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Cholinergic modulation of ion channels in the CNS Tai, Chao 2009

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Cholinergic Modulation Of Ion Channels In The CNS  by  Chao Tai  B.Sc., Peking University, P. R. China, 2002  A THESIS SUBMITTED fl%4 PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY  in  THE FACULTY OF GRADUATE STUDIES (Neuroscience)  UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  September, 2009  © Chao Tai, 2009  Abstract The cholinergic system is one of the most important modulatory neurotransmitter systems in the tpermable ion 2 CNS. In this dissertation, I report novel cholinergic modulations of three Ca channels, including R-type voltage-gated calcium channels (VGCCs), TRPC5 channels and NMDA receptors, in hippocampal CA 1 pyramidal neurons and the potential functional roles of these modulations in both physiological and pathophysiological conditions. I first studied the “toxin-resistant” R-type VGCCs, and found that muscarinic activation specifically enhances R-type, but does not affect T-type, Ca 2 currents in hippocampal CAl pyramidal neurons. The muscarinic stimulation of R-type Ca 2 channels is mediated by M1/M3 tindependent PKC pathway. Furthermore, the 2 receptors and requires the activation of a Ca enhancement of R-type Ca 2 currents resulted in remarkable changes in the firing pattern of the de novo R-type Ca 2 spikes, which could fire repetitively in the theta frequency. Therefore, muscarinic  enhancement of R-type Ca2+ channels could play an important role in the intrinsic resonance .  .  .  properties of neurons. Next, I studied the muscarinic-induced prolonged seizure-like depolarizations called plateau potentials (PPs) in CAl pyramidal neurons. I found that muscarinic stimulation significantly and specifically triggered rapid translocation of TRPC5 channels into plasma membrane. Moreover, TRPC channels contribute to the generation of PPs, the underlying tail currents  (‘tail)  and the  associated dendritic Ca 2 influx in CA 1 pyramidal neurons, via a calmodulin- and PI K-dependent 3 pathway. Thus the muscarinic-induced membrane insertion of TRPC5 channels could contribute to the generation of PPs and the prolonged neuronal depolarization during the ictal discharges in epilepsy. And finally, I report that muscarinic modulation of NMDA-evoked current  (INA)  in CA 1  pyramidal neurons is age-dependent. I found that muscarinic stimulation potentiated Iti in both young and old animals. However, in young animals, muscarinic stimulation potentiated Itr through a [Ca ]-independent but PKC- and Src-dependent pathway. While in old animals, 2 muscarinic stimulation potentiated  through a [Ca ]-dependent but PKC-independent 2  pathway. Interestingly, the activity of the Gctq-coup led Mi-like muscarinic receptors was required ii  for the potentiation of  in both cases. These findings may provide a crucial mechanism by  which cholinergic input modulates learning and memory.  111  Table of Contents Abstract  .  Table of Contents List of Figures List of Symbols, Abbreviations and Nomenclature  ii  iv viii x  Acknowledgements  xiv  Dedication  xv  Co-Authorship Statement  Chapter One: Introduction The Cholinergic System Of The CNS 1.1. General Anatomy 1.2. Cholinergic Forebrain Projections Cholinergic Projections In Hippocampus and The Septohippocampal Pathway 1.2.1. 1.3. Brain stem Projections 1.4. Cholinergic Interneurons 2. Cholinergic Receptors In The CNS 2.1 Nicotinic Cholinergic Receptors Distribution of Neuronal nAChRs 2.1.1. Channel Properties of Neuronal nAChRs 2.1.2. Physiology of Neuronal nAChRs 2.1.3. Pathophysiology of Neuronal nAChRs 2.1.4. Muscarinic Cholinergic Receptors 2.2. G-Protein-Coupled Receptors 2.2.1. 2.2.2. Distribution of mAChRs In The CNS 2.2.3. Signal Transduction Pathways Of mAChRs 2.2.4. Physiology of Muscarinic Receptors In The CNS 2.2.4.1. Muscarinic Modulation of Ion Channels 2.2.4.1.1. Voltage Gated Sodium Channels 2.2.4.1.2. Voltage Gated Potassium Channels 2.2.4.1.3. Voltage Gated Calcium Channels 2.2.4.1.4. Non-Selective Cation Channels 2.2.4.2. Muscarinic Modulation of Synaptic Transmission 2.2.4.2.1. Presynaptic Modulation 2.2.4.2.2. Postsynaptic Modulation 1.  xvi  1 1 1 2 2 6 6 7 7 8 9 10 11 12 12 14 15 17 17 18 19 21 23 24 24 26 iv  2.2.4.3. Muscarinic Modulation of Synaptic Plasticity 2.2.4.3.1. Long-Term Potentiation 2.2.4.3.2. Long-Term Depression 2.2.4.4. Muscarinic Receptors in Theta Rhythm 2.2.5. Pathophysiology of Muscarinic Receptors In The CNS 2.2.5.1. Alzheimer’s Disease 2.2.5.2. Epilepsy 3. R-type VGCCs Structures and Properties 3.1. Distribution 3.2. Physiology 3.3. Pathophysiology 3.4. 4. TRPC Channels Structures and Properties 4.1. Distribution 4.2. 4.3. Physiology 4.4. Pathophysiology 5. NMDA Receptors Structures and Properties 5.1. Distribution 5.2. Physiology 5.3. Neuronal Survival 5.3.1. Synaptic Transmission and Plasticity 5.3.2. 5.4. Pathophysiology 5.4.1. Ischemic Stroke Neuropathic Pain 5.4.2. 6. Thesis Hypotheses and Objectives 7.  References  27 27 28 28 30 30 31 33 36 38 39 40 41 41 47 48 49 50 51 55 56 57 57 59 60 61 61 63  Chapter Two: Muscarinic Enhancement of R-type Calcium Currents in Hippocampal 99 CAl Pyramidal Neurons 99 Introduction 100 Materials and Methods 100 2.1. Hippocampal Slice Preparation 101 Whole-cell Patch-clamp Recordings 2.2. 102 Extracellular Recording 2.3. 102 Reagents 2.4. 102 Data Analysis 2.5. 102 3. Results 3.1. Carbachol Enhances Toxin-resistant High-Voltage-Activated (HVA) R-type But Does 102 2 Currents Not Affect Low-Voltage-Activated (LVA) T-type Ca  1. 2.  v  3.2. M1/M3 Cholinergic Receptors Mediate Muscarinic Stimulation of R-type VGCCs... 108 3.3. Muscarinic Modulation of R-type VGCCs Is Mediated by a Ca -independent PKC 2 Pathway 113 3.4. Muscarinic Enhancement of R-type Ca Spikes 2 116 3.5. Muscarinic Enhancement of R-type Ca 2 Spikes Contributes to Carbachol-induced Theta Burst Oscillations 119 125 4. Discussion 4.1. Mechanisms Underling Muscarinic Enhancement of R-type VGCCs 125 4.2. 126 R-type Verses Other VGCC Types R-type Spikes and Theta Oscillations 128 4.3. 130 5. References  Chapter Three: Translocation of Cholinergic-Induced Plateau Potentials  TRPC5  Channels  Contributes  to  the 137  1. 2.  Introduction 137 138 Materials and Methods 138 2.1. Hippocampal Slice Preparation 139 Whole-cell Patch-clamp Recordings 2.2. 140 Biotinylation of Surface Protein and Western Blotting 2.3. 140 2.4. Data Analysis 141 3. Results Translocation of TRPC5 Channels. 141 Muscarinic Activation Induced Rapid Membrane 3.1. Cholinergic-Induced Plateau Potentials and Tail Currents are Inhibited by TRPC 3.2. 141 Channel Blockers Calmodulin Activity Is Required for the Generation of Cholinergic-Induced Tail Current 3.3. 146 as well as Membrane Translocation of TRPC5 Channels 3.4. 3 Kinase Activity Is Required for Membrane Translocation of TRPC5 Channels as P1 151 well as The Generation of the PPs 152 4. Discussion 155 5. References  Chapter Four: Cholinergic Modulation of NMDA Current In Hippocampal CAl 161 Pyramidal Neurons Is Age-Dependent 1. 2.  Introduction Materials And Methods 2.1. Hippocampal Slice Preparation Whole-cell Patch-clamp Recordings 2.2. 2.3. NMDA Current Induction 2.4. Reagents 2.5. Data Analysis  161 162 162 163 163 164 164 vi  Results 3.1. Muscarinic Stimulation Potentiates ‘A in Young Animals 3.2. Muscarinic Stimulation Potentiates Ig in Old Animals 3.3. Downstream Pathway of Muscarinic Potentiation of iNuA in Young Animals 3.4. Downstream Pathway of Muscarinic Potentiation of ‘NtA in Old Animals 4. Discussion 4.1. Downstream Mechanisms Underlying the Muscarinic Modulations of 4.2. The Difference of ‘NMDA Obtained from Young and Old Animals 4.3. Physiological Relevance of the Developmental Pattern of the Modulation of 5. References 3.  .  Chapter Five: General Discussion  164 164 169 169 176 183 183 184 186 188  193  How Many Pathways 1. Downstream Pathways Coupled to Cholinergic Stimulation 9 There 2. Physiology of Cholinergic Stimulation The Involvement of New Players Neuronal Bursting and Afterdepolarization 2.1. 2.2. Long Term Potentiation 2.3. ThetaRhythm 3. Pathophysiology of Cholinergic Stimulation The Involvement of New Players —  —  —  3.1. Epilepsy 3.2. Ischemia/Stroke 4. Future Directions 5. References  Appendix 1: UBC Animal Care Certificate  Are  194 195 198 199 200 202 202 204 206 213 223  vii  List of Figures Figure 1.1. Schematic diagrams showing the cholinergic projections in the rat brain and the main 3 connections of the septohippocampal pathway in the hippocampal CAl region Figure 1.2. Schematic summary of the physiological, pharmacological and structural properties of 34 calcium channels Figure 1.3. Schematic representation of the evolutionary development, classification and structural 43 properties of TRP channels Figure 1.4. Schematic representation of the structure of a NMDA receptor with highlighting binding 53 sites for numerous regulatory molecules  104 2 currents Figure 2.1. Carbachol enhances toxin-resistant HVA Ca 106 2 current Figure 2.2. Muscarinie activation enhances R-type but not T-type Ca 109 Figure 2.3. T-type Ca current is not affected in carbachol 111 2 current Figure 2.4. M1/M3 musarinic subtypes mediate the stimulation of R-type Ca 114 Figure 2.5. Mechanisms underling muscarinic stimulation of R-type VGCCs 117 Figure 2.6. R-type VGCC-dependent spikes are enhanced by carbachol 121 2 spikes fire repetitively at theta frequency Figure 2.7. Muscarinic enhanced R-type Ca Figure 2.8. Carbachol-induced theta burst oscillations observed with extracellular recordings of 123 2 sensitive field potentials are Ni Figure 3.1. Muscarinic stimulation triggered rapid membrane insertion of TRPC5 channel proteins. 142 1 ] 2 Figure 3.2. TRP channel antagonists significantly depressed muscarinic-induced PP, ‘tail and [Ca 144 elevation K 3 is sensitive to a calmodulin inhibitor W-7 and a PI Figure 3.3. The muscarinic-induced 147 inhibitor wortmannin K inhibitor wortmannin and calmodulin inhibitor W-7 both significantly 3 Figure 3.4. The PI 149 depressed the muscarinic-induced rapid membrane insertion of TRPC5 channel proteins Figure 4.1. Muscarinic stimulation potentiates INMA in CAl pyramidal cells in young animals... 165 167 in old animals Figure 4.2. Muscarinic stimulation potentiates 170 i-independent 2 Figure 4.3. Muscarinic potentiation of INMA in young animals is [Ca 172 Figure 4.4. PKC activity is required for muscarinic potentiation of ‘DA in young animals Figure 4.5. Non-receptor tyrosine kinase activity is required for muscarinic potentiation of iitA in 174 young animals 177 ]-dependent 2 Figure 4.6. Muscarinic potentiation of ‘A in old animals is [Ca 179 in old animals Figure 4.7. PKC activity is not required for muscarinic potentiation of viii  Figure 4.8. Developmental expression pattern of NMDAR subtypes in CAl pyramidal neurons.. 181 Figure 5.1. Proposed downstream pathways linking muscarinic receptors and their target ion 196 channels Figure 5.2. Two-photon imaging of intracellular calcium dynamics in apical dendrites and spines of 207 CAl pyramidal neurons 2 uncaging in Figure 5.3. Two-photon imaging of intracellular calcium dynamics induced by Ca 211 CAl pyramidal neurons  ix  List of Symbols, Abbreviations and Nomenclature 2-APB 4-AP 4-DAMP AC ACh AChE ACSF AD ADP AED AHP ALS AMPA AP AP5 ATP BAPTA BDNF °C CA CaBP CaM CAN CCK cAMP 1 ] 2 [Ca CaMKII CCh ChAT CIRB CNG CNS CRAC DAG DG DHPG DR DMSO DSI EC eCB EEG EGTA  2-aminoethoxydiphenyl borate 4-aminopyridine 4-diphenylacetoxy-N-methylpiperidine methiodide adenylate cyclase acetylcholine acetylcholinesterase artificial cerebrospinal fluid Alzheimer’s disease afterdepolarization antiepileptic drug afterhyperpolarization amyotrophic lateral sclerosis a-amino-3 -hydroxy-5-methyl-4-isoxazole propionic acid action potential L-2-amino-5-phosphonovalerate adenosine triphosphate bis(2-aminophenoxy)ethane-J\JVN ‘,N’-tetra-acetic acid brain-derived neurotrophic factor degrees centigrade cornu Ammonis calbindin calmodulin -sensitive non-selective cation channel 2 Ca cholecystokinin cyclic adenosine monophosphate intracellular calcium concentration calcium/calmodulin-dependent protein kinase II carbachol choline acetyltransferase calmodulin- and IP3R-binding domain cyclic nucleotide-gated central nervous system calcium release activated channels diacylglycerol dentate gyrus (RS)-3 ,5-Dihydroxyphenylglycine dorsal raphé dimethyl sulfoxide depolarization-induced suppression of inhibition entorhinal cortex endocannabinoid electroencephalogram ethylene glycol-bis(f3-aminoethyl ether)-A}VN’,N’-tetraacetic x  EM EPSC EPSP ER GABA GC GDP GF 109203 x GIRK Go 6976 GPCR GTP HJPP HD hdB HEPES hr HVA Hz I ‘NaT ‘NaP  ‘K  ICj IP 3 1P R 3 IP IPSC IPSP kHz LC LDT LTD LTP LVA mAChR MAGUK MF mg mGluR mm  acid electron microscopy excitatory postsynaptic current excitatory postsynaptic potential endoplasmic reticulum 7-aminobutyric acid granule cell guanosine diphosphate 2-[ 1 -(3 -dimethylaminopropyl)indol-3 -yl]-3 -(indol-3 -yl) maleimide G protein-activated, inwardly rectif’ing K current 1 2-(2-cyanoethyl)-6,7, 12,1 3-tetrahydro- 13 -methyl-5-oxo-5Hindolo(2,3-a)pyrrolo(3 ,4-c)-carbazole G protein coupled receptor guanosine triphosphate hippocampus Huntington’s disease horizontal limb nuclei of the diagonal band of Broca N-2-hydroxyethylpiperazine-N’ -2-ethanesulfonic acid hour high-voltage-activated hertz current hyperpolarization-activated cation currents transient sodium current persistent sodium current M current delayed rectifier-like K current islands of Calleja interpeduncular nucleus inositol 1 ,4,5-triphosphate inositol 1 ,4,5-triphosphate receptor inhibitory postsynaptic current inhibitory postsynaptic potential kilohertz locus ceruleus laterodorsal tegmental nucleus long-term depression long-term potentiation low-voltage-activated muscarinic acetylcholine receptor membrane-associated guanylate kinases mossy fibres milligram metabotropic glutamate receptor minute xi  ml mm mM M2 MS ms mV nA nAChR NBM nif nM NMDA NPY pA PD PFC K 3 PI 2 PIP PKA PKC PLC PNS PP PP2 PPT pS PTKs PV REM RGS Ro 31-8220 RSA RT-PCR s Sch SN S.O. SOCE SOM S.P. S.R. SUB  micromolar micrometer milliliter millimetre millimolar megaohm medial septum millisecond millivolt nanoamp nicotinic acetylcholine receptor nucleus basalis magnocellularis nifedipine nanomolar N-methyl-D-aspartate neuropeptide Y picoamp Parkinson’s disease prefrontal cortex phosphoinositide 3-kinase phosphatidylinositol 4,5-bisphosphate cAMP-dependent protein kinase protein kinase C phospholipase C peripheral nervous system plateau potential protein phosphatase 2 pedunculopontine tegmental nucleus pico Siemens protein tyrosine kinases parvalbumin rapid eye movement regulators of G protein signaling 3 -[1—3 -(amidinothio)-propyl-’H-indol-3 .-yl].-3 -(1-methyl-i 3 -yl)maleimide rhythmical slow activity single-cell reverse-transcription polymerase chain reaction second Schaffer collateral substantia nigra stratum oriens store-operated calcium entry somatostatin stratum pyramidale stratum radiatum subiculum xli  TEA TPM trans-ACPD TRP TRPC TTX V vdB VGCC VGSC VIP Vm  tetraethylammonium chloride topiramate (±)- 1 -Aminocyclopentane-trans- 1,3 -dicarboxylic acid transient receptor potential transient receptor potential classical tetrodotoxin volt vertical limb nuclei of the diagonal band of Broca voltage-gated calcium channel voltage-gated sodium channel vasoactive intestinal peptide membrane potential  xlii  Acknowledgements I sincerely thank Dr. Brian A. MacVicar for his exceptional supervision, insight, encouragement and friendship throughout the course of this dissertation. I also thank my supervisory committee members, Drs. Yu Tian Wang, Terry Snutch, and Tim Murphy for their valuable advice and assistance. I also thank all of the past and present members of the MacVicar lab, for assistance and friendship throughout the course of this degree. I would particularly like to thank Drs. Hyun Boem Choi, Dustin Hines, Brent Kuzmiski, Ms. Ning Zhou and Ms. Denise Feighan. I also thank all of my family and friends that accompanied me for the precious six years during my graduate school. Finally, I would like to thank HSFC, Savoy Foundation, UGF and UBC for financial support during my PhD studies.  xiv  Dedication Th my parents, my loving wife, Shanshan, for everything  xv  Co-Authorship Statement  I designed and performed all of the experiments and analyzed the experimental results for all experiments, except for those shown in Figure 2.6, Figure 2.7, and Figure 2.8. Dr. Joseph Brent Kuzmiski performed the extracellular recording and most of the current-clamp recording experiments in Chapter Two.  In terms of manuscript preparation, I wrote the entire manuscripts for Chapter One, Three, Four and Five with subsequent editing from Dr. Brian MacVicar. For Chapter Two, I wrote all sections except for section 2.3.4 and 2.3.5 (written by J.B. Kuzmiski). I offered editing and feedback on the sections that I did not write for Chapter 2.  xvi  Chapter One: Introduction  Chapter One: Introduction 1. The Cholinergic System Of The CNS Aeetylcholine (ACh) was the first neurotransmitter to be identified (Henry Dale in 1914 and Otto Loewi in 1936) in mammalian nervous system (Valenstein, 2002). In the peripheral nervous system (PNS), ACh activates muscles and is a major neurotransmitter in the autonomic nervous system. In the CNS, the cholinergic system is considered to be one of the most important neuromodulatory neurotransmitter systems in the brain. ACh is synthesized from acetyl-CoA and choline in nerve terminals, in a reaction catalyzed by the cytosolic enzyme choline acetyltransferase (ChAT). ACh is then packaged into presynaptic vesicles by vesicular cholinergic transporters and released into the synaptic cleft via exocytosis. Once released into the synaptic cleft, the concentration of ACh quickly declines by the hydrolytic enzyme acetylcholinesterase (AChE) into choline and acetate. The choline is then rapidly transported back into the synaptic terminal via a high-affinity sodium-dependent reuptake system to replenish ACh stores in the presynaptic terminals. In the CNS, the cholinergic system plays a key role in modulating neuronal excitability, synaptic plasticity and neuronal intrinsic properties (Jerusalinsky et al., 1997; Lucas-Meunier et al., 2003; Hasselmo, 2006). It is also implicated in many brain disorders including Alzheimer’s disease (Fodale et aL., 2006) and epilepsy (Turski et al., 1989). The diverse impact of the cholinergic system arises from the extensive number of ion channels that are modulated by acetylcholine (Lanzafame et al., 2003). In this section, I will briefly review the principles of the cholinergic system in the CNS, the cholinergic modulations of different ion channels, and the functions of the cholinergic system in both physiological and pathophysiological conditions.  1.1. General Anatomy ACh and the associated neurons form the cholinergic neurotransmitter system. The development of immunohistochemical methods and in situ hybridization histology for ChAT has helped people to accurately identify the localization and organization of cholinergic neurons in the brain. The projections of cholinergic system were determined using the retrograde or anterograde 1  Chapter One: Introduction tracing techniques combined with immunohistochemisty, and the regional measurement of cholinergic markers following localized lesions. These studies, combined with ultrastructural investigations have led to a greater understanding of the synaptic connectivity of cholinergic neurons and their target areas. Cholinergic neurons are distributed in a variety of different nuclei in the brain (Fig. 1. 1A). Two major groups of the cholinergic neurons located in the basal forebrain and pedunculopontine area are especially prominent and project extensively to the cortex and thalamus. Striatal structures, such as nucleus accumbens, caudate-putamen complex and olfactory tubercle, contain abundant intrinsically organized cholinergic intemeurons that project locally.  1.2. Cholinergic Forebrain Projections The basal forebrain contains several cholinergic neuron groups with widespread projections throughout the entire allocortex and isocortex (Butcher et al., 1993; Page and Sofroniew, 1996). These nuclei include the medial septal nucleus (MS), vertical (vdB) and horizontal (hdB) limb nuclei of the diagonal band of Broca, and the nucleus basalis magnocellularis (NBM) (Fig. 1.1 A). The axons from the MS predominately innervate the hippocampus, whereas those of the vdB and HUB project into the anterior cingulate cortex and olfactory bulb, respectively. The vdB also  projects to the hippocampal formation. The NBM pathway has widespread projections innervating the amygdaloid complex, the neocortex, and both reticular and mediodorsal nuclei of the thalamus (Fig. 1.1 A). The ability to selectively lesion the basal forebrain cholinergic neurons in experimental animal models using specific immunotoxins has greatly advanced our knowledge of the cognitive functions of these neurons (Baxter and Chiba, 1999). The basal forebrain cholinergic neurons are considered to be essential for controlling selective attention, working memory and demnesia (especially AD) (Perry et al., 1999).  1.2.1.  Cholinergic Projections In Hippocampus and The Septohippocampal Pathway  The mammalian hippocampal formation consists a group of brain areas including the dentate gyrus (DG), hippocampus, subiculum, presubiculum, parasubiculum, and entorhinal cortex (EC). 2  Chapter One: Introduction  Figure 1.1. Schematic diagrams showing the cholinergic projections in the rat brain and the main connections of the septohippocampal pathway in the hippocampal CA 1 region. (A) Schematic representation of the distribution of cholinergic neurons and their projections in the rat brain. Note the cholinergic projections to the hippocampus from the medial septum (MS) and vertical limb nucleus of the diagonal band of Broca (vdB). Additional abbreviations: hdB, horizontal limb nucleus of the diagonal band of Broca; NBM, nucleus basalis magnocellularis; PPT, pedunculopontine tegmental nucleus; LDT, laterodorsal tegmental nucleus; PFC, prefrontal cortex; ICj, islands of Calleja; SN, substantia nigra; IP, interpeduncular nucleus; DR, dorsal raphe; LC, locus ceruleus. Modified from Everitt and Robbins, 1997. (B) Schematic representation of the main connections between septal complex and the CA 1 region of the hippocampus (HIPP). Cholinergic neurons (ACh; black circle) in the medial septum and nucleus of the diagonal band of Broca (MS-DB) terminate on the soma and proximal dendrites of pyramidal neurons (triangle) and on GABAergic interneurons (grey circle). GABAergic neurons containing parvalbumin (PV) terminate on different populations of GABAergic interneurons in the hippocampus. Some of these interneurons contain neuropeptide Y (NPY), somatostatin (SOM), calbindin (CaBP), vasoactive intestinal peptide (ViP), and cholecystokinin (CCK). CaBP positive interneurons project back to the MS-DB and innervate cholinergic neurons and PV containing GABAergic neurons. Pyramidal neurons in CAl project on GABAergic neurons in the lateral septum (LS). Both medial and lateral septa receive other inputs, especially from brain stem and hypothalamus. Additional abbreviations: S.R., stratum radiatum; S.P., stratum pyramidale; S.0, stratum oriens. Modified from Dutar et al., 1995.  3  Chapter One: Introduction  A  B  4  Chapter One: Introduction Due to its outstanding structure with a large, bulb-like shape protruding into the lateral ventricles, the hippocampus has played a central role in neuroscience since the very beginning of brain investigations. In 1957, Scoville and Milner found that the removal of the hippocampus from the patient H.M. to treat intractable epilepsy resulted in profound memory deficit (Scoville and Milner, 1957). This striking discovery has triggered intense research focusing on the role of hippocampus in memory for the last half century. Moreover, the hippocampus has also attracted the interest of modern neuroscientists in the study of many neurological disorders including stroke, epilepsy and Alzheimer’s disease. Recent advanced research technologies, such as electrophysiological recordings and two-photon imaging techniques, have led to a better understanding of the mechanisms underlying hippocampal functions under both physiological and pathophysiological conditions. The hippocampus receives its cholinergic innervations mainly from the septum. The septohippocampal system is one of the best-characterized eholinergic pathways in the brain (Fig. 1 .1A, 1 .1B) (Dutar et al., 1995) and originates from septal neurons located in the MS and VDB. The septal neurons project mainly to the ipsilateral hippocampal formation via the fimbria-fomix, and the fibers of these neurons have been identified in all layers in the Cornu Ammonis (CA) and the DG. Cholinergic axons target granule cells in the DG and pyramidal neurons in the hippocampus proper and form a three-dimensional network around the cell bodies of these cells. Cholinergic axons also target non-pyramidal neurons in both regions. Cholinergic fibers have been demonstrated as unmyelinated axons that form both asymmetric and symmetric synaptic contacts with pyramidal neurons and granule cells. Interestingly, the asymmetric contacts are found on postsynaptic spines and dendrites, while the symmetric contacts are found more often on cell bodies and dendritic shafts (Clarke, 1985; Frotscher and Leranth, 1985; Frotseher et al., 1986; Gartner et al., 2001). GABAergic interneurons in the hippocampus also receive symmetric cholinergic inputs from the septohippocampal pathway. The septohippocampal pathway also contains GABAergic projections that target mainly the GABAergic basket interneurons in hippocampus and their activity likely leads to disinhibition of pyramidal neurons (Freund and Antal, 1988; Toth et al., 1997). Interestingly, GABAergic neurons within the hippocampus in turn project back mainly on to septal GABAergic 5  Chapter One: Introduction projection neurons but not ChAT-positive neurons (Alonso and Kohler, 1982; Toth et al., 1993). The main connections between the septal complex and the CA 1 area of the hippocampus have been represented in Figure 1.1B.  1.3. Brainstem Projections Another major group of cholinergic neurons is localized to the brainstem and contains the laterodorsal tegmental nucleus (LDT) and the pedunculopontine tegmental nucleus (PPT; Fig. 1.1 A) (Butcher et al., 1993; Farris et al., 1995). These nuclei display both ascending and descending diffuse projections. The LDT selectively projects to the anterior, central medial and mediodorsal nuclei, which are closely associated with the limbic system. While the PPT cholinergic neurons show widespread projection to almost all thalamic nuclei, especially the specific relay nuclei. Both the LDT and PPT also innervate the lateral septum, the lateral hypothalamus, amygdala and the basal forebrain (Fig. 1.IA). Over 90 percent of brainstem projections to the thalamus are cholinergic. These ascending cholinergic projections are thought to play an important role in the reticular activating system and likely function in the regulation of conscious awareness. Both the PPT and LDT also display descending projections that innervate pontine and medullary targets via several pathways (Butcher et al., 1993; Farris et al., 1995). The most prominent pathway descends through the ventromedial branch of the lateral tegmentoreticular tract. The major targets of the descending pathways within the pontine include the medial pontine reticular formation and the gigantocellular field in the medulla. Additional brainstem targets include the superior colliculus, the rostral ventrolateral medulla and the nucleus raphe magnus. Descending cholinergic projections are thought to have a crucial role in the mediation of muscle atonia during rapid eye movement (REM) sleep. They have also been implicated in the waking state that provide feedback into the thalamus and striatum (Inglis and Winn, 1995).  1.4. Cholinergic Interneurons Aside from the extensively projecting cholinergic neurons in basal forebrain and brain stem, there are also abundant local projection cholinergic interneurons distributed in the brain. 6  Chapter One: Introduction Interestingly, cholinergic interneurons are normally distributed in areas with a dense dopaminergic afferents, such as the dorsal and ventral striatum, where dopaminergic innervation displays a powerful control over cholinergic transmission. These giant aspiny cholinergic interneurons are autonomous pacemakers and have richly arborizing axons with large terminal fields. Dopamine (DA) inhibits the autonomous spiking of striatal cholinergic interneurons and their release of ACh. In the striatum, the crosstalk between DA and ACh plays a principal role in the pathophysiological changes in brain function underlying several movement disorders, such as Parkinson’s disease (PD), Huntington’s disease (HD), dystonia and Tourette syndrome. In Parkinson’s disease, degradation of the striatal dopaminergic neurons leads to increased release of ACh by these interneurons, distorting network function and inducing structural changes that strongly contribute to the symptoms. While in Huntington’s disease, there is a decrease in striatal cholinergic markers. Cholinergic interneurons are also considered to play an important role in striatal synaptic plasticity and in reward learning and motivation (Pisani et al., 2007; Langmead et al., 2008).  2. Cholinergic Receptors In The CNS Like many other ligand-activated neurotransmitter receptors, there are two major subtypes of ACh receptors: the ionotropic nicotinic (nAChR) and the metabotropic muscarinic cholinergic receptors. To date, the activity of muscarinic receptors has been extensively studied in the CNS, but the physiological roles of nAChRs in the brain are still largely unknown.  2.1 Nicotinic Cholinergic Receptors Nicotinic receptors are fast ionotropic cationic channels sensitive to activation by nicotine. These channels are found in both peripheral and central neurons. Based on their major site of expression, nAChRs are subdivided into muscle or neuronal subtypes. Muscle nAChRs consist of five subunits: c1 and four non-ct subunits, 131, y, 6, and c. Only two receptor combinations have been identified from this multiple subunit pool: the subunit composition of ctl, 131, 6  and 6 or al, 131,  and 6, each with the stoichiometry of 2:1:1:1. The relative level of expression of these two  receptors is based on species and muscle innervation. In the CNS, the neuronal nAChRs can be both 7  Chapter One: Introduction homopentamers and heteropentamers. To date, eight tx-like subunits,cL2, x3, a4, ct5, cL6, ct7, a9 and CL 10  (a8 subunit is not found expressed in mammals), and three non-cL subunits (132,  133  and 134) have  been cloned from mammalian neuronal tissues. Each subunit has an extracellular C-terminal and an extracellular N-terminal region and four transmembrane domains. Neuronal nAChRs display great diversity in subunit combinations. For instance, functional nAChRs formed with the stoichiometry of two  CL  and three f3 subunits, or three ct and two  13  subunits, or five homomeric cc7, c8 and a9  subunits have all been identified. The diversity of nAChR subunit combinations is a major determinant of the specialized properties and functions of the receptors, such as the distribution patterns and the distinct pharmacological and physiological profiles of these receptors in the brain (Albuquerque et al., 2009).  2.1.1.  Distribution of Neuronal nAChRs  Multiple technologies have been used to determine the localization of neuronal nAChRs, including electron microscopy (EM), pharmacological methods, electrophysiological recordings, H]-nicotine and [‘ 3 1]-cL-bungarotoxin, and in situ hybridization techniques. In 25 autoradiography of [ the adult mammalian brain, the distributions of different subunits are heterogeneous and various subunits are particularly abundant in the cortex, interpeduncular nucleus, SN pars compacta, somatosensory cortex, medial habenula, olfactory area, cerebellum, and locus coerulus. At the subcellular level, evidence from a multitude of studies converges to the conclusion that nAChRs are located at one of five primary locations: the cell soma, dendrites, preterminal axon regions, axon terminals,  and  myelinated  axons  on  the  neurons.  In  the  hippocampus,  single-cell  reverse-transcription polymerase chain reaction (RT-PCR) analysis has detected the expression of mRNA for cL4, ct5, cL7 and 132-4 subunit in stratum radiatum interneurons, cL2-4, cL7, and f32-3 subunit in stratum oriens interneurons and cL4, cL5, 132-4 as well as low levels of cL7 subunit in pyramidal neurons (Sudweeks and Yakel, 2000). Using EM immunolabeling, people have found that in CAl stratum radiatum cL7 subunits are highly abundant and present at both GABAergic and glutamatergic synapses (Fabian-Fine et al., 2001). Interestingly, numerous reports have also provided evidence that cL7 nAChRs are abundantly present on glutamatergic presynaptic terminals in hippocampus and 8  Chapter One: Introduction could profoundly modify synaptic transmissions.  Neuronal nAChRs are also expressed in other  cell types, such as glia cells and endothelial cells (Sudweeks and Yakel, 2000).  2.1.2.  Channel Properties of Neuronal nAChRs  nAChRs with different subunit compositions display different channel properties, such as their ion selectivities and permeabilities, channel dynamics and current rectification. Nicotinic receptors are non-selective cationic channels permeable to Nat, K and Ca . The Ca 2 /Na permeability ratio 2 of neuronal nAChRs is more than 1 (10 for x7-containing nAChRs), much higher than that of muscle nAChRs (0.1-0.3) (Vernino et a!., 1992). The high Ca 2 permeability of nAChRs is of particular interest because Ca 2 influx through neuronal nAChRs can result in the activation of -dependent processes (Mulle et a!., 1992), and that neuronal nAChRs could allow Ca 2 2 multiple Ca influx even at hyperpolarized potentials. Another distinction of neuronal nAChRs is the strong inward rectification of the channels due to the voltage-dependent block by intracellular polyamines (Haghighi and Cooper, 1998). Polyamines bind to negatively charged residues near the intracellular 2 ions (Haghighi and Cooper, mouth of the pore that also functions as a selectivity filter for Ca 2000). In the presynaptic terminals the polyamine-dependent inward rectification provides a voltage-dependent mechanism to prevent shunting of action potentials. Diverse subunit combinations of nAChRs also result in different pharmacological characterizations, which make it possible to differentiate different subtypes of neuronal nAChRs with specific agonists and/or antagonists. nAChRs display several basic conformational states, such as the closed state at rest, the open state and the desensitized state. Most of the single channel properties, including the rate of activation, the channel open time and probability, and the rates of desensitization and recovery from desensitization, as well as the open channel conductance are also dependent on the subunit composition of neuronal nAChRs. This also provides another means to differentiate different subtypes of neuronal nAChRs, electrophysiologically. For instance, single channel analysis of nAChRs in CA 1 stratum radiatum interneurons has revealed two types of nAChRs with varying open channel conductance levels and mean open times, depending on the subunit types 9  Chapter One: Introduction (ci7-containing and non-ct7-containing) (Shao and Yakel, 2000). Another study showed that stratum radiatum interneurons express rapidly activating and rapidly desensitizing nAChRs, whereas stratum oriens interneurons expressed slowly activating and slowly desensitizing nAChRs (Sudweeks and Yakel, 2000), probably also dependent on varying subunit types.  2.1.3.  Physiology of Neuronal nAChRs  The relatively high Ca 2 permeability of neuronal nAChRs (even at hyperpolarized potentials) allows these receptors to play a role in many Ca -dependent physiological processes. In 2 mammalian sympathetic neurons, nAChR activation induces the opening of a nonselective cation channel that leads to membrane depolarization and a secondary activation of voltage-gated Ca 2 tdependent kinases, including 2 channels (VGCCs). nAChR activation also stimulates several Ca P13 K, protein kinase C (PKC), protein kinase A (PKA), calmodulin-dependent protein kinase II (CAM kinase II), and extracellular signal-regulated kinases (ERKs). The Ca 2 flux directly through nAChR channels or indirectly via VGCCs, and the downstream pathways plays a primary role in the nicotinic modulation of transmitter release, synaptic plasticity, as well as neuronal viability, differentiation, and migration. For instance, presynaptic nAChRs activation enhances synaptic transmission in the hippocampus. The Ca 2 flux through the ct -containing nAChRs was required for 7 the enhancement of EPSCs in CA3 pyramidal neurons (Gray et al., 1996). In the CAl region, -containing nAChRs located in glutamatergic axons also enhanced EPSCs in stratum 7 non-cL radiatum interneurons (Alkondon and Albuquerque, 2002; Alkondon et al., 2003). In addition to the presynaptic regulatory effects, nAChRs located on the postsynaptic membrane in hippocampal interneurons could directly generate fast excitatory synaptic transmission (Albuquerque et al., 1998; Frazier et a!., 1998; Heffi et al., 1999). Functional, native ct7 nAChRs are expressed in cultured hippocampal neurons and electrical stimulation of the hippocampus elicits cc7-containing nAChR-dependent EPSCs in area CA 1 interneurons. The activation of the x7-containing nAChRs on the GABAergic interneurons strongly inhibits currents in pyramidal neurons and depresses hippocampal activity (Ji and Dani, 2000; Buhler and Dunwiddie, 2002).  10  Chapter One: Introduction 2.1.4.  