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Purinergic modulation of neurotransmission Brust, Tyson Brennan 2006

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Purinergic Modulation of Neurotransmission by Tyson B reman Brust A THESIS S U B M I T T E D T O T H E F A C U L T Y O F G R A D U A T E S T U D I E S I N P A R T I A L F U L F I L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F D O C T O R OF P H I L O S O P H Y in T H E F A C U L T Y OF G R A D U A T E S T U D I E S ( N E U R O S C I E N C E ) T H E U N I V E R S I T Y OF B R I T I S H C O L U M B I A June, 2006 © Tyson Brennan Brust, 2006 Abstract Signalling through adenosine- and adenine-nucleotide-gated receptors (purinoceptors) regulates a vast array of physiological processes in the central nervous system, including synaptic transmission. The research presented here describes a role for mitogen-activated protein kinases ( M A P K s ) in mediating molecular signalling cascades initiated by activation of purinoceptors in the hippocampus. In particular, it is proposed that M A P K activation is a requirement for purinoceptor-mediated presynaptic inhibition of neurotransmission. The three primary findings presented here are: 1) P 2 X 7 receptors are localized on mossy fiber terminals where they function to inhibit mossy fiber-CA3 synaptic transmission in a pathway requiring p38 M A P K activation; 2) Adenosine A i receptors exist in a complex with p38 M A P K in which A i receptors activate p38 M A P K to decrease Schaffer-collateral-CAl synaptic transmission; and 3) Sequential activation of C-Jun N-terminal kinase ( INK) following p38 M A P K activation is also required for A i receptor-mediated synaptic depression. Immunocytochemistry was used to demonstrate that P 2 X 7 receptors are abundant on presynaptic terminals of mossy fiber synapses in the rat hippocampus. Western blotting was used to show increases in the phosphorylation state of p38 M A P K and J N K following A i receptor stimulation in the C A 1 region. Co-immunoprecipitation showed that A i receptors are physically associated with p38 M A P K and J N K in the hippocampus. Synaptic function was assessed using field excitatory postsynaptic potentials (fEPSPs) evoked in stratum lucidum in the C A 3 region or in stratum radiatum in the C A 1 region of rat hippocampal slices. Selective stimulation of P 2 X 7 receptors with the agonist B z - A T P potently decreased mossy fiber-CA3 synaptic depression and this was blocked by the P 2 X 7 antagonist oxidized-ATP, but not by the i i P2Xi-3,5,6 antagonist, P P A D s or the P 2 Y antagonist, R B 2 . Bz-ATP-induced synaptic depression was blocked by the p38 M A P K inhibitor SB203580. Stimulation of A i receptors with exogenous adenosine, endogenous adenosine released during hypoxia, or the agonist N 6-cyclopentyladenosine (CPA) depressed evoked fEPSPs in the C A 1 region. These inhibitory responses were blocked with the A i receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine ( D P C P X ) , the p38 M A P K inhibitors SB203580 and SB202190, and the I N K inhibitors SP600125 and I N K Inhibitor V . These results suggest that the inhibitory actions of purinoceptors requires the activation o f p38 M A P K and I N K in the hippocampus. 1 i i i Table of Contents Abstract i i Table of Contents iv List o f Figures v i i List o f Symbols, abbreviations, and pharmacological agents x Acknowledgements x i i i Dedication xiv Co-authorship statement xv Chapter One: Introduction 1 1.1. Hypothesis and Obj ectives 2 1.2. Introduction 5 1.3. Purinergic receptors in the central nervous system 5 1.3.1. Adenine nucleotide-gated receptors 6 1.3.1.1. P 2 X receptors 6 1.3.1.2. _P2Y receptors 8 1.3.2. Adenosine receptors 8 1.4. Sources of extracellular adenosine 9 1.4.1. Extracellular conversion o f adenine nucleotides 10 1.4.2. Release of adenosine through facilitated diffusion transporters 10 1.5. Removal of extracellular adenosine 11 1.6. Functional roles of A T P and adenosine in the C N S 12 1.6.1. Pain 12 1.6.2. Sleep and arousal 14 1.6.3. Epilepsy 16 1.6.4. Alcoholism 17 1.6.5. Neuroprotection 18 1.6.5.1. Mechanism of A i receptor-mediated neuroprotection 19 1.6.5.2. Ischemic preconditioning 22 1.7. Mitogen-activated protein kinases 22 1.7.1. Classic M A P K cascade 23 1.7.2. Signaling by G protein-coupled receptors to M A P K s 23 1.7.3. Involvement of M A P K s in synaptic plasticity 25 1.7.4. Role of M A P K s in cardioprotection and neuroprotection 28 1.7.4.1. The role of A i receptors in p38 MAPK-media ted ischemic preconditioning 29 1.8. Conclusion 30 1.9. References 30 Chapter Two: Activation of P2X7-Like Receptors Depresses Mossy Fiber-CA3 Synaptic Transmission Through p38 Mitogen-Activated Protein Kinase 50 2.1. Introduction 51 2.2. Materials and methods 52 2.2.1. S D S - P A G E & western blotting 52 2.2.2. Immunocytochemistry 53 2.2.3. Electrophysiology 54 iv 2.3. Results 56 2.3.1. P2X7 receptors were found on mossy fiber terminals 56 2.3.2. P2X7 receptor activation depressed mossy fiber-CA3 synaptic transmission 61 2.3.3. Bz-ATP-induced synaptic depression was blocked by o -ATP 66 2.3.5. P2X7 receptor activation increased paired-pulse facilitation 69 2.3.6. P2X7 receptor-mediated synaptic depression required activation o f p 3 8 M A P K 69 2.3.7. Inhibitory effects of adenosine were not mediated through p38 M A P K 71 2.4. Discussion 75 2.5. References 79 Chapter Three: p38 Mitogen-Activated Protein Kinase Contributes to Adenosine Ai-Receptor-Mediated Synaptic Depression in Area CA1 of the Rat Hippocampus 85 3.1. Introduction 86 3.2. Materials and methods 88 3.2.1. Hippocampal Slice Preparation 88 3.2.2. Electrophysiology 88 3.2.3. Immunoprecipitation, Co-immunoprecipitation and Western Blot Analysis . 89 3.2.4. Drugs 92 3.3. Results 92 3.3.1. p38 M A P K activation increases following Ai-receptor stimulation 92 3.3.2. The adenosine A i receptor and phospho-p38 M A P K are physically associated 95 3.3.3. A1-Receptor-dependent increases in p38 M A P K activation are blocked by p38 M A P K inhibition and A l receptor antagonism 96 3.3.4. Ai-Receptor activation induced translocation of protein phosphatase 2a to the plasma membrane 99 3.3.5. Adenosine-induced depression of synaptic transmission is mediated by the A i receptor subtype and is sensitive to p38 M A P K inhibition 103 3.3.6. Neither the inactive analogue SB202474 nor the E R K 1/2 M A P K inhibitor PD98059 decreased adenosine-induced synaptic depression 106 3.3.7. Ai-receptor-mediated synaptic depression was decreased by p38 M A P K inhibition 109 3.3.8. Hypoxia-induced synaptic depression was mediated by the Al-receptor and was attenuated by p38 M A P K inhibition 113 3.4. Discussion 116 3.5. References 122 Chapter Four: Ai Receptor-Mediated Synaptic Depression is Mediated by Sequential Activation of p38 MAPK and C-Jun N-Terminal Kinase in the Rat Hippocampus 132 4.1. Introduction 133 4.2. Materials and methods 135 4.2.1. Hippocampal Slice Preparation 135 4.2.2. Electrophysiology 136 4.2.3. Western Blot Analysis 137 4.2.4. Immunoprecipitation and Co-Immunoprecipation 138 v 4.2.5. Drugs 139 4.3. Results 140 4.3.1. J N K inhibitors attenuate the synaptic depression induced by adenosine, hypoxia, and C P A 140 4.3.2. Ai-receptor-mediated synaptic depression is decreased by SP600125 and J N K Inhibitor V , but not by an inactive analogue 143 4.3.3. J N K inhibition increases the excitability of rat hippocampal slices 147 4.3.4. A i receptor stimulation increases J N K phosphorylation 147 4.3.5. The A i receptor is physically associated with J N K , but not phospho-JNK .151 4.3.6. CPA-dependent increases in J N K phosphorylation are blocked by A i receptor antagonism 152 4.3.7. CPA-dependent increases in J N K phosphorylation are blocked by inhibition of J N K and p38 M A P K 152 4.3.8. CPA-dependent increases in p38 M A P K phosphorylation are blocked by p38 M A P K inhibition but not J N K inhibition 155 4.4. Discussion 158 4.5. References 163 Chapter Five: General Discussion 168 5.1. Introduction 169 5.2. P 2 X 7 receptor-mediated synaptic depression 169 5.2.1. Possible limitations 171 5.2.2. Future Directions 176 5.3. Adenosine A j receptor signalling through p38 M A P K 177 5.3.1. Possible limitations 178 5.3.2. Future Directions 179 5.4. Adenosine A\ receptor signalling through J N K 180 5.4.1. Possible limitations 181 5.4.2. Future Directions 183 5.5. The physiological relevance o f ambient adenosine 187 5.6. A revised role for p38 M A P K and J N K in neuroprotection 191 5.6.1. Whether p38 M A P K and J N K activation is neuroprotective or neurodegenerative may be a matter of timing 191 5.7. Presynaptic inhibition revisited 197 5.7.1. Classic G protein modulation o f neuronal calcium channels 197 5.7.2. M A P K modulation of neuronal calcium channels 199 5.8. Conclusion 202 5.9. References 205 v i List of Figures Figure 1.1. G protein-coupled receptor-dependent activation o f M A P K s 26 Figure 2.1. P 2 X 7 receptors are located on presynaptic terminals of mossy fiber synapses 57 Figure 2.2. P 2 X 7 immunoreactivity was not co-localized with dendritic M A P - 2 immunoreactivity 59 Figure 2.3. P 2 X 7 immunoreactivity was co-localized with presynaptic syntaxin 1A/B immunoreactivity 60 Figure 2.4. The P 2 X 7 agonist, B z - A T P , depressed mossy fiber fEPSPs but had no detectable effect on the presynaptic fiber volley 62 Figure 2.5. Activation of presynaptic P 2 X 7 receptors with B z - A T P selectively depressed synaptically evoked mossy fiber currents in C A 3 65 Figure 2.6. The selective P 2 X 7 antagonist o -ATP blocked B z - A T P induced depression of the mossy fiber-CA3 synaptic responses 67 Figure 2.7. Activation of presynaptic P 2 X 7 receptors increased mossy fiber paired-pulse facilitation (PPF, 50msec) 70 Figure 2.8. P 2 X 7 receptor mediated mossy fiber synaptic depression required p38 M A P K activity 72 Figure 2.9. The p38 antagonist, SB203580, blocked the actions of B z - A T P but not the inhibition by adenosine (30pM) 74 Figure 3.1. C P A increased p38 M A P K phosphorylation in membrane fractions and decreased p38 M A P K phosphorylation in cytosolic fractions 93 v i i Figure 3.2. The increase in p38 M A P K phosphorylation induced by C P A was blocked by p38 M A P K inhibition and A l receptor antagonism 97 Figure 3.3. C P A increases PP2a levels in the membrane fraction 100 Figure 3.4. Adenosine-induced depression of C A 1 fEPSPs is mediated by the A l -receptor subtype and sensitive to p38 M A P K inhibition 104 Figure 3.5. Adenosine-induced synaptic depression was attenuated by SB203580, but not SB202474 (an inactive analogue) nor PD98059 ( E R K 1/2 M A P K inhibitor) 107 Figure 3.6. CPA-mediated depression of C A 1 fEPSPs was attenuated by p38 M A P K inhibition I l l Figure 3.7. p38 M A P K inhibition attenuated hypoxia-mediated depression o f C A 1 fEPSPs 114 Figure 4.1. Synaptic depression induced by adenosine, hypoxia, and C P A is attenuated by I N K inhibition 141 Figure 4.2. Adenosine A ] receptor-mediated synaptic depression is attenuated by I N K inhibition with SP600125 and I N K inhibitor V , but not an inactive analogue 145 Figure 4.3. Stimulation of adenosine A i receptors, which are physically associated with I N K , increases I N K phosphorylation 149 Figure 4.4. The increase in I N K phosphorylation induced by C P A was blocked by A ] receptor antagonism 153 Figure 4.5. Adenosine A i receptor stimulation sequentially activates p38 M A P K and J N K 157 Figure 5.1. Mode l of adenosine A i receptor-MAPK signalling in the nerve terminal 182 Figure 5.2. The A i receptor agonist C P A induces a form of long-term depression that is insensitive to J N K inhibition 186 Figure 5.3. Summary of purinergic signalling through M A P K s in the Hippocampus 204 ix List of Symbols, abbreviations, and pharmacological agents A / C Associative/commissural A i Gj/ 0 protein-coupled adenosine receptor A 2 A Gs/oif protein-coupled adenosine receptor A 2 B G s protein-coupled adenosine receptor A 3 Gi3/ q protein-coupled adenosine receptor aCSF Artificial cerebrospinal fluid A M P Adenosine monophosphate A M P A a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid A R L 67156 A n ecto-ATPase inhibitor A S K 1 Apoptosis signal-regulating kinase 1 ( J N K M A P K K K ) A T P Adenosine triphosphate B z - A T P 2 ' 3' -0-(4-benzoylbenzoyl)-ATP C A 1 Cornu Ammonis; field 1 C A 3 Cornu Ammonis; field 3 C A D O 2-chloroadenosine; A i receptor agonist c A M P Cycl ic adenosine monophosphate C a v Voltage-dependent calcium channel C C P A 2-chloro-N 6-cyclopentyl-adenosine; A ] receptor agonist Cdc42 Small GTPase belonging to the Rho family C H A Cyclohexyladenosine; A i receptor agonist C K 2 Casein Kinase II C N S Central Nervous System COS-7 African Green Monkey SV40-transf d kidney fibroblast cell line C P A N 6-cyclopentyl-adenosine; A i receptor agonist Cterm-ab P 2 X 7 antibody directed towards the intracellular C-terminus D H P G 3,5-dihydroxyphenylglycine; group I m G l u R agonist D M S O Dimethyl sulfoxide D P C P X l,3-dipropyl-8-cyclopentylxanthine; A i receptor agonist EctoAb-1 P2X7 antibody (recognizes an extracellular epitope) EctoAb-2 P 2 X 7 antibody (recognizes an extracellular epitope) E D T A Ethylenediaminetetraacetic acid; divalent cation chelator E E G electroencephalogram E G T A Ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-t etraacetic acid; C a 2 + chelator E N T 1 Gene encoding equilibrative nucleoside transporter 1 E P S C Excitatory postsynaptic current E R K Extracellular signal-regulated kinase ffiPSP Field excitatory postsynaptic potential G protein Guanine nucleotide binding protein G A B A y-aminobutyric acid G D P Guanosine diphosphate Gi Inhibitory; inhibits adenylate cyclase x GIRK G protein-activated inwardly rectifying K + channel Glaxo P2X 7 7 " Transgenic mouse with lacZ gene inserted in beginning of exon 1 GluR Glutamate receptor GR79236 A] receptor agonist G s Stimulatory; activates adenylate cyclase to increase cAMP GTP Guanosine triphosphate GW-493838 A] receptor agonist G a G protein alpha subunit: GTPase domain alpha-helical domain G P y G protein beta-gamma complex h Dentate hilus H C N Ffyperpolarization-activated cyclic nucleotide-gated cation channels H E K Human embryonic kidney I-II linker Cytoplasmic loop connecting domain Ca v a l domains I and II rp Immunoprecipitation ITP Inosine triphosphate I N K C-Jun N-terminal kinase I N K Inhibitor V l,3-Benzothiazol-2-yl-(2-((2-(3-pyridinyl)ethyl)amino)-4-pyrimidinyl)acetonitrile; I N K inhibitor kDa Kilodalton L-CCG-I 2-(carboxycyclopropyl)glycine; group II mGLuR agonist L T D Long-term depression LTP Long-term potentiation luc Stratum lucidum M A P K Mitogen-activated protein kinase M A P K K Mitogen-activated protein kinase kinase (a.k.a. M E K ) M A P K K K Mitogen-activated protein kinase kinase kinase (a.k.a. M E K K ) M E K M A P / E R K kinase (a.k.a. M A P K K ) M E K K M E K kinase (a.k.a. M A P K K K ) M F Mossy fiber MKK3/6 Member of M E K family; phosphorylates p38 M A P K MKK4/7 Member of M E K family; phosphorylates I N K M L K 4 mixed-lineage kinase 4 (INK M A P K K K ) MOPS 3-(N-Morpholino)propanesulfonic acid; 4-Morpholinepropanesulfonic acid; buffer mRNA Messenger ribonucleic acid Na v Voltage-dependent sodium channel N B Q X l,2,3,4-Tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide; AMPA/Kainate receptor antagonist N M D A N-methyl-D-aspartate o-ATP Periodate oxidized ATP PI Adenosine-gated G protein-coupled receptor P2 Adenine nucleotide-gated receptor P2X Ionotropic Adenine nucleotide-gated receptor P2Y Metabotropic Adenine nucleotide-gated receptor p38 M A P K p38 mitogen-activated protein kinase PD98059 2 -Amino-3'-methoxyflavone; M E K inhibitor XI Pfizer P2X7"A Transgenic mouse lacking the C-terminus of the P2X7 gene Phospho-JNK Phosphorylated I N K Phospho-p38 Phosphorylated p38 PI3K Phosphatidylinositol 3-kinases PKA cAMP-dependent protein kinase PKC Protein kinase C PMSF Phenylmethylsulfonyl fluoride PP Protein phosphatase PP2a Protein phosphatase 2a PPADS Pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid Purinoceptor PI or P2 receptor PVDF Polyvinylidene fluoride Racl Small GTPase belonging to the Rho family Rap Member of Ras superfamily of small GTPases Ras Member of Ras superfamily of small GTPases RB2 Reactive blue 2 REM Rapid eye movement Rho Ras-homologous GTPase (Superfamily of small GTPases) SB202190 4-(4-Fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)lH-imidazole; p38 MAPK inhibitor SB202474 4-Ethyl-2(p-methoxyphenyl)-5-(4'-pyridyl)-IH-imidazole; negative control for SB203580 and SB202190 SB203580 4-(4-Fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl) 1H-imidazole; p38 MAPK inhibitor SB239063 trans-1 -(4-Hydroxycyclohexyl)-4-(4-fluorophenyl)-5-(2-methoxypyrimidin-4-yl)imidazole; p38 MAPK inhibitor SEM Standard error of the mean Small GTPase G protein; alternately binds GTP and GDP SP600125 1,9-pyrazoloanthrone; I N K inhibitor T-62 A i receptor allosteric enhancer TAB1 TAK1 binding protein 1 TAK1 Transforming-growth factor P-activated kinase 1 TBS Tris buffered saline TBST Tris buffered saline with Tween 20 TNF-a Tumor necrosis factor-alpha TTX Tetrodotoxin WB Western blot YO-PRO-1 Quinolinium,4-[(3-methyl-2(3H)-benzoxazolylidene)methyl]-l-[3-(triemthylammonio) propyl]-diiodide; fluorescent dye Xll Acknowledgements I would to thank my supervisor and mentor Dr. Brian Mac Vicar for his encouragement, i enthusiasm, and unwavering support. I was also fortunate to have had the opportunity to collaborate with two talented postdoctoral fellows: Franscisco S. Cayabyab and John N . Armstrong. Thanks for imparting your knowledge and your friendship. I would like to thank my supervisory committee members Dr. Steven R. Vincent, Dr. Steven Pelech, and Dr. Y u Tian Wang for their helpful ideas and comments. I would also like to thank my St. John's College friends (you know who you are) and the members of the Mac Vicar lab for helping to maintain my sanity: Dustin Hines, Brent Kuzmiski, Sean Mulligan, Charles (Chucky) Stringer, Chao Tai, Roger Thompson, and Ning Zhou. In addition, I would like to thank Tom Smith for proofreading an earlier version of this manuscript. Finally, I would like to acknowledge financial support from the Natural Science and Engineering Research Council, the Alberta Heritage Foundation for Medical Ressearch, and the NeuroScience Canada Foundation. Xll l Dedication To Walter and Susan Co-authorship statement Chapter 2: Activation of P2X7-Like Receptors Depresses Mossy Fiber-CA3 Synaptic Transmission Through p38 Mitogen-Activated Protein Kinase For this study, I contributed field excitatory postsynaptic potential (fEPSP) recordings for figures 2, 4, 6, and 7. Chapter 3: p38 Mitogen-Activated Protein Kinase Contributes to Adenosine Ai-Receptor-Mediated Synaptic Depression in Area CA1 of the Rat Hippocampus I was the lead researcher on this study. I did all the fEPSP recordings and approximately half of the Western Blotting. I also assisted with the co-immunoprecipitation experiments. Chapter 4: C-Jun N-Terminal Kinase Regulates Adenosine Ai Receptor-Mediated Synaptic Depression in the Rat Hippocampus I took a lead role in this study. I contributed all the fEPSP recordings and approximately half of the Western Blot experiments. I assisted with the co-immunoprecipitation experiments as well . xv C h a p t e r O n e : I n t r o d u c t i o n i 1.1. Hypothesis and Objectives The purpose of this research was to investigate the mechanisms by which adenosine triphosphate (ATP) and adenosine modulate neurotransmission in the hippocampus. The three main areas of investigation were: 1) modulation of mossy fiber-CA3 synaptic transmission by ATP acting at P2X 7 receptors; 2) modulation of CA1-CA3 synaptic transmission by adenosine A i receptors signalling through p38 mitogen-activated protein kinase (MAPK); and 3) modulation of CA1-CA3 synaptic transmission by adenosine A) receptors signalling through C-Jun N-terminal kinase (JNK). These areas were chosen because signalling by adenosine and adenine nucleotides (purinergic signalling) is crucial for brain function in both physiological and pathophysiological conditions. The hippocampal slice preparation was chosen for these studies because it exhibits well defined forms of synaptic plasticity at identified synapses, is specifically vulnerable to ischemia, and its anatomical organization makes it particularly suitable for extracellular electrophysiological recordings. The hippocampus is not only an ideal model for studying the mechanisms underlying synaptic transmission, but it is also of great functional relevance because it is required for the formation of new memories. 2 Hypothesis 1: P2X 7 receptors are expressed on mossy fiber terminals where they function to decrease mossy f iber-CA3 synaptic transmission. Object ives: 1. Image the cellular and subcellular distribution of P 2 X 7 receptors in the hippocampus using immunohistochemistry. 2. Determine whether P 2 X 7 receptor activation modulates synaptic transmission by recording whole-cell currents and extracellular field excitatory postsynaptic potentials (fEPSPs) evoked in stratum lucidum of hippocampal slices. Hypothesis 2: Adenosine A i receptors decrease C A 3 - C A 1 synaptic transmission through p38 mitogen-activated protein kinase ( M A P K ) . Object ives: 1. Quantify changes in the phosphorylation state o f p38 M A P K caused by adenosine A i receptor stimulation using Western blot analysis. 2. Resolve whether A l receptors and p38 M A P K are physically associated using co-immunoprecipitation. 3. Determine whether inhibiting p38 M A P K decreases the magnitude of A\ receptor-dependent depression of fEPSPs evoked in the C A 1 region of rat hippocampal slices. 3 Hypothesis 3: Adenosine Ai receptors decrease CA3-CA1 synaptic transmission through p38 M A P K mediated activation of C-Jun N-terminal kinase (INK). Objectives: 1. Determine whether inhibiting INK modulates the magnitude of Ai receptor-dependent depression of fEPSP amplitudes in the CA1 region of rat hippocampal slices 2. Quantify changes in the phosphorylation state of JNK in response to Ai receptor stimulation using Western blot analysis. 