brain (Gu, 2002), and ev idence suggests the 5-HT6 receptor may be especially critical for certain forms of memory (Hirst et al., 2006; Lieben et al., 2005). However, the in 1  volvement of the 5-HT6 receptor in synaptic plasticity in the hippocampus has not previously been reported. I hypothesized that a 5-HT6 antagonist can modulate different types of hippocampal synaptic plasticity. Experi ments were therefore designed to characterize the modulation of LTP and LTD by a 5-HT6 antagonist in different areas of the adult rat hippocampus. The second major aim of this work (described in Chapter 3) was to examine the effects of different conditioning protocols on LTP in the CAl region of the hippocampus in adult and aged mice. While many studies have looked at synaptic plasticity in aged rats, the effects of aging on hippocainpal plasticity remains elusive in mice. The few studies on LTP in aged mice use a variety of different conditioning protocols and the threshold to observe LTP deficits in aged mice still needed to be established. I hypothesized that age-related reductions in synaptic plasticity would exhibit a frequency dependency, and that changes in the capacity to induce synaptic plasticity would be reflected by changes in the contributing mechanisms to this process. Electrophysiology experiments were designed to assess different conditioning protocols on the magnitude of LTP in adult and aged mice. The third major aim of this work (described in Chapter 3) was to ex plore the contribution of oxidative stress to age-related impairments in LTP in aged mice, with a focus on glutathione (GSH), the major endogenous antioxidant produced by cells. GSH levels are reported to decrease with age, making it an attractive candidate for mediating age-related changes in synaptic plasticity. I hypothesized that supplementing aged mice with N-acetylcysteine (NAC), a precursor for the formation of GSH, can reverse the age-induced deficits in LTP. I therefore designed a supplementation pro 2  tocol for aged mice and used electrophysiology and imaging to study the relationship between GSH and LTP.  1.2  Introduction to the hippocampus  The work described in Chapters 2 and 3 was entirely carried out in the hippocampus, a brain region critical for some forms of memory. The hip pocampal formation is a group of brain areas consisting of the dentate gyrus (DC), the hippocampus proper (containing areas Cornu Ammonis 1 (CAl), CA2 and CA3), the subiculum, presubiculum and parasubiculum, and the enthorinal cortex. The hippocampal formation belongs to the limbic system and is located inside the medial temporal lobe, beneath the cortical surface. Initially thought to play an important role in olfaction due to the fact that macrosmatic animals, who have a highly developed sense of smell, have a prominent hippocampus, the hippocampal formation is now known to be important for different types of memory and spatial navigation. The hippocampus proper consists of six layers, namely the pyramidal cell layer, the stratum oriens (containing basal dendrites of pyramidal cells and interneurons), the thin, fiber-containing alveus, the stratum  lucidum  (containing mossy fibers, only found in the CA3), the stratum radiatum, and the stratum lacunosum-moleculare, where fibers from the enthorinal cortex projections terminate. The hippocampus receives extrinsic projections from the enthorinal cortex, the neocortex and amygdaloid complex, the basal forebrain, the hypothalamus, the thalamus and the brain stem. In terms of intrinsic connections, the major input to the CAl region is the Schaffer  3  collaterals from the CA3 region. An interesting property of the hippocampal formation is the unidirectionality of its inputs and outputs, in a way that leads excitation to flow from one structure to the next with a minimum amount of feedback.  1.3  Synaptic plasticity: changes in synaptic transmission  Synaptic plasticity, which is believed to represent a cellular model for pro cesses underlying learning and memory, has been extensively studied as LTP and LTD of glutamatergic synaptic transmission of Schaffer collateral CAl synapses in the developing and mature rodent hippocampus (Bliss & Collingridge, 1993; Bliss  Lomo, 1973). LTP is a persistent increase in  synaptic strength in response to a strong conditioning stimulus. It has been detected at many excitatory synapses in the brain and has been reported to last up to several months (Malerika  Bear, 2004). LTP is achieved exper  imentally by strong, often high-frequency electrical stimulation of afferent inputs to a given region (Bliss & Collingridge, 1993; Rose & Dunwiddie, 1986). In contrast, LTD is a persistent decrease in synaptic strength that can be induced by low frequency electrical stimulation (Thiels et al., 1996). LTP and LTD are most commonly studied at the CA3-CA1 synapse and typically require calcium influx through N-methyl-D-aspartate (NMDA) glu tamate receptors for induction (Dudek & Bear, 1992; Mulkey & Malenka, 1992).  However, in recent years, different, NMDA receptor-independent  forms of synaptic plasticity have been discovered, relying on varying modes 4  of calcium entry such as metabotropic glutamate receptors (mGluRs) for LTD and L-type calcium channels for LTP (Otani & Connor, 1998; Shankar et aL, 1998).  1.4  Synaptic plasticity in the mammalian hippocampus  1.4.1  Long-term potentiation  Synaptic plasticity in the adult nervous system generally refers to a per sistent (> 30 mm) change in synaptic efficacy following a strong electrical stimulation. This change in synaptic strength is modulated by changes in postsynaptic currents and potentials. Synaptic plasticity is classically stud ied in the CAl region of the mammalian hippocampus, and the most stud ied form of synaptic plasticity is NMDA receptor-dependent LTP. NMDA receptor-dependent LTP can by characterized by three experimentally dis tinct properties: input specificity, cooperativity, and associativity. The in put specificity property arises from the fact that potentiation only occurs at active synapses (Andersen et al., 1977; Lynch et al., 1977). Cooperativity refers to the need for exciting multiple afferents to achieve LTP induction. Experimentally, this has been shown by demonstrating that a weak condi tioning protocol does not trigger LTP (McNaughton et al., 1978). Similarly, it is thought that a relationship exists between the intensity and frequency of the stimulation and the duration of LTP: the stronger the stimulation, the more afferents are recruited, and the longer the duration of LTP (Lovinger  5  & Routtenberg, 1988; Malenka, 1991). Finally, associativity refers to the observation that the induction of LTP can be influenced by activity at other synapses. When a weak stimulation of a single pathway is insufficient to induce LTP, simultaneous stimulation of neighboring pathways can induce LTP at both pathways (Levy & Steward, 1979; McNaughton et al., 1978). The combination of those three properties ensures that synapses undergo LTP only when they are active and when the dendrite has been sufficiently depolarized (Bliss & Collingridge, 1993). The molecular basis for the three properties of NMDA receptor-dependent LTP lies in the voltage-dependence of the NMDA receptor which allows it to act as a postsynaptic coincidence detector for presynaptic signal and postsynaptic depolarization. NMDA re ceptors are opened by glutamate release from presynaptic terminals, but this can oniy occur in the presence of a sufficiently strong postsyiiaptic de polarization in order to relieve the magnesium block in the NMDA receptor pore (Nowak et al., 1984). Therefore, NMDA receptors are perfectly suited for detecting correlated pre- and postsynaptic activity, and play an essential role in LTP induction. The properties of the NMDA receptors also explain how the modulation of LTP can occur following either electrical changes or changes in intracellular signalling.  1.4.2  Molecular mechanisms of LTP induction and maintenance  LTP is classically divided into two phases: an early phase (E-LTP), which lasts in the order of hours (Frey & Morris, 1997), and a late phase (L-LTP), which requires protein synthesis and has been reported to last up to sev 6  eral months (Malenka & Bear, 2004). While the molecular mechanisms of hippocampal LTP are still being studied, a few key pathways have been identified. Initially, for the induction of LTP, there is a requirement for cal cium influx through NMDA receptor channels. Once inside the cells, calcium ions bind to calmodulin and this interaction leads to the activation of the calcium/calmodulin dependent kinase II (CaMKII) (Kennedy et al., 2005). The activated CaMKII then phosphorylates proteins nearby the calcium in flux site at NMDA receptors and this is thought to represent the mechanism for E-LTP expression. One key target of CaMKII activity is the c-amino-3hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptor. AMPA recep tors are responsible for a large component of the excitatory postsynaptic current (EPSC) following synapse activation. Accordingly, LTP leads to an increase in AMPA receptor channel conductance via phosphorylation by CaMKII and also leads to an increase in postsynaptic AMPA receptor inser tion (Malinow & Malenka, 2002). More specifically, CaMKII activation leads to the phosphorylation of the G1uR1 subunit of AMPA receptors and this directly increases the single channel conductance of AMPA receptors (An drsfalvy & Magee, 2004). In addition, CaMKII activation also promotes the insertion of GluRl-containing AMPA receptors, which leads to an increase in the number of postsynaptic AMPA receptors (Shi et al., 2001). Over all, it is widely accepted that CaMKII plays a key role in E-LTP and this is supported by a high postsynaptic concentration of this kinase (Malenka & Nicoll, 1999). In addition, LTP cannot be achieved if CaMKII is either pharmacologically blocked or genetically deleted (Malenka et al., 1989; Ma linow et al., 1989). Finally, the presence of a constitutively active CaMKII 7  occludes hippocampal LTP (Pettit et al., 1994). L-LTP is a persistent potentiation that requires gene transcription as well as protein synthesis. Both protein synthesis inhibitors such as anisomycin and transcriptional inhibitors such as actinomycin block L-LTP (Malenka Bear, 2004). Interestingly, only a strong conditioning stimulus can induce L LTP (Malenka, 1991). One hypothesis that has been put forward to explain this phenomenon is that strong stimulation activates postsynaptic molecu lar mechanisms that are not needed for E-LTP. For example, only a strong stimulus can trigger a signal that travels to the nucleus and lead to the synthesis of proteins required for L-LTP. While protein synthesis and tran scriptional inhibitors only block L-LTP, kinase inhibitors are able to block both E-LTP and L-LTP (Hanse & Gustafsson, 1994).  Therefore, kinase  activity is an attractive candidate to mediate the messenger pathway that moves the signal from the initial site of calcium influx to the nucleus. Poten tial kinases that have been investigated and are suggested to play a signaling role for L-LTP include CaMKIV, cAMP-dependent protein kinase (PKA) and mitogen-activated protein kinase (MAPK). These kinases can activate CREB (cAMP-response element binding protein), a key transcription fac tor, and immediate early genes (lEGs) (Abraham & Williams, 2003; Lynch, 2004; Pittenger & Kandel, 2003). A strong case is made for the involvement of CREB in L-LTP due to reports that its downstream target, the nuclear transcription factor CRE (calcium/calmodulin and cAMP-dependent) is ac tivated by a strong but not a weak LTP conditioning stimulus (Impey et al., 1996). Another hallmark of L-LTP is the structural remodeling and growth of 8  spines that may support the maintenance of long-term changes in synap tic strength.  Morphological changes associated with L-LTP include the  addition of new dendritic spines, the growth of existing spines and possi bly the splitting of existing spines into two separate synapses (Abraham & Williams, 2003; Yuste & Bonhoeffer, 2001) (but see Maletic-Savatic et al., 1999) (Maletic-Savatic et al., 1999).  1.4.3  Long-term depression  LTD is similar to LTP but leads to a decrease in synaptic strength as opposed  to an increase. Like LTP, LTD can be NMDA receptor-dependent (Dudek & Bear, 1992) and requires calcium influx for its induction (Mulkey & Malenka, 1992). NMDA receptor-dependent LTD has been observed in various brain regions but is predominantly studied in the hippocampus. Since both LTP and LTD require calcium influx through NMDA receptors for their induc tion, a model has emerged that suggests a small calcium elevation leads to LTD while a larger one leads to LTP (Castellani et al., 2005).  1.4.4  Molecular mechanisms of LTD induction and maintenance  While LTP requires the phosphorylatiori of specific postsynaptic proteins for its induction, LTD is thought to require the dephosphorylation of post  synaptic proteins (Hrabetova & Sacktor, 2001; Kameyama et al., 1998; Lee et al., 1998; van Dam et al., 2002). Evidence for the involvement of PKA dephosphorylation in LTD has been shown by inhibiting postsynaptic PKA, which prevents LTD and leads to a decrease in synaptic transmission. In ad  9  dition, while postsynaptic activation of PKA does not affect basal synaptic transmission, it can block previously induced LTD (Kameyama et al., 1998). Interestingly, CaMKII substrate phosphorylation is unaltered during LTD. This may be explained by a selective recruitment of protein phosphatases (protein phosphatase one (PP1)) to specific substrates (PKA) (Morishita et al., 2001). This is supported by evidence showing that postsynaptic in hibition of protein phosphatases such as PP1 can prevent LTD (Kirkwood & Bear, 1994; Mulkey et al., 1994). The expression of LTD, like that of LTP, is thought to be mediated by changes in AMPA receptor phosphorylation. However, specific phosphory lation and dephosphorylation patterns help distinguish between LTP and LTD. During LTD, AMPA receptor open channel probability is decreased due to the dephosphorylation of ser-845 on the GluRl subunit. This phos phatase activity leads to a reduced expression of AMPA receptors via inter nalization (Malinow 8 Malenka, 2002). Conversely, during LTP, CaMKII mediated changes in phosphorylation occur at ser-831 (Shukla et al., 2007; Whitlock et al., 2006). As with LTP, LTD can be divided into two phases, an early phase (E LTD) and a protein-synthesis dependent late phase (L-LTD) (Kauderer & Kandel, 2000) However, unlike L-LTP which requires both mRNA transla tion and transcription, L-LTD only requires translation. While it remains unclear which specific newly synthesized proteins are required for the main tenance of LTD, it is probable that structural modifications of synaptic elements are needed for L-LTD expression. One theory is that LTD results from the loss of slot proteins such as postsynaptic density 95 (PSD-95) while 10  LTP results from a recruitment of these same proteins (Malinow & Malenka, 2002). This theory is supported by evidence showing that overexpression of PSD-95 increases the number of AMPA receptors at the synapse (Schnell et al., 2002) while removal of PSD-95 from the synapse through depalmitoy lation decreases the number of AMPA receptors at the synapse (El-Husseini et al., 2002).  1.4.5  Short-term forms of plasticity  In addition to different forms of long-term synaptic plasticity, hippocampal neurons can also exhibit short-term changes in synaptic strength. A first transient modification of synaptic responses is the paired-pulse facilitation or depression phenomenon. Paired-pulse facilitation has been described in numerous brain regions including the hippocampus and occurs when the postsynaptic response to a second pulse delivered a few milliseconds after a first pulse is increased. However, if the time separating the two pulses is in the order of seconds, the postsynaptic response to the second pulse is decreased (paired-pulse depression). The mechanism underlying pairedpulse facilitation relies on an increase in neurotransmitter release during the second pulse due to a higher concentration of presynaptic calcium. This higher concentration of calcium comes from residual synaptic calcium from the first pulse (Wu  Saggau, 1994). Accordingly, synapses that display  high levels of paired-pulse facilitation are thought to have a low probability of release initially. The mechanism underlying paired-pulse depression relies on the depletion of vesicular content as well as feedback mechanisms that lead to inhibition such as the activation of presynaptic 7-aminobutyric acid 11  (GABA) receptors. A second form of transient modification of synaptic responses is post tetanic potentiation. In this case, repeated synaptic stimulation leads to a short-term facilitation of the postsynaptic responses. This effect has been observed to last in the order of minutes. The mechanism underlying post tetanic potentiation is thought to be similar to that of paired-pulse facilita tion and to rely on an increase in presynaptic calcium concentration.  1.5  Induction protocols  Experimentally, many different conditioning protocols can induce LTP and LTD. The two most commonly used forms of LTP induction at afferent fibers are tetanic electrical stimulation and patterned electrical stimulation. However, there is a great variability in the specific parameters of electrical stimulation protocols used in various studies.  1.5.1  Induction protocols used to induce LTP  In area CAl, one of the most common protocols is high-frequency stimula tion (HFS) of presynaptic axons. This type of protocol usually consists of trains of 1 s and a frequency of 100 Hz (Bliss & Collingridge, 1993). These high-frequency trains can be repeated, generally up to six times, with in tertrain intervals ranging from seconds to minutes (Raymond, 2007). HFS causes a large postsynaptic depolarization, leading to the calcium influx nec essary for potentiation through the removal of the NMDA receptor block, the activation of voltage-sensitive calcium channels and the calcium-induced  12  calcium release (CICR) from intracellular stores. Theta-burst stimulation (TBS) was devised in an attempt to use more physiogically relevant stimuli. This type of conditioning protocol is thought to mimic the firing patterns observed in the hippocampus during learning (Otto et al., 1991). TBS generally involves short bursts of four or five pulses at a frequency of 100 Hz. These bursts are repeated at 5 to 8 Hz (theta) in trains of typically 10 bursts. Often, more than one train is used, and they are usually separated by 10-60 s (Raymond, 2007). TBS, like HFS, leads to a large postsynaptic depolarization. In rat area CAl, maximal LTP can be induced by 8-16 TBS trains (Abraham & Huggett, 1997). Finally, it is possible to induce LTP using a lower frequency if a pairing protocol is used (Gustafsson et al., 1987). This type of conditioning stimuli usually consist of stimulating a single afferent  using  repeated low frequency  pulses (0.1 to 1 Hz) while pairing with a depolarization of the postsynaptic cell (typically to 0 to 5 mV for 100 ms). This pairing of presynaptic stim ulation with postsynaptic depolarization is then repeated multiple times (generally up to 100).  1.5.2  Induction protocols used to induce LTD  LTD is typically induced using a low frequency stimulation protocol. A frequently used protocol consists of 900 pulses at 1 Hz for 15 mm  (Thiels  et al., 1994). Similarly, LTD can be induced at a slightly higher frequency, for example 900 pulses at 5 Hz for 3 mm  (Kauderer & Kandel, 2000). These  types of protocols produce LTD that is blocked by the NMDA receptor antagonist D-amino-5-phosphonovalerate (APV). This blockade prevents the 13  calcium influx that is responsible for the signaling cascade that leads to LTD as well as LTP (Cavazzini et al., 2005).  1.5.3  Spike-timing protocols  Spike-time dependent plasticity (STDP) refers to a conditioning protocol similar to the pairing protocol described above. However, the depolariz ing pulse is limited to the minimum stimulation needed to elicit a single postsynaptic action potential. LTP occurs when the action potential backpropagates in the dendrites after the stimulation of the afferents has pro duced an excitatory postsynaptic potential (EPSP) within the target cell. The time window for these events to occur and lead to LTP is typically less than 50 ms. As the interval between the presynaptic stimulation and postsynaptic action potential increases, the degree of LTP decreases (Bi & Poo, 2001). Evidence suggests that a STDP protocol can induce LTP with only a single pairing (Vislay-Meltzer et al., 2006). Interestingly, if the order of events is reversed and the action potential is induced prior to the presy naptic stimulation (again in a window of less than 50 ms), this results in LTD (Debanne et al., 1999).  