Pathophysiology of Neuronal nAChRs  Rodent animals, as well as humans, exhibit a striking age-related decline in nAChR expression, which is not rescued by long-term treatment of nicotine. ciA nAChR expression can decrease by more than 80% in the AD brain. Possible impacts of selective nAChR loss on the aging or AD brain were provided by evidence that in primary cultures ciA-containing nAChRs protect neurons against toxic fragments of the amyloid  peptides while cL7 nAChRs protect against excitotoxic challenges  like NMDA. Obviously, more future work is required to clarify the role of nAChR in aging and AD (Albuquerque et al., 2009). Epidemiological studies have reported that heavy smokers are less likely to experience Parkinson’s Disease (PD). This protection is real and not related to selective diminishment of the smoking population through early death related to other side effects of smoking. Moreover, in primates, oral nicotine reduces the nigrostriatal neuronal loss observed in chemically induced PD. But again, more future work is required to clarify the role of nAChR in PD (Albuquerque et al., 2009). And finally, as one of the major compounds in cigarettes, nicotine is perhaps the most addictive drug that is widely used. With the use of recent advanced genetic approaches, the role of different nAChR subunits in nicotine addiction has been extensively studied. For instance, genetic manipulation experiments provide direct evidence of the participation of ciA nAChR subunits in several components of nicotine addiction (Flores et al., 1992; McCallum et al., 2006; Albuquerque et al., 2009). In these experiments, a knock-in mouse that exchanged a highly conserved leucine in TM2 with an alanine in the ciA nAChR subunit was developed. The mutant subunit increased the sensitivity of the receptor to nicotine. More importantly, this mutation and enhanced receptor activation alone can trigger nicotine reinforcement, sensitization, and the development of tolerance. In addition, people have demonstrated that nicotine-induced upregulation requires at least the 2 nAChR subunit, development of tolerance to nicotine requires neither the 2 nor the ci7 nAChR subunit; instead, it appears to be modulated by a ciA-containing nAChR and to require a p4-containing subunit. These result suggested that nAChRs play a central role in nicotinic addiction, and more genetic studies on other subunits would be helpful in the future for a better insight of the 11  Chapter One: Introduction connection between nAChRs and nicotinic addiction.  2.2. Muscarinic Cholinergic Receptors  Muscarinic acetylcholine receptors (mAChRs) are members of the guanine nucleotide-binding protein (G-protein)-coupled receptors (GPCR5) superfamily. To date, molecular-cloning studies have revealed five distinct mammalian musarinic genes (ml-m5) that encode for mAChR subtypes Ml to M5 (Bonner et al., 1987; Bonner et al., 1988). As a member of the seven transmembrane GPCRs, the mAChRs are single subunit integral proteins with seven transmembrane domains, three extracellular loops, three intracellular loops, an extracellular amino terminus, and an intracellular carboxy tail (Hulme, 1990). G proteins bind to the second and third intracellular loops of mAChRs (Haga et al., 1993; Wess et al., 1995). mAChRs are generally divided into two classes according to their signal transduction pathways. The Mi-like receptors, including Mi, M3 and M5, selectively 3 bind to G-proteins of the Gq/1 1 family and stimulate phosphatidylinositol turnover and produce 1P and DAG. The M2-like receptors, including M2 and M4, selectively couple to G-proteins of the Go/i family, inhibit adenylate cyclase and reduce the intracellular concentration of cAMP (Mutschler et al., 1995).  2.2.1.  G-Protein-Coupled Receptors  GPCRs are integral membrane proteins, which are coupled to heterotrimeric G-proteins and involved in the transmission of extracellular signals to the cytoplasm. Various ligands and stimuli (e.g. neurotransmitters, hormones, cytokines, odorants, light, pH, and growth factors) can activate intracellular signaling cascades via specific GPCRs. They are the largest and most versatile group of cell surface receptors, and also the most common targets for currently approved drugs. It has been reported that nearly a third of prescription drugs are GPCR ligands (Kotecha and MacDonald, 2003). To date, over one thousand members of GPCRs have been identified, more than two percent of the total human genes (Baldwin, 1994; Sanders et al., 2008). GPCRs have a common structural model that consist of seven transmembrane helices, similar to that of the mAChRs. The third intracellular loop is the most variable region among this group of receptors (Baldwin, 1994), which has been 12  Chapter One: Introduction shown to be primarily responsible for the interaction of the receptor with G-proteins (Kobilka et a!., 1998). Other intracellular segments may also interact with other accessory proteins and regulate signal transmission. Under the resting conditions the receptors are loosely associated with the heterotrimeric G-protein, with the Oct subunits bound to GDP. In response to agonist binding, GPCRs transduce a signal by undergoing a profound conformational change in the transmembrane and cytosolic regions of the receptors, leading to exposure of the linked G-protein that results in the release and exchange of GDP from the ct subunit to GTP (Hamm and Gilchrist, 1996; Freissmuth et al., 1999). The activated GTP-binding Oct subunit and GJ y dimer then dissociate, and could both 3 independently activate appropriate downstream signaling cascades and regulate separate cellular effectors. The GTP bound to Oct is then hydrolyzed to GDP by the intrinsic GTPase, which results in the deactivation and return to the basal state of Oct and its reassociation with Ofry. Accessory proteins, called “regulators of 0 protein signaling” (ROS), could bind directly to activate Oct subunits and stimulate the GTPase activity leading to the deactivation of G-proteins and termination of downstream signals (Hollinger and Hepler, 2002). The heterotrimeric 0-proteins consist three parts: the ct-,  1-  and ‘y-subunits. In mammals,  molecular cloning has defined 33 genes encoding G-protein subunits, 16 for Gct subnits, 5 for GJ3 subunits, and 14 Gy subunits (Hurowitz et a!., 2000; Sanders et al., 2008; Smrcka, 2008). The ct-subunit is the one that binds to GDP or GTP depending on its conformational status. Four subfamilies of 0-proteins have been classified according to the primary sequence similarity and the coupling biochemical cascades of the ct-subunits: Gct,  Gctilo, GcLq,1i,  and Gct . For example, the 12  110 and members of the Oct, family activate adenylyl cyclases, leading to increased cAMP; the Gct Oct proteins inhibit adenylyl cyclases but activate 0-protein-coupled inwardly rectif’ing potassium (GIRK) channels (Milligan and Kostenis, 2006); the  Gctq/11  family regulates the activity of  12 proteins can phospholipase C (PLCf) and its downstream effectors like P3 and DAG; and Gct cause activation of the Rho small 0-proteins (Sah et al., 2000). The Of3 and Gy subunits function as a dimer, called the Gfry subunits. Initially Gfry was thought to simply regulate Oct activity, which is required for the proper targeting of Oct to the membrane and the GPCR catalyzed nucleotide exchange. However, recent studies have clearly shown that Gfry could act as a signal transduction 13  Chapter One: Introduction molecule itself by direct binding to effector molecules. To date, the list of molecules that have been reported to be regulated and interact to Gfiy continues to grow. Particularly, one of the most interesting actions of the Gfry subunit is the direct binding and regulation of certain ion channels, such as the GIRK channels and the voltage-gated calcium channels (Krapivinsky et al., 1995; Smrcka, 2008). Interestingly, recent studies have shown that the activities of the “G-protein-coupled” GPCRs do not necessarily require the involvement of the heterotrimeric G-proteins. A growing body of evidence has suggested that GPCRs can transduce signals independent of G-protein activation. For example, it has been shown that activation of the group I mGluRs triggers a nonselective cation current rat hippocampal CA3 cells through both a G-protein dependent and a G-protein independent pathway (Gee et al., 2003); in another study it was also shown that with GTP-yS, which permanently activates G proteins, could not occlude the mGluR-mediated inward current (Guerineau et al., 1995); in addition, in CA3 pyramidal cells people showed that the activation of mGluRl potentiates NMDA current via a G-protein-independent mechanism involving Src kinase activation (Benquet et al., 2002). The physical interactions between receptors and ion channels, such as the dopaminergic Dl receptor interaction with NMDA receptors (Lee et a!., 2002), the dopaminergic D5 interaction with GABA(A) receptors (Liu et al., 2000), and the Dl interaction with N-type VGCCs (Kisilevsky et al., 2008), have also been studied extensively.  2.2.2.  Distribution of mAChRs In The CNS  mAChRs are widely expressed throughout the CNS and in various peripheral tissues. Interestingly, virtually all tissues and cell types express at least two mAChR subtypes. Each of the five mAChR subtypes exhibits a distinct pattern of distribution. Several technologies have been used to identify the localization of mAChRs in the CNS, including autoradiography of  H]-propylbenzilylcholine, in situ hybridization and the use of subtype selective and/or 3 [ non-selective immunocytochemistry. All five mAChR subtypes (both mRNAs and proteins) have been localized in the CNS with distinct expression profiles (Brann et al., 1993). The predominant mAChR in the brain is Ml, which is expressed in the cortex, striatum, thalamus and hippocampus, 14  Chapter One: Introduction mainly postsynaptically (Ellis et al., 1993). M2 receptors are also widely expressed in the CNS. M2 mAChRs are predominantly expressed in the brainstem and thalamus, as well as in the cortex, striatum and hippocampus, on the cholinergic synaptic terminals (Rouse et al., 1997). M3 receptors are found in the thalamus, cortex and hippocampus. M4 receptors are found predominantly in the striatum, but also expressed in the cortex and hippocampus. The expression of the M5 receptors is considered to be the most spatially restricted with a very discrete localization in the substantia nigra, as well in the hippocampus and striatum (Langmead et al., 2008). In the hippocampus, all of the five mAChR subtypes are identified (Vilaro et al., 1993), four of which are abundantly expressed (M1-M4) (Rouse et al., 1999). Interestingly, the hippocampus is the only brain region that expresses all five types of mAChRs (Levey et al., 1995). Each of the receptors has a distinct localization pattern in the hippocampus proper and dentate gyrus (Levey et al., 1995). Ml receptors are widely expressed in the cell bodies and dendrites of pyramidal neurons and granule cells. M2 receptors are expressed mostly in the axon terminals of both cholinergic and non-cholinergic neurons in CAl (Rouse et al., 2000b), as well as in the dendrites and axon terminals of interneurons (Hajos et al., 1998). M3 receptors are abundantly expressed in the molecular layer of the dentate gyrus and pyramidal neuron dendrites, especially in stratum lacunosum-moleculare (Levey et al., 1995). M4 receptors are also found enriched in the molecular layer of the dentate gyrus, as well as in the nonpyramidal neurons (Levey et a!., 1995).  2.2.3.  Signal Transduction Pathways Of mAChRs  As a member of the seven transmembrane GPCRs, muscarinic receptors also bind heterotrimeric G-proteins and act as guanine nucleotide exchange factors. As mentioned above, Gctj Gaq,ii, and GcL , 12 (Hepler and G-proteins could be divided into four groups, termed Gx, 10  Gilman, 1992). Two drugs are extensively used in defining distinct Gx protein function: the bacterial cholera and pertussis toxins. Cholera toxin catalyzes ADP-ribosylation of an arginine residue near the GTP binding site specifically on Gct, locks the G protein in its GTP-bound form, thereby continually activates  and stimulates adenylate cyclase to produce cAMP. On the other  hand, pertussis toxin catalyzes ADP-ribosylation of a cysteine residue on the carboxy terminus 15  Chapter One: Introduction specifically on GcL , locks GcL 110 110 in its inactive state and thereby prevents the G-protein-receptor interaction. The activity of Gctqiii and Gct 12 are resistant to both pertussis and cholera toxins. According to their signal transduction pathways, mAChRs are generally divided into two classes: the Mi-like receptors (Ml, M3 and M5) and the M2-like receptors (M2 and M4). Generally, Mi, M3, and M5 receptors couple efficiently through Gqii to stimulate phospholipase C (PLC), which  hydrolyzes  membrane  phosphatidylinositol  4,5-bisphosphate  ) 2 (PIP  into  inositol  i ,4,5-triphosphate (P ) and the membrane associated fatty acid diacyiglycerol (DAG) (Caulfield 3 and Birdsall, 1998). Cytosolic 1P 3 binds to and triggers the release of Ca 2 through the 1P 3 receptors R), from the endoplasmic reticulum (ER). The elevation of intracellular Ca 3 (IP 2 concentration could in turn activate many Ca -sensitive sensor molecules, such as calmodulin that in turn activate 2 downstream effectors including Ca /calmodulin-dependent protein kinases (CaMK5), PI 2 K, 3 calcineurin, protein kinase C (PKC), adenylate cyclases and transcription factors. DAG activates PKCs directly or together with intracellular Ca , and PKCs in turn can phosphorylate a variety of 2 downstream targets. Interestingly, PIP 2 itself can function to signal transmission by anchoring numerous signaling molecules and cytoskeleton at the cell membrane. Recently, many ion 2 (Hilgemann et al., 2001). transporters and channels have been discovered to be regulated by PIP 2 have been proposed to play the The Mi-induced PLC activity and the subsequent turnover of PIP  central role in the muscarinic-induced depression of M-current rather than from the effect of a product of PIP 2 hydrolysis as previously thought (Hernandez et al., 2008). In a recent paper, Hildebrand et al., reported the Gccq,ii-coupled Mi receptors depressed Cav3 .3 calcium channels, , Ca 3 2 and PIP , suggesting 2 through a novel to-be-defined pathway that is independent of PKC, 1P the existence of other downstream mechanisms of GcLq,j i proteins (Hildebrand et al., 2007). M2 and M4 receptors generally couple to GcL j proteins, which inhibit adenylate cyclase and 0 reduce the intracellular concentration of cAMP (Ashkenazi et al., 1987; Peralta et al., 1988; Caulfield and Birdsall, i998). The decrease of intracellular cAMP leads to the inhibition of the phosphorylation of cAMP-dependent protein kinase (P1(A), and therefore reduce the activites of the downstream effectors of PKA. Another two well-known consequences of M2/4 activation are mediated by the Gf3y subunits released from Gci 1 proteins. Firstly, the released Gfry subunits directly 16  Chapter One: Introduction bind to and activate or inhibit different potassium currents, and modulate the excitability of different cells (Krejci and Tucek, 2002). Secondly, in neuronal cells, mainly on synaptic terminals, the released Gfry subunits directly interact with high voltage-activated calcium channels and inhibit calcium influx into nerve terminals, which results in depression of synaptic transmission (Catterall, 2000; Mark and Herlitze, 2000). The activation of M2 receptors can also activate a Rap 1 GTPase-activating protein through an interaction with GcLi, which stimulates a Ras-ERKIIvIAPK mitogenic pathway (Mochizuki et al., 1999). A recent study also showed that M2 activity indirectly enhances Ca1 .2 currents that couple sequentially to Gfry, PI K, a novel PKC, and c-Src (Callaghan 3 et a!., 2004), suggesting a greater complexity of the signaling transduction pathways of muscarinic systems.  2.2.4.  Physiology of Muscarinic Receptors In The CNS  Muscarinic cholinergic system is one of most important modulatory neurotransmitter system, which plays a key role in modulating neuronal excitability, synaptic plasticity and neuronal intrinsic properties (Jerusalinsky et al., 1997; Lucas-Meunier et a!., 2003; Hasselmo, 2006). Muscarinic activity results in a number of positive and negative modulations of neuronal excitability. The effect of ACh is generally excitatory, such as to accelerate the action potential firing rate, via the depolarization of neurons. But ACh can also suppress excitatory as well as inhibitory neurotransmission. Thus, the net effect of ACh is that although the neurons are depolarized, the communication among neurons is suppressed. The complexity of the ACh actions likely contributes to both normal and abnormal hippocampal neuronal functioning.  2.2.4.1. Muscarinic Modulation of Ion Channels Neuronal excitability is controlled and regulated by different ion channels across the cytoplasm membrane, which are permeable to particular ions. The properties of these ion channels can be modulated by ligands, membrane potential, cytoplasmic second messengers or even mechanical stress. The diverse impact of the cholinergic system arises from the extensive number of ion channels that are modulated by ACh. I will briefly review the muscarinic modulations of several 17  Chapter One: Introduction types of sodium, potassium, calcium and mixed cation channels in this section.  2.2.4.1.1.  Voltage Gated Sodium Channels  Voltage-gated sodium channels (VGSC5) are the basis for action potential initiation and propagation, and are therefore an important point to regulate the neuronal excitability and signal transduction. Previous studies led to the idea that VGSCs were not subject to neuronal modulation, but a growing body of evidence has shown that the properties and functions of VGSCs are also affected by neurotransmitters, such as ACh. In recombinant systems, the alpha subunit of VGSCs has been reported to be phosphorylated by PKA (Costa et al., 1982; Costa and Catterall, 1984b, a) and PKC (Costa and Catterall, 1 984b; Murphy and Catterall, 1992). The activity of PKA and PKC both significantly depressed sodium currents. Intracellular recordings from CAl pyramidal neurons in hippocampal slices showed that muscarinic activity reduces the action potential amplitude and rate of rise, and the spike threshold is raised (Figenschou et al., 1996). Moreover, voltage clamp recordings from acutely isolated pyramidal neurons have revealed that muscarinic receptor activation reduced the peak transient sodium current  (‘NaT)  and slowed the rate of Na channel  tsensitive PKC-dependent pathway (Cantrell et al., 1996; 2 inactivation, via the activation of a Ca Cantrell and Catterall, 2001). Cholinergic input to the hippocampus, acting through muscarinic receptors, inhibits intrinsic bursting activity of CA 1 neurons, converting their firing pattern from phasic bursting separated by quiescent periods to tonic firing of single spikes. These effects are thought due to the reduction of persistent sodium current  (I,p)  in CA 1 neurons, also via a  PKC-dependent mechanism (Azouz et al., 1994; Alroy et al., 1999). Reduction in  ‘NaT  and  ‘NaP  current amplitude would reduce neuronal excitability and protect neurons from hyperexcitability. On the other hand, in dorsal root ganglion neurons, which express high level of TTX-resistant Nay 1.8 and Nay 1.9 channels, neurotransmitters that activate PKA or PKC (such as ACh) increase the peak Na currents and the action potential firing rate. The neurotransmitter-induced upregulation of Na channel function has been considered an essential component of the hyperalgesic response, and the TTX-resistant sodium channels have become novel therapeutic targets for the treatment of intractable pain (Desaphy et al., 2009). 18  Chapter One: Introduction  2.2.4.1.2.  Voltage Gated Potassium Channels  In 1980, Brown and Adams first described a muscarinic inhibition of a voltage- and time-dependent K current in sympathetic ganglionic neurons (Brown and Adams, 1980). This current, termed M current (Halliwell and Adams, 1982).  (IM), ‘M  was subsequently observed in hippocampal pyramidal cells  shows no inactivation and contributes to a steady-state membrane  conductance at depolarized membrane potential. Functionally, the activation of  stabilizes the  ‘M  membrane potential and opposes prolonged depolarizations. Molecular cloning studies have shown that members of the KCNQ gene family encode for M channels (Wang et al., 1998), with KCNQ2/315 subunits contributing to IM in hippocampal neurons (Shah et al., 2002).  M 1  also shows  slow activation and deactivation properties, which limits its ability to contribute to rapid action potential repolarization. However, the activation of  M T  does stabilize the membrane potential and  opposes prolonged depolarizations. Despite decades of study, the identity of signaling elements that couple muscarinic activation to M current modulation has remained unknown. Bertil Hille termed the unknown factor mediating M current inhibition the “mystery messenger”. Until recently, people 2 itself is a crucial determinant of M channel modulation, and that found that the breakdown of PIP the phospholipid-ion channel interactions might play a more important role in the regulation of ion channel than previous realized (Suh and Hille, 2002). Muscarinic-induced inhibition of IM causes membrane depolarization and a decrease in membrane conductance, which increases neuronal excitability (Adams et a!., 1982; Halliwell and Adams, 1982). A transient K current, termed the A-current (IA) is also reported to be modulated by muscarinic stimulation (Nakajima et a!., 1986).  ‘A  shows rapid activation and rapid inactivation responding to  depolarizations, and facilitates the repolarization for action potentials. It has been shown that the apical dendrites of CAl pyramidal neurons display a higher density of 1 A than the soma (Hoffman et a!., 1997).  IA  reduces the amplitude of transient EPSPs and the amplitude of backpropagating action  potentials, and increases the threshold of action potential initiation in the dendrites (Hoffman et a!., 1997; Magee and Carruth, 1999). In the hippocampus, muscarinic activity suppresses  ‘A  through a  PKC-dependent mechanism and increases dendritic action potential amplitude (Hoffman and 19  Chapter One: Introduction Johnston, 1998, 1999). Activation of muscarinic receptors also regulates a voltage-activated, delayed rectifier-like K current (IK) (Zhang et a!., 1992; ifrench-Mullen et al., 1994).  ‘K  is activated by membrane  depolarization to potentials positive to —40 mV and shows little inactivation with long lasting depolarization. In the hippocampus, muscarinic stimulation potentiates  ‘K,  which facilitates action  potential repolarization and reduces sustained depolarizations (Zhang et al., 1992). While on the ventromedial hypothalamic neurons, muscarinic stimulation inhibits  ‘K  and enhances the neuronal  excitability (ifrench-Mullen et al., 1994). Muscarinic stimulation also inhibits a voltage-independent leak K current, termed  ‘leak,  in  hippocampal pyramidal neurons and basolateral amygdala neurons (Womble and Moises, 1992; Guerineau et al., 1994; Selyanko and Sim, 1998). Suppression of  ‘leak  leads to neuronal  depolarization and an increase in input resistance. The depression of ‘leak is considered to play a role in the generation of the cholinergic slow EPSPs observed during strong electrical stimulation in hippocampal slices (Cole and Nicoll, 1984). Genetic studies showed that MI receptors are required for this modulation (Rouse et al., 2000a). -activated, voltage-independent slow afterhyperpolarizing current (I) plays a central 2 The Ca role in spike-frequency adaptation and the associated slow afterhyperpolarization (Alger and Nicoll, 1 via influx of ] 2 1980; Madison et al., 1987). This K conductance is activated by elevations in [Ca ’ stores (Lancaster and Adams, 1986; Sah and 2 2 through VGCCs and/or release from internal Ca Ca Bekkers, 1996). Muscarinic stimulation suppresses the  results in increased action potential  firing frequency and prolongs repetitive firing of neurons (Cole and Nicoll, 1984; Madison et al., 1987). The activity of M 1/3 receptors and CaMXII are required for the depression of Ip (Rouse et al., 2000b; Krause et al., 2002). And finally, in the hippocampus muscarinic stimulation potentiates G protein-activated, inwardly rectifying K channels (GIRKs) (Breitwieser, 2005). Four distinct mammalian genes have been cloned for GIRKs (termed GIRK 1 -GIRK4 or Kir3.1 -Kir3 .4) (Dascal, 1997). The opening of GIRKs causes hyperpolarization of the membrane potential and decreases membrane resistance (Seeger and Alzheimer, 2001). GIRKs are opened by the activation of Gx-coupled GPCRs, and the 20  Chapter One: Introduction released Gfry dimers directly bind and gate the channel (Sadja et al., 2003). Since the identification of GIRK genes, many other regulators of GIRKs have also been reported, including AT Na, and 2 (Breitwieser, 2005). GcLq-coupled GPCRs also suppress GIRKs via the depletion of PIP PIP , 2 activation of PKC and the phosphorylation of GIRK channels (Breitwieser, 2005; Brown et al., 2005). Activation of GIRKs attenuates postsynaptic excitability and EPSPs in CAl pyramidal neurons (Luscher et al., 1997; Seeger and Alzheimer, 2001).  2.2.4.1.3.  Voltage Gated Calcium Channels  Calcium entry through voltage-gated calcium channels (VGCCs) triggers many different physiological events, such as muscle contraction, gene transcription, presynaptic neurotransmitter release, and synaptic plasticity (Catterall et al., 2005; Jarvis and Zamponi, 2007). Molecular cloning experiments have revealed a number of different calcium channel subtypes (L-type, N-type, P-type, Q-type, R-type and T-type VGCC5), based on their specific structural and functional characteristics (Catterall, 2000). T-type VGCCs are also called low-voltage-activated (LVA) VGCCs, while the others are high-voltage-activated (HVA) VGCCs. Muscarinic stimulation has been reported to profoundly modify all these types of VGCCs. HVA VSCCs in hippocampal neurons were suppressed by muscarinic receptor stimulation  (Gahwiler and Brown, 1987; Toselli et al., 1989; Toselli and Lux, 1989). Single channel recordings have revealed two signaling pathways of the muscarinic inhibition of HVA currents. One is a cytoplasmic pathway that involves a diffusible second messenger, which is activated by agonist applied to the neuronal membrane on the outside of the cell-attached patch (Fisher and Johnston, 1990). This pathway involves the activation of GfLq,iicOupled mAChRs (Ml, M3 and M5), and inhibits L- and N-type VGCCs (Stewart et al., 1999; Bannister et al., 2002). This pathway is voltage-independent and takes many seconds to turn on. Despite extensive research however, the diffusible second messengers have long remained unidentified. However, similar to the studies on 2 depletion might mediate this slow, M current, recent evidence also suggests that PIP  voltage-independent inhibition of HVA VGCCs in neurons (Wu et al., 2002; Gamper et a!., 2004). 2 also stabilizes the activity of HVA VGCCs and produces a voltage-dependent inhibition that PIP  21  Chapter One: Introduction  can be antagonized by protein kinase A (PKA) phosphorylation (Wu et al., 2002; Michailidis et al., K promotes 3 2007). Moreover, increased production of another phosphoinositide PIP 3 by PI trafficking of HVA VGCCs to the plasma membrane (Michailidis et al., 2007). The second pathway -coupled muscarinic 1 is observed in excised patches (Toselli and Taglietti, 1994), which involves Ga receptors (M2 and M4) and is independent of diffusible second messengers. This pathway inhibits N- and P/Q-type VGCCs by shifting their gating to more depolarized potentials (Bemheim et al., 1992; Shapiro et al., 2001), and is voltage-dependent and fast (maximal within seconds). Gfry 2 dimers reLeased from G-proteins directly interact with the VGCC cLl subunit to inhibit the Ca 2 is also involved in this fast pathway is current (Herlitze et al., 1996; Dolphin, 2003). Whether PIP still controversial (Michailidis et al., 2007). Unlike other types of HVA VGCCs, the “toxin-resistant” R-type VGCCs are potentiated by GxqIiicoup1ed muscarinic receptors. In expression systems, R-type current generated from overexpression of neuronal Cav2 .3 (a 1 E) subunits (Piedras-Renteria and Tsien, 1998; Sochivko et al., 2002) are both stimulated and inhibited by the activation of muscarinic receptors (Meza et al., 1999; Melliti et al., 2000; Bannister et al., 2004; Kamatchi et al., 2004). Similar to other HVA channels, muscarinic inhibition of Cav2.3 currents depends on the pertussis toxin-sensitive (M2 and M4), Gfry-dependent fast pathway (Meza et al., 1999; Bannister et al., 2004). In contrast, stimulation of Cav2.3 depends on muscarinic receptors that coupled to Gctqjii (Ml, M3 and M5) and the activation of PKC (Meza et al., 1999; Melliti et al., 2000; Bannister et al., 2004; Kamatchi et al., 2004). Similar modulation of R-type VSCCs by muscarinic receptors in native neurons has not been reported yet. The LVA T-type VGCCs are activated by small depolarizations and are important for rhythmic burst firing (Perez-Reyes, 2003). All three T-type VGCC subunits (Cav3.1, Cav3.2, and Cav3.3) are expressed in hippocampal pyramidal neurons (Talley et a!., 1999). However, their modulation and functional impact on hippocampal pyramidal neurons are not yet known. Compared with HVA channels, T-type VGCCs are more stable and are less likely to be modulated (Huang et al., 2005). 2 currents, Muscarinic activation has been reported to increase, decrease, or not affect the T-type Ca depending on the cell type and experimental conditions (Yunker, 2003). Muscarinic stimulation of 22  Chapter One: Introduction hippocampal neurons enhances T-type Ca 2 currents via a pertussis toxin-insensitive pathway (Toselli and Lux, 1989; Fraser and MacVicar, 1991). In an expression system, M3 and M5 receptor activation and increased PKA activity stimulated T-type VGCCs (Pemberton et al., 2000). A recent study aiming to study the muscarinic modulation on individual subtypes of T-type VGCCs found that Ml activity selectively depressed cdl but not alG or cL1H currents (Hildebrand et al., 2007).  2.2.4.1.4.  Non-Selective Cation Channels  Acetyicholine acting on muscarinic receptors induces a remarkable transformation of the electrophysiological properties and spiking activity of hippocampal pyramidal neurons. In addition to the well-known depression of potassium channels and most high threshold calcium channels, there is profound enhancement of R-type calcium channels (Bannister et al., 2004; Tai et al., 2006) and several nonselective cation conductances (Fraser and MacVicar, 1996; Kuzmiski and MacVicar, 2001; Lee et al., 2003; Takai et al., 2004), including the hyperpolarization-activated current (I), a -dependent non-selective cation conductance 2 Ca (ITIUC)  (‘CAN).  and a transient receptor potential current  (Colino and Halliwell, 1993; Guerineau et al., 1995; Strubing et al., 2001).  Hyperpolarization-activated cation currents  (Ih)  have been implicated in rhythmogenesis in both  heart (DiFrancesco, 1993) and brain (Robinson and Siegelbaum, 2003).  ‘h  is a mixed cation current  that typically activates with hyperpolarizing steps to potentials negative to —60 mV (Robinson and Siegelbaum, 2003). Activation of  ‘h  at resting potentials results in a net inward current and  depolarization of the membrane potential that facilitate action potential firing (Crepel and Penit-Soria, 1986; Congar et al., 1997). It has been shown that antagonists of  ‘h  suppress  muscarinic-induced theta oscillations in vivo (Kocsis and Li, 2004). We have shown that the muscarinic-induced plateau potentials (PPs) are mediated by a -sensitive non-selective cation channel (CAN) (Fraser and MacVicar, 1996). The CAN 2 Ca conductance is also present in a broad range of cell types, including cardiac cells, secretory cells, and neurons (Partridge and Swandulla, 1988). A CAN conductance underlies the PPs in rat substantia nigra GABAergic neurons (Lee and Tepper, 2007). Nigral dopaminergic neurons exhibit PPs in response to depolarizing current pulses, which are also conducted by a CAN channel (Ping 23  Chapter One: Introduction and Shepard, 1999). The CAN conductance may also contribute to PPs observed in neostriatal medium spiny neurons (Hernandez-Lopez et al., 1994). In most cases, the exact type of channel underlying the PPs is not clear yet. Recently, muscarinic receptor stimulation has been shown to activate ion channels from the TRPC channel family. TRPC channels are widely distributed in the brain and they are non-selective cation channels activated by increases in intracellular Ca 2 and phospholipase C-coupled receptors. They are “receptor-operated channels” that are activated by stimulation of G-protein-coupled receptors (GPCRs) and tyrosine kinase receptors (Venkatachalam and Montell, 2007). In hippocampus TRPC4 and TRPC5 channels were the predominant TRPC subtypes expressed (Chung et al., 2006). Muscarinic activation enhances TRPC protein translocation and insertion in the plasma K-dependent pathway (Bezzerides et al., 3 membrane of cultured hippocampal neurons, through a PI 2004). TRPC5 generates both homomeric (TRPC5) and heteromeric (TRPC 1/TRPC5) channels (Strubing et al., 2001; Montell, 2005; Schaefer, 2005). Although both TRPC1 and TRPC5 are expressed in the soma, dendrites and axons, TRPC 1 is selectively excluded from synaptic structures, whereas TRPC5 is present in nascent synapses and growth cones (Strubing et al., 2001; Greka et al., 2003). The exact physiological roles of either homomeric or heteromeric TRPC5 channels are not clear yet.  2.2.4.2. Muscarinic Modulation of Synaptic Transmission As the most important modulatory neurotransmitter, ACh can modulate synaptic efficacy at both excitatory and inhibitory synapses, and at both pre- and postsynaptic sites.  2.2.4.2.1.  Presynaptic Modulation  2 entry through presynaptic VGCCs initiates the release of neurotransmitters. Presynaptic Ca VGCCs form a large signaling complex that directly interacts with presynaptic vesicles for efficient release (Catterall and Few, 2008). N- and P/Q-type VGCCs are primarily responsible for initiating synaptic transmission at fast conventional synapses (Olivera et al., 1994; Dunlap et al., 1995). Consistent with the muscarinic inhibition of these HVA VGCCs, presynaptic inhibition of glutamate 24  Chapter One: Introduction release by muscarinic stimulation has also been reported in the hippocampus (Hounsgaard, 1978; Valentino and Dingledine, 1981). Muscarinic stimulation reduced the evoked EPSPs, but did not change postsynaptic currents responding to exogenous kainate or quisqualate (Markram and Segal, 1 990b). The postsynaptic membrane conductance and the presynaptic volley are not affected by muscarinic activity. On the other hand, muscarinic stimulation increased paired-pulse facilitation, indicating a depression of presynaptic neurotransmitter release. Calcium imaging of the presynaptic terminals of CA3-CA1 synapses revealed that the muscarinic-induced depression of presynaptic VSCCs underlies the depression of synaptic transmission (Qian and Saggau, 1997). Recent studies at the calyx of Held with parallel measurements of Ca 2 currents and synaptic transmission also directly proved that G-protein-induced depression of synaptic transmission is due to the presynaptic inhibition of HVA VGCCs (Takahashi et al., 1996; Takago et al., 2005). This muscarinic-induced depression of synaptic transmission could serve a compensatory protective role to balance the enhancement of postsynaptic activities. Similar to glutamatergic synapses, muscarinic stimulation also decreases evoked GABAergic IPSPs (Pitler and Alger, 1992; Behrends and ten Bruggencate, 1993). The frequency of miniature IPSPs (mIPSPs) were decreased by muscarinic stimulation, suggesting a presynaptic mechanism (Behrends and ten Bruggencate, 1993; Parra et al., 1998). The activity of M2 receptors and Gfry dimers are required for the muscarinic depression of GABAergic transmission (Hajos et al., 1998). At the calyx of Held with parallel measurements of VGCC currents and synaptic transmission, GPCR-induced depression of GABAergic transmission is also reported via the presynaptic inhibition of HVA VGCCs (Kajikawa et al., 2001). The cholinergic suppression of GABA release is also observed in vivo, following the stimulation of the septohippocampal pathway (Ben-An et al., 1981;Krnjevicetal., 1981). Muscarinic stimulation can also inhibit GABAergic transmission indirectly via the endocannabinoid (eCB) system. eCBs act as retrograde messengers at inhibitory synapses at many regions of the brain and contribute to the generation of the depolarization-induced suppression of inhibition (DSI) of synaptic transmission (Wilson and Nicoll, 2001, 2002). A postsynaptic depolarization evokes a large increase in intracellular Ca , triggers the generation and release of 2 25  Chapter One: Introduction  eCBs from postsynaptic sites, which act retrogradely at CB 1 receptors on the presynaptic terminals and reduces the probability of GABA release. Bath application of muscarinic agonists or synaptically evoked ACh enhance the DSI of IPSCs (Pitler and Alger, 1994; Martin and Algei 1999; Martin et al., 2001; Kim et al., 2002). It is also reported that at lower concentration of muscarinic agonist suppresses evoked IPSCs exclusively via an eCB-dependent mechanism (Kim et al., 2002).  2.2.4.2.2.  