3. Analyze the dependence of JNK activation on p38 M A P K activation using pharmacology and Western blot. 4. Determine whether JNK (or phosphorylated JNK) are physically associated with the A] receptor in the hippocampus using co-immunoprecipitation. 4 1.2. Introduction Extracellular A T P and adenosine are ubiquitous transmitters of chemical signals between neurons. A T P is present in vesicles containing both peptides and small-molecule neurotransmitters and is released by exocytosis at virtually all synapses. Adenosine is a degradation product of A T P metabolism and is arguably the most widespread and potent inhibitor of excitatory synaptic transmission in the nervous system. A T P and adenosine transmission, or purinergic transmission, is highly conserved across species and cell types. A T P and adenosine play functional roles in an array of neural processes that include: regulating sleep and arousal; analgesia; alcohol addiction; epilepsy; and neuroprotection (Dunwiddie and Masino, 2001; Burnstock, 2004). Although the actions of A T P and adenosine in the nervous system have been known for some time, the signalling by which purinergic transmission occurs has not been studied in detail. 1.3. Purinergic receptors in the central nervous system A T P produces diverse physiological effects by activating multiple P2 receptors. Adenosine is generated following hydrolysis of A T P by the ecto-nucleotidase cascade (Zimmermann and Braun, 1 9 9 9 ) , which terminates P2 receptor signalling and stimulates G protein-coupled PI receptors. PI and P2 receptors are co-expressed in most cells (Matsuoka and Ohkubo, 2004). Considerable effort has gone into distinguishing PI and P2 receptor-mediated effects using a number of pharmacological and biochemical tools such as metabolically stable P2-receptor agonists, PI and P2 receptor antagonists, adenosine uptake inhibitors, and adenosine deaminase. Nonetheless, the actions of specific purinoceptor subtypes can be difficult to isolate because of overlapping 5 pharmacological profiles. For example, it can be a challenge to distinguish P2X4 and P2X 7 receptor-mediate effects because of the lack of specific antagonists (North and Surprenant, 2000). 1.3.1. Adenine nucleotide-gated receptors P2 receptors include ionotropic P2X and G protein-coupled P2Y receptors. Both receptors exhibit various effects at neuronal and glial cells, and are distributed widely in the central nervous system (Abbracchio and Burnstock, 1994; Ralevic and Burnstock, 1998; North, 2002). In astrocytes, P2Y receptors predominate over P2X receptors and mediate short-term increases in intracellular C a 2 + and eventually effects such as proliferation and apoptosis. Both classes of P2 receptors are also expressed in neurons, where P2X receptors mediate fast synaptic responses to ATP and P2Y mediates slow changes in membrane excitability in response to either non-synaptically released ATP or interactions with other receptors. Neuronal P2 receptors also regulate trophic effects such as neurite outgrowth, neuronal maturation, and the expression of new receptors. 1.3.1.1. P2X receptors P2X receptors form a family of at least seven subunits (P2Xi_7) and occur as stable trimers (three subunits), hexamers (six subunits), or multimeric assemblies of P2X 2 /P2X 3 , P2X4/P2X6, or P2X]/P2X 5 . P2X 7 receptors form homomeric channels. Each subunit is comprised of two transmembrane domains, an ATP binding site contained on a large extracellular loop, and intracellular N and C termini (North, 2002). Traditionally, it was thought that P 2 X i . 6 receptor subtypes were predominantly localized in neurons, whereas P2X 7 receptors were reported only in activated microglia, 6 macrophages, or lymphocytes, which is consistent with its known role in repairing brain damage due to immune insult, inflammation, or infarction (North, 2002). In recent years, it has been proposed that P2X7 receptors are also present in presynaptic excitatory terminals in the spinal cord and brain (Deuchars et al., 2001; Armstrong et al., 2002; Atkinson et al., 2002; Lundy et al., 2002; Sperlagh et al., 2002; Ishii et al., 2003; Cavaliere et al., 2004), although these findings have since generated controversy (see discussion page 169). Both P 2 X 2 and P2X4 are unequivocally localized at postsynaptic specializations of synapses in the cerebellum and hippocampus, where the P 2 X 2 receptor facilitates inward currents in C A 1 interneurons (Khakh et al., 2003). In general, P 2 X receptors mediate neuronal inward currents throughout the C N S (Hies and Ribeiro, 2004), supporting the notion that A T P is a fast synaptic transmitter. P 2 X receptors may also play a role in synaptic plasticity. When A T P (that is coreleased with glutamate) acts at postsynaptic Ca 2 +-permeable P 2 X receptors, calcium enters the cell and inactivates N M D A receptors, a process that tonically inhibits the effectiveness of N M D A receptors in inducing L T P (Pankratov et al., 2002). Indeed, inhibition of P 2 X receptors by P P A D S greatly facilitates the induction of L T P (Pankratov et al., 2002). Ca 2 +-dependent inactivation of N M D A receptors by P 2 X receptors provides a mechanism to prevent weak stimuli from inducing L T P . Astrocytic release of A T P , glutamate, and G A B A may also modulate synaptic transmission. The stimulation of P2X7 receptors on astrocytes leads to astrocytic release of both glutamate (Duan et al., 2003) and G A B A (Wang et al., 2002a), providing a link between the secretion of A T P and excitatory or inhibitory neurotransmitters and thus dynamically regulating synaptic transmission. 7 1.3.1.2. P 2 Y r e c e p t o r s P 2 Y receptors form a family of ten cloned and functionally defined subtypes, o f which P 2 Y j , P 2 Y 2 , P 2 Y 6 , P 2 Y n , P 2 Y j 2 , P 2 Y , 3 , and P 2 Y , 4 are expressed in the C N S . P 2 Y receptors contain the seven hydrophobic transmembrane domains typical of G protein-coupled receptors. The diversity of P 2 Y receptor-mediated functions is probably attributable to the differential expression of each subtype in different brain regions and cell types. For example, P2Yj is localized in neurons, and particularly abundant in nucleus accumbens, striatum, caudate nucleus, putamen, globus pallidus, cerebellum, hippocampus, and throughout the cerebral cortex, whereas P 2 Y ] 2 is predominantly expressed in glia (Illes and Ribeiro, 2004). P2Y] and P 2 Y 2 receptors are also expressed in astrocytes where they mediate the propagation of Ca 2 + waves between astrocytes induced by the release o f A T P (Haydon, 2001). 1.3.2. Adenosine receptors PI receptors include the A i , A 2 A , A 2 B , and A 3 subtypes, each of which has a distinct tissue distribution, pharmacological profile, and effector coupling. For example, the Gj protein-coupled A j receptor and G s protein-coupled A 2 A receptor have opposing effects on adenylyl cyclase activity. The stimulant effects of adenosine receptor antagonists such as caffeine are due to antagonism of A i and A 2 A receptor subtypes. The A 2 A receptor is particularly abundant in the striatum, where it functionally interacts with dopamine receptor signalling. The A i receptor is the predominant subtype found in the C N S , and is abundant in the spinal cord, cerebellum, hippocampus, and cerebral cortex. A j receptor stimulation causes decreased release o f every classical neurotransmitter, including glutamate^ G A B A , 8 acetylcholine, norepinephrine, 5-hydroxytryptamine, dopamine, and others. The effect of A i stimulation is most dramatic on excitatory synaptic transmission, which can be completely blocked by adenosine. A] receptors are expressed both pre- and postsynaptically. Presyaptic A i receptors are resistant to desensitization whereas postsynaptic A i receptors are not. The mechanism by which A i receptor stimulation inhibits neurotransmission appears to depend on a G protein-coupled inhibition of N-type C a 2 + channels in presynaptic terminals, although adenosine also inhibits spontaneous Ca 2 +-independent release o f neurotransmitter (Scanziani et al., 1992). Other mechanisms may contribute as well , as stimulation of postsynaptic A i receptors activates G protein-dependent inwardly rectifying K + channels (GIRKs) causing hyperpolarization of the resting membrane potential. 1.4. Sources of extracellular adenosine A basal purinergic tone, maintained by tonic activation of A i and A 2 A receptors, permeates most tissues. The concentration of extracellular adenosine under normal conditions is in the range of 25-250 n M . Basal adenosine tone reflects a balance between mechanisms that increase extracellular adenosine pitted against mechanisms that remove adenosine through uptake or metabolism. Unlike A T P , adenosine is not released in vesicles like a classical neurotransmitter. Rather, adenosine reaches the extracellular space by two primary mechanisms: 1) dephosphorylation of adenine nucleotides by ecto-nucleotidases; and 2) release of adenosine through nucleoside transporters. 9 1.4.1. Extracellular conversion of adenine nucleotides Ecto-nucleotidases, ecto-phosphodiesterases, and apyrases are widely expressed and rapidly (in less than a second (Dunwiddie et al., 1997)) dephosphorylate any adenine nucleotide to 5 ' - A M P , which is then dephosphorylated to adenosine by 5'-nucleotidase (Zimmermann, 2000). Adenine nucleotides accumulate in the extracellular space due to co-release o f A T P with classical neurotransmitters (White, 1977; Fredholm et al., 1982) or release of c A M P by a probenecid-senstive transporter (Rosenberg and L i , 1995), both of which are sufficient to cause large increases in extracellular adenosine following hydrolysis by ecto-enzymes (Dunwiddie et al., 1992; Brundege et al., 1997). A T P is also released from glial cells. The rapid and localized production of adenosine by ecto-nucleotidases in close physical proximity to presynaptic inhibitory A i receptors on the membrane surface is an important determinant of cellular responses to A T P . 1.4.2. Release of adenosine through facilitated diffusion transporters Equilibration of adenosine concentrations across cellular membranes occurs through facilitated diffusion nucleoside transporters (Boumah et al., 1994). Nucleoside transporters are bidirectional and passive; that is, they do not depend on energy derived from A T P or ionic gradients to transport adenosine. Adenosine concentrations inside cells are normally low due to the high activity of intracellular adenosine kinase (which converts adenosine to 5 ' - A M P ) , resulting in a net inward flux through these transporters. Adenosine is released into the extracellular space by nucleoside transporters when intracellular adenosine concentrations rise, as occurs during pathological conditions such as hypoxia/ischemia. Because nucleoside transporters are bidirectional, the regulation of 10 extracellular adenosine is intimately linked to intracellular adenosine concentrations. A s intracellular adenosine concentrations rise, adenosine can no longer be taken up from the extracellular space by nucleoside transporters, and when the concentration of adenosine becomes greater inside the cell than outside the cell, direct efflux of adenosine occurs. Adenosine formation within cells depends on the breakdown of A T P , and occurs as a result of the action of cytosolic-5'-nucleotidase on 5 ' - A M P (Zimmermann and Braun, 1996). Another source of 5 ' - A M P is metabolism of c A M P by c A M P phosphodiesterase. In general, adenosine is released when the brain's ability to synthesize A T P is outstripped by its energy requirements. The large increase in energy requirements that occurs during seizure, or the loss of metabolic substrates that occurs during ischemia, reduces A T P levels and increases the level of adenine nucleotides and adenosine. 1.5. Removal of extracellular adenosine The primary mechanism by which adenosine is cleared from the extracellular space is by transport of adenosine into cells followed by either deamination to inosine by adenosine deaminase or phosphorylation to A M P by adenosine kinase (Latini and Pedata, 2001). Uptake inhibitors dramatically increase the concentration of extracellular adenosine, indicating that under normal conditions the majority of adenosine in the extracellular space is cleared by uptake (Dunwiddie and Diao, 1994). In support of this concept, the nucleoside transport inhibitors such as propentofylline protect against brain damage after global ischemia by increasing extracellular adenosine (Fredholm et al., 1994). During hypoxia and ischemia, adenosine transporters are not able to remove adenosine, allowing adenosine deaminase activity to assume the primary role in removing adenosine (Lloyd and Fredholm, 1995; Barankiewicz et al., 1997; Dupere et al., 1999). 11 1.6. Functional roles of ATP and adenosine in the C N S Purinergic transmission plays a functional role in a vast array o f physiological and pathological processes in the C N S . Adenine nucleotide-gated P2 receptors have been implicated in the pain.associated with cancer, reflex sympathetic dystrophy, angina, causalgia, lumbar, and migrane, as well as inflammation, seizure induction, astrocyte proliferation, and astrogliosis associated with ischemia and neurodegenerative disorders (Burnstock, 2004). Observations that awareness and learning were enhanced by caffeine, which is the most widely used psychoactive drug in the world and a classical adenosine receptor antagonist, has long been an impetus into studying the role of adenosine in the nervous system. Numerous lines of evidence now show that adenosine PI receptors have functions in sleep and arousal, seizure susceptibility, analgesia, locomotor effects, neuroprotection, Parkinson's disease, anxiety, and alcohol and drug addiction (Dunwiddie and Masino, 2001). Some of the most important functions of A T P and/or adenosine in normal and pathological physiology are discussed in further detail below. 1.6.1. Pain Systematic administration of A T P elicits pain responses. The antinociceptive properties of non-selective P2 receptor antagonists (e.g. suramin) may be due to antagonism of P2X3 receptors located on sensory neurons in trigeminal, nodose, and dorsal root ganglia (Burnstock, 1996). In support of a role for P 2 X 3 receptors in pain, nociception and inflammation are reduced in P 2 X 3 knockout mice (Cockayne et al., 2000). 12 Adenosine also influences pain transmission through multiple mechanisms at peripheral and spinal sites. The A i receptor is abundant in the dorsal horn of the spinal cord, where the density o f receptors is almost as high as it is in the hippocampus (Johansson et al., 2001). A i receptors are responsible for the analgesic effects of intrathecally administered A i agonists, which cause antinoception by reducing c A M P levels in sensory nerve terminals (Sawynok, 1998). A i receptor knockout mice show increased hyperalgesia, and loss of the analgesic effect of adenosine characteristic of wild-type animals (Johansson et al., 2001), although A l receptors might be more important in chronic pain than acute pain (Wu et al., 2005). In humans, adenosine infusion into the spinal cord decreases post-operative pain (Gordh et al., 1995). Adenosine A] receptor activation might be an effective treatment for migraine and cluster headache because it inhibits neurotransmission without concomitant vasoconstriction (Giffin et al., 2003). Two A ! receptor agonists (GR79236 and G W -493838) and an A i receptor-selective allosteric enhancer (T-62) are of clinical interest for the treatment of migraine and neuropathic pain (Giffin et al., 2003; L i et al., 2003; Zambrowicz et al., 2003). It should be noted that caffeine has analgesic effects against some (but not all) types of pain (Fredholm et al., 1999), which is contrary to what might be expected based on the above studies showing that A] receptor agonists are antinociceptive. This discrepancy probably arises because caffeine acts at A 2 A as well as A i receptors (Ledent et al., 1997; Huang et al., 2005), and A 2 A receptors have different effects on pain transmission than A i receptors. For example, A 2 A knockout mice exhibit slower responses to hot plate, tail-flick, and tail immersion tests than wild-type mice (Ledent et 13 al., 1997; Berrendero et al., 2003). In the spinal cord, adenosine released following opioid stimulation acts at both A) and A 2 A receptors to elicit antinociception (Sweeney et al., 1987; De Lander and K e i l , 1994), whereas in the periphery, activation o f A 2 A receptors stimulates nociceptive nerve terminals (McQueen and Ribeiro, 1986). Thus, the increased nociceptive threshold (i.e. increased tolerance for pain) in A 2 A knockout mice suggests that peripheral nociceptive A 2 A receptor-mediated nociception predominates over spinal A 2 A receptor-mediated antinociception. The above observations indicate that adenosine exerts a wide variety of effects on pain depending on the site of action and the subtype ( A i or A 2 A ) o f receptor that is activated. 1.6.2. Sleep and arousal Because caffeine promotes wakefulness, it is logical to assume that adenosine plays a role in sleep and the regulation o f arousal. The sleep-inducing effects of adenosine were first described in cats by Feldberg and Sherwood (1954), and have since been repeatedly demonstrated in a variety of species (Haulilca et al., 1973; Dunwiddie and Worth, 1982; Virus et al., 1983; Radulovacki et al., 1984; Radulovacki et al., 1985). Adenosine levels progressively increase during prolonged wakefulness and decrease during recovery sleep (Huston et al., 1996; Porkka-Heiskanen et al., 1997; Porkka-Heiskanen, 1999). The oscillation of adenosine concentrations through the circadian rhythm is consistent with evidence that adenosine release is directly proportional to metabolism and neural activity (Pull and Mcl lwa in , 1972; V a n Wylen et al., 1986; Tobler and Scherschlicht, 1990). Consistent with this idea is the fact that metabolic rate during wakefulness is approximately 30% higher than during n o n - R E M sleep (Madsen et al., 1991; Maquet et al., 1992; Madsen, 1993). Because one of the functions of sleep is probably energy 14 restoration, it has been proposed that adenosine, as a by product of energy metabolism, may play a role in sleep-wake behaviour by serving as a homeostatic regulator of energy in the brain during sleep (Chagoya de Sanchez et al., 1993; Benington and Heller, 1995). The A i receptor is the primary receptor subtype involved in regulating sleep, as sleep is induced by A i receptor agonists and reduced by antagonists (Virus et al., 1990; L i n et al., 1997; Portas et al., 1997). In addition, an A] receptor antisense construct decreases R E M sleep and increases wakefulness when infused into the basal forebrain (Thakkar et al., 2003). Possible mechanisms by which A i receptors induce sleep include tonically inhibiting the firing of long cholinergic neurons by increasing K + conductances (Rainnie et al., 1994) and disinhibiting the G A B A e r g i c input to ventrolateral preoptic neurons (Chamberlin et al., 2003). In support of these mechanisms, adenosine dialysis into cholinergic nuclei or the preoptic area in vivo promotes sleep and reduces the level of arousal as measured by E E G activity (Rainnie et al., 1994; Portas et al., 1997). However, it should be noted that the amount of sleep or rebound after sleep deprivation is identical in A) receptor knockout mice as in wild-type controls, even though an A i receptor antagonist reduced sleep in control animals (Stenberg et al., 2003). Thus, it appears that A i receptors are not absolutely required for sleep because other unknown adaptive regulatory mechanisms are capable of taking over in their absence. In summary, adenosine is an important endogenous homeostatic sleep factor that inhibits wake promoting neurons in the cholinergic basal forebrain via A i receptor activation. 15 1.6.3. Epilepsy A role for adenosine as an endogenous modifier of seizures was long suspected because adenosine is a well known inhibitory neuromodulator that suppresses repetitive firing. In support for this notion, seizure susceptibility was reported to increase in the brain when adenosine levels were decreased (Gouder et al., 2004). It is well established that adenosine receptor agonists have anticonvulsant effects (Dunwiddie and Worth, 1982; Barraco et al., 1984; Zhang et al., 1990), whereas antagonists have proconvulsant effects (Dunwiddie and Hoffer, 1980; Aul t et al., 1987; Alzheimer et al., 1989). The anticonvulsant effects o f adenosine are mainly mediated by A i receptors (Murray et al., 1992; Zhang et al., 1994), although the A 2 A receptor may play a role in some brain regions as well (De Sarro et al., 1999). The above evidence suggests that raising the extracellular concentration of adenosine in the brain may be a viable therapeutic intervention against seizures. This is indeed the case, as transplanting adenosine-producing cells into rat brain decreases seizure susceptibility (Huber et al., 2001; Boison et al., 2002). A chronic reduction of A i receptors, and hence a loss of tonic inhibition, also occurs in epileptic tissue in both rats and humans, which might explain the hyperexcitability and recurrent seizures that are characteristic of epilepsy. In further support of a clinical relevance for local adenosine release in epilepsy, mouse myoblasts were recently engineered to release adenosine by genetic inactivation of adenosine kinase and grafted into the lateral brain ventricles of rats kindled in the hippocampus (Guttinger et al., 2005). These grafts conferred complete long-term protection (up to 8 weeks) from the convulsive seizures observed in rats with wild-type 16 grafts, suggesting that adenosine release from cellular implants is a feasible option for the long-term treatment of focal epilepsies. 