1.6  Particularities of plasticity in hippocampal dentate gyrus  The specific mechanisms underlying synaptic plasticity are not identical throughout the different regions of the hippocampus. The DG is a C-shaped structure that lies close to the amygdala and extends in rodents from a dorso 14  medial position to a ventro-lateral position. The DG receives its inputs from the enthorinal cortex via the perforant path fibers and in turn serves as the input for the CA3 region of the hippocampus via the mossy fibers. Anatomically, the DG is formed of three layers. The middle layer con tains the major cells of the DG, the granule cells. Granule cells lack basal dendrites, are smaller than pyramidal cells, and display a high threshold for activation (Jung  McNaughton, 1993). The dendrites of the granule cells  extend into the molecular layer while the axons, the main components of the mossy fibers, extend towards the CA3 region. Finally, the polymorphic layer harbors mossy cells. While synaptic plasticity has been studied most extensively in the CAl region of the hippocampus, it was originally discovered and described in the DG, at the synapses between perforant path fibers and granule cells. LTP in the DG is of two different types, reflecting the fact that enthorinal cortex fibers divide into lateral and medial tracts, each with different character istics. Electrophysiological differences observed between the two pathways include the rising phase of the field excitatory postsynaptic current (fEPSC), which is slower for the lateral input, and paired-pulse plasticity, which is fa cilitated through the lateral input but depressed through the medial input. LTP and LTD at the medial perforant path synapses are similar to the typical forms observed in the CAl region in that they are NMDA receptor dependent.  However, plasticity at the lateral perforant path is NMDA  receptor-independent and, contrary to that of the medial path, is opioid sensitive (Xie  Lewis, 1991). In addition, while traditional LTP in the  CAl region and in the medial perforant path of the DG can be induced 15  using pairing protocols, these are not effective at the lateral perforant path (Colino & Malenka, 1993). An additional distinguishing feature of the DG is its ability to support neurogenesis into adulthood. New granule cells are born on a daily basis, and a proportion of those survive and develop into mature granule cells and are integrated into the existing neuronal network (Shors et al., 2001; Hastings & Gould, 2003). Neurogenesis is thought to be modulated both by genetic factors (Kempermann & Gage, 2000) as well and environmental factors, such as exercise (van Praag et al., 1999). This particularity of the DC to give rise to new granule cells can have an impact on synaptic plasticity as the threshold for induction of LTP has been shown to be lower for immature granule cells (Schmidt-Hieber et al., 2004). This effect is thought to be attributed to the presence of T-type calcium channels and increased input resistance in these cells.  1.7  Modulation of LTP by neurotransmitter systems  The magnitude of LTP achieved in a given set of experimental conditions is subject to modulation. For example, several neurotransmitter systems have been shown to act to adjust or regulate the degree of hippocampal LTP. In this section, I will focus on the serotonergic system, as it is the neurotransmitter system studied in Chapter 2.  16  1.7.1  Serotonergic system arid receptors in the hippocampus  The expansive serotonergic system is thought to exert a mainly modulatory influence on its numerous targets in the brain (Jacobs & Azmitia, 1992). Dysregulation of the serotonergic system in the central nervous system has been linked to a variety of diseases ranging from depression to nausea. Serotonin (5-hydroxytryptamine, 5-HT) is a monoamine neurotransmit ter synthesized from the amino acid tryptophan by a metabolic pathway comprised of two enzymes: tryptophan hydroxylase (rate-limiting enzyme) and amino acid decarboxylase. Serotonergic neurons are located in raphe nuclei the brain stem (Azmitia & Segal, 1978) and are the principal source of serotonin release in the brain. Serotonin, once released in the extracel lular space, can diffuse over 20 Itm and activates specific 5-HT receptors on nearby neurons. The adult hippocampus in rodents receives substantial innervations from serotonergic projections originating in the rostral raphe nuclei (Kohler & Steinbusch, 1982). Experiments using electron microscopy report that the terminals of the hippocampal 5-HT projections make synap tic contact with both pyramidal cells and GABAergic interneurons (DeFelipe et al., 1991). Over 16 different 5-HT receptors have been cloned to date, all of which are expressed in the central nervous system and most of which can be found in the hippocampus. These receptors are organized into 7 families, 5-HT1 to 5-HT7. With the exception of the 5-HT3 receptor, which is a ligand-gated cation channel, all other 5-HT receptors are G-protein coupled receptors  17  (GPCRs). The 5-HT receptors can mediate excitatory or inhibitory neuro transmission depending on the type of G-protein they are coupled to and the downstream signaling pathways. Receptor families 5-HT1 and 5-HT5 are coupled to Gi/Go protein, and their downstream pathways lead to a decrease in the cellular levels of cAMP. Therefore, the activation of these re ceptors mediates mostly inhibitory neurotransmission. The 5-HT2 receptors are Gq/G11 protein coupled, and their mechanism of action is through an in crease in the cellular levels of inositol triphosphate (1P3) and diacylglycerol (DAG), making the activation of these receptors lead to excitatory neuro transmission. Activation of the ligand-gated 5-HT3 cation channel leads to depolarization of the membrane, and these receptors are thought to mediate excitatory neurotransmission. Finally, 5-HT4, 5-HT6 and 5-HT7 receptors are all coupled to Gs protein, and their activation leads to an increase in cel lular levels of cAMP via the action of adenylyl cyclase, mediating excitatory neurotransmission as well.  1.7.2  Modulation of hippocampal synaptic plasticity by serotonin  In many types of preparations including hippocampal slices, serotonin has been shown to attenuate or block LTP in both the CAl region and the DG region (Corradetti et al., 1992; Stewart & Reid, 2000).  This effect  has been confirmed using selective serotonin reuptake inhibitors (SSRIs), which increase the levels of extracellular serotonin and lead to decreased LTP in both CAl and DG (Stewart & Reid, 2000; Mnie-Filali et al., 2006). In addition, experiments using agonists and antagonists of specific 5-HT 18  receptors have implicated different 5-HT receptor types as mediators of the modulation of synaptic plasticity by serotonin. The effects of serotonin on hippocampal LTP are mostly attributed to its action at the 5-HT1a receptor (Izquierdo & Medina, 1997). Activation of the 5-HT1a receptor results in an inhibition of adenylyl cyclase, a decrease in cAMP levels and an impairment of the activity of PKA, a kinase involved in hippocampal LTP (Izquierdo 8 Medina, 1997). 5-HT1a receptor activa tion is also thought to inhibit CaMKII activity, which in turn affects the phosphorylation of AMPA receptors and their subsequent increased activity (Schiapparelli et al., 2005), a mechanism that is necessary for the expres sion of hippocampal LTP. Furthermore, serotonin has been shown to inhibit TBS-induced activation of NMDA receptor currents in hippocampal slices from rats (Staubli  Otaky, 1994). This effect is hypothesized to be me  diated by a direct hyperpolarization of the pyramidal neurons in the CAl region via activation of the 5-HT1a receptors (Andrade  Nicoll, 1987). In  addition to modulating LTP, the 5-HT1a receptor most likely modulates LTD. Applying a 5-HT1a agonist on slices lead to a decrease in LTD in the CAl region (Normann et al., 2000). The 5-HT3 receptor, the ligand-gated cation channel, has also been shown to act as a modulator of hippocampal LTP. In freely moving rats, a 5-HT3 antagonist enhanced LTP in the CAl region (Stubli & Xu, 1995). A significant body of evidence shows that activation of the 5-HT3 receptor can increase the excitability of GABAergic interneurons in the hippocampus (Oleskevich & Lacaille, 1992). Under normal conditions, GABAb synapses in the hippocampus lead to a prolonged hyperpolarization. Blocking this 19  GABAb-mediated hyperpolarization enhanced the magnitude of LTP in duced by TBS (Arai & Lynch, 1992). Therefore, it is hypothesized that the 5-HT-mediated enhancement in GABAergic transmission is in part re sponsible for the decrease in NMDA receptor currents and LTP observed following TBS (Staubli & Otaky, 1994). Not all 5-HT receptors have inhibitory actions on hippocampal synaptic plasticity. A study of LTP in the DG of freely moving rats revealed that blocking the 5-HT4 receptor led to a decrease in LTP (Kulla & Manahan Vaughan, 2002). In accordance with this study, a recent report suggests that the activation of the 5-HT4 receptor serves to consolidate certain forms of LTP (Huang & Kandel, 2007). Finally, some families of 5-HT receptors still don’t have a clearly estab lished role in the modulation of hippocampal LTP. Experiments using mice lacking the 5-HT2c receptor show decreased LTP in the DG, but normal LTP in the CAl (Tecott et al., 1998). Conversely, a study using a 5-HT2a antagonist applied on hippocampal slices showed increased LTP in the CAl (Wang & Arvanov, 1998). While these two receptors share some common signaling pathways, the specific mechanisms that take place following their activation and how these mechanisms may modulate synaptic plasticity are unclear. Overall, 5-HT is thought to negatively modulate LTP in the hippocam pus. However, this modulation greatly depends on the types of receptors activated and the model studied.  20  1.7.3  The 5-HT6 receptor  The 5-HT6 receptor has been shown to be involved in memory processes (see section 1.7.4), and thus in Chapter 2 the involvement of this receptor in hippocampal synaptic plasticity was investigated. The 5-HT6 receptor was first isolated from the rat brain in 1993 (Monsma et al., 1993), and the human 5-HT6 receptor was cloned for the first time in 1996 (Kohen et al., 1996). Like most other 5-HT receptors, the 5-HT6 receptor is a 7 transmembrane domains G-protein coupled receptor (Monsma et al., 1993). While it displays strong homology to the other 5-HT GPCR, the 5-HT6 receptor also has distinguishing features. Unlike the other 5-HT receptors, the 5-HT6 subtype has a glycosylation site on its N-terminal tail as well as conserved cysteine residues that may lead to the formation of a disulfide bond that would link the first and the second extracellular loops (Woolley et al., 2004). In addition, the third intracellular loop and the Cterminal region of the 5-HT6 receptor contain a number of sites destined to phosphorylation by protein kinase C (PKC) (Woolley et al., 2004). The 5-HT6 receptor gene contains two introns but generates no functional splice variants (Woolley et al., 2004). The 5-HT6 receptor is positively coupled to adenylyl cyclase, and its activation leads to an accumulation of cAMP (Monsma et al., 1993; Ruat et al., 1993), and a subsequent activation of PKA (Woolley et al., 2004). In terms of distribution, the 5-HT6 receptor is localized almost exclu sively in the central nervous system (Woolley et al., 2004).  The 5-HT6  receptor is particularly abundant in brain regions involved in regulating  21  cognitive processes and is present in all regions of the hippocampus (CAl, CA2, CA3, DG) (Ruat et al., 1993). The distribution of the 5-HT6 receptor has been shown to be very similar in rats and in humans (Woolley et al., 2004). However, the levels of 5-HT6 receptors in the mouse brain are sig nificantly reduced when compared to those in the rat (Hirst et al., 2003), which is an important consideration when using mouse models to study this receptor. Since its discovery in the 1990s, several agonists and antagonists of the 5-HT6 receptor have been discovered and developed. Interestingly, the phar macological profile of this receptor demonstrates a high affinity for a vari ety of typical and antitypical antipsychotic compounds, such as loxapine and clozapine, as well as for tricyclic antidepressants (Monsma et al., 1993; Woolley et al., 2004). These characteristics suggest a role for the 5-HT6 receptor in mood and emotion regulation.  1.7.4  The 5-HT6 receptor and memory  Due to the discovery that a polymorphism (C267T) of the 5-HT6 receptor may confer an increased risk for Alzheimer’s disease (Tsai et al., 1999), there has been widespread interest in the preclinical and clinical study of the ef fects of pharmacological manipulation of the 5-HT6 receptor on cognition, memory and behavior. This interest is strengthened by the localization of the 5-HT6 receptor in limbic and cortical regions. Initial studies looking at the role of the 5-HT6 receptor in memory used 5-HT6 receptor antisense oligonucleotides to knock down the receptor (Upton et al., 2008; Woolley et al., 2001). In rats, this treatment led to an enhancement of both learning 22  and memory in the Morris water maze task (Upton et al., 2008; Woolley et aL, 2001). The improved memory retention in this paradigm was con firmed by using a 5-HT6 receptor antagonist (Woolley et al., 2001). The Morris water maze was also used to assess age-induced memory impair ments. In aged rats, treatment with a 5-HT6 receptor antagonist reversed the impairments in both acquisition and retention in the water maze task (Upton et al., 2008; Foley et al., 2004). However, it is important to men tion that the results obtained with the water maze task, either in adult or aged rats, were not easily replicated by other groups (Russell & Dias, 2002; Lindner et al., 2003; Upton et al., 2008). Using dfflérent behavioral tasks and experiment al paradigms, several studies have confirmed the role of 5-HT6 receptor antagonists as cognitive enhancers. For example, two different 5-HT6 receptor antagonists signifi cantly increased the performance of normal adult rats on the novel object recognition task (King et al., 2004).  Interestingly, the administration of  an NMDA receptor antagonist prior to the novel object recognition task in 5-HT6 receptor antagonist-treated rats blocked the effect of the 5-HT6 antagonist (King et al., 2004). This result highlights the possibility that the 5-HT6 receptor mediates its effects on cognition via the modulation of glutamatergic transmission. In humans, preliminary data from clinical trials suggest that not only are 5-HT6 antagonists safe and well-tolerated, they may also improve both cognitive and global function in patients suffering from Alzheimer’s disease. The mechanisms mediating the effects of the 5-HT6 receptor are still unclear, but microdialysis studies have shown that blockade of the 5-HT6 23  receptor leads to an enhancement of both cholinergic and glutamatergic neurotransmission in the hippocampus (Zhang et al., 2007; Dawson et al., 2000, 2001). In addition, 5-HT6 receptors are known to be expressed on GABAergic neurons in several areas of the brain including the hippocam pus (Woolley et al., 2004), and administration of a 5-HT6 receptor agonist leads to increases in extracellular GABA in the hippocampus (Upton et al., 2008). Overall, a model has been suggested where tonic activation of the 5-HT6 receptors located on GABAergic hippocampal neurons leads to the extracellular release of GABA and the subsequent decrease in the activity of nearby glutamatergic and cholinergic neurons (Upton et al., 2008).  1.8  LTP in the aged mammalian hippocampus  Among the numerous factors that can affect hippocampal synaptic plasticity, aging is known to play a key role in the modulation of LTP. The impact of aging on hippocampal LTP is studied in Chapter 3.  1.8.1  Impact of aging on the hippocampus  It was initially believed that age-related cognitive and memory impairments  were due, at least in part, to a decrease in neuron numbers in the brain, including in the hippocampus (Brody, 1955). However, the recent develop ment of improved stereological techniques has shown that significant neil ronal loss in the hippocampus and cortex is not a characteristic of normal aging (Burke & Barnes, 2006). Morphologically, aging is thought to affect the hippocampus differently depending on which specific region is studied.  24  In the DG, aging leads to fewer synapses between the axon collaterals com ing from the enthorinal cortex and the granule cells (Kelly et al., 2006). However, there appears to be a compensatory mechanism in place as the re maining synaptic contacts are more powerful for a given input (Kelly et al., 2006). In area CAl, while there appears to be no change in the total num ber of synapses with aging, there is evidence for a reduction in functional synaptic contacts, but no change in the efficacy of individual synapses (Kelly et al., 2006). In contrast with the changes described above, some age-related mor phological changes occur uniformly in the hippocampus. For example, ag ing leads to an increase in gap-junction connectivity between hippocampal neurons, which in turn may lower the threshold for action potential firing (Barnes et al., 1987). While the hippocampus is particularly vulnerable to the process of aging, many physical properties of hippocampal neurons remain unchanged with age, including resting membrane potential, input resistance, height of the action potential, time constant and EPSP rise time and half-width (Kelly et al., 2006). However, a number of studies report an increase in calcium conductance in aged hippocampal neurons, which led to the calcium dys regulation hypothesis of aging (Thibault et al., 2007). A first evidence for elevated neuronal calcium influx is the increased amplitude and duration of the afterhyperpolarization (AHP) that follows action potentials in CAl neu rons (Landfield & Pitler, 1984). This larger AHP could suggest that CAl neurons are less excitable, but no age-related changes in the in vivo firing rates of pyramidal neurons have been found (Burke 8 Barnes, 2006). This 25  may be due to either compensatory mechanisms or other age-related changes such as the increased gap-junction connectivity described above (Kelly et al., 2006). Studies using single-channel patch clamp recordings from neurons in hip pocampal slices and measurements of calcium potentials and currents con sistently show elevated L-type calcium currents with aging (Pitler & Landfield, 1990). Furthermore, this increased activity of L-type calcium channels seems to be relevant at a functional level, because blocking these channels with antagonists can improve learning and memory in aged animals (Deyo et al., 1989; Disterhoft et al., 2004). It is thought that this increase in the activity of the L-type calcium channels is the main mechanism underlying age-related calcium dysregulation. In addition, recent evidence suggests a direct, physical link that favors the alignment of the L-type calcium channel with ryanodine receptors (RyR) (Kim et al., 2007). During aging, increased activity of L-type calcium channels may lead to enhanced CICR from RyRs and an amplification of calcium transients, further contributing to calcium dysregulation (Thibault et al., 2007). While there appears to be no age-related decline in the density of hip pocampal N1VIDA receptors, calcium influx via these receptors is reported to be reduced in aged animals (Barnes et al., 1997; Magnusson, 1998; Shankar et al., 1998; Rosenzweig & Barnes, 2003). This may be explained by the age-related decrease in NMDA binding on the receptor in the hippocampus, a finding that has been documented in a number of animal models ranging from mice to monkeys (Tamaru et al., 1991; Wenk et al., 1991; Magnusson & Cotman, 1993). 26  1.8.2  Impact of aging on hippocampal LTP  Aging is thought to affect synaptic plasticity in aged animals but only un der certain conditions. Four variables need to be considered when studying synaptic plasticity in aged animals: 1) the intensity of the induction stimu lus; 2) whether the induction or the maintenance is being measured; 3) the region studied (for example, CAl region or DG) and 4) the model (in vitro or in vivo) (Rosenzweig & Barnes, 2003).  Induction of LTP Experimental protocols using robust, high-frequency stimulation show that LTP is intact in the hippocampus of aged rats (Diana et al., 1994; Landfield & Lynch, 1977). However, this is a controversial finding, as other studies also using a high-frequency protocol show deficits in both CAl and DG LTP (Griffin et al., 2006). The variability of these results could be attributed to differences in rat strains and stimulation protocols, as a number of variations on the standard HFS and TBS protocols are used in the literature (see Table 1.1). When weaker conditioning stimuli are used, involving either fewer stimulus pulses or lower amplitude currents to induce LTP, aged rats consistently show deficits in CAl LTP (Deupree et al., 1993; Rosenzweig et al., 1997).  Maintenance of LTP When the maintenance of LTP is studied in a context where robust, high intensity stimulation is used, there seems to be no age-related change in LTP  27  decay rates during the first hour post-conditioning (Landfield & Lynch, 1977; Deupree et al., 1993). However, age-related deficits in LTP maintenance start to appear when a longer time scale is studied. For example, a study looking at LTP in vitro in aged mice uncovered LTP maintenance deficits starting at 3 hours post-conditioning (Bach et al., 1999).  