Postsynaptic Modulation  A predominant postsynaptic effect of muscarinic stimulation in CAl pyramidal neurons is the potentiation of currents through NMDA receptors. Henry Markram and Menahem Segal first discovered that ACh potentiates NMDA responses (Markram and Segal, 1 990b), via the activation of mAChR receptors (Markram and Segal, 1 990a; Marino et al., 1998). This potentiation in CA 1 neurons was identified in acute slices (Markram and Segal, 1992), in dissociated cells (Lu et al., 1999), and also in the other cell types such as striatal spiny neurons (Calabresi et al., 1998), and in auditory neocortical cells (Aramakis et al., 1999). Ml specific toxins blocked the carbachol-induced potentiation (Marino et al., 1998), suggesting that the muscarinic potentiation is likely dependent on Ml subtype. Consistent with this finding, Ml receptors were shown to co-localize with NR1A at particular postsynaptic sites (Marino et al., 1998). However, the mechanism underlying the potentiation has long remained mystery. Markram and Segal first reported that the muscarinic -dependent pathway, while no 2 3 and intracellular Ca potentiation of NMDA current requires P protein kinase activity is needed (Markram and Segal, 1992). In contrast, Calabresi et al. reported that the activity of PKC potentiates NMDAR currents using phorbol esters (Calabresi et al., 1998). A recent study showed that in cultured CAl pyramidal neurons, phorbol esters or muscarinic agonist could enhance NMDA currents, and surprisingly, through the Src tyrosine kinase activity (Lu et al., 1999). However, the mechanism underlying the muscarinic potentiation of NMDA current in many other experimental systems still remains elusive. Interestingly, muscarinic-induced depression of NMDA current is also reported in CA3 hippocampal pyramidal cells via -dependent activation of tyrosine phosphatase (Grishin et al., 2004; Grishin et a!., 2005). In 2 Ca contrast, ACh failed to affect responses to kainate or quisqualate (Markram and Segal, 1 990b). 26  Chapter One: Introduction  2.2.4.3. Muscarinic Modulation of Synaptic Plasticity It is generally believed that a long-term change in synaptic efficiency, termed the synaptic plasticity, is the cellular basis of learning and memory (Brown et al., 1988; Kandel, 1997). In the mammalian CNS, the most extensively studied examples of synaptic plasticity are long-term potentiation (LTP) and long-term depression (LTD). Synaptic plasticity is one of the most important neurochemical foundations of learning and memory. Muscarinic activity has been shown profoundly modify both LTP and LTD.  2.2.4.3.1.  Long-Term Potentiation  LTP is a persistent enhancement of excitatory synaptic strength that results from a short period of synaptic activity but lasts at least several hours and often longer (Bliss and Gardner-Medwin, 1973; Bliss and Lomo, 1973). As the most common form of synaptic plasticity, it is believed to be a important mechanism for the cellular basis of learning and memory (Lisman and Harris, 1993). The induction of LTP in CAl pyramidal neurons normally requires the activity of NMDA receptors, which triggers Ca 2 entry and the activation of several downstream enzymes like CaMKII and induces AMPA receptor membrane insertion. The activity of VGCCs is also reported required for the induction of several forms of LTP (Magee and Johnston, 1997; Malenka and Nicoll, 1999). Generally, the early phase of LTP is dependent on the phosphorylation and trafficking of AMPA receptors, while the late phase of LTP requires gene transcription and protein synthesis (Malenka and Nicoll, 1999). It has been reported that the induction of hippocampal LTP is enhanced by the application of cholinergic agonists (Blitzer et a!., 1990; Auerbach and Segal, 1996) on acute brain slices. Muscarinic stimulation via the medial septohippocampal pathway also facilities LTP induction in vivo (Galey et al., 1994; Markevich et al., 1997). The muscarinic enhancement of LTP induction in hippocampus is considered due to the depression of K conductances, inhibition of GABAergic transmission and enhancement of NMDA receptor-mediated currents (Auerbach and Segal, 1996). A recent study showed that Ml, M2 and M4 receptors, but not M3 and M5 receptors, are required for 27  Chapter One: Introduction the muscarinic facilitation of LTP induction in hippocampus (Luo et al., 2008). Muscarinic activity is also reported to directly induce LTP. Auerbach and Segal first reported that application of lower concentration (submicromolar) of muscarinic agonist carbachol induces LTP termed LTPm (Auerbach and Sega!, 1994). LTPm is mediated via activation of M2 mAChRs and mainly caused by an enhancement of the postsynaptic NMDA current (Auerbach and Sega!, 1994). In a recent paper, Fernandez de Sevilla et a!. showed that ACh induced a Ca 2 wave and synaptic enhancement mediated by insertion of AMPA receptors in spines. Activation of Ml mAChRs and Ca 2 release from 1P -sensitive internal calcium stores are required for this type of 3 3 has not been LTP (LTP ) (Fernandez de Sevilla et al., 2008). This Ml mAChR-mediated LTP 3 p 1 repeated yet, and the discrepancy between LTPm and LTP 3 is not fully understood.  2.2.4.3.2.  Long-Term Depression  LTD is a persistent weakening of synaptic strength that lasts several hours to days (Dudek and Bear, 1992). In hippocampus, low-frequency stimulation (LFS) induces an NMDA receptor (NMDAR) dependent LTD, while the activity of metabotropic glutamate receptor (mGluR) can also induce LTD. Both forms of LTD require postsynaptic calcium signaling. Recently, people have identified a novel muscarinic-induced LTD (mLTD) at CA3—CA1 synapses mediated by Ml muscarinic receptors (McCutchen et al., 2006). mLTD is both activity and NMDAR dependent. Interestingly, mLTD is independent from other forms of synaptic plasticity, such as HFS-induced LTP or LFS-induced LTD (McCutchen et a!., 2006). Similarly, in the visual cortex, muscarinic stimulation via Ml receptors also induces LTD that is activity dependent and partially NMDAR dependent (Kirkwood et a!., 1999; McCoy and McMahon, 2007).  2.2.4.4. Muscarinic Receptors in Theta Rhythm Theta rhythms are one of several characteristic electroencephalogram waveforms associated with various sleep and wakefulness states. During exploratory behaviors and in REM sleep, the hippocampus of rodents and many other mammals shows an oscillation of its local field potential in the range of 4—12 Hz, frequency range referred to as theta rhythm (Inanaga, 1998). Many 28  Chapter One: Introduction hippocampal principal neurons (pyramidal and granule cells) synchronously fire action potentials that are phase-locked to this rhythm (Maurer and McNaughton, 2007). Theta rhythms are observed in awake children under the age of 13 years, and in adults during some sleep states, and in states of quiet focus, such as meditation (Aftanas and Golosheykin, 2005). Theta rhythms can also be seen in cases of focal or generalized subcortical brain damage and epilepsy (Colom, 2006). Despite extensive investigation, the mechanisms involved in theta rhythm generation are not clear. In the hippocampus, cholinergic system plays a key role in rhythmic activity, because theta rhythm is abolished by lesion or inactivation of medial septal neurons, the major cholinergic input of hippocampus (Petsche and Stumpf, 1960). Furthermore, muscarinic antagonists could completely block theta in anaesthetized animals, and injection of muscarinic agonists in the hippocampus of both anaesthetized and awake animals produces theta (Bland and Bland, 1986). Application of muscarinic agonists to hippocampal slices in vitro also induces theta-like slow waves (Konopacki et al., 1987). However, although a large body of evidence indicates the key role of muscarinic system in theta, the cholinergic mechanisms underlying theta are still elusive. In hippocampus, cholinergic stimulation profoundly modulated numerous ion channel conductances and thus changes the intrinsic properties and firing patterns of the neurons, as discussed above. However, it is still not clear what conductance plays the most important role in theta generation. Theta rhythms are also correlated with many learning and memory tasks. Extensive evidence shows that theta rhythm is likely involved in spatial learning and navigation (Buzsaki, 2005). Theta rhythms are very strong in rodent hippocampi during learning and memory retrieval (Maurer and McNaughton, 2007), and are believed to be vital to synaptic plasticity, e.g. the induction of LTP and long-term depression LTD, the only potential cellular mechanisms of learning and memory (Hasselmo et al., 2002; Hasselmo, 2005). Interestingly, in hippocampal slices, electrical stimulation delivered on the positive peaks of cholinergic-induced theta in CA 1 in vitro favors the induction of LTP while when delivered on the negative peaks results in LTD (Huerta and Lisman, 1993, 1995, 1996). However, the mechanisms underlying the potentiation and depression of synaptic efficiency during theta are still elusive. A possible explanation is that at the positive peaks of theta, muscarinic stimulation facilitates backpropagating action potentials and triggers larger depolarization and 29  Chapter One: Introduction dendritic Ca 2 spikes, which in turn facilitate Ca 2 influx via VGCCs and relieve NMDA receptors of Mg 2 block, conditions favoring the induction of LTP (Tsubokawa and Ross, 1997; Golding et al., 2002). While at the negative peaks of the theta, the membrane potential of the dendrite falls, reducing Ca 2 levels and favoring the induction of LTD (Kamondi et al., 1 998b). In summary, the timing of the excitatory inputs and postsynaptic situation during the theta rhythms controls the directions of the change in synaptic efficacy.  2.2.5.  Pathophysiology of Muscarinic Receptors In The CNS  Muscarinic cholinergic activity is implicated in a number of neurological disorders including Alzheimer’s disease (AD), epilepsy, schizophrenia, Parkinson’s disease (PD), and Huntington’s disease (HD). Alzheimer’s disease is associated with the degeneration of the cholinergic system of the basal forebrain, whereas excessive cholinergic stimulation is implicated in epilepsy. Central muscarinic and dopaminergic pathways are functionally and anatomically interconnected in many regions of the brain. Cholinergic neurons that project from mesopontine cholinergic nuclei (LDT and PPT) to the dopamine-containing cells of the substantia nigra and the ventral tegmental area (VTA) are thought play a central role in schizophrenia. And a proper balance between striatal cholinergic interneurons and dopaminergic neurons is required for coordinated locomotor control, and is involved in the pathophysiology of PD and HD. The lack of subtype-specific agonists and antagonists makes it very hard to define the roles of individual mAChR subtypes. Recent genetic studies using mutant mouse strains deficient in each of the five mAChRs have led to a wealth of new information in the physiological roles of them and the development of novel subtype-specific muscarinic drugs.  2.2.5.1. Alzheimer’s Disease  AD is the most common neurodegenerative disorder that affects the elderly. AD is characterized by extensive neuron death particularly in the cortex and hippocampus, which result in profound memory loss and cognitive dysfunction (Fillit et al., 2002). The etiology of AD is characterized by two hallmarks: neurofibrillary tangles composed of abnormally hyperphosphorylated tau proteins,  30  Chapter One: Introduction and the senile plaques containing the peptide amyloid-f3 peptide (Af3) (Brion, 1998). It is generally accepted that the loss of cholinergic neurons plays a central role in the cognitive impairment in AD. It has been shown that the progression of AD is accompanied by loss of cholinergic neurons in the basal froebrain (Muir, 1997), and that the aggregation of Af3 causes a variety of acute and chronic neuronal death in cholinergic neurons (Kar and Quirion, 2004). Thus the depletion of cholinergic neurons is hypothesized to significantly contribute to the cognitive and memory deficits associated with AD, called the “cholinergic hypothesis” (Bartus, 2000). Consistent with this hypothesis, mAChR antagonists and specific lesions of cholinergic projections in the basal forebrain cause cognitive impairment, and the drugs that potentiate central cholinergic function have been proven to be the most effective forms of therapeutic treatment against AD. Recent genetic studies using knockout mouse suggests that Ml, M2 and M5 receptors might play important roles in the induction of hippocampal long-term potentiation, learning and working memory (Bartus, 2000), and that muscarinic agonists with selectivity for two or more mAChR subtypes might be more efficient in the treatment of AD.  2.2.5.2. Epilepsy  Epilepsy is a common chronic neurological disorder that is characterized by recurrent unprovoked seizures. These seizures are transient signs and symptoms due to abnormal, excessive or synchronous neuronal activity in the brain. Epilepsy includes a group of syndromes with vastly divergent symptoms that all involve episodic abnormal electrical activity in the brain (Pellock, 2007). Seizures lasting longer than thirty minutes might result in severe brain injury and even death (Corsellis and Bruton, 1983). Epilepsy affects one to three percent of the population, and approximately ten percent of the population will have one or more seizures at some time in their life (Shorvon, 1996). Temporal lobe epilepsy is the very common type of epilepsy, and the majority of complex partial seizures originate in the temporal lobe. Neuronal networks in limbic system structures such as the hippocampus are implicated in the generation of seizures with temporal lobe epilepsy (Schwartzkroin, 1994). Acetylcholine muscarinic system plays an important role in epileptogenesis, especially in 31  Chapter One: Introduction temporal lobe epilepsy (Friedman et al., 2007). Muscarinic receptor agonists generate limbic seizures and are used to generate a model for temporal lobe epilepsy (Leite et al., 2002). Hippocampal sclerosis created by infusion of cholinergic agonists accurately mimics the neuropathology of human status epileptics (Majores et al., 2007). It was also reported that in epileptic patients the levels and activity of ACh, AChE and ChAT were up regulated. Based on the convulsant property of ACh, local or systemic administration of cholinergic receptor agonists (e.g. pilocarpine) or AChE inhibitors (e.g. physostigmine, soman) have been used to induce a pattern of repetitive limbic seizures and status epilepticus (SE) (Turski et al., 1989). SE triggers neuropathological changes such as neuronal death in the hippocampus that closely mimics hippocampal sclerosis from patients with temporal lobe epilepsy. More interestingly, using the pilocarpine-induced SE model, it has been reported that injection of the muscarinic antagonists atropine or pirenzepine (Cruickshank et al., 1994) prevents the onset of seizures, but not the established seizures (Wasterlain and Jonec, 1983; Cruickshank et al., 1994), suggesting that muscarinic receptors are involved in the initiation but not the maintenance of seizures. Furthermore, the central role of Ml receptors are supported by the fact that the Ml knockout mice were resistant to pilocarpine-induced SE (Hamilton et a!., 1997), whereas M2-M5 receptor knockout mice are still sensitive to pilocarpine (Hamilton et al., 1997; Bymaster et al., 2003). 2 influx Our laboratory has reported that muscarinic stimulation in combination with Ca generates prolonged depolarizations called plateau potentials (PPs) in CAl hippocampal pyramidal neurons (Fraser and MacVicar, 1996). PP is an attractive candidate for a major intrinsic conductance generating the prolonged depolarization observed during ictal phase of seizures (Dichter and Ayala, 1987; Fraser and MacVicar, 1996). PPs are novel regenerative events that are generated by -activated cation conductances that are activated by calcium influx through high threshold 2 Ca calcium channels (Kuzmiski and MacVicar, 2001). The conductance that generates the PP is still not identified yet. CNG channels and TRP channels could play important roles in the generation of PPs. It has been reported that the PP is depressed by anticonvulsants such as topiramate and phenytoin (Kuzmiski et al., 2005). The mechanisms underlying the generation of PPs involves M1/M3 receptors coupled to  CLI  proteins and the activation of serine/threonine protein phosphatases and 32  Chapter One: Introduction CaMKII (Fraser and MacVicar, 1996; Alberi et al., 2000; Fraser et al., 2001). Similar PPs have been observed in a broad range of cell types, including cardiac cells, secretory cells, and neurons (Partridge and Swandulla, 1988). In the brain, PPs have been reported in many types of neurons including pyramidal neurons in other cortical regions including the subiculum (Kawasaki et al., 1999), layer V pyramidal neurons in prefrontal cortex (Haj-Dahmane and Andrade, 1998), entorhinal cortex layer II neurons (Klink and Alonso, 1997), substantia nigra GABAergic neurons (Lee and Tepper, 2007), and nigral dopaminergic neurons (Ping and Shepard, 1999).  3.  R-type VGCCs The “toxin-resistant” R-type VGCC is the least understood VGCCs in terms of physiology,  pharmacology and clinical relevance. Pharmacologically, R-type VGCCs were originally identified as the high voltage-activated (HVA) Ca 2 currents that were resistant to the antagonists, -conotoxin MVIIC, ci-conotoxin-GVIA, cü-agatoxin WA and the dihydropyridines (Zhang et al., 1993; Randall and Tsien, 1995). To date, ten different pore-forming c 1 subunits of VGCCs have been cloned, and they are divided into HVA L-type (Cavl .1 LVA T-type Ca 2 channels (Cav3.1  -  -  1.4) and non-L-type (Cav2. 1  -  2.3) Ca 2 channels, and  3.3) (Fig. 1 .2A). It is generally believed that in the CNS Cav2.3  encoded Ca 2 channels are responsible for the generation of R-type Ca 2 current. Cav2.3 was first cloned from rabbit (Niidome et al., 1992) and rat (Soong et al., 1993), and the primary sequence of two alternative splice variants was identified in human brain in 1994 (Schneider et al., 1994; Williams et al., 1994). On the recombinant systems, overexpression of Cav2.3 was initially reported related to LVA T-type (Soong et al., 1993) but later to HVA R-type (Schneider et al., 1994; Williams et al.,  1994) Ca 2 current.  In cerebellar granular cells, experiments using antisense  oligonucleotides to knockdown Cav2.3 demonstrated that three cerebellar R-type currents with different pharmacological properties are all encoded by the a( 1 E) gene (Piedras-Renteria and Tsien, 1998; Tottene et al., 2000). Cav2.3 knockout studies also showed that Cav2.3 subunits underlie a significant portion of the R-type current in hippocampal CA 1, dentate granule neurons, cortical neurons, and amygdala neurons (Lee et al., 2002; Sochivko et al., 2002).  However, it should  be noted that in cerebellar granule cell and dorsal root ganglion cell cultures prepared from Cav2.3 33  Chapter One: Introduction  Figure 1.2. Schematic summary of the physiological, pharmacological and structural properties of calcium channels. (A) Schematic summary of the classification, distribution and pharmacology of the ten different pore-forming ctl subunits of VGCCs that have been cloned, which are divided into HVA L-type (Cavl.1  -  1.4) and non-L-type (Cav2. 1 2.3) Ca2+ channels, and LVA T-type Ca 2+ channels (Cav3 .1 -  -  3.3). Notice the lines on the left showing the sequence similarity of the ctl subunits of VGCCs. (B) Subunit structure of a high-voltage-gated (HVA) calcium channel, which is composed of a pore-forming al subunit and several accessory subunits, including an intracellular f3 subunit, a transmembrane disulfide-linked complex of 2 and ö subunits, and in some cases a transmembrane y subunit. This model fits available biochemical and molecular biological results for Cavi channels and Cav2 channels. Modified from Catterall et al., 2005.  34  Chapter One: Introduction  A Genes  Distribution  Antagonist  Ca1.4(a1F)  skeletal muscle, elsewhere cardiac, smooth muscle, elsewhere neuronal, heart, elsewhere retina, sensory neurons  Ca2.I (a IA)  neuronal (elsewhere)  Ca2.2(a1B)  neuronal  Ca2.3(aIE)  neuronal (elsewhere)  Ca1.1 (alS) Ca1.2(a1C) Ca1 .3 (aID)  Type  _DHP  L  w-Agatoxin IVA  P/Q  w-Conotoxin GVIA  N  SNX-482  R  Ca3.I (alG) brain (elsewhere)  T  Ca3.2(a1H) brain (elsewhere)  T  brain (elsewhere)  T  Ca3.3(a1l)  B y  al  a2 8  N 3 •H  2 c0  35  Chapter One: Introduction knockout mice only a minor portion of the R-type current results from expression of Cav2.3 (Wilson et al., 2000), suggesting a non- Cav2.3 component might also contribute to R-type Ca 2 current. The lack of selective blockers is one of the biggest challenges in the study of R-type VGCCs. A peptide toxin originated from the venom of Hysterocrates gigas, SNX-482, has been reported as the only selective antagonist for R-type VGCCs on expression systems as well as in human neuroendocrine tumor cells (Mergler et al., 2003). However, SNX-482 failed to block R-type currents in many native cells. For example, in cultured cerebellar granule cells people identified three R-type VGCC components, with one highly sensitive to SNX-482, one sensitive to higher concentration of the toxin, and one resistant to SNX-482 (Tottene et al., 2000). Similarly, in another study, it is reported that the majority of R-type current in cerebellar granule cells and dorsal root ganglion cells are SNX-482-resistant (Wilson et al., 2000). In hippocampal CAl pyramidal neurons, R-type current is also significantly less sensitive to SNX-482 (Sochivko et al., 2002). Therefore 2 at low concentrations is the most selective until better pharmacological tools are developed Ni blocker of R-type current in CAl neurons.  3.1. Structures and Properties Similar to other HVA VGCCs, R-type VGCC is composed of a pore-forming ciA subunit (Cav2.3, also called alE) and several accessory subunits, including an intracellular j3 subunit, a transmembrane disult’ide-linked complex of a2 and  subunits, and in some cases a transmembrane  y subunit (Fig. 1.2B). Numerous splice variants of the alE subunit have been reported (Weiergraber et al., 2006), which play a key role in controlling the pharmacological and electrophysiological properties of the channels, as well as the regulations of the channels. For example, the exon 19 encoded arginine rich insert 1 in the intracellular Il-Ill loop is required for the PKC-induced downregulation of inactivation and upregulation of recovery from inactivation (Pereverzev et al.,  2002; Krieger et al., 2006). The  I subunit in encoded by four non-allelic genes (p1-4), and also with  multiple alternatively spliced variants (Hidalgo and Neely, 2007). It binds with high affinity to the a 1 subunit via the alpha interaction domain (AID) located on the I-IT linker. The f3 subunit has been 36  Chapter One: Introduction reported to modify the electrophysiological properties of the channels by regulating the voltage dependence of activation and inactivation, and the channel opening probability at the single channel level (Birnbaumer et aL., 1998). It also plays a role in targeting ni subunit to the plasma membrane (Jarvis and Zamponi, 2007). To date 4 n26 (n2M-4) subunits have been cloned. The ct28 subunit is a highly glycosylated protein that is encoded by a single gene. The transmembrane  part anchors the  extracellular n2 part to the membrane via many disulfide connections (Klugbauer et a!., 2003). The ct26 subunit also shows multiple spliced variants in a tissue-specific manner (Klugbauer et al., 2003). Coexpression of ct26 together with n 1 E increased the current density and shifts the voltage dependence of activation to more hyperpolarized potentials (Hobom et a!., 2000), but the underlying mechanisms are not well understood yet (Klugbauer et al., 2003). The  ‘  subunit of VGCCs is first  identified in muscle with L-type VGCCs. Since then, 8 members (yl-8) have been identified. There is evidence that y2, y3 and y4 could associate with alA and alB VGCCs in the brain using immunoprecipitation methods (Kang et a!., 2001; Sharp et al., 2001). However, whether native neuronal VGCCs contain y subunits, and the functions of the VGCC y subunits are still elusive. Modulation of R-type Ca 2 currents could have profound impact on dendritic excitability through modification of intrinsic firing patterns and the integrative properties of dendrites. Brief trains of back-propagating action potentials have been shown to depress Ca 2 entry through R-type VSCCs located in dendritic spines and thereby block theta-burst-induced long-term potentiation (LTP) (Yasuda et al., 2003). In expression systems, R-type Ca 2 currents due to recombinant Cav2.3 VGCCs (Piedras-Renteria and Tsien, 1998; Sochivko et a!., 2002; Bannister et al., 2004a) are stimulated by the activation of coexpressed Gq-coup!ed muscarinic (Meza et a!., 1999; Melliti eta!., 2000; Bannister et al., 2004a) or metabotropic glutamate receptors (mGkiR) (Stea et a!., 1995), through a Ca -independent PKC pathway. Similar to other HVA VGCCs, R-type VGCCs are also 2 inhibited by Gi/o-coup!ed GPCRs, such as M2 and M4 muscarinic receptors, probably via the activity of the released Gfry dimers (Bannister et a!., 2004a). Interestingly, Ca 2 influx through R-type VGCCs could modulate the channel activity itself at lower cytosolic Ca 2 concentrations. A positive feedback mechanism slows down inactivation and speeds up recovery from inactivation of R-type VGCCs via a Ca - and PKC-dependent pathway, leading to tonic activation of these 2 37  Chapter One: Introduction channels (Leroy et al., 2003; Kiockner et a!., 2004). This calcium-dependent facilitation pathway might be inhibited when the cytosolic Ca 2 concentration increases and turns on a negative feedback mechanism that involves the N-lobe CaM-dependent calcium-dependent inhibition pathway (Liang et al., 2003).  3.2. Distribution R-type VGCCs are widely expressed in the CNS and the peripheral system, as well as other organs, such as the endocrine and cardiovascular system (Weiergraber et al., 2006). In the brain, R-type VGCCs are distributed both presynactically and postsynaptically. Immunohistochemistry experiments showed that R-type VGCCs were detected abundantly in axon terminals of many types of neurons, such as in the presynaptic boutons of mossy fiber, in the globus pallidus neurons, and in the neuromuscular junctions (Pagani et a!., 2004). Interestingly, it is reported that in the presynaptic terminals, only a small portion of R-type VGCCs is restricted to the active zone, and the majority of R-type channels localize more distant from the release sites (Wu et a!., 1999), so that R-type VGCCs might not contribute to the baseline synaptic transmission but be involved in the induction of synaptic plasticity (Dietrich et al., 2003; Kamp et al., 2005). R-type VGCCs also display a prominent expression on the neuronal bodies and dendrites. It has been revealed that these channels are preferentially distributed on the soma and dendritic arbor. Only certain nuclei and particular cell types exhibit apical dendritic distribution of the channel, such as the CAl hippocampal pyramidal neurons. In hippocampal CA 1 pyramidal neurons, R-type and LVA T-type VGCCs are highly expressed in distal dendrites, while other types of HVA VGCCs are mainly expressed in the soma and basal dendrites (Christie et al., 1995; Magee and Johnston, 1995). Furthermore, it has been reported that R-type VGCCs are selectively distributed in the apical dendritic spines, and are thought to be primarily responsible for Ca 2 influx in apical dendrites and spines (Sabatini and Svoboda, 2000; Yasuda et a!., 2003).  38  Chapter One: Introduction 3.3. Physiology Neurotransmitter release at presynaptic axon terminal in the CNS and neuromuscular junction is mediated via the Ca 2 influx through N-type and/or P/Q-type VGCCs. R-type VGCCs normally do not contribute directly to baseline neurotransmission. However, interestingly, in Cav2.1-deficient mice R-type VGCCs contribute to transmitter release in the neuromuscular junction (Urbano et al., 2003; Pagani et al., 2004), suggesting that R-type but not N-type VGCCs replace P/Q-type VGCCs in the active zone. Similarly, antisense knockdown of Cav2. 1 channels in cerebellar Pukinje neurons leads to upregulation of R-type VGCCs (Gillard et al., 1997). In another study, Wu et al. showed that R-type VGCCs mediated the transmitter release in the calyx of Held from young mice (Wu et al., 1998). Overexpression of Cav2 .3 channels in superior cervical ganglion neurons also mediate cholinergic neurotransmission (Mochida et al., 2003). These data indicate that R-type VGCCs are capable of initiating exocytosis of neurotransmitter vesicles. A recent study showed that R-type VGCCs underlie a non-linear modulation of synaptic transmission, via a regulatory loop that requires opening of Cav2.3 channels, voltage-gated Na channels, small conductance Ca-activated potassium (SK) channels, and NMDA receptors (Bloodgood and Sabatini, 2007), suggesting that postsynaptic R-type VGCCs could also play a role in synaptic transmission. Recently, R-type VGCCs have been shown to be required for LTP induced by brief tetanus stimulation in several brain areas. In the CA3 region of hippocampus, presynaptic Ca 2 entry through Cav2.3 channels contributes to the induction of mossy fiber LTP by brief trains of presynaptic action potentials, while they do not contribute to fast synaptic transmission, paired-pulse facilitation, or frequency facilitation (Breustedt et al., 2003; Dietrich et al., 2003). Knockout experiments also showed that Cav2.3-deficient mice display increased threshold for inducing mossy fiber LTP (Breustedt et al., 2003; Dietrich et al., 2003). Except for this NMDA-independent presynaptic plasticity in the mossy fiber terminals, R-type VGCCs are also reported to play a role in the NMDA-dependent postsynaptic LTP in CAl pyramidal neurons. Interestingly, LTP induced from the basal dendrites and soma is not affected by R-type VGCCs antagonists, but only the LTP recorded from the apical dendrites is dependent on R-type VGCCs, consistent with the subcellular distribution of R-type VGCCs along the dendrites of CAl pyramidal 39  Chapter One: Introduction neurons (Isomura et al., 2002). Interestingly, R-type VGCC itself also displays plasticity. Brief trains of backpropagating action potentials have been shown to induce a long-term depression of Ca 2 entry through R-type VGCCs located in dendritic spines, which thereby block theta burst-induced LTP in CA 1 pyramidal neurons (Yasuda et al., 2003).  3.4. Pathophysiology The clinical relevance and pathophysiological role of R-type VGCCs are largely unknown. Recently advanced technologies with Cav2.3 knockout mice have provided novel insight into the functional roles of this channel. It has long been proposed that R-type VGCCs play a role in the etiology and pathogenesis of seizures in many types of epilepsy. R-type VGCCs contribute to the generation of plateau potentials (PPs) (Kuzmiski et al., 2005) and afterdepolarizations (ADPs) (Metz et a!., 2005) and facilitate triggering epileptiform discharges. Traditional pharmacological experiments have revealed that many antiepileptic drugs specifically target Cav2.3 could inhibit epileptiform burst activity (Hainsworth et al., 2003; Kuzmiski et al., 2005). Interestingly, initially people did not find any ictal-like discharges in Cav2.3 knockout mice. However, later it was revealed that the seizure susceptibility was dramatically decreased in the knockout animals (Weiergraber et al., 2006), supporting the pharmacological studies showing that R-type VGCCs play an important role in generating epileptiform activity. However, further investigations are required before we can clarify the relationship between R-type VGCCs and epilepsy and develop new antiepileptic drugs targeting on Cav2.3 channels (Weiergraber et a!., 2006). In addition to the functional role in epileptogenesis, knockout experiments also showed that R-type VGCCs might also exert a protective role in ischemic neuronal injury (Toriyama et a!., 2002). Cav2.3 knockout mice also exhibited less response to acute cocaine administration, suggesting a novel pathway leading to cocaine rewarding and addiction (Han et al., 2002). R-type VGCCs are also reported involved in fear (Lee et al., 2002) and pain behavior (Saegusa et al., 2000). 40  Chapter One: Introduction  4.  TRPC Channels TRP ion channels are a large class of non-selective cation channel subunits that share a primary  structure of six transmembrane domains that assemble as tetramers to form the Ca -permeable 2 pores. TRP channels are widely distributed in mammalian tissues, but their specific physiological functions are largely unknown. More than 30 genes have been identified as TRP channel proteins, and the mammalian TRP superfamily is divided into six subfamilies: classical (TRPC), vanilloid (TRPV), melastatin (TRPM), muclopins (TRPML), polycystin (TRPP), and ANKTM1 (TRPA) (Clapham et al., 2001; Venkatachalam and Montell, 2007) (Fig. 1 .3A). TRP channels display much greater diversity in activation mechanisms and selectivities compared to any other ion channel families. Although TRPVs are the only TRPs known to have a specific agonist (vallinoid), TRP channels can response to a wide range of environment signals including temperature, touch, pain, osmolarity, taste, pH, pheromones and other stimuli (Clapham, 2003). However, how TRP channels are gated and what are their functions are two remaining enigmas. The first identified mammalian TRP channel was the canonical TRPC1 channel (Wes et al., 1995). Since then 7 genes have been recognized in this group and can be divided into four subgroups, TRPC1, TRPC2, TRPC3/6/7, and TRPC4I5, on the basis of sequence homology and functional similarities (Ramsey et al., 2006). TRPC channels are widely distributed in the brain and they are non-selective cation channels activated by increases in intracellular Ca 2 and PLC-coupled receptors. However, the downstream mechanisms underlying receptor operation of TRPC channels, and the physiological functions of these channels in the CNS are still largely unknown.  4.1. Structures and Properties Similar to other TRP channels, all TRPC channel subunits have a predicted primary structure of six transmembrane(TM) domains, homologous to the topology of Shaker potassium channels (Clapham et al., 2001; Ramsey et al., 2006). Just like Shaker channels, biochemical (Phelps and Gaudet, 2007) and atomic force microscopy studies (Barrera et al., 2007) have shown that TRP proteins also assemble as tetramers. Each subunit contains a cytosolic N-terminal, a cytosolic 41  Chapter One: Introduction C-terminal and a pore-forming domain (P-loop) wedged between the fifth and sixth TM domains that allows the passage of cations. The large N- and C-terminal cytosolic domains contain putative protein interaction and regulatory motifs and have distinct features in different TRP subfamilies (Gaudet, 2008). Ankyrin repeats are present in the N-terminal cytosolic region of TRPC channels. The biochemical function of ankyrin repeat domains is not clear, but it has been reported to interact with ligands, although the nature of ligands is highly diverse, from small molecules to unusual oligonucleotides and large proteins (Gaudet, 2008). The cytosolic C-terminal of TRPC channels contains several conserved sequences and regulatory motifs, such as the TRP box (EWKFAR in TRPC), coiled-coil domain, a putative calmodulin- and IP3R-binding domain (CIRB), canonical tbinding domain, and PDZ-binding domain (Clapham, 2003; Gaudet, 2008). 2 helix-loop-helix Ca The cytosolic C-terminal could also form the lower gate that controls the opening and closing of the channel. The P-loops, contributed from each of the four subunits, are thought to form the channel pore and the selectivity filter of permeable ions. The selectivity filter itself might also regulate the gating properties of the channel (Clapham, 2003; Gaudet, 2008) (Fig. 1.3B). All TRPC channels are non-selective cation channels with PCa/PNa < 10 (Venkatachalam and Montell, 2007). TRPC channels have the strongest sequence homology to the founding member of the TRP superfamily, the Drosophila TRP (30-40% identity).  Just like Drosophila TRP channels, TRPC  channels also function as receptor-operated channels, activated by the stimulation of GPCRs and receptor tyrosine kinases (Venkatachalam and Montell, 2007). activation of all TRPC channels (Montell, 2005). activity and channel stimulation are variable.  PLC activity is required for the  However, the mechanisms coupling to PLC  Homomeric TRPC 1 channels have been reported to  be activated by Ca 2 store-depletion, conformational coupling and mechanical stretch (Ramsey et al., 2006). On recombinant systems, TRPC 1 could also form heteromeric channels with TRPC3, TRPC4 and TRPC5, and functions as part of a Gq/11-coupled cation channel (Strubing et al., 2001). Several TRPCs require DAG for channel activation, including TRPC2, TRPC6 and TRPC7 (Venkatachalam and Montell, 2007).  In primary pontine neurons, the native TRPC3 currents  are activated through a pathway involving the brain-derived neurotrophic factor (BDNF) receptors that requires the activity of PLC,  an elevation of intracellular Ca 2 concentration and 1P 3 receptors 42  Chapter One: Introduction  Figure 1.3. Schematic representation of the evolutionary development, classification and structural properties of TRP channels. (A) Phylogenetic tree of the mammalian TRP channel superfamily, which is divided into six subfamilies: classical (TRPC), vanilloid (TRPV), melastatin (TRPM), muclopins (TRPML), polycystin (TRPP), and ANKTM1 (TRPA). The TRPN proteins are not found in mammals, although they are expressed in some vertebrates, such as worm, Drosophila, and zebrafish. Modified from Nilius, B. et al., 2007. (B) Structure of TRPC channels. The following domains are indicated on the left: the six-transmembrane domains, the pore-forming ioop (P), intracellular N and C-domains, ankyrin repeats, coiled-coil domain, protein kinase binding domains, TRP box, and Homer- and PDZ-binding domains. Also shown on the right are the tetrameric structure of TRPC channels, the passage of cations (Na and Ca ), and the antagonists of TRPC channels. 2  43  Chapter One: Introduction  A  TRPML (mucolipin  TRPC TRPC5  TRPP  TRPC3 TRPC7 TRPC6  IRPP5 TRPP3  (polycystin)  (canonical)  TRPP2  TRPC2 TRPV1  TRPM5  TRPV2  TRPM4 TRPM&  TRPM  TRPV3 TRPV4  TRPM2  (melastatin)  TRPV5 TRPM6  TRPMI TRPM3  TRPV (vail in oi d)  TRPA1  TRPA (ankyrin)  Na*  B  2*  3 L  .  