1.6.4. Alcoholism The popular belief that caffeine can counteract the intoxicating effects of alcohol has led to the hypothesis that adenosine receptors, and A 2 A receptors in particular, play a role in mediating the intoxication resulting from acute alcohol administration. In support of this hypothesis, there is a growing body of evidence that adenosine mediates the neuronal responses to ethanol, and thus may be involved in a number of physiological effects such as alcohol-induced ataxia and alcohol addiction (Mailliard and Diamond, 2004). Extracellular adenosine increases in response to ethanol exposure because ethanol inhibits a specific nucleoside transporter (ENT1) preventing adenosine uptake (Diamond et al., 1991; Krauss et al., 1993). This increase in extracellular adenosine activates G s -coupled A 2 A receptors, leading to increased c A M P , activation of P K A I & II, P K A II translocation to the nucleus, and cAMP-induced gene expression (Mailliard and Diamond, 2004). Addictive drugs such as ethanol increase dopamine release in the nucleus accumbens, a brain region intimately involved in reward and reinforcement (Hyman and Malenka, 2001). Activation of the dopamine D 2 receptor in particular has been implicated in the behaviour of alcohol consumption (Phillips et al., 1998). The microinjection of dopamine D 2 receptor agonists or antagonists into the nucleus accumbens can increase or decrease ethanol self-administration (Hodge et al., 1997). Interestingly, the most abundant expression of A 2 A receptors in the brain occurs in the i medium spiny neurons of the nucleus accumbens (Jarvis et al., 1989; Fredholm et al., 17 1998). D 2 receptors and A 2 A receptors are coexpressed in these neurons (Fink et al., 1992) , where they synergistically activate P K A and cAMP-dependent gene expression (Yao et al., 2002). Ordinarily, G s coupled A 2 A and Gj coupled D 2 receptors are antagonistic. The synergistic action of A 2 A and D 2 receptors depends on the release of Gj/o py dimers (Yao et al., 2002) that potentiate cAMP production via adenylate cyclase isoforms II and IV (Tang and Gilman, 1991; Federman et al., 1992; Inglese et al., 1994; Baker et al., 1999). Blocking Py dimers by expression of a Py inhibitor in the nucleus accumbens decreases voluntary consumption of alcohol (Yao et al., 2002), suggesting that the signalling pathways responsible for A 2 A / D 2 synergy could be targeted for treating alcoholism. 1.6.5. Neuroprotection The brain is extraordinarily vulnerable to ischemic events due to its high metabolic rate. A 5 minute interruption of cerebral blood flow is sufficient to kill neurons, whereas it takes 20-40 minutes of ischemia to kill cardiac or kidney cells (Lee et al., 2000a). The very intracellular and intercellular signalling mechanisms that normally make the brain so specialized for information processing become its bane under ischemic conditions, accelerating cell death by rapid energy depletion and excessive excitation (Lee et al., 2000a). Adenosine is thought to be an endogenous neuroprotective agent in the CNS because it attenuates damage caused by electrical activity (Arvin et al., 1989; Lloyd et al., 1993) , excitotoxicity (Arvin et al., 1988; Arvin et al., 1989; Finn et al., 1991; MacGregor and Stone, 1993), hypoxia (Gribkoff and Bauman, 1992; Fowler, 1.993b, 1993a), ischemia (Lloyd et al., 1993; Latini et al., 1999a), and even mechanical stimulation 18 (Mitchell et al., 1995) and methamphetamine-induced neurotoxicity (Delle Donne and Sonsalla, 1994). Increasing extracellular adenosine by preventing its uptake or inhibiting its degradation (by administering adenosine kinase and deaminase inhibitors) protects against excitotoxic or ischemic brain injury (DeLeo et al., 1988; Phill is and O'Regan, 1989; Dux et al., 1990; L i n and Phillis, 1992; Gidday et al., 1995; Johnson et al., 1998). Similar results are obtained by stimulating A i (Rudolphi et al., 1992) receptors or blocking A 2 A receptors (Jones et al., 1998), whereas A i receptor antagonists exacerbate the resulting neuronal damage (Rudolphi et al., 1992; Zhou et al., 1994). In animal models of global or focal ischemia, local administration of C A D O , an adenosine analogue, attenuates cell death in the C A 1 region of the hippocampus (Evans et al., 1987). Specific A) receptor activation during global forebrain ischemia (with the agonists C H A , C P A , or C C P A ) reduces mortality and neuronal loss in the hippocampus and improves neurological deficits (von Lubitz et al., 1988; Januszewicz von Lubitz et al., 1989; von Lubitz and Marangos, 1990; V o n Lubitz et al., 1994; Zhou et al., 1994; V o n Lubitz et al., 1996). In contrast, acute administration of A i receptor antagonists such as theophylline or D P C P X during ischemia potentiates mortality and degeneration in hippocampal cells and leads to memory impairment (Rudolphi et al., 1987; Boissard et al., 1992; Zhou et al., 1994; Phill is, 1995). 1.6.5.1. M e c h a n i s m of receptor-mediated neuroprotect ion There are three cellular mechanisms by which A i receptor activation leads to neuroprotection: 1) inhibition of voltage-dependent C a 2 + channels and decreased C a 2 + influx; 2) inhibition of neurotransmitter release (particularly glutamate); and 3) activation o f postsynaptic G I R K s leading to membrane hyperpolarization (Dunwiddie and Masino, 19 2001). These actions are neuroprotective both because they limit Ca entry, which is a key trigger of excitotoxic damage (Sattler and Tymianski, 2000), and because they reduce metabolic demand, thus preserving A T P stores necessary for pumping C a 2 + out o f cells. Interruption of cerebral blood flow results in a rapid rise in adenosine levels in cortical areas in a number o f species (Rudolphi et al., 1992; de Mendonca et al., 2000), which can then activate presynaptic A] receptors leading to decreased C a 2 + influx through N-type voltage-dependent C a 2 + channels (Mogul et al., 1993; Yawo and Chuhma, 1993; Mynl ief f and Beam, 1994; W u and Saggau, 1994; Ambrosio et al., 1997; Zhang and Schmidt, 1999; Brown and Dale, 2000; Park et al., 2001; Sun et al., 2002; Wang et al., 2002b; Manita et al., 2004) and thus decreased glutamate release. Excitatory amino acids such as glutamate, which are released immediately after ischemic injury, induce cell death by causing excessive membrane depolarization and a rise in intracellular C a 2 + (Simpson et al., 1992; Martin et al., 1994; Arundine and Tymianski, 2003). The net result of presynaptic A i receptor activation is a decrease in N M D A receptor activation and less NMDA-media ted C a 2 + influx into neurons, which is vitally important in achieving neuroprotection. In addition to activating presynaptic A] receptors, adenosine also mediates neuroprotection by activating postsynaptic A i receptors. The hypothesis that postsynaptic A i receptors are involved in neuroprotection has been tested in a number of models in which A] receptor agonists were found to attenuate the neuronal damage induced by N M D A , kainate, quisqualate, or ibotinate (Arvin et al., 1988; A r v i n et al., 1989; Finn et al., 1991; MacGregor and Stone, 1992; MacGregor et al., 1993; MacGregor et al., 1996b; MacGregor et al., 1996a; MacGregor et al., 1997; MacGregor et al., 1998). 20 Moreover, A t receptor antagonists potentiate kainic aeid-induced neurotoxic effects in the hippocampus (MacGregor and Stone, 1992; MacGregor et al., 1993; MacGregor et al., 1996b; MacGregor et al., 1996a; MacGregor et al., 1997; MacGregor et a l , 1998). Postsynaptic A i receptor stimulation reduces the excitotoxic effects of excitatory amino acids by activating G I R K s and thus counteracting excessive membrane depolarization by increasing K + efflux (Dunwiddie and Masino, 2001). The result is decreased neuronal C a 2 + influx through voltage dependent C a 2 + channels. Adenosine may act as a neuroprotector by elevating the threshold for N M D A receptor channel opening and thus maintaining C a 2 + homeostasis in postsynaptic neurons. Given the anti-neuroprotective effects of adenosine receptor antagonists, it would be reasonable to predict that chronic consumption of caffeine would potentiate ischemic injury. Surprisingly, this is not the case. In contrast to the damaging effects of acute administration of caffeine or D P C P X , chronic administration (2-4 weeks prior to ischemic insult) of these compounds lowers neuronal injury as assessed by magnetic resonance and histology (Rudolphi et al., 1989; Sutherland et al., 1991; V o n Lubitz et al., 1994; Rudolphi and Schubert, 1997). The beneficial effects of chronic administration of A i receptor antagonists may be due to upregulation of A i receptors (Rudolphi et al., 1989), although such a mechanism is still the subject of debate (Jacobson et al., 1996). Finally, it should be noted that there is no difference in the severity of ischemic damage between A] receptor knockout and wi ld type mice, even though acute administration of an A i receptor antagonist potentiated ischemic damage in vivo in response to the same ischemic insult (Olsson et al., 2004). This is surprising considering the amount of evidence supporting a role for the A i receptor in neuroprotection, and 21 highlights one o f the pitfalls of using knockout mice to study receptor function: the emergence of compensatory mechanisms. 1.6.5.2. I s c h e m i c p r e c o n d i t i o n i n g Cerebral ischemic preconditioning is a phenomenon in which a brief episode of sublethal ischemia increases the brain's tolerance for subsequent longer and more severe ischemic insults, presented hours or even days later (Lee et al., 2000a). Ischemic preconditioning was initially described in the heart (Swain et al., 1984), but it occurs in a variety of organs (Hawaleshka and Jacobsohn, 1998). The primary mechanisms underlying ischemic preconditioning in the brain are thought to be adenosine release, activation of Ai receptors, activation of M A P K s , and the opening of ATP-dependent K + channels (Heurteaux et al., 1995). Adenosine's involvement in ischemic preconditioning highlights its central role in endogenous neuroprotection. 1.7. Mitogen-activated protein kinases Integrated function of the cell is achieved through highly interactive networks of protein kinases and other second messenger systems. This is partly achieved by activation or deactivation of regulatory proteins through modulation of protein phosphorylation states. Mitogen-activated protein kinases ( M A P K s ) are a ubiquitous family o f conserved signal transducers within the cellular signalling network. There are three important members of this family: 1) the extracellular signal-regulated kinase ( E R K ) ; 2) p38 M A P K ; and 3) C -Jun N-terminal kinase (JNK), which are classified according to their phospho-acceptor motif (T /SEY, T G Y , and T P Y respectively). Each is activated by dual threonine and tyrosine phosphorylation. The E R K group consists of E R K 1 (p44 M A P K ) , E R K 2 (p42 22 M A P K ) , E R K 3 a , E R K 3 P , E R K 5 , and E R K 7 . There are three J N K gene products ( JNK1, J N K 2 , and J N K 3 ) and four p38 M A P K gene products (p38a, p38p, p38y, and p38a). Classic M A P K functions include regulation of cell differentiation, cell proliferation, embryogenesis, and cell death. However, a substantial amount of evidence has emerged over the course of the past decade that also implicates M A P K s in ion channel regulation, cardioprotection, neuroprotection, synaptic plasticity, and learning and memory. 1.7.1. Classic MAPK cascade M A P K activation is rapid, allowing cells to respond to environmental changes in a tightly controlled series of three sequential intracellular protein kinase activation steps. The M A P K cascade is initiated by activation of M A P K kinase kinase ( M A P K K K ) , which in turn phosphorylates (and thus activates) M A P K kinase ( M A P K K ) , which then finally activates the specific M A P K . M A P K s are proline-directed kinases, meaning that they regulate a wide array o f protein kinases, nuclear proteins, and transcription factors through phosphorylation of serine or threonine residues that are adjacent to proline. 1.7.2. Signaling by G protein-coupled receptors to MAPKs Receptors that transduce signals through activation of heterotrimeric GTP-binding proteins (G proteins), or G Protein-coupled receptors, form the largest superfamily of cell surface receptors encoded by the human genome, with over 1000 members (Flower, 1999). G protein-coupled receptors are activated by a wide array of stimuli, such as polypeptides, chemoattractants, growth factors, hormones, neurotransmitters, phospholipids, odorants, photons, and taste ligands. G protein-coupled receptors contain 23 a conserved structural motif consisting of seven a-helical membrane spanning regions, which undergo significant changes upon receptor activation (Dixon et al., 1986; Dohlman et al., 1987). The G a subunit is G D P bound and closely associated with the GPy heterodimer in the absence o f ligand. The Ga-GDP/G(3y heterotrimer is associated with the cytosolic loops of a G protein-coupled receptor. Binding o f a ligand to the G protein-coupled receptor induces a conformational change in the G a subunit which results in the release of G D P and binding of G T P in its place. G a - G T P dissociates from the G complex exposing effector interaction sites in the Py heterodimers. Both G a - G T P and GPy are capable of signalling to their respective effectors. G a hydrolysis of the gamma-phosphate moiety of G T P returns the cycle to the basal state (Lambright et al., 1994; Sondek et al., 1994; Lambright et al., 1996; Sondek et al., 1996; Hamm, 1998; McCudden et al., 2005). There are four major families of G protein a subunits based on their primary sequence similarity: G s , G i , G q , and G i 2 (Wilkie et al., 1992). Each regulates the activity o f several second messenger systems. For example, G s a subunits activate adenylyl cyclases whereas Gj a subunits inhibit adenylyl cyclases and thus oppositely influence intracellular concentrations of adenosine 3',5'-monophosphate ( c A M P ) (Simonds, 1999). GPy dimers themselves regulate numerous signalling molecules, such as receptor kinases, adenylyl cyclases, phospholipases, phosphatidylinositol 3-kinases (PI3Ks), and ion channels (Clapham and Neer, 1997). There is extensive evidence that G protein-coupled receptors are capable of activating M A P K s via an intricate signalling network (Gutkind, 2000). The complexity of the signal transduction network leading to M A P K activation is a reflection of the role 24 of M A P K s as multitasking mediators able to generate a specific response to a diversity of upstream stimuli, depending on the receptor, agonist, and cellular background. The details of the signalling pathways linking G protein-coupled receptors to E R K 1 / 2 activation are known in more detail than signalling to p38 M A P K or J N K . Generally, E R K is phosphorylated by M E K (its M A P K K ) , which acts downstream of the protein kinase Raf. Signals transduced by G protein-coupled receptors converge on this three kinase module through Ras-GTPase, which activates Raf to stimulate M E K (Kolch, 2000) (Fig. 1.1). G protein-coupled receptor-mediated stimulation of p38 M A P K and J N K is well documented, but the exact mechanism of activation is not yet clear. Activation of J N K by Py subunits appears to occur through M K K 4 or M K K 7 (JNK-specific M A P K K s ) (Yamauchi et al., 1999) following activation of two small GTPases of the Rho family, R a c l and Cdc42 (Collins et a l , 1996; Mitsui et al., 1997), whereas Ga]2 stimulates J N K through Src-like kinases (Nagao et al., 1999). There is even less knowledge on the mechanism of activation of p38 M A P K by G protein-coupled receptors. Both G a subunits and Py dimers can activate p38 M A P K by initiating signalling cascades that converge on a tri-kinase phosphorelay module that is specific to p38 M A P K (Yamauchi et al., 1997). This module phosphorylates p38 M A P K after M K K 3 or M K K 6 (p38-specific M A P K K s ) are phosphorylated by M L K 3 , M E K K 4 , A S K 1 , or T A K 1 (p38-specific M A P K K K s ) (Gallo and Johnson, 2002). 1.7.3. Involvement of MAPKs in synaptic plasticity After M A P K s were first identified and found to play a role in response to growth factors and other mitogens to regulate proliferation and differentiation, it was observed that 25 Figure 1.1. G protein-coupled receptor-dependent activation of MAPKs. JNK is phosphorylated by M K K 4 and M K K 7 , which can be activated by py subunits in a pathway involving the small GTPases Rac and Cdc42. Although less is known about the molecular mechanisms connecting G protein-coupled receptors and p38 M A P K , it has been shown that p38 M A P K can be activated by both a and Py subunits through M K K 3 and M K K 6 (Gutkind, 2000). G P C R 26 M A P K s , their upstream regulators, and many of their downstream targets are highly expressed in mature neurons (Boulton et al., 1991), when proliferation and differentiation are no longer required. The discovery that glutamatergic signalling activates E R K s (Fiore et al., 1993; Kurino et al., 1995; X i a et al., 1996) suggested that M A P K s play a role in the normal functioning of the mature nervous system, and led many researchers to explore the role of M A P K s in synaptic plasticity. M A P K involvement in numerous forms of synaptic plasticity in the mammalian brain has since been reported (Thomas and Huganir, 2004). Long lasting changes in synaptic transmission can occur at C A 3 - C A 1 synapses when brief periods of synaptic activity lead to NMDA-sens i t ive glutamate receptor opening, increased postsynaptic C a 2 + concentration, activation of intracellular signalling pathways, enlargement of dendritic spines, the appearance o f new filopodia, and synaptic delivery and removal of AMPA-sensi t ive glutamate receptors (Malinow and Malenka, 2002; Sheng and K i m , 2002; Bredt and Nico l l , 2003; Thomas and Huganir, 2004). A l l three M A P K families have now been implicated in one or more of the above processes underlying synaptic plasticity. E R K inhibition with M E K inhibitors or dominant-negative Ras expression prevents activity-dependent dendritic enlargement and the appearance o f new filopodia (Wu et al., 2001). E R K 1 / 2 is also activated by Ras to signal delivery of A M P A receptors containing G l u R l and G l u R 2 L (long cytoplasmic termini) subunits during L T P (Zhu et al., 2002a). p38 M A P K is activated by Rap l to control the removal of GluR2 and GluR3 during L T D (Zhu et al., 2002a). J N K activation is required for Rap2-dependent removal of G l u R l and G l u R 2 L during depotentiation (Zhu et a l , 2005b). 27 M A P K s are also involved in other postsynaptically expressed forms o f synaptic depression. For example, p38 M A P K and E R K 1 / 2 are required for the induction of L T D by chemical stimulation of group I metabotropic glutamate receptors (Bolshakov et al., 2000; Gallagher et al., 2004). In addition, J N K regulates electrically induced L T D in the dentate gyrus (Curran et al., 2003). M A P K s may also be involved in presynaptic changes in synaptic transmission. Blocking J N K activity increases baseline synaptic transmission and depresses paired-pulse facilitation (Costello and Herron, 2004), which suggests that endogenous J N K may be involved in the regulation of neurotransmitter release. Moreover, inhibition of J N K and p38 M A P K activity attenuates A p - and interleukin-ip-mediated impairment of L T P (Vereker et al., 2000a; Curran et al., 2003; Costello and Herron, 2004). Interestingly, the increases in p38 M A P K and J N K phosphorylation associated with interleukin-ip treatment corresponds to decreased glutamate release from dentate gyrus synaptosomes (Vereker et al., 2000a), further implicating p38 M A P K and J N K in presynaptic mechanisms of synaptic transmission and plasticity in the brain. 1.7.4. Role of MAPKs in cardioprotection and neuroprotection In heart tissue, numerous studies show that M A P K cascades are involved in cardioprotection. A l l three M A P K s are activated by ischemia in the heart, but p38 M A P K is the most readily activated by ischemia. p38 M A P K is markedly and transiently activated after only a few minutes o f ischemia, and generally returns to control levels after 30 minutes (Y in et al., 1997; Shimizu et al., 1998). A number of studies have reported that p38 M A P K activity is necessary for acute and delayed ischemic and pharmacological preconditioning in the heart (Fryer et al., 2001a; Fryer et al., 2001b; 28 Schulte et al., 2004; Lasley et al., 2005). However, it has also been reported that p38 M A P K inhibition is neuroprotective (Mackay and Mochly-Rosen, 1999, 2000; Marais et al., 2001; Martin et al., 2001). 1.7.4.1. The role of A-i receptors in p38 MAPK-mediated i schemic precondit ioning There is evidence that p38 M A P K is activated by adenosine A i receptor stimulation in several tissues, including heart (Haq et al., 1998; Robinson and Dickenson, 2001; L i u and Hofmann, 2003). Adenosine A i receptor-dependent p38 M A P K activation has been implicated in delayed ischemic preconditioning. The p38 inhibitor SB203580 blocks A i receptor agonist-induced late phase preconditioning in mouse heart (Zhao et al., 2001b) and human atrial tissue (Carroll and Yel lon , 2000; Loubani and Galinanes, 2002). In addition, A i receptor stimulation induces p38 M A P K activity in rabbit myocardium (Dana et al., 2000). A] receptor-dependent activation of p38 M A P K is also necessary for delayed preconditioning of rat myocardium in vivo (Lasley et al., 2005). The role of p38 M A P K in ischemic preconditioning in the brain is less clear. p38 M A P K is activated within minutes of transient forebrain ischemia in vivo (Sugino et al., 2000), where it induces tolerance to a serious ischemic insult following a brief sublethal ischemic insult in the gerbil hippocampus (Nishimura et al., 2003). However, whether p38 M A P K is specifically activated by the A i receptor during cerebral ischemia has not yet been tested. Another type of preconditioning that occurs in the brain is called anaesthetic preconditioning. Volatile anaesthetics, i f applied minutes or days before an ischemic insult, can precondition the brain against subsequent ischemic injuries (Kapinya et al., 29 2002; Zhao and Zuo, 2004; Bickler et al., 2005; Gray et al., 2005). Interestingly, cerebral anaesthetic preconditioning requires the activation of both A) receptors (Liu et al., 2006) and p38 M A P K (Zheng and Zuo, 2004). Whether A] receptors specifically activate p38 M A P K to elicit anaesthetic preconditioning is not known. 