L-type calcium channel-dependent LTP In 1990, Grover and Teyler discovered a new form of LTP that was inde pendent of NMDA receptors (Grover & Teyler, 1990). This form of LTP can be induced in the presence of an NMDA receptor antagonist using a high-intensity conditioning protocol. It was established that this type of LTP depends on calcium influx through voltage-dependent L-type calcium channels (Grover & Teyler, 1990). This new form of LTP shares many im portant characteristics of NMDA receptor-dependent LTP such as synapse specificity (Grover & Teyler, 1992). However, it is also distinct in some ways. For example, while NMDA receptor-mediated LTP requires the activation of serine-threonine kinases (Malinow et al., 1988; Malenka et al., 1989), L-type calcium channel-dependent LTP requires the activation of tyrosine kinases (Cavus & Teyler, 1996). It is suggested that in most experimental condi tions, a standard high-intensity conditioning protocol will elicit a compound LTP containing an NMDA receptor-mediated component as well as an L type calcium channel-mediated component, though the contribution of each mechanism may vary. In aged animals, there is an increase in the magnitude of L-type cal ciurn channel-mediated LTP (Shankar et al., 1998). This increase is con 28  sistent with the elevated L-type calcium channel currents in aged animals described above. Interestingly, when either L-type calcium channel block ers such as nifedipine or tyrosine kinase inhibitors are used to block this form of LTP, an age-related impairment in NMDA receptor-mediated LTP is uncovered (Rosenzweig  Barnes, 2003). This finding suggests that the  initial studies looking at LTP in aged animals using a strong stimulation protocol may have failed to detect an impairment because the decrease in NMDA receptor-mediated LTP was compensated by an increase in L-type calcium channel-mediated LTP. When a lower-intensity conditioning proto col is used, however, no L-type calcium channel-dependent LTP is elicited, and an impairment in NMDA receptor-dependent LTP can be detected in aged animals (Deupree et al., 1993; Rosenzweig et al., 1997). Overall, the results from studies of LTP in aged rodents suggest that there is a shift in plasticity mechanisms with aging, going from mostly NMDA receptordependency in adult animals to mostly L-type calcium channel-dependency in aged animals.  1.9  Modulation of LTP by oxidative stress  1.9.1  Introduction to oxidative stress in the brain  One theory of aging stipulates that an increase in oxidative stress in the brain is the contributing factor to cognitive and memory impairments. In Chapter 3, this theory is explored by looking at synaptic plasticity during aging while modulating the levels of antioxidants in neurons. In humans, the brain represents approximately 2% of total body weight 29  but accounts for 20% of the oxygen requirements in the body. It is therefore no surprise that the brain is one of the organs generating large quantities of reactive oxygen species (ROS). A portion of the oxygen consumed by mito chondria is diverted to form superoxide, an anion and free radical (Chance et al., 1979). The enzyme superoxide dismutase (SOD) can convert superoxide to hydrogen peroxide, which is then converted to water and oxygen by either GSH peroxidase (GPx) or catalase (Dringen, 2000). However, hy drogen peroxide can also lead to the formation of lipid peroxidation-causing hydroxyl radicals by reacting with iron (Youdim et al., 1989). The brain also harbors large amounts of nitric oxide (NO), one of the few gaseous signaling molecules known, produced mainly by the neuronal NO synthase (nNOS). While neither superoxide nor NO is toxic, the reaction of the two compounds together generates peroxynitrite, a highly reactive and toxic ox idant agent (Pacher et al., 2007). Accumulation of peroxynitrite leads to many detrimental effects in cells, including oxidation of proteins, DNA and lipids, nitration of amino acids such as tyrosine, and inactivation of mito chondrial enzymes, which leads to decreases in energy production (Pacher et al., 2007). The brain is particularly vulnerable to oxidative stress due to low SOD, catalase and GPx activities and due to its abundance of targets for lipid peroxidation (Dringen, 2000). The brain also has a low OSH concentration compared with the liver, kidney, spleen or small intestine (Commandeur et al., 1995). GSH acts as a major antioxidant in the brain (Dringen, 2000) and the intracellular GSH levels are essential in limiting oxidative stress induced neuronal injury. Decreases in brain GSH concentration leads to 30  increased production of superoxide, hydrogen peroxide and hydroxyl radicals (Gupta et al., 2000).  1.9.2  Glutathione  Glutathione synthesis and metabolism GSH is a tripeptide formed of glutamate, cysteine and glycine. The synthesis of GSH occurs in two enzymatic steps, each requiring adenosine triphosphate (ATP). ‘y-glutamylcysteine ligase (GCL) catalyzes the first reaction between glutamate and cysteine to form ‘y-glutamylcysteine (7GluCys), a dipeptide (Dringen, 2000). This is the rate-limiting enzymatic step. Subsequently, -yGluCys reacts with glycine to produce GSH via the GSH synthetase enzyme (GS) (Dringen, 2000). Feedback inhibition of GCL by GSH allows the GSH synthesis to be self-regulating (Richman & Meister, 1975). The rate-limiting substrate for the neuronal synthesis of GSH is cysteine (Dringen et al., 1999). When GSH is involved in the detoxification of ROS, two types of reac tions can occur. In the first type, GSH reacts in a non-enzymatic manner with radicals (such as hydroxyl radical or NO) (Clancy et al., 1994). In the second type of reaction, GSH acts as an electron donor for the reduction of peroxides (Chance et al., 1979). The final product of GSH oxidation is glutathione disulfide (GSSG). From GSSG, GSH can be regenerated by an NADPH-requiring reaction catalyzed by the glutathione reductase (GR) en zyme (Dringen, 2000). The ratio of reduced GSH over oxidized GSSG can serve as an indicator of the cellular redox level (Schafer & Buettner, 2001). For an overview of GSH metabolism, see Figure 1.1.  31  Functions of glutathione in the brain In Chapter 3, the relationship between neuronal levels of GSH, the main endogenous antioxidant in the brain, and hippocampal LTP during aging is assessed. GSH is quite abundant in the nervous system and can be found in concen trations in the low millimolar range in tissues (Slivka et al., 1987). GSH acts as the major antioxidant in the brain and has many functions in maintain ing a healthy redox balance (Dringen, 2000). First, as previously mentioned, GSH reacts in a non-enzymatic manner as a scavenger of free radicals, in cluding NO (Clancy et al., 1994), superoxide (Winterbourn  Metodiewa,  1994), hydroxyl radical (Bains & Shaw, 1997) and peroxynitrite (Koppal et al., 1999). Because there is no enzymatic defense against hydroxyl rad icals, GSH has a very crucial role in scavenging these radicals (Bains & Shaw, 1997). A second role for GSH is to act as an essential cofactor for different enzymes. For example, GSH is involved in the reduction of hy drogen peroxide by acting as an electron donor for the catalytic action of GPx (Chance et al., 1979). GSH also contributes to the detoxification of the cellular environment by reacting with highly reactive aldehydes (Xie et al., 1998). Third, GSH serves as a form of cysteine storage. Cysteine can me diate many toxic effects such as increasing extracellular levels of glutamate, which leads to an increased activation of NMDA receptors, and the gener ation of free radicals (Janky et al., 2000). Therefore, GSH plays a crucial role by forming a non-toxic storage system for cysteine. A fourth role for GSH in the brain is the maintenance of redox homeostasis by acting as a  32  major antioxidant. Through S-glutathionylation, a process by which GSH reversibly forms disulfides between protein groups, GSH helps prevent ir reversible oxidation of proteins (Giustarini et al., 2004). Finally, GSH can act as a neurotransmitter, namely by binding to NMDA receptors (Janky et al., 1999). GSH can act as an agonist or an antagonist for neuronal sig nals mediated through NMDA receptors. This interaction will be reviewed in greater detail in the following section.  Glutathione modulation of NMDA receptors GSH as an agonist/antagonist of NMDA receptors GSH can modulate the activity of NMDA receptors by directly interact ing with these receptors and acting as either an agonist or an antagonist. Pharmacological studies have shown that GSH can displace the binding of [ H]glutamate to synaptic membranes of the rat brain (Koller & Coyle, 3 1985). In addition, GSH can also inhibit the binding of DL-(E)-2-aminoj CGP 39653), a labeled radioligand ([ H 4-propyl-5-phosphono-3-pentenoate 3 (Ogita et al., 1998). Thus, GSH displays affinity for the NMDA recognition domain of the receptor. It remains unclear whether GSH acts as an agonist Hjglutamate 3 or an antagonist at the NMDA receptor. GSH inhibition of [ HJCGP 39653 3 binding is of greater magnitude that GSH inhibition of [ (Ogita et al., 1998). Under normal conditions, NMDA receptor agonists HICGP 3 Hjglutamate binding than of [ 3 lead to a greater displacement of [ 39653. Conversely, NMDA receptor antagonists are more potent at displac ing [ H]CGP 39653 binding (Zuo et al., 1993). Pharmacological studies have 3 confirmed that GSH-mediated potentiation of NMDA receptors cannot be 33  attributed to glutamate derived from the cleavage of GSH (Ogita et al., 1998). These findings lead to the suggestion that GSH acts as an agonist at the NMDA receptor. However, the specific action at the receptor may depend on the concentration of GSH. At low concentrations, GSH has been found to act as an agonist or an antagonist, but at high concentrations, GSH acts as an agonist (Janky et al., 1999). The discrepancy between the results of various laboratories on whether GSH acts as an agonist or an antagonist at NMDA receptor can also possibly be attributed to the different NMDA receptor subunits that can be found in different cell types (Nakanishi Masu, 1994), but this has not yet been studied in detail.  GSH as a reducing agent of NMDA receptors In addition to acting as an agonist or an antagonist, GSH can impact the activity of NMDA receptors through its action as a reducing agent. NMDA receptor activity is sensitive to the redox environment and can be decreased by oxidizing agents and increased by reducing agents (Tang  Aizenman,  1993; Choi & Lipton, 2000). While several reports show that GSH depletion has no effect of basal synaptic transmission (Steullet et al., 2006; Almaguer Melian et al., 2000), GSH depletion does cause a significant decrease in NMDA receptor-mediated field excitatory postsynaptic potentials (fEPSPs) (Steullet et al., 2006). These effects are thought to be caused by a major oxidation of the redox-sensitive sites of NMDA receptors which then leads to a decrease in the activity of these receptors. Additional pharmacological experiments using DTNB, a membrane-impermeable thiol-oxidizing agent, have confirmed this by showing that the redox sites of NMDA receptors are maximally oxidized in a GSH-depleted environment (Steullet et al., 2006). In 34  addition, control experiments have confirmed that GSH depletion does not lead to a decrease in the total number of NMDA receptors in the hippocam pal preparation, which could have explained some of the effects observed (Steullet et al., 2006). Amino acids in the primary structure of the NMDA receptor are thought to be the entities responsible for redox modification of the receptor. More specifically, three pairs of cysteine residues in the extracellular region of subunit NMDAR1 and NMDAR2A are thought to be susceptible to redox modulation (Sullivan et al., 1994). The precise site and mechanism of redox modulation of the NMDA receptor will be discussed in greater detail in the following section.  GSH as a mode of S-nitrosylation prevention Oxidizing intracellular agents, as opposed to reducing agents, lead to the formation of NO, a potent inhibitor of NMDA receptor activity via 5nitrosylation. Since GSH is one of the main reducing agents in the brain, it plays a role not only as a reducing agent of NMDA receptors but as a modulator for other types of redox modifications at the NMDA receptors. 5-nitrosocysteine (SNOC), an endogenous NO donor, has been shown to decrease NMDA receptor-mediated currents through S-nitrosylation (Lip ton et al., 1993). In normal conditions, the addition of L-arginine to the extracellular environment as a substrate for the generation of NO by the nitric oxide synthase (NOS) leads to a downregulation of NMDA recep tor activity (Takahashi et al., 2007). Electrophysiological experiments have uncovered six cysteine residues on the extracellular portion of the NMDA receptor involved in redox modulation by reducing agents as well as by ox idizing agents, such as NO (Lipton et al., 2002). These cysteine residues 35  are thought to work in pairs, as mutagenesis experiments have shown that mutating one member of a given pair leads to the same outcome as mutat ing both members of the cysteine residues pair (Choi et al., 2000). These results suggest that the formation of a disulfide bond occurs between the members of cysteine pairs (Choi et al., 2000). The specific cysteine residues are the following: Cys79 and Cys308 (NR1 subunit), Cys744 and Cys798 (NR1 subunit) and Cys87 and Cys320 (NR2A subunit) (Lipton et al., 2002). All these residues are located on the N-terminal regulatory domain of the NMDA receptor, the portion of the receptor that controls its activity (Choi et al., 2000). During oxidative stress, S-nitrosylation of the NMDA recep tor on those critical cysteine residues leads the formation of disulfide bonds between the residues and this in turn leads to a decrease in the activity of the receptor.  Manipulation of GSH levels in the brain Several pharmacological compounds allow for the manipulation of the GSH content in both astroglial cells and neurons (Dringen, 2000). In cultures and in slices, inhibitors of GSH synthesis such as buthionine sulfoxide (BSO) as well as sulfhydryl reagents such as ethacrynic acid are commonly used to decrease GSH levels in both astroglial cells and neurons (Dringen & Ham precht, 1997; Huang & Philbert, 1996; Wllner et al., 1999). Conversely, un der the same experimental conditions, glutamylcysteine is commonly used to increase GSH levels in astroglial cells and neurons (Pileblad et al., 1991; Dringen et al., 1999), and cysteine and NAC have been shown to increase GSH levels specifically in neurons (Dringen & Hamprecht, 1999; Kranich 36  et al., 1996). In whole animal studies, BSO is the most commonly used compound to deplete glutathione levels in a variety of organs (Watanabe et al., 2003). As for supplementation, many considerations need to be taken. If administered orally or intravenously, GSH is rapidly hydrolyzed (Aebi et al., 1991). While some reports suggest the presence of a transporter for GSH at the bloodbrain barrier (Kannan et al., 1996), the mechanism of this transport is un known and it is generally accepted that GSH does not cross the blood-brain barrier readily (Jam  et al., 1991). Cysteine, the rate-limiting enzyme for  the synthesis of glutathione, is also catabolized in the gastrointestinal tract and in blood plasma (Lavoie et al., 2008). Furthermore, cysteine is poten tially toxic, and can lead to overactivation of NMDA receptors, which leads to increased glutamate neurotoxicity (Puka-Sundvall et al., 1995). Recent studies have therefore been using NAC as a cysteine donor and precursor for GSH synthesis (Figure 1.2). When administered orally, NAC is absorbed rapidly and crosses the blood-brain barrier. To date, several animal studies have confirmed that systemic administration of NAC can protect the brain against a depletion of GSH levels (Aydin et al., 2002; Ercal et al., 1996; Fu et al., 2006; Kamboj et al., 2006).  1.9.3  Oxidative stress and GSH in the aged brain  The oxidative stress theory of aging stipulates that an increase in the damage caused by ROS is a critical determinant of aging pathophysiology and life span (Beckman & Ames, 1998).  During aging, the increase in oxidative  stress can be explained by an increase in oxidant generation, a decrease in 37  antioxidant defenses, or a combination of both. A first source of increased oxidative stress in aging is dysfunctional mito chondria. One theory suggests that mitochondrial DNA (mtDNA) lies very close to the cell’s major generators of free radicals and is therefore particu larly susceptible to oxidative damage. This proximity leads to an increase in mutations in mtDNA, which in turn leads to aerobic respiration dysfunc tion and the generation of ROS (Genova et al., 2004). This vicious cycle is thought to play a central role in the oxidative stress theory of aging. There are also non-mitochondrial sources of increased oxidants in aging. For example, aging is associated with an increase in iron content in the body, which favors the Fenton reaction, a non-enzymatic conversion of hydrogen peroxide which leads to the generation of DNA and RNA-damaging hydroxyl radicals (Liu et al., 2004). In addition, hippocampal slices from aged rats produce more hydrogen peroxide than slices from younger rats (Auerbach & Segal, 1997). In parallel with an increase in oxidants, aging may lead to a decrease in antioxidants. However, studies looking at enzymatic antioxidant agents such as catalase, SOD and GSH peroxidase are inconsistent and inconclusive. In creases, decreases and lack of change in antioxidant levels have been found in aged animals (Serrano & Klann, 2004). To further complicate these findings, an increase in antioxidant levels in aged animals could be interpreted as an adaptation to counteract an increased production of oxidants. In contrast with enzymatic antioxidant agents, a clear picture emerges when looking at non-enzymatic antioxidant agents. Aging leads to a decrease in the levels of critical antioxidants such as GSH, melatonin and tocopherol (Gilca et al,, 38  2007). The age-related decrease in GSH levels is thought to result from a decrease in the activity of the activity of GCL (Sandhu & Kaur, 2002). More specifically, the decrease in activity of GCL is suggested to be caused by a decrease in the level of the transcription factor Nrf2, which is involved in the induction of the genes encoding GCL (Suh et al., 2004). Therefore, in aged rats, the basic transcriptional mechanism for the production of GSH is still present, but turned down (Maher, 2005). In addition to the decrease in GCL activity, is it also possible that the age-related GSH depletion results from a change in GS activity (Liu & Dickinson, 2003). Overall, aging leads to an imbalance between the cellular generation of ROS and the antioxidant defenses that remove them. The accumulation of oxidative damage in aged animals leads to enhanced lipid peroxidation (Calabrese et al., 2004), enhanced protein oxidation (Forster et al., 1996), and increased damage to nuclear and mitochondrial DNA (Hamilton et al., 2001).  1.9.4  Impact of oxidative stress on LTP  Over the recent years, the involvement of ROS in hippocampal synaptic plasticity in young and adult animals has been demonstrated. Evidence is especially consistent for the role of superoxide as a cellular messenger dur ing LTP. In hippocampal slices from rats, the activation of NMDA receptors leads to an increase in the generation of superoxide (Bindokas et al., 1996). Furthermore, scavengers of superoxide can block or attenuate HFS-induced hippocampal LTP (Klann, 1998). Conversely, incubating hippocampal slices with a superoxide-generating system leads to an increase in synaptic trans 39  mission that occludes LTP (Knapp  Klann, 2002).  In addition to superoxide, hydrogen peroxide may also play a role in synaptic plasticity. Incubating hippocampal slices with catalase decreases hippocampal LTP (Thiels et al., 2000). However, the involvement of hy drogen peroxide in LTP is somewhat controversial and may depend on the concentration of hydrogen peroxide in the preparation. When high concen trations (0.5-1 mM) of hydrogen peroxide are applied on hippocampal slices, LTP is impaired (Auerbach & Segal, 1997). However, when low concentra tions (1 M) are applied on hippocampal slices, LTP is enhanced (Kamsler & Segal, 2003). Overall, these findings suggest that the involvement of ROS in hippocampal LTP in young to adult animals is variable and depends on which ROS is studied and at what concentration. In aged animals, there is an increase in the levels of ROS in the brain, and this is suggested to lead to the age-related impairments in hippocampal synaptic plasticity. Some evidence suggests that application of hydrogen peroxide on aged hippocampal slices reverses the age-related impairments in LTP seen in control slices (Kamsler & Segal, 2003). However, other studies report that LTP in aged animals appears to be unchanged by exogenously applied hydrogen peroxide, even when high concentrations are used (Watson et al., 2002). The discrepancy between those results may be explained by the type of conditioning protocol used to elicit LTP (HFS versus TBS), as different stimulation protocols may trigger different signaling cascades which are then differentially susceptible to hydrogen peroxide. In spite of this inconsistency, the application of ROS onto aged hippocampal slices did not alter LTP as was seen in younger animals (Kamsier & Segal, 2003). 