Na 2 Ca  TRPC  Na*  •  2-APB  SKF-96365  2 Ca  Ankyrin repeat  TRPbox  R (CIRB) 3 CaM & IP  Coiled-coil domain  Homer  PDZ (TRPC4,5)  PKA,PKC,CaMK  PI3K-SH3 (TRPC3,5,6,7)  44  Chapter One: Introduction (Li et al., 1999). Interestingly, DAG is not required in this pathway, which activates TRPC3 current in recombinant system (Venkatachalam and Montell, 2007). As to TRPC4 and TRPC5 channels, PLC is required for channel activation, while neither DAG nor 1P 3 alone is sufficient to activate the channels (Schaefer et a!., 2000; Strubing et a!., 2001). A unique property of TRPC4 and TRPC5 among TRP channels is that micromolar concentrations of the trivalent lanthanide cations La 3 and 3 could potentiate the activation of these monomeric channels (Schaefer et al., 2000; Strubing et Gd al., 2001). However, the details of mechanism underlying receptor-operated channel activation remain fundamentally mysterious to date. Recently, a growing body of evidence has emerged showing that regulated vesicular trafficking mechanisms might play a critical role in controlling their surface expression as well as their activation in response to different stimuli (Montell, 2005; Ambudkar, 2007). A number of cytosolic molecules such as second messengers and scaffolding proteins are involved in the trafficking of TRPC channels (Ambudkar, 2007). For example, regulated trafficking of TRPC1 to the membrane is reported in endothelial cells (Mehta et a!., 2003), following stimulation with thrombin. TRPC1 is R and RhoA. Actin polymerization and the interaction of TRPC 1 3 assembled in a complex with IP with RhoA are required for the assembly of this complex and translocation of the channel to the plasma membrane (Mehta et al., 2003; Ambudkar, 2007). The interaction of TRPC3 with PLOy regulates its trafficking and cell surface expression (Patterson et al., 2005). Moreover, it is reported that TRPC3 channels localize in mobile intracellular vesicles and that muscarinic stimulation increases plasma membrane expression of TRPC3, via the activity of vesicle-associated membrane protein VAMP2, suggesting that fusion of intracellular vesicles is involved in regulated translocation of TRPC3 (Singh et a!., 2004). Similarly, homomeric TRPC5 channels are also localized in vesicles. Rapid trafficking of these vesicles to the plasma membrane is activated in response to stimulation of hippocampal and other neuronal cells with EGF and nerve growth factor K, GTPase, and 3 (NGF). The mechanism underlying the rapid trafficking involves the activity of PI Racl (Bezzerides et al., 2004). It should be noted here that the heteromeric TRPC1  +  TRPC5  channels do not display similar vesicular trafficking property. Translocation of TRPC4 to the plasma membrane was also reported recently, in response to EGF stimulation (Odell et al., 2005), via the 45  Chapter One: Introduction activation of protein tyrosine kinases such as Fyn. TRPC6 proteins are also reported to translocate -independent 2 to the plasma membrane upon stimulation with muscarinic agonists by a Ca mechanism (Cayouette et al., 2004). It should be noted that TRPCs could interact with each other to form heteromeric proteins. However, any direct impact of TRP multimerization on TRPC3/6 trafficking is not yet known. It is also not clear whether the multimerization of TRPC channels affects the trafficking properties of the channels, and whether the trafficking of homomeric channels differ from that of heteromeric channels (Arnbudkar, 2007). An interesting feature of many members of the TRP superfamily is that closely related TRP channels could form heteromers (Venkatachalam and Montell, 2007). Heteromerization could effectively modulate the distribution, biophysical properties and functions of the interacting channels. Heteromultimeric interactions normally only happen between members that are closely related to each other (Venkatachalam and Montell, 2007). The best example is the TRPC I channels. TRPC 1 is most related to TRPC4 and TRPC5 proteins. Indeed TRPC 1 displays overlapping distribution with TRPC4 and TRPC5 in the hippocampus, and forms heteromeric channels with TRPC5  and  TRPC4  when  coexpressed  in  cultured  cells  (Strubing  et  al.,  2001).  Heteromultimerization between TRPC 1 and TRPC5 leads to the generation of novel nonselective cation conductances with biophysical properties distinct from those of either TRPC 1 or TRPC5 homomers (Strubing et al., 2001). Biochemical studies also revealed TRPC1 and TRPC4 or TRPC5 heteromers in rat embryonic brains, which form channels with novel biophysical properties when expressed in vitro (Strubing et al., 2003). Interestingly, TRPC1 can also form heteromers with TRPC3I6/7 subfamily as well (Lintschinger et al., 2000; Strubing et al., 2003). Native TRPC3 and TRPC6 interact with TRPC1  in the rat embryonic brain (Strubing et al., 2003). The  heteromerization between TRPC3 and TRPC 1 is required for the generation of a novel conductance in cultured cells (Lintschinger et al., 2000; Strubing et al., 2003). Thus the heteromerization between different TRPC proteins could dramatically amp1if’ the diversity of the channels with distinct biophysical properties and biological functions.  46  Chapter One: Introduction 4.2. Distribution TRPC channels are widely expressed throughout many organisms, including the CNS and PNS, heart and other tissues like ovary, liver, testis and spleen. All TRPC channels are distributed in the brain. TRPC2 is distinct from other TRPC subfamily members in that its human gene encodes a nonfunctional truncated protein and is considered a pseudogene (Vannier et al., 1999). In rodent the TRPC2 gene has been reported to express functional TRCP2 channels, which is activated by DAG (Lucas et al., 2003). In the rodent vomeronasal organ, TRPC2 is localized to neuronal microvilli and appears to be required for neuronal excitability in pheromone signal transduction (Liman et al., 1999). It is reported that BDNF-triggered TRPC3-dependent conductance were observed in primary pontine neurons (Li et a!., 1999) and hippocampal pyramidal neurons (Amaral and Pozzo-Millei 2007). Immunohistological experiments showed that TRPC3 also display a preferable distribution in non-excitable cells in the brain, including the oligodendrocytes and astrocytes (Li et a!., 1999; Fusco et al., 2004). TRPC6 is most prominently expressed in lung tissues (Corteling et a!., 2004). Three splice variants with shorter amino termini were additionally cloned from rat lung (Zhang and Saffen, 2001). TRPC6 expression in brain is lower than that of TRPC3. Biochemical data revealed TRPC6 expression exclusively in the dentate granule cell layer of the adult mouse brain (Otsuka et al., 1998). In the rat carotid body only TRPC7 is expressed in glial cells, while carotid sinus nerve fibers express other TRPC channels (Buniel et al., 2003). In human arteries, TRPC7 is expressed in endothelial cells but not in smooth muscle cells, while other types were expressed in both cells (Yip et al., 2004). All of the TRPC3/6/7 subfamily are highly expressed in smooth and cardiac muscle cells (Inoue et al., 2001). TRPC4 and TRPC5 are also widely expressed but particularly abundant in brain. In rat hippocampus, the pyramidal cell bodies of CAl, CA3 and the granule cell bodies of the dentate gyrus expressed high level of TRPC1, TRPC4 and TRPC5 (Chung et al., 2006). Biochemical methods like in situ hybridization, Western blotting, and immunocytochemistry have revealed prominent expression of TRPC4 in the hippocampal formation and cortical plate. Analysis of the adult hippocampa! formation has revealed TRPC4 expression in hippocampus from CA 1 to CA3 and also in the dentate gyrus (Zechel et al., 2007). TRPC1 and TRPC5 channels showed overlapping distributions in the hippocampus. Both TRPC1 and TRPC5 are expressed in the soma, 47  Chapter One: Introduction dendrites and axons, but TRPC 1 is selectively excluded from synaptic structures, whereas TRPC5 is present in nascent synapses and growth cones (Strubing et al., 2001; Greka et al., 2003).  4.3. Physiology Despite extensive studies on TRPC channels, the physiological roles of these channels are still largely unknown. TRPC1 is known to be involved in several important processes in the brain. Metabotropic glutamate receptor mGluRl -evoked slow excitatory postsynaptic conductance (EPSC) is mediated by TRPC1 in Purkinje cells (Kim et al., 2003). Similar currents that sensitive to TRPC inhibitors were also recorded in hippocampal pyramidal neurons (Gee et al., 2003; Rae and Irving, 2004) and midbrain dopamine neurons (Tozzi et al., 2003). TRPC1 is also involved in netrin-1 and BDNF-mediated growth cone guidance (Shim et al., 2005; Wang and Poo, 2005). In cultured neurons, TRPC5 insertion and TRPC5-mediated Ca 2 influx is an important determinant of hippocampal neurite outgrowth rates and growth cone morphology (Greka et al., 2003; Bezzerides et al., 2004). BDNF triggered a TRPC3-dependent conductance in primary pontine neurons and hippocampal pyramidal neurons via the activity of TrkB, which is required for the BDNF-induced enhancement of dendritic spine density and development (Li et al., 1999; Amaral and Pozzo-Miller, 2007). A recent report suggests that the activity of mGluRs triggers endocytosis of TRPC4 and TRPC5 channels in CA 1 pyramidal neurons, which may contribute to alterations of seizure discharges (Wang et al., 2007). Recently, the TRPC5 knockout mice have been developed, which exhibit diminished innate fear levels in response to innately aversive stimuli. The responses mediated by synaptic activation of Group I metabotropic glutamate and cholecystokinin 2 receptors are also significantly reduced in neurons of the amygdala. Synaptic strength at afferent inputs to the amygdala was also diminished in young null mice. These experiments provide genetic evidence that TRPC5 has an essential function in innate fear (Riccio et al., 2009). 2 influx. SOC channels are Store-operated calcium entry (SOCE) is a major mechanism for Ca 2 stores (primarily the ER). defined by their activation in response to depletion of the internal Ca Influx through SOC channels is necessary for the replenishment of the Ca 2 stores. TRPC channels have long been proposed as candidates for SOC channel. However, despite extensive studies, the 48  Chapter One: Introduction  role of TRPC channels in the conduction of SOCE still remains highly controversial. A number of studies have revealed that Orai and STIM proteins form the calcium release activated channels (CRAC), the first identified and best described SOC current (Peinelt et al., 2006; Prakriya et al., 2006; Lioudyno et al., 2008; Penna et al., 2008).  Functional interactions between STIM1, Orail  and TRPCs have been reported. CRAC channels are proposed to be composed of TRPC pore-forming subunits and Orai regulatory subunits that transduce the store depletion signal from STIMI to TRPCs (Huang et al., 2006; Liao et al., 2007; Worley et al., 2007). Consistent with this model, a dynamic assembly between TRPC1, STIM1 and Orail has been reported to be required for SOC channel function (Ambudkar et al., 2007). Lipid raft domains are also shown to be important for functional interaction of STIM 1 with TRPC 1 and activation of SOCE after store depletion (Alicia et al., 2008; Jardin et al., 2008; Pani et al., 2008). Physical interactions between TRPCs and Rs are also reported, but the functional role of this interaction in still elusive. A recent study 3 IP R is required for the maintenance of 3 showed that the coupling between TRPC 1 and the type II IP SOCE in human platelets, where STIM1 and Orai proteins are not observed in these cells (Lopez et al., 2006; Jardin et al., 2008).  4.4. Pathophysiology The clinical relevance and pathological functions of TRPC channels are largely unknown to date. The activity of TRPC channels might play a role in ischemic cell death. The first demonstration that oxidative stress causes constitutive activity of a TRP was obtained for TRPC3 in aortic endothelial cells (Balzer et al., 1999). A recent study showed that overexpression of TRPC3 increases apoptosis but not necrosis in response to ischemia-reperfusion in adult mouse cardiomyocytes (Shan et al., 2008). It is also reported that TRPC3 and TRPC4 channels are redox-sensitive and could be activated by oxidants in non-neuronal cells (Groschner et al., 2004). TRPC channel blockers like 2-APB is also reported to depress several acute ischemic responses in hippocampal CA 1 neurons, such as the negative shifts of extracellular DC potential, the rate of the 2 influx, and tissue swelling (Lipski et al., 2006). initial slow membrane depolarization, Ca Intracellular ATP is reported to inhibit TRPC5 channel activity, suggesting that the activity of 49  Chapter One: Introduction TRPC5 could be involved in the Ca 2 overload occurring during and after ischemia when ATP is depleted (Dattilo et al., 2008). Mutation in TRPC6 causes a type of kidney disorder called focal and segmental glomerulosclerosis (FSGS), which is characterized by a breach in the permeability barrier between capillary and kidney (Reiser et al., 2005; Winn et al., 2005). The mutation of TRPC6 induced the disruption of this permeability barrier leading to proteinuria and end-stage renal disease. FSGS patients also display hypertension, a clinical manifestation consistent with higher blood pressure in the TRPC6 knockout mice (Dietrich et al., 2005). However, the mechanism of TRPC6 activation in FSGS is also mystery.  5. NMDA Receptors  Glutamate is the major excitatory neurotransmitter in the CNS. Early pharmacological studies revealed multiple types of ionotropic glutamate receptors. In the 1 970s, selective agonists that could distinguish different subtypes of glutamate receptor were developed (Watkins et al., 1990), including N-methyl-D-aspartate (NMDA), cc-amino-3 -hydroxy-5 -methylisoxazolepropionic acid (AMPA), kainate, and quisqualate, which have been used extensively to characterize the ionotropic glutamate receptor family (Watkins et al., 1990). Three ionotropic glutamate receptor subfamilies have been defined: NMDA receptors (NMDARs), AMPA receptors and kainate receptors, with distinct pharmacological, electrophysiological and functional properties. Quisqualate is unique in its capability of activating both ionotropic and metabotropic glutamate receptors subtypes (Hollmann and Heinemann, 1994). Both AMPAR and kainate receptors are rapidly activated by high concentrations of glutamate with a high probability of opening, and both possess low-affinity glutamate binding and deactivate quickly. Thus the gating of AMPA and kainate receptors by glutamate is extremely fast while NMDARs display high affinity to glutamate and slow activation and deactivation kinetics. It is generally believed that AMPA and kainate receptors underlie the fast excitatory neurotransmission while NMDARs underlie the late phase of the transmission and the plasticity of the changes in transmission efficiency (Dingledine et al., 1999). NMDA receptor is an important ionotropic glutamate receptor, hallmarked by its large 50  Chapter One: Tntroduction -permeability and the voltage-dependent Mg 2 Ca 2 blockade. The NMDARs have several unique features different from most other ligand-gated ion channels. For example, binding of glycine as a coagonist is required for effective channel opening, and the membrane potential must be depolarized to remove the tonic block of the channel by extracellular Mg , making NMDAR a 2 “coincidence detector” of presynaptic glutamate release and postsynaptic depolarization. Since the early 1 980s, it has been found that NMDAR antagonist AP5 displays neuroprotective and anticonvulsant functions (Choi, 1998; Dingledine et al., 1990), and the Ca 2 entry through NMDARs is essential in development, synaptic plasticity and in learning and memory (Maren and Baudry, 1995; Asztely and Gustafsson, 1996). Since then, extensive studies on NMDAR have revealed its role in a variety of neurological disorders, including ischemia, epilepsy and neurodegenerative disorders such as Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD) and amyotrophic lateral sclerosis (ALS) (Gerber and Vallano, 2006; Planells-Cases et al., 2006).  5.1. Structures and Properties  Molecular cloning revealed that NMDARs are encoded by at least three gene families (Dingledine et al., 1999). The first cDNA encoding for a subunit of NMDAR was identified by Nakanishi and colleagues (Moriyoshi et al., 1991), which is termed NR1. Since then, seven distinct subunits of NMDAR have been cloned: one NR1, four NR2 (NR2A-D) (Kutsuwada et al., 1992; Monyer et al., 1992), two NR3 (NR3A-B) (Ciabarra et al., 1995; Nishi et al., 2001). The NR1 gene encodes a 920 amino acid polypeptide that has a molecular weight of 103 kDa. NR2 subunits have larger carboxyl terminus and their molecular mass are 3 0-63 kDa greater than NR1. Despite extensive studies, little data are available about the subunit stoichiometry of NMDARs to date. Early evidence favored a pentameric structure similar to that of nAChRs (Brose et al., 1993; Premkumar et al., 1997), but later there were reports favoring a tetrameric composition of NMDAR2 (Behe et al., 1995; Doyle et al., 1998; Laube et al., 1998). The transmembrane topology of the individual NMDAR subunits was also determined (Kuner and Schoepfer, 1996). Against initial expectation, NMDAR subunits contain only three transmembrane domains, a cytosolic 51  Chapter One: Introduction re-entrant membrane ioop, an extracellular N-terminal and an intracellular C-terminal, which is largely different from the transmembrane topology of nAChRs. Residues in this re-entrant second membrane ioop are thought to control permeation properties of the ion channel (Dingledine et al., 1999). The NR1 subunit contains three alternatively spliced exons, one in the N-terminal and two in the C-terminal, resulting in eight splice variants with distinct properties in binding with different proteins, regulation by different secondary messengers, and targeting of NMDARs to the plasma membrane (Wenthold et al., 2003). A functional NMDAR is normally considered to contain two glycine-binding NR1 subunits and two glutamate-binding NR2 subunits. The glycine-binding NR3 subunits could take the place of NR2 to create functional excitatory glycine receptors with NR1 tpermeability and resistant to Mg 2 (Chatterton et al., 2002), with very low Ca , glutamate, NMDA 2 and MK-801 (Chatterton et al., 2002). At the postsynaptic site, NMDARs are linked to a large, multi-protein complex via the cytoplasmic C-terminal of NR1 and NR2 subunits (Collins et al., 2006) (Fig. 1.4). This complex is supposed to anchor NMDARs in specific areas such as the postsynaptic density (PSD) and to facilitate the assembly of a variety of downstream signaling molecules for NMDAR activities. The C-terminal of NR2 subunits link to a group of proteins called membrane-associated guanylate kinases (MAGUKs), including PSD-95, SAP-102 and PSD-93. These proteins contain several PDZ-binding domains to interact with other proteins. A number of other proteins have also been shown to interact with the C-terminals of both NR1 and NR2 subunits, such as AP-2 adaptor proteins, spectrin, tubulin, Src and so on. And the extracellular N-terminals might also display physical interactions with other proteins, like EphB receptor and the extracellular matrix protein Reelin (Dalva et al., 2000; Groc et al., 2007). In addition to extracellular Mg 2 and glycine, NMDA receptors are also modified by numerous 2 and Cu 2 inhibit NMDA receptors other endogenous molecules (Fig. 1.4). Metal ions such as Zn by both a voltage-dependent and voltage-independent mechanism (Smart et al., 1994; Trombley and Shepherd,  1996). NMDA receptors are also inhibited by more physiologically relevant  concentrations of extracellular protons, through a voltage- and agonist-independent reduction in the single-channel opening frequency (McBain and Mayer, 1994).  Extracellular polyamines, such as 52  Chapter One: Introduction  Figure 1.4. Schematic representation of the structure of a NMDA receptor with highlighting binding sites for numerous regulatory molecules. A functional NMDA receptor normally contains two glycine-binding NR1 subunits and two glutamate-binding NR2 subunits. Each NMDA receptor subunit contains three transmembrane domains, a cytosolic re-entrant membrane loop, an extracellular N-terminal and an intracellular C-terminal. The channel property of NMDA receptor could be regulated by numerous molecules, including agonists (glutamate and NMDA), co-agonists (glycine and D-serine), antagonists (D-APV, , memantine, PCP and 2 ifenprodil, Ro 25-6981, NVP-AAMO77 and Zn ), channel blockers (Mg 21 MK-80 1) and other modulators such as polyamines and histamine. The activity of NMDA receptor could also be modified by phosphorylation.  53  Chapter One: Introduction  Agonists  Open-channel blockers  • Glutamate, NMDA  • Mg 2  Coagonists  * MK/801, Memantine, PCP  • Glycine, D-serine  Competitive antagonists  o •  Channel modulators • Polyamine, Histamine  2 Zn D-APV  -  Phosphorylation site  Ifenprodil, Ro 25-6981 (NR2B) NVP-AAM 077 (NR2A)  54  Chapter One: Introduction  spermidine and spermine, have multiple effects on NMDARs, including voltage-dependent inhibition, glycine-dependent potentiation, and voltage- and glycine-independent potentiation (Rock and Macdonald, 1995; Williams, 1997). The activity of NMDARs is also profoundly modified by many second messenger systems. NMDARs can be phosphorylated by several serine and threonine kinases including PKA, PKC, and CaMKII. In cultured neurons, PKC activators can enhance NMDA current indirectly through a PKC-dependent activation of the non-receptor tyrosine kinase (Src) signaling cascade (Lu et al., 1999), while direct phosphorylation of NMDARs by PKC increases the sensitivity of NMDA channel inactivation to intracellular Ca 2 (MacDonald et al., 2001). Little is known about the regulations of NMDARs by CaMKII and PKA. CaMKII can link with NR1, NR2A and NR2B. The association of CaMKII and NMDARs is believed to occur following autophosphorylation of CaMKII due to Ca 2 entry from activated NMDARs, which brings CaMKII into close proximity with AMPA receptors (Wenthold et al., 2003). On the other hand, serine and threonine phosphatases 1, 2A and 2B (calcineurin) could inhibit NDMAR function. Other important kinases such as protein tyrosine kinases (PTKs) and cyclin-dependent kinase-5 (Cdk5) are also reported to modifi the activity of NMDARs (Wenthold et al., 2003).  5.2. Distribution Glutamate receptors, including NMDARs, are mainly expressed in neurons in the CNS. NMDARs are also distributed in other types of cells, including neurons in the PNS, glia cells, and other non-excitable cells. For example, it is reported that NMDA as well as non-NMDA receptors are expressed in un-myelinated sensory nerve terminals in the skin (Carlton et al., 1995), suggesting that peripheral glutamate receptors might be associated with forms of pain and inflammation. NMDARs are also identified in brain microglia, astrocytes, and oligodendrocytes (Verkhratsky and Kirchhoff, 2007). Functional NMDARs are also found in mast cells, taste receptor cells, cardiac ganglia cells, pancreatic islet cells and so on (Dingledine et a!., 1999). The four NR2 subunits show a distinct distribution in the CNS with different expression patterns during development (Monyer et al., 1994; Standaert et al., 1996). NR2B (and NR2D) 55  Chapter One: Introduction subunits are abundantly expressed in many principal neurons at early developmental stages, but decline postnatally, while the expression of NR2A and NR2C subunits are progressively enhanced during development. In the adult brain, NR2A is ubiquitously expressed, NR2B is restricted to the forebrain, NR2C is highly enriched in the cerebellum, while NR2D is expressed in restricted brain regions in the midbrain and hindbrain (McBain and Mayer, 1994; Kohr, 2006; Papadia and Hardingham, 2007a). NR3B is expressed predominantly in motor neurons whereas NR3A is more widely distributed (Chatterton et al., 2002). Interestingly, in NR3A knockout mice NMDA currents in cortical neurons were increased about 3-fold as was synaptic spine density (Das et al., 1998), suggesting that NR3 subunits might have a regulatory role by their co-assembly with NR1 and NR2, leading to the reduction in both whole-cell current (Ciabarra et al., 1995; Nishi et al., 2001) single channel conductance (Das et al., 1998), and lower Ca 2 permeability (Perez-Otano et al., 2001). In neurons, NMDARs have been detected at both presynaptic and postsynaptic sites (Tzingounis and Nicoll, 2004; Kohr, 2006). In the postsynaptic membrane of excitatory neurons, the density of NMDARs is higher in dendritic spines, within the postsynaptic density (P SD), than in the dendritic shaft and somatic membrane. The subcellular location of NMDARs might profoundly affect the functions of NMDARs. Developing neurons have internal pools of NMDARs at both synaptic and extrasynaptic locations, which signal very differently (Papadia and Hardingham, 2007a). Over the past few years, several studies have suggested that the differences in synaptic and extrasynaptic signaling could rise from different subunit composition of the NMDARs. It has been revealed that extrasynaptic NMDARs are preferentially enriched with NR2B-containing receptors. However, NR2B subunits do not exclusively segregate to extrasynaptic compartments but also present in the synaptic position (Fujisawa and Aoki, 2003; Kohr et al., 2003). Furthermore, NR2A is not exclusively synaptic but is also found extrasynaptically (Mohrmann et al., 2002; Thomas et al., 2006). Thus the segregation of NR2B and NR2A to extrasynaptic and synaptic sites is not completely determined yet.  5.3. Physiology  NMDAR activity is vital for brain function. NMDARs play a central role in synaptic 56  Chapter One: Introduction transmission and in many forms of synaptic plasticity that are thought to underlie the formation of learning and memory. A growing body of evidence has shown that physiological levels of synaptic NMDAR activity display a neuroprotective function that promote survival of many types of neurons.  5.3.1.  Neuronal Survival  Neuronal survival is dependent on physiological levels of electrical synaptic activity, supported by the fact that blocking electrical activity in vivo or in vitro causes cell death (Catsicas et al., 1992; Sherrard and Bower, 1998). Ca 2 influx via NMDARs is believed to play a central role in the neuroprotective effects of electrical activity. For example, in vivo blockade of NMDAR activity causes extensive apoptosis in many brain areas including hippocampus and cerebellum (Ikonomidou et al., 1999; Monti and Contestabile, 2000), and enhances trauma-induced injury in developing neurons (Ikonomidou et al., 1999; Adams et al., 2004). NMDAR blockade also prevents the survival of newborn neurons in the adult dentate gyrus (Tashiro et al., 2006). Several NMDAR-dependent prosurvival pathways have been identified. The PI K-Akt kinase cascade is a 3 key signaling pathway responsible for prosurvival effects of NMDAR activity (Lafon-Cazal et al., 2002; Papadia et al., 2005), which promote cell survival and growth via phosphorylation and activation or inactivation of its many targets, such as GSK3f3, FOXO and BAD (Papadia and Hardingham, 2007a). Ca 2 influx via NMDARs also activates the Ras/ERK pathway in the cytoplasm and nuclear 2 /Ca calmodulin-dependent protein kinases, especially CaMKTV both activate and enhance CREB activation, which is necessary for cell survival (Hardingham et al., 2001; Papadia and Hardingham, 2007a).  5.3.2.  Synaptic Transmission and Plasticity  The NMDAR channel pore is blocked by extracellular Mg 2 in a voltage dependent manner. Release of glutamate causes Na influx through AMPA receptors and depolarization in the postsynaptic plasma membrane, resulting in removal of the Mg 2 block of NMDARs. The activated NMDAR is permeable to Na that also contributes to postsynaptic depolarization and 57  Chapter One: Introduction high-frequency synaptic transmission, but more crucially, the Ca 2 entry through NMDARs mediates most of the physiological effects of NMDAR activity. NMDARs are the most important trigger for the long-term modification of synaptic strength. Two paradigmatic examples of long-term changes in synaptic strength are long-term depression (LTD) and long-term potentiation (LTP), which are recorded in a variety of brain regions with diverse stimulation protocols (Malenka and Bear, 2004). The induction of many forms of LTD and LTP requires NMDAR activation and the subsequent Ca 2 influx that triggers numerous downstream events. In the CNS, LTD is often associated with postsynaptic AMPA receptor endocytosis, while LTP associated with exocytosis of AMPA receptors into postsynaptic membranes (Malenka and Bear, 2004). LTD and LTP are also reported to be associated with changes in spine morphology and number (Matsuzaki et al., 2004; Zhou et al., 2004). The direction of the plasticity is largely controlled by the kinetics and amount of Ca 2 influx through NMDARs, and different downstream pathways that coupled to NMDARs. There is evidence that the activity of CaMKII, PKA and P13K are involved in LTP induction, while protein phosphatases and PKC are involved in  the induction of LTD. These kinases and phosphatases could all be activated by the Ca 2 influx via NMDARs, however, the details about how NMDARs couple to different signaling pathways and control the direction of the plasticity (weakening or strengthening) via the same second messenger, 2 influx, are still largely unknown. One explanation is that NMDARs at different locations Ca (synaptic and extrasynaptic) activate different signaling pathways. There is evidence that synaptic NMDARs support LTP while extrasynaptic NMDARs mediate LTD in the mature brain (Lu et al., 2001; Massey et al., 2004), although this remains controversial. Another explanation suggests that different subunit composition of NMDARs (NR2A- or NR2B-containing receptors), which have distinct single channel properties and different coupling protein partners, control different downstream signaling pathways and the direction of plasticity. NR2B-containing NMDARs display longer currents, carry more Ca 2 per unit of current, and interact with CaMKII with a higher affinity compared to NR2A-containing NMDARs (Strack and Colbran, 1998), leading to the hypothesis that NR2B subtypes are more likely to favor the induction of LTP compared to NR2A subtypes, which is supported by several studies (Barria and Malinow, 2005; Berberich et al., 2005). Contrary to these 58  Chapter One: Introduction findings, it has been reported that NR2A-containing, but not NR2B-containing, NMDARs mediate LTP (Liu et al., 2004; Massey et al., 2004; Bartlett et a!., 2007). As to LTD induction, it is even more controversial. Several studies demonstrated that NR2B antagonists completely block hippocampal LTD (Liu et a!., 2004), while several studies showed the both NR2A and NR2B are involved in LTD induction, and there is also evidence that hippocampa! LTD is insensitive to or even enhanced by NR2B blockade in the CA 1 region of the hippocampus (Hendricson et al., 2002; Morishita et al., 2007). Given that LTD and LTP are complicated cellular processes involving many signaling proteins and are expressed by different mechanisms among brain regions and developmental stages (Malenka and Bear, 2004), more future work are required before we could determine how NMDARs control the directions and levels of synaptic plasticity. Interestingly, the NMDAR-mediated synaptic transmission itself can display long-term changes, a process termed metaplasticity. On the plasma membrane of neurons there are mobile and immobile pools of NMDARs, at both synaptic and extrasynaptic sites (Benke et al., 1993; Groc et al., 2004). NMDARs can move from extrasynaptic to synaptic sites on the cell surface, so the number of NMDARs at synapses might be rapidly modulated by lateral diffusion in the plasma membrane (Tovar and Westbrook, 2002; Groc et a!., 2004). However, the mechanisms by which NMDARs can rapidly move between the intracellular and extracellular compartments of neurons are now being elucidated. Furthermore, an increase in NMDAR surface expression has been reported after the induction of LTP at adult CA 1 synapses, while a form of LTD that is induced by mGluR activation is associated with the internalization of NMDARs (Collingridge et al., 2004). These data suggest that NMDAR surface expression can be rapidly regulated by activity, indicating another molecular mechanism for metaplasticity. The association of NMDARs with other protein partners such as PSD-95 and AP2, and the regulation of NMDARs by second messengers like PKC and Src might also play a role in the generation of metaplasticity (Collingridge et al., 2004).  5.4. Pathophysiology A dysfunction of NMDAR is implicated in numerous neurological disorders, such as stroke, schizophrenia, and neuropathic pain. Furthermore, NMDAR antagonists tested in animal models 59  Chapter One: Introduction have been shown to be protective against ischemic stroke, neuropathic pain, and several neurodegenerative diseases, although the clinical use of NMDAR antagonists as effective therapeutic agents is still largely limited because of strong side effects.  5.4.1.  Ischemic Stroke  Excessive Ca 2 entry through pathologically activated NMDARs is a major cause of neuronal death following acute excitotoxic damage, such as brain ischemia, hypoxia and mechanical trauma (Arundine and Tymianski, 2004; Papadia and Hardingham, 2007a). The brain damage induced by over excitation of glutamate receptors, which is termed excitotoxicity, results from an increase in glutamate efflux (from presynaptic terminals and astrocytes) and a decrease in glutamate re-uptake (due to reduced glutamate transporter activity). The excessive calcium influx via NMDARs leads to cell death, through several mechanisms. The first one is the mitochondrial dysfunction caused by excessive Ca 2 uptake into the mitochondria through the potential-driven uniporter (Stout et al., 1998; Nicholls, 2004), which results in less production but more depletion of cytosolic AT and more production and release of reactive oxygen species and cytochrome c, all leading to apoptosis. The second mechanism involves the activation of calpains and other Ca -dependent proteases 2 triggered by excessive Ca 2 entry through NMDARs, which cleave a major isoform of Na/Ca 2 exchanger on plasma membrane, severely impairing Ca 2 efflux and removal from cytoplasm (Schwab et al., 2002; Bano et al., 2005; Pottorf et al., 2006). The third mechanism is that NMDAR-dependent Ca 2 influx triggers production of both NO and superoxide, which combine to form ONOO and activate TRPM7 channel, another highly Ca -permeable channel, resulting in 2 more Ca 2 influx (Aarts et al., 2003). Stress-activated protein kinases such as p38 and JNKs are also involved in the NMDAR-induced neuronal death (Cao et al., 2004; Semenova et al., 2007). However, although numerous studies have shown the role of NMDAR activity in neuronal loss following ischemia, many clinical trials with different NMDAR antagonists for the treatment of ischemic stroke have failed (Ikonomidou and Turski, 2002; Muir, 2006). The major reason for the failure is proposed to be that the global treatment of NMDAR antagonists also block the physiological NMDAR activity, which activates several “prosurvival” signaling pathways and 60  Chapter One: Introduction exerts a neuroprotective function (Ikonomidou and Turski, 2002; Papadia and Hardingham, 2007a). Therefore the new concept of designing novel antiexcitotoxic drugs would be to block the excitotoxic consequences of NMDAR activation while leaving prosurvival signals intact.  5.4.2.  Neuropathic Pain  Neuropathic pain is a chronic pain state resulting from peripheral or central nerve injury, which often persists long after tissue injury has subsided. In the pain pathway, activation of NMDARs accentuates the sustained depolarization and contributes to an increase in the discharge of dorsal horn nociceptive neurons in a process called “windup”. The pursuit of an NMDA receptor antagonist for the relief of neuropathic pain dates from the late 1 980s, when it was shown that NMDA antagonists inhibit the “windup” response (Davies and Lodge, 1987; Dickenson and Sullivan, 1987). To date, the most optimistic results in terms of overall pain relief have been obtained with compounds that provide potent blockade of the NMDARs and readily penetrate the CNS (Chizh and Headley, 2005). For example, low doses of ketamine, an open-channel NMDAR blocker, markedly reduce chronic pain associated with spinal cord injuries (Eide et al., 1995). Other NMDAR antagonists, such as dextromethorphan, memantine, and amantadine, also reduce neuropathic pain in patients (Hewitt, 2000). However, these compounds failed to display widespread clinical use because of their psychotomimetic side effects (Childers and Baudy, 2007). Therefore, the search for effective therapeutic agents for neuropathic pain is still continuing, and the clinical trials with newer NMDA antagonists such as glycineB and NR2B-selective modulators are of great interest (Childers and Baudy, 2007).  6. Thesis Hypotheses and Objectives The hippocampal formation is a major target for cholinergic projections. Cholinergic stimulation has been shown to modulate a wide variety of cellular processes, both physiologically  and phathophysiologically.  Elucidating the mechanisms  that underlie  cholinergic-induced excitation may provide significant insight into not only the physiological functions of the brain, such as neuronal intrinsic bursting and rhythmic activity, but also pathological activities, such as epileptogenesis. Thus, the hypotheses of this dissertation were: 61  Chapter One: Introduction 1)  Cholinergic stimulation enhances R-type calcium currents and spikes in CAl  pyramidal neurons that contribute to hippocampal rhythmic activity. 2)  Rapid  membrane  translocation  of  TRPC5  channels  contributes  to  the  cholinergic-dependent plateau potential in hippocampal CA 1 pyramidal neurons and represents a novel target for therapeutic treatment of epilepsy. 3)  In hippocampal CAl pyramidal neurons cholinergic stimulation regulates the  properties of NMDA channels in an age-dependent manner.  In this dissertation, we will use a combination of research approches, including electrophysiology, molecular biology and biochemistry assays, pharmacology and two-photon microscopy, to address the above hypotheses, and the following objectives were generated: 1)  To identify the non-selective cation conductance and to characterize the downstream  mechanisms of the cholinergic-dependent plateau potential and their role in epileptogenesis. 