1.8. Conclusion Adenosine and ATP, acting at PI and P2 purinoceptors, respectively, are implicated in a diverse array of brain functions in both physiological and pathophysiological conditions. 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J Auton Pharmacol 16:397-400. Zimmermann H , Braun N (1999) Ecto-nucleotidases—molecular structures, catalytic properties, and functional roles in the nervous system. Prog Brain Res 120:371-385. 49 C h a p t e r T w o : A c t i v a t i o n o f P 2 X 7 - L i k e R e c e p t o r s D e p r e s s e s M o s s y F i b e r - C A 3 S y n a p t i c T r a n s m i s s i o n T h r o u g h p 3 8 M i t o g e n - A c t i v a t e d P r o t e i n K i n a s e A version of this chapter was previously published: Armstrong J N , Brust T B , Lewis R G , Mac Vicar B A (2002) Activation of presynaptic P2X7-l ike receptors depresses mossy fiber-CA3 synaptic transmission through p38 mitogen-activated protein kinase. J Neurosci 22:5938-5945. 50 2.1. Introduction Adenosine 5'-triphosphate (ATP) is released from synapses throughout the peripheral and central nervous system (White, 1977; White, 1978; Jahr and Jessell, 1983; Edwards et al., 1992; Edwards and Gibb, 1993), where it can act on P2X receptors to modulate neurotransmission. P2X receptors are ligand-gated, calcium permeable cation channels (Khakh, 2001) that are activated by extracellular ATP. There are seven known P2X receptor subunits, P2Xi_ 7 . Of the seven subunits only P2X 7 subunits are thought to function exclusively as homomeric receptors (North and Surprenant, 2000). Activation of P2X 7 receptors can lead to the initiation of signaling cascades through second messengers such as phospholipase D (Kusner and Adams, 2000), p38 M A P K (Hu et al., 1998; Hide et al., 2000; Panenka et al., 2001) or the transcription factor NF-kappaB (Ferrari et al., 1997). Recent data suggests that the initiation of these signaling cascades could be mediated through putative protein interactions with the long cytoplasmic C-terminus of the P2X 7 subunit (Denlinger et al., 2001; Kim et al., 2001). In some circumstances the pore formed by the P2X 7 receptor may allow permeation of large cations (North and Barnard, 1997; North and Surprenant, 2000) that may eventually lead to cytolysis (Di Virgilio, 1995; Baricordi et al., 1999; Mutini et a l , 1999). P2X 7 receptors are only activated by high extracellular concentrations of ATP. Low concentrations (nM) of ATP that are ineffective at activating P2X 7 receptors are known to increase neuronal excitation and synaptic activity in the nervous system. For example, ATP-induced activation of P2X receptors can evoke single channel cation currents from chick ciliary ganglion nerve terminals (Sun and Stanley, 1996) and enhance the frequency of miniature endplate currents at the frog neuromuscular junction (Fu and 51 Poo, 1991). Activation of P 2 X receptors has also been shown to increase the frequency o f miniature postsynaptic currents in D R G dorsal horn neuronal cocultures (Gu and MacDermott, 1997; MacDermott et al., 1999) and increase excitation in the hippocampus (Wieraszko and Seyfried, 1989; Inoue et al., 1992; Inoue et a l , 1995). On the other hand, high concentrations of A T P (uM) are known to induce a long lasting form of synaptic depression that cannot be explained by the degradation of A T P into adenosine (Wieraszko and Seyfried, 1989). The synaptic depression mediated by high concentrations of A T P could be explained by the activation of presynaptic P 2 X 7 receptors. Recently Deuchars et al. (2001) reported the presynaptic localization of P 2 X 7 receptors in the spinal cord and brainstem of the rat. We subsequently investigated the anatomical distribution of P 2 X 7 receptors in the rat hippocampus. When we discovered that P 2 X 7 receptors were abundant on hippocampal mossy fiber terminals, we used whole-cell and extracellular field recordings to determine the actions of P 2 X 7 receptor activation on neurotransmission at this synapse. In the following study we provide physiological and pharmacological evidence that activation of these presynaptic P 2 X 7 receptors results in rapid and long lasting synaptic depression that is mediated through a p38 mitogen-activated protein ( M A P K ) signaling cascade. 2.2. Materials and methods 2.2.1. SDS-PAGE & western blotting Hippocampal proteins were isolated from Sprague-Dawley rats (3-4 weeks old) and homogenized in 0.32 M sucrose. Small (P2) and large (P3) mossy fiber synaptosomal fractions were then isolated according to previously published methods (Terrian et al., 52 1988; Terrian et al., 1989). Proteins were subjected to SDS-PAGE on 10% gels and probed with the following antibodies; rabbit anti-P2X7 polyclonal (1:18,000, Alomone Laboratories, Jerusalem, Israel), rabbit ant i-NMDARl (1:3,000, Chemicon Temecula, CA) mouse anti-P-tubulin (1:6,000, Sigma, St. Louis, MO) and rabbit anti-synaptoporin (1:30,000, Synaptic Systems, Gottingen, Germany) Immunoreactive signals were visualized using peroxidase-labeled goat secondary antibodies (1:10,000, Jackson Immunoresearch, WestGrove, PA) and enhanced chemiluminesence (Lumi-Light p l u s, Roche Diagnostics, Mannheim, Germany). 2.2.2. Immunocytochemistry For immunocytochemistry, rats were anaesthetized and transcardially perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Brains were sectioned in the coronal plane (50 pm) on a vibrating microtome (VT100, Leica, Willowdale, Ont.) and processed for immunocytochemistry using standard procedures (Sloviter et al., 1996). The following primary antibodies were used: rabbit anti-P2X7 (1:3,000, Alomone Laboratories, Jerusalem, Israel) mouse anti-microtubule associated protein (MAP)-2 (1:20,000; Sigma, St. Louis, MO) or anti-syntaxin 1A/B (1:5,000; Stressgen, Victoria, BC). The following secondary antibodies were used: biotinylated SP-donkey anti-mouse or rabbit IgG, Cy2-conjugated donkey anti-mouse IgG and Cy3-conjugated donkey anti-rabbit IgG or Cy5-conjugated donkey anti-rabbit IgG (1:1000 all from Jackson Immunoresearch, WestGrove, PA). Sections were imaged on an Axioskop LSM510 Laser Scanning Microscope (Carl Zeiss Microscopy, Jena, Germany). 53 2.2.3. Electrophysiology Hippocampal slices (300 pm thick) were obtained from 10-30 day old rats, immersed in ice cold artificial cerebrospinal fluid (aCSF; see below) and incubated in a submersion chamber for at least 1 hr at room temperature. For recordings individual slices were transferred to either an interface chamber (Fine Science Tools, Foster City, CA) for extracellular recordings or a submersion chamber for whole cell voltage-clamp recordings. A l l recordings were done at room temperature. In either chamber, slices were superfused (2 ml/min) with aCSF consisting of (in mM): 119 NaCl, 2.5 KC1, 1.3 MgSC-4, 26 NaHCC-3, 1 N a H 2 P 0 4 , 2.5 CaCl 2 , and 10 glucose, aerated with 95%0 2/5%CO 2. Extracellular recordings were obtained with glass micropipettes filled with HEPES-buffered aCSF (resistance 1-3 MCI). Extracellular recordings were filtered at 5 kHz, digitized at 10 kHz using a Digidatal200 interface (Axon Instruments, Foster City, CA) and stored on a Pentium III computer for later analysis using Clampfit (Axon Instruments, Foster City, CA). A bipolar tungsten-stimulating electrode was used to stimulate dentate granule cells, thereby activating mossy fibers. Mossy fiber-CA3 synaptic responses were measured in the stratum lucidum of the CA3 region and distinguished by their characteristic short latency, rapid rise time, large paired-pulse facilitation and >70% inhibition by mGluR agonist L-CCG-I. Whole cell recordings were obtained using patch pipettes filled with 100 m M cesium methanesulfonate, 10 m M cesium-Bapta, 40 m M HEPES, 5 m M QX-314, adjusted to pH 7.3 with cesium hydroxide (resistance 1-3 M Q CaCl 2 and MgSC>4 were increased to 4 mM in the aCSF for all whole cell recordings. During paired-pulse facilitation experiments picrotoxin (10 u.M) was included in the patch pipette to block 54 G A B A A receptors (Nelson et al., 1994; Xiang and Brown, 1998). Series resistance in all recordings was < 20 M O and data was excluded i f series resistance varied more than 15%. A l l recordings were digitized at 5-10kHz and filtered at 2kHz. Statistics. A l l statistics were carried out using a paired (correlated groups) Mest except for the comparison between the effect of B z - A T P and adenosine on slices incubated with SB203580. In this case, a simple one-way analysis of variance ( A N O V A ) was used. 55 2.3. Results 2.3.1. P2X7 receptors were found on mossy fiber terminals Western blotting and immunocytochemistry revealed that P2X 7 receptors were abundant on presynaptic terminals of the rat hippocampus. We used a P2X 7 antibody that was raised against amino acid residues 576-595 of the rat P2X 7 receptor subunit. This antibody recognized a single 70 kDa band in western blots of proteins isolated from the hippocampus (H; Figure 1A-B), small (P2) hippocampal synaptosomes and large, mossy fiber (P3) synaptosomes (Figure 2.1 B). Inclusion of the P2X 7 antigenic peptide (1:1) with the antibody blocked detection of the 70kDa P2X7-immunoreactive band. We did not detect any signal in the P3 fraction with an antibody against iba-1, a protein selectively expressed in microglia (Ito et al., 1998). This indicates that microglia did not contaminate our P3 synaptosome preparation (data not shown). Immunocytochemistry with this P2X 7 selective antibody revealed dense immunoreactive terminals throughout mossy fiber termination zones in the dentate hilus (h) and stratum lucidum (luc) of CA3 (arrows in Figure 2.1 Q . Fainter staining was also observed throughout the hippocampus and may represent immunoreactivity of other presynaptic terminals, or glial cells, such as microglia (Ferrari et al., 1996; Chessell et al., 1997; Di Virgilio et al., 1999) or astrocytes (Kukley et al., 2001; Panenka et al., 2001). Confocal microscopy confirmed that the P2X7-immunoreactive boutons were presynaptic mossy fiber terminals because P2X 7 -immunoreactivity was co-localized with presynaptic syntaxin 1 A/B-immunoreactivity (Bennett et al., 1992; Ruiz-Montasell et al., 1996) (Figure 2.3 G-l) but not dendritic MAP-2-immunoreactivity (Figure 2.2 D-F). 56 Figure 2.1. P2X 7 receptors are located on presynaptic terminals of mossy fiber synapses. A, Western blot of proteins isolated from the rat hippocampus showed that the P2X 7 antibody recognized a single band of protein at an approximate molecular weight of 70 kDa. Lane 1 contains Amido Black (AB) stained proteins that were first immunoblotted in lane 2. B, P2X7-immunoreactive bands of protein were present in proteins isolated from whole hippocampus (H) as well as small (P2) synaptosomal and large (P3) mossy fiber synaptosomal preparations. The presence of synaptoporin and relatively low abundance of NMDAR1 and P-tubulin indicates that P2X 7 receptors were highly enriched in the fraction containing mossy fiber terminals (P3). C, Immunocytochemistry with this P2X 7 selective antibody revealed dense immunoreactivity throughout mossy fiber termination zones in the dentate hilus (h) and stratum lucidum (luc) of CA3 (arrows). Fainter staining was also observed throughout the hippocampus and may represent immunoreactivity of other presynaptic terminals. Abbreviations: rad, stratum radiatum; 1-m, stratum lacunosum-moleculare; m, molecular layer; dgc, dentate granule cell layer. Scale bar=2 mm in C. 57 58 Figure 2.2. P2X7 immunoreactivity was not co-localized wi th dendritic M A P - 2 immunoreactivity. D-F, P2X7-immunoreactivity (blue) was found throughout stratum lucidum, however, dendritic MAP2-immunoreactivity (green) did not colocalize with the punctate P2X7-immunoreactivity. Cell bodies were counterstained with ethidium bromide (red). Scale bar=70 pm in D, 50 pm in E, and 30 pm in F. 59 Figure 2.3. P 2 X 7 immunoreactivity was co-localized with presynaptic syntaxin 1A /B immunoreactivity. G-7, Presynaptic syntaxin 1A/B immunoreactivity (green) was colocalized with the punctate P2X7-immunoreactivity (red) demonstrating that the mossy fiber terminals contained P 2 X 7 receptors. Scale bar=100 pm. 6 0 2.3.2. P2X7 receptor activation depressed mossy fiber-CA3 synaptic transmission Next, we investigated the effect of P 2 X 7 receptor activation on synaptic transmission at mossy fiber synapses. First, we recorded evoked postsynaptic field potentials (fEPSPs) in stratum lucidum of C A 3 following stimulation of the dentate granule cells (Figure 2.4). To ensure that we were recording from mossy fiber-CA3 synapses we first applied L -C C G - I (20 u.M), an m G l u R agonist that selectively depresses mossy fiber inputs onto C A 3 pyramidal cells (Manzoni et al., 1995; Schmitz et al., 2000). Bath application of L -C C G - I reversibly depressed the amplitude of the fEPSP (Figure 2.4 A). Subsequent bath application of the P 2 X 7 receptor agonist, 2'3'-0-(4-benzoylbenzoyl) - A T P ( B z - A T P ; 30 uM) also depressed the fEPSP (Figure 2.4 A). However, application of L - C C G - I during the peak of the B z - A T P response did not result in any further depression of the synaptic response (fEPSP amplitude after B z - A T P was 0.22 ± 0 . 1 2 of control versus 0.17 ± 0.09 o f control in B z - A T P + L - C C G - I , mean ± s.e.m, n=3) indicating that P 2 X 7 receptor activation depressed the same population of synaptic inputs as L - C C G - I . B z - A T P was also applied alone to monitor the time course of the P2X 7-mediated synaptic depression without prior L - C C G - I application (Figure 2.4 B). This prevented any potential interactions between progressive drug applications. A s shown in Figure 2.4 B, B z - A T P caused a rapid and long lasting (> 2 hours) statistically significant [t(5)=10.37,/? < 0.01] decrease in the fEPSP (fEPSP amplitude following B z - A T P was 0.30 ± 0.05 of control amplitude, mean ± s.e.m., n=6). In separate experiments, the postsynaptic response was blocked by N B Q X and the presynaptic fiber volley was monitored following B z - A T P application (Figure 2.4 Q . 61 Figure 2.4. The P 2 X 7 agonist, Bz-ATP, depressed mossy fiber fEPSPs but had no detectable effect on the presynaptic fiber volley. A, Averaged sample traces and plots of mossy fiber-CA3 synaptic responses recorded extracellularly from the stratum lucidum during one experiment at the indicated time points. L-CCG-I (20 pM) reversibly depressed the postsynaptic component of the field potential (indicated by asterisk in the first trace). Bz-ATP (30 pM) depressed the L-CCG-I-sensitive component of the fEPSP and coapplication of L-CCG-I did not cause further depression. B, Summary of separate experiments in which Bz-ATP was applied without preapplication of L-CCG-I. Averaged sample traces are shown before and after Bz-ATP application. Plot shows the mean values obtained from 6 slices. Bz-ATP depressed the mossy fiber fEPSP amplitude for greater than 2 hours. C, Single plot and mean sample traces from a single experiment where the field response was recorded in the presence of N B Q X (20 pM) to monitor the presynaptic fiber volley. Bz-ATP had no effect on the presynaptic fiber volley. D, Summary of the effects of Bz-ATP on the fEPSP (n=6) and the presynaptic fiber volley (n=5). Asterix indicates statistical significance using a pair t-test,^ ? <0 .01. Scale bar A : 0.2 mV, 20 msec B: 0.5 mV, 10 msec C: 0.3 mV, 20 msec. 62 A 01 I 1.5 "5. E ni CL 1.0 CD CL LLi A. J\ 3 k L-CCG1 Bz-ATP L-CCG1 te^ NBQX 2 0 4 0 6 0 Time (minutes) 63 There were no significant [t(4)=-1.13,/? > 0.05] alterations in the presynaptic fiber volley as a result of Bz-ATP application (n=5; summarized in Figure 2.4 D). Furthermore, we visualized mossy fiber terminals using a laser scanning microscope to see i f Bz-ATP induced the uptake of YO-PRO-1 via pore dilation and cell lysis (e.g. Virginio et al., 1999). We did not observe any uptake of YO-PRO-1 after Bz-ATP application (data not shown; n=2). These data suggest that activation of P2X7-receptors with Bz-ATP does not induce cytolysis of mossy fiber terminals. Next, we obtained whole-cell voltage clamp recordings from CA3 pyramidal neurons to determine whether Bz-ATP selectively depressed mossy fiber-CA3 synaptic transmission or had a postsynaptic effect on A M P A receptors. As shown in Figure 2.5, Bz-ATP significantly [t(5)=22.36,/? < 0.01] depressed the amplitude of voltage-clamped mossy fiber EPSCs (mossy fiber EPSC amplitude after Bz-ATP was 0.33 ± 0.04 of control, mean + s.e.m., n=6) but had no statistically significant [t(4)=2.02,/? > 0.05)] effect on associative/commissural EPSCs (assoc/comm EPSC amplitude after Bz-ATP was 0.81 +0.10 of control, mean ± s.e.m., n=5). Associational/commissural responses were evoked by stimulation of stratum radiatum in the presence of L-CCG-I to block mossy fiber synapses. Bz-ATP also had no significant effect on the CA3 whole cell conductance (308 ± 34 pS before versus 288 ± 60 pS after Bz-ATP) or holding current (65.8 ± 8.2 pA prior to Bz-ATP application versus 71.6 ± 7.9 pA after Bz-ATP). Therefore, P2X 7 receptor activation selectively depressed mossy fiber synapses and had no direct postsynaptic effect on CA3 neurons. 64 Figure 2.5. Act ivat ion of presynaptic P 2 X 7 receptors wi th B z - A T P selectively depressed synaptically evoked mossy fiber currents in C A 3 . A, Plots of mean whole cell voltage-clamp recordings in CA3 pyramidal neurons following stimulation of the mossy fiber (MF) pathway (n=6 slices) or the associative/commissural (A/C) pathway (n=5 slices). Bz-ATP significantly depressed the amplitude of voltage-clamped M F EPSCs but had no significant effect on A / C EPSCs. B, Summary of the data presented in A. Asterix indicates statistical significance using a paired t-test, p < 0.01. C, Average sample traces from evoked responses following stimulation of the MF pathway or the A / C pathway. Scale bar C: 200 pA, 50 msec; 250 pA, 50 msec. T ime (minutes) 65 2.3.3. Bz-ATP-induced synaptic depression was blocked by o-ATP To further delineate P2X 7 receptor involvement in this Bz-ATP-induced effect we assessed the ability of Bz-ATP to induce synaptic depression of mossy fiber-CA3 fEPSPs in the presence of the nonselective P2Y antagonist reactive blue 2 (RB2, 30 uM, Figure 2.6 A) or the P2X]_3,5,6 receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 10 uM, Figure 2.6 B). As shown in Figure 2.6, both RB2 (n=5) and PPADs (n=4) failed to block the effect of Bz-ATP on mossy fiber fEPSPs [t(4)=13.81,p < 0.01 and t(3)=26.55jp < 0.01 respectively]. This data suggests that Bz-ATP-induced synaptic depression was not mediated by the nonselective activation of P2Y receptors or postsynaptically located P2X]-3,5,6 receptors. To determine i f Bz-ATP-induced synaptic depression was mediated by activation of P2X 7 receptors we applied Bz-ATP in the presence of the P2X 7 receptor antagonist oxidized periodate-ATP (o-ATP, 100 uM, Murgia et al., 1993; Visentin et al., 1999). Consistent with Bz-ATP acting at presynaptic P2X 7 receptors, Bz-ATP-induced synaptic depression was potently inhibited by 2 hour preincubation with the P2X 7 antagonist o-ATP (in matched slices fEPSP amplitude after Bz-ATP was 0.30 ± 0.05 of control versus 0.81 ±0.12 of control in slices pre-incubated with o-ATP, mean ± s.e.m, see Figure 2.6 C; n=5 for both). This P2X 7-like pharmacological profile combined with our inability to detect a postsynaptic current in CA3 pyramidal cells suggests that Bz-ATP acted presynaptically at P2X 7 receptors to mediate mossy fiber synaptic depression. 66 Figure 2.6. The selective P 2 X 7 antagonist o - A T P blocked B z - A T P induced depression of the mossy f ibe r -CA3 synaptic responses. A, Plots and average sample traces of mossy fiber-CA3 synaptic responses recorded extracellularly from the stratum lucidum of CA3 in the presence of the nonselective P2Y antagonist reactive blue 2 (RB2, 30 pM, n=5 slices). RB2 failed to block the effect of Bz-ATP on mossy fiber fEPSPs. B, Similarly, the P2Xi_3 i5 i6 receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADs) failed to block the effect of Bz-ATP on mossy fiber fEPSPs (n=4 slices). C, However, preincubation of the slices with the P2X 7 receptor antagonist o-ATP (100 pM, n=5 slices) significantly reduced the magnitude of the Bz-ATP induced depression compared to control (n=5 slices). D, Summary of data present presented in A-C. Asterix indicates significance using a paired t-test,/? < 0.01. Scale bar A : 0.5 mV, 10 msec. 67 Time (minutes) 0 20 40 60 Time (minutes) 68 2.3.4. P2X7 receptor activation increased paired-pulse facilitation If Bz-ATP depresses mossy fiber synaptic transmission by activating presynaptic P2X 7 receptors, then Bz-ATP-induced depression should be associated with an increase in paired-pulse facilitation (PPF, e.g. Regehr et al., 1994; Salin et al., 1996). We monitored PPF while recording whole-cell synaptic evoked currents in CA3 neurons. Consistent with Bz-ATP activating presynaptic P2X 7 receptors, we observed a significant [t(4)=-4.65, p<0.01] increase in the ratio of the 2 n d EPSC amplitude to the 1 s t EPSC amplitude y immediately after application of Bz-ATP (ratio before Bz-ATP was 1.74 ± 0.05, ratio after was 2.14 ± 0.09, mean ± s.e.m., n=5 slices, Figure 2.7). This data indicates that Bz-ATP decreased the probability of release at mossy fiber synapses. 