40  Several mechanisms that might explain the link between ROS and agerelated LTP impairments have been investigated. One theory stipulates that ROS can affect signaling enzymes such as phosphatases, and thereby impact LTP. For example, the activity of the phosphatase calcineurin is increased in aged animals, and this increase appears to lead to a decrease in LTP but an increase in LTD (Foster, 2002). Another ROS-mediated mechanism that could explain LTP impairments in aged animals is lipid peroxidation. Lipid membranes are an important target for lipid peroxidation and this leads to changes in the properties of the membranes, which can in turn affect LTP (Lynch, 1998). Finally, insights into the mechanisms of ROS-mediated LTP impairments were gained from studies looking at aged animals fed with antioxidant supplements. While control aged animals show increases in the levels of ROS, c-Jun N-terminal kinase (JNK) and p38, antioxidant-fed aged animals show significant decreases in the levels of these three compounds compared with aged animals (Martin et al., 2002).  Consequently, it has  been suggested that ROS-mediated increases in JNK and p38, which leads to a decrease of glutamate release, could be involved in causing age-related LTP impairments (Martin et al., 2002).  Overall, a number of key LTP  components may be modulated by an increase in ROS during aging.  41  1.10  Figures  42  cell membrane  GSSG  Y Glu  rvGluCvs Gly Cys  GSH  GSY  GS-Y  Y-CysGly GSH yGluX X  Figure 1,1: Metabolism of glutathione. X represents an acceptor of the glutamyl moiety transferred by -yGT from GSH, Y represents a substrate of GSH-S-transferases. 1, 7-glutamylcysteine ligase; 2, GSH synthetase; 3, GSH peroxidase; 4, GSH reductase; 5, GSH-S-transferase; 6, ‘y-glutamyl transpeptidase; 7, ectopeptidase. Adapted from Dringen (2000) -  43  yGIuX Y-CysGIy  Figure 1.2: Impact of N-acetylcysteine on glutathione metabolism. N acetylcysteine acts as a cysteine donor for the formation of GSH. X repre GT from GSH, sents an acceptor of the 7-glutamyl moiety transferred by 7 Y represents a substrate of GSH-S-transferases. 1, 7-glutamylcysteine hg ase; 2, GSH synthetase; 3, GSH peroxidase; 4, GSH reductase; 5, GSH-S transferase; 6, y-glutamyl transpeptidase; 7, ectopeptidase. Adapted from Dringen (2000) Dringen (2000).  44  1.11  Tables  45  ia K  Modelfage  Reference  LTP (compared to uug  Conditioidnaa protocol(s)  Area  -  •  Pc  Moore et a]. 1993  Fischer 344 24-2t months  Diana et at 1994  SpragueDawley and Fischer 344 20-24 months  DO  Fischer 344 4 mon ths  CAl  Fischer 344 24 months  C’’ 1  Fischer344 24 months  CAl  -,  Norris et a!.  (Al  -—  .  Rosenzsveig 199 4 Shankaretal. 1998 uonget  15t111  ‘  Kumar and Foster 2005  Fischer 344 2_-4 months  Billiard and Rottaud 2007  CAl  LTP(l): 4pulses at 200Hz. primed (primed burstj LTP(2):lsat200Hz LTP: is at 100Hz LTP: 2 X 100 pulses at 100Hz (lOs mterval) LTD 900 pulses. 1Hz LI’? (11): Primed burst (see Moore et at) LTP (2): 4 pulses at 100Hz LTP: 40.Ssninsat 200Hz (5s interval) LTP:lOX2opulsesat 200Hz (2s interval) LTD: 900 pulses at 3Hz  touttols)  LTD (compared to .oung controls)  Pnmed burst protocol. impaired protocol: mtact —  intact  intact  intact Impaired with both protocoic he ac Abolished  Intact intact  CAl  LTD 900 pulses at 1Hz  SpragueDa’wley 23-27 months  (‘Al  LIT: S X 4 pulses at 100Hz (200 ms interval) repeated 4 rimes JBS) LTD: 900 pulses at 1Hz  Impaired  Impaired  Authors  Modeliaae a  Area  Conditiomna protocol(s)  LTP  LTD  Watabe and O’Dell 2003  C7BLi6 , 4-_8 months  (‘Al  150 pulses at 5Hz  Intact  Frocetal. 2003  CS7BL’ó 12-24 months  DO  LTP:4X50 pulses at 100Hz (30s interval)  Abolished  t W atson et al. 2006  C’57BL16 22-26 months  Table 1.1: ticity  in  aij CM  X is at 100Hz  impaired  Relevant studies looking at the effects of aging on synaptic plas  vitro  compared  .‘  in wild-type animals.  with  Effects on  LTP  and  LTD  described as  young controls,  46  Chapter 2  The Role of the 5-HT6 Receptor in Hippocampal Synaptic Plasticity in the rat 2.1  Introduction  Serotonin (5-hydroxytryptamine, 5-HT) regulates a variety of functions and is implicated in numerous pathologies ranging from nausea to depression. In addition, serotonin is thought to exert critical modulatory influence on learning and memory processes by acting on numerous receptors and in teracting with other neurotransrnitter systems including glutamatergic and cholinergic transmission. The serotonergic neurons of the raphe nuclei are the principal source of serotonin release and extent their projections to most brain regions, includ ing the hippocampus (Jacobs & Azmitia, 1992). The expansive serotonergic system can have inhibitory or excitatory actions depending on the target brain region, the receptor subtype activated, and the receptor-effector cas cades activated (Jacobs & Azmitia, 1992).  47  Serotonin has been implicated in the processes underlying memory for mation in the mammalian brain (Gu, 2002), and evidence suggests the 5HT6 receptor may be especially critical for certain forms of memory. In the rat, the 5-HT6 subtype is mainly expressed in the CNS, with increased expression in the hippocampus, the cortex, the striatum, the olfactory tu bercules and the nucleus accumbens (Ruat et al., 1993; Grard et al., 1996). This specific distribution pattern suggests an involvement of the 5-HT6 re ceptor in cognitive processes. In recent years, there has been increasing interest for this receptor as it has been shown to be implicated in cognition (Mitchell & Neumaier, 2005), memory performance (Lieben et al., 2005) and pathologies such as Alzheimer’s disease (Tsai et al., 1999), a disorder char acterized by memory deficits. A recent study showed that in aged rats, oral administration of a 5-HT6 antagonist reversed the age-dependent deficits in the water maze task, suggesting that this receptor is involved in spatial memory (Hirst et al., 2006). Furthermore, this same antagonist reversed scopolamine-induced deficits in a novel object recognition task (Hirst et al., 2006). Long-term potentiation (LTP) and long-term depression (LTD) are phys iological models of learning and memory (Bliss & Collingridge, 1993). Both 5HT receptor agonists and antagonists have been used to study the in volvement of different 5-HT receptor subtypes in LTP and LTD. In general, increasing serotonin levels in the hippocampus appears to decrease LTP expression in both the CAl and dentate gyrus (DG) subfields of the hip pocampus (Stewart & Reid, 2000; Mnie-Filali et al., 2006), while lesions to the raphe nucleus increase LTP expression (Ohashi et al., 2003). This 48  general effect has been shown mostly using selective serotonin reuptake in hibitors (SSRIs). These drugs lead to an increase in extracellular serotonin and have been shown to decrease the magnitude of LTP in both the CAl and the DG (Stewart  Reid, 2000; Mnie-Filali et al., 2006).  When looking at specific subtypes of 5-HT receptors, the situation is less clear. Pharmacological studies using agonists and antagonists to assess the involvement of a variety of 5-HT receptor subtypes in hippocampal synaptic plasticity have yielded mixed results. For example, activation of the 5HT1a receptor subtype decreases LTP and has been shown to block LTD induc tion in hippocampal slices (Normann & Clark, 2005; Normann et al., 2000; Izquierdo & Medina, 1997). In contrast, both 5HT2c and 5HT4 receptor antagonists appear to facilitate LTP expression in the CAl subfield (Stubli & Xu, 1995; Wang & Arvanov, 1998) while decreasing it in the DG (Kulla & Manahan-Vaughan, 2002). Given the evidence for a role for the 5-HT6 receptor subtype in memory paradigms and the modulation of LTP and LTD by different serotonin re ceptor subtypes, the goal of the present study was to investigate the role of the 5-HT6 receptor in synaptic plasticity in both the CAl and DG subfields in vivo.  2.2 2.2.1  Experimental procedures In vitro receptor binding  The selectivity of ((S)—7-Benzenesulfonyl-2 ,3-dihydrobenzol [1,4] dioxin-2-ylmethyl)methylamine (R9237)  (  1 M) was determined from its ability to displace 49  radioligand binding to a diverse collection of 85 receptors, transporters and ion channels using widely reported assay methods (Kambhampati et al., 2008). Under these conditions, less than 50% inhibition was observed ex cept at the 5-HT receptors. Binding affinities for R9237 at various 5-HT receptors are depicted in Table 2.1.  2.2.2  Subjects  Eighty-one male Sprague-Dawley rats (100-300g; 24-60 days of age) were used for this study.  Animals were maintained on a 12/12HR light/dark  cycle, and all electrophysiological testing was carried out during the light phase of the cycle. The colony was maintained at 21 ± 1°C and animals were given ad libitum access to Purina Rat Chow and water. All experiments were performed in accordance with UBC and Canadian standards for animal care. Animals were anesthetized with urethane (1.5g/kg, i.p.) and placed in a stereotaxic apparatus (Kopf Instruments). Surgical plane of anesthesia was maintained by administering supplemental doses of urethane as necessary. Body temperature was maintained at 37 + 1.0°C throughout experiments using a grounded homeothermic blanket (Harvard Instruments, MA, USA).  2.2.3  Electrophysiology  A 75 im stainless-steel recording electrode (A-M Systems, Inc., WA, USA) was directed through a trephine hole into the DG (3.5 mm posterior, 2.0  mm lateral to bregma) or the CAl stratum radiatum area of the hippocam pus (3.0 mm posterior and 2.5 mm lateral to bregma). A 75 im monopolar 50  stimulating electrode (A-M Systems, Inc.) was directed through another trephine hole to activate the perforant path input to the DG (7.4 mm pos terior, 3.0 mm lateral to bregma) or through the same hole as the record ing electrode for the CAl region (3.4 mm posterior and 2.2 mm lateral to bregma). The final depth of both electrodes was determined by adjusting each electrode to obtain the maximal field excitatory postsynaptic poten tial (fEPSP) using minimal stimulation (< 100 microamps). Field EPSP responses were elicited with single-pulse stimuli of fixed amplitude and du ration (120 ,us) at 0.2-0.33 Hz. Signals were amplified (Getting Instruments), filtered (1 Hz to 3 Hz) and digitized at 5 kHz. Baseline responses were stan dardized to 50% of maximal response size to record from the steep portion of the input-output (I/O) curve. Each recording was required to exhibit a stable baseline for a minimum of 30 minutes to be included for analysis. At the beginning and at the end of each recording, I/O curves were generated by delivering pulses of increasing amplitude. Similarly, paired-pulse stimuli of varying interpulse intervals (50, 100, 500 ms) were administered to assess the contribution of presynaptic mechanisms. LTP was elicited in the CAl region using a theta-burst stimulation (TBS) consisting of 4 100 Hz pulses administered 8 times at 200 ms intervals. Two protocols were used to in duce LTP in the DG. The strong theta-burst stimulus (sTBS) consisted of 5 pulses delivered as 400 Hz bursts. There were 10 bursts per train adminis tered at an interburst interval of 200 ms. The administration of this protocol was repeated 4 times. The weak theta-burst stimulus (wTBS) consisted of 8  trains of theta-patterned conditioning stimuli. Stimuli were delivered as 10 bursts of 5 pulses at 100 Hz with a 200ms interburst interval. Animals used 51  for LTD experiments were subjected to acute stress. Prior exposure to acute stress is known to increase the capacity of the hippocampus to undergo LTD in vivo in male animals (Titterness & Christie, 2008). In the CAl region,  long-term depression (LTD) was induced in younger animals (100-200g) fol lowing a stress paradigm during which rats were put on an elevated platform for 30 minutes. The conditioning stimuli consisted in 900 pulses adminis tered at 3 Hz. For DG recordings, neither the stress-3Hz protocol nor the 900 pulses at 1Hz protocol produced LTD. However, LTD could be induced by administering 900 pulses at 1 Hz twice with a 20-mm  2.2.4  interval.  Drugs  R9237 (Figure 2.1) was obtained from Roche Pharmaceuticals (Palo Alto, CA) and was injected in the intraperitoneal cavity 30 minutes before the start of electrophysiological recordings. Based on studies using similar com pounds, we estimate that at 3 mg/kg injected i.p., R9237 is occupying more than 70% of the 5-HT6 receptors in the brain (Sleight et al., 1998).  2.2.5  Data analysis  Data was collected and analyzed using custom-written software (Lee Camp bell; Getting Instruments). The slope of the rising phase of the fEPSP was used to determine changes in synaptic efficacy. Following the conditioning stimuli, single-pulse stimuli were administered for a minimum period of lh (LTP) or 2h (LTD). All EPSP slope data are presented as the mean percent change from the pre-conditioning stimuli baseline SEM. Data was analyzed using ANOVA, t-tests and post-hoc tests as appropriate. 52  2.3 2.3.1  Results Role of 5-HT6 receptors in LTP in the CAl region in vivo  In order to assess the involvement of 5-HT6 receptors in hippocampal synap tic plasticity, we used a specific 5-HT6 antagonist, R9237. We initially ex amined the relationship between the dose of R9237 administered and the magnitude of LTP in the CAl region in vivo in order to determine the con centration to use for subsequent experiments. We used a standard TBS protocol at 100Hz to induce LTP in control animals and animals that had been previously injected with 1, 3, 5 or 10mg/kg of the specific 5-HT6 recep tor antagonist R9237 (Figure 2.2). Interestingly, none of the concentrations tested, with the exception of 3mg/kg, produced a significant change in the magnitude of LTP compared to controls. However, at 3mg/kg, R9237 signif icantly increased the magnitude of LTP obtained (73.38 + 7.04%) compared to control conditions (36.32 ± 7.26%, p=O.OO1 ). Post-hoc Tukey t-tests re 4 vealed significant differences only between the 3mg/kg groups and all the other groups (p  <  0.05). A dose of 3mg/kg of R9237 was used for all sub  sequent experiments. We examined a role for the 5-HT6 receptor in LTP in the CAl region using control animals and i.p. injections of R9237 (Figure 2.3). For each recording, I/O curves were constructed both before and after LTP induc tion to confirm that our conditioning protocols had rio detrimental effects on the stimulated fibers (Figure 2.4). The TBS protocol used in control con ditions elicited significant LTP (36.32 ± 7.26%, p=O. ). R9237 injected 0001 53  at 3mg/kg also produced significant LTP (73.38 ± 7.04%,  pO.OOOO).  Com  pared to control conditions, R9237 significantly enhanced LTP in the CAl region at the 60 minute time point (prrO.0035, Figure 2.3). In order to assess the magnitude of short-term potentiation (STP), we also analyzed data at the one-minute post-conditioning stimulus time point. At this time point, R9237 induced a significantly larger magnitude of post-tetanic potentiation (p=O.0081).  2.3.2  Role of 5-HT6 receptors in LTD in the CAl region in vivo  Since LTD is not readily induced in adult rats (Errington et al., 1995), younger animals were used for the LTD experiments. In addition, a stress paradigm was used to ensure reliable and significant LTD in control con ditions (Titterness  Christie, 2008). We used i.p. injections of R9237 to  examine its effects on stress-induced LTD using a 3Hz protocol (Figure 2.5). As with LTP experiments, I/O curves were constructed pre- and 120 min utes post-recording (Figure 2.6). In order to be consistent with our LTP experiments and in accordance with evidence for a narrow window of selec tivity for pharmacological modulators of 5-HT6 receptors (Pitsikas et al., 2008), we used the same dose of 3 mg/kg for LTD experiments. Using a 30mm  stress paradigm coupled with 3Hz stimulation, we were able to induce  significant LTD in control animals (-13.57 ± 5.52%, p=O.O2S3). R9237 injec tions of 3mg/kg completely blocked stress-induced LTD as measured after a 120 mm  decay period (6.92 ± 3.57%, p=O.O3l2, Figure 2.5). However, at  one minute post-conditioning stimulus, the magnitude of the response for 54  the 5-HT6 antagonist group was not significantly different from that of the control group (p=O.5770). In order to determine if the effects of the 5-HT6 receptor antagonist were mediated through presynaptic release changes, we conducted pairedpulse tests using 3 different interpulse intervals before baseline and after LTD induction (Figure 2.7). None of the experimental conditions produced significant changes in the paired-pulse ratios (p  2.3.3  >  0.05).  Role of 5-HT6 receptors in LTP in the dentate gyrus region in vivo  We next examined if R9237 exerted different effects on synaptic plasticity in the granule cells of the DG. We first looked at the effects of the 5-HT6 antag onist on LTP using a 400 Hz theta-burst pattern stimulation (Figure 2.8). As described for the CAl experiments, pre- and post-recording I/O curves were constructed in these experiments (Figure 2.9). In control conditions, the theta-burst protocol induced significant LTP in the DG (24.46 ± 4.07%, p=O.0000; Figure 4A). R9237-treated animals also showed significant LTP (27.14 ± 6.15%, prrO.OOl7), and the magnitude of LTP at the 60 mm  time  point in those animals was not significantly different from that observed in control animals (p=O.7O97). Similarly, the magnitude of post-tetanic poten tiation did not differ significantly from control animals (prrO.9429).  55  2.3.4  Role of 5-HT6 receptors in LTD in the dentate gyrus region in vivo  We were unable to induce LTD in the DG using the stress protocol that we employed in the CAl region, but did find that the application of 2 trains of 900 pulses at 1Hz (20-mm. intertrain interval) produced significant LTD (Figure 2.10). As previously, I/O curves were again constructed prior to and following each experiment to confirm that this extensive low-frequency pro tocol did  not  damage fibers (Figure 2.11). The application of wo trains of 1  Hz stimuli produced small, but significant, LTD (-22.26 ± 6.65%, pO.OO4O) that could be completely arid significantly reversed at 120 mm  by the ad  ministration of R9237 (p=O.OO18, Figure 2.10). Indeed, in this instance the R9237 actually revealed a significant LTP (28.70 ± 13.24%; p=O.O514) at 120 minutes. In contrast, LTD at 5 minutes was not significantly different between the control and the 5-HT6 antagonist group (p=O.Oll8).  2.3.5  Role of 5-HT6 receptors in weak-stimulus induced LTP in the dentate gyrus region in vivo  Since LTD was reversed by the 5-HT6 antagonist in both regions examined and that LTP was enhanced in the CAl region, we decided to test the hypothesis that R9237 exerts its effects by lowering the threshold for LTP induction. Using the same dose of the antagonist, we looked at the effects of a weaker LTP stimulation (Figure 2.12). In the control group, this weak TBS elicited LTP that was of smaller magnitude than the strong stimulus 5). In the 5-HT6 6 induced LTP (11.15 ± 4.37%) but still significant (p=rO.0l  56  antagonist-treated group, it appeared that there was a trend for LTP being slightly enhanced (18.38 ± 2.00%) but the results were not significant at  the 60 mm  time point (p=0.1970) or at the post-tetanic potentiation level  (p=O.6921, Figure 2.12). As with other groups, I/O curves were analyzed  before and after each recordings (Figure 2.13). As for the CAl region, paired-pulse analysis pre- and post-recording with 3 different interpulse interval times was conducted for all experimental con ditions to examine if the 5-HT6 antagonist effects observed in the DG could be attributed to presynaptic transmitter release. None of the conditions exhibit significant changes in the paired-pulse analysis.  