2)  To examine the cholinergic modulation of different types of VGCCs in CA 1 pyramidal  neurons and in particular, to determine the cholinergic modulation of R-type currents and spikes and their role in hippocampal rhythmic activity. 3)  To examine the developmental pattern of the cholinergic modulation of NMDA  currents in CAl pyramidal neurons.  62  Chapter One: Introduction  7.  References:  Aarts M, Iihara K, Wei WL, Xiong ZG Arundine M, Cerwinski W MacDonald JF, Tymianski M (2003) Akey role for TRPM7 channels in anoxic neuronal death. 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J Neurosci 12:4510-4520.  97  Chapter One: Introduction Zhou  Q, Homma KJ, Poo MM (2004) Shrinkage of dendritic spines associated with long-term depression of  hippocampal synapses. Neuron 44:749-757.  98  Chapter Two: Muscarinic Modulation of R-type VGCCs  Chapter Two: Muscarinic Enhancement of R-type Calcium Currents in Hippocampal CAl Pyramidal Neurons.  *  1. Introduction R-type voltage sensitive Ca 2 currents were originally identified as the high voltage-activated (HVA)  2 currents Ca  that  were  “resistant”  to  the  antagonists,  co-conotoxin  MVIIC,  co-conotoxin-GVIA, 0)-agatoxin WA and the dihydropyridines (Zhang et al., 1993; Randall and Tsien, 1995).  In hippocampal CAl pyramidal neurons, R-type voltage-gated Ca 2 channels  (VGCCs) are highly expressed in distal dendrites (Christie et al., 1995; Magee and Johnston, 1995) and are thought to be primarily responsible for Ca 2 influx in dendrites and spines (Sabatini and Svoboda, 2000; Yasuda et al., 2003). R-type Ca 2 currents are involved in generating action potential bursts and afterdepolarizations (Magee and Carruth, 1999; Metz et al., 2005), and in the induction of synaptic plasticity (Isomura et al., 2002; Breustedt et al., 2003; Dietrich et al., 2003; Yasuda et al., 2003). Modulation of R-type Ca 2 currents could have profound impacts on dendritic excitability through modification of intrinsic firing patterns and the integrative properties of dendrites.  Brief  trains of back-propagating action potentials have been shown to depress Ca 2 entry through R-type VGCCs located in dendritic spines and thereby block theta-burst-induced long-term potentiation (LTP) (Yasuda et al., 2003). In expression systems, R-type Ca 2 currents due to recombinant Cav2.3 (alE) VGCCs (Piedras-Renteria and Tsien, 1998; Sochivko et al., 2002; Bannister et a!., 2004a) are stimulated by the activation of coexpressed muscarinic (Meza et al., 1999; Melliti et a!., 2000; Bannister et al., 2004a) or metabotropic glutamate receptors (mGIuR) (Stea et al., 1995). However, the modulation of R-type Ca 2 currents by muscarinic or metabotropic receptors has not been examined in native neurons in brain slices or in vivo.  Therefore, we used whole cell recordings to  examine whether Ca 2 currents due to R-type VGCCs are enhanced in hippocampal brain slices by muscarinic activation.  We show that both R-type Ca 2 currents and spikes are enhanced by the  stimulation of muscarinic receptors in CA 1 pyramidal neurons.  This is in striking contrast to the  *  A version of this chapter has been published. Tai C, Kuzmiski JB, MacVicar BA (2006) Muscarinic enhancement of R-type calcium currents in hippocampal CAl pyramidal neurons. JNeurosci 26:6249-6258. 99  Chapter Two: Muscarinic Modulation of R-type VGCCs extensively studied depression of N-, P-/Q- and L-type Ca 2 currents by activation of muscarinic receptors (Gahwiler and Brown, 1987; Shapiro et a!., 1999; Stewart et al., 1999; Shapiro et al., 2001). Furthermore, initiation of dendritic Ca 2 spikes has been suggested to play a role in generating or shaping neuronal network oscillations (Kamondi et a!., 1998b; Buzsaki, 2002). Interestingly, we found that muscarinic stimulation leads to remarkable and novel changes in the R-type Ca 2 spike firing pattern.  Following muscarinic receptor stimulation, enhanced R-type  2 spikes repetitively fired at theta frequencies (6-10 Hz), and blocking R-type VGCCs depressed Ca carbachol-induced spontaneous field potential theta oscillations, suggesting that enhanced R-type calcium spikes play a role in dendritic bursting and network oscillations.  Therefore, muscarinic  receptor activation in hippocampal neurons will profoundly alter dendritic integration and intrinsic resonance properties by shifting the normal pattern of Ca 2 entry from the slowly inactivating N-, P/Q- and L-type VGCCs to domination by the high voltage-activated, rapidly inactivating R-type VGCCs.  2. Materials and Methods 2.1. Hippocampal Slice Preparation. Hippocampal slices were prepared from Sprague Dawley rats aged postnatal days 13-16, according to standard procedures (Fraser and MacVicar, 1996). Our experiments were approved by the Canadian Council for Animal Care and the University of British Columbia Animal Care Committee. All experiments were conducted in strict accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Briefly, rats were deeply anesthetized with halothane and rapidly decapitated. The brain was quickly removed and horizontal hippocampal slices  (-- 400 jim) were cut with a vibratome (VT100; Leica, Willowdale, Ontario, Canada) in  chilled (0-4°C) slicing solution containing (in mM): 75 sucrose, 87 NaCI, 25 NaHCO , 25 3 D-glucose, 2.5 KC1, 1.25 , 4 P 2 NaH O 0.5 CaCl , 7.0 MgCl 2 , pH 7.3. Then slices were transferred to a 2 storage chamber with fresh ACSF containing (in mM): 126 NaC1, 2.5 KC1, 2.0 MgC1 , 2.0 CaCI 2 , 2 1.25 , 4 P 2 NaH O 26 NaHCO , and 10 D-glucose, pH 7.3, and incubated at room temperature for more 3 than 1 hour before recording. All solutions were saturated with 95% 02 and 5% CO . 2 100  Chapter Two: Muscarinic Modulation of R-type VGCCs  2.2. Whole-cell Patch-clamp Recordings Whole-cell voltage-clamp or current-clamp recordings from CAl neurons within hippocampal slices (Blanton et a!., 1989) were obtained at room temperature (22-24°C). Individual slices were transferred to a recording chamber located on an upright microscope (Axioskop; Zeiss, Oberkochen, Germany) and perfused rapidly with oxygenated ACSF (2 mI/mm). Patch electrodes (3-5 MQ) were pulled from 1.5 mm outer diameter thin-walled glass capillaries (150F-4; World Precision Instruments, Sarasota, FL) in three stages on a Flaming-Brown micropipette puller (model P-97; Sutter Instruments, Novato, CA) and filled with intracellular solution containing (in mM): 115 Cs-methanesulphonate, 25 TEA-Cl, 10 HEPES, 1.1 EGTA, 0.1 CaCl , 4 Mg-ATP and 0.5 2 Na-GTP, pH 7.2. Intracellular 2 {Ca was calculated to be 16 nM. In some experiments, BAPTA ] was substituted for EGTA, as described. In the BAPTA experiments stable whole cell recordings were maintained for more than 15-20 mm dendritic regions.  before recording to ensure that BAPTA diffused into  R-/T-type Ca 2 currents were pharmacologically isolated by preincubating slices  in a cocktail containing w-conotoxin MVIIC (2 tM), o-conotoxin-GVIA (2 jiM), and o -agatoxin WA (0.4 jiM) to block N-, P- and Q-type Ca 2 currents, and Cytochrome C (0.1 mglml) to block nonspecific toxin binding, for more than 1 hour at room temperature. Nifedipine (20 jiM) and tetrodotoxin (1.2 jiM; TTX) were bath applied to block L-type Ca 2 currents and Na currents, respectively. 2 mM CsCl and 1 mM 4-AP were also bath applied to block residue K channels. Membrane potentials and currents were monitored with an Axopatch 200B amplifier (Axon Instruments, Foster City, CA), acquired through a Digidata 1200 series analog-to-digital interface onto a Pentium computer using Clampex 9.0 software (Axon Instruments). Data were sampled at 10  kHz and most were low-pass filtered (four-pole Bessel) at 1 kHz.  Data were not filtered for tail  current analysis. For voltage-clamp recordings, leakage and capacitive currents were subtracted using a P/-4 protocol (four negative correction pulses, amplitude 1/4 of that of the test pulse). In current-clamp recordings, bridge balance and capacitance compensation were performed. Access resistance was continuously monitored, and only cells with access resistance <20 M2 were used.  101  Chapter Two: Muscarinic Modulation of R-type VGCCs 2.3. Extracellular Recording For extracellular recordings, slices were transferred to an interface chamber (32-34°C) (Fine Science Tools, Vancouver, BC). We recorded extracellular potentials with glass micropipettes filled with ACSF (1-3 M). Recording electrodes were positioned in the CA 1 pyramidal cell layer and signals were acquired using an A-M Systems amplifier (low filter, 1.0 Hz; high filter, 5.0 kHz; xl000) (Model 1800 Microelectrode, A-M Systems, Sequim, WA). Before spontaneous field activity was recorded, slice viability and stability was verified by recording evoked field EPSPs. Glass micropipettes filled with ACSF were used to electrically stimulate the Schaffer-Collateral pathway (0.03 Hz). Stable recordings (--20 mm) with a population spike that had peak-to-peak amplitudes of> lOmV were used.  2.4. Reagents TTX, o-conotoxin-GVIA and -agatoxin WA were purchased from Alomone Labs (Jerusalem, Israel), co-conotoxin MVIIC from Bachem (Torrance, CA), nickel (II) chloride from J.T. Baker (Paris, KY), pirenzepine, PP2, GF 103209x from Tocris (Ellisville, MO), BAPTA from Molecular Probes (Eugene, OR), Ro 3 1-8220, KN-93, Go 6976 from Calbiochem (San Diego, California) and all other reagents from Sigma (St. Louis, MO).  2.5. Data Analysis Data were analyzed using Clampfit 9.0 (Axon Instruments). In all cases, t tests were used for statistical comparisons with p  3.  <  0.00 1 considered significant. Values are reported as mean ± SEM.  Results  3.1. Carbachol Enhances Toxin-resistant High-Voltage-Activated (HVA) R-type But Does Not Affect Low-Voltage-Activated (LVA) T-type Ca 2 Currents. We tested the hypothesis that muscarinic receptor stimulation enhances R-type VGCCs in CA 1 pyramidal neurons similar to the muscarinic enhancement of recombinant Cav2.3 VGCCs described in HEK 293 cells (Melliti et al., 2000; Bannister et al., 2004a). We performed voltage-clamp 102  Chapter Two: Muscarinic Modulation of R-type VGCCs recordings to characterize the cholinergic modulation of the toxin-resistant Ca 2 currents, which were isolated with a cocktail containing TTX, nifedipine and VGCC toxins (as described in Methods). CA 1 pyramidal neurons from hippocampal slices were patch clamped with a Cs and TEA-based intracellular solution to suppress K channels and minimize muscarinic mediated effects on K channels.  The ACSF contained Cs and 4-AP to block potassium current and to  increase the space clamp efficiency. Under these conditions, we observed an enhancement of FIVA 2 currents. In the presence of the VGCC blocker cocktail, “toxin resistant” low (T-type) and high Ca (R-type) voltage-activated transient Ca 2 currents were recorded in all cells (Fig. 2. 1A).  The  classification of currents was based on the potentials for activation, toxin resistance and Ni 2 sensitivity. Only the HVA R-type current was dramatically enhanced following application of carbachol (Fig. 2.1B; n=13). This carbachol-mediated stimulation was reversible (Fig 1C; n6) and the currents were sensitive to 50 i1 2 (Fig. 2.lD; n=5). Strikingly, the I-V curve of the Ca M Ni 2 currents showed that the T-type component (observed as a shoulder in the IV at -60 to -30 mV) was not affected while the peak of the R-type current was dramatically enhanced in carbachol (Fig. 2.1 E). The averaged I-V curve before and after carbachol treatment was shown in Figure 2.2A (n7). The peak amplitudes of T-type and R-type currents are plotted in Figure 2.2B showing that T-type component did not change (99.6 increased significantly (154  ±  ±  2% of control; p=0.76; n10) whereas the R-type component  4% of control; p<O.0001; n13). The example traces for T-type and  R-type are shown in Figure 2.2C. The enhanced amplitude of the R-type Ca 2 current was consistently associated with a significant shift of the voltage-dependent activation curve to negative potentials, with a change in the voltage for half-maximal activation (Vact) (AVact -4.2  ±  0.7 mV;  p<O.OO1; n=7; Fig. 2.2D). The enhancement of R-type Ca 2 currents was reversible and blocked by atropine (1 jtM), demonstrating that it was due to muscarinic receptor activation by carbachol (Fig. 2.2E; n=6). Pooled data for muscarinic modulation of R-type and T-type VGCCs are shown in Figure 2F (for each group, n5). These results suggest that in CAl pyramidal neurons, muscarinic activation enhances only the HVA R-type Ca 2 currents but not the low voltage-activated (LVA) T-type Ca 2 currents. To verify further that T-type Ca 2 currents were not modulated by muscarinic activation, we first 103  Chapter Two: Muscarinic Modulation of R-type VGCCs  Figure 2.1. Carbachol enhances toxin-resistant HVA Ca 2 currents. Whole-cell patch clamp recordings were performed with a Cs and TEA based intracellular solution. Slices were preincubated with toxins (as described in Methods) and perfused with TTX (1.2 jiM) and nifedipine (20 jiM). (A) In voltage-clamp mode, a well-clamped Ca 2 current was recorded. (B) The current was enhanced by application of carbachol (CCH, 30 jiM). This enhancement was reversible (C) and sensitive to Ni 2 (50 jiM; D). (E) I-V relationships of this cell before and after treatment, showing that the T-type component (observed as a shoulder at -60 to -30 mV in the I-V) was not affected while the peak of the R-type current was dramatically enhanced in carbachol.  104  Chapter Two: Muscarinic Modulation of R-type VGCCs  A Control  B Carbachol  C Washout  D Nickel (TI  lOOms  E  Voltage (mV) -50  -25  0  25  0. 4-’  C-) -1  —0—Control -.-CCH Washout —ó—Nckel  105  Chapter Two: Muscarinic Modulation of R-type VGCCs  Figure 2.2. Muscarinic activation enhances R-type but not T-type Ca 2 current. (A-C) HVA but not LVA component was stimulated by muscarinic activation. The mean I-V relationships before and after carbachol treatment from 7 cells were shown in (A). The peak current amplitudes and sample traces are shown in (B) and (C). (D) The Ca 2 channel activation curve was shifted significantly to the left. The tail currents were normalized to the maxim um and plotted against the voltage of the step. The result was fit with a Boltzmann equation. (E) Time course of the peak HVA Ca 2 current showed that carbachol stimulation of R-type VGCC was reversible and blocked by the muscarinic receptor antagonist atropine (1 tM). (F) Mean data for the muscarinic modulation of the LVA and HVA Ca 2 current components. All recordings were obtained in TTX and VGCC blockers (as described in Methods). Asterisk (*) indicates significant change (p 0.001).  106  <  Chapter Two: Muscarinic Modulation of R-type VGCCs  A  :  I::  D  Voltage (mV) 0  Contro,,zAe  <0.50  ocntroi\j, 40  HVA  B  -30  LVA  2600  HVA  )  400  -10  0  10  Voltage (mV)  E 500  -20  2000  CCH  2000  CCH  Nickel  Atropine 300  1600  200  1000  100  500 w  r  \  Q. 1000  I  \  4%  .f  c’  c$’  0  .  I  I  10  20  30  I  I  40  50  Time (mm)  /ontro  lOOms  11  lOOms OmV I  I  -4OrnV  i  J  I  I  L  0  Z 0.0  •0  Z 0.0  107  Chapter Two: Muscarinic Modulation of R-type VGCCs blocked all HVA Ca 2 currents by perfusing 30 jiM Cd 2 instead of the strategy employed above using toxins and nifedipine. This is effective for isolating T-type Ca 2 currents, because the HVA 2 currents are much more sensitive to low concentrations of Cd Ca 2 (Ozawa et al., 1989; Mogul and Fox, 1991; Avery and Johnston, 1996; Huguenard, 1996). Under these conditions, a LVA component was observed with no apparent HVA component (Fig. 2.3A; n7). Application of Cd 2 (30 jiM) only slightly decreased T-type Ca 2 currents (91 while the R-type Ca 2 currents were abolished (11  ±  ±  6% of control; p=O.68; n=7; Fig. 2B,C),  7% of control; 0 p<O.0 0 1; n=7; Fig. 2.3B,D).  We then tested the modulation of 2 -Cd iso1ated T-type Ca 2 currents by muscarinic receptor activation. In agreement with our results described above we did not observe any significant alteration of the isolated T-type Ca 2 currents by carbachol (104  ±  7% of control; p=O.5 ; n=6; Fig. 7  2.3E,F,G). The averaged I-V curves for carbachol effects on T-type Ca 2 currents are shown in Figure 2.3F.  Application of Ni 2 (50 pM) suppressed this current by approximately 50% (n=5;  p<O.0001; Fig. 2.3E,G).  Since low micromolar concentrations of Ni 2 only block alH subunits but  not xlG or cdl (Kiockner et a!., 1999; Lee et al., 1999; Perez-Reyes, 2003), our results suggest the existence of multiple subunits of T-type VGCCs in CA 1 pyramidal neurons, which is consistent with previous in situ hybridization work (Talley et al., 1999). These results confirmed that in CAl pyramidal neurons, muscarinic activation had no effect on T-type Ca 2 currents.  3.2. Ml /M3 Cholinergic Receptors Mediate Muscarinic Stimulation of R-type VGCCs. The ability of atropine (1 pM) to block carbachol’s stimulation of R-type Ca 2 currents demonstrates that muscarinic receptors mediate this enhancement. In the hippocampus, four of the muscarinic subtypes (M1—M4) are expressed abundantly (Vilaro et al., 1993; Levey et a!., 1995; Rouse et al., 1999). To further determine the muscarinic receptor subtypes involved in the enhancement of R-type Ca 2 currents, we tested the antagonists and agonists for Ml -M4 cholinergic receptors. We found that both pirenzepine (1 pM; n5), a Ml specific antagonist and 4-DAMP (1 pM; n=4), an antagonist with equal affinity to both Ml and M3 receptors, could reverse the carbachol enhancement of R-type Ca 2 currents (Fig. 2.4 A,B,C). In contrast the M2/M4 antagonists, methoctramine (1 pM) and  tropicamide (1 pM) had no effect  (n=5; Fig. 2.4C).  To confirm the 108  Chapter Two: Muscarinic Modulation of R-type VGCCs  Figure 2.3. T-type Ca 2 current is not affected in carbachol. 2 (30 1 Cd iM) was used to isolate the LVA VGCCs. (A) Mean I-V curve of Cd -isolated Ca 2 2 current (n=7).  Mean data for Cd 2 effects in (B) show that the LVA component was slightly  decreased while HVA component was nearly diminished by Cd 2 (30 1 iM). Sample traces for LVA and HVA currents before and after Cd 2 treatment were shown in (C) and (D). The Cd -isolated 2 LVA Ca 2 current was not affected by carbachol (CCH) or during washout, but was partially blocked by subsequent treatment with Ni 2 (50 jiM; E). (F) Mean I-V curves for carbachol effects on the -isolated T-type Ca 2 Cd 2 currents (n=5). (G) Mean data for muscarinic modulation of Cd -isoIated 2 T-type VGCCs (for each group, n5). Asterisk (*) indicates significant change (p  <  0.00 1).  109  Chapter Two: Muscarinic Modulation of R-type VGCCs  A  B  Voltage(mV) 6040200  LVA  :1  4-,  t  L.  0 0  -0.5  N  Cu  EI.. 0  z  -1.0  HVA  °°  20  —o-Contro ---Cadrnium  ç&.  C)  D  C)  E 4. Nickel  / 2. Cadmium \tCadmium 1. Control 3. Washout CCH  g  Lms  ‘4,  0  g  lOOms OmV  -4OmV  F  lOOms  L G  Voltage (mV) -50  -25  0  -4OmV  1.26 25  4-’  1.00  =  0.75 0  -100  0.60  E 0  0.25  z  -200  urn -300  Washout CCI-l  C) *0  110  Chapter Two: Muscarinic Modulation of R-type VGCCs  Figure 2.4. M1/M3 musarinic subtypes mediate the stimulation of R-type 2 current. Ca (A-C) The Ml antagonist pirenzepine (1 .tM) reversed the stimulation of R-type VGCCs by carbachol (CCH).  I-V curves and sample traces are shown in (A) and (B). (C) Mean data for the  effects of pirenzepine (1 jtM), 4-DAMP (1 jiM; Ml /M3 antagonist), methoctramine and tropicamide (1 jiM; M2 and M4 antagonists) on the enhancement of R-type VGCCs. (D-F) Ml agonist McN-A-343 (100 jiM) mimicked and occluded the stimulation of R-type VGCCs by carbachol.  I-V curves and sample traces are shown in (D) and (E). (F)  Mean data for the effects  of McN-A-343 and the subsequent application of carbachol on R-type VGCC s.  All recordings  were obtained in TTX and VGCC blockers (as described in Methods). Asteris (*) k indicates significant change (p <0.001).  111  Chapter Two: Muscarinic Modulation of R-type VGCCs  A  Voltage (mV) -60  -40  D  -20  Voltage (mV) -60  -40  -20  0  20  -200 C  C)  -400 —0— Contro’  -600  -.-CCH Pirerizepine  3.CCH lOOms  OmV  -j C  -  QmV  JOmj D0.52  F C?  zQ 40  1  20  E C  -c  C LU  x  112  Chapter Two: Muscarinic Modulation of R-type VGCCs involvement of M1IM3 subtypes, we tested the Ml agonist McN-A -343 (100 j.tM). We found that McN-A-343 mimicked the carbachol-mediated enhancement of R-type 2 currents (n=6), and Ca when applied first McN-A-343 occluded further enhancement by the subsequent treatment of carbachol (30 jiM; n=4; Fig. 2.4D,E,F). Thus the muscarinic enhanc ement of R-type Ca 2 currents is mediated by Ml/M3 subtypes.  3.3. Muscarinic Modulation of R-type VGCCs Is Mediated by a 2 -C independent a PKC Pathway. In recombinant systems, the stimulation of Cav2.3 Ca 2 currents is dependent on phosphorylation mediated by a pathway coupled to a pertussis toxin-insensi tive Gut subunit (Gutqiii) (Bannister et al., 2004a). M1/M3 receptors couple to Gctq subunits to stimul ate phospholipase C13 1 (PLC), which initiates phosphatidylinositol (PIP ) turnover. This leads to the production of 2 diacylglycerol (DAG) and 3 -1P mediated Ca 2 release, which in turn activates protein kinase C (PKC). Basically there are three groups of PKCs, the Ca - and DAG-dependent isofonns (Group I), 2 the 2 -C independent a but DAG-dependent isoforms (Group II), and the atypical isoform s (Group III). We examined the signaling mechanisms underlying the enhancement of R-type 2 currents Ca by first testing the Ca 2 dependence and then by examining the sensitivity to different PKC inhibitors. High concentration of high-affinity Ca 2 chelator BAPTA (10 mM) was used in the pipette solution, which has proved sufficient to completely block carbac hol-induced plateau potentials or tail currents in our lab (Fraser and MacVicar, 1996; Kuzmiski and MacVicar, 2001). We found that 10 mM BAPTA could not prevent the carbachol enhancement of R-type VGCCs (Fig. 2.5A,D; n=5), suggesting it involves a 2 -C independent a pathway. To investigate the potential involvement of PKC in the enhancement of R-type VGCCs, we preincubated slices for more than 30 minutes with the broad-spectrum PKC antagonists GF103209x (10 1 iM; n5) or Ro 3 1-8220 (10 jiM; n5). In the presence of these PKC antagonists carbachol did not enhance R-type Ca 2 currents (Fig. 2.5B,E), suggesting that PKCs are involved in this modulation. Interestingly, in the presence of PKC inhibitors (GF 1 03209x or Ro 31-8220), a significant suppre ssion of R-type Ca 2 currents was observed by muscarinic stimulation (Fig. 2.5B,E). This inhibit ory effect probably results from the activation of pertussis toxin-sensitive G proteins coupled M2/M 4 receptors and Gfl’y 113  Chapter Two: Muscarinic Modulation of R-type VGCCs  Figure 2.5. Mechanisms underling muscarinic stimulation of R-type VGCCs. (A) 2 -Ca independence of muscarinic enhancement of R-type VGCCs. In a CAl pyramidal neuron recorded with 10mM BAPTA based intracellular solution, the R-type Ca 2 current was enhanced in carbachol (CCII).  Mean data for the effects of intracellular solutions containing either  1.1 mM EGTA or 10 mM BAPTA on the muscarinic enhancement of R-type VGCCs are shown in (D). (B and C) A 2 -Ca independent PKC pathway is involved in muscarinic modulation of R-type VGCC. (B) Preincubation of the broad spectrum PKC blocker, GF103209x (10 aiM) abolished the muscarinic stimulation of R-type Ca 2 currents and conversely, resulted in current depression. (C) After preincubation of the Group I PKC 2 (Ca dependent isoforms) inhibitor, Go 6976 (10 1 iM), muscarinic stimulation of R-type VGCCs was not affected. (E) Mean data for GF 1 03209x (10 jiM), Ro 3 1-8220 (10 jiM; broad spectrum PKC blocker), Go 6976 (10 jiM), KN-93 (10 jiM; CaMKII blocker), and PP2 (10 jiM; Src tyrosine kinase inhibitor) effects on muscarinic modulation of R-type VGCCs (for each group, n5). All recordings were obtained in TTX and VGCC blockers (as described in Methods). Asterisk (*) indicates significant change (p <0.001).  114  Chapter Two: Muscarinic Modulation of R-type VGCCs  IOmMBAPTA  A  Voltage (my)  ::  IOpMGF1O92O3x  B  Voltage (mV)  tro  .1200  1OpMGo6976  C -60  40-200  20 •  <  -260  Contro’  \r 2. CCH  controi\j  0 my 7Omj  D  pO27  E  idfln ; nfl 60  Co  I  50  CC  JI  cb  115  Chapter Two: Muscarinic Modulation of R-type VGCCs  subunit-mediated inhibition (Meza et al., 1999; Bannister et a!., 2004a). Since we observed a stable enhancement of R-type currents in normal conditions, we did not study this inhibitory effect further. -dependent) 2 To determine which group of PKCs is involved, we applied the specific Group I (Ca PKC inhibitor, Go 6976 (10 tM; n=5). Application of Go 6976 did not block the carbachol-mediated  enhancement  of  R-type  2 Ca  currents  (Fig.  2.5C,E).  /calmodulin-dependent protein kinase II (CaMXII) and Src-family signaling pathways have 2 Ca also been reported to be involved in some muscarinic-activated pathways.  However, we found that  M; n=6) and Src-kinase (PP2 lOjiM; n=5) had no effect on lO i inhibitors of CaMKII (KN-93 1 2 currents at concentrations that have reported to be effective muscarinic stimulation of R-type Ca in brain slices (Fig. 2.5E) (Zhao et al., 2004; Grishin et al., 2005; Huang et al., 2005). This pattern of sensitivity to PKC antagonists and insensitivity to BAPTA suggests the involvement of 21 currents. -independent PKCs in the muscarinic effect on R-type Ca 2 Ca  2 Spikes 3.4. Muscarinic Enhancement of R-type Ca Current-clamp recordings were also performed to characterize the cholinergic modulation of the 2 spikes. CAl toxin-resistant Ca  pyramidal neurons from hippocampal slices were also  patch-clamped with a Cs-based intracellular solution to suppress K channels and minimize muscarinic mediated effects on K channels. When recordings were obtained from CAl neurons in 2 spikes were 2 currents, large amplitude, prolonged plateau Ca TTX without blocking any Ca evoked by injection of brief (40 ms) intracellular current pulses (n=7; Fig. 2.6A,B). Carbachol 2 spikes consistent with a muscarinic depression of L-, N- and P/Q-type depressed the plateau Ca 2 spike. A significant portion of this calcium spike in VGCCs leaving only a transient HVA Ca 2 sensitive (n=4). These results 2 currents because it was Ni carbachol could be due to R-type Ca suggested that muscarinic receptor activation in hippocampal neurons could shift the normal pattern of Ca 2 entry from the slowly inactivating N-, PIQ- and L-type VGCCs to domination by the HVA, rapidly inactivating R-type VGCCs. 2 spikes, we isolated To rigorously delineate carbachol’s direct action on the toxin-resistant Ca them with a cocktail containing TTX,  nifedipine and toxins,  as shown in previous voltage-clamp 116  Chapter Two: Muscarinic Modulation of R-type VGCCs  Figure 2.6. R-type VGCC-dependent spikes are enhanced by carbachol. 2 spikes evoked by current injection before and after carbachol application. (B) (A) Ca 2 spikes in (A) before and after carbachol in TTX but no VGCC blockers. In Expanded view of Ca 2 spike remained. (C) Silent neuron in the presence of 1.2 iM TTX, 20 tM carbachol a HVA Ca nifedipine, co-conotoxin IvWIIC, co-conotoxin-GVIA and co-agatoxin [VA, as described in voltage-clamp experiments. (D) Activation of R-type VGCC-dependent spikes were observed only 2 (R-type VGCC blocker) depressed iM Ni after application of carbachol. (E) Application of 50 1 2 spikes. (F) Plot of V-I relationship before and after carbachol. Note carbachol-enhanced R-type Ca iM 2 (n=20), 20 1 2 spikes in carbachol. (G H) Effects of 50 lIM Ni the activation of R-type Ca nimodipine (L- and T-type VGCC blocker; nimo; n=5) and membrane depolarization to —60 mV (n=5) on the peak amplitude or the maximum rate of rise (dVmldtmax) of the carbachol-enhanced R-type Ca 2 spikes compared to spikes evoked in carbachol. Asterisk (*) indicates significant depression (p< 0.001).  117  00  a  a a a  •o .  •o  99  0  p  0  0 t\)  p  o  F%)  /  o  .  S  Nimo  I’) 0 I  -  0  0  .  0) 0 I  0) 0 I  o  0)  -1*  —  % Depression dVm/dtm  c  0)  % Depression Spike Amplitude  NI  0• 0O 0O  0  0)  9_9  cx.  -6OmV  9  o  3  ‘!  Gil  <-Ti  CD  C)  z  m  0  C)  1  a  C)  0  0  -‘  0 D  C)  C)  3  I\)  C) C) Cd)  CD  0  C  C  C)  C)  Cd)  C  H  CD  z..  Chapter Two: Muscarinic Modulation of R-type VGCCs experiments. Under these conditions, depolarization of neurons with intracellular current injection often led to the generation of a transient, HVA Ca 2 spike (n50169). The Ca 2 spike inactivated during the depolarizing command pulses consistent with a Ca 2 spike mediated by rapidly inactivating, HVA R-type Ca 2 currents (Randall and Tsien, 1997). However, even with strong current injection (0.5 nA) numerous cells (28%, n19/69) remained silent. As shown previously with current-clamp recordings (Kuzmiski et al., 2005), subsequent bath application of carbachol resulted in persistent enhancement of R-type Ca 2 spiking in both the spiking and initially silent neurons (Fig. 2.6C,D).  The initially silent neurons exhibited transient Ca 2 spikes in the presence  of carbachol (Fig. 2 .6C,D; n 19).  In neurons that initially showed an R-type Ca 2 spike, carbachol  p<O.0001;  induced an increase in spike amplitude (A AP  =  the threshold for spike activation  0.8 mV; p<O.000l; n50), and a reduction in the  (AAPTH  -6.7  ±  5.7  current injection required to evoke a spike (AI= -80  ±  ±  0.7 mV;  n50), a decrease in  12 pA; p<O.0001; Fig. 2.6F; n=50). These  enhanced spikes were blocked by the application of Ni 2 (50 jtM; n=20; Fig. 2.6E,GH), which at this concentration is selective for R- and T-type VGCCs. This enhancement was not caused by a muscarinic-mediated increase in input resistance (R) (control Rp 115.7 R 118.0  ±  ±  5.9 M vs. carbachol  6.1 M2; pO.’77; n=69). The application of high concentrations of nimodipine (20 jiM)  2 sufficient to partially block T-type VGCCs had no effect on the carbachol-enhanced HVA Ca spikes (% AAP 3.5  ±  2.6%; p=O.25; %  z  dVm/dtmax -5.2  ±  9.1 %; pO.38; n5; Fig. 2.6GH). In  addition, the Ca 2 spikes were only weakly voltage dependent when held at depolarized voltages (—70 mV to —60 mV)(% AAP 2.9  ±  4.8 %; pO.82; %  L  dVmldtmax -14.6 ± 10.6%; p=O.24; n5;  Fig. 2.6GH). Consistent with our voltage-clamp results, we concluded that R-type but not T-type 2 currents generated the toxin resistant Ca Ca 2 spikes.  2 Spikes Contributes to Carbachol-induced Theta Burst 3.5. Muscarinic Enhancement of R-type Ca Oscillations. The cholinergic system is implicated in the generation of theta both in vitro and in vivo (Buzsaki, 2002). During theta in vivo, HVA Ca 2 spikes oscillate rhythmically at theta frequencies in the dendrites of CAl pyramidal neurons and may contribute to current generation and amplification of 119  Chapter Two: Muscarinic Modulation of R-type VGCCs  theta (Kamondi et al., 1998b; Buzsaki, 2002). However, the VGCC subtypes that contribute to dendritic  oscillations  during  theta  are  unknown.  We  examined  the  possibility  that  muscarinic-enhanced R-type Ca 2 spikes can resonate at theta frequency and thereby contribute to 24 spikes, the generation of dendritic oscillations. To evaluate the firing frequencies of R-type Ca long (0.5-1.0 sec) depolarizing steps of current were injected in neurons in the presence of TTX, VGCC toxins and nifedipine as described above. Under these conditions cells were either silent or only a single spike could be evoked even with strong current injection (Fig. 2.7A).  After addition  of carbachol to the perfusate, R-type Ca 2 spiking was enhanced and now multiple spikes could be 2 spiking displayed a regular rhythm in the evoked (Fig. 2.7B). Interestingly, repetitive R-type Ca theta frequency range (6-9 Hz; n=11; Fig. 2.7C). Application of K channel blockers (4-AP 5mM and linopirdine, lOjiM) failed to mimic this repetitive firing pattern (n=3; Fig. 2.7D), suggesting that the ability of the R-type Ca 2 spikes to fire at theta frequency is due to direct actions on R-type VGCCs. To avoid the generation of plateau potentials (Fraser and MacVicar, 1996), our recordings were limited to  <  2 spiking could Is. These findings indicate that carbachol-enhanced R-type Ca  contribute to the intrinsic resonant depolarizations of CA 1 pyramidal neurons in the theta frequency. To examine the potential contribution of muscarinic enhanced R-type VGCCs to theta burst oscillatory activity, spontaneous extracellular events were also recorded from area CAl. In the majority of naive hippocampal slices, theta frequency oscillations were generated after continuous bath application of carbachol (Fig. 2.8A). Field potential recordings showed a peak power spectra 0.0071 mV IHz at a mean frequency of 8.3 2  ±  0.6 Hz (n=8; Fig. 2.8B). The spontaneous  oscillatory bursts occurred at regular intervals of 101.4  ±  10.9 s (n=8) and lasted for 21.4  of 0.016  ±  ±  2.6 sec.  Theta burst oscillations closely resembled those previously described (Bland et al., 1988). 2 depressed the oscillatory activity as shown by Subsequent application of a low concentration of Ni an almost complete abolition of the power in the theta frequency range (n8/8; Fig. 2.8A,B). In 5/8 2 reduced the slices, the oscillatory bursts were completely abolished. In the other three slices, Ni amplitude of the theta burst oscillations and increased the interburst interval without significant effects on burst durations (19.3  ±  2.8 sec; n=3) or the frequency of the oscillations (8.3  ±  0.4 Hz).  The disruption of the theta bursts was often accompanied by a change in the field events to a slower, 120  Chapter Two: Muscarinic Modulation of R-type VGCCs  2 spikes fire repetitively at theta frequency. Figure 2.7. Muscarinic enhanced R-type Ca (A) R-type Ca 2 spike evoked by injection of a long (1.0 s) depolarizing current pulse. (B) Repetitive R-type Ca 2 spiking evoked during a long current pulse following application of 2 spikes after carbachol carbachol. (C) Instantaneous firing frequency of repetitive R-type Ca treatment (n=1 1). (D) Application of 5 mM 4-aminopyridine (K channel blocker; 4-AP) and 10 tM 2 spiking (n=3). linopirdine (M-current blocker) failed to induce the repetitive R-type Ca  121  Chapter Two: Muscarinic Modulation of R-type VGCCs  A  B  Control  Carbachol  2OmV 0.25  D  C  Linopirdine  12-  c,) C  .  +  S  N  10-  ci,  8-  ci)  6-  >, 0’-’ C  cts Ca)  4-  0 C  2-  0  0-  I 2OmV 0.25 I  2  I  I  4 3 Spike Number  S  5  122  Chapter Two: Muscarinic Modulation of R-type VGCCs  Figure 2.8. Carbachol-induced theta burst oscillations observed with extracellular recordings of field potentials are Ni 2 sensitive. (A) Extracellular recordings in area CAl of spontaneous oscillatory bursts induced by perfusion . The solid bar indicates the 2 with 40 jiM carbachol before and after application of 100 jiM Ni application of Ni . (a) Expanded trace from (A) showing a spontaneous burst activated in carbachol. 2 The arrow in (A) marked a indicates the location of the burst. (b) Expanded trace of the field 2 indicated by the arrow in (A) marked b. (B) Mean potential recording after application of Ni power spectra of the field potential oscillations in carbachol showing the peak at theta frequency is abolished by Ni 2 (n8).  123  Chapter Two: Muscarinic Modulation of R-type VGCCs  a  A  b Nickel  II h  a  ‘r’  1 U LIj jj,j U U u  r rj H H U I  “  j  I I [1  [Ti ‘•!  i  ‘  5 mm  B  Carbachol  80  N  Carbachol 6 .A”  >  2.Os  Nickel  J ‘  ‘it  E  a)  a-  .dIi ‘  i..  1 0.5 mV  1:45’O Frequency (Hz)  124  Chapter Two: Muscarinic Modulation of R-type VGCCs more interictal-like pattern. Since we demonstrated that T-type VGCCs are not affected by cholinergic stimulation, these results suggest that Ni 2 suppressed carbachol-induced spontaneous field potential theta oscillations by depressing R-type VGCCs. Therefore, our results indicate that in CA 1 pyramidal neurons enhancement of R-type VGCCs could contribute to muscarinic-induced theta frequency oscillations.  4. Discussion Our results show that muscarinic activation stimulates R-type Ca 2 currents in hippocampal 2 spikes. CA 1 neurons which can lead to de novo activation of R-type dependent Ca  This  tindependent PKC pathway because it is blocked by 2 enhancement is likely mediated through a Ca tdependent PKC, and it is 2 broad spectrum inhibitors to PKC, but not by an inhibitor of Ca 2 by BAPTA. In contrast to the enhancement of the insensitive to chelation of intracellular Ca 2 currents by muscarinic receptor activation. R-type current, we found no modulation of T-type Ca 2 spikes Furthermore, we show that the muscarinic-mediated enhancement of transient R-type Ca 2 spikes and could contribute to theta result in remarkable changes in the firing pattern of R-type Ca oscillations.  4.1. Mechanisms Underling Muscarinic Enhancement of R-type VGCCs. The R-type Ca 2 currents in hippocampal neurons are due to Cav2.3 subunits (Sochivko et al., 2002; Sochivko et al., 2003).  In recombinant systems Ca 2 currents due to expressed Cav2.3  subunits are enhanced by Gaq,i i-coupled muscarinic receptor activation (Bannister et al., 2004a; Kamatchi et al., 2004).  2 currents in Here we show a similar enhancement of R-type Ca  hippocampal neurons that was mediated by stimulation of muscarinic M1/M3 receptors. Hippocampal pyramidal neurons express high levels of postsynaptic Ml and M3 receptors (Vilaro et al., 1993; Levey et al., 1995; Rouse et al., 1999).  These receptors are Ga,ii coupled and their  activation results in the generation of DAG and 1P3 through PLC activation. DAG and 1P3 in turn activate PKC and IP R pathways, respectively. In agreement with previous work done on HEK cells 3 and Xenopus oocytes (Bannister et al., 2004a; Kamatchi et al., 2004), we found that in hippocampal 125  Chapter Two: Muscarinic Modulation of R-type VGCCs  CAl neurons, muscarinic stimulation of R-type VGCCs was independent of intracellular Ca 2 and tindependent Group II PKC(s). All three groups of PKCs are expressed in 2 required activation of Ca rat hippocampus (Naik et al., 2000). It is possible that PKC 6 from among the Group II PKCs ( 6, ‘t and 0) is the isoform of PKC involved.  