2.3.5. P2X7 receptor-mediated synaptic depression required activation of p38 MAPK Recent evidence suggests that M A P K activity is potently activated by synaptic activity and is essential for some forms of synaptic plasticity (Impey et al., 1999). For example, erkl/erk2 M A P K activation is essential for the induction of long-term potentiation and p38 M A P K activity is essential for the induction of long-term depression in CA1 of the hippocampus (Bolshakov et al., 2000). We have recently shown that activation of P2X 7 receptors in cultured astrocytes leads to activation of p38 and ERK1/2 M A P K (Panenka et al., 2001). To determine whether M A P K activity was necessary for the synaptic depression induced by Bz-ATP we preincubated the slices (2 hours) with the p38 M A P K inhibitor SB203580 (25 uM) or the ERK1/2 M A P K inhibitor PD98059 (50 uM). Bz-ATP-induced synaptic depression of the L-CCG-I sensitive mossy fiber-CA3 69 Figure 2.7. Act ivat ion of presynaptic P 2 X 7 receptors increased mossy fiber paired-pulse facilitation (PPF , 50msec). PPF was monitored by recording whole-cell synaptic currents evoked in CA3 neurons (n=5 slices). A, Mean PPF ratio before and after application of Bz-ATP. Asterix indicates statistical significance using a paired t-test, p < 0.01. We observed a significant increase in the PPF ratio following Bz-ATP application, which is consistent with Bz-ATP acting on presynaptic P2X 7 receptors. B, Average sample traces before and after Bz-ATP application. The lower trace on the right was rescaled so that the first current was the same size after Bz-ATP as it was in control. Scale bar B; 200 pA, 50 msec, rescaled traces 70 pA. 70 postsynaptic response was significantly [t(3)=0.19, p>0.05] blocked by inhibition of p38 M A P K activity with SB203580 (fEPSP amplitude after Bz-ATP was 0.93 ± 0.12 of control amplitude in slices preincubated with SB203580, n=4, Figure 2.8 A). In contrast, preincubation of the slices with the ERK1/2 M A P K activity inhibitor PD98059 failed to block [t(3)=8.27,/? < 0.01] Bz-ATP-induced mossy fiber synaptic depression (fEPSP amplitude after Bz-ATP was 0.24 ± 0.11 of control in slices preincubated with PD98059, n=4, Figure 6B). These data demonstrate that activation of p38 M A P K was necessary for P2X 7 receptor-mediated depression of mossy fiber-CA3 synaptic transmission. 2.3.6. Inhibitory effects of adenosine were not mediated through p38 MAPK ATP and some of its analogs can be rapidly degraded into adenosine by the actions of ectonucleotidase (Dunwiddie et al., 1997; Cunha et al., 1998). Thus, ATP application can inhibit synaptic transmission indirectly through adenosine formation and the activation of presynaptic Ai-receptors (Dunwiddie et al., 1997; Cunha et al., 1998; Cunha and Ribeiro, 2000; Dunwiddie and Masino, 2001). We could not use an A] antagonist such as DPCPX because blocking A i receptors results in persistent seizure activity in the CA3 region making it impossible to record stable mossy fiber responses as previously reported (Thummler and Dunwiddie, 2000). Therefore, to determine indirectly whether Bz-ATP-induced mossy fiber-CA3 synaptic depression was mediated through degradation of Bz-ATP into adenosine, we tested whether adenosine inhibits synaptic transmission through p38 M A P K activity. As shown in Figure 2.9, preincubation of the slices with SB203580 (25 pM) blocked Bz-ATP induced mossy fiber synaptic depression (n=4) but failed to have any effect on adenosine (30 pM) mediated mossy fiber 71 Figure 2.8. P 2 X 7 receptor mediated mossy fiber synaptic depression required p38 M A P K activity. A-B, Plots of mean mossy fiber-CA3 synaptic responses recorded extracellularly from stratum lucidum of CA3. A, Preincubation of the slices in the p38 M A P K inhibitor SB203580 (25pM) completely blocked Bz-ATP-induced synaptic depression but had no effect on the L-CCG-Tinduced depression of the mossy fiber fEPSP (n=4 slices). B, Preincubation of the slices with the erkl/erk2 M A P K inhibitor PD98059 (50pM) failed to have any effect on Bz-ATP-induced synaptic depression (n=4 slices). C, Summary of data presented in A-B. Asterix indicates significance using a paired t-test, p<0.01. Scale bar^4: 0.5 mV, 10 msec. 72 Figure 2.9. The p38 antagonist, SB203580, blocked the actions of B z - A T P but not the inhibi t ion by adenosine (30uM). A, Plot of an extracellular recording in which the p38 M A P K inhibitor SB203580 blocked the Bz-ATP-induced mossy fiber synaptic depression but failed to block the adenosine-induced inhibition of the mossy fiber fEPSP. B, SB203580 differentially affected the depression induced by Bz-ATP and adenosine. Therefore, adenosine does not exert its inhibitory actions through p38 M A P K and the presynaptic actions of Bz-ATP cannot be explained by the degradation of Bz-ATP into adenosine by ectonucleotidase activity. Asterix indicates statistically significance using a one-way A N O V A , / ? < 0.01. 74 synaptic depression (n=4) [in slices preincubated with SB203580 the fEPSP amplitude following Bz-ATP was 0.95 ± 0.15 of control versus 0.15 ± 0.09 of control in adenosine; F(l,6)=21.58,/> < 0.01]. Therefore, adenosine does not exert its inhibitory actions through p38 M A P K and the presynaptic actions of Bz-ATP cannot be explained by the degradation of Bz-ATP into adenosine. 2.4. Discussion The results of the present study demonstrate that activation of presynaptic P2X 7 receptors results in the inhibition of neurotransmission at mossy fiber-CA3 synapses through a p38 M A P K -signaling pathway. First, we have used immunocytochemistry to demonstrate that P2X 7 receptors are abundant on presynaptic terminals of mossy fiber synapses in the rat hippocampus. Immunocytochemistry with a specific P2X 7 antibody resulted in the labeling of small terminal-like puncta throughout the hippocampus. P2X 7 -immunoreactivity was particularly dense throughout the termination zones of hippocampal mossy fibers where it was completely colocalized with the presynaptic marker syntaxin 1A/B but not the dendritic marker MAP-2 (see Figures 2.2 & 2.3). Syntaxin 1A is known to be present in the presynaptic mossy fiber terminals and syntaxin IB is present in the mossy fiber axons (Ruiz-Montasell et al., 1996). As demonstrated in Figure 2.3, all of the observed P2X 7 -immunoreactivity in stratum lucidum was colocalized with the syntaxin 1A labeling of the presynaptic terminal. These data demonstrate that P2X 7 receptors are located presynaptically in stratum lucidum of the rat hippocampus. The specific presynaptic P2X 7 receptor localization shown here contrasts with the postsynaptic location of other known P2X receptors in the hippocampus of the 75 rat (e.g. P2X 2 , P2X4 and P 2 X 6 , Le et al., 1998; Rubio and Soto, 2001). These postsynaptically located receptors are likely to contribute to the increase in excitation that is observed in hippocampus following application of low doses of ATP (Wieraszko and Seyfried, 1989). Consistent with their presynaptic localization, activation of P2X 7 receptors with Bz-ATP completely depressed the L-CCG-I sensitive mossy fiber-CA3 synaptic response in extracellular field recordings. However, no direct effects of Bz-ATP on postsynaptic CA3 pyramidal neurons were observed when the conductance and holding current were monitored during whole-cell voltage clamp recordings. Furthermore, we found no significant effect of Bz-ATP on A M P A receptor mediated associative/commissural synaptic transmission in CA3. This observation is consistent with our conclusion that Bz-ATP selectively activates presynaptic P2X 7 receptors and suggests that at this concentration (30 uM), Bz-ATP did not activate other known postsynaptic P2X receptors (e.g. P2X 2 , P2X4, P2X6). Although our conclusions support the involvement of P2X 7 receptors in presynaptic depression, the possible contribution of P2X4 cannot be totally eliminated. The enhancement of PPF during the Bz-ATP-induced synaptic depression is also consistent with a presynaptic site of action (Regehr et al., 1994; Salin et al., 1996) similar to what has been observed during mGluR-mediated depression in CA1 (Fitzjohn et al., 2001). Bz-ATP induced synaptic depression was not blocked by the P2Y receptor antagonist RB2 (30 uM) or the P 2 X i . 3 j 5 . 6 antagonist PPADS (10 uM). Other P2X receptors are antagonized by PPADS at this concentration whereas P2X 7 receptors are not (Surprenant et al., 1996). However, Bz-ATP mediated synaptic depression required P2X 7 receptor activation because little or no synaptic depression was observed when the slices 76 were preincubated with the irreversible P2X 7 receptor antagonist o-ATP (see Figure 2.6; Murgia et al., 1993; Visentin et al., 1999). Bz-ATP-induced synaptic depression also cannot be explained by the degradation of Bz-ATP into adenosine by local ectonucleotidases because adenosine mediated synaptic inhibition was not blocked by p38 M A P K inhibition whereas Bz-ATP's actions were (see below). Mossy fiber synapses contain vesicular ATP, and synaptosomes prepared from mossy fiber synapses release ATP in a Ca dependent manner in response to K -induced depolarization (Terrian et al., 1989). However, it is not known whether ATP is normally released from mossy fiber synapses or whether P2X 7 receptors are normally activated during the evoked release of neurotransmitters from mossy fiber terminals. It is possible that presynaptic mossy fiber P2X 7 receptors might only be activated during intense periods of mossy fiber activity such as that observed during tetanus or seizure when ATP release might reach levels high enough to activate P2X 7 receptors. Therefore P2X 7 receptors might play an important role in limiting synaptic transmission when mossy fiber synaptic transmission is unusually high. The results of the present study demonstrate that activation of p38 M A P K is necessary for P2X 7 receptor mediated depression of mossy fiber-CA3 synaptic transmission. Maruyama et al. (2000) have recently demonstrated that p38 M A P K is abundant in the terminals of mossy fiber synapses. As shown in Figure 2.8, Bz-ATP-induced synaptic depression was completely blocked by preincubation of the slices with the p38 M A P K activity inhibitor SB203580 but not the ERK1/2 M A P K activity inhibitor PD98059. The presynaptic mechanism by which P2X 7 receptor-dependent p38 M A P K activity depresses mossy fiber synaptic transmission remains to be determined. However, 77 recent evidence suggests that p38 M A P K activity is necessary for the inhibition of N-type calcium currents in neuroblastoma cells following bradykinin application (Wilk-Blaszczak et al., 1998). Therefore, it is possible that P 2 X 7 receptor-dependent p38 M A P K activity depresses mossy fiber synaptic transmission through inhibition of calcium channels. However, mossy fiber terminals exhibit predominantly P-type calcium channel-dependent evoked neurotransmitter release and contain few N-type channels (Castillo etal., 1994). Recent evidence suggests that M A P K activity is potently activated by synaptic activity and is essential for some forms of neuronal plasticity (Impey et al., 1999; Bolshakov et al., 2000). For example, translocation of erkl/erk2 M A P K to the nucleus of the presynaptic neuron is essential for long-term facilitation in Aplysia neurons (Martin et al., 1997) and p38 M A P K is essential for the induction of mGluR-receptor dependent long-term depression in C A 1 of the hippocampus (Bolshakov et al., 2000). Interleukin-i p has also been shown to increase p38 activation and modify long-term potentiation in perforant path synapses (Vereker et al., 2000). In the present study, we have shown that the rapid and reversible depression of mossy fiber synaptic transmission by the m G l u R agonist L - C C G - I is unaffected by preincubation of the slices with the p38 M A P K inhibitor SB203580. Therefore, the mGluR-induced inhibition o f mossy fiber synapses is apparently not mediated by p38 as it is in C A 1 . In conclusion, we have provided evidence that P 2 X 7 receptor subunits are abundant on presynaptic terminals of mossy fiber synapses in the rat hippocampus. 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Complex synaptic current waveforms evoked in hippocampal pyramidal neurons by extracellular stimulation of dentate gyrus. J Neurophysiol 79: 2475-84. 84 C h a p t e r T h r e e : p 3 8 M i t o g e n - A c t i v a t e d P r o t e i n K i n a s e C o n t r i b u t e s t o A d e n o s i n e A i - R e c e p t o r - M e d i a t e d S y n a p t i c D e p r e s s i o n i n A r e a C A 1 o f t h e R a t H i p p o c a m p u s A version of this chapter has been submitted for publication: Brust, Tyson B . ; Cayabyab, Francisco, S.; Zhou, Ning; and Mac Vicar , Brian A (2006). p38 mitogen-activated protein kinase contributes to adenosine A1-receptor dependent synaptic depression in area C A 1 of the rat hippocampus. J neurosci Feb 21. 85 3.1. Introduction Extracellular adenosine concentrations in the brain can increase 30-100 fold following pathological trauma such as head injury, epileptic seizures, and hypoxia/ischemia (During and Spencer, 1992; Bell et al., 1998; Von Lubitz, 1999). Adenosine inhibits glutamate release via A i receptors (Fowler, 1989; Katchman and Hershkowitz, 1993; Zhu and Krnjevic, 1993), and may act as an endogenous neuroprotectant by preventing glutamate excitotoxicity (Dunwiddie and Masino, 2001). There is evidence that p38 mitogen-activated protein kinase (MAPK) can be activated by the adenosine A i-receptor via a G-protein-dependent pathway (Robinson and Dickenson, 2001). p38 M A P K is a member of a large family of M A P K s that mediate inflammation, cell proliferation, and apoptosis (Seger and Krebs, 1995; Cobb, 1999). M A P K s are also activated by synaptic activity and are essential for some forms of synaptic plasticity (Impey et al., 1999). In rat hippocampus, p38 M A P K activation mediates metabotropic glutamate-receptor-dependent long-term depression (LTD) (Bolshakov et al., 2000; Rush et al., 2002), as well as Rap-dependent removal of A M P A receptors during LTD (Zhu et al., 2002a). p38 M A P K also mediates inhibition of long-term potentiation by (3-amyloid (Saleshando and O'Connor, 2000; Wang et al., 2004) and interleukin-lp (Coogan et al., 1999; Vereker et al., 2000a). In heart tissue, p38 M A P K is activated by adenosine (Haq et al., 1998), and plays an important role in cardioprotection during ischemia (Weinbrenner et al., 1997; Baines et al., 1998; Baines et al., 1999). Ai-receptor activation triggers early and delayed ischemic preconditioning (Thornton et al., 1992; Tsuchida et al., 1993) and mediates 86 myocardial protection by activating p38 M A P K (Zhao et al., 2001b; Schulte et al., 2004). Recent studies have confirmed that A i receptor-mediated delayed preconditioning against myocardial infarction is dependent on p38 M A P K in vivo (Lasley et al., 2005). Two independent lines of evidence also suggest that A\ receptor-mediated neuroprotection is also dependent on p38 M A P K activity in the brain. A brief exposure to volatile anesthetics such as isoflurane protects the brain against subsequent ischemic insults (Kapinya et al., 2002; Zhao and Zuo, 2004). Isoflurane tolerance against focal cerebral ischemia is dependent on an A i receptor-mediated pathway (Liu et al., 2006). Interestingly, isoflurane tolerance against cerebral ischemia requires the activation of p38 M A P K in vivo (Zheng and Zuo, 2004). Adenosine A i receptor depresses synaptic transmission in the hippocampus by inhibiting voltage dependent calcium channels in presynaptic nerve terminals (Wu and Saggau, 1994; Manita et al., 2004). p38 M A P K has also been implicated in presynaptic inhibition, as p38 M A P K activation is necessary for inhibition of N-type calcium current in a G protein-dependent pathway (Wilk-Blaszczak et al., 1998). Although both A i -receptors (Lee et al., 1983; Fastbom et al., 1987; Tetzlaff et al., 1987; Ochiishi et al., 1999) and p38 M A P K (Lee et al., 2000b; Maruyama et al., 2000) are widely expressed in brain tissue, it is not known whether Ai-receptors can activate p38 M A P K in the brain, or whether p38 M A P K activation plays a role in A i-receptor-mediated synaptic depression. In the present study, we have used electrophysiology, Western blot analysis, and co-immunoprecipitation to test whether p38 M A P K activation contributes to the synaptic depression induced by A] receptor stimulation in area CA1 of the rat hippocampus. We showed that selective A i receptor stimulation in hippocampal slices rapidly elevated 87 phospho-p38 M A P K and that A i receptors and phospho-p38 M A P K both exist in the same signaling complex. We also found that protein phosphatase 2a (PP2a) rapidly translocates from the cytosol to the membrane after A i receptor stimulation in a p38 MAPK-dependent pathway. Finally, we demonstrated that p38 M A P K contributes to adenosine Ai-receptor-mediated depression of CA3-CA1 synaptic transmission. 3.2. Materials and methods 3.2.1. Hippocampal Slice Preparation Sprague Dawley rats (p21-p28) were anaesthetized with halothane and decapitated according to protocols approved by the University of British Columbia committee on animal care. Brains were rapidly extracted and placed into ice-cold oxygenated dissection medium containing the following (in raM): 87 NaCl, 2.5 K C l , 2 NaFbPO^ 7 MgCl2, 25 NaHC03, 0.5 CaCl2, 25 D-glucose, and 75 sucrose. Hippocampal slices (400pm thick) were cut using a vibrating tissue slicer (VT1000S, Leica, Nussloch, Germany) and maintained for 1-5 hours at 24°C in aCSF containing (in mM): 119 NaCl, 2.5 K C l , 1.3 MgS04, 26 NaHC0 3 , 2.5 CaCl 2 , and 10 D-glucose, and aerated with 95% 02/5% C O 2 . For electrophysiological recordings, slices were transferred to a submerged recording chamber and allowed to equilibrate for at least 1 hour. The bath solution was perfused with aerated aCSF at a rate of 1.5 - 2 mL/min. 3.2.2. Electrophysiology Field excitatory postsynaptic potentials (fEPSPs) were evoked by orthodromic stimulation of the Schaffer collateral pathway using a bipolar tungsten-stimulating 88 electrode. Glass micropipettes filled with aCSF (resistance 1-3MO) were used to measure CA1 fEPSPs in stratum radiatum. fEPSP signals were amplified 1000 times with an A C amplifier, band-pass filtered at 0.1-100 Hz, digitized at 10 kHz using a Digidata 1320A interface board (Axon Instruments, Foster City, CA), and transferred to a computer for analysis. Data were analyzed using Clampfit 9.0 (Axon Instruments). Baseline synaptic responses were established by evoking fEPSPs every 30s (0.03Hz) for at least 20 min. The fEPSP slope was normalized to the mean of the 20 sweeps (10 min) immediately preceding drug perfusion. The mean normalized fEPSP slope was plotted as a function of time with error bars representing the standard error of the mean (SEM). Sample traces are the average of 5 sweeps from a recording that was included in the plot of the mean normalized fEPSP slope. A l l bar graphs show the mean normalized percent inhibition from baseline ± SEM. Statistical significance was assessed using a Student t-test {p < 0.05). 3.2.3. Immunoprecipitation, Co-immunoprecipitation and Western Blot Analysis For biochemical studies, rat hippocampal slices were first incubated with various treatments (see below) and then lysed in a solubilization buffer (30 min, 4 °C) that contained 1% NP-40, 20 m M MOPS (pH 7.0), 5 m M EDTA, 2 mM EGTA, 1 m M phenylmethylsulfonyl fluoride (PMSF), 10 ug/ml aprotinin, 10 ug/ml leupeptin, 10 ug/ml pepstatin A, 1 m M N a 3 V 0 4 , 30 m M NaF, 40 m M p-glycerophosphate (pH 7.2), 20 m M sodium pyrophosphate, and 3 mM benzamidine. The tissue homogenates were then centrifuged at 13,000 x g (20 min, 4 °C) to remove cellular debris, then protein concentrations of the crude lysates were determined by performing a Bradford assay with 89 the DC Protein Assay dye (Bio-Rad, Mississauga, ON, Canada). In some experiments, the membrane and cytosolic fractions from hippocampal slices were separated by centrifugation at 13,000 x g for 1 hr at 4°C by omitting the detergent from the solubilization buffer. The proteins from the particulate (membrane) fraction were resolved in normal solubilization buffer (as above) after removal of the cytosolic extract. Hippocampal homogenates were diluted with I X Laemmli sample buffer and boiled for 5 min. The proteins were resolved in 10% polyacrylamide gel and electrotransferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Cambridge, ON, Canada). The blots were blocked with 5% non-fat milk in TBST for 1 hr, and the membranes were incubated with primary antibody (see below) overnight at 4 °C. Following four washes with TBST, the membranes were incubated with the appropriate secondary antibody conjugated to horseradish peroxidase (1 hr, room temperature). The membranes were then washed 3-4 X (15 min) with TBST, and proteins were visualized using enhanced chemiluminescence (ECL, Amersham Bioscience, Arlington Heights, IL ). To examine interactions between adenosine A i receptors and phospho-p38 M A P K , co-immunoprecipitation was performed by first incubating 1 mg extract from hippocampal homogenates with a goat or rabbit IgG (1 hr, 4 °C), then goat or rabbit IgG agarose beads (Sigma) were added to the homogenates for a further 1 hr. In some experiments, ~ 250 pg of lysates from the membrane or cytosolic fractions were used for the co-immunoprecipitation. The agarose beads were removed by pulse spinning at 6000 rpm for 5 s, and the supernatant was subsequently reacted with an immunoprecipitating antibody overnight at 4 °C. A l receptor was immunoprecipitated using either a polyclonal goat anti-Al receptor (5 pg, Santa Cruz, CA) or a polyclonal rabbit anti-Al 90 receptor (5 pg, Sigma). After overnight pre-incubation of lysates with a polyclonal rabbit anti-phospho p38 M A P K antibody (5 pg, Cell Signalling, Beverly, M A ) , the p38 M A P K antigen was immunoprecipitated by incubating the immune complexes for >6 hrs at 4 °C with agarose beads conjugated to secondary antibody (rabbit or goat anti-IgG). Agarose beads were then collected by pulse spins, and washed 4 times with wash buffer (solubilization buffer containing 0.1 % NP-40). Proteins from the agarose beads were eluted with 60 pL of I X Laemmli sample buffer (Bio-Rad), boiled for 5 min, and resolved in 10% polyacrylamide gels. Proteins were then electrotransferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Cambridge, ON, Canada). PVDF membranes were blocked with 5% non-fat milk in TBS (50 m M Tris (pH 7.4), 150 m M NaCl) containing 0.1 % Tween-20 (TBST). The membrane was incubated overnight (4 °C) with the appropriate primary antibody diluted in 5% non-fat milk in TBST containing 0.025% sodium azide. The antibody dilutions are as follows: polyclonal rabbit anti-Al receptor (1:1000, Sigma), polyclonal rabbit anti-phospho p38 M A P K (1:500, Cell Signalling), polyclonal rabbit pan-specific p38 M A P K antibody (1:500, Cell Signalling), and mouse anti-PP2a (1:500, Cell Signalling). To normalize the protein bands from the membrane and cytosolic fractions, we used a polyclonal rabbit anti-p-actin (1:8000, RBI) or a polyclonal goat anti-GAPDH (1:300, Santa Cruz). The PVDF membranes were washed 3 X 1 5 min with TBST and then incubated with a mouse, goat, or rabbit horse radish peroxidase-conjugated secondary antibody against IgG (1:3000, Santa Cruz) in 5% non-fat milk. After 3-4 washes with TBST, labelled protein bands were visualized using ECL) and Fluor-S-Max Imaging Software. Densitometry analysis was carried out using ImageJ Software. 