2.4  Discussion  In summary, the present results indicate a role for 5HT-6 receptors in LTD in both the CAl region and the DG region of the adult rat hippocampus. In addition, a reliable enhancement of LTP was also observed in the CAl region, but only with a dose of 3 mg/kg. A similar enhancement was not observed in the DG, suggesting that the capacity for serotonin to modulate synaptic plasticity is different  iii  these two regions. We also observed that  our effects were likely not due to presynaptic mechanisms, which is in ac cordance with studies showing that the 5-HT6 receptor is mostly located postsynaptically (Grard et al., 1996). This is, to our knowledge, the first report of the involvement of the 5HT6 receptor in synaptic plasticity in the hippocampus. However, other  studies have shown the involvement of different serotonin receptors in LTP  57  and LTD in the hippocampus. Interestingly, studies using antagonists for serotonin receptors also coupled to stimulatory G proteins yield a variety of results. For example, Wang and Arnavov showed that blockade of the 5HT2a receptor leads to increased LTP magnitude in CAl slices (Wang & Arvanov, 1998). This is similar to what we observed in vivo and might suggest similar downstream signaling pathways for the 5-HT2a and 5-HT6 receptors. In contrast, mutation of the 5-HT2c receptor or pharmacological blockade of the 5-HT4 receptor both lead to decreased LTP in the DG in two different models (Tecott et al., 1998; Kulla & Manahan-Vaughan, 2002). This suggests that even though different 5-HT receptors are coupled to stim ulatory G proteins, their modulation of hippocampal synaptic plasticity can differ, either through specific downstream signaling pathways, through the modulation of different receptors, or even through the modulation of differ ent subsets of neurons. One main concern when comparing these studies is the selectivity of the antagonists, as many serotonergic antagonists are known to block, at least to some degree, more than one subtype of receptor. R9237 is no exception, as it has some selectivity for 5-HT2a, 5-HT2c and 5-HT1a (see Table 2.1). However, compared to the affinity for the 5-HT6 receptor, these affinities are negligible (R9237 is more than 200 times more selective for the 5-H6 receptor than for the next most selective target). In addition, while some of the values in Table 2.1 refer to affinity for the human 5-HT receptors, the rodent and human 5-HT6 receptors share 89% overall amino acid homology (Woolley et al., 2004) and have binding sites that exhibit nearly identical pharmacological profiles (Hirst et al., 2003). Based on this evidence, we 58  consider our effects to be most likely mediated through the 5-HT6 receptor when using a dose of 3 mg/kg. It is unclear why R9237 would exert its effect only at a concentration of 3mg/kg (see Figure 2.2). It may be that this pharmacological agent has a narrow window of selectivity or that in greater concentrations it activates different compensatory mechanisms. Given the selectivity data described in Table 2.1, it is possible that the action of R9237 at other 5-HT receptors when administered at higher doses may account for the narrow window of effect of this drug. In this paper we compared the effects of a 5-HT6 antagonist on synap tic plasticity in the CAl region and in the DG. However, one should be careful when comparing results obtained in each region. Slightly different stimulation protocols were used in each hippocampal region to elicit strong and reliable LTP. However, the same frequency of stimulation was used (100 Hz), a similar pattern was used, and comparable levels of LTP were ob tained. Therefore, we propose that the difference in the modulation of LTP by 5-HT6 depends on which region is studied and this implies that LTP in the CAl region and in the DG is controlled by different mechanisms. LTD is notoriously difficult to obtain in vivo in the DG of anesthetized rats using low-frequency stimulation (LFS) (Doyre et al., 1996). Previous studies have relied on priming, the activation of group II metabotropic glu tamate receptors prior to LFS (Manahan-Vaughan, 1997) to elicit a robust LTD, or studying depotentiation as an alternative to long-term depression of synaptic activity. While recent studies in freely moving animals suggest that it is possible to obtain LTD in vivo using LFS (Pschel & Manahan 59  Vaughan, 2007), we were unable to elicit a stable and reliable LTD in our rat model using a standard LFS protocol. Therefore, we developed a doublestimulation protocol to obtain LTD in the DG. Thus, we can attribute dif ferences between the effects of R9237 in the CAl region and in the dentate gyrus to three potential causes: 1) differential modulation of LTD depend ing on brain region; 2) differences in the stimulation protocols inducing the activation of different mechanisms; and 3) the use of a stress paradigm to elicit robust LTD in the CAl region might also activate mechanisms that modulate serotonergic transmission (Homberg  Contet, 2009).  Future directions will focus on determining the potential mediators of the effects of 119237 on synaptic plasticity. Studies show that blocking the 5-HT6 receptor in rats leads to increased extracellular levels of both gluta mate (Dawson et al., 2001) and acetylcholine (Shirazi-Southall et al., 2002) in the hippocampus. An interesting question to answer will be whether sero tonin acts solely through its receptor and associated downstream signaling pathways or if the effects of 119237 can be attributed to the modulation of other receptors through serotonergic transmission. In conclusion, this is the first report showing an involvement of the 5-HT6 receptor in LTP and LTD in two regions of the rat hippocampus. Since more and more evidence links this receptor to cognitive functions and pathologies, it is potentially a key player in the development of new therapeutic strategies and much can be gained from understanding its involvement in models of learning and memory.  60  2.5  Figures  61  00 N H  Me  ((S)-7-Benzenesulfonyl-2 ,3of R9237, Figure 2.1: Structure dihydrobenzo[1 ,4] dioxin-2-ylmethyl)-methylamine  62  *  90 80 C)  o,  70  60 50 C) 0.  o  40  (I, 0. (I)  a-  LU  20 10 0— 0  1  3 Dose (mg/kg)  5  10  Figure 2.2: Dose-response for the effect of R9237 on CAl LTP. Effect of dif ferent concentrations of the 5-HT6 antagonist R9237 (1, 3, 5 and 10mg/kg) on in vivo LTP in the CAl region of the hippocampus of adult rats.  63  14C •i927 (n=6) • .ontrol (n) I 2(  i:c  i w  20 0  ..I.i..,’.II.  -20 -15  0  15  30 Time (mm)  45  60  Figure 2.3: R9237 increases the magnitude of LTP in the CAl region. CAl fEPSP slope in response to TBS stimulation applied at t=O in adult rats in control conditions (squares, n=9) and rats previously injected of 3 mg/kg R9237 (diamonds, n=6).  64  B  Control  R9237  -1 C  E o  a  L  12  121 101  —  Ore I-’ost  -.Rre I-’ost  bc 0 80  80 60  0  40  40 0,  j  20  ii  0  20 0  0  0.05  0.1  0.15  0.2  0.25  Pulse width (ms)  0.3  0  0.05  0.1  0.15  0.2  0.25  0.3  Pulse width (ms)  Figure 2.4: Input-output curves for CAl LTP. A) I/O curves pre- and post LTP induction in control animals. B) I/O curves pre- and post-LTP induc tion in animals treated with 3mg/kg R9237. Averaged fEPSP traces inset where 1 represents the averaged trace for the last 5 mm of the baseline and 2 represents the averaged trace for the last 5 mm of the decay. Scale bars: 1 mV, 5 is.  65  60  • Control (n=8) .R9237 (n=4)  20 i) 0 U) 0 C,)  0-b  o -2C  w  -4C  -15  0  15  30  45 75 60 Time (mm)  90  105  120  Figure 2.5: R9237 completely blocks LTD in the CAl region. CAl fEPSP slope in response to LFS stimulation applied at t=rO in adult rats in control conditions (squares, n=8) and rats previously injected of 3 mg/kg R9237 (diamonds, n=4).  66  B  Control  A  R9237  VL 120  —  120 Ere I-’ost 100  Ore F-’ost  K K  100  0  80  ‘  80  a)  60  a  60  8  8  0. 0  a Cl) a LU  0  40  40 Cl)  lb  20 00  0.05  0.15 0.2 0.1 Pulse width (ms)  0.25  0.3  20 0  0  0.05  0.1 0.15 0.2 Pulse width (ms)  0.25  0.3  Figure 2.6: Input-output curves for CAl LTD. A) I/O curves pre- and post-LTD induction in control animals. B) I/O curves pre- and post-LTD induction in animals treated with 3mg/kg R9237. Averaged fEPSP traces inset where 1 represents the averaged trace for the last 5 mm of the baseline and 2 represents the averaged trace for the last 5 mm of the decay. Scale bars: 1 mV, 5 its.  67  ____ A  LTP Control  B  LTP R9237  1o :bt 70  ‘UI  °80  Interpulse interval (ms 3  Interpulse interval (ms 3  D  C Stress LTD Control  Interpulse interval (ms 3  Stress LTD R9237  Interpulse interval (md)  Figure 2.7: Paired-pulse analysis from the CAl region. R9237 does not in duce presynaptic release changes in LTP or stress-induced LTD in the CAl region. A. Paired-pulse ratios generated pre- and post-LTP induction in con trol animals. B. Paired-pulse ratios generated pre- and post-LTP induction in animals treated with 3mg/kg R9237. C. Paired-pulse ratios generated pre- and post-LTD induction in control animals. D. Paired-pulse ratios gen erated pre- and post-LTD induction in animals treated with 3mg/kg R9237.  68  6( a)  Control (n=7) .R9237(n=6)  j 2( o hImfi’ w -15  15 30 Time (mm)  45  60  Figure 2.8: 119237 does iiot affect LTP in the dentate gyrus. DG fEPSP slope in response to TBS stimulation applied at t=O in adult rats in control conditions (squares, nrr7) and rats previously injected of 3 mg/kg R9237 (diamonds, n=6).  69  Aj  B  L  12( i-’ost  1101 8 8 80 60 s 0.  o 60 0  0  40  40  a)  (0 0  20 0  j  0.05  0.1 0.15 0.2 0.25 Pulse width (ms)  Control  0.3  20 0  3  0.05  0.1 0.15 0.2 0.25 Pulse width (ms)  0.3  R9237  Figure 2.9: Input-output curves for DG LTP. A) I/O curves pre- and post LTP induction in control animals. B) I/O curves pre- and post-LTP induc tion in animals treated with 3mg/kg R9237. Averaged IEPSP traces inset where 1 represents the averaged trace for the last 5 mm of the baseline and 2 represents the averaged trace for the last 5 mm of the decay. Scale bars: 5 mV, 5 /.ts.  70  •Control (n=1O) • R9237 (n=7) a) c5) C-)  ci  a  0 Co  0 Cl) 0  w  Time (mm)  Figure 2.10: R9237 reverses LTD in the dentate gyrus. DG fEPSP slope in response to 2 LFS stimulation protocols applied at t=0 in adult rats in control conditions (squares, n=10) and rats previously injected of 3 mg/kg R9237 (diamonds, n=7).  71  A$j 12  2L 121  -.Pre  -.-ost  -.-Post 101  10 0  a) -  ‘6  80  0)  60  -  80 60  16  CO  40  40  0  Q  20  20 0  0 0  0.05  0.1 0.15 0.2 0.25 Pulse width (ms)  Control  0.3  0  0.05  0.1 0.15 0.2 0.25 Pulse width (ms)  0.3  R9237  Figure 2.11: Input-output curves for DG LTD. A) I/O curves pre- and post-LTD induction in control animals. B) I/O curves pre- and post-LTD induction in animals treated with 3mg/kg R9237. Averaged fEPSP traces inset where 1 represents the averaged trace for the last 5 mm of the baseline and 2 represents the averaged trace for the last 5 mm of the decay. Scale bars: 5 mV, 5 is.  72  4C • Control (n7 .R9237 (n=4 a3 2C 0 ci) 0  Cl) 0  w  -‘  -15  0  15 30 Time (mm)  45  60  Figure 2.12: R9237 does not significantly affect LTP induced using a weak stimulus in the dentate gyrus. DG fEPSP slope in response to a weak TBS stimulation applied at tO in adult rats in control conditions (squares, n=7) and rats previously injected of 3 mg/kg R9237 (diamonds, n=4).  73  B  AL 120  120  Ore  I-’ost  gioo  .‘10c 8 ‘6 80  8 80  60  60  40  40  20  20  0  0  O.05  0.1 0.15 0.2 Pulse width (ms)  Control  0.26  0.3  0.05  0.1  0.15  0.2  0.25  0.3  Pulse width (ms)  R9237  Figure 2.13: Input-output curves for DG LTP induced using a weak stimulus. A) I/O curves pre- and post-LTP induction in control animals. B) I/O curves pre- and post-LTP induction in animals treated with 3mg/kg R9237. Averaged IEPSP traces inset where 1 represents the averaged trace for the last 5 mm of the baseline and 2 represents the averaged trace for the last 5 mm of the decay. Scale bars: 5 mV, 5 ts.  74  —1  riD  CD  Table 2.1: In vitro binding affinities for 119237 at various 5-HT receptors.  KI (nM) Receptor 5-HT6 (human) 0.19 ± 0.07 42 ± 18 5-HT2A (human) 200 ± 67 5-HT1D (bovine) 252 ± 96 5-HT2C (human) 631 ± 210 5-HT1A (human) 5-HT3 (human) > 10000 (human) > 100000 5-HT4 5-HT5 (human) > 100000 > 100000 5-HT7 (human) deviation (n4). ± standard mean Values represent  76  Chapter 3  Glutathione Alters the Mechanism of Synaptic Memory in Aged Mice 3.1  Introduction  While the hippocampus is particularly vulnerable to the process of aging, hippocampal neuronal numbers are not significantly decreased in normal, aged animals (Burke & Barnes, 2006). However, several studies report an increase in the amplitude and duration of the calcium-mediated afterhypopo larization (AHP) following action potentials in the CAl region (Landfield & Pitler, 1984). This finding, combined with reports showing a significant increase in L-type calcium currents in aged animals, have led to the cal cium dysregulation hypothesis of aging, which stipulates that alterations in calcium-dependent processes during aging affect calcium signaling and con sequently impair neuronal functions dependent on this signaling (Thibault et al., 2007). One such calcium-dependent process that is affected in aging is synaptic  77  plasticity. Hippocampal synaptic plasticity is thought to represent a cellular model for learning and memory (Bliss & Lomo, 1973). Long-term poten tiation (LTP), a sustained increase in the efficacy of synaptic transmission following an electrical conditioning stimulus, has been studied extensively in the CAl region of the hippocampus and has been demonstrated to rely on the activation of N-methyl-d-aspartate (NMDA) receptors and the sub sequent calcium influx in hippocampal neurons (Bliss & Collingridge, 1993). The calcium dyregulation observed in aged animals predicts impaired synaptic plasticity in the hippocampus. Interestingly, many studies report this is not the case, and that LTP induction is intact in the hippocampus of aged rats (Diana et al., 1994; Landfield & Lynch, 1977). However, these studies used strong, high-frequency conditioning stimuli. When stimulation protocols involving fewer pulses or lower current amplitudes are used to elicit LTP in aged rats, impairments are unmasked (Deupree et al., 1993; Rosenzweig et al., 1997). This may be explained by the fact that aging leads to an increase in the magnitude of LTP mediated by L-type cal cium channels (Grover & Teyler, 1990).  Conversely, normal aging leads  to hypofunction of NMDA receptors, and an associated decrease in NMDA receptor-mediated LTP (Rosenzweig & Barnes, 2003). Because L-type cal cium channel-dependent LTP can only be induced using a robust, high frequency conditioning stimulus, initial reports studying LTP in aged ani mals using a strong conditioning protocol may have failed to detect deficits in LTP because the increase in L-type calcium channel-dependent LTP com pensated for the decrease in NMDA receptor-mediated LTP. However, it is important to note that while many studies have looked at synaptic plastic 78  ity in aged rats, the effects of aging on hippocampal LTP and LTD remain unclear in mice. A contributing factor for this lack of established knowledge is the variety of different conditioning protocols and mice models used to assess the impact of aging on synaptic plasticity. Aside from the calcium dysregulation hypothesis of aging, other mecha nism have been proposed to explain various impairments observed in mod els of aging. The oxidative stress theory of aging suggests that age-related pathophysiology can be explained by an increase in damage caused by reac tive oxygen species (ROS) (Beckman  Ames, 1998). During aging, there is  an increase in oxidative stress caused by both an increase in oxidant gener ation as well as a decrease in oxidant defenses. Several reports suggest that the levels of glutathione (GSH), a glutamate, cysteine and glycine tripep tide, are sigruficantly depleted in models of natural aging (Gilca et al., 2007; Sandhu  Kaur, 2002). GSH is an abundant, major antioxidant in the brain  and as a potent reducing agent plays a central role in the maintenance of a healthy redox balance in neurons (Dringen, 2000). In addition, through its role as a reducing agent, GSH can modulate the function and the activ ity of several components of neuronal function such as protein kinases and phosphatases, and a variety of receptors. The activity of the NMDA receptor, in animals at all stages of life, can be modulated by oxidizing agents as well as reducing agents (Tang & Aizen man, 1993).  Oxidizing agents lead to a reduction in the activity of the  NMDA receptor while reducing agents lead to an increase in the activity of the NMDA receptor (Tang & Aizenman, 1993; Choi & Lipton, 2000). GSH depletion leads to significant decreases in NMDA receptor-mediated field 79  excitatory potentials (fEPSPs), and this effect is thought to be mediated through the oxidation of redox-sensitive sites on the extracellular portion of the NMDA receptors (Steullet et al., 2006). These sites have been identified as three distinct pairs of cysteine residues located on the extracellular por tion of NR1 and NR2A subunits. In a healthy redox environment, reducing agents promote free thiols on these residues. However, an oxidizing envi ronment, such as one created by GSH depletion, leads to the formation of disulfide bonds between members of a given pair of cysteine, which in turns leads to a decreased activity of the receptor (Sullivan et al., 1994). For the work described in this chapter, we propose to first establish the impact of aging on hippocampal synaptic plasticity in our mouse model. We hypothesize that age-related deficits in LTP will be dependent on the fre quency of the conditioning protocol. Next, we will explore the contribution of GSH to age-related impairments in LTP in aged mice. We hypothesize that supplementing aged mice with N-acetylcysteine (NAC), a precursor for the formation of GSH, will reverse the aged-related LTP deficits.  3.2  Experimental procedures  3.2.1  Animals and feeding protocol  All procedures were approved by the Canadian Council for Animal Care and the University of British Columbia Animal Care and Use Committee. Adult (2-4 months old) and naturally aged (14-18 months old) C57BL/6 mice were used for this study. Adult and aged animals used for the stimulation protocol study did not undergo any feeding treatment. Aged animals used for the 80  study of the impact of GSH supplementation on synaptic plasticity were fed NAC, a cysteine donor and precursor for the formation of GSH. Unlike GSH, NAC is readily absorbed and easily crosses the blood-brain barrier. In addition, several reports confirm that orally administered NAC protects the brain against GSH depletion (Aydin et al., 2002; Ercal et al., 1996; Fu et al., 2006; Kamboj et al., 2006). Aged NAC-fed mice were fed once daily for 21 days with NAC at 200mg/kg incorporated into lmL flavoured gelatin, in addition to their standard, ad libitum mouse chow. Feeding was monitored daily and only the mice that ate the entire portion everyday for the duration of the protocol were included. In order to control for the possibility that the feeding protocol itself has an impact on synaptic plasticity, a subset of aged mice (control-fed group) was fed once daily for 21 days with lmL flavoured gelatin, also in addition to their ad libitum mouse chow. Animals undergoing a feeding protocol were sacrificed and used for experiments the day following their last feeding.  3.2.2  Hippocampal brain slice preparation  C57BL/6 mice were anaesthetized with halothane (Sigma) and rapidly de capitated. Hippocampal transverse slices (400 tim, from either adult or aged animals as described above) were prepared using a vibratome (Leica, Wil lowdale, Ontario, Canada) in a chilled (0-4°C) sucrose buffer that contained (in mM) sucrose (230), NaHCO 3 (26), KC1 (2.5), glucose (10), 4 PO 2 NaH (1.25), MgSO 4 (10) and CaCl 2 (0.2) that was constantly oxygenated with /5%CO After collection, slices were transferred to a storage cham 95%O . 2 ber containing an oxygenated (with 2 /5%C0 artificial cerebrospinal 95%0 ) 81  fluid (aCSF) solution for at least 1 hr. The aCSF contained (in mM): NaC1 4 (1.3) PO (1.25), MgSO 2 NaH (120), NaHCO 3 (26), KC1 (3), glucose (10), 4 and CaC1 2 (2), pH 7.3. Slices were allowed to recover at room temperature for at least one hour before recording. All solutions were saturated with . 2 95% 02/5% Co  3.2.3  Electrophysiology  For extracellular LTP or LTD recordings, slices were submerged in a record ing chamber perfused with oxygenated aCSF. Field excitatory post-synaptic potentials (fEPSPs) were evoked by stimulation of the Schaffer collateral pathway using a bipolar stimulating electrode. Glass micropipettes filled with aCSF were used to measure CAl fEPSPs in stratum radiatum. In dividual fEPSPs were evoked and recorded every 15 s and a stable 15 mm baseline was required in all experiments. After baseline recording, one of two different protocols was used to induce LTP. The high-frequency stimulus (HFS) consisted of 4 times 50 pulses at 100 Hz, with 30 s between each set of 4 pulses. The theta-burst stimulus (TBS) consisted of 7 trains of 4 bursts of 4 pulses each delivered at 100 Hz, with an interburst interval of 200 ms and an intertrain interval of 7 s. The TBS protocol was designed to roughly match the number of pulses and the duration of the HFS protocol, with the only distinguishing features to be the pattern of stimulation and the fre quency of the trains. After application of the conditioning protocol, fEPSPs were again recorded for 1 h. A standard low-frequency protocol was used to elicit LTD (900 pulses at 1 Hz). All drugs were bath-applied 15 mm  prior  to conditioning stimuli. The slopes of the initial phase of the fEPSP wave 82  forms were computed as a measure of change in synaptic strength. Results were processed for statistical analysis using Clampfit (Axon Instruments, Molecular Devices). Data are presented as means and SEM. For whole-cell LTP recordings, whole-cell patch clamp recordings from CAl neurons within hippocampal slices were obtained at room tempera ture. Individual slices were transferred to a recording chamber located on an upright microscope and rapidly perfused with oxygenated aCSF. Patch electrodes (4-6 M resistance) were pulled from thin-walled glass capillaries in three stages on a micropipette puller (Sutter Instruments, Novato, CA) and filled with a pipette solution containing (in mM): Cs-methanesulfonate 2 (5), 4Cs-BAPTA (5), TEA (5), HEPES (20) at pH (100), CsCl 2 (25), NaCl 7.2. For some experiments, 10 mM GSH was added to the pipette solution. Whole-cell voltage-clamp recordings were obtained from CAl pyramidal cells under microscopic guidance.  