PKC 6 is highly expressed in rat hippocampal  CAl pyramidal neurons (McNamara et al., 1999; Tang et al., 2004) and in response to muscarinic stimulation PKC 6 is activated and translocated to plasma membrane (Brown et al., 2005). In recombinant systems, coexpression of the regulatory domain of PKC 6 (a dominant-negative) blocked muscarinic stimulation of Cav2.3 subunits, while the blocker of PKC  could not  (Bannister et al., 2004a). However, we could not definitively rule out the contribution of other 2 isoforms. Interestingly, in the presence of PKC inhibitors, we observed a suppression of R-type Ca currents by muscarinic stimulation. This suggested the existence of other inhibitory pathways, which most likely result from the activation of pertussis toxin-sensitive G proteins coupled M2/M4 receptors and Gfry subunit-mediated inhibition (Meza et al., 1999; Bannister et al., 2004a).  4.2. R-type Verses Other VGCC Types. The muscarinic enhancement of R-type VGCCs is strikingly opposite to the depression of all the other HVA VGCCs (N-type, P-/Q-type, and L-type) by muscarinic receptor activation (Gahwiler and Brown, 1987; Shapiro et al., 1999; Stewart et al., 1999; Shapiro et al., 2001). With pharmacological and high-speed fluorescence imaging techniques, Christie et al. (1995) have shown that in the soma and basal dendrites  (  50 tm) all the HVA VGCCs as well as LVA VGCCs  2 entry, while in apical dendrites contribute to spike-triggered Ca  ( 100  2 p.m) T-type and Ni  2 entry (Christie et al., 1995). sensitive R-type VGCCs predominantly undergo spike-triggered Ca Single channel analysis has confirmed the pattern of channel distribution: all VGCC types were recorded in the soma, while in dendrites T- and R-type VGCCs were the predominant types (Magee and Johnston, 1995). On the basis of voltage dependence and pharmacological sensitivity, it has been proposed that action potential or depolarization-induced Ca 2 influx in dendrites and spines is predominantly mediated by R-type VGCCs (Sabatini and Svoboda, 2000; Yasuda et al., 2003). The distinct distribution in apical dendrites and the unique modulation of R-type VGCCs suggested that 126  Chapter Two: Muscarinic Modulation of R-type VGCCs they play a different role and underlie distinct cellular functions from other types of VGCCs, such as synaptic integration and plasticity. Interestingly, despite the general suppression of Ca 2 currents, muscarinic activation also induced a paradoxical increase in intracellular Ca 2 accumulations in the dendrites and spines responding to depolarization or synaptic stimulation (Muller and Connor, 1991, 1992; Tsubokawa and Ross, 1997; Beier and Barish, 2000). The blockade of K conductances and the P3-mediated release of Ca 2 from intracellular stores are thought to contribute to this accumulation (Muller and Connor, 1991, 1992; Tsubokawa and Ross, 1997; Beier and Barish, 2000). However, our results 2 currents might also significantly contribute to the suggest that the stimulation of R-type Ca 2 accumulations. muscarinic mediated intracellular Ca All three T-type VGCC subunits (cdG ctlH and 1I) are expressed in hippocampal pyramidal neurons (Talley et al., 1999). However their modulation and functional impact on hippocampal pyramidal neurons are not yet known. Compared to HVA channels, T-type VGCCs are more metabolically stable and are less likely to be modulated (Huang et al., 2005). Muscarinic activation 2 currents, depending on the cell has been reported to increase, decrease or not affect the T-type Ca type and experimental conditions (Yunker, 2003). There are three subunit types of T-type VGCCs and the possible differential modulation of each subunit is still unknown. In the present study we show that T-type Ca 2 currents in hippocampal pyramidal neurons are not affected by muscarinic stimulation. A major effect of muscarinic stimulation is the suppression of K channels, such as M channels (Brown and Adams, 1980; Halliwell and Adams, 1982), which enhance the excitability of neurons. 2 currents is not likely due to K However in the present study, the enhancement of R-type Ca channel depression. High concentrations of potassium channel blockers were applied both intracellularly and extracellularly (see “Methods”) and most K currents were observed to be 2 (1 mM), muscarinic activation 2 currents were first blocked with Cd blocked. When the HVA Ca did not affect the residue K currents (data not shown).  2 In addition, the repetitive R-type Ca  spike firing we observed in carbachol was not elicited with K channel inhibitors. Furthermore, T-type Ca 2 currents were found to be unaltered by carbachol application. Under normal 127  Chapter Two: Muscarinic Modulation of R-type VGCCs physiological conditions, it is likely that the suppression of K channels and enhancement of R-type VGCCs both contribute to the muscarinic stimulation of neuronal excitability. A concern for this type of study is the possibility of poor voltage control during voltage clamp experiments. To achieve well clamped currents, high concentrations of K channel blockers were applied both intra- and extracellularly (Colino and Halliwell, 1993) and younger animals (13-16 days) were used so that calcium currents would be smaller and dendritic processes less extensive. Also, large electrode tips were employed (3-5 M-ohm) and only cells with access resistances less than 20 M-ohm were included in the study. The voltage clamp appeared to be sufficient in most experiments because there was a graded turn-on of the currents, and channel activation properties of the currents are similar to those observed in cultured neurons or recombinant systems (Bannister et al., 2004a).  4.3. R-type Spikes and Theta Oscillations. Muscarinic receptor stimulation in the hippocampus in vitro generates robust oscillations, which share a common frequency with theta rhythm observed in vivo. The activation of oscillatory intrinsic conductances contribute importantly to theta current generation (Buzsaki, 2002). Voltage-dependent oscillations have been described in the somata (Leung and Yim, 1991) and dendrites of pyramidal neurons (Kamondi et al., 1 998b). During theta induction, the somatic membrane hyperpolarizes, whereas dendrites depolarize. When the dendritic depolarization is 2 spike dependent sufficiently strong, the resonant property of the membrane leads to a HVA Ca self-sustained oscillation in the theta frequency range (Kamondi et al., 1998b). Nickel ions have been shown to selectively abolish dendritic calcium spikes through blocking R- and/or T-type VGCCs (Gillies et al., 2002; Isomura et al., 2002). Here we have demonstrated that -sensitive R-type Ca 2 2 spikes can fire repetitively at theta frequencies. carbachol-enhanced, Ni Given that T-type VGCCs are not affected by carbachol, our results suggest that R-type VGCCs contribute to the amplification of the theta oscillations. Based on pharmacological sensitivity, two types of theta could be distinguished: atropine-sensitive and atropine-resistant (Kramis et al., 1975; Buzsaki, 2002). Our results show that 128  Chapter Two: Muscarinic Modulation of R-type VGCCs 2 blocked carbachol-induced spontaneous theta burst oscillations. This is in agreement with a Ni previous study that demonstrated atropine-resistant theta oscillations in response to mGluR stimulation were also sensitive to Ni 2 (100 .tM) (Gillies et al., 2002). During the oscillations, tdependent spikes were generated in the distal dendrites at theta frequencies. Interestingly, we 2 Ni found that the mGluR agonists trans-ACPD (50 iM) and DHPG (50 riM) resulted in enhanced R-type Ca 2 spiking similar to that observed with carbachol (data not shown) (Kuzmiski and MacVicar. Soc. Neurosci Abstr. 258.11, 2003). These results suggested that a similar contribution of R-type VGCCs to both atropine-sensitive and resistant forms of theta might exist. In the hippocampus, Ca 2 spikes are generated during highly synchronous excitatory input that is associated with behaviour in vivo. The ability of carbachol enhanced R-type Ca 2 spikes to resonate at theta frequencies suggests that it is an intrinsic property of the neuronal membrane that plays a key role in the generation of neuronal network oscillations. The resonance property of R-type VGCCs may also be important for synaptic plasticity induced by theta burst stimulation (Ito et al., 1995), the enhanced induction of theta during cholinergic stimulation (Huerta and Lisman, 1993) and the formation of accurate spatial memory (Kubota et al., 2001).  129  Chapter Two: Muscarinic Modulation of R-type VGCCs  5.  References:  Avery RB, Johnston D (1996) Multiple channel types contribute to the low-voltage-activated calcium current in hippocampal CA3 pyramidal neurons. J Neurosci 16:5567-5582.  Bannister RA, Melliti K, Adams BA (2004) Differential Modulation of CaV2.3 Ca2+ Channels by G {alpha} q/l 1-Coupled Muscarinic Receptors. Mo! Pharmacol 65:381-388.  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J Neurosci 24:76-84.  136  Chapter Three: Muscarinic Modulation of TRPC5 Channels  Chapter Three: Translocation of TRPC5 Channels Contributes to the Cholinergic-Induced Plateau Potentials. 1.  *  Introduction Acetyicholine acting on muscarinic receptors induces a remarkable transformation of the  electrophysiological properties and spiking activity of hippocampal pyramidal neurons. In addition to the well-known depression of potassium channels (Delmas and Brown, 2005; Hemandez et al., 2008) and most high threshold calcium channels (Gahwi!er and Brown, 1987; Shapiro et al., 1999; Shapiro et al., 2001), there is profound enhancement of R-type calcium channels (Bannister et a!., 2004b; Tai et al., 2006) and a calcium-activated nonselective cation current (Fraser and MacVicar, 1996; Kuzmiski and MacVicar, 2001; Lee et al., 2003; Takai et al., 2004). The non-selective cation current causes a prolonged depolarization called a plateau potential (PP) that likely contributes to the generation of seizure activity (Dichter and Ayala, 1987; Fraser and MacVicar, 1996). Previously we showed the contribution of cyclic-nucleotide gated channels to the later phase of PPs but there was a substantial cation current that was unidentified (Kuzmiski and MacVicar, 2001).  In this  paper, we studied muscarinic activation and regulation of a TRPC5 channel (Ramsey et a!., 2006; Venkatachalam and Montell, 2007) in CAl pyramidal neurons using acutely prepared hippocampal slices to see if this channel contributes to the nonselective cation current underlying the PP. There are 7 types of TRPC channels (TRPC1-7) that as a family are considered “receptor-operated channels” due to their activation by G-protein-coupled receptors (GPCRs), especially Gctqiii group, and tyrosine kinase receptors (Li et al., 1999; Monte!!, 2001; Strubing eta!., 2001; Minke and Cook, 2002). Recent work indicated that regulated vesicular trafficking mechanisms might play a critical role in the activation of these channels (Monte!l, 2005; Ambudkar, 2007). In cultured hippocampal pyramidal neurons growth factors have been shown to induce P13K-dependent vesicular trans!ocation of TRPC5, thereby rapidly and dramatically increasing membrane expression of TRPC5 channels and functional TRPC5 currents (Greka et al., 2003; Bezzerides et a!., 2004). Other studies have reported regulated vesicular trafficking of TRPC 1  A version of this chapter has been submitted for publication. Tai C, Hines DJ, Choi HB, MacVicar BA (2009) Translocation of TRPC5 channels contributes to cholinergic-induced plateau potentials. *  137  Chapter Three: Muscarinic Modulation of TRPC5 Channels (Mehta et al., 2003), TRPC3 (Singh et al., 2004), TRPC4 (Odell et al., 2005) and TRPC6 (Cayouette et al., 2004) in other systems in response to different stimuli. TRPC5 is abundantly expressed in the brain (Freichel et al., 2004; Ramsey et al., 2006), and forms both homomeric (TRPC5) and heteromeric (TRPC1/TRPC5) channels, each with distinct electrophysiological properties and subcellular distributions (Strubing et al., 2001), but only homomeric TRPC5 channels displayed rapid vesicular translocation following growth factor stimulation (Strubing et al., 2001; Bezzerides et al., 2004). In cultured neurons, TRPC5 insertion and TRPC5-mediated Ca 2 influx is an important determinant of hippocampal neurite outgrowth rates and growth cone morphology (Greka et al., 2003; Bezzerides et al., 2004). In the current study, we report that the muscarinic-induced translocation and activation of TRPC5 channels plays a key role in generating cholinergic-induced PPs in CAl pyramidal neurons, through a CaM- and P13K-dependent pathway.  Our results  identify the TRPC5 channel as a novel player in the generation of PP and suggest that TRPC5 trafficking to the membrane may contribute to the neurotransmitter-induced epileptiform discharges that are observed in seizures and epilepsy.  2.  Materials and Methods  2.1. Hippocampal Slice Preparation. Hippocampal slices were prepared from Sprague Dawley rats (age postnatal day 14-20), according to standard procedures (Fraser and MacVicar, 1996). Our experiments were approved by the Canadian Council for Animal Care and the University of British Columbia Animal Care Committee. All experiments were conducted in strict accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Briefly, rats were deeply anesthetized  with halothane and decapitated. The brain was quickly removed and horizontal hippocampal slices  (—  400 jim) were cut with a vibratome (VT 100; Leica, Willowdale, Ontario, Canada) in chilled  , 25 D-glucose, 2.5 3 (0-4°C) slicing solution containing (in mM): 75 sucrose, 87 NaCl, 25 NaHCO , pH 7.3. Then slices were transferred to a storage 2 KCI, 1.25 4 PO 0.5 CaCI 2 NaH , , 7.0 MgCl 2 , 2 chamber with fresh ACSF containing (in mM): 126 NaCl, 2.5 KCI, 2.0 MgCI , 2.OCaC1 2 138  Chapter Three: Muscarinic Modulation of TRPC5 Channels 1.25 4 PO 26 NaHCO 2 NaH , , and 10 D-glucose, pH 7.3, and incubated at room temperature for more 3 than 1 hour before recording. All solutions were saturated with 95% 02 and 5% CO . 2  2.2. Whole-cell Patch-clamp Recordings Whole-cell voltage-clamp or current-clamp recordings were performed on CAl pyramidal neurons within hippocampal slices (Blanton et al., 1989). Individual slices were transferred to a recording chamber located on an upright microscope (Axioskop; Zeiss, Oberkochen, Germany) and perfused rapidly with oxygenated ACSF (2 ml/min). Patch electrodes were pulled from 1.5 mm outer diameter thin-walled glass capillaries (150F-4; World Precision Instruments, Sarasota, FL) in three stages on a Flaming-Brown micropipette puller (model P-97; Sutter Instruments, Novato, CA) and filled with intracellular solution containing (in mM): K-gluconate (115), KC1 (20), Na-phosphocreatine (10), N-2-hydroxyethylpiperazine-N’ -2-ethanesulfonic acid (HEPES; 10) and ethylene glycol-bis(-aminoethyl-ther)-N,N,N’ ,N’ -tetraacetic acid (EGTA; 1.1), CaCl 2 (0.1). pH 7.25. When filled with intracellular solution, patch electrode resistance ranged from 4 to 7 M2. In  voltage-clamp recordings, K-gluconate was replaced by Cs-methanesulphonate.  2.3. Two-photon calcium imaging. CA 1 pyramidal neurons in acute hippocampal slices were loaded with dyes during whole-cell patch recordings for at least 20 minutes before imaging. Measurements of calcium transients in the basal apical dendrites were taken at distances of 50-100 tm from the soma. We used a two-photon laser-scanning microscope (Zeiss-Axioskop-2 fitted with a 40X-W/0.8NA objective lens) directly coupled to a Ti:sapphire laser (Mira; Coherent) providing 100 fs pulses at 80 MHz and pumped by a 5W laser source (Verdi; Coherent). The wavelength of the laser was set at 810 nm. The iM) and Alexa-594 (red, 20 jiM) were detected fluorescence from Fluo-5F (green, 200 1 simultaneously with 2 photomultiplier tubes in separate fluorescence channels. EGTA was not included in intracellular solutions with the calcium indicator dye. Linescan fluorescence measurements of the basal apical dendrites were performed at 50 Hz. We calculated the area under  139  Chapter Three: Muscarinic Modulation of TRPC5 Channels the fluorescence measurements obtained with the linescan traces to quantify the muscarinic-induced elevation in [Ca ] and the effect of the TRPC antagonists. 2  2.3. Biotinylation of Surface Protein and Western Blotting. Hippocampal slices were treated with carbachol (CCH, 30 tM) in the presence or absence of inhibitors and then were cooled to 4°C and labeled for 45 mm with sulfo-NHS-SS-Biotin (1 mg/mI, Pierce, Rockford, IL).  Non-reacted biotinylation reagents were quenched using quenching buffer  for 10 mm and subsequently washed three times with cold ACSF and collected in lysis buffer. The tissue homogenates were then centrifuged at 13,000 x g (5 mm, 4 °C) to remove cellular debris, then protein concentrations of the lysates were determined by performing a Bradford assay with the DC Protein Assay dye (Bio-Rad, Mississauga, ON, Canada).  Cell lysates were incubated  overnight with streptavidin beads (500 jtg protein), washed two times and diluted with 1X Laemmli sample buffer and boiled for 5 mm.  The proteins were resolved in 10% polyacrylamide gel and  transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Cambridge, ON, Canada). The blots were blocked with 5% bovine serum albumin in TBST for 1 h, and the membranes were incubated overnight at 4 °Cwith primary antibodies against TRPC1, TRPC5, TRPC4 (rabbit polyclonal antibodies, 1:200 dilution, Alomone Labs, Jerusalem, Israel) or the transferrin receptor (Ta) which was used as surface membrane control to quantify the TRPC proteins (mouse monoclonal antibody TfR, 1:200 dilution, Zymed Laboratories, South San Francisco, CA). Following four washes with TBST, the membranes were incubated with the anti-rabbit or anti-mouse secondary antibodies conjugated to horseradish peroxidase (1 h, room temperature). The membranes were then washed 3-4 times (15 mm) with TBST, and proteins were visualized using enhanced chemiluminescence (ECL, amersham Bioscience, Arlington Heights, IL).  2.4. Data Analysis.  Data from electrophysiology experiments were analyzed using Clampfit 9.0 (Axon Instruments). Statistical analyses were performed using a two tailed Student’s t test or ANOVA where applicable, with p  <  0.01 considered significant. Values are reported as mean ± SEM. 140  Chapter Three: Muscarinic Modulation of TRPC5 Channels  3.  Results  3.1. Muscarinic Activation Induced Rapid Membrane Translocation of TRPC5 Channels. Bezzerides et al (2004) showed that growth factor stimulation could activate functional TRPC5 current through rapid vesicular translocation of TRPC5 channels to the plasma membrane. To test whether TRPC channels also display rapid translocation in brain slices in response to muscarinic stimulation, we carried out a biotinylation assay to analyze the membrane protein expression level of TRPC channels.  We found that the surface expression of TRPC5 channels was significantly  increased after the treatment with CCH (30 tM, 10 mm), (197± 9% of control; p <0.001; n8; Fig. 3.1A, B) compared to another membrane control protein, the transferrin receptor (TfR) which showed no changes in the same western blot membranes (Fig. 3.1A). Atropine (1 jiM), the muscarinic antagonist, inhibited the enhancement of TRPC5 surface expression by CCH (CCH, 197± 9% of control;  +  atropine, 121± 8% of control; p <0.001; n=5; Fig. 3.1A, B). TRPC5 proteins  generate channels by homomeric or heteromeric interaction with other TRPC channels, like TRPC 1 (Strubing et al., 2001). Interestingly, we only observed the enhancement of surface expression of TRPC5 by muscarinic stimulation, but not TRPC4 (94 (98  ±  6% of control; p> 0.2; n=4) (Fig 3.1C, D).  ±  4% of control; p> 0.2; n5), or TRPC1  Our data suggest that TRPC5 channels were  rapidly and selectively inserted into plasma membrane after muscarinic stimulation.  3.2. Cholinergic-Induced Plateau Potentials and Tail Currents are Inhibited by TRPC Channel Blockers. We tested the inhibitory actions of two TRPC antagonists to determine if there was a contribution of TRPC channels to the generation of the PPs and  hails.  As we previously reported  chohnergic stimulation in conjunction with Ca2+.influx in CAl pyramidal neurons from acute hippocampal slices could generate long-lasting inward tail currents  (hail)  under voltage-clamp  conditions (Kuzmiski and MacVicar, 2001) (Fig. 3.2A,B,C) or long-lasting depolarizations called plateau potentials (PPs) under current-clamp conditions (Fraser and MacVicar, 1996) (Fig. 3.2D,E,F).  We examined ‘tail at a holding potential of-70 mV after a voltage step to 0 mV for 800 141  Chapter Three: Muscarinic Modulation of TRPC5 Channels  Figure 3.1. Muscarinic stimulation triggered rapid membrane insertion of TRPC5 channel proteins. Biotinylation experiment is performed on acute hippocampal slices. (A) A representative western blotting result after biotinylation showing that CCH (30 tM, 10 mm) stimulation strongly enhanced membrane expression of TRPC5 channels. This enhancement is antagonized by muscarinic antagonist atropine (1 jiM), showing that the effect of CCH is dependent on muscarinic receptors. Transferrin receptor is also plotted as control, showing no change with CCH stimulation or atropine treatment. (B) Pooled data for the CCH-induced enhancement of membrane expression of TRPC5 proteins and atropine’s antagonism. (C) A sample western blotting result after biotinylation showing that CCH (30 jiM, 10 mm) and atropine (1 jiM) did not affect membrane expression of TRPC4 channels. Pooled data for TRPC4 are shown in (D). Asterisks (* *) indicate significant change (p  <  0.001) compared to Control, and pounds (##) indicate significant change  compared to CCH-treated group (p  <  0.00 1).  142  Chapter Three: Muscarinic Modulation of TRPC5 Channels  A  C  Biotinylation of TRPC5 channels  Biotinylation of TRPC4 channels a)  c5< C)  C)  TRPC5  TRPC4  L_  Transferrin R  Transferrin R  B ci) >  D  2.5  ri  .  2.5 ci  ci)  -J C  > ci) -J  **  .  ci  0  2.O  0 I  0  TRPC4  0  TRPCI  C-)  c a) .  a)  1.0•  N  z  1.0  z 0.5  0  0  Control  CCH  +  Atropine  /  0  C)0  cf  ,C) x  143  Chapter Three: Muscarinic Modulation of TRPC5 Channels  Figure 3.2. TRP channel antagonists significantly depressed muscarinic-induced PP;  ‘tail  and [Ca ] 2  elevation. (A) Typical responses of a hippocampal CA 1 pyramidal neuron under voltage-clamp conditions. In the absence of CCH (control), an 800 msec depolarizing voltage pulse to 0 mV from a holding potential of -70 mV resulted in unclamped Ca 2 currents. At the offset of the pulse, observed. (B) In the presence of CCH (30 1 iM), a long-lasting inward  ‘tail  ‘tail  was not  was induced at the offset  of the depolarizing voltage pulse under voltage-clamp conditions. (C) TRPC channel antagonist 2-APB (100 jiM) significantly inhibited the CCH-induced  ‘tail.  (D) Typical responses of a  hippocampal CAl pyramidal neuron under current-clamp conditions. In the absence of CCH (control), an 800 msec depolarizing current injection elicited robust action potential spikes, and the membrane potential immediately returned to baseline levels after cessation of the current pulse. (E) In the presence of CCH (30 jiM), identical stimuli resulted in a long-lasting PP. (F) TRPC channel antagonist 2-APB significantly inhibited the CCH-induced PP. (G) Typical responses of a hippocampal CAl pyramidal neuron illustrating the transient fluorescence increase that was evoked in basal apical dendrite by the depolarization illustrated in (J). (H) In the presence of CCH (30 jiM), identical stimuli resulted in a long-lasting elevation in [Ca ]. (I) TRPC channel antagonist 2-APB 2 significantly inhibited the CCH-induced enhancement of  j. (J) (1) 3D reconstruction of a 2 [Ca  recorded neuron from a Z series stack of two-photon images. (2)-(5) Linescan images (dashed line in (1)). Green fluorescence (Fluo-5F, (3) and (5)) and red fluorescence (Alexa-594, (2) and (4)) were recorded simultaneously. Arrows indicate the onset of the voltage step to OmV to evoke CCH. of  ‘tail.  (K) (Left) Pooled data for the area of CCH-induced  ‘tail  ‘tail  in  and the effects of 2-APB on the area  (Right) Pooled data for the CCH-induced enhancement of [Ca2+ ], and the effect of 2-APB  (normalized to control). (L) The area of CCH-induced  ‘tail  is calculated before and after the  application of 2-APB, and another TRP channel antagonist, SKF96365 (100 jiM). Asterisk indicates significant change (*,p <0.01; **,p <0.001).  144  Chapter Three: Muscarinic Modulation of TRPC5 Channels  A  C  Control  +  2-APB  pA  I sec OmV  D  OmV  OmV  L  -70  -70 rjj/f1  -70 mV  E  Control  -70 mV  -70  -70 mV  F  +CCH PP  +  2-APB  L!T” 2 sec  0.3 nA  0 nAIL  G  0.3 nA 0 nAJL  0 nA  H  Control  0.3 nA OnAJ]  0 nA  +CCH  OnA  +2-APB  LI C  L()  L  K  J  2 sec  1  Au I  I  I 4  —.-  U  a ‘°2sec  4-  0  C  OE C) C  -  3OpM  I  6 OmV -70 mVfl -70 mV  oxr_  0’’  (è  cy  x  C  w  145  Chapter Three: Muscarinic Modulation of TRPC5 Channels  msec before and after CCH application (30 tM) (Fig. 3 .2A, B).  ‘tails  were quantified by measuring  the area of the inward current after the end of the voltage command pulse, as described before (Kuzmiski and MacVicar, 2001). Consistent with our previous data (Kuzmiski and MacVicar, 2001), significant increases in ±  ‘tail  areas were induced by CCH (control, 0.035  0.119 nA sec;p <0.001; n  =  ±  0.01 nA see; CCH, 1.332  17) (Fig. 3.2A, B, K, L). Both TRPC channel antagonists, 2-APB  (100 jiM) and SKF96365 (100 jiM) (Boulay et al., 1997; Bootman et al., 2002; Wang et al., 2007), significantly depressed the generation of ‘tail (CCH, 1.332  ±  nA sec;p <0.001; n11) (Fig. 3.2C, K, L) (CCH, 1.332 0.147 nA see; p <0.001; n6) (Fig. 3.2K, L).  ‘tails  0.119 nA see;  ±  +  0.119 nA see;  2-APB, 0.433 +  ±  0.049  SKF96365, 0.5 11  ±  partially recovered after 10 minutes washout  (data not shown). Similarly, under current-clamp conditions, the prolonged PPs that we observed in the presence of CCH were depressed by the TRP channel blockers (Fig. 3 .2D, E, F; n6). TRPC channels are Ca -permeab1e non-selective cation channels. To confirm the Ca 2 -permeability of ‘tail, 2 we performed two-photon calcium imaging experiment on the basal apical dendrites of CA 1 pyramidal neurons on acute hippocampal slices (Yasuda et al., 2003; Kisilevsky et al., 2008). -sensitive dye (Fluo-5F, green) to monitor [Ca 2 Neurons were loaded with a low-affinity Ca J, and 2 -insensitive dye (Alexa-594, red) to show the morphology of the cell (Fig. 3.2J) (Yasuda et al., 2 a Ca 2003). Consistent with the electrophysiological results, we found that CCH (30 jiM) greatly enhanced the depolarization-induced Ca 2 influx (Fig. 3 .2H). This enhancement was significantly depressed by the TRPC antagonist 2-APB (100 jiM; Fig. 3.21, K) similar to the depression of the PP (Fig. IF) and  ‘tail  (Fig. 3.2C). Taken together, these pharmacological data strongly suggest that  TRPC channels contribute to the generation of the cholinergic-induced  ‘tail  and PPs, consistent with  their rapid insertion into plasma membrane after muscarinic stimulation.  3.3. Calmodulin Activity Is Required for the Generation of Cholinergie-Induced Tail Current as well as Membrane Translocation of TRPC5 Channels. We have previously reported that the muscarinic induction of PP and  ‘tail  requires an elevation  of intracellular Ca 2 concentration (Fraser and MacVicar, 1996; Kuzmiski and MacVicar, 2001). Interestingly, it is also shown that the calcium binding protein, calmodulin (CaM) is involved in the 146  Chapter Three: Muscarinic Modulation of TRPC5 Channels  Figure 3.3. The muscarinic-induced  ‘tail  is sensitive to a calmodulin inhibitor W-7 and a PI K 3  inhibitor wortmannin. (A) Typical responses of a hippocampal CA 1 pyramidal neuron under voltage-clamp conditions. In the absence of CCH (control), a depolarizing voltage pulse to 0 mV from a holding potential of -70 mV did not result in long-lasting inward Muscarinic-induced  ‘tail ‘tail  ‘tail  at the offset of the pulse. In the presence of CCH (30 tM), a  was induced at the offset of the depolarizing voltage pulse. (B)  is sensitive to calmodulin inhibitor W-7. Preincubation of W-7 (50 jiM) for  lh significantly depressed 30 jiM CCH-induced significantly inhibited the muscarinic-induced wortmannin on the area of CCH-induced  ‘tailS  ‘tail.  ‘tajI  (C) Preincubation of wortmannin (1 jiM)  (D) Pooled data for the effects of W-7 and  Asterisk (*) indicates significant change (p <0.001).  147  Chapter Three: Muscarinic Modulation of TRPC5 Channels  A  Control Control  +CCH  N  pA  tail  1 sec  W-7  B  +CCH  Control  N C  Wortman ni n +CCH  Control  N tail  QmV -70 myfl  -70 mV  OmV  -70 mV  -70 m(f1  D crj 1.5 C-) C) 1.0 C  **  0.5 **  1 .  0.0  c’. 00  148  Chapter Three: Muscarinic Modulation of TRPC5 Channels  Figure 3.4. The PI K inhibitor wortmannin and calmodulin inhibitor W-7 both significantly 3 depressed the muscarinic-induced rapid membrane insertion of TRPC5 channel proteins. Biotinylation experiment is performed on acute hippocampal slices. (A) A representative western blotting result after biotinylation showing that CCH (30 jiM, 10 mm) stimulation transiently enhanced membrane expression of TRPC5 channels, and that this enhancement is significantly depressed with the pretreatment of W-7 (50 jiM) or wortmannin (1 jiM). (B) Pooled data for the CCH-induced enhancement of membrane expression of TRPC5 proteins and the effects of W-7 and wortmannin. Asterisks (**) indicate significant change (p <0.001) compared to Control, and pounds (##) indicate significant change compared to CCH-treated group (p <0.001).  149  Chapter Three: Muscarinic Modulation of TRPC5 Channels  A  Biotinylation of TRPC5 channels  00  (j  TRPC5 Transferrin R  B ci) > a)  -J  a.)  **  2.0  4-  0  LO  C-,  1.5 ##  I  0  x )(  150  Chapter Three: Muscarinic Modulation of TRPC5 Channels  activation of muscarinic-induced activation of TRPC5 current (Kim et al., 2006; Shimizu et al., 2006; Kim et a!., 2007).  CaM-binding sites have been identified on the C-terminals of TRPC5  channels (Ordaz et al., 2005). The amplitude of TRPC5 currents activated by muscarinic stimulation is also increased by intracellular application of CaM, while decreased by CaM antagonists, such as W-7 (Kim et al., 2006). To determine whether CaM is involved in the generation of PP as well as the translocation of TRPC5 channels, we performed both patch clamping and biotinylation experiments on acute hippocampal slices after preincubation of W-7. We found that pretreatment with W-7 (50 tM) for more than 1 hr significantly inhibited muscarinic-induced 0.119 nA  sec;  +  W-7, 0.145  ±  ‘tails  (CCH, 1.332  ±  0.059 nA sec; n5;p <0.001; Fig. 3.3A, B, D), suggesting that  CaM activity is involved in the generation of PP and  ‘tail.  Consistent with the electrophysiological  recordings, pretreatment with W-7 also antagonized the muscarinic-induced enhancement of membrane expression of TRPC5 proteins (CCH, 197 p  <  ±  9% of control;  +  W-7, 117  ±  12% of control;  0.00 1; n=5; Fig. 3.4A, B), suggesting that CaM activity is also involved in the translocation of  TRPC5 channels. These data indicate that muscarinic-induced activation of CaM may contribute to translocation of TRPC5 channels and the generation of cholinergic PPs.  3.4. P1 3 Kinase Activity Is Required for Membrane Translocation of TRPC5 Channels as well as The Generation of the PPs. In their original report Bezzerides et al. (2004) also showed that P13K activity is required for the rapid translocation of TRPC5 to plasma membrane triggered by growth factor stimulation. To determine whether P13K activity is also involved in the muscarinic-induced TRPC5 translocation K inhibitor wortmannin (1 jiM) in both biotinylation 3 and PP we examined the effects of the PI experiments in brain slices and in patch clamp experiments in CAl pyramidal neurons. We found that pretreatment with wortmannin significantly inhibited muscarinic-induced 0.119 nA sec;  +  wortmannin, 0.388  ±  ‘tails  (CCH, 1.332  ±  0.114 nA see; n=8;p <0.001; Fig. 3.3A, C, D), suggesting  that P13K activity is involved in the generation of PP and  ‘tail.  In addition, pretreatment with  wortmannin also significantly inhibited the CCH-indueed enhancement of the membrane expression of TRPC5 channels (CCH, 197 ± 9% of control;  +  wortmannin, 93  ±  10% of control;p <0.001; n=5; 151  Chapter Three: Muscarinic Modulation of TRPC5 Channels Fig. 3.4A, B).  These data suggest that PI K activity is also involved in muscarinic-induced 3  translocation of TRPC5 leading to the generation of PPs.  4. Discussion In the present study, we report that muscarinic receptor activation triggers the membrane insertion of TRPC5 channels thereby profoundly altering the electrophysiological properties of hippocampal neurons in acute hippocampal slices.  The activation of TRPC5 currents after  insertion triggers prolonged inward currents and long-lasting depolarizations, which may play an important role in seizure-like discharges observed in epilepsy and stroke-induced seizures (Dichter and Ayala, 1987; Congar et al., 2000; Desmond et al., 2002). We conclude that TRPC5 channels are involved in generating PPs because of several observations. Firstly, antagonists of TRPC5 channels, 2-APB and SKF96365, significantly inhibited PPs and  ‘tails.  Secondly, TRPC5 channels and not  TRPC4 or TRPC 1 channels underlie this current because only TRPC5 displayed rapid membrane insertion in the biotinylation experiments. In addition, we identified the intracellular pathway linking the receptors and the channels: the activation of GcLq,ii-coupled muscarinic receptors increased intracellular Ca 2 concentration and subsequently the activity of CaM and P13K, leading to the rapid translocation of TRPC5 channels and the generation of PPs. tpermeable nonselective cation channels activated by GPCRs and 2 TRPC5 channels are Ca receptor tyrosine kinases. Previous reports have shown that TRPC5 channels can be activated by a number of different GPCRs, via Gaq or Ga 11 (Schaefer et al., 2000; Plant and Schaefer, 2003, 2005; Zhang et al., 2008). Hippocampal pyramidal neurons express high levels of postsynaptic Gaqiii-coupled Ml and M3 receptors (Vilaro et al., 1993; Levey et al., 1995; Rouse et al., 1999) and these receptors are also required for muscarinic-induced PPs (Fraser and MacVicar, 1996). Intracellular Ca 2 elevation is also required for receptor-mediated channel activation of TRPC5 (Schaefer et al., 2000; Strubing et al., 2001), as well as for the generation of PPs and  ‘tails  (Fraser  and MacVicar, 1996; Kuzmiski and MacVicar, 2001). Muscarinic activation dramatically increases intracellular Ca 2 accumulations in soma, dendrites and spines of neurons from depolarization or synaptic stimulation, by inhibiting K conductance, enhancing R-type voltage-gated calcium 152  Chapter Three: Muscarinic Modulation of TRPC5 Channels channels (VGCCs), and inositol 1 ,4,5-triphosphate (1P )-mediated Ca 3 2 release from intracellular stores (Muller and Connor, 1991, 1992; Tsubokawa and Ross, 1997; Beier and Barish, 2000; Tai et al., 2006). A target for intracellular Ca 2 is CaM, which has been reported to be required for the activation of TRPC5 current (Kim et al., 2006; Shimizu et al., 2006; Kim et a!., 2007). CaM also directly binds to and activates P13K (Joyal et al., 1997), which is required for the trans location TRPC5 channels in cultured pyramidal neurons (Bezzerides et al., 2004; Ambudkar, 2007). In addition both Ml and M3 muscarinic receptors have been reported to signal through P13K activation (Murga et al., 1998; Murga et al., 2000; Tang et al., 2002; Hirota et al., 2004). We report in this study that CaM and P13K activity are also required for the translocation of TRPC5 in hippocampal neurons in brain slices. The activation of muscarinic-induced PPs also required CaM and P13K activity consistent with our conclusion that the translocation of TRPC5 channels contributes to the generation of muscarinic-induced prolonged depolarizations. A recent report suggests that the activity of another GCLq -coupled receptor, the group I mGluRs, also triggers 111 trafficking of TRPC5 channels in CA3 pyramidal neurons, which may contribute to alterations of seizure discharges (Wang et al., 2007). TRPC5 is predominantly expressed in the central nervous system (CNS) (Freichel et al., 2004; Montell, 2005), and the transcripts and proteins of TRPC5 are especially abundant in hippocampus (Greka et al., 2003). TRPC5 generates both homomeric (TRPC5) and heteromeric (TRPC1/TRPC5) channels (Strubing et al., 2001; Montell, 2005; Schaefer, 2005). Although both TRPC1 and TRPC5 are expressed in the soma, dendrites and axons, TRPC1 is selectively excluded from synaptic structures, whereas TRPC5 is present in nascent synapses and growth cones (Strubing et al., 2001; Greka et al., 2003). The exact physiological roles of either homomeric or heteromeric TRPC5 channels are not clear yet. TRPC5 homomers might play a role in the regulation of the neurite length and morphology of growth cones of cultured hippocampal pyramidal neurons (Greka et al., 2003). In this study, we found that muscarinic stimulation triggered rapid membrane insertion of TRPC5, but not TRPC 1 channels into plasma membrane, suggesting that only homomeric TRPC5 display rapid translocation. This is consistent with previous evidence showing that only TRPC5 homomers, but not TRPC 1/TRPC5 heteromers, display rapid vesicular trans location following 153  Chapter Three: Muscarinic Modulation of TRPC5 Channels growth factor receptor stimulation (Bezzerides et al., 2004). These results indicate that homomeric TRPC5 channels contribute to the generation of the cholinergic-induced long-term depolarizations, indicating a novel physiological role for these channels. TRPC5 channels, and the translocation of these channels might be a novel therapeutic target for the treatment of epilepsy. The hippocampal formation receives cholinergic afferents mainly from the medial septum and the diagonal band (Wainer et al., 1993). This septohippocampal pathway plays a critical role in epileptogenesis, especially temporal lobe epilepsy (Friedman et al., 2007). Muscarinic stimulation is effective in generating limbic seizures and injection of cholinergic agents is used as a model for temporal lobe epilepsy (Lothman et al., 1991). A generalized seizure in the whole animal involves a prolonged depolarization, which is termed the tonic component of the ictal seizure (Dichter and Ayala, 1987). The muscarinic-induced long-term depolarization (PP or ‘tail) is an excellent candidate for the major intrinsic conductance underlying the prolonged depolarization observed during the ictal phase of seizures (Dichter and Ayala, 1987; Fraser and MacVicar, 1996; Kuzmiski and MacVicar, 2001). The identity of the conductance underlying PP has long remained elusive although there is a contribution of cyclic nucleotide gated channels (Kuzmiski and MacVicar, 2001). 