91 3.2.4. Drugs Adenosine, N6-cyclopentyladenosine (CPA), and 8-cyclopentyl-l,3-dipropylxanthine (DPCPX) were obtained from Sigma-Aldrich Inc. (St. Louis, M O , U.S.A.). Tetrodotoxin (TTX) was purchased from Alomone Labs (Jerusalem, Israel). SB203580, SB239G63, SB202190, SB202474, and PD98059 were obtained from Calbiochem (San Diego, CA, U.S.A.), and made up as stock in DMSO before being added to aCSF. The final concentration of DMSO was always < 0.1%. 3.3. Results 3.3.1. p38 MAPK activation increases following A^receptor stimulation The selective A( receptor agonist N6-cyclopentyladenosine (CPA) activates p38 M A P K in rat ventricular myocytes within minutes (Liu and Hofmann, 2003). In the present study, we tested whether CPA treatment increased the level of phosphorylated p38 M A P K in rat hippocampus. Hippocampal slices were treated with either normal aCSF or CPA (500 nM) for time periods ranging from 2 min to 30 min. The activation of p38 M A P K was determined using an antibody that only recognizes p38 M A P K when it is dually phosphorylated at threonine 180 and tyrosine 182. When samples of homogenized hippocampal slices were centrifuged to separate the membrane and cytosolic fractions, we observed that p38 M A P K activation increased in the membrane fraction and decreased in the cytosolic fraction (Fig. \A,B). In the membrane fraction, the response was maximal after 10 min of CPA exposure (500 nM) and returned partly back to 92 Figure 3.1. C P A increased p38 M A P K phosphorylation in membrane fractions and decreased p38 M A P K phosphorylation in cytosolic fractions. Hippocampal slices were exposed to either normal aCSF or CPA (500 nM) for 2, 5, 10, or 30 min. The membrane and cytosolic fractions of hippocampal homogenates were separated by centrifugation for Western blot analysis. Whole-cell hippocampal lysates were used for immunoprecipitation. A, Representative Western blots showing phosphorylated p38 M A P K (p-p38) and P-actin in the membrane and cytosolic fractions. B, Quantitative representation of multiple Western blots showing phospho-p38 M A P K immunoreactivity (mean ± SEM) in the membrane fraction [control (n=5), 2 min (n=5), 5 min (n=l 1), 10 min (n=l 1) and 30 min (n=5)] and cytosolic fraction [control (n=5), 2 min (n=5), 5 min (n=15), 10 min (n=9), and 30 min (n=5)]. Data were normalized to the level of protein phosphorylation at time 0 after CPA treatment, and P-actin immunoreactivity was used as a loading control. C, upper panel, Representative Western blot showing that the total amount of p38 did not change in the membrane fraction in response to CPA treatment (500nM; 10 min). C, lower panel, Quantitative representation of total p38 immunoblots (mean ± SEM) showing no significant change in the total amount of p38 (n=5). D, Co-immunoprecipitation showing an association between p-p38 and the adenosine A l Receptor (AIR) (lane 3). E, Co-immunoprecipitation showing an association between the A I R and p-p38 (lane 2, reverse of E). No association between these proteins was detected when the immunoprecipitating antibodies were omitted (lane 1 of D and E). F, phospho-p38 M A P K immunoprecipitates from membrane (lane 1) and cytosolic (lane 2) hippocampal lysates also showed immunoreactivity to Ai receptor 93 antibody. Statistical significance compared with control was assessed using a Student t-test, where * denotes p < 0.05 and ** denotes p < 0.01. p-p38 (3-actin p-p38 p-actin Time of CPA treatment (min) 0 2 5 10 30 J ! 48 48 48 B Membrane Fraction Con CPA 48 p38 . — — ~Z (3-actin — —» Cytosolic Fraction D g O 5s? 0 10. Con CPA O <5 ~o # ^ of of of 4' 4' 4' 48 33 WB:A1 Receptor o -t 175 c o * 150 o g . 125 CO £ 100 o a. 75 </> o £ 50 • Membrane o Cytosol 5 10 15 20 25 Time (minutes) 30 -<y -<y -o Of of of C 4' 48 mm <_33_ WB: Phospho-p38 C ? 48 33 WB: A1R IP: Phospho-p38 94 baseline by 30 min (the longest time point tested). In the cytosolic fraction, phospho-p38 M A P K decreased maximally at 30 min. Specifically, in membrane fraction CPA increased phosphorylated-p38 (phospho-p38) M A P K immunoreactivity to 148 ± 12% (n=5; p < 0.01) of baseline after 2 min, 135 ± 6% (n=l\\p < 0.01) after 5 min, 158 ± 15% (n=l \;p < 0.05) after 10 min, and 124 ± 35% (n=5;p > 0.05) after 30 min. In the cytosolic fraction, CPA decreased phospho-p38 M A P K to 98 ± 6% (n=5; p > 0.05) of baseline after 2 min, 85 ± 5% (n=15; p > 0.05) after 5 min, 78 ± 6% (n=9; p < 0.05) after 10 min, and 64 ± 5% (n=5;p < 0.01) after 30 min. To test whether the total amount of p38 M A P K was increasing in the membrane fraction, we performed a parallel experiment using a pan-specific p38 M A P K (pan-p38 M A P K ) antibody. We normalized the pan-p38 M A P K signal to |3-actin immunoreactivity and found that CPA (500 nM; 10 min) did not change the total amount of p38 present in the membrane fraction (100 ± 11% of control; n=5; p > 0.30) (Fig 1C). 3.3.2. The adenosine >A7 receptor and phospho-p38 MAPK are physically associated We performed co-immunoprecipitation experiments to determine whether the adenosine A l receptor exists in the same signalling complex as phospho-p38 M A P K . The adenosine A i receptor corresponds to a band at approximately 37 kDa in the rat hippocampus (Rebola et al., 2003). Immunoprecipitation with the adenosine A\ receptor antibody pulled down the adenosine A i receptor in whole-cell hippocampal lysates (Fig. ID, lane 2). Immunoprecipitation with the phospho-p38 M A P K antibody (probed with a polyclonal rabbit anti-Ai receptor antibody) revealed the A i receptor (Fig. \D, lane 3). 95 No such bands were present when the immunoprecipitating antibody was omitted (Fig. ID, lane 1). The reverse immunoprecipitation also confirmed the presence of phospho-p38 M A P K in the adenosine A i receptor immunoprecipitates (Fig. IE, lane 2). A phospho-p38 M A P K band is also present in the positive control lane (Fig. IE, lane 3). Immunoprecipitation with anti-phospho-p38 M A P K pulled down the A i receptor in lysates prepared from both membrane and cytosolic fractions (Fig. IF). These data suggest that the adenosine A] receptor and p38 M A P K physically interact in the hippocampus. 3.3.3. A1-Receptor-dependent increases in p38 MAPK activation are blocked by p38 MAPK inhibition and A1 receptor antagonism The pyridinyl imidazole compound SB203580 is a relatively selective inhibitor of p38 M A P K activity (Cuenda et al., 1995). In heart tissue, increases in p38 M A P K phosphorylation due to adenosine A i receptor activation are blocked by SB203580 (Liu and Hofrnann, 2003). We tested whether A] receptor-induced phosphorylation of p38 M A P K was also blocked by SB203580 in the brain. Hippocampal slices were either incubated in normal aCSF or SB203580 (25 pM) for 1 hour. Slices from each condition were either left untreated or treated with 40 nM CPA or 500 nM for 10 minutes, and then homogenized and centrifuged to separate the membrane and cytosolic fractions. 40 nM CPA (10 min) and 500 nM CPA (10 min) increased phospho-p38 M A P K immunoreactivity to 118 ± 7.2% (n=4;p < 0.05) and 151 ± 11.7% (n=l \;p < 0.05) of control respectively. SB203580 by itself decreased phospho-p38 M A P K immunoreactivity to 65.8 ± 8.4% (n=4) of control (p < 0.01) (Fig. 2A,B). Neither 40 96 Figure 3.2. The increase in p38 M A P K phosphorylation induced by CPA was blocked by p38 M A P K inhibition and A i receptor antagonism. Hippocampal slices were either preincubated with normal aCSF or SB203580 (25 uM) for 1 hour and then exposed to CPA (40 n M or 500 nM) for 10 min. The membrane fraction was separated by centrifugation for Western blot analysis. A, Representative Western blots showing phosphorylated p38 M A P K (p-p38) and P-actin in response to CPA in the presence and absence of SB203580 (25 pM).. B, Quantified phosphorylated p38 M A P K immunoreactivity (mean ± SEM) for control (n=4), control + SB203580 (n=4), 40 nM CPA (n=4), 40 n M CPA + SB203580 (n=4), 500 nM CPA (n=15), and 500 nM CPA + SB203580 (n=4). C, Representative Western blots showing that the CPA-induced increase in phospho-p38 M A P K was blocked in the presence of DPCPX. D, Quantified phospho-p38 M A P K immunoreactivity for control (n=10), 500 nM CPA (n=10), and 500nM DPCPX (n=10). Data were normalized to P-actin immunoreactivity. Statistical significance compared with control was assessed using a Student Mest, where * denotesp < 0.05 and ** denotesp < 0.01. 97 * f £ <f £ ^5 p-p38 B oo O co o <T # * >T <^ * J 48 48 Con SB CPA+SB CPA+SB (40nM) (500nM) p-p38 p-actin 48 48 Con CPA +DPCPX 98 nM CPA nor 500 nM CPA had an effect in slices pre-treated with SB203580, as phospho-p38 M A P K immunoreactivity decreased to 63.1 ± 6.4% (n=4) and 62.3 ± 7.8% (n=4) of control respectively. Neither condition was statistically different from slices treated with SB203580 alone (p > 0.30). We also tested whether the A i receptor antagonist DPCPX prevented the CPA-induced increase in the amount of phospho-p38 M A P K in the membrane fraction. Because DPCPX increases glutamate release in brain slices (Sehmisch et al., 2001; Marcoli et al., 2003), which could potentially activate p38 M A P K through metabotropic glutamate receptors (Rush et al., 2002), we treated slices with tetrodotoxin (TTX) (1.2 uM) for 20 min to block action potentials and thus prevent DPCPX-induced increases in glutamate release. We found that in the presence of TTX, CPA (500 nM; 10 min) increased phospho-p38 M A P K to 143 ± 15% (n=10; p < 0.01) of control levels in the membrane fraction (Fig. 2C,D). This CPA-induced increase in phospho-p38 M A P K was completely blocked (98 ± 15% of control; n=10; p > 0.80) by pre-treatment with DPCPX (500 nM; 20 min) for 20 min prior to CPA application (Fig. 2C,D). 3.3.4. A^Receptor activation induced translocation of protein phosphatase 2a to the plasma membrane Adenosine A i receptor activation causes protein phosphatase 2a (PP2a) translocation and activation in ventricular myocytes (Liu and Hofmann, 2002) in a pathway requiring p38 M A P K (Liu and Hofmann, 2003). We tested whether CPA treatment activated PP2a in rat hippocampal slices. Slices were treated with either normal aCSF or CPA (500 nM) for time periods ranging from 2 min to 30 min, and homogenized and centrifuged to isolate the membrane fraction. Using an antibody recognizing the C-subunit of PP2a, we 99 Figure 3.3. C P A increases PP2a levels in the membrane fraction. Hippocampal slices were exposed to either normal aCSF or CPA (500 nM) for 2, 5, 10, or 30 min. The membrane fraction was extracted by centrifugation for Western blot analysis. A, Representative Western blot showing the time course of CPA-induced changes in PP2a immunoreactivity. B, Quantified PP2a immunoreactivity, showing that PP2a increases within minutes in response to CPA treatment [control (n=7), 2 min (n=7), 5 min (n=7), 10 min (n=15) and 30 min (n=7)]. C, Representative Western blot showing that the CPA-induced increase in PP2a levels was blocked in the presence of DPCPX (500 nM). D, Quantification of C (n=8). E, Western blot showing that the p38 M A P K inhibitor SB239063 (15 uM) prevented CPA from increasing PP2a levels. F, Quantification of E (n=6). A l l data were normalized to p-actin immunoreactivity. Values are means ± SEM. Statistical significance compared with control was assessed using a Student Mest, where * denotesp < 0.05 and ** denotesp < 0.01. 100 101 determined that CPA treatment increased PP2a immunoreactivity within minutes in membrane fractions (Fig 3A,B). Specifically, PP2a immunoreactivity increased to 123 ± 10% (n=7; p < 0.05) of baseline after 2 min, 145 ± 13% (n=7; p < 0.05) after 5 min, 139 ± 13% (n=15;p < 0.05) after 10 min, and 121 ± 12% (n=7; p > 0.05) after 30 min. The response was maximal after 5 min of CPA exposure and returned partly back to baseline by 30 min. In addition, we tested whether DPCPX prevented the increase in PP2a immunoreactivity in membrane fractions induced by treating slices with CPA. A l l slices were incubated in T T X (as above) and then exposed to either CPA (500 nM; 10 min) alone or in the presence of D P C P X (500 nM). CPA increased the amount of PP2a in the membrane fraction to 145 ± 21% (n=8; p < 0.05) of control (Fig. 3C,D). However, in the presence of DPCPX, the CPA-induced increase in PP2a was blocked (86 ± 16% of control; n=8;p > 0.30) (Fig. 3C,D). Finally, we also determined that p38 M A P K inhibition prevented the CPA-induced increase in PP2a immunoreactivity in membrane fractions (Fig. 3E,F). We incubated slices in either normal aCSF or the second generation p38 M A P K inhibitor SB239063 (15 pM) for 1 hour prior to CPA treatment (500 nM; 10 min). In slices treated with SB239063, CPA failed to increase levels of PP2a in the membrane fraction (91 ± 6% of control; n=6; p > 0.15). Together, these data suggest that p38 activation is required for the A] receptor mediated translocation of PP2a to the membrane. 102 3.3.5. Adenosine-induced depression of synaptic transmission is mediated by the Ai receptor subtype and is sensitive to p38 MAPK inhibition To investigate the functional significance of A!-receptor-mediated p38 M A P K activation in the brain, we used extracellular fEPSP recordings to monitor the effect of p38 M A P K inhibition on the action of adenosine in area CA1 of the rat hippocampus. We confirmed that adenosine-induced depression of synaptic transmission in area CA1 is mediated by the Ai-receptor subtype in area CA1 (Dunwiddie and Fredholm, 1989; Wu and Saggau, 1994; Johansson et al., 2001). In normal aCSF, perfusion of 20 u M adenosine onto the slice for 10 min decreased the mean normalized fEPSP slope to 33 ± 4% (p < 0.01; n=8) of baseline (Fig. 4A). Perfusion of 100 nM DPCPX, a specific Al-receptor antagonist (Bruns et al., 1987; Haleen et al., 1987), onto the same slice for 20 min increased the fEPSP slope to 125 ± 4% (p < 0.01; n=8) of baseline, presumably due to removal of tonic Ai-receptor-mediated inhibition of glutamate release. Perfusion of 20 uM adenosine in the presence of 100 nM DPCPX had no effect on fEPSPs (98 ± 3% of baseline; p > 0.15; n=8), indicating that the Ai-receptor subtype mediates the inhibition of fEPSPs caused by adenosine in rat CA1. Perfusion of the p38 inhibitor, SB203580 (25 uM; 40 min) slightly increased the mean fEPSP slope to 111 ± 4.6% of baseline (p < 0.05; n=5) (Fig. 4B). The magnitude of fEPSP depression induced by adenosine (10 uM) was attenuated by SB203580 (adenosine decreased the mean fEPSP slope by 55.0 ± 3.8% in normal aCSF but by only 27.3 + 2.6% after SB203580 treatment; n=5;p < 0.01) (Fig. 4B,D). Because SB203580 was dissolved in DMSO, we ensured that 0.1% DMSO (Fig. 4 Q had no effect on 103 Figure 3.4. Adenosine-induced depression of CA1 fEPSPs is mediated by the A l -receptor subtype and sensitive to p38 MAPK inhibition. A-C Averaged sample traces and plot of the mean fEPSP slope (± SEM) over time normalized to baseline. fEPSPs (evoked every 30 sec) were recorded in CA1 stratum radiatum. A, Adenosine reversibly decreased the mean normalized fEPSP slope. After recovery to baseline, the A1-receptor antagonist D P C P X (100 nM) was bath applied, resulting in an increase in the mean fEPSP slope. A second perfusion of adenosine in the presence of 100 nM D P C P X had no effect on the mean normalized fEPSP slope (n=8). B, Perfusion of SB20380 (25 pM) slightly increased baseline synaptic transmission and partially inhibited the ability of adenosine (10 pM) to decrease fEPSPs (n=5). C, The effect of adenosine (10 pM) was the same whether applied in the presence of 0.1% DMSO (50 min perfusion) or normal aCSF (n=10). D, Summary of A-C showing that SB203580 attenuates adenosine-induced depression whereas DMSO does not. Calibration: 10msec, lmV. *Denotes statistical significance using a Student t-test (p < 0.01). 104 20|iM Adenosine 100nM DPCPX CD 125-Q. „—„ O CO line 100-Q. CO ase 75-CL LD M— o 50-c CO CP 25-2 0-20uM Adenosine a* 1 V V V V J 1 r> A 1 B CD CL s O CD to £ Q_ CD CO CD CL _Q LU o CD 0 — 125 100 75 50 25 0 20 40 60 80 Time (minutes) 10(iM Adenosine 25uM SB203580 10uM Adenosine 1 0 20 40 60 80 Time (minutes) D 100 120 0.1% DMSO 0 20 40 60 80 100 120 Time (minutes) 105 baseline synaptic transmission nor the magnitude of adenosine induced depression (adenosine decreased the mean fEPSP slope by 51.0 ± 2.4% in normal aCSF and 51.3 ± 3.5% after 50 min of DMSO treatment in the same slice; n=10; p > 0.30) (Fig. AB.D). 3.3.6. Neither the inactive analogue SB202474 nor the ERK 1/2 MAPK inhibitor PD98059 decreased adenosine-induced synaptic depression The health of hippocampal slices, as determined by the slope of evoked fEPSP slopes, was not adversely affected by prolonged exposure to SB203580 (over 50 min in the bath perfusate). Therefore in the next set of experiments we preincubated slices in SB203580 instead of prolonged bath perfusions. In all subsequent experiments matched slices from the same animal were either pre-incubated in SB203580 (25 pM; 1-2 hours) or normal aCSF. Perfusion of adenosine (10 pM; 10 min), depressed the mean normalized fEPSP slope by 61.7 ± 2.7% (n=5) in control, and only 30.6 ± 3.0% (n=6) in slices pre-incubated in SB203580 (p < 0.01) (Fig. 5A,D). Thus, the effects of SB203580 on adenosine-mediated synaptic depression were similar in both pre-incubation and perfusion experiments. SB202474 is an inactive pyridinyl imidazole compound that has been used as a negative control in p38 M A P K studies (Lee et al., 1994; Y u et al., 2000). Slices were either pre-incubated with 25 p M SB202474 or with normal aCSF (control). There was no difference in the action of adenosine in slices treated with SB202474 compared with control slices (Fig. 5B,D). In control, perfusion of adenosine (10 pM; 10 min) depressed the mean fEPSP slope to 52.2 ± 6.4% (n=7). In slices that had been pre-treated with 106 Figure 3.5. Adenosine-induced synaptic depression was attenuated by SB203580, but not SB202474 (an inactive analogue) nor PD98059 (ERK 1/2 MAPK inhibitor). A-C, Averaged sample traces and plot of the mean normalized fEPSP slope (± SEM) over time. A, Matched slices were either pre-treated with SB203580 for 1-2 hours (n=7) or normal aCSF (n=13). Adenosine-induced depression of fEPSPs was attenuated in the presence of SB203580. B, Matched slices were either pre-treated with SB202474 for 1-2 hours (n=5) or normal aCSF (n=7). SB202474 did not affect adenosine-induced depression of fEPSPs. C, Matched slices were either pre-treated with PD98059 (n=5) or normal aCSF (n=4). Adenosine-induced depression of fEPSPs was not attenuated by PD98059. D, Summary of A-C showing the mean maximal effect of adenosine expressed as percentage inhibition from baseline. Calibration: 10msec, lmV. *Denotes statistical significance compared with control using a Student Mest (p < 0.01). 107 10u.M Adenosine 10 20 30 40 Time (minutes) 10u.M Adenosine 10 20 30 40 Time (minutes) B 10u.MI Adenosine 10 20 30 40 Time (minutes) <# <??> „<>dr& r # <# 108 25 uM SB202474, adenosine depressed the mean fEPSP slope by 67.0 ± 3.4% (n=5), which was not statistically different from control (p > 0.10). The extracellular signal-regulated kinase (ERK) 1/2 M A P K pathway is activated by synaptic activity and is required for the induction of long-term potentiation of synaptic transmission in area CA1 of the hippocampus (Impey et al., 1999; Bolshakov et al., 2000). The E R K 1/2 M A P K pathway is also required for metabotropic glutamate receptor-dependent long term depression (Gallagher et al., 2004). We tested whether E R K 1/2 M A P K also played a role in adenosine-mediated synaptic depression. Slices were either pre-treated with the selective E R K 1/2 M A P K inhibitor PD98059 (Pang et al., 1995) (50 pM) or with normal aCSF (control). In control, adenosine (10 p M ; 10 min) depressed the mean fEPSP slope to by 52.5 ± 5.7% (n=4) of baseline (Fig. 5C,D). In slices that were pre-treated with 50 p M PD98059, adenosine depressed the mean fEPSP slope by 49.0 ± 3.0%> (n=5) of baseline, which was not statistically different from control (p > 0.30). These data indicate that inhibiting E R K 1/2 M A P K does not modify adenosine-mediated synaptic depression in area CA1. 