A bipolar electrode was placed on Schaffer  collaterals, 250 pm away from the soma of the recorded cell. Responses were evoked with monophasic voltage pulses, and membrane potential was clamped at -70 mV. A few minutes after gaining whole-cell access, LTP was induced through a paired-stimuli protocol. Three bursts of 50 pulses delivered at 100 Hz and separated by 5 s were coupled to a membrane de polarization to +5 mV. After induction protocol, stimulation was restarted at basal conditions for 30 minutes. For all electrophysiology experiments, a minimum of 5 animals per group was used.  83  3.2.4  Glutathione assay  GSH levels were measured using an assay kit (BioVision) based on the reac tion between 5,5’-dithiobis (2-nitrobenzoic acid) (DTNB) and OSH to gen erate 2-nitro-5-thiobenzoic acid and oxidized GSH (GSSG). Since 2-nitro-5thiobenzoic acid is yellow, the GSH concentration in a sample solution can be determined by optical density measurement at 412 nm absorbance. The concentration of GSH was calculated from formula and expressed as nmol per milligram protein. Hippocampi were dissected and homogenized. The ho mogenate was centrifuged for 10 mm at 10,000 rpm at 4 °C and supernatant was used for the GSH assay according to the manufacturer’s instructions.  Protein content was measured by Coomasse blue protein-binding method using bovine serum albumin as standard.  3.2.5  Imaging  Monochlorobimane (MCB) was purchased from Fluka and made as a 100 mM stock solution in Me SO and stored at -20 °C. Two-photon excitation 2 of GSH-MCB conjugates was achieved using a Coherent Chameleon laser pumped by a 5-watt Verdi laser tuned to 780 nm. Images were acquired using LSM software and by using a Zeiss water-immersion objective (40; 0.80 numerical aperture). Fluorescence was collected using photo-multiplier tube 1 (PMT1) for the GSH-MCB signal (512-562 urn). Images (512 x 512 pixels) were acquired using 8-line averaging between 50 and lOOILm deep into the slice in area CAl. GSH z-stacks (one control and then one treatment) were acquired once per slice and fluorescence intensity was normalized to a known  84  fluorescent compound concentration value (1 mM Lucifer Yellow) inside a sealed pipette positioned in the field of view of the imaged region. The measurements were made at the depth that gave the identical fluorescence intensity of the Lucifer Yellow standard. Fluorescence intensity will change with depth because of the scattering of the brain tissue. With increasing depth fluorescence excitation is reduced and the fluorescence images are fainter (Oheim et al., 2001). The fluorescence standard allowed us to ensure that the plane of imaging in each experiment was constant for the standard and therefore we could coiripare fluorescence intensities of stained cells at this imaging depth. For calcium imaging, hippocampal slices from aged mice were loaded using the cremophor loading technique (Yuste  Bonhoeffer, 2001) with  10 uM of calcium indicator Rhod-2 for a maximum of 45 mm  at room  temperature in aCSF. Rhod-2 fluorescence was visualized by excitation at 835 nm. A baseline was established by giving 3 stimulations of the Schaffer collaterals at 100 Hz, mimicking the LTP conditioning protocol used in electrophysiological experiments, in normal ACSF. Subsequently, either 10 rM nimodipine or 50 tM APV was applied to the bath and the conditioning protocol was re-applied to evaluate the contribution of NMDA receptors and L-type calcium channels to the calcium signal.  85  3.3 3.3.1  Results HFS-induced LTP is of similar magnitude in adult and aged mice  We initially wanted to determine if our mouse model of aging displayed any hippocampal synaptic plasticity deficits compared to adult mice. In order to carry out this comparison, we used extracellular field recordings to induce LTP and LTD in the CAl region from hippocampal slices prepared from adult and aged animals. Two protocols were used to elicit LTP: a high-frequency stimulation (HFS), which consisted of 4 trains of 50 pulses at 100 Hz with an intertrain interval of 30 s, and a theta-burst stimulation (TBS) protocol, consisting of 7 trains of 4 bursts of 4 pulses delivered at 100 Hz, with an interburst interval of 200 ms and an intertrain interval of 7 s. These two protocols, because they are roughly equal in duration and number of pulses but of different patterns, would allow us to uncover any frequency-dependent impairment in LTP in aged animals. In adult animals (2-4 months old), HFS induced a reliable LTP (53% ± 12%) (Figure 3.1). The TBS protocol also induced LTP (30% ± 13%) (Figure 3.1), however this was significantly reduced compared with the LTP induced by the HFS protocol (p=O.O ). In aged animals (14-18 months old), 2 both HFS and TBS protocols induced LTP of similar magnitude to that in adult animals (HFS: 58% ± 9%, TBS: 52% ± 15%) (Figure 3.2). induced  using  LTP  HFS arid LTP induced using TBS were riot of significantly  different magnitudes (p=O.49), although it is important to note that the TBS protocol in aged mice led to very variable results. Most importantly,  86  the HFS protocol in adult and aged mice elicited LTP of similar magnitude  (pO.lO).  Therefore, we decided to use this protocol for all subsequent LTP  experiments carried out using extracellular field recordings. We were unable to obtain a significant amount of LTD in either adult or aged mice using a standard, low-frequency protocol (900 pulses at 1 Hz). In both adult and aged animals, we obtained a small, non significant amount of LTP (adult: 8% ± 6%, aged: 8% ± 9%) (Figure 3.3). Some reports studying LTD in aged animals suggest that LTD is easier to obtain when the ratio of calcium to magnesium in the aCSF is higher than 1.5 (Norris et al., 1996). Because our ratio in our normal aCSF was of 1.5, we attempted to obtain LTD using aCSF with a ratio of 2 (4 mM calcium, 2 mM magnesium). However, this new aCSF did not help with the induction of LTD. As with the normal aCSF, our low-frequency stimulation protocol only induced a small LTP in both adult and aged animals (adult: 6% ± 26%, aged: 10% ± 5%) (Figure 3.4).  3.3.2  LTP is NMDA receptor-dependent in adult mice but L-type calcium channel-dependent in aged mice  We next wanted to establish the mechanisms underlying LTP in both adult and aged mice.  Field excitatory postsynaptic potentials (fEPSPs) were  recorded in area CAl of hippocampal slices from adult (2-4 months old) and aged (14-18 months old) C57BL/6 mice and our standard HFS protocol was used to elicit LTP. In the adult hippocampus, LTP was blocked by a bath application of 50 M APV, an NMDA receptor channel blocker (control: 53% ± 12%, APV: 12% ± 8%, p  =  0.01) (Figure 3.5). Conversely, LTP in 87  the adult hippocampus was not significantly affected by a bath application of an L-type calcium channel blocker, nimodipine (10 tiM) (control: 53% ± 12%, nimodipine: 49% ± 10%, p  =  0.81) (Figure 3.6). A quantitative  representation of these results can be found in Figure 3.7. In contrast to the results obtained in adult animals, LTP in the hip pocampus of naturally aged mice was not significantly blocked by the ap plication of 50 1 iM APV (control: 58% ± 9%, APV: 46% ± 13%, p  0.48)  (Figure 3.8). However, the application of 10 1 M nimodipine significantly decreased the magnitude of LTP in these animals (control: 58% + 9%, ni modipine: 26% ± 6%, p  0.01) (Figure 3.9). A quantitative representation  of these results can be found in Figure 3.10. Taken together, these find ings confirm that in our mouse model, HFS-induced LTP in the CAl region of the adult hippocampus is NMDA receptor-dependent while in the aged hippocampus, LTP is partially dependent on L-type calcium channels.  3.3.3  Oral NAC supplementation leads to higher levels of neuronal GSH in the hippocampus  The concentrations of GSH, the most abundant endogenous antioxidant in the brain, is known to be reduced in aged animals and humans. To assess the impact of reduced GSH concentrations on synaptic plasticity in aged mice, we designed a protocol for increasing GSH concentrations in mice by diet. Our aged mice (14-18 months) were fed daily with 200 mg/kg NAC, a cysteine donor and precursor for the formation of GSH. Consequently, we assessed the validity of this model by determining if NAC supplementation leads to increased levels of OSH in the brain. 88  We initially used a GSH assay to demonstrate that our NAC feeding pro tocol led to an increase in hippocampal levels of GSH. GSH concentration in the hippocampi of control-fed aged and NAC-fed aged animals were mea sured and we found that our NAC supplementation protocol significantly increases the levels of GSH in the hippocampi of aged mice (Figure 3.11). As GSH can be found in both astroglial cells and neurons, we next wanted to confirm that oral NAC supplementation specifically increases neuronal levels of GSH. In order to do so, we used MCB (60 1 iM), a compound that enters cells and forms a GSH-MCB adduct through the action of the GSH-s transferase. This adduct can then be measured fluorometrically, and used to quantify intracellular levels of GSH. Once slices were loaded with MCB, we used two-photo imaging to assess the fluorescence intensity in both neurons and astrocytes in the CAl region of hippocampal slices from control-fed and NAC-fed aged mice. Because the images were taken at a certain depth in the slice and fluorescence intensity decreases with depth (Oheim et al., 2001), we used a sealed pipette filled with a known concentration of a fluorescent compound (1 mM Lucifer Yellow) to control for the depth at which selected images could be used for analysis to compare between control-fed mice and NAC-fed mice (Figure 3.12). Measurements were made at depths where the fluorescence intensity matched that of our Lucifer Yellow standard. This standard allowed us to ensure that the fluorescence intensity was constant between experiments. Using this MCB labeling and imaging technique, we established that NAC-fed aged mice show significantly more intense labeling of the neuronal cell bodies and dendritic layer in the CAl region compared with control-fed aged mice (p < 0.001, Figures 3.13 and 3.14). In astrocytes, 89  while the NAC-fed aged mice did show an increase in the intensity of the labeling, this was not significant (pr=O.O9) (Figures 3.13 and 3.14). These results validate NAC supplementation in aged mice as a way to increase levels of GSH in the hippocampus, and more specifically in hippocampal neurons.  3.3.4  Oral NAC supplementation restores the NMDA receptor-dependence of LTP in aged mice  The reported impacts of changing redox levels on NMDA receptors in neu rons on raised the interesting possibility that GSH levels can modulate synaptic plasticity in the hippocampus.  Thus we repeated the LTP ex  periments described in section 2.2 above in both NAC-fed aged mice and control-fed aged mice to assess the contributions of NMDA receptors and L type calcium channels to HFS-induced LTP in mice with altered hippocam pal GSH levels. Interestingly, in the NAC-fed mice, the mechanisms under lying HFS-induced LTP were restored back to NMDA receptor-dependence as seen in adult mice. In NAC-fed mice, HFS-induced LTP was completely blocked by 50 pM APV (control: 58% ± 2%, APV: 2% ± 6%, p  <  0.001)  (Figure 3.15). In addition, LTP in NAC-fed aged mice remained largely unaffected by bath application of 10 ,uM nimodipine (control: 58% ± 2%, nimodipine: 55% ± 8%, p = 0.75) (Figure 3.16). A quantitative represen tation of these results can be found in Figure 3.17. We hypothesized that the enrichment due to the feeding protocol of gelatin only (without NAC) would be insufficient to alter the mechanisms underlying LTP in aged control-fed mice. As predicted, the magnitude of 90  LTP in control-fed aged mice was not significantly blocked by 50 tM APV, even though a small decrease was observed (control: 57% ± 8%, APV: 39% ± 13%, p < 0.24) (Figure 3.18). In addition, the presence of 10 tM nimodipine in the bath lead to a significant decrease in the magnitude of LTP in control-fed aged mice (control: 57% ± 8%, nimodipine: 18% ± 7%, p  =  0.002) (Figure 3.19). A quantitative summary of these results can be  found in Figure 3.20. These results confirm that LTP in aged control-fed mice (flavored gelatin and no NAC) is mediated by the same mechanisms observed in aged unfed mice.  3.3.5  Oral NAC supplementation restores NMDA receptor-mediated calcium signals in aged mice following an LTP protocol  To further test the impact of NAC supplementation on synaptic plasticity in aged mice, we used two-photon imaging to record hippocampal dendritic calcium signals elicited by a HFS LTP-inducing stimulation protocol. Be cause standard AM dye loading works well in astroglial cells but poorly in neurons, we used the cremophor loading technique developed by Yuste and al. to allow maximal loading in hippocampal neurons (Yuste & Bonhoeffer, 2001). Once the slices were loaded with the calcium indicator, we applied three LTP stimuli and measured the resulting dendritic calcium signals in the CAl region. The area under the curve for these signals was calculated and the values were compared between NAC-fed aged mice and control-fed aged mice. In NAC-fed aged mice, consistent with the mechanisms observed in electrophysiological field recordings, the calcium signals elicited by an LTP 91  stimuli are partially blocked by the bath application of 50 itM APV but unaffected by the bath application of 10 fLM nimodipine (APV: 78% ± 4%, nimodipine: 97% + 14%) (Figures 3.21 and 3.22). In contrast, the calcium signals generated by an LTP stimuli in slices from control-fed aged mice were partially blocked by nimodipine but unaffected by APV (APV: 100% ±8%, nimodipine: 81% ± 3%) (Figures 3.23 and 3.24). These results act as a powerful confirmation that supplementation with a GSH precursor can restore the NMDA receptor-dependence of hippocampal LTP.  3.3.6  Whole-cell LTP in aged mice can be restored by adding GSH to the intrapipette solution  It is notoriously difficult to observe hippocarnpal LTP in older animals in a whole-cell recording configuration. One potential explanation for this dif ficulty may be that important mediators of LTP are dialyzed out of the neurons. In light of our findings that NAC supplementation can restore the NMDA-receptor dependence of LTP in aged mice, we hypothesized that LTP in the whole-cell configuration may also be restored if GSH is added di rectly in the intrapipette solution. Thus, whole-cell recordings of excitatory postsynaptic currents (EPSCs) were carried out in CAl region of hippocam pal slices from aged unfed mice. To be consistent with field recordings, a high-frequency LTP protocol was used to elicit LTP, and was paired with a depolarization of the postsynaptic neuron. Using a normal intrapipette solution, we were unable to produce LTP (Figure 3.25). However, adding GSH (10 mM) to the intrapipette solution restored a significant magnitude of LTP in hippocampal slices from aged mice (control (n=6): -17% + 14%, 92  GSH in the pipette solution (n=9): 54% + 25%,  pO.O3)  (Figures 3.25 and  3.26). These results suggest that the intracellular redox environment may play a crucial role in the modulation of hippocampal synaptic plasticity.  3.4  Discussion  In summary, our results show that in mice, the aging process leads to a shift in the mechanisms underlying LTP from NMDA receptor-dependency to L-type calcium channel-dependency. In addition, we show that this shift in mechanisms can be reversed by oral supplementation of NAC, a precursor for the formation of the antioxidant glutathione.  3.4.1  Synaptic plasticity in the aged mouse  Several studies report that using a weak conditioning protocol to elicit LTP in aged rats leads to age-related LTP deficits (Deupree et al., 1993; Rosen zweig et al., 1997). The TBS form of conditioning protocol, while thought to represent a weaker, more physiologically relevant form of stimulus (Otto et al., 1991), typically uses more stimulation pulses and covers a longer du ration of stimulation when compared with the standard HFS protocol. In order to address these seemingly contradictory features of the TBS protocol, we devised a new TBS protocol that roughly matches the number of pulses and the duration of the HFS protocol and that differs only in pattern. In our adult mice, the use of this new TBS protocol to induce LTP led to a sig nificant decrease in the magmtucle of LTP wlieii compared with that elicited by a standard HFS protocol. We conclude that, as per previous reports, the  93  magnitude of LTP in these animals displays frequency-dependency (Ray mond, 2007). In contrast, in our model of naturally aged mice, the new TBS protocol induced LTP of similar magnitude to HFS-induced LTP. Sev eral variables could explain this finding. First, while several studies look at synaptic plasticity in aged rats, there are significantly fewer studies in mice. Because rats and mice exhibit some differences in their hippocampal structure (van Groen et al., 2002), it is possible that aging affects synaptic plasticity differently in mice. This possible discrepancy highlights the im portance of establishing the ground work for age-related deficits in synaptic plasticity in mice as this species is increasingly used to look at age-related diseases.  Second, the definition of a weaker conditioning stimulus varies  greatly across different studies.  In addition to changes in the frequency  of the pulses, other variables of the conditioning protocol might need to be modulated to observe age-related deficits in LTP, such as using fewer stimulus pulses, or using lower amplitude currents to elicit LTP. Finally, TBS-induced LTP in our model of aging was very variable, with individual recordings of the magnitude of LTP at 60 mm  ranging from -10% up to  +130%. It is possible that the variability of this LTP is preventing us from observing a clear deficit. The reason why the TBS protocol is leading to more variable LTP compared with the HFS protocol is not clear. One possi bility is that the naturally occurring theta rhythms, themselves modulated by the process of aging (Yordanova et al., 1998), somehow interact with the TBS protocol, since this protocol essentially mimics naturally-occuring theta rhythms. We were unable to induce LTD in our model of naturally aged mice, even 94  when using a calcium/magnesium ratio in our aCSF that was previously reported to help induce LTP in older rats (Norris et al., 1996). This results are not surprising, however, because while LTD has been shown to be more easily induced in aged rats compared with adult rats, this does not appear to be the case in mice (Milner et al., 2004). This difference may be attributed to slightly different hippocampal anatomy between the two species, to the use of a variety of different strains of both rats and mice, or to the presence of different modulators of hippocampal synaptic plasticity in the brains of rats and mice.  3.4.2  GSH depletion in aged animals  Many studies confirm that various models of aging show a decrease in GSH levels (Maher, 2005). However, whether the levels of reduced glutathione (GSH) or oxidized glutathione (GSSG) are changed by the aging process has proven very difficult to determine as it is very challenging to accurately measure levels of GSSG in tissue. There are two potential reasons to explain this difficulty. First, GSSG levels are normally very low (the GSH/GSSG ratio in normal, adult conditions is around 100), which makes it difficult to measure accurately. Second, during tissue preparation (slicing, mechan ical disruptions, biochemical disruptions, etc.), a large amount of GSH is oxidized, making it difficult to assess the GSSG levels pre-manipulations. However, a few studies managed to carefully measure levels of GSSG, and an age-dependent increase in the levels of GSSG was observed in the brains of both rats and mice (Palomero et al., 2001; Rebrin et al., 2007). Because GSH is a main antioxidant in the brain, the overall decrease in OSH and 95  increase in GSSG in aged animals strongly suggests that aging leads to a significant alteration of the redox environment in the brain. What is the cause of GSH depletion in aging? The answer to this ques tion could be a decrease in the production of GSH, an increase in the con sumption of GSH, or a combination of both. Strong correlations have been made between the decrease in GSH levels and a decrease in the activity of the glutamyl-cysteine ligase (GCL), the rate-limiting enzyme in the forma tion of GSH (Sandhu & Kaur, 2002). In turn, the decrease in the activity of GCL can be linked to a decrease in the levels of GCL protein and mRNA (Liu, 2002). In the liver, where an age-related decrease in GSH levels also occurs, this process has been linked to a decrease in the level of the Nrf2 transcription factor, responsible for the induction of the GCL genes (Suh et al., 2004). It is currently unknown if a similar decrease occurs in the brain.  