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Wang M, Bianchi R, Chuang SC, Zhao W, Wong RK (2007) Group I metabotropic glutamate receptor-dependent TRPC channel trafficking in hippocampal neurons. J Neurochem 101:411-421.  Yasuda R, Sabatini BL, Svoboda K (2003) Plasticity of calcium channels in dendritic spines. Nat Neurosci 6:948-955.  Zhang C, Roepke TA, Kelly MJ, Ronnekleiv OK (2008) Kisspeptin depolarizes gonadotropin-releasing hormone neurons through activation of TRPC-like cationic channels. 3 Neurosci 28:4423-4434.  160  Chapter Four: Muscarinic Modulation of NMDA Receptors  Chapter Four: Cholinergic Modulation of NMDA Current In Hippocampal CAl Pyramidal Neurons Is Age-Dependent. 1.  *  Jntroduction The cholinergic system is one of the most important neuromodulatory neurotransmitter systems  in the CNS. Both animal and human studies indicate cholinergic system plays a key role in modulating neuronal excitability, synaptic plasticity and neuronal intrinsic properties (Jerusalinsky et al., 1997; Lucas-Meunier et al., 2003; Hasselmo and Giocomo, 2006). One of the predominant effects of cholinergic agonists on hippocampal CA 1 neurons is potentiation of currents through the ionotropic glutamatergic N-methyl-d-aspartate (NMDA) receptors (NMDARs) (Markram and Segal, 1990b, 1992; Marino et al., 1998; Lu et al., 1999), which play a central role in synaptic transmission and in mediating synaptic plasticity, learning and memory (Dingledine et al., 1999; Papadia and Hardingham, 2007a). A dysfunction of NMDARs or their regulation is implicated in diverse brain disorders (Lau and Zukin, 2007; Papadia and Hardingham, 2007a). The cholinergic potentiation of NMDAR could then provide a crucial mechanism by which cholinergic input modulates learning and memory, and provide novel therapeutic targets for the treatment of diverse brain disorders, such as stroke and Alzheimer’s disease. However, the mechanisms underlying the cholinergic modulation of NMDARs has long remained mysterious and controversial. Markram and Segal first discovered that ACh potentiates NMDA-evoked current (INA) (Markram and Segal, 1 990b), via the activation of Gctq coupled muscarinic receptors (Markram and Segal, 1 990b, a; Marino et al., 1998). This potentiation in hippocampal pyramidal CA 1 neurons was observed in acute slices (Markram and Segal, 1 990b; Marino et al., 1998), in dissociated cells (Lu et al., 1999), and also in other cell types such as striatal spiny neurons (Calabresi et al., 1998), and in auditory neocortical cells (Aramakis et al., 1999). Markram and Segal also first reported that the downstream pathway of the muscarinic potentiation of  ‘DA  -dependent pathway, while no protein 2 required activation of 1P 3 and intracellular Ca  kinase activity was needed (Markram and Segal, 1992). Tn contrast, several recent studies showed *  A version of this chapter will be submitted for publication. Tai C, MacVicar BA (2009) The Age-Dependence of the Cholinergic Modulation of NMDA Current In Hippocampal CAl Pyramidal Neurons. 161  Chapter Four: Muscarinic Modulation of NMDA Receptors that phorbol esters or muscarinic agonist could enhance  INMA,  surprisingly, via PKC and Src  tyrosine kinase activity (Lu et al., 1999; Kotecha and MacDonald, 2003; Salter and Kalia, 2004). Therefore the mechanisms underlying the cholinergic modulation of NMDA receptor activated currents remains still elusive. In the present study, we report a pronounced difference in the muscarinic modulation of ItDA in CA 1 pyramidal neurons at two different development stages. We found that muscarinic agonist carbachol potentiated  in both young (5-10 postnatal days) and old (6-8 postnatal weeks)  animals, but strikingly, via distinct downstream pathways. In young animals, muscarinic stimulation potentiated  ‘DA  through a [Ca -independent but PKC- and Src-dependent pathway. In contrast, 1 ] 2  in old animals, muscarinic stimulation potentiated  through a [Ca -dependent but 1 ] 2  PKC-independent pathway. Interestingly, the activity of the GcLçcoupled Mi-like muscarinic receptors was required for the potentiation of  in both young and old animals. Our results  indicate a novel developmental pattern of cholinergic modulation of  in CA 1 pyramidal  neurons involving different downstream mechanisms. These findings may have important implications in the induction and regulation of NMDA-dependent synaptic plasticity at different development stages, and may provide a crucial mechanism by which cholinergic input modulates learning and memory.  2. Materials And Methods 2.1. Hippocampal Slice Preparation Hippocampal slices were prepared from Sprague Dawley rats, aged postnatal 5-i 0 days (young animals) or postnatal 6-8 weeks (old animals), according to standard procedures (Fraser and MacVicar, 1996). Our experiments were approved by the Canadian Council for Animal Care and the University of British Columbia Animal Care Committee. All experiments were conducted in strict accordance with National Institutes of Health Guide for the Care and Use of Laboratory Animals. Briefly, the rats were anesthetized deeply with halothane and decapitated rapidly. The brain was removed quickly, and horizontal hippocampal slices (‘-400 j,tm) were cut with a vibratome (VT i 00, Leica, Willowdale, Ontario, Canada) in chilled (0-4°C) slicing solution containing the following (in 162  Chapter Four: Muscarinic Modulation of NMDA Receptors  PO4, 0.5 CaC1 2 mM): 75 sucrose, 87 NaCl, 25 NaHCO , 25 D-glucose, 2.5 KC1, 1.25 NaH 3 , and 7.0 2 , pH 7.3. Then the slices were transferred to a storage chamber with fresh artificial CSF 2 MgCl , 2.0 CaC1 2 , 1.25 2 (ACSF) containing the following (in mM): 126 NaC1, 2.5 KC1, 2.0 MgC1 PO4, 26 NaHCO 2 NaH , and 10 D-glucose, pH 7.3, and were incubated at room temperature for >1 3 h before recording. All solutions were saturated with 95% 02/5% Co . 2  2.2. Whole-cell Patch-clamp Recordings Whole-cell voltage-clamp recordings from CA 1 neurons within hippocampal slices (Blanton et al., 1989) were obtained at room temperature (22-24°C). Individual slices were transferred to a recording chamber located on an upright microscope (Axioskop, Zeiss, Oberkochen, Germany) and perfused rapidly with oxygenated ACSF (2 ml/min). Patch electrodes (3-5 M2) were pulled from 1.5 mm outer diameter thin-walled glass capillaries (1 50F-4, World Precision Instruments, Sarasota, FL) in three stages on a Flaming-Brown micropipette puller (model P-97, Sutter Instruments, Novato, CA) and were filled with intracellular solution containing the following (in mM): 140 , 4 Mg-ATP and 0.5 Na-GTP pH 7.2. Intracellular 2 K-gluconate, 10 HEPES, 1.1 EGTA, 0.1 CaC1 ] was calculated to be 16 nM. In some experiments BAPTA was substituted for EGTA, as 2 [Ca described. In the BAPTA experiments stable whole-cell recordings were maintained for >15—20 mm before recording to ensure that the BAPTA diffused into dendritic regions. Tetrodotoxin (TTX; 1.2 jiM) was bath applied to block Na currents. Membrane potentials and currents were monitored with an Axopatch 200B amplifier (Molecular Devices, Union City, CA), acquired via a Digidata 1200 series analog-to-digital interface onto a Pentium computer with Clampex 9.0 software (Molecular Devices). Data were sampled at 10 kHz, and most were low-pass filtered (four-pole Bessel) at 1 kHz. Access resistance was monitored continuously, and only cells with access resistance <20 Mf2 were used.  2.3. NMDA Current Induction For measurement of NMDA-evoked currents, NMDA (500 jiM) was pressure ejected from a low-resistance pipette positioned  100 jim from the soma of the recorded cells via a Picospritzer III 163  Chapter Four: Muscarinic Modulation of NMDA Receptors  (General Valve, Fairfield, NJ) pressure ejection device. NMDA-evoked currents were recorded from a holding potential of —60 mV and were completely blocked by AP5 (50 jiM). Stable inward NMDA currents (50—100 pA) were induced at 1 -mm intervals. The hippocampal slices were bathed in ACSF containing 1 jiM TTX. Percent potentiation was defined by using the ratio of maximum peak current amplitude during carbachol application to average current amplitude of three trials immediately preceding drug application (referred to as “baseline”). When testing the effects of different extracellular Ca 2 concentrations, the concentration of Mg 2 was maintained at 2 mM.  2.4. Reagents  TTX was purchased from Alomone Labs (Jerusalem, Israel); pirenzepine, protein phosphatase 2 (PP2), and 2-[ 1 -(3 -dimethylaminopropyl)indol-3 -yl]-3-(indol-3 -yl) maleimide (GF 1 09203x) from Tocris  (Ellisville,  MO);  BAPTA  from  Molecular  Probes  (Eugene,  OR);  3-[ 1—3-(amidinothio)-propyl- 1H-indol-3-yl]-3-( 1 -methyl- 1H-indol-3-yl)maleimide (Ro 31-8220) from Calbiochem (La Jolla, CA); all other reagents were purchased from Sigma (St. Louis, MO).  2.5. Data Analysis Data were analyzed with Clampfit 9.0 (Molecular Devices). In all cases Students t tests were used for statistical comparisons, with p  <  0.00 1 considered significant. Values are reported as the  mean ± SEM.  3.  Results  3.1. Muscarinic Stimulation Potentiates To study the muscarinic modulation of  in Young Animals. at different developmental stages, we performed  whole-cell voltage clamp recordings on hippocampal CAl pyramidal neurons in acute brain slices from both young (5-10 postnatal days) and old rats (6-8 postnatal weeks). Stable inward INiA (50—100 pA) were induced at 1 -mm  intervals at a holding potential of —60 mV using a pressure  ejection (puffing) system. Bath application of APV (50 jiM) completely blocked the induced currents, confirming the involvement of NMDARs (data not shown). 164  Chapter Four: Muscarinic Modulation of NMDA Receptors  Figure 4.1. Muscarinic stimulation potentiates  ‘A  in CAl pyramidal cells in young animals.  Bath application of CCh (20 jiM) induces a potentiation of currents evoked by NMDA application. (A) The time course of CCh-induced potentiation of  ‘‘fA  and the washout of this  effect. (B) Single traces obtained before CCh application, at the peak of CCh-induced potentiation, and after washout. (C) Pooled data for the CCh-induced potentiation of  and the effects of  atropine (1 jiM), pirenzepine (1 jiM), and methoctramine (1 jiM) on the enhancement  ‘NMtA.  Asterisk (**) indicates significant change (p <0.001).  165  Chapter Four: Muscarinic Modulation of NMDA Receptors  A I.  G)  I I  C.)  z 1.0 .  N  CCH  E I  0  Z 0.5  -5  0  5  10  15  Time (mm)  B Control  +CCH  Washout  C .1-I  C  a) I I..  z  0  z D  a)  1.0  N  E I 0  ZO.5  -  LH1  -  0 C) 166  Chapter Four: Muscarinic Modulation of NMDA Receptors  Figure 4.2. Muscarinic stimulation potentiates I, in old animals. Bath application of CCh (20 jiM) induces a marked potentiation of currents evoked by NMDA application. (A) The time course of CCh-induced potentiation of 1 r and the washout of this effect. (B) Single traces obtained before CCh application, at the peak of CCh-induced potentiation, and after washout. (C) Pooled data for the CCh-induced potentiation of  and the effects of  atropine (1 jiM), pirenzepine (1 jiM), and methoctramine (1 jiM) on the enhancement Asterisk (**) indicates significant change (p <0.001).  167  Chapter Four: Muscarinic Modulation of NMDA Receptors  A C) 2.0 1.5 N  to  EI  CCH  0  Z 0.5  -5  0  5  10  15  Time (mm)  B +CCH  Control  Washout  Vh4V VLA 5 sec  C a) I  Z  C.)  2  z .1. N Cu  E 0  zo. C  nfl  0  C,’, 0  168  Chapter Four: Muscarinic Modulation of NMDA Receptors  We first studied the muscarinic modulation of ‘A in young animals. muscarinic agonist, (141  ±  Bath application of a  carbachol (CCh, 20 1 .tM) produced a significant increase in  ‘A  amplitude  8% of control; Fig. 4.1A,B). This potentiation peaks at about 3 mm after the start of CCh  treatment and is reversible after 10 mm of washing (Figure 4.1A,B). The non-selective muscarinic antagonist, atropine (1 jiM) blocked the CCh-induced enhancement of  ‘NA,  demonstrating that  muscarinic receptors mediate this enhancement. Furthermore, we found that pirenzepine (1 jiM; n 5), a specific antagonist for Mi-like receptors, could reverse the CCh enhancement of 4.1 C). In contrast, the M21M4 antagonist methoctramine (1 1 iM) had no effect (n These results indicate that the muscarinic enhancement of  =  =  (Fig.  5) (Fig. 4.1 C).  is mediated by the Giqcoup1ed  Mi-like muscarinic receptors, consistent with previous studies using Mi specific toxins (Marino et al., 1998).  3.2. Muscarinic Stimulation Potentiates  ‘DA  in Old Animals.  We then studied the muscarinic modulation of application of CCh (20 jiM) strongly potentiated  ‘DA  ‘A  4.2A,B). This potentiation also peaks at about 3 mm reversible after 10 mm  in old animals. We found that bath  amplitude (211  ±  11% of control; Fig.  after the start of CCh treatment and is  of washing (Fig. 4.2A,B). Similar to the results obtained from young  animals, the muscarinic potentiation of 1 A is also blocked by atropine (1 jiM) and the Mi-like antagonists pirenzepine (1 jiM), but not M2-like antagonist methoctramine (1 jiM) (Fig. 4.2C), suggesting that the muscarinic enhancement of  iMA  in old animals is also mediated by Mi-like  receptors.  3.3. Downstream Pathway of Muscarinic Potentiation of ‘NA in Young Animals. We next tested the downstream mechanisms underlying the muscarinic potentiation of ‘DA at the two developmental stages. In young animals, we found that intracellular application of a high concentration of high-affinity Ca 2 chelator BAPTA (10 mM), which has proved to be sufficient to block completely the  muscarinic-induced  plateau potentials or  tail currents in CA 1 pyramidal 169  Chapter Four: Muscarinic Modulation of NMDA Receptors  Figure 4.3. Muscarinic potentiation of ‘A in young animals is 2 [Ca 1 i independent. (A)-(B) In CA 1 pyramidal neurons recorded with 10 mM BAPTA-based intracellular solution, the  A 1  is irreversibly enhanced in CCh (20 jiM). (A) The time course of CCh-induced  potentiation of  ‘NA.  (B) Single traces obtained before CCh application, at the peak of  CCh-induced potentiation, and after washout. Notice that the potentiation in not reversible after washout with BAPTA treatment. (C)-(D) In CA I pyramidal neurons recorded with 5 mg/mL heparin-based intracellular solution, the  ‘A  is still enhanced in CCh (20 jiM). (C) The time  course of CCh-induced potentiation of ‘NA. (D) Single traces obtained before CCh application, at the peak of CCh-induced potentiation, and after washout.  170  Chapter Four: Muscarinic Modulation of NMDA Receptors  A  C  BAPTA Effect  —.  c  Heparin Effect 1.5  z .  .  CCH  E o  0  Z 0.5  Z  -5  10 5 Time  0  CCH  E 15  0.5 -5  0  5 10 Time  15  D  B Control  [A 5 sec  +  CCH  Washout  Control  +  CCH  Washout  vvov LPA 5 sec  171  Chapter Four: Muscarinic Modulation of NMDA Receptors  Figure 4.4. PKC activity is required for muscarinic potentiation of  in young animals.  Preincubation of the broad spectrum PKC blocker GF 103209x (10 jiM, (A)-(B)) and Ro 31-8220 (10 jiM, (C)-(D)) abolishes the muscarinic stimulation of CCh-induced modulation of  ‘NA.  iu. T  (A) The time course of  (B) Single traces obtained before, during and after CCh  application, in the presence of GF 1 09203x. (C) The time course of CCh-induced modulaton of ‘NMDA.  (D) Single traces obtained before, during and after CCh application, in the presence of Ro  31-8220.  172  Chapter Four: Muscarinic Modulation of NMDA Receptors  A  GFlO32O9xEffect  C 4-.  4  a) 1 C.)  Ro 31-8220 Effect 5  0 1.0  ZI. .  a)  . .  .  E  CCH  o  CCH  E 0  Z 0.5  •  -5  0  10 5 Time  15  B Control  Z 0.5  -5  10 5 Time  0  15  D +  CCH  Washout  vvv 5sec  vv  Control  +  Washout  CCH  pA  5sec  173  Chapter Four: Muscarinic Modulation of NMDA Receptors  Figure 4.5. Non-receptor tyrosine kinase activity is required for muscarinic potentiation of  in  young animals. Preincubation of the broad spectrum tyrosine kinase blockers genistein (50 jiM, (A)-(B)) or lavendustin A (10 jiM, (C)-(D)) abolishes the muscarinic stimulation of of CCh-induced modulation of  NA. 1  (A) The time course  (B) Single traces obtained before, during and after CCh  application, in the presence of genistein. (C) The time course of CCh-induced modulation of ‘NA. (D) Single traces obtained before, during and after CCh application, in the presence of lavendustin A. Notice that in the presence of these inhibitors bath application of CCh induced a depression of ‘NMDA.  174  Chapter Four: Muscarinic Modulation of NMDA Receptors  A  C  Genistein Effect  a)  Lavendustin A Effect  a)  I 1..  I I  C.)  CCH  CCH  C.)  z  z  N (U  (U  E I  E I  0  z  0  -5  0  10  5  15  Z 0.5  -5  Time  15  Time  B Control  10  5  0  D +  CCH  Washout  5 sec  Control  +  CCH  Washout  5 sec  175  Chapter Four: Muscarinic Modulation of NMDA Receptors  neurons in our lab (Fraser and MacVicai 1996; Kuzmiski and MacVicar, 2001), could not prevent the muscarinic enhancement of  ‘A  (n  tindependent pathway. Similarly, 1P 2 Ca 3  =  5) (Fig. 4.3A,B), suggesting the involvement of a  receptor  antagonist  heparin (5 mg/mL) could not  prevent potentiation either (Fig. 4.3C,D), suggesting that the 1P —induced Ca 3 2 release in not involved in the enhancement. In addition, to investigate the potential involvement of PKC in the enhancement of ‘NA, we preincubated slices for> 1 hr with the broad spectrum PKC antagonists GF 109203x (10 1 iM; n  5) or Ro 3 1-8220 (10 tM; n  antagonists CCh did not enhance  ‘tA  =  5). In the presence of these PKC  (Fig. 4.4), suggesting that PKCs are involved in this  modulation. And finally, to investigate the role of the non-receptor tyrosine kinases in the muscarinic potentiation of ‘MA, we preincubated slices for> 1 hr with the broad-spectrum tyrosine kinase inhibitors genistein (50 jiM) and lavendustin A (10 jiM). In the presence of these inhibitors, CCh failed to potentiate  ‘NA,  consistent with a previous study showing that muscarinic receptors  act via PKC and Src kinases to regulate NMDA receptors (Lu et al., 1999). Interestingly, in the presence of these inhibitors (genistein or lavendustin A) a significant suppression of  currents  was observed by muscarinic stimulation (Fig. 4.5). This inhibitory effect probably results from the /calmodulin-dependent activation of protein tyrosine phosphatases, which results in the 2 Ca depression of I, (Grishin et al., 2004; Grishin et al., 2005). Because we observed a stable enhancement of ‘A in normal conditions, we did not study this inhibitory effect further. Taken together, our data suggested that in young animals, muscarinic stimulation potentiates  ‘A  through a Ca -independent, but PKC- and Src tyrosine kinase-dependent pathway. Our results from 2 young animals are consistent with several previous studies obtained from cultured or recombinant systems (Lu et al., 1999; Kotecha and MacDonald, 2003; Salter and Kalia, 2004).  3.4. Downstream Pathway of Muscarinic Potentiation of 1 DA in Old Animals. 2 chelator Unlike in young animals, however, intracellular application of the high-affinity Ca BAPTA (10 mM) significantly reduced the muscarinic potentiation of I\’fA in old animals (Fig. -dependent pathway. However, the muscarinic 2 4.6), suggesting the involvement of a Ca potentiation of ‘flA was unlikely due to PKC activation in old animals  because preincubation of 176  Chapter Four: Muscarinic Modulation of NMDA Receptors  Figure 4.6. Muscarinic potentiation of  in old animals is {Ca -dependent. 1 J 2  CAl pyramidal neurons were recorded with 10 mM BAPTA-based intracellular solution in both young and old animals. (A) Single traces obtained in young animals before CCh application, at the peak of CCh-induced potentiation, and after washout. (B) Single traces obtained in old animals before CCh application, at the peak of CCh-induced potentiation, and after washout. (C) Pooled data for the effects of BAPTA on the CCh-induced enhancement  in young and old animals.  Asterisk (*) indicates significant change (p <0.001).  177  Chapter Four: Muscarinic Modulation of NMDA Receptors  A  BAPTA Effect in Young Animals  Control  +  Washout  CCH  5 sec  B  BAPTA Effect in Old Animals  Control  C  +  Washout  CCH  5 sec  Old  Young  I 100  N.S. 50  N  C)  C) 178  Chapter Four: Muscarinic Modulation of NMDA Receptors  Figure 4.7. PKC activity is not required for muscarinic potentiation of I1JA in old animals. CAl pyramidal neurons were recorded from both young and old animals in the presence of the broad spectrum PKC blocker GF 109203x (10 tM). (A) Single traces obtained in young animals before CCh application, at the peak of CCh-induced potentiation, and after washout. (B) Single traces obtained in old animals before CCh application, at the peak of CCh-induced potentiation, and after washout. (C) Pooled data for the effects of GF 1 09203x on the CCh-induced enhancement ‘NMDA  in young and old animals. Asterisk (*) indicates significant change (p  <  0.00 1).  179  Chapter Four: Muscarinic Modulation of NMDA Receptors  A  GF 103209x Effect in Young Animals Control  +  CCH  Washout  V V 5 sec  B  GF 103209x Effect in Old Animals Control  +  CCH  Washout  5 sec  C Young C 0  Old N.S.  **  z C)  z C 0  E a)  C.) C C  LU  x 2o0 C o C’)  —  4  c_)  C LI  180  Chapter Four: Muscarinic Modulation of NMDA Receptors  Figure 4.8. Developmental expression pattern of NMDAR subtypes in CAl pyramidal neurons. (A)-(C) NR2B-containing NMDARs predominantly mediated  in young but not in old animals.  Single traces showed that NR2B specific antagonist Ro 25-6981 blocked Irp. in young (A) but not in old (B) animals. (C) Pooled data for the effects of Ro 25-698 1 on  ‘‘fA  in young and old animals.  (D)-(F) NR2A-containing NMDARs predominantly mediated  in old but not in young animals.  Single traces showed the effects of NR2A specific antagonist NVP-AAM 077 on Jgp at different concentrations (0.1 jiM and 0.4 jiM) in young (D) and old (E) animals. (F) Pooled data for the effects of different concentrations of NVP-AAM 077 on (**) indicates significant change (p  <  ‘A  in young and old animals. Asterisk  0.00 1).  181  Chapter Four: Muscarinic Modulation of NMDA Receptors  A  Young Animals Control  +  Ro 25-6981  D  Young Animals  Control  +  NVP-AAMO77  5 sec 5 sec  E  Old Animals  B  Control  +  0.1 uM  Ro 25-6981  0.4 uM  Old Animals  Control  ÷ NVPAAMO77  5 sec  0.1 uM  5 sec  0.4 uM  F  C Old  Young ***  C.)  0.5 N  z 0.5  Young —o- Old  —.—  EI E 0  Z  0.0  ri —  •‘  0  z  C,  40’  00  182  Chapter Four: Muscarinic Modulation of NMDA Receptors  the broad spectrum PKC antagonists GF 109203x (10 pM; n >  =  5) or Ro 31-8220 (10 jiM; n  5) for  1 hr failed to block the potentiation (Fig. 4.7). These data suggested that in old animals,  muscarinic stimulation potentiates  ‘flA  through a  distinct downstream mechanism from that in  tdependent and PKC-independent pathway. 2 young animals in requiring the activity of a Ca Interestingly, our results from old animals are also consistent with several previous reports obtained in acute hippocampal slices from adult animals (Markram and Segal, 1990b, 1992).  4.  Discussion In the present study, we compared the mechanisms of the muscarinic modulation of  i1tA  in  CAl pyramidal neurons at different development stages. We found that muscarinic stimulation enhances  ‘]A  in both young and old animals, and both via the activation of GcLq-coupled Mi-like  muscarinic receptors. However, the same receptors coupled different downstream pathways to potentiate  ‘NA•  In young animals, muscarinic stimulation potentiated  through a  -independent but PKC- and Src-dependent pathway. In old animals, however, muscarinic 1 2 [Ca stimulation produces a stronger potentiation of ‘DA through a different mechanism, requiring the 2 elevation but not PKC. Therefore, we report a novel developmental activity of intracellular Ca pattern of the cholinergic modulation of In in CAl pyramidal neurons, which could be essential for the role of the cholinergic system in modulating synaptic plasticity, learning and memory at different development stages.  4.1. Downstream Mechanisms Underlying the Muscarinic Modulations of ‘N1A. According to their signal transduction pathways, muscarinic receptors are generally divided into two classes: the MI-like receptors (Ml, M3 and M5) and the M2-like receptors (M2 and M4) (Langmead et al., 2008). Previous studies have shown that Ml specific toxins blocked the muscarinic induced potentiation of  (Marino et al., 1998), suggesting that the muscarinic  potentiation is likely dependent on Mi-like subtype. Here we show a similar enhancement of 1 DA in hippocampal neurons also mediated by muscarinic M1IM3 receptor stimulation, in both young and old animals. Consistent with this finding, Ml receptors were shown to co-localize with NR1A 183  Chapter Four: Muscarinic Modulation of NMDA Receptors  at particular postsynaptic sites (Marino et al., 1998). Hippocampal pyramidal neurons express high levels of postsynaptic Ml and M3 receptors (Vilaro et al., 1993; Levey et al., 1995; Rouse et al., 1999). These receptors are Gcq/11 coupled, and their activation results in the generation of DAG 3 receptors and 3 activates 1P 3 via PLC activation. DAG in turn activates PKC, while 1P and 1P 2 stores. Previous studies on the muscarinic modulation of triggers Ca 2 release from the internal Ca ‘NMDA  3 2 released from P have generated controversial results. Initially it was considered that the Ca  receptors is required for the muscarinic enhancement of ‘NMrA, while protein kinase activity was not involved (Markram and Segal, 1990b, 1992). However, later studies showed that the activity of PKC and Src tyrosine kinase are required for the muscarinic enhancement of In (Lu et al., 1999; Kotecha and MacDonald, 2003; Salter and Kalia, 2004). In the present study, we found a developmental pattern of the muscarinic modulation of ‘NTA, providing a novel explanation for the controversy obtained from different experimental systems. 2 influx through NMDARs could trigger a negative feedback It has been reported that Ca tCa dependent proteins, such as process that inactivates NMDARs, via the activation of various 2 -dependent phosphatases (Kotecha et al., 2003; Kotecha and 2 calmodulin and several Ca MacDonald, 2003). Indeed, the activation of GcLq-coupled metabotropic and muscarinic receptors is also reported to reduce  ‘flA  2 release, calmodulin and tyrosine via a pathway requires Ca  phosphatase (Grishin et al., 2004; Grishin et al., 2005). Interestingly, consistent with these reports, with the tyrosine kinase activity blocked we also observed a muscarinic induced depression of ‘NMDA  In the present study, we report that muscarinic stimulation could potentiate  via  1 elevation might have distinct ] 21 different downstream pathways. The muscarinic induced [Ca impact at different development stages. Thus we suggest that the activity of NMDARs is controlled by a multiple-pathway signaling network, which could be changed and tuned during development.  4.2. The Difference of ‘EA Obtained from Young and Old Animals. It is still not clear why and how the same muscarinic receptors couple to different second messenger systems to activate the same target proteins at different developmental stages. One possibility is due to the distinct developmental expression pattern of different subtypes of NMDARs. 184  Chapter Four: Muscarinic Modulation of NMDA Receptors  There are several subtypes of NMDARs expressed in the mammalian brain, dependent on the different types of NR2 subunits that they inherit (Paoletti and Neyton, 2007). Each subtype of NMDARs has distinct pharmacological and physiological properties (McBain and Mayer, 1994; Kohr, 2006; Papadia and Hardingham, 2007a), and among them, the NR2A and NR2B-containing NMDARs (NR2ARs and NR2BRs, respectively) are the most abundant ones (Wenthold et al., 2003). Interestingly, people found that the expression pattern of NR2 subunits changes during development: NR2BRs are predominately expressed in early stages of development but decrease with time, while the expression of NR2ARs dramatically increases with developmental age (Monyer et al., 1994; Watanabe et al., 1994; Standaert et al., 1996). Recent studies also indicate that NR2ARs and NR2BRs play different roles in cell death processes: the NR2BRs could promote cell death, while paradoxically the NR2ARs could promote cell survival (Liu et al., 2007). There is also evidence suggesting that different NMDA subtypes play distinct roles in synaptic plasticity induction (Liu et al., 2004; Bartlett et al., 2007). In the present study, using subtype specific antagonists of NR2AR- and NR2BR-containing  ‘NA,  I studied the developmental expression  pattern of NMDA subtypes in CAl pyramidal neurons. I found that Ro 25-698 1, a NR2B antagonist (Gogas, 2006), blocked more than 80 percent of total Ir in young animals, but only blocked less than 10 percent of IflA in old animals (Fig. 4.8A, B, C). On the other hand, NVP-AAIvIO77, an antagonist preferentially blocks NR2A-containing IIoA,(Liu et al., 2004; Frizelle et al., 2006), blocked most of  in old animals, but not in young animals (Fig. 4.8D, E, F). These data  confirmed that NR2BR predominantly expressed in young animals, while NR2AR predominantly expressed in old animals, providing a potential mechanism underlying the age-dependence of the cholinergic modulation of  It should be noted that other factors might also affect the  age-dependence of the muscarinic modulation of INA, including the expression level of muscarinic receptors during development (Aubert et al., 1996), the location of muscarinic receptors and NMDARs (Wenthold et al., 2003; Langmead et aL, 2008), the protein partners that bind to the receptors and NMDARs (Wenthold et al., 2003), and so on. Future studies will be needed to rigorously test these potential mechanisms.  185  Chapter Four: Muscarinic Modulation of NMDA Receptors  4.3. Physiological Relevance of the Developmental Pattern of the Cholinergic Modulation of ‘NA• At present, the physiological roles of the muscarinic potentiation of  ‘NMrA  and the  age-dependence of this potentiation are not entirely clear (Marino et al., 1998). The hippocampal CA 1 pyramidal neurons receive a prominent and diffuse innervation of ChAT-positive terminals that rarely make defined synaptic formations (Umbriaco et al., 1995). It is generally believed that modulation by ACh occurs through a diffuse transmission that relies on the nonsynaptic elevation of ambient ACh levels (Umbriaco et al., 1995; Descarries et al., 1997). It is not known whether ambient levels of ACh are sufficient to affect  ‘A  and glutamatergic transmission, although  several in vivo studies have shown that stimulation of cholinergic afferents increases the excitability of CAl pyramidal neurons (Cole and Nicoll, 1984; Morton and Davies, 1997). A definitive statement as to the physiological role of the age-dependence of the cholinergic modulation of ‘rA must await further study. Cholinergic stimulation has been reported to facilitate the induction of hippocampal LTP (Blitzer et al., 1990; Auerbach and Segal, 1996) on acute brain slices. Muscarinic stimulation via the medial septohippocampal pathway also facilities LTP induction in vivo (Galey et al., 1994; Markevich et al., 1997). The muscarinic enhancement of LTP induction in hippocampus is considered due to the depression of K conductances, inhibition of GABAergic transmission and enhancement of  (Auerbach and Segal, 1996). It is still not known whether the muscarinic  facilitation of LTP induction is age-dependent yet. In a recent paper, Sevilla et al. showed that in young animals ACh induced a Ca 2 wave and synaptic enhancement mediated by insertion of AMPA receptors in spines (Fernandez de Sevilla et al., 2008). Activation of Ml muscarinic -sensitive internal calcium stores are required for this type of 3 receptors and Ca 2 release from P 13 is not observed in old animals, LTP (LTP ) (Fernandez de Sevilla et al., 2008). In contrast, LTP 3 and instead, application of lower concentration (submicromolar) of CCH induces LTP (termed LTPm) in acute hippocampal slices from old animals, which is mediated via activation of M2 muscarinic receptors (Auerbach and Segal, 1994, 1996). The discrepancy between LTPm and LTP 3 is not fully understood yet, and our results suggest that the age-dependence of the muscarinic modulation of 186  Chapter Four: Muscarinic Modulation of NMDA Receptors  ‘NMDA  might contribute to the induction of different types of LTP.  The present study might also have implications in the treatment of a number of neurological disorders. NMDAR-mediated excitotoxicity has been implicated in several disorders, such as stroke, trauma, and certain neurodegenerative disorders (Lau and Zukin, 2007; Papadia and Hardingham, 2007a). Muscarinic induced potentiation of ‘NA, especially in old animals, via the turning on of t and 1P 2 -sensitive pathway might strongly potentiate excitotoxicity. It is plausible to 3 the Ca propose that the blockade of Mi-like muscarinic receptors might be used for reducing NMDAR-mediated excitotoxicity in some conditions. Thus the Mi-like muscarinic receptors, and their coupling downstream pathways might provide novel therapeutic targets for the treatment of these diseases.  187  Chapter Four: Muscarinic Modulation of NMDA Receptors  5.  References:  Aramakis VB, Bandrowski AE, Ashe JH (1999) Role of muscarinic receptors, 0-proteins, and intracellular messengers in muscarinic modulation of NMDA receptor-mediated synaptic transmission. 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Annu Rev Pharmacol Toxicol 43 :335-358.  192  Chapter Five: General Discussion  Chapter Five: General Discussion In this dissertation, I report the novel muscarinic-mediated modulations of several -permable ion channels, R-type VGCCs, TRPC5 channels and NMDA receptors, in the 2 Ca hippocampal CAl pyramidal neurons. The potential functional roles of these modulations in both physiological and pathophysiological conditions are also described. I first studied the muscarinic modulation of the “toxin-resistant” R-type VGCCs. I report that 2 currents in hippocampal CA 1 neurons that muscarinic activation specifically stimulates R-type Ca 2 currents were not 2 spikes. T-type Ca can lead to de novo activation of R-type dependent Ca similarly modulated by muscarinic receptor activation. This enhancement is mediated via a 2 currents resulted in -independent PKC pathway and moreover, the enhancement of R-type Ca 2 Ca 2 spikes and could contribute to theta remarkable changes in the firing pattern of R-type Ca oscillations. Therefore, muscarinic receptor activation in hippocampal neurons will alter profoundly 2 the dendritic integration and intrinsic resonance properties by shifting the normal pattern of Ca entry from the slowly inactivating N-, P/Q-, and L-type VSCCs to domination by the HVA rapidly inactivating R-type VGCCs. I next studied the cholinergic-induced prolonged depolarizations called plateau potentials in CA 1 pyramidal neurons that might play an important role in the generation of prolonged neuronal depolarization during the ictal discharges associated with epilepsy. I report that muscarinic receptor activation triggers the membrane insertion of TRPC5 channels thereby profoundly altering the electrophysiological properties of hippocampal neurons in acute hippocampal slices. Antagonists of TRPC5 channels significantly inhibited PPs and  ‘tails,  and only TRPC5 displayed rapid membrane  insertion in the biotinylation experiments. The activation of GcLq,ii-coupled muscarinic receptors K, leading to 3 2 concentration and subsequently the activity of CaM and PI increased intracellular Ca the rapid translocation of TRPC5 channels and the generation of PPs. The translocation of TRPC5 currents will then trigger prolonged inward currents and long-lasting depolarizations that may play an important role in seizure-like discharges observed in epilepsy. Finally, I compared the mechanisms of the muscarinic modulation of 1 A in CAl pyramidal neurons at different development stages. I found that muscarinic stimulation enhances Irs. in both 193  Chapter Five: General Discussion young and old animals by the activation of GcLq-coupled Mi-like muscarinic receptors, but interestingly via different downstream pathways. In young animals, muscarinic stimulation potentiated  -independent but PKC- and Src-dependent pathway. In older 1 ] 2 through a [Ca  animals, howevei muscarinic stimulation produces a stronger potentiation of ‘tA via the activity of intracellular Ca 2 elevation but not PKC. Therefore, I report a novel developmental pattern of the cholinergic modulation of  in CAl pyramidal neurons, which could be essential for the role of  the cholinergic system in modulating synaptic plasticity, learning and memory at different development stages. Taken together, using a combination of whole-cell patch clamping, biochemistry, and two-photon microscopy imaging techniques, I have identified three novel regulatory targets of cholinergic stimulation in the CAl region of hippocampus. We studied the downstream pathways linking the receptors and the effectors, as well as the potential physiological roles of these modulations. Our data indicate that cholinergic input to the hippocampus contributes to excitation and stronger Ca 2 influx into the postsynaptic space via the activation of different ion channels. The -permeable ion channels might provide novel understanding of the 2 stimulation of these higher Ca physiological and pathophysiological roles of cholinergic systems in the CNS and might also provide novel therapeutic targets for brain disorders concerning cholinergic systems, such as epilepsy and Alzheimer’s disease.  1.  Downstream Pathways Coupled to Cholinergic Stimulation  —  How Many Pathways Are There?  