3.3.7. Ai-receptor-mediated synaptic depression was decreased by p38 MAPK inhibition The physiological ligand adenosine, acts at multiple receptor subtypes, and is rapidly degraded by ecto-nucleotidases and/or removed from the extracellular space via nucleoside transporters (Dunwiddie and Masino, 2001). To avoid these issues, we next used the Ai-receptor agonist N6-cyclopentyladenosine (CPA). We tested whether synaptic depression induced by CPA was also attenuated by the p38 M A P K inhibitor 109 SB203580. Recordings were obtained from slices that were either incubated in 25 uM SB203580 (1-2 hours) or normal aCSF (control). In control, a 10 min perfusion of 40nM CPA decreased the mean fEPSP slope by 72.8 ± 2.1% (n=4) (Fig. 6A,B,E), confirming that selective A i -receptor activation decreases synaptic transmission. However, in SB203580, CPA perfusion only decreased the mean fEPSP slope by 10.4 ± 2.0% (n=5; p < 0.01) (Fig. 6A,B,E). These results provide additional evidence that p38 M A P K activity is necessary for A1-receptor-mediated synaptic depression. To further confirm that p38 M A P K activity was required for Ai-receptor-mediated depression of fEPSPs, we carried out an experiment using another pyridinyl imidazole compound, SB202190 (50 uM), which also blocks the activity of p38 M A P K (Davies et al., 2000). Recordings were obtained from slices that were either incubated in 50 uM SB202190 (1-2 hours) or normal aCSF (control). In control, a 15 min perfusion of CPA (40 nM) decreased the mean fEPSP slope by 74.3 ± 2.3% (n=5) (Fig. 6C,E). However, in SB202190, CPA perfusion decreased the mean fEPSP slope by only 25.0 ± 6.0% (n=5) of baseline (p < 0.01) (Fig. 6C.E). To control for possible non-specific effects of the pyridinyl imidazole p38 M A P K inhibitors SB203580 and SB202190, we tested the effects of the inactive pyridinyl imidazole analogue SB202474 on the depression of CA1 fEPSPs induced by CPA. Recordings were obtained from slices that were either incubated in 25 uM SB202474 (1-2 hours) or normal aCSF (control). In control, a 15 min perfusion of 40nM CPA decreased the mean fEPSP slope 74.8 ± 3.4% (n=l 1) (Fig. 6C.E). In SB202474-incubated slices, a 15 min perfusion of 40nM CPA decreased the mean normalized fEPSP 110 Figure 3.6. CPA-media ted depression of C A 1 fEPSPs was attenuated by p38 M A P K inhibi t ion. A, Representative fEPSP traces before (1) and after (2) CPA (40 nM) treatment in slices either in normal aCSF (top) or preincubated in 25 uM SB203580 (bottom). Calibration: 10 msec, 1 mV. B-D, Plots of the mean normalized initial fEPSP slope (± SEM) over time. B, Slices were either preincubated with SB203580 (n=5) or normal aCSF (n=4) prior to treatment with CPA for 10 min. C, Slices were either preincubated with the p38 M A P K inhibitor SB202190 (50 uM) (n=5) or normal aCSF (n=5) and then treated with CPA (10 min). D, Slices were either preincubated with the inactive analogue SB202474 (25 uM) (n=8) or normal aCSF (n=l 1) and then treated with CPA (10 min). E, Summary of B-D showing the magnitude of CPA-induced depression of fEPSP slope (± SEM) as a percentage decrease from baseline in normal aCSF, SB203580, SB202190, or SB202474. CPA-mediated depression of fEPSPs was attenuated by both p38 M A P K inhibitors (SB203580 and SB202190) but not by the inactive analogue (SB202474). *Denotes statistical significance using the Student Mest (p < 0.01). I l l 112 amplitude to 70.6 ± 4.4% (n=8), which was not statistically different from control (p > 0.30) (Fig. 6D,E). 3.3.8. Hypoxia-induced synaptic depression was mediated by the A1-receptor and was attenuated by p38 MAPK inhibition Hypoxia releases adenosine into the extracellular space (Fowler, 1993a; Dale et al., 2000), where it activates Ai-receptors. During extended periods of hypoxia, adenosine release contributes to -50% of the hypoxia-induced synaptic depression (Fowler, 1989). In contrast to long hypoxic insults, the synaptic depression induced by short hypoxic insults (e.g. 2-3 min) is almost completely mediated by the Apreceptor (Latini et al., 1999b; Sebastiao et al., 2000). In the present study, we found that the synaptic depression induced by a brief (5 min) hypoxic insult was virtually blocked in the presence of the Ai-receptor antagonist DPCPX (100 nM) (Fig. 1A-C). Hypoxia was induced by perfusion of aCSF bubbled with 95%N 2 /5%C0 2 . In normal aCSF, 5 min perfusion of hypoxic solution decreased the mean fEPSP slope by 46.9 ± 4.3% (n=5) (Fig. 1A-C). In the presence of DPCPX, 5 min hypoxia did not have a significant effect, only decreasing the fEPSP slope by 5.6 ± 1.7% (n=5; p > 0.30). This was significantly less depression than occurred in normal aCSF (n=5;p < 0.01) (Fig. 1A-C). To test the contribution of p38 M A P K activity to hypoxia-induced synaptic depression, we pre-treated slices with 25 uM SB203580 (1-2 hours) and exposed them to hypoxia for 5 min. In SB203580-treated slices, 5 min hypoxia decreased the mean fEPSP slope by 21.0 ± 1.8% (n=5), which was significantly less depression than occurred in matched control slices (p < 0.01) (Fig. 1A-C). Given that the synaptic depression induced 113 Figure 3.7. p38 MAPK inhibition attenuated hypoxia-mediated depression of CA1 fEPSPs. A, Representative fEPSP traces before (1) and after (2) treatment with hypoxia (5 min). Hypoxia was induced by bath application of aCSF bubbled with 95%N2/5%C02. Slices were either treated with 100 nM DPCPX, 25 p M SB203580, or normal aCSF prior to exposure to hypoxia. Calibration: 10 msec, 1 mV. B, Plot of the mean normalized initial fEPSP slope (± SEM) over time. A1-receptor inhibition with DPCPX (n=5) and p38 M A P K inhibition with SB203580 (n=5) attenuated hypoxia-induced synaptic depression versus matched controls (n=5). C, Summary of B showing the mean fEPSP slope (± SEM) as a percentage of baseline following 5 min hypoxia in normal aCSF, DPCPX, or SB203580. *Denotes statistical significance using a Student /-test (p < 0.01). 114 5 min hypoxia ^ ' K / 1+2 +DPCPX +SB203580 VJ V VJ B 100-CD Q. ^ O CO line 75-CL CD PS bas 50-UJ o c CD 25-CD Hypoxia . Control » SB203580 * D P C P X 10 20 30 Time (minutes) > j& 40 115 by 5 min hypoxia was almost entirely blocked by the A] antagonist DPCPX, this finding indicates that p38 M A P K contributes to the synaptic depression induced by Ai-receptor activation due to hypoxia. Thus, the neuroprotective action of adenosine released during oxygen deprivation may result from A i-receptor-mediated activation of p38 M A P K . 3.4. Discussion In this study, we discovered a novel interaction between the adenosine A i-receptor and p3 8 M A P K in the central nervous system. We present biochemical and electrophysiological evidence showing that p38 M A P K is activated by A i receptor stimulation and that p38 M A P K inhibition significantly attenuates Ai-receptor mediated synaptic depression. Furthermore, co-immunoprecipation revealed that phospho-p38 M A P K and the adenosine A] receptor are physically associated in the hippocampus. Our results suggest that A i receptors and p38 M A P K form a signaling complex, and that adenosine A i receptor-mediated synaptic depression is at least partly dependent on the activation of p38 M A P K in the rat hippocampus. Although adenosine Aj-receptors and p38 M A P K are expressed both presynaptically (Maruyama et al., 2000) and postsynaptically (Bolshakov et al., 2000; Zhu et al., 2002a) in the hippocampus, the primary mechanism underlying adenosine-induced synaptic depression is inhibition of calcium influx into presynaptic nerve terminals (Dunwiddie and Masino, 2001). In area CA1 of the hippocampus, as well as other areas, the adenosine A i-receptor subtype decreases neurotransmitter release from presynaptic nerve terminals primarily by inhibiting N-type calcium channels (Mogul et al., 1993; Yawo and Chuhma, 1993; Mynlieff and Beam, 1994; Wu and Saggau, 1994; Ambrosio et al., 1997; Zhang and Schmidt, 1999; Brown and Dale, 2000; Park et al., 116 2001; Sun et al., 2002; Wang et al., 2002b; Manita et al., 2004). Therefore, our study suggests that p38 M A P K activation is a necessary step for A\ receptor activation to decrease synaptic transmission by downregulating presynaptic voltage-dependent calcium channels. p38 M A P K activation is necessary for the depression of N-type calcium currents in neuroblastoma X glioma cells in response to bradykinin application (Wilk-Blaszczak et al., 1998). However, it is not yet determined that p38 M A P K activation modulates calcium channel function in the hippocampus. A possible link between p38 M A P K and calcium channel modulation is our finding that A]-receptor stimulation induced PP2a translocation to the membrane requires p38 M A P K , which is in agreement with previous studies in cardiomyocytes (Liu and Hofmann, 2002, 2003). PP2a is also activated by p38 M A P K in the brain where it can modulate serotonin transporters (Zhu et al., 2005a). However, it is not known whether PP2a plays a role in presynaptic inhibition. PP2a binds to the C-terminus of alpha-lC calcium channels, and reverses PKA-mediated phosphorylation of serine 1928 located inside this C-terminal region (Davare et al., 2000). A recent report has also demonstrated direct interaction of the protein phosphatase 2Ca (PP2Ca) to the C-terminus of both N -and P/Q-type calcium channels, and that PP2Ca reverses phosphorylation of these neuronal calcium channels by P K C (Li et al., 2005). However, it is unclear whether PP2a also physically associates with and alters the function of presynaptic alpha IB (N-type) calcium channels. In the present study, we demonstrated that CPA-mediated synaptic depression accompanied increased activation of p38 M A P K and PP2a. Future biochemical and functional studies are needed to address whether this decreased synaptic 117 transmission may be due, in part, to neuronal calcium channel inhibition by channel interaction with PP2a. Other voltage-gated ion channels, including sodium channels and hyperpolarization-activated cyclic nucleotide (HCN)-gated channels, are newly discovered potential targets for the A i receptor-mediated p38 M A P K activation. A recent report demonstrated that the sodium channel N a v l .6 and p38 M A P K co-localized in rat cerebellar Purkinje cells and co-immunoprecipitated from rat brain (Wittmack et al., 2005). Activation of p38 M A P K decreased the sodium current density by phosphorylating a serine residue within the first cytoplasmic loop of this channel. Since the sodium channel N a v l .6 mRNA and proteins are expressed in the hippocampal presynaptic and postsynaptic sites (Schaller et al., 1995; Caldwell et al., 2000), it is possible that downregulation of Nav1.6 function by p38 M A P K activation contributes to decreased membrane excitability and leads to synaptic depression. Another possibility for p38 modulation is the H C N channel. p38 M A P K inhibition with SB203580 has recently been shown to decrease H C N current in hippocampal pyramidal neurons, which would be predicted to increase membrane excitability (Poolos et al., 2002; Poolos, 2005). Indeed we found that SB203580 caused a modest (10-20%) increase in field potential slopes (Figure 45), which could be attributed, in part, to the modulation of H C N channels. It is interesting that this p38 M A P K inhibitor did not affect the N a v l .6 current density (Wittmack et al., 2005). It remains to be established whether adenosine Aj receptor-mediated p38 M A P K activation accompanies Nav1.6 channel downregulation and/or H C N channel upregulation to play additional roles in decreasing membrane excitability and synaptic depression. 118 Canonical M A P K activation occurs via a cascade of three kinases in a module consisting of M A P K kinase kinase ( M A P K K K ) , M A P K kinase (MAPKK) , and M A P K . M K K 3 and M K K 6 are the M A P K K s that activate p38a M A P K . Pyridinal imidazoles such as SB203580 inhibit p38a M A P K by binding to both the active and inactive enzyme in an ATP-competitive manner, and thus prevent p38 M A P K from phosphorylating its downstream substrates (Kumar et al., 2003). SB203580 does not inhibit M K K 3 or M K K 6 (Kumar et al., 1999). Nonetheless, treatment with SB203580 results in the abolition of CPA-induced increases in p38 M A P K phosphorylation in the heart (Liu and Hofmann, 2003) and brain (Fig. 2; current study). SB203580 also prevents phosphorylation of p38a in a number of other experimental systems (Frantz et al., 1998; Galan et al., 2000; Matsuguchi et al., 2000; Zhuang et al., 2000). These observatoins imply that phosphorylation of p38 M A P K can occur due to intrinsic kinase activity. Indeed, it was reported that binding of T A B 1 (transforming growth factor-b-activated protein kinase 1 (TAKl)-binding protein 1) to p38a causes autophosphorylation and subsequent activation of the kinase in a pathway independent of M K K 3 and M K K 6 (Ge et al., 2002). Moreover, TABl-induced phosphorylation of p38a is sensitive to SB203580 both in vitro and in vivo, whereas T A B 1-independent phosphorylation of p38a is not (Ge et al., 2002). Thus, the sensitivity of CPA-induced p38 M A P K phosphorylation to SB203580 may indicate that A i receptor-dependent p38 M A P K activation occurs via autoactivation of p38 M A P K facilitated by interaction with a regulatory molecule (e.g. TAB1) in the hippocampus. Excessive synaptic release of glutamate during trauma such as hypoxia/ischemia is a major mechanism underlying neuronal cell death (Kass and Lipton, 1982; Rothman, 119 1983; Rothman, 1984). Adenosine acts as an endogenous neuroprotectant during hypoxia/ischemia, likely by decreasing glutamate release in the brain via A i receptors (Dunwiddie and Masino, 2001). The importance of adenosine as an endogenous neuroprotective agent is exemplified by the observation that brief insults of hypoxia/ischemia are more deleterious when repeated (Tomida et al., 1987; Kato et al., 1989) likely because of adenosine depletion (Pearson et al., 2001b). However, a role for p38 M A P K in adenosine-mediated neuroprotection has not been described in the brain. Previous studies have found that the p38 M A P K pathway is activated following hypoxia/ischemia in brain tissue (Walton et al., 1998; Zhu et al., 2002b). However, there is evidence that p38 M A P K activity is deleterious, mediating the cell death induced by hypoxia (Zhu et al., 2002b), oxygen glucose deprivation, magnesium withdrawal, and glutamate receptor agonist exposure (Legos et al., 2002). In contrast, in heart tissue, p38 M A P K plays an important role in cardioprotection during ischemia (Weinbrenner et al., 1997; Baines et al., 1998; Baines et al., 1999; Zhao et al., 2001a; Lasley et al., 2005). The results of the present study indicate that p38 M A P K could act as a neuroprotectant by mediating the actions of adenosine in the brain. The apparent contradiction between our results showing that p38 M A P K activation may be neuroprotective, and previous findings showing that p38 M A P K inhibition may be neuroprotective (Barone et al., 2001; Legos et al., 2002) is probably attributable to differences in the time scales analyzed. We found that A] receptor stimulation phosphorylated p38 M A P K within minutes (Fig. \A,B). However, most reports linking p38 M A P K activity and excitotoxic injury have analyzed cell survival in culture hours and/or days following trauma (Walton et al., 1998; Barone et al., 2001; 120 Legos et al., 2002; Zhu et al., 2002b). Our results suggest that, in contrast to its longer-term deleterious actions, p38 M A P K activation may have a short-term neuroprotective role during transient oxygen deprivation. Therefore, a viable therapeutic intervention to prevent ischemic brain damage might involve the use p38 M A P K inhibitors only after reperfusion. We have shown previously that p38 M A P K is involved in presynaptic inhibition, as it mediates the potent and long-lasting synaptic depression induced by 2',3'-0-(4-benzoylbenzoyl)-ATP (Bz-ATP) at mossy fiber-CA3 synapses (Armstrong et al., 2002). However, in mossy fiber-CA3 synapses, there is no evidence that adenosine-induced synaptic depression is attenuated by SB203580 (Armstrong et al., 2002). Thus, although p38 M A P K is involved in presynaptic inhibition at both mossy fiber-CA3 and CA3-CA1 synapses, the pathway that activates p38 M A P K is different in these two distinct areas of the hippocampus. It is unlikely that SB203580 exerts its effects on mossy fiber-CA3 synaptic depression by blocking adenosine transporters as previously suggested (Kukley et al., 2004). The inactive analogue SB202474 (Sweeney et al., 1999) is reported to have a similar effect on nucleoside tranporters as SB203580 (Huang et al., 2002). Therefore to rule out the possibility that SB203580 and SB202190 were exerting their effects by blocking nucleoside transporters, we tested whether SB202474 affected adenosine- or CPA-induced synaptic depression. We found that SB202474 failed to affect adenosine Ai-receptor-mediated synaptic depression in area CA1 (Fig 5B,D and 6D,E). In addition, inhibitors of nucleoside transporters in brain slices actually enhance the concentration of extracellular adenosine during hypoxia or electrical stimulation (Fredholm et al., 1994). 121 This indicates that the net effect of nucleoside transporters is the uptake of adenosine from the extracellular space (Latini and Pedata, 2001). Thus, blockade of nucleoside transporters would be expected to decrease synaptic transmission due to a build-up of excess adenosine in the extracellular space as has been reported (Dunwiddie and Diao, 2000). In contrast, we observed that SB203580 increased synaptic transmission (Fig. 45). 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J Biol Chem 275:25939-25948. 131 C h a p t e r F o u r : A i R e c e p t o r - M e d i a t e d S y n a p t i c D e p r e s s i o n i s m e d i a t e d b y s e q u e n t i a l a c t i v a t i o n o f p 3 8 M A P K a n d C -J u n N - T e r m i n a l K i n a s e i n t h e r a t h i p p o c a m p u s A version of this chapter was submitted for publication: Brust, Tyson B.; Cayabyab, Francisco S.; and MacVicar, Brian A. (2006). C-Jun N -terminal kinase regulates adenosine A l receptor-mediated synaptic depression in the rat hippocampus. Neuropharm Apr 2. 132 4.1. Introduction Adenosine A i receptors are G protein-coupled receptors that rapidly cause potent depression of synaptic transmission in the central nervous system. The level of ambient adenosine in the extracellular space gates synaptic transmission and plasticity in the hippocampus by inhibiting glutamate release (de Mendonca et al., 1997; Nicoll and Schmitz, 2005). Because the concentration of adenosine in the extracellular space increases dramatically during brain trauma, such as stroke and seizure, it may act as an endogenous neuroprotectant by preventing excessive excitation (Dunwiddie and Masino, 2001). Despite the importance of A i receptor activation in mediating presynaptic inhibition, the detailed signalling pathway leading from A] receptor stimulation to decreased neurotransmitter release are unclear. Mitogen-activated protein kinases (MAPKs) are a large family of serine/threonine kinases widely expressed in brain that regulate diverse physiological processes such as cell survival, synaptic plasticity, and inducible gene expression (Nozaki et al., 2001; Pearson et al., 2001a). There are three subfamilies of M A P K s : 1) extracellular signal-regulated kinases; 2) p38 M A P K ; and 3) the c-Jun N-terminal kinase (JNK). p38 M A P K and JNK are activated when phosphorylated by M A P K kinases MKK3/6 and MKK4/7 respectively. This occurs in response to diverse cellular stresses such as ischemia, osmotic shock, cytokines, growth factors, ultraviolet light, and via activation of G protein-coupled receptors (Marinissen and Gutkind, 2001). Numerous studies suggest that both p38 M A P K and JNK play roles in synaptic depression. For example, p38 M A P K mediates the long-term depression (LTD) induced by chemical stimulation of metabotropic glutamate receptors (Bolshakov et al., 2000), as 133 well as removal of A M P A receptors during LTD (Zhu et al., 2002a). JNK is involved in low frequency stimulation-dependent L T D in the dentate gyrus (Curran et al., 2003), and is required for the removal of synaptic A M P A receptors during depotentiation (Zhu et al., 2005b). JNK activation may also depress synaptic transmission through a presynaptic mechanism. The inhibitory effect of interleukin -1 p and the beta-amyloid peptide on long term potentiation (LTP) was reported to involve JNK and/or p38 M A P K activation (Vereker et al., 2000a; Costello and Herron, 2004), and increased JNK and p38 M A P K phosphorylation is associated with decreased glutamate release in the hippocampus (Vereker et al., 2000a). Together, these findings suggest that activation of endogenous JNK and p38 M A P K initiates signalling cascades that inhibit glutamate release from presynaptic terminals. In support of this hypothesis, we have recently described a role for p38 M A P K in adenosine A i receptor-mediated depression of CA3-CA1 synaptic transmission in the rat hippocampus (Brust et al., 2005). In the present study, we tested whether JNK activation also played a role in adenosine A i receptor-mediated inhibition of neurotransmission. We recorded fEPSPs in area CA1 of rat hippocampal slices to monitor the effect of JNK inhibitors on adenosine-induced synaptic depression. Western blot analysis was used to quantify changes in the phosphorylation state (and thus activation) of JNK in hippocampal lysates. Co-immunoprecipitation was used to investigate an association between the A i receptor and JNK. We report that the synaptic depression induced by selective A i receptor activation was attenuated by JNK inhibition, and that A i receptor stimulation induced a rapid and 134 transient phosphorylation of JNK through a mechanism that may involve the association of A i receptors and JNK in the plasma membrane. 