3.4.3  Antioxidant properties of NAC  The role of NAC as a cysteine donor for the formation of GSH has been well established (Aydin et al., 2002; Ercal et al., 1996; Kamboj et al., 2006). However, NAC is also an antioxidant itself, and may also modulate the re dox properties of NMDA receptors (Lavoie et al., 2008). Like GSH, NAC can impact cellular oxidative damage through its action as a free scavenger, by promoting a reaction between its thiol groups and ROS (Aruoma et al., 1989). Oral administration of NAC can prevent protein oxidation in mi tochondria in the brain without affecting GSH levels, which confirms an antioxidant property specific to NAC (Martinez et al., 1996). In addition,  96  NAG can activate redox-sensitive transcription factors such as nuclear factor ,B, which in turn can modulate the gene regulation of detoxifying enzymes (Cotgreave, 1997). In our feeding model, oral NAC supplementation leads to a significant increase in levels of GSH in hippocampal neurons, so we conclude that NAC is impacting synaptic plasticity at least partially via its cysteine donor properties. However, we cannot unequivocally rule out the contribution of other NAG-mediated mechanisms of antioxidant action.  3.4.4  Agonist properties of GSH  GSH can impact the activity of the NMDA receptors through the modu lation of redox-sensitive sites on the extracellular portion of the NR1 and NR2A subunits (Steullet et al., 2006). However, it has also been shown that GSH can impact the activity of NMDA receptor by non-redox mechanisms, namely by acting directly with the NMDA receptor as an agonist or an an tagonist. GSH can displace the binding of both radiolabelled agonists and radiolabelled antagonists of the receptor in synaptic membrane preparations (Koller & Coyle, 1985; Ogita et al., 1998). While it is still unclear if GSH acts specifically as an agonist or an antagonist, pharmacological studies report that GSH is more potent at displacing agonists, and therefore, likely acts as an agonist at the NMDA receptor (Ogita et al., 1998). However, whether GSH acts as an agonist or an antagonist at the NMDA receptors may de pend on the subunit composition of the receptor or on the concentration of GSH (Janky et al., 1999). Given these findings, it becomes clear that in our aged animals, an in crease in GSH could consequently increase the activity of NMDA receptors 97  either by the modulation of redox-sensitive sites on the receptors or by act ing as an agonist. Further experiments looking specifically at the redox state of the NMDA receptor with or without GSH supplementation would be required to determine to what extent this redox modulation accounts for changes in NMDA receptor activity in our model. However, since it has been extensively demonstrated that oxidative stress is increased in models of aging (Serrano & Klann, 2004) and that NMDA receptors are more oxidized in this environment (Aoyama et al., 2008), we propose that the increase in function of the NMDA receptor we observed was at least partially due to a GSH-mediated modulation of redox sensitive sites on the receptor.  3.4.5  Intracellular and extracellular impact of GSH on NMDA receptors  In addition to the modulation of NMDA receptors on the extracellular side, the intracellular levels of GSH may also affect some mechanisms involved in regulating the activity of NMDA receptors. Even though NMDA receptors do not appear to contain redox-sensitive sites in the intracellular compart ment (Kohr et al., 1994), changes in the intracellular redox environment can alter several enzymatic mechanisms involved in modulating the function of NMDA receptors (Sommer et al., 2002, 2000). For example, a change in the intracellular redox environment has been shown to affect the activity of the nitric oxide synthase enzyme, which regulates the production of nitric oxide (NO). In turn, NO can diffuse to the extracellular compartment and affect the activity of the NMDA receptors by S-nitrosylation of free thiol groups on the redox-sensitive cysteine sites (Takahashi et al., 2007). 98  Another example of how of changes in the intracellular environment can lead to altered NMDA receptor activity is the interaction between GSH and zinc. A decrease in intracellular GSH levels may hinder the normal state of binding of zinc by the protein metallothionein (Chen & Maret, 2001). Metallothionein is a cysteine-rich protein involved in the uptake, transport and regulation of zinc and may play an important role in the synaptic vesicle trafficking of zinc as well (Knipp et al., 2005). Thus, it is possible that intracellular GSH depletion would lead to changes in the synaptic release of zinc following stimulation. This could in turn modify the activity of NMDA receptors as zinc is an endogenous inhibitory regulator of these receptors (Smart et al., 2004). Overall, we propose that the GSH-induced impact on NMDA receptor activity and synaptic plasticity is caused by a combination of mechanisms, including both redox-dependent and redox-independent mechanisms, and involving both intracellular and extracellular locations of action.  3.4.6  Other pathways involved in LTP in aged animals  While much of our study focused on the age-related hypofunction of NMDA receptors, another type of age-related process occurs in parallel and also impacts synaptic plasticity: an increase in L-type calcium channel activ ity (Thibault et al., 2001). Interestingly, the state of the redox environment can also impact this pathway. L-type calcium channels are thought to physi cally interact with ryanodine receptors (RyRs) on the endoplasmic reticulum (ER), and may act as a preferred source of calcium to activate RyRs (Wang et al., 2001). In turn, the activation of RyRs leads to calcium-induced cal 99  cium release (CICR) from the ER and this serves to further amplify calcium transients. During aging, the increase in calcium influx through the L-type calcium channels and subsequent amplification of this calcium transient via the activation of nearby RyBs may contribute significantly to the calcium dysregulation and associated impacts on hippocampal synaptic plasticity (Thibault et al., 2007). Like NMDA receptors, RyRs can be modulated through the redox state of critical cysteine residues (Pessah et al., 2002). A recent study looking at an animal model of ischemia established that during ischemia, the redox environment is altered in the brain and there is a significant decrease in the GSH/GSSG ratio (Bull et al., 2008). In turn, this change in the GSH/GSSG ratio leads to an increase in S-glutathionylation of RyRs, which allows these receptors to sustain CICR (Bull et al., 2008). In light of these findings, and in light of the evidence that confirms that aging, like ischemia, leads to alterations in the redox environment in the brain, we can hypothesize that during aging, the change in the redox environment affects not only NMDA receptors but also RyRs, which leads to a further increase in calcium dysregulation and thus impacts hippocampal synaptic plasticity.  100  3.5  Figures  101  ADULT 250  -200 •HFS 15O  TVTB5  1oo 20 40 Time(min)  60  Figure 3.1: Effect of stimulation protocol on LTP in adult mice. LTP in the CAl region of the hippocampus of adult mice is of significantly larger magnitude when induced with a high-frequency stimulation (HFS) proto col (blue circles, n=13) then when induced using a theta-burst stimulation (TBS) protocol (red triangles, nr=1O).  102  AGED 250  200 • HFS v TBS  150  20 40 Time (mm)  60  Figure 3.2: Effect of stimulation protocol on LTP in aged mice. LTP in the CAl region of the hippocampus of aged mice is of similar magnitude when induced with a high-frequency stimulation (HFS) protocol (blue cir cles, n=7) and when induced with a theta-burst stimulation (TBS) protocol (red triangles, nrrrl2).  103  NORMAL ACSF 250  • ADULT vAGED  ioo 50  0  20 40 Time (mm)  60  Figure 3.3: LTD in adult and aged mice. Using a standard low-fre quency stimulation, no significant LTD was elicited in the CAl region of the hip pocampus of either adult (blue circles, n=6) or aged (red triangles, n=7) mice.  104  4:2 ACSF 250 200 a)  • ADULT vAGED  c2-  cL LU  1 50  0  20 40 Time (mm)  60  Figure 3.4: LTD in adult and aged mice using an altered cal cium/magnesium ratio in the aCSF. When using an aCSF solution con taining an increased ratio of calcium to magnesium (4 mM calcium, 2 mM magnesium), a standard low-frequency protocol still failed to elicit LTD in the CAl region of the hippocampus from adult (blue circles, n=4) and aged (red triangles, n=4) mice.  105  150 -  I  V.)  1100  0  50  I  0 Time (mm)  4060  Figure 3.5: APV blocks LTP in the CAl region of adult mice. CM fEPSP slope in response to HFS stimulation applied at t=O in adult mice in control conditions (blue circles, n=13) and during NMDA receptor blockade using APV (50 1 iM, red triangles, nrrlO). Averaged IEPSP traces inset. Scale bars: 5 ms, 0.2 mV.  106  150 ci) C  ci)  V.,  2  100  0 ci) C  $  •LTP V NIMO  50  0’  ci) a 0  cI  V., uJ  0  20 40 Time (mm)  60  Figure 3.6: LTP is unaffected by nimodipine in the CAl region of adult mice. CAl fEPSP slope in response to HFS stimulation applied at t=0 in adult mice in control conditions (blue circles, n=13) and during L-type calcium channel blockade using nimodipine (10 pM, red triangles, nr=8). Averaged fEPSP traces inset. Scale bars: 5 ms, 0.2 mV.  107  ADULT 180 *  160  -14 0  120  100  LW  L  ARI NIMO  Figure 3.7: LTP is NMDA receptor-dependent in adult mice. Quantitative representation of the data from Figures 3.5 and 3.6.  108  150 C)  ci) V.)  1100 •LTP VAPV  50 I-)  0  C) .  20 40 Time (mm)  60  Figure 3.8: LTP is unaffected by APV in the CAl region of aged mice. CAl fEPSP slope in response to HFS stimulation applied at t=O in aged mice in  control conditions (blue circles, n=7) and during NMDA receptor blockade using APV (50 1 tM, red triangles, n=8). Averaged fEPSP traces inset. Scale bars: 5 ms, 0.2 mV.  109  __  a)  150  V)  1100 •LTP YNIMO  [0  0  20 40 Time (mm)  60  Figure 3.9: LTP is significantly decreased in the presence of nimodipine in the CAl region of aged mice. CAl fEPSP slope in respon se to HFS stimulation applied at t=O in aged mice in control conditions (blue circles, n=7) and during L-type calcium channel blockade using nimodipine (10 tM, red triangles, n=6). Averaged fEPSP traces inset. Scale bars: 5 ms, 0.2 mV.  110  AG6D 180  *  160  -140  12O  100  LW  AR’ NIMO  Figure 3.10: LTP is L-type calcium channel-dependent in aged mice. Quan titative representation of the data from Figures 3.8 and 3.9.  111  *  0.6  1  0.5 0.4b -J  0.3 0.2 0.1 0  Cfl  NAC  Figure 3.11: NAC supplementation in aged mice leads to increased levels of hippocampal GSH. NAC-fed aged mice (n=5) had a significantly higher hippocampal GSH content compared with control-fed aged mice (n=5).  112  deep  shallow  —  Figure 3.12: Example of MCB labeling within a single stack of images . Sample of two images from a same stack showing the decreasing intensity of the MCB labeling with increasing depth into the stack. Scale bar: 50 jim.  113  Figure 3.13: The intensity of MCB labeling is increased in NAC-fed aged mice. Brain slices, loaded with 60 M MCB to visualize the GSH level, show ing CAl region of the hippocampus. Upper: an aged, control-fed mouse. Lower: an aged, NAC-fed mouse. Scale bar: 50 ILm.  114  Stratum radiatum  Astrocytes  *  100  4-.  200  80  ‘f)  4-’  C  C  ci) I’)  C ci)  ci) U C ci) U  60  I-) V.)  V.)  ci)  ci)  0  0  I-.  40  100  D  ci) >  ci) >  4-,  ci)  150  4-,  (U ci)  20 0  CTRL  NAC  50  0  CTRL  NAC  Figure 3.14: NAC supplementation in aged mice leads to increased levels of GSH in hippocampal neurons. Fluorescence intensity of GSH labeling with MCB in the stratum radiatum (S.R., dendritic region) and in the astrocyte somas. The fluorescence intensity is normalized to a fluorescent pipette tip within slice (see Figure 3.12). NAC-fed mice (n=6) showed significantly more GSH in the dendritic region compared with control-fed mice (n=5).  115  150 cI  C ci)  1100 •LTP VAPV 0  20 40 Time (mm)  60  Figure 3.15: APV blocks LTP in the CAl region of NAC-fed aged mice. CAl fEPSP slope in response to HFS stimulation applied at trr0 in NAC fed aged mice in control conditions (blue circles, nrrrT) and during NMDA receptor blockade using APV (50 jtM, red triangles, n=6). Averag ed fEPSP traces inset. Scale bars: 5 ms, 0.2 mV.  116  150 ci) ci)  V)  1100 •LTP YNIMO  I° 0  20 40 Time (mm)  60  Figure 3.16: LTP is unaffected by the presence of nimodipine in the CAl region of NAC-fed aged mice. CAl fEPSP slope in response to HFS stim ulation applied at t=O hi NAC-fed aged mice in control conditions (blue circles, n=7) and during L-type calcium channel blockade using nimodipine (10 jIM, red triangles, n=9). Averaged fEPSP traces inset. Scale bars: 5 ms, 0.2 mV.  117  NAC 180 *  160 a) 0  120  AR! NIMO Figure 3.17: LTP is NMDA receptor-dependent in NAC-fed aged mice. Quantitative representation of the data from Figures 3.15 and 3.16.  118  150 cl) C  a) 1100 •LTP VAPV  I° 0  20 40 Time (mm)  60  Figure 3.18: LTP is not significantly blocked by APV in the CAl region of control-fed aged mice. CAl fEPSP slope in response to HFS stimulation applied at t=O in control-fed aged mice in control conditions (blue circles, n=8) and during NMDA receptor blockade using APV (50 tM, red triangles, n=r5). Averaged fEPSP traces inset. Scale bars: 5 ms, 0.2 mV.  119  150  -  100  2  •  LTP VNIMO  TThTTTTTTT1I  U 0”  G) ci o -i;:;  —  r .1  0  20 40 Time (mm)  60  Figure 3.19: LTP is significantly decreased in the presence of nimodipine in the CAl region of control-fed aged mice. CAl fEPSP slope in response to HFS stimulation applied at t=O in control-fed aged mice in control condi tions (blue circles, n=8) and during L-type calcium channel blockade using nimodipine (10 jiM, red triangles, n=7). Averaged fEPSP traces inset. Scale bars: 5 ms, 0.2 mV.  120  c11180 *  160  I  0  120  100  LW  AR! NIMO  Figure 3.20: LTP is L-type calcium channel-dependent in control-fed aged mice. Quantitative representation of the data from Figures 3.18 and 3.19.  121  4  4  4  4  Figure 3.21: NMDA receptor blockade reduces HFS-induced calcium signals in NAC-fed mice. Calcium signal traces in control (blue) and during APV (red) or during nimodipine (green) application in the stratum radiatum from a NAC-fed mouse in response to HFS. Scale bars: F/F , 3 s. 0  122  _______  NAC 120  100  0  (‘3 C  80  V)  E 60 (‘3 L)  0  40  (‘3  ci)  4-.’  C  20 0  APV  NIMO  Figure 3.22: NAC supplementation restores NMDA receptor-mediated cal cium signals in aged mice. Quantitative representation of the data from Figure 3.21. Only NMDA receptor blockade reduces the calcium signal.  123  +  +  +  +  +  Figure 3.23: L-type calcium channel blockade reduces HFS-induced calcium signals in control-fed mice. Calcium signal traces in control (blue) and during APV (red) or during nimodipine (green) application in the stratum radiatum from a control-fed mouse in response to HFS. Scale bars: F/Fo, 3 5.  124  CTRL 120 100 0  (‘3 C  E D  60  (‘3 U  040 (‘3 G)  20 0  NIMO  Figure 3.24: Control-fed mice exhibit nimodipine-sensitive calcium signals. Quantitative representation of the data from Figure 3.23. Only k-type cal cium channel blockade reduces the calcium signal.  125  250 200 • Normal vGSH  150 E CD  U  1  w  -10  0  10 20 Time (mm)  30  Figure 3.25: GSH in the intrapipette solution leads to significant LTP in whole-cell conditions. Whole-cell EPSC amplitude in slices from aged mice recorded with either a normal intrapipet solution (blue circles, n=6) or one supplemented with 10 mM GSH (red triangles, n=9) in response to HFS stimulation applied at t=0.  126  *  l O 8 r 160 14O  D  IOOF  20  Figure 3.26: GSH restores whole-cell LTP. Quantitative summary of the data from Figure 3.25. Introducing intracellular GSH restored LTP in aged slices.  127  Chapter 4  General Discussion 4.1  Summary of findings  The results described in Chapters 2 and 3 clearly demonstrate that hip pocampal synaptic plasticity is subject to modulation by both exogenous agents, such as a 5-HT6 receptor antagonist, and endogenous agents, such as the levels of glutathione (GSH), a major antioxidant in the brain. As hippocampal synaptic plasticity is thought to represent a cellular model of learning and memory, it is critical to establish the impact of various mod ulators in animals of all ages. Modulation of LTP and LTD is especially important to study in aged animals as more information in this field could lead to new therapeutic avenues for age-associated cognitive diseases.  4.1.1  Involvement of the 5-HT6 receptor in hippocampal synaptic plasticity  The research presented in Chapter 2 looked at the involvement of the 5-HT6 receptor in the modulation of synaptic plasticity in two different regions of the hippocampus. In the CAl region of the hippocampus of adult rats, treatment with a 5-HT6 receptor antagonist, R9237, lead to a significant  128  increase in the magnitude of LTP. In the dentate gyrus (DG) region of the hippocampus, however, this enhancement was not observed, and the magnitude of LTP was comparable in control rats and in treated rats. In addition, our results show a role for the 5-HT6 receptor in the modulation of LTD in both the CAl and the DG, as in both regions, LTD was completely blocked by the treatment with R9237. In the DG, treatment with the 5-HT6 antagonist lead to a reversal of LTD into a small but significant amount of LTP, thus I investigated whether R9237 was acting to lower the threshold for LTP by using a weak, LTP-inducing protocol. However, I did not observe an enhancement in DG LTP even when using a weak conditioning stimulus. These results arc the first report of the modulation of hippocampal synaptic plasticity by 5-HT6 receptors and highlight differences in the modulation of LTP in the CAl region and in the DO.  4.1.2  Impact of aging and glutathione on hippocampal synaptic plasticity  The research presented in Chapter 3 looks at hippocampal synaptic plastic ity in aged mice. In the CAl region of the hippocampus, HFS-induced LTP is of similar magnitude in adult male mice and in our model of naturally aged mice. However, in adult mice, LTP is mostly NMDA receptor-dependent, while in aged mice, LTP is mostly L-type calcium channel dependent. These results confirm in mice the age-related shift in the mechanisms underlying LTP observed initially in rats. Next, a model of GSH supplementation was developed in aged mice us ing oral N-acetylcysteine (NAC) administration, and showed that this pro129  tocol increases GSH levels in the hippocampal neurons of aged mice. Using this model, the effects of NAC supplementation on hippocampal LTP were investigated. Interestingly, NAC-fed aged mice displayed NMDA receptordependent LTP similar to adult mice, while control-fed aged mice displayed L-type calcium channel-dependent LTP similar to unfed aged mice. There fore, I propose that a GSH deficit in aged mice leads to hypofunction of NMDA receptors and that this in turn impacts synaptic plasticity. To con firm the shift in mechanisms underlying LTP observed in electrophysiologi cal experiments, calcium imaging was used to investigate calcium signals in response to an LTP stimulus in control-fed and NAC-fed aged mice. The re suits showed that NAC supplementation restored NMDA receptor-mediated calcium signais in response to an LTP conditioning protocol. To further confirm the involvement of GSH in LTP, whole-cell recordings were used and showed that the addition of GSH to the intrapipette solution restores whole-cell LTP in hippocampal slices from aged, unfed mice. Overall, our results suggest an important role for GSH as a modulator of hippocampal synaptic plasticity during aging.  4.2  Impact of oxidative stress on the L-type calcium channel pathway  In Chapter 3, it was demonstrated that the process of aging leads to a decrease in NMDA receptor-mediated LTP and a concurrent increase in L type calcium channel-mediated LTP. While much of our study focused on the age-associated hypofunction of NMDA receptors, the increase in L-type 130  calcium channel activity during aging is also thought to play an important role in the calcium dysregulation which leads to synaptic plasticity impair ments in the hippocampus (Thibault et al., 2007). The functional impor tance of the increase in the activity of L-type calcium channels during aging has been demonstrated by experiments showing that treating aged animals with L-type calcium channel blockers leads to improvements in learning and memory in these animals (Deyo et al., 1989; Disterhoft et al., 2004). Can oxidative stress modulate the activity of L-type calcium channels like it modulates the activity of NMDA receptors? Several studies looking at L-type calcium channels in the heart suggest that indeed, redox modulation call affect the activity of L-type calcium channels (Scragg et al., 2008; Zima & Blatter, 2006). Much like NMDA receptors, cardiac L-type calcium chan nels have redox-sensitive cysteine residues that can modulate the activity of the channel (Scragg et al., 2008). However, it remains unknown whether L-type calcium channels in the brain can be modulated in a similar way. In addition to L-type calcium channels, redox modulations can impact the activity of another key player in this pathway: the ryanodine receptors (RyRs). RyRs are thought to interact directly with L-type calcium channels (Kim et al., 2007). Because of this alignment, calcium influx from L-type calcium channels may become the preferred source of calcium for the acti vation of CICR via RyRs (Wang et al., 2001). During aging, the increased activity of the L-type calcium channels leads to an increase in the activa tion of CICR and an amplification of calcium dysregulation (Thibault et al., 2007). The activity of RyRs, like that of NMDA receptors, can be influenced by 131  the redox environment (Pessah et al., 2002). RyRs contain redox-sensitive cysteine residues that can be modulated by reducing and oxidizing agents (Pessah et al., 2002).  