In hippocampal CAl pyramidal neurons, muscarinic receptors have been reported to link to numerous downstream signalling mechanisms. The GcLq-coupled Mi-like muscarinic receptors 2 into 1P 3 and the membrane couple efficiently to stimulate PLC, which hydrolyzes membrane PIP 3 binds to and triggers the associated fatty acid DAG (Caulfield and Birdsall, 1998). Cytosolic 1P 3 receptors from the endoplasmic reticulum (ER). The elevation of release of Ca 2 through the IF -sensitive sensor molecules, such as calmodulin 2 intracellular Ca 2 concentration activates many Ca /calmodulin-dependent protein kinases 2 that in turn activate downstream effectors including Ca (CaMKs), PI K, calcineurin, adenylate cyclases and transcription factors. DAG activates PKCs 3 194  Chapter Five: General Discussion directly (Group II PKCs) or together with intracellular Ca 2 (Group I PKCs), and PKCs in turn can 2 itself can function to signal transmission by phosphorylate a variety of downstream targets. PIP anchoring numerous signaling molecules and cytoskeleton at the cell membrane (Hilgemann et al., 2001; Hernandez et al., 2008). Ml receptors also depresses Cav3.3 calcium channels through a , suggesting the 2 novel to-be-defined pathway that is independent of PKC, 1P , Ca and PIP 3 existence of other downstream mechanisms of Gnqjii proteins (Hildebrand et al., 2007). Muscarinic receptors can also transduce signals independent of G-protein activation (Gee et al., 2003). In this dissertation, I report that cholinergic stimulation via Gctq-coupled muscarinic receptors tCa permeable ion channels, and interestingly, via distinct enhances the activity of three 2 downstream pathways. Muscarinic enhancement of R-type VGCCs is mediated via a tindependent Group II PKC pathway, the enhancement of TRPC5 channels is mediated via a 2 Ca K-dependent pathway, while the muscarinic enhancement of NMDA receptors 3 -, CaM-, and PI 2 Ca is mediated via a Ca - and PKC-independent but Src-dependent pathway in the young animals, 2 -independent but PKC-dependent pathway in old animals. A schematic summary of 2 while via a Ca the distinct pathways linking muscarinic receptors and their effectors is shown in Fig 5.1. It is still not clear how the same receptors couple to so many different downstream pathways and in turn modulate so many target proteins in different manners. The subcellular localization of the receptors and the target effectors, the structures and conformational states of the target ion channels, and the molecules that link the receptors and effectors might underlie these different pathways (Fig. 5.1). However, further work is required before we can attempt to unify these pathways. My data and others indicate that the cholinergic stimulation in the hippocampus results in a net upregulation of 2 influx and neuronal excitability at the postsynaptic sites via the upregulation of the Ca tpermeable cation channels and the downregulation of K conductances. 2 Ca  2. Physiology of Cholinergic Stimulation  —  The Involvement of New Players  As one of the most important modulatory neurotransmitters, ACh plays important role in numerous physiological processes. However, the mechanisms underlying the cholinergic impact have proven elusive.  In the present study, I have identified three novel modulatory targets of the 195  Chapter Five: General Discussion  Figure 5.1. Proposed downstream pathways linking muscarinic receptors and their target ion channels. Please refer to the text for explanation of the pathways. Abbreviations: ACh, acetyicholine; Glu, , phosphatidylinositol 4,5-bisphosphate; DAG, 2 glutamate; Gly, glycine; PLC, phospholipase C; PIP R, inositol 1,4,5-triphosphate receptor; PKC, 3 , inositol 1,4,5-triphosphate; 1P 3 diacyglycerol; 1P K, phosphoinositide 3-kinase; 3 protein kinase C; NMDAR, NMDA receptor; CaM, calmodulin; PI VGCC, voltage-gated calcium channel; M1/M3, muscarinic receptor.  196  Chapter Five: General Discussion  ACh  M1/M3  Glu  Gly  Ca2.3 VGCC  A  PLC  Na* Ca 2  Na Ca 2 ??  K 3 P1  197  Chapter Five: General Discussion  cholinergic stimulation that might provide novel insight into the functions of the cholinergic system in the brain.  2.1. Neuronal Bursting and Afterdepolarization Bursting activity is characterized by two or more action potentials (APs) superimposed on a single slow depolarizing component. Burst firing has been observed in a variety of brain regions and has been reported to play a number of roles. Bursting of APs increases the probability of release per event, allowing activity to propagate more reliably through the network (Lisman, 1997). Bursting has also been reported to enhance synaptic plasticity at the Schaffer collateral to CAl synapse (Thomas et al., 1998; Pike et al., 1999; Fortin and Bronzino, 2001), and to be involved in goal identification and approach, as well as rest and sleep (Suzuki and Smith, 1985). In principal neurons of the CNS, especially in hippocampal CAl pyramidal neurons, single APs are often followed by an afterdepolarization (ADP) (Schwartzkroin, 1975; Storm, 1987), which provides a prolonged somatic depolarization necessary for the initiation of multiple spikes (Wong and Prince, 1981; Jensen et al., 1996; Magee and Carruth, 1999). Muscarinic stimulation has been reported to enhance the backpropagation of APs into the dendrites (Tsubokawa and Ross, 1997; Hoffman and Johnston, 1999), and to enhance the bursting activity and ADP in many regions of the brain (McQuiston and Madison, 1999; Whalley et al., 2005; Zhang et al., 2005; Moore et al., 2009). The mechanisms underlying the muscarinic modulation of ADP and bursting are still not clear. Interestingly, recent data showed that the activity of R-type VGCCs plays a central role in ADP and bursting in hippocampal CA 1 pyramidal neurons (Metz et -activated non-selective cation channels, including members of the TRP channel 2 al., 2005). Ca family, may also play a role in ADP and bursting (Moore et al., 2009). In this dissertation, I have shown that muscarinic stimulation strongly and specifically enhances the activity of R-type VGCCs and TRPC5 channels, which provide a novel understanding of the generation and regulation of ADP bursting, and the excitability of the pyramidal neurons. In CA 1 pyramidal neurons, highly synchronous excitatory synaptic input can also induce bursting APs that are observed during sharp waves in vitro (Moore et al., 2009) and in vivo (Ranck, 198  Chapter Five: General Discussion  1973; Kamondi et aL, 1998a), which are also followed by ADPs (Golding et al., 1999). NMDA receptor activity has been reported to contribute to the ADP in CAl pyramidal neurons during synaptic stimulation (Wu et al., 2004). My results and others showed that muscarinic stimulation could potentiate the activity of NMDA receptors and thus enhance the late phase of synaptic transmission and Ca 2 influx, which might play a role in the muscarinic regulation of synaptic transmission and neuronal excitability. It should be noticed that muscarinic stimulation might also enhance bursting activity and ADP by other mechanisms by suppressing A-type or D-type K channels (Golding et al., 1999; Wu and Barish, 1999),  2.2. Long Term Potentiation The induction of LTP in CA 1 pyramidal neurons normally requires the activity of NMDA receptors, which triggers Ca 2 entry and the activation of several downstream enzymes like CaMKII and induces AMPA receptor membrane insertion. The activity of VSCCs is also reported required for the induction of several forms of LTP (Magee and Johnston, 1997; Malenka and Nicoll, 1999). The induction of hippocampal LTP is enhanced by the application of cholinergic agonists in rat hippocampus by lowering the threshold for induction (Blitzer et al., 1990; Auerbach and Segal, 1996). In vivo studies showed similar results (Galey et al., 1994; Markevich et al., 1997). Apparently, as shown in Chapter Four, the enhancement of NMDA receptors plays an important role in the muscarinic facilitation of LTP induction (Auerbach and Segal, 1996). However, my studies on R-type VGCCs and TRPC channels might provide more information about this phenomenon. Recently, R-type VGCCs have been shown to be required for the induction of presynaptic LTP in the CA3 region of hippocampus (Breustedt et al., 2003; Dietrich et al., 2003). It is also reported that R-type VGCCs play a role in the NMDA-dependent postsynaptic LTP in CA 1 pyramidal neurons (Isomura et al., 2002). Thus the muscarinic enhancement of R-type VGCCs might facilitate LTP induction. Moreover, it is also possible that high frequency tetanic stimulation could activate the cholinergic-induced PP that is contributed by the membrane insertion of TRPC5 channels, and facilitate LTP induction by further Ca 2 influx. 2 influx. The generation of PPs requires Ca Muscarinic stimulation strongly enhances the activity of R-type VGCCs and NMDA receptors 199  Chapter Five: General Discussion allowing more Ca 2 influx that facilitates the generation of PPs, which might in turn facilitate the induction of LTP. The location of these channels also provides insights into the roles of these channels in the muscarinic facilitation of LTP induction. In the CA 1 pyramidal neurons, the NMDAR-dependent postsynaptic LTP occurs mainly in dendritic spines. Interestingly, similar to NMDA receptors, R-type VGCCs are also largely distributed in the dendritic spines, and are thought to be primarily responsible for Ca 2 influx in apical dendrites and spines (Sabatini and Svoboda, 2000; Yasuda et al., 2003), while other types of HVA VGCCs are mainly expressed in the soma and basal dendrites (Christie et al., 1995; Magee and Johnston, 1995). TRPC5 channels are also expressed in the soma, dendrites and axons, as well as in nascent synapses and growth cones (Strubing et al., 2001; Greka et al., 2003). It is plausible to hypothesize that muscarinic stimulation enhances the activity of all ] concentrations in subcellular microdomains, such as 2 these three channels and increases local [Ca dendritic spines, and dramatically regulate the generation of synaptic plasticity.  2.3. Theta Rhythm Hippocampal theta rhythm, also termed rhythmical slow activity (RSA), is characterized by oscillations of membrane potential at 4-12 Hz frequency range (Bland and Bland, 1986). In the hippocampus, the cholinergic system plays a key role in rhythmic rhythm activity, because theta rhythm is abolished by lesions or inactivation of medial septal neurons, the major cholinergic input of hippocampus (Petsche and Stumpf, 1960). Cholinergic stimulation of hippocampal slices induces robust oscillations at similar frequency range to spontaneously occurring theta in vivo (Konopacki et al., 1987; Bland et al., 1988; MacVicar and Tse, 1989; Garcia-Munoz et al., 1993). During theta 2 spikes were observed to oscillate rhythmically at theta frequencies in the activity in vivo HVA Ca dendrites of CA 1 pyramidal neurons and may contribute to current generation and amplification of theta (Kamondi et al., 1 998b; Buzsaki, 2002). However, the exact VGCC subtypes that contribute to dendritic oscillations during theta have been elusive. As shown in this dissertation, muscarinic 2 spikes while depressing all stimulation enhances R-type VGCCs and generates R-type HVA Ca 2 also strongly depresses the other types of HVA VGCCs. The R-type VGCC antagonist Ni 200  Chapter Five: General Discussion cholinergic-induced theta oscillations at low concentrations, suggesting that R-type VGCCs play a central role in theta generation. Consistent with this, both atropine-resistant and atropine-sensitive theta oscillations are inhibited by low concentrations of Ni 2 (Kramis et al., 1975; Buzsaki, 2002; Gillies et al., 2002). Interestingly, intracellular recordings from CA 1 pyramidal neuron dendrites in vivo have shown that the dendritic membrane potential displays long-lasting slow depolarization during the cholinergic-induced theta (Kamondi et al., 1998b). The cholinergic-induced non-selective cation conductance has been shown to underlie a prolonged membrane depolarization that supports sustained spiking activity in rat prefrontal cortex (Haj-Dahmane and Andrade, 1996, 1998). We show that the cholinergic-induced PP could reflect the slow depolarization during theta in the 2 influx and ACh release, hippocampus. Simultaneous synaptic stimulation could evoke large Ca which could in turn induce the rapid translocation of TRPC5 channels and the generation of PPs, and facilitate the generation of theta oscillations. NMDA receptors might also play important in the generation of theta ryhthm. Blockade of NMDA receptor by APV inhibited theta activity in vivo (Leung and Desborough, 1988) and the spread from the CA3 into CAl hippocampal region of cholinergic-induced theta in vitro (Williams and Kauer, 1997). In addition, glutamate either applied iontophoretically or released via synaptic stimulation of Schaffer collateral in vitro can induce an intracellular theta-like rhythm via activation of NMDARs (Bonansco et al., 2002), which shares common properties with theta recorded in vivo (Fujita and Sato, 1964; Ylinen et al., 1995; Kamondi et al., 1998b). The NMDA-induced theta does not depend on circuit interactions and relies on the voltage dependence of the NMDA channel to 2 spikes to boost the depolarizing phase of initiate the oscillations, and on dendritic HVA Ca oscillations (Bonansco and Buno, 2003). Thus the interactions between NMDA receptors and intrinsic membrane conductances, including R-type VGCCs and TRPC5 channels, might play a critical role in the generation of theta in CA 1 pyramidal neurons. Thus the cholinergic stimulation of NMDA currents, R-type HVA Ca 2 spikes, as well as TRPC5-dependent PPs, might work together to facilitate the generation of theta rhythms in hippocampal CAl neurons.  201  Chapter Five: General Discussion 3. Pathophysiology of Cholinergic Stimulation  —  The Involvement of New Players  tCa permeable channels plays important As discussed above, muscarinic stimulation of highly 2 roles in the regulation of membrane excitability, synaptic plasticity and intrinsic properties of CA 1 pyramidal neurons. However, over-activation of these channels might trigger excessive Ca 2 influx into pyramidal neurons, which could be pathological leading to neuronal damage.  3.1. Epilepsy Well-documented experimental evidence from both in vitro and in vivo models of epilepsy has supported the critical involvement of the cholinergic system in epileptogenesis, especially in temporal lobe epilepsy (Leite et al., 2002; Friedman et al., 2007; Majores et al., 2007). It is widely believed that the impact of the cholinergic system in epileptogenesis arises from its• regulation of numerous types of ion channels, although the mechanisms are not clear. The enhancement of the -permeable ion channels described in this dissertation might provide novel understanding 2 three Ca of the roles of cholinergic system in epileptogenesis. During seizures, neurons undergo prolonged ictal events characterized by tonic membrane depolarizations accompanied by trains of fast APs, separated by interictal spiking. The transition from interictal events to ictal depolarizations likely involves multiple mechanisms (Shin and McNamara, 1994). The types of ionic conductances underlying the ictal depolarizations are still not clear. We have shown that the cholinergic-induced PP shares common properties as ictal events recorded from human epileptic hippocampus in vivo or rat hippocampal slices in vitro (Lothman et al., 1991; Nagao et al., 1996), and might contribute to the intrinsic mechanisms underlying ictal depolarizations (Fraser and MacVicar, 1996). In this dissertation, I propose that the activity of TRPC5 channels significantly contributes to PP and the tonic ictal depolarization. Enhanced synaptic transmission and repetitive burst discharges lead to stronger neuronal excitability and elevated release of ACh and glutamate, both of which could activate PPs. It is plausible to 2 influx hypothesize that cholinergic or metabotropic glutamatergic stimulation, together with Ca from R-type VGCCs, triggers the rapid translocation and activation of TRPC5 channels, resulting in long lasting PPs and ictal-like depolarization waveforms. It should be noted that TRPC5 channel is 202  Chapter Five: General Discussion not the only player in the generation of PP or ictal depolarizations. The ictal phase of the epileptic discharges is complicated and likely to involve multiple mechanisms. Indeed, we found that CNG channels might also contribute to the generation of PP. It has been proposed that R-type VGCCs play a role in the etiology and pathogenesis of seizures in many types of epilepsy. R-type VGCCs contribute to the generation of plateau potentials (PPs) (Kuzmiski et al., 2005) and afterdepolarizations (ADPs) (Metz et al., 2005) and facilitate triggering epileptiform discharges. Pharmacological experiments have revealed that some antiepileptic drugs specifically target Cav2.3 and that could explain some of their actions to reduce epileptiform burst activity (Hainsworth et al., 2003; Kuzmiski et al., 2005). Seizure susceptibility was dramatically decreased in the R-type VGCCs knockout animals (Weiergraber et al., 2006), supporting the pharmacological studies that this type of calcium channel plays an important role in epileptiform 2 influx from R-type VGCCs is both sufficient and activity. We have previously shown that the Ca required for the generation of PPs (Kuzmiski et al., 2005). Elevated levels of extracellular ACh or 2 spikes during excitatory synaptic glutamate would promote the initiation of dendritic R-type Ca 2 spikes might input, leading to increased [Ca 1 accumulation. The cholinergic-induced R-type Ca ] 2 then play a critical role in the transition from interictal to ictal activity. However, future investigations are required to clarify the relationship between R-type VGCCs and epilepsy and to develop new antiepileptic drugs targeting Cav2.3 channels (Weiergraber et al., 2006). Synaptically released glutamate acting on ionotropic glutamate receptors is also well recognized as a critical player in many forms of interictal and ictal epileptiform activity (Rogawski, 2008). Repeated seizures could induce a movement of NMDA receptors from subsynaptic sites to the synaptic membrane, causing further hyperexcitability (Chen et al., 2007; Wasterlain and Chen, 2008). Many studies have demonstrated that NMDA receptor antagonists, either noncompetitive channel blockers like ketamine and MK-80 I or competitive antagonists such as AP5 are powerful anticonvulsants in animal models (Walker and Ni, 2007; Rogawski, 2008). In this dissertation, I propose that repetitive burst discharges and increased synaptic activity trigger stronger neuronal excitability and elevated release of ACh and glutamate which could potentiate NMDA receptor activity leading to further Ca 2 influx and over-excitability. The cholinergic-induced stimulation of 203  Chapter Five: General Discussion R-type VGCCs and TRPC5-dependent PPs might also facilitate the activity of NMDA receptors, especially in older animals, where the cholinergic modulation of NMDA receptors appears -dependent. 2 Ca  3.2. IschemiaJStroke Neurodegeneration in cerebral ischemic stroke is a leading cause of death and disability worldwide. Ischemic insults to the brain in stroke produce transient excessive release of neurotransmitters, such as glutamate and ACh from depolarized presynaptic terminals (Chung et al., 2 entry into postsynaptic neurons and 2002; Muir and Lees, 2003), stimulating massive Ca activating a biochemical cascade that results in cell death. A number of different mechanisms are involved in cell death in cerebral ischemia, including excitotoxicity, oxidative stress, apoptosis and necrotic cell death (Won et al., 2002). Interestingly, all these mechanisms require monovalent and 2 channels and/or non-selective cation channels divalent cation influx into neurons, implicating Ca (Simard et al., 2007). Excessive Na entry can induce necrotic cell swelling that results in necrotic cell death, and excessive Ca 2 entry can trigger both apoptotic and necrotic cell death (Simard et al., 2 influx and lead to neuronal 2007). However, the initial routes that allow excessive Na and Ca death are not fully understood. An overwhelming body of evidence from both in vitro and in vivo animal studies has consistently demonstrated that overaactivation of the NMDA subtype of glutamate receptors is the primary step leading to neuronal injury after insults of stroke and brain trauma (Arundine and Tymianski, 2004; Liu et al., 2007; Papadia and Hardingham, 2007b). The excessive release of ACh and glutamate could profoundly modify the properties of NMDA receptors. As shown in Chapter Four, cholinergic stimulation could potentiate NMDA currents, in a age-dependent manner. In older -dependent. This 1 ] 2 animals, we observed a stronger potentiation of NMDA current, which is [Ca could change the vulnerability of the brain to ischemic insults during development. There is little doubt that NMDA receptors play a central role in glutamatergic excitotoxicity and ischemic neuronal death (Liu et al., 2007; Papadia and Hardingham, 2007b). However, clinical trials with agents that block these channels in patients with acute ischemic stroke has shown little 204  Chapter Five: General Discussion benefit (Horn and Limburg, 2000; Kemp and McKernan, 2002). Recently, a growing body of evidence has suggested the involvement of newly identified non-selective cation channels in cerebral ischemia (Simard et al., 2007), including the transient receptor potential (TRP) channels (Aarts et al., 2003). TRP channels form a recently identified superfamily of non-selective cation channels that display great diversity of activation mechanisms and selectivities. They are widely distributed in mammalian tissues and respond to a wide range of environmental signals (Ramsey et al., 2006). Several in vitro studies have indicated the involvement of some TRP channels, including TRPM2, TRPM7 and TRPC4 (Aarts et al., 2003; Simard et al., 2007), in ischemic stroke. We also have evidence that 0 /glucose deprivation (OGD) can activate some cation conductances in acutely 2 isolated neurons, which probably consist a class of TRP channels (TRPC4/5) (Thompson et al., 2006). In acute brain slices, we have recorded similar conductances. In brain slices, we also found that cholinergic stimulation or OGD could induce rapid membrane translocation of TRPC5 channels, which could contribute to the prolonged depolarization (PPs) of neurons in stroke-like conditions. These results strongly suggest that TRP channels might play a much more important role in ischemic stroke than previously realized. TRP channels might therefore be novel therapeutic targets for the treatment of stroke. Another major pathway of Ca entry into depolarized nerve cells is through VGCCs. A large 2 fraction of this Ca entry is mediated through R-type VGCCs. As shown in Chapter Two, most high 2 threshold calcium channels (L-, P-IQ- and N-type) are decreased, while R-type VGCCs are selectively enhanced, by metabotropic or muscarinic activation. This change profoundly alters 2 entry from the slowly inactivating dendritic integration by shifting the normal pattern of Ca VSCCs to domination by the high threshold, rapidly inactivating R-type VSCCs (Tai et al., 2006). Thus the excessive release of glutamate and ACh in stroke could make R-type VSCCs the dominant route of Ca 2 entry. The activity of R-type currents and spikes is also critical in the generation of PPs together with cholinergic or glutamatergic stimulations (Kuzmiski et al., 2005), which might in turn trigger more Ca 2 entry and neuronal damage.  205  Chapter Five: General Discussion 4. Future Directions An interesting question raised in this dissertation is the location of the muscarinic modulations. The patch-clamp recordings and biochemistry methods that we used could only provide information about the macro whole-cell currents but not the subcellular location of the channels and the muscarinic modulations. Two-photon laser scanning microscopy (TPLSM) imaging of intracellular 2 dynamics, together with electrophysiological recordings, would allow us to identify the Ca 2 currents, PP genesis and NMDA currents. subcellular location of muscarinic-enhanced R-type Ca The high temporal and spatial resolution of two-photon imaging would make it possible to test whether the muscarinic-induced R-type Ca 2 spikes, PPs, and upregulation of NMDA currents occur in soma, basal dendrites and spines and in the distal dendrites. As shown in Figure 5.2, two-photon 2 dynamics at different imaging could be performed in CA 1 pyramidal neurons to monitor the Ca locations with Ca -sensitive dyes that are loaded using simultaneous whole-cell patch clamp 2 2 influx into the apical dendrites techniques in the soma. Back-propagating APs could induce Ca and spines of CAl pyramidal neurons, where R-type VGCCs are the predominant route of the depolarization-induced Ca 2 influx (Yasuda et al., 2003). Muscarinic agonists and antagonists will then be bath applied to test the location of the enhancement of R-type current and the generation of 2 transient is also observed correlating R-type spikes. Similarly, a large regenerative dendritic Ca with the activation of  ‘tail  (Fig. 3.2). Future experiments will be designed to determine the  2 influx. contribution of R-type VSCCs and TRPC channels to the elevations in dendritic Ca Moreover, the advanced technique of two-photon glutamate uncaging, together with whole-cell 2 imaging, will allow the stimulation of measurements of synaptic currents and two-photon Ca NMDA receptors on apical dendrites and spines while measuring the amplitudes of uncaged 2 influx (Sobczyk and Svoboda, 2007). Using this approach it glutamate-evoked currents and Ca should be possible to determine the subcellular location of the muscarinic modulation of NMDA receptors, in both young and old animals. Another major question in this dissertation is whether the muscarinic stimulation directly enhances R-type VGCCs and NMDA receptors, or indirectly via K channel depression. Although we have used high levels of K channel  inhibitors,  both intracellularly and extracellularly,  the 206  Chapter Five: General Discussion  Figure 5.2. Two-photon imaging of intracellular calcium dynamics in apical dendrites and spines of CAl pyramidal neurons. To study the properties of VGCCs in apical dendrites and spines, we imaged secondary- or higher-order dendrites and their spines (within 200 im from the soma) of CAl pyramidal neurons. -insensitive (red, 2 -sensitive (green, Fluo-5F, 200 jiM) and a Ca 2 Neurons were loaded with a Ca Alexa-594, 20 jiM) dye and imaged using 2-photon laser scanning microscopy. (A) A typical hippocampal CA 1 pyramidal neuron whole-cell voltage clamped and loaded with fluorescent dyes using 3D reconstruction from a Z series stack of two-photon images. (B) The morphology of apical dendrites and spines. (C) & (D) Linescan images (dashed line in (B)) in an apical spine (C) and its , indicated 1 j 2 parent dendrite (D). Backpropagating action potetials triggers transient changes in [Ca by Fluo-5F (red trace in (C) and blue trace in (D)), but do not change the fluorescence of Alexa-594 1 and is ] 2 (black traces in (C) and (D)). The ratio of green to red fluorescence is proportional to [Ca used to quantify the change in [Ca . The wavelength of the laser was set at 810 urn. 1 ] 2  207  Chapter Five: General Discussion  A  B  D.  Dendrite  5Oms  208  Chapter Five: General Discussion muscarinic enhancement of R-type and NMDA currents could still rise from the depression of residual K currents. Single-channel recordings may help us to solve this problem. Cell-attached recordings performed on the membrane of soma, basal dendrites and apical dendrites could verify whether muscarinic agonist directly modify the single properties of the channels, such as single-channel conductance, channel activation and inactivation dynamics, and desensitization and deactivation dynamics. Excised inside-out recordings could be used to confirm the involvement of intracellular second messengers in the muscarinic modulation of these channels. Moreover, as shown in Figure 5.3, two-photon Ca 2 uncaging together with whole-cell recordings allows us to test whether the elevation of intracellular Ca 2 concentration could directly induce  ‘tail,  or modulate  the property of R-type currents and NMDA receptors. Finally, genetic manipulation of R-type VGCCs, TRPC channels and NMDA receptors will help to clarify the roles of these channels in the physiological and pathophysiological functions of the cholinergic system in the brain. Indeed, one of the biggest challenges in this dissertation was the lack of selective pharmacological tools to specifically block or activate R-type VGCCs, TRPC channels and different subtypes of NMDA receptors. This forced us to rely on antagonists that are likely to have actions on multiple ion channel types. The only selective blocker of R-type VGCCs in expression systems, the spider toxin SNX-482, failed to block R-type currents in many native cells, especially in hippocampal CAl pyramidal neurons, where R-type current is significantly less sensitive to SNX-482 (Sochivko et al., 2002; Sochivko et al., 2003). Until better pharmacological tools are developed, genetic methods will be the best option to selectively block the target channels. Genetic knockout mice have been developed recently for Cav2.3 channels, TRPC5 channels, and different subunits of NMDA receptors (Forrest et al., 1994; Kutsuwada et al., 1996; Sprengel et al., 1998; Breustedt et al., 2003; Dietrich et al., 2003; Townsend et a!., 2003; Riccio et al., 2009). Future experiments will test for alterations in the cholinergic-induced theta burst activity using the Cav2.3 VGCC knockout mice (Breustedt et a!., 2003; Dietrich et al., 2003), as well as test the generation of PPs on brain slices and the epileptogenesis in animal models using the TRPC5 channels knockout mice (Riccio et al., 2009). The muscarinic modulation of different types of NMDA receptors at different development stages using the NR2A or NR2B knockout mice could also be examined 209  Chapter Five: General Discussion  (Sakimura et al., 1995; Kutsuwada et al., 1996; Sprengel et a!., 1998).  210  Chapter Five: General Discussion  Figure 5.3. Two-photon imaging of intracellular calcium dynamics induced by Ca 2 uncaging in CA 1 pyramidal neurons. (A) 3D reconstruction from a Z series stack of two-photon images of a typical hippocampal -sensitive fluorescent dye 2 CA 1 pyramidal neuron whole-cell voltage clamped and loaded with a Ca (red, Rhod-2, 200 jiM) and NDBF-EGTA (100 jiM), an efficient uncaging compound of calcium. (B) , 1 ] 2 Linescan imagings from the soma of the neuron. Depolarization triggers transient changes in [Ca 2 release from indicated by Rhod-2. (C) Two-photon laser stimulation at 700 nM triggers Ca , also indicated by Rhod-2. 1 ] 2 uncaged NDBF-EGTA and induces transient elevations in [Ca  211  Chapter Five: General Discussion  A. Two-photon imaging of a CAl cell  B. Depolarization-induced calcium signal  U U  C  LC)  50s O.5s  1.Os  C. Uncaged-NDBF-induced calcium signal  O.6s  1.2s  2.4s  2.4s  212  Chapter Five: General Discussion  5. 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J Neurophysiol 92:2346-2356.  221  Chapter Five: General Discussion Yasuda R, Sabatini BL, Svoboda K (2003) Plasticity of calcium channels in dendritic spines. Nat Neurosci 6:948-955.  Ylinen A, Soltesz I, Bragin A, Penttonen M, Sik A, Buzsaki G (1995) Intracellular correlates of hippocampal theta rhythm in identified pyramidal cells, granule cells, and basket cells. Hippocampus 5:78-90.  Zhang L, Liu Y Chen X (2005) Carbachol induces burst firing of dopamine cells in the ventral tegmental area by promoting calcium entry through L-type channels in the rat. J Physiol 568:469-481.  222  Appendix 1: Certificates  Appendix 1: UBC Animal Care Certificate  223  https;f/rise.ubc.cj/rise/Doc/O/6MCQGH4GLONKJ2TABE4VAFT929/...  USC  THE UNWERSITY OF BRITISH COLUMBIA  ANIMAL CARE CERTIFICATE Application Number: A06-0429 Investigator or Course Director: Brian MacVicar Department: Psychiatry Animals:  Rats Sprague-Dawley  Start Date:  10001  August 17, 2006  Approval  December 12, 2008  Funding Sources: Funding Agency: Funding Title:  Heart and Stroke Foundation of British Columbia and Yukon  Funding Agency: Funding Title:  Canadian Institutes of Health Research (CIHR)  Cellular mechanisms underlying ischemia induced neuronal necrosis  NEURO Reseich program  Funding Agency: Funding Title:  Heart and Stroke Foundation of Canada  Funding Agency:  Heart and Stroke Foundation of Canada  Funding Title’  A novel pathway in astrocytes providing a neuronal energy substrate during ischemic conditions  R-type VGCCs and TRP channels contribute to stroke-induced neuronal death  Canadian Stroke Network (C SN) Networks of Centres of Excellence (NICE) -  Funding Title:  Targeting cell death cascades in the neuro vascular-inflammatory unit  7/16/2009 10:55AM  https://rise.ubc.qIrise/Doc/0/6MCQGH4GL0NKJ2TABB4VAFr929/...  Funding Agency:  Leducq Foundation  Funding ‘fitle  Mechanisms matching the brian’s vascular energy supply to neural activity, and their failure in dise,..  Funding Agency: Funding Title:  Signaling pathways underlying spreading depression and Ischemic Depolarization  Funding Agency: Funding Title:  Michael Smith Foundation for Health Research  Funding Agency: Funding Title:  Alberta Heritage Foundation for Medical Research  Signalings pathway underlying spreading depression and ischemic depolarization  Neuronal astrocyte interactions underlie cerebral vasculature control  Funding Agency: Funding Title:  Heart and Stroke Foundation of Canada  Funding Agency: Funding Title:  Heart and Stroke Foundation of Canada  Funding Agency:  Leducq Foundation  Funding Title  Mechanisms matching the brian’s vascular energy supply to neural activity, and theft failure in dise...  •  Funding Agency: Funding TitJe  2of5  Heart and Stroke Foundation of Canada  Signaling pathways underlying spreading depression and Ischemic Depolarization  R-type VGCCs and TRP channels contribute to stroke-induced neuronal death  Heart and Stroke Foundation of Canada •  A novel pathway in astrocytes providing a neuronal energy substrate during ischesnic conditions  Funding Agency: Funding Title:  Heart and Stroke Foundation of Canada  Funding Agency: Funding Title:  Canadian Stroke Network (C SN) Networks of Centres of Excellence (NCE)  Microglia Cells limit the spread of neurotrauma in stroke model -  Targeting cell death cascades in the neuro vascular-inflammatory unit  Funding Agency: Funding Title:  Canadian Institutes of Health Research (CIHR)  Funding Agency:  Canadian Institutes of Health Research (CuR)  Synaptic and nonsynaptic modulation of neuronal excitability  7/16/2009 10:55AM  https://rise.ubc.ca/rise/Doc/O/6MCQGH4GLONKJ2TABB4VAFt’929/...  Funding Title:  Development of molecular mechanisms underlying neuron-astrocyte control of cerebral blood flow  Funding Agency:  Canadian Institutes of Health Research (CIHR)  Funding Title:  Development of molecular mechanisms underlying neuron-astrocyte control of cerebral blood flow  Funding Agency: Funding Title:  Heart and Stroke Foundation of British Columbia and Yukon  Funding Agency: Funding Title:  Alberta Heritage Foundation for Medical Research  Funding Agency: Funding Title: Funding Agency: Funding Title:  Cellular mechanisms underlying ischenila induced neuronal necrosis  Neuronal astrocyte interactions underlie cerebral vasculature control Heart and Stroke Foundation of Canada Microglia Cells limit the spread of neurotrauma in stroke model Canadian Institutes of Health Research (CIHR) NEURO Research program  Fnnding Agency: Funding Title:  Canadian Institutes of Health Research (CIHR)  Funding Agency: Funding Title:  Michael Smith Foundation for Health Research  Funding Agency:  Canadian Stroke Network (CSN) Networks of Centres of Excellence (NCE)  Synaptic and nonsynaptic modulation of neuronal excitability  Signalings pathway underlying spreading depression and ischemic depolarization  -  Funding Title:  Xin Wang: CSN Summer Studentship Contribution of Hemichanncl opening to Ischemic Damage in Brain Slices  Funding Agency:  Networks of Centres of Excellence (NCE)  Funding Title:  -  Theme III Adnthi. Support Molecular mechanisms of neuronal injury: Development of a therapeutic strategy for stroke-induced damage -  Fnnding Agency: Funding Title:  Canadian Institutes of Health Research (CIHR)  Funding Agency:  Networks of Centres of Excellence (NCE)  Synaptic function and plasticity  7/16/2009 10:55 AM  https://rise.ube.cJrisefDoc/O/6MCQGH4GLONKJ2TABB4VAFT929/.,.  Funding Title’ FlIfld1II  Fundin g Title  CSN Project #9: Molecular mechanisms of neuronal injury: Development of a therapeutic strategy for stroke-induced damage Networks of Centres of Excellence (NCE) CSN project #9: Molecular mechanisms of neuronal injury: Development of a therapeutic strategy for stroke-induced damage  Funding Agency; Funding Title;  Canadian Institutes of Health Research (CIHR)  Funding Agency: Funding Title:  UBC Faculty of Medicine  Funding Agency: Funding Title:  Michael Smith Foundation for Health Research  Synaptic function and plasticity  Start Up Funding  Neuronal astrocyte interactions underlie cerebral vasculature control  Funding Agency; Funding Title:  Michael Smith Foundation for Health Research  Funding Agency: Funding Title:  Canadian Institutes of Health Research (CIHR)  Funding Agency: Funding Title:  Michael Smith Foundation for Health Research  Funding Agency; Funding Title:  UBC Start Up Funds  Frndrng  Canadian Stroke Network (C SN) Networks of Centres of Excellence (NCE)  Synaptic and non-synaptic modulation of neuronal excitability  Calcium signaling in astrocytes  Synaptic and non-synaptic modulation of neuronal excitability  Synaptic and non-synaptic modulation of neuronal excitability -  4 015  Funding Title:  Neuroprotection: Preventing cell death and neuronal damage from stroke  Funding Agency: Funding Title:  Canadian Institutes of Health Research (CIHR)  Funding Agency: Funding Title:  Michael Smith Foundation for Health Research  Synaptic and non-synaptic mechanisms in controlling neuronal excitability .  Synaptic and non-synaptic modulation of neuronal excitability  7/16/2009 10:55 AM  

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