In conditions of oxidative stress, the oxidation of  these critical cysteine residues on RyRs leads to an increase in RyR activity. Because aging leads to an increase in oxidative stress, this phenomenon may contribute to further calcium dysregulation during the aging process. Since RyRs can be modulated by the GSSG/GSH ratio (Bull et al., 2008), an attempt was made to study the redox modulation of RyR through S-glutathionylation in our model of naturally aged mice. I was hoping to show that aging, like ischemia, leads to a change in the GSSG/GSH ratio that affects RyRs via S-glutathionylatiori. Unfortunately, the very large size of the RyR (565 kDa) made it difficult to study using Western blots in our model of hippocampal slices. Using our model, I was unable to reproduce the findings from Bull et al. (2008) that RyRs are glutathionylated in conditions of oxidative stress (Bull et al., 2008). However, Bull et al. (2008) had used a preparation of isolated endoplasmic reticulum (ER) vesicles, which may explain why they were more successful at isolating RyRs on Western blots. Overall, aging leads to an increase in oxidative stress and this change in the redox environment can affect not only the NMDA receptor pathway, but also the L-type calcium channel/RyR pathway.  132  4.3 4.3.1  Clinical relevance of work Alzheimer’s disease  Alzheimer’s disease (AD) is the most common neurodegenerative disorder, with over 26 million sufferers worldwide (Brookmeyer et al., 2007). The clinical features of this disease include a progressive cognitive decline, im pairments in daily life activities and a variety of behavioral symptoms such as depression arid aggression. Biologically, AD leads to significant neuronal loss and plaque and tangle pathology. The specific etiology behind these changes in the brain is still unclear, and several neurotransmitter pathways have been implicated, including GABAergic, glutamatergic and serotoner gic pathways (Upton et al., 2008). The current therapies for AD, such as cholinesterase inhibitors and memantine (an NMDA receptor blocker), seem to have only small effects in some patients and none in others, in addition to leading to unwanted side effects (Birks  Harvey, 2006). Therefore, there is  an ever-present need to develop novel therapeutic avenues for AD. 5-HT6 receptors in Alzheimer’s disease  Soon after its discovery, the 5-HT6 receptor became of interest for AD due to its abundance in brain regions involved in regulating cognitive processes (Woolley et al., 2004) and its polymorphism potentially associated with an increased risk of dementia (Tsai et al., 1999). In addition, early studies investigating the role of 5-HT6 receptors led to the discovery that 5-HT6 receptor blockade leads to an increase in yawning and stretching behavior that can be reversed by antagonists of muscarinic receptors (Bourson et al., 133  1995).  This finding suggests that 5-HT6 receptors may act to suppress  cholinergic transmission. Since the cholinergic system was already known to be involved in several cognitive processes, 5-HT6 receptors were hypoth esized to play a role in cognition as well and were investigated further in behavioral cognition and memory paradigms. Experiments using both antisense oligonucleotides or pharmacological antagonists of the 5-HT6 receptor showed that blockade of this receptor in young, unimpaired rats leads to improved spatial learning and memory in the Morris water maze (Woolley et al., 2001). While later studies failed to replicate the effects on learning acquisition, the effect of 5-HT6 receptor blockade on retention of information was confirmed (Rogers & Hagan, 2001). However, other groups have not been able to replicate either finding (Lindner et al., 2003; Russell & Dias, 2002), and more work is needed to define under what specific conditions the modulation of 5-HT6 receptors can enhance cognitive performance in the Morris water maze. The cognitive enhancing properties of 5-HT6 receptor antagonists have been confirmed using a variety of different behavioral cognitive tasks, such as novel object recognition and social recognition tasks (King et al., 2004; Loiseau et al., 2008).  In addition, several studies looking at age-related  cognitive impairment find improvements when animals are treated with a 5HT6 antagonist, notably in the Morris water maze (Hirst et al., 2006; Foley et al., 2004). The cognitive enhancing properties of 5-HT6 antagonists have also been demonstrated in aged nonhuman primates in a delayed matching to-sample task (Upton et al., 2008). To date, no study has evaluated the effect of 5-HT6 receptor blockade on animal models of AD, however the 134  successes observed when treating animals exhibiting age-associated memory impairments with 5-HT6 antagonists suggests that these pharmacological tools could be of use to treat AD as well.  Glutathione in Alzheimer’s disease Several lines of evidence suggest that oxidative stress is an important con tributor to the pathogenesis of AD. One such line of evidence lies in the fact that oral aipha-tocopherol supplementation can strengthen antioxidant defenses and may delay the progression of AD in patients exhibiting mod erate to severe impairments (Sano et al., 1997). In addition, the brains of AD patients show a significant increase in lipid peroxidation compared with age-matched controls (Marcus et al., 1998). However, it remains unclear whether the pathophysiology of AD is a cause or an effect of an increase in oxidative stress (Smith et al., 1996). The ratio of oxidized GSH (GSSG) to reduced GSH (GSH), also known as the GSH redox status, is thought to represent an accurate and reliable measure of oxidative stress in most types of tissues, cells and organelles (Via et al., 2004). This ratio is significantly increased in peripheral cells in AD patients compared with age-matched controls (Via et al., 2004). In contrast, the brain levels of total GSH do not appear to change in AD (Perry et al., 1987). However, it is difficult to interpret this finding as levels of GSSG could be increased while levels of reduced GSH decreased. Changes in the enzymes involved in the metabolism of glutathione are equally unclear. Some groups find increases in brain levels of GSH peroxidase (GPx) and GSSC reductase (CR) (Lovell et al., 1995), while others find no change (Marcus et al., 1998). 135  In terms of behavior studies, several reports suggest that NAC, the pre cursor for the formation of GSH, leads to improvements in cognitive per formance in a wide variety of models of cognitive impairments, including aluminium exposure (Prakash & Kumar, 2009), sepsis (Barichello et al., 2007), hypobaric hypoxia (Jayalakshmi et al., 2007), and carbofuran ex posure (Kamboj & Sandhir, 2007). Perhaps most interesting in the con text of AD is a report showing that a combination of antioxidants includ ing NAC enhances or maintains cognitive function in mice models of ag ing and neurodegeneration, including mice expressing human apolipoprotein E4 (ApoE4) (Chan & Shea, 2007), a genetic risk factor for some forms of AD. Overall, while oxidative stress plays an important role in AD, the spe cific contribution of the GSH system remains unknown. However, findings from behavior studies suggest that GSH supplementation may constitute a promising therapeutic avenue for cognitive impairments.  4.3.2  Other conditions  Clinical relevance of 5-HT6 receptor antagonists To date, several 5-HT6 receptor antagonists, including pharmacological com pounds still being developed, are being investigated as potential treatments for several diseases (schizophrenia, obesity, AD). Overall, there is a majority of clinical trials looking at treating AD using 5-HT6 receptor antagonists. In healthy volunteers (Phase I clinical trials), all the 5-HT6 antagonists tested so far have shown adequate safety and tolerability profiles, even fol lowing the administration of repeated doses. One compound, SB-742457  136  (GlaxoSmithKline), was recently tested in two phase II trials with patients diagnosed with mild to moderate AD. The results from these two clini cal trials suggest that not only is SB-742457 well tolerated, it also leads to improvements in cognitive function as well as daily activity function as assessed by the Alzheimer’s Disease Assessment Scale-Cognitive Subscale (ADAS-cog) and the Clinician’s Interview-Based Impression of Change-plus Caregiver Input (CIBIC+) (Maher-Edwards et al., 2009). While this effect was comparable to the effect of donepezil, an acetylcholinesterase inhibitor, it represents an interesting new therapeutic avenue for the treatment of AD.  Clinical relevance of N-acetylcysteine NAC, under various formulations, is already commonly used to treat a vari ety of ailments. First, due to its mucolytic properties, NAC is often indicated for respiratory conditions that involve excessive production of mucus, such as emphysema or tuberculosis. The use of NAC to treat cystic fibrosis is debated, however a recent publication suggests NAC could be effective for this condition (Tirouvanziam et al., 2006). In addition, several clinical trials are currently underway to evaluate the possibility of treating cystic fibrosis with NAC. Another common pharmacological use of NAC is as a treatment for paracetamol (acetaminophen) overdose. In this condition, NAC serves to protect hepatocytes from paracetamol-induced toxicity. In addition, NAC can act as a nephroprotective agent and is commonly used in renal impair ment patients for the prevention of acute renal failure. In addition to the current applications of NAC treatment, there are sev eral clinical trials currently underway to assess the efficacy of NAC in a wide 137  variety of diseases and conditions. For example, NAC is being investigated in contexts ranging from autism spectrum disorders to chemotherapy-induced toxicity. Another potentially interesting use of NAC is in the treatment of a number of addictions. Clinical trials are currently looking at NAC as a treatment for nicotine addiction, alcohol dependency, cocaine addiction, and pathological gambling. These trials are based on a recent body of literature pertaining to NAC and addiction in animal studies (Moussawi et al., 2009). Most relevant to the research described in Chapter 3, NAC was also investigated as a potential treatment for AD. During the controlled, doubleblind clinical trial, NAC or a placebo was administered to patients meeting the criteria for probable AD, arid efficacy tests were conducted after three and six months of treatment. A comparison between the placebo group and the NAC group showed that NAC treatment improved almost every outcome measure.  However, these differences were only significant for a  subset of cognitive tasks (Adair et al., 2001). These results in combination with research showing that NAC decreases mitochondrial oxidative stress in AD patients (Moreira et al., 2007), suggest that antioxidant therapies involving NAC may be a promising new therapeutic avenue for the treatment of AD.  138  4.4 4.4.1  Future directions Mechanisms involved in the modulation of synaptic plasticity  The modulation of hippocampal plasticity by the 5-HT6 receptor The precise role of the 5-HT6 receptor in the brain is still unknown, and there are several ways in which the activation of the 5-HT6 receptor could modu late hippocampal LTP and LTD. In the hippocampus, agonists of the 5-HT6 receptor elicit significant increases in extracellular GABA levels (Schechter et al., 2008).  Conversely, administration of a 5-HT6 receptor antagonist  leads to elevated levels of both extracellular acetycholine and glutamate in the hippocampus of freely moving rats (Zhang et al., 2007; Dawson et al., 2000, 2001). One possible hypothesis for the tonic modulation of hippocam pal activity through the 5-HT6 receptor stipulates that activation of the 5HT6 receptors present on GABAergic neurons leads to elevated extracellular levels of CABA and consequently a decrease in the activity of glutamatergic neurons (Upton et al., 2008). This proposed model could explain our results that show increased LTP in the CAl region of 5-HT6 antagonist-treated rats. However, our findings also show that this decrease does not occur in the DG. Further experiments are needed to assess whether the 5-HT6 re ceptor acts to modulate synaptic plasticity at the cellular level via its effect on cAMP levels and PKA activation or whether it acts on a broader level by exerting a tonic modulation of hippocampal activity. Mice have lower levels of 5-HT6 receptors relative to rats and so are poor  139  models to study this receptor. This is unfortunate because while there are many mice models of AD, this is not the case in rats. Nonetheless, it would be interesting to look at the effects of our 5-HT6 receptor antagonist on hippocampal synaptic plasticity in a rat model of AD involving intracere broventricular injections of the peptide amyloid beta, the main constituent of the amyloid plaques present in the brains of AD patients. This study would prove very valuable to assess the involvement of this receptor in amy bid beta-mediated plasticity impairments.  The modulation of hippocampal plasticity by glutathione Our findings on GSH and synaptic plasticity in the aged mouse suggest that the redox modulation of the NMDA receptor is at least in part responsi ble for the age-related impairments in NMDA receptor-dependent synaptic plasticity. However, several questions remain. First, it would be interesting to measure the state of the oxidation of the NMDA receptors in our model of naturally aged mice, and to compare this state between young adult mice, control-fed mice and NAC-fed mice. This would allow us to confirm that GSH depletion in aged animals directly leads to NMDA receptor hypofunc tion. In addition, further studies will be required to describe the impact of the GSH supplementation on the activity of L-type calcium channels, as some evidence suggests they may also be subject to redox modulation (Scragg et al., 2008). As well, it would be interesting to investigate the impact of changing GSH levels on RyR activity. Finally, it would be inter esting to investigate whether oral supplementation with NAC can restore LTD in aged animals. Overall, these further directions would allow us to 140  paint a comprehensive picture of the effect of GSH supplementation on the main pathways involved in hippocampal synaptic plasticity.  4.4.2  Behavior studies  5-HT6 antagonists as cognitive enhancers While several studies already looked at the effect of various 5-HT6 antag onists on cognition and memory (Upton et al., 2008; Woolley et al., 2001), there are some contradictions and inconsistencies in the literature regard ing the efficacy of 5-HT6 antagonists as cognitive enhancers. Therefore, it would be important to evaluate the effect of our antagonist in cognitive and memory tasks that depend on the hippocampus such as the Morris water maze. These experiments in concert with our findings described in Chapter 2 would allow us to evaluate the impact of changes in synaptic plasticity on behavior. Furthermore, given the interest for 5-HT6 antagonists as a poten tial treatment for AD, it would be interesting to first assess this possibility in animal models. As previously discussed, mice are not good candidates for the study of 5-HT6 receptors, however rat models of AD are available. Further directions should include experiments assessing the efficacy of our 5-HT6 antagonist at treating amyloid beta-induced cognitive and memory deficits.  NAC as a cognitive enhancer In Chapter 3, the magnitude of hippocampal LTP was demonstrated to be similar in young adult mice and in aged mice, but the mechanisms un  141  denying this LTP are different. This suggests that the level of achievable plasticity in the CAl region of the hippocampus remains constant, possi bly due to a compensatory mechanism in aged animals. It is important to note that even though the magnitude of LTP was similar in adult and aged animals, one cannot conclude that L-type calcium channel-mediated LTP does not lead to memory impairments in aged animals. The relationship between the different mechanisms underlying LTP and memory has yet to be described. L-type calcium channel-dependent LTP and NMDA receptordependent LTP are thought to activate different downstream mechanisms (Cavus & Teyler, 1996) and may lead to the regulation of different genes (Bading et al., 1993). This differential gene regulation may in turn lead to different levels of functional cognitive processes (Cavus & Teyler, 1996). Therefore, an interesting experiment would be to assess the cognitive per formance of our mouse model of natural aging in memory tasks. 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(1993). Further evidence for multiple forms of an n-methyl-d-aspartate recognition domain in rat brain using membrane binding techniques.  J Neurochem, 61(5),  1865—1873.  180  Appendix A  Research Ethics Approval  181  liSps //rise,ubcca/risefDoc/O/EEHSOA3V7OD4ROIHQ2EV63RF77/frorn..  The University of British Colunibia  Animal Care Certificate Application Number:  A04-l0l0  Investigator or Course Director: Brian R. Clsristie Psychology, Department of  Department:  Animals Approved:  Start Date:  Mice C57 based transgenics 250 RatsSD25O  December 1, 2004  Approval Date: January 4, 2006  Funding Sources:  Funding Agency: Funding Title: Funding Agency: Funding Title:  Natural Sciences and Engineering Research Council Neurogenesis and synaptic plasticity in the adult brain. Roche Phannaceuticals Role of 5HT Receptors in Hippocampal Synaptic Plasticity  Funding Agency:  Canadian Institutes of Health Research  Funding Title  Role of kainate receptors in synaptic physiology in the hippocampal dentate gyrus,  Funding  Agency:  Scottish Rile Charitable Fdn of Canada (see Roeher Inst)  Funding Title:  Fragile X Syndrome: Effects of Fmrl deletion on hippocampal structure and function.  Unfunded title:  n/a  The Animal Care Committee has examined and approved the use of animals for the above experimental project.  This certificate is valid for one year from the above start or approval date (whichever is later) provided there  is no change in the experimental procedures. Annual review is required by the CCAC and some granting agencies.  1 of2  2/6/2010 2:37 PM  182  https://rise,ubc,ca/riseJDoe/0/EEHSOA3V7004ROIHQ2EV63RF77/frorn..  A copy of this certificate must be displayed in your animal facility  Office of Research Services and Administration 102, 6190 Agronomy Road, ‘vhncouver, V6T 1Z3 Phone: 604-827-5111 Fax: 604-822-5093  2 of2  2/6/20102:37 PM  183  https//rise.ubc.caJrise/Doc/O/8S8ATVFO2OD4RFFOJ5BWfT99O4/fromSt..  UBC  THE UNIVERSITY OF BRITISH COLUMBIA  ANIMAL CARE CERTIFICATE Application Number: A04-0296 Investigator or Course Director: Brian R. Christie  Department: Psychology, Department of Animals:  Rats 280 Mice 280  Start Date:  September 1, 2004  Approval  September 25, 2008  Funding Sources: Funding II Agency: Funding Title: Funding Agency: Funding Title:  Fragile-X Syndrome: Effects of FMRI deletion on hippocampal structure and function Fragile X Research Foundation of Canada Synaptic changes in Fragile X Syndrome  Funding Agency:  Canadian Institutes of Health Research (CIHR)  Funding Title:  Influence of glutamate receptor topology on structural and functional plasticity in the hippocampua aging  Funding Agency: Funding Title: Funding Agency: Funding Title:  I of2  Scottish Rite Charitable Foundation of Canada  British Columbia Miriiatiy of Children and Family Development Ultrastructural Analysis of Neuronal and Synaptic Development in Fragile-X Syndrome Canadian Institutes of Health Research (CIHR) Effects of exercise on stntctural and functional plasticity in the aging hyppocampus  2/6/20t02:38PM  184  hUps://rise.ubc.cs/Tise/Docf0/8S8ATVFO2OD4RFFOJ5BHtJT9904/fronSt..  tidinfi Funding Title:  Unfunded title:  British Columbia Ministry of Children and Family Development through HELP How does ABCG1 Over-expression in Down’s Syndrome Impact the Structural and Functional Development of the Hippocampus.  N/A  The Animal Care Committee has examined and approved the use of animals for the above experimental  project. This certificate is valid for one year from the above start or approval date (whichever is later) provided there is no change in the experimental procedures. Annual review is required by the CCAC and some granting agencies.  A  copy  of  this certificate must be displayed in your animal facility.  Office 102,  of  6190  Research  Agronomy  Phone:  2of2  Services  Road,  604-827-5111  and  Administration  Vancouver,  Fax:  BC  V6T  1Z3  604-822-5093  2/6/201t2:35PM  185  

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