4.2. Materials and methods 4.2.1. Hippocampal Slice Preparation Sprague Dawley rats (p21-p28) were anaesthetized with halothane and decapitated according to protocols approved by the University of British Columbia committee on animal care. Brains were rapidly extracted and placed into ice-cold oxygenated dissection medium containing the following (in mM): 87 NaCl, 2.5 K C l , 2 NaH 2 P0 4 , 7 M g C l 2 , 25 NaHCC>3, 0.5 CaCl 2 , 25 D-glucose, and 75 sucrose. Hippocampal slices (400pm thick) were cut using a vibrating tissue slicer (VT1000S, Leica, Nussloch, Germany) and maintained for 1-5 hours at 24°C in aCSF containing (in mM): 119 NaCl, 2.5 K C l , 1.3 MgS04, 26 NaHC0 3 , 2.5 CaCl 2 , and 10 D-glucose, and aerated with 95%0 2/5% C 0 2 (or 95%N 2/5%C02 for hypoxia). For electrophysiological recordings, slices were transferred to a submerged recording chamber and allowed to equilibrate for at least 1 hour. The bath solution was perfused with aerated aCSF at a rate of 1.5 - 2 mL/min. 135 4.2.2. Electrophysiology Field excitatory postsynaptic potentials (fEPSPs) were evoked by orthodromic stimulation of the Schaffer collateral pathway using a bipolar tungsten-stimulating electrode. Glass micropipettes filled with aCSF (resistance 1-3MO) were used to measure CA1 fEPSPs in stratum radiatum. fEPSP signals were amplified 1000 times with an A C amplifier, band-pass filtered at 0.1-100 Hz, digitized at 10 kHz using a Digidata 1320A interface board (Axon Instruments, Foster City, CA), and transferred to a computer for analysis. Data were analyzed using Clampfit 9.0 (Axon Instruments). Baseline synaptic responses were established by evoking fEPSPs every 30s (0.03Hz) for at least 20 min. The fEPSP slope was normalized to the mean of the 20 sweeps (10 min) immediately preceding drug perfusion. The mean normalized fEPSP slope was plotted as a function of time with error bars representing the standard error of the mean (SEM). Sample traces are the average of 5 sweeps from a recording that was included in the plot of the mean normalized fEPSP slope. Input-Output curves were generated by systematically increasing the voltage delivered by the stimulating electrode (4V - 10V in increments of IV), and measuring the resulting fEPSP slope. A l l bar graphs show the mean normalized percent inhibition from baseline ± SEM. Statistical significance was assessed using a Student /-test (p < 0.05). 136 4.2.3. Western Blot Analysis For biochemical studies, rat hippocampal slices were first incubated with various treatments (see below) and then lysed in a solubilization buffer (30 min, 4 °C) that contained 1% NP-40, 20 m M MOPS (pH 7.0), 5 m M EDTA, 2 m M EGTA, 1 m M phenylmethylsulfonyl fluoride (PMSF), 10 pg/ml aprotinin, 10 ug/ml leupeptin, 10 pg/ml pepstatin A , 1 m M N a 3 V 0 4 , 30 m M NaF, 40 m M p-glycerophosphate (pH 7.2), 20 m M sodium pyrophosphate, and 3 m M benzamidine. The tissue homogenates were then centrifuged at 13,000 x g (20 min, 4 °C) to remove cellular debris, then protein concentrations of the crude lysates were determined by performing a Bradford assay with the DC Protein Assay dye (Bio-Rad, Mississauga, ON, Canada). Membrane and cytosolic fractions from hippocampal slices were separated by centrifugation at 13,000 x g for 1 hr at 4°C by omitting the detergent from the solubilization buffer. The proteins from the particulate (membrane) fraction were resolved in normal solubilization buffer (as above) after removal of the cytosolic extract. Hippocampal homogenates were diluted with I X Laemmli sample buffer and boiled for 5 min. The proteins were resolved in 10% polyacrylamide gel and electrotransferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Cambridge, ON, Canada). The blots were blocked with 5% non-fat milk in TBST (50 m M Tris (pH 7.4), 150 m M NaCl, 0.1 % Tween-20) for 1 hr, and the membranes were incubated overnight at 4 °C with primary antibody diluted in TBST containing 5% non-fat milk or 5% bovine serum albumin and 0.025%) sodium azide. The antibody dilutions are as follows: polyclonal rabbit anti-Ai receptor (1:1000, Sigma), polyclonal rabbit anti-phospho JNK (1:1000, Cell Signalling), and polyclonal rabbit pan-137 specific JNK antibody (1:1000, Cell Signalling). To normalize the protein bands from the membrane and cytosolic fractions, we used polyclonal rabbit anti-(3-actin (1:8000, RBI). Following four washes with TBST, the membranes were incubated with a secondary antibody against rabbit IgG conjugated to horse radish peroxidase (1:3000, Santa Cruz) diluted in 5% non-fat milk (1 hr, room temperature). The membranes were then washed 3-4 X (15 min) with TBST, and proteins were visualized using enhanced chemiluminescence (ECL, Amersham Bioscience, Arlington Heights, IL) using Quantity One Fluor S - M A X imaging software (Bio-Rad, Hercules, CA). Densitometry analysis was carried out using ImageJ Software (National Institutes of Health, USA). 4.2.4. Immunoprecipitation and Co-lmmunoprecipation To examine interactions between adenosine A ! receptors and JNK and phosphorylated-JNK (phospho-JNK), co-immunoprecipitation was performed by first incubating 1 mg extract from hippocampal homogenates with rabbit IgG (1 hr, 4 °C). Next goat anti-rabbit IgG antibody covalently linked to agarose beads (Sigma) was added to the homogenates for a further 1 hr. The agarose beads were removed by pulse spinning at 6000 rpm for 5 s, and the supernatant was subsequently reacted with an immunoprecipitating antibody overnight at 4 °C. A i receptor was immunoprecipitated using a polyclonal rabbit anti-A] receptor antibody (5 pg, Sigma). JNK and phospho-JNK were immunoprecipitated with a polyclonal rabbit anti-pan JNK and anti-phospho JNK antibody respectively (5 pg, Cell Signalling, Beverly, MA) . After overnight pre-incubation of lysates with the immunoprecipitating antibodies, the immune complexes were collected by incubating the lysates for >6 hrs at 4 °C with agarose beads conjugated to anti-rabbit IgG antibody. Agarose beads were then collected by pulse spins, and 138 washed 4 times with wash buffer (solubilization buffer containing 0.1% NP-40). Proteins from the agarose beads were eluted with 60 pL of I X Laemmli sample buffer (Bio-Rad), boiled for 5 min, resolved in 10% polyacrylamide gels, and subjected to Western blot analysis as described above. 4.2.5. Drugs Adenosine, N6-cyclopentyladenosine (CPA), and 8-cyclopentyl-l,3-dipropylxanthine (DPCPX) were obtained from Sigma-Aldrich Inc. (St. Louis, MO, U.S.A.). Tetrodotoxin (TTX) was purchased from Alomone Labs (Jerusalem, Israel). SP600125, JNK inhibitor V , JNK inhibitor II negative control (N'-methyl-l^-pyrazoloanthrone), and SB239063 were obtained from Calbiochem (San Diego, CA, U.S.A.), and made up as stock in DMSO before being added to aCSF. The final concentration of DMSO was always < 0.01%. 139 4.3. Results 4.3.7. JNK inhibitors attenuate the synaptic depression induced by adenosine, hypoxia, and CPA First, we determined whether JNK activation contributed to synaptic depression induced by exogenously applied adenosine, endogenous adenosine released by hypoxia, or an A1 receptor agonist in area CA1 of the rat hippocampus. We tested the effects of the JNK inhibitor SP600125 (Shin et al., 2002) on the magnitude of fEPSPs depression in area CA1 of the rat hippocampus. Hippocampal slices were pre-treated with the JNK inhibitor SP600125 (20uM), which selectively blocks JNK activity at a concentration of 20uM in hippocampal slices (Vereker et al., 2000a; Curran et al., 2003) or with vehicle alone (0.01% DMSO) for at least 1 hour prior to activation of A l receptors. After a stable fEPSP baseline was achieved, adenosine, CPA (an A i receptor agonist), or hypoxic aCSF (which releases adenosine; Fowler, 1989; Zhu and Krnjevic, 1997) was perfused onto the slice to activate A i receptors. We used 5 min exposures to hypoxic ACSF because, we have previously shown that the synaptic depression during this brief period of hypoxia is totally blocked by A i receptor antagonism (Brust et al., 2005), which is in agreement 140 Figure 4.1. Synaptic depression induced by adenosine, hypoxia, and C P A is attenuated by J N K inhibi t ion. A, Sample traces and plot of the mean fEPSP slope (±SEM), normalized to baseline, showing that pre-incubation of slices in the JNK inhibitor SP600125 (20uM) decreases the magnitude of adenosine-induced synaptic depression. B, Sample traces and plot of the mean fEPSP slope (±SEM) showing that adenosine-induced synaptic depression was suppressed in slices pre-incubated in SP600125. C, Sample traces and plot of the mean fEPSP slope (±SEM) showing that synaptic depression induced by the adenosine A) receptor agonist CPA was attenuated in slices pre-incubated with SP600125. D, Summary of A-C showing that the maximal magnitude of depression of fEPSP slope (±SEM) induced by adenosine, hypoxia, and CPA was lower in slices incubated in SP600125 than slices incubated in vehicle. *Statistical significance assessed using the Student t-test, p< 0.01. Calibration: lOmV/lOms. 141 Adenosine • Normal aCSF + 0.2% DMSO ° 20^MSP600125 ~~ 10 20 30 Time (minutes) 40 B CD C 0) CL o CD CL CO D . L U 100 75 50 25 0 0 Hypoxia D M S O Hypoxia SP600125 1+2 Hypoxia • Normal aCSF + 0.2% DMSO ° 20|iM SP600125' 10 20 30 Time (minutes) 40 C P A ca • Normal aCSF + 0.2% DMSO ° 20uM SP600125 ~ 10 20 30 40~ Time (minutes) 50 T 9 sr ^ .05- „c§> 142 with other studies (Latini et al., 1999b; Sebastiao et al., 2000). Adenosine, CPA, and hypoxia all depressed the fEPSP slope (Fig. 1). When JNK was inhibited with SP600125, the reduction of the fEPSP slope by these agonists was significantly attenuated. Adenosine (+vehicle) decreased the fEPSP slope by 53.0 ± 1.7% (n=6). In slices pretreated with SP600125 (Fig. IA.D), however, adenosine only depressed the fEPSP slope by 19.7 ± 5.4% (n=6), which was significantly less than the in vehicle alone (p < 0.01). JNK inhibition also decreased the magnitude of fEPSP depression induced by a brief hypoxic insult. In slices pretreated with SP600125, hypoxia decreased the fEPSP slope by only 18.3 ± 6.5% (n=5; p < 0.01) (Fig. \B,D), which was significantly less than in vehicle alone (53.6 ± 7.4% depression of fEPSPs; n=5). We also tested whether synaptic depression induced by selective A i receptor activation (with CPA) was sensitive to JNK inhibition. CPA decreased the fEPSP slope by 67.6 ± 4.7% (n=5) and this was reduced to 29.0 ± 7.8% (n=6; p < 0.01) when slices were pretreated with SP600125 (Fig. 1 C,D). These data suggest adenosine A i receptor-induced synaptic depression is mediated in part by JNK activation. 4.3.2. A^receptor-mediated synaptic depression is decreased by SP600125 and JNK Inhibitor V, but not by an inactive analogue To ensure that the effect of SP600125 on adenosine A i -mediated synaptic depression was due specifically to JNK inhibition, we tested a second JNK inhibitor (JNK Inhibitor V) , as well as an inactive analogue (N'-methyl-l^-pyrazoloanthrone), which has been used as a negative control for SP600125 at 5 p M (Shin et al., 2002). It is important to note that N'-methyl-1,9-pyrazoloanthrone can inhibit JNK activity with an IC50 of 24pM, but is ineffective at lower doses. Therefore, to compare the effects of the JNK inhibitors with 143 the negative control we used 5uM for all compounds. We recorded from matched slices (i.e. from the same animal) that were pre-incubated in either of 5uM SP600125 (instead of 20uM as in Fig. 1), JNK Inhibitor V (5uM), the negative control compound (5uM), or control aCSF. CPA (40nM; 10 min) was used to selectively activate A i receptors. SP600125 at 5uM had a similar effect as SP600125 at 20uM (see above) in decreasing the magnitude of the CPA-induced depression of fEPSPs. In slices pre-treated with 5uM SP600125, CPA decreased the mean normalized fEPSP slope by 41.4 ± 2.7% (n=8), which was significantly less than CPA-alone in matched control slices (70.2 ± 1.0%; n=5 p < 0.01) (Fig. 2A,B,D). To ensure that SP600125 was exerting its effects by selectively blocking JNK activity, we also monitored CPA responses in slices that had been pre-incubated with an inactive analogue (5uM JNK Inhibitor II negative control compound, N1-methyl-1,9-pyrazoloanthrone). CPA similarly decreased the magnitude of the mean fEPSP slope in slices pre-incubated with the inactive analogue (63.0 ± 4.0%; n=9) and normal aCSF (70.2 ± 1.0%; p > 0.20) (Fig. 2A,B,D). We next tested a second JNK inhibitor (JNK inhibitor V), and found that like SP600125, JNK Inhibitor V attenuated CPA-induced synaptic depression. In slices pre-treated with 5uM JNK inhibitor V , CPA depressed the mean fEPSP slope by 28.6 ± 1.0% (n=8), which was significantly less than control (70.2 ± 1.0%; n=5;p < 0.01) (Fig. 2A,C,D). These results suggest that JNK activity contributes to Ai-receptor-mediated synaptic depression. 144 Figure 4.2. Adenosine A i receptor-mediated synaptic depression is attenuated by JNK inhibition with SP600125 and JNK inhibitor V, but not an inactive analogue. A, Sample traces showing that decreases in ffiPSP amplitude caused by 40nM CPA was reduced in slices pre-treated with 5uM SP600125 (JNK Inhibitor II) or 5pM JNK inhibitor V compared with control, but not in slices pre-treated with 5pM JNK Inhibitor II negative control. B, Plot of the mean fEPSP slope (±SEM), normalized to baseline, showing that CPA-induced synaptic depression was attenuated in slices pre-treated with 5pM SP600125 compared with slices pre-treated with 5pM JNK Inhibitor II negative control or normal aCSF. C, Plot of the mean fEPSP slope (±SEM) showing that pre-treating slices with 5pM JNK inhibitor V decreased the efficacy of CPA in decreasing synaptic transmission. D, Summary bar chart of B and C showing the mean magnitude of CPA-induced depression of fEPSP slopes (±SEM). *Statistical significance assessed using the Student t-test, p < 0.01. Calibration: lOmV/lOms. 145 4.3.3. JNK inhibition increases the excitability of rat hippocampal slices It has been reported that the JNK inhibitor, SP600125 (20pM) increases baseline synaptic transmission in rat hippocampal slices (Costello and Herron, 2004), suggesting that tonic A l receptor activation mediates inhibition of basal glutamate release. Consistent with this notion, inhibiting JNK by pre-treating slices with 5pM JNK Inhibitor V (n=8) led to increased excitability as indicated by an increase in the average magnitude fEPSPs compared to matched controls (n=l 1). This occurred at stimulus intensities ranging from 4 to 10 V without a change in the apparent threshold for activation (~ 6 V ; Fig. 2E). However, the responses elicited by a given stimulus intensity were significantly larger in slices that had been pre-treated with JNK inhibitor V (5pM) (Fig 2E). On average, pre-treatment with JNK inhibitor V (5uM; 1-3 hours) increased the mean fEPSP slope by 89 ± 3.2% (p < 0.01) at suprathreshold stimuli. These data indicate that JNK inhibition increases fEPSP magnitude, possibly due to removal of tonic adenosine inhibition in the slice. 4.3.4. A1 receptor stimulation increases JNK phosphorylation The sensitivity of adenosine A i receptor-mediated synaptic depression to JNK inhibitors suggests that JNK activity is a necessary step in adenosine-induced inhibition of synaptic transmission. Therefore we tested whether A i receptor activation increased JNK activity in hippocampal slices by quantifying the amount of activated (i.e. phosphorylated) JNK. Slices were treated with either CPA (500nM), to selectively activate A i receptors, or with 147 normal aCSF for 2, 5, 10, or 30 min and the amount of phosphorylated JNK compared by Western blotting. Using centrifugation, we extracted the membrane fraction from hippocampal lysates. Western blot analysis was performed using a rabbit polyclonal antibody that only recognizes JNK when it is dually phosphorylated at threonine 183 and tyrosine 185. Of the three JNK isoforms encoded by different genes, JNK1 and JNK2 are ubiquitously expressed, whereas JNK3 is selectively expressed in the nervous system and heart (Gupta et al., 1996; Martin et al., 1996; Kuan et al., 1999). The antibody against phosphorylated JNK recognizes a band at 46 kDa which consists of all three JNK isoforms (JNK1, JNK2, and JNK3), and a band at 54 kDa which consists of only the JNK2 and JNK3 isoforms (Kuan et al., 2003). Densitometry analysis of the 54 kDa band (representing JNK2 and JNK3), normalized to B-actin, revealed that A i receptor stimulation with CPA increased JNK phosphorylation to 154 ± 14.3% of baseline within 2 minutes (n=6; p < 0.01; Fig. 3 A). Phospho-JNK immunoreactivity remained significantly elevated after CPA treatment for 5 min (144 ± 7.0% of baseline; n=6; p < 0.05) and 10 min (165 ± 19.4% of baseline; n=5; p < 0.01). By 30 min of CPA treatment, JNK phosphorylation had returned towards baseline levels (121 ± 17.9%; n=6; p > 0.20) (Fig 3A). Densitometry analysis of the 46 kDa band, which contains all three JNK isoforms (Fig. 35) revealed that JNK phosphorylation significantly increased after 2 min of CPA treatment (134 ± 12.3%; n=6; p < 0.05). Although the mean phospho-JNK immunoreactivity trended higher than control after 5 min (121.7 ± 11.9%; n=6; p = 0.09) and 10 min (137 ± 16.6%; n=9; p = 0.09) of CPA treatment, this trend failed to reach statistical significance. By 30 min, which was the longest time point tested, phospho-JNK immunoreactivity had returned to control levels (102 ± 12.0% of baseline; n=5). 148 Figure 4 .3 . Stimulation of adenosine Ai receptors, which are physically associated with JNK, increases JNK phosphorylation. Hippocampal slices were exposed to either normal aCSF or CPA (500 nM) for 2, 5, 10, or 30 min. The membrane and cytosolic fractions of hippocampal homogenates were separated by centrifugation for Western blot analysis. A (upperpanel), Representative Western blots showing phosphorylated JNK 2/3 (phospho-JNK 2/3) and p-actin in the membrane fraction at time points ranging from 0 to 30 min. Lower panel, Quantitative representation of multiple Western blots showing phospho-JNK 2/3 immunoreactivity (mean ± SEM) in the membrane fraction at time 0 min (n=6), 2 min (n=6), 5 min (n=6), 10 min (n=5) and 30 min (n=5). Data were normalized to the level of protein phosphorylation at time 0 after CPA treatment (control), and B-actin immunoreactivity was used as a loading control. B (upperpanel), Representative Western blots showing phosphorylated JNK 1/2/3 and p-actin immunoreactivity in the membrane fraction at time points ranging from 0 to 30 min. Lower panel, Quantitative representation of multiple Western blots showing phospho-JNK 1/2/3 immunoreactivity (mean ± SEM) in the membrane fraction at time 0 min (n=6), 2 min (n=6), 5 min (n=6), 10 min (n=5) and 30 min (n=5). C, Representative Western blots and mean immunoreactivity (normalized to P-actin; ± SEM) using a pan-specific JNK antibody showing that CPA treatment does not change the total amount of JNK immunoreactivity in the membrane fraction (n=6). D, Western blot showing that immunoprecipitation with a pan-specific JNK antibody pulls down the Ai receptor (lane 1). Lane 2 shows the presence of the Ai receptor in whole-cell hippocampal lysate. E, Western blot showing that immunoprecipitation with a phospho-JNK antibody does not pull down the Ai receptor (lane 1). Lane 2 shows the 149 presence of the A i receptor in whole-cell hippocampal lysate. Statistical significance assessed using the Student /-test, where * denotesp < 0.05 and ** denotes p < 0.01. Phospho-JNK 2/3 (54 kDa) _ P-actin Time in CPA (minutes) 0 2 5 10 30 48* S 200 c 8 180 S 160 co 140-1 CSJ * 120 o CL w O x: C L 100 80 0 5 10 15 20 25 30 Time of CPA treatment (minutes) Control CPA Total JNK 2/3 P-actin f 125 8 100 6 7 5 CO CN 50 3 ,o 25 0 48 48 Control CPA B Time in CPA (minutes) 0 2 5 10 30 48 Phospho-JNK 1/2/3 (46 kDa) mm mm mm mm* — 48 s? p-actin § 160 o ^ 140 C O 5 120 Q. to o 100 80 0 5 10 15 20 25 30 Time of CPA treatment (minutes) W B : A-j Receptor 33 IP: JNK Lysate W B : Ai Receptor IP: Phospho-JNK Lysate 150 We next determined i f A i receptor activation changed the amount of JNK associated with the plasma membrane. Using a pan-specific antibody, we determined that total JNK immunoreactivity in the membrane fraction did not change in response to CPA treatment (105 ± 8.0% of baseline; n=6; p > 0.50) (Fig. 3 Q . Thus, western blot analysis of phospho-JNK immunoreactivity suggests that A i receptor stimulation increased the activity of JNK in the hippocampus. This finding is consistent with our electrophysiological data (see above) which show that blocking JNK activity reduced the ability of A i receptors to inhibit synaptic transmission. 4.3.5. The receptor is physically associated with JNK, but not phospho-JNK We performed co-immunoprecipitation experiments to determine whether the adenosine A i receptor exists in the same signalling complex as JNK or phospho-JNK. The adenosine A] receptor corresponds to a band at approximately 37 kDa in the rat hippocampus (Rebola et al., 2003). Immunoprecipitation with the JNK antibody (probed with a polyclonal rabbit anti-Ai receptor antibody) revealed the Aj receptor (Fig. 3D, lane 1). Lane 2 shows the presence of the same band in whole-cell lysates. Immunoprecipitation with the anti-phospho-JNK antibody failed to pull down the A i receptor (Fig. 3E, lane 1). Lane 2 shows that the A i receptor was detectable in whole-cell lysates. These data suggest that the adenosine A i receptor is physically associated with JNK, but not phospho-JNK, in the hippocampus. 151 4.3.6. CPA-dependent increases in JNK phosphorylation are blocked by 4? receptor antagonism To determine whether the increase in JNK phosphorylation that we observed in response to CPA treatment (see above) was specifically mediated by A i receptor stimulation, we tested whether the A i receptor antagonist DPCPX blocked the CPA-induced increases in phospho-JNK immunoreactivity. As reported above, the 54 kDa band showed sustained significant increase in the level of phospho-JNK after 10 min of CPA treatment, but the 46 kDa band was not significantly increased at this time point. We therefore focused our analysis on the 54 kDa band. Because DPCPX has been reported to increase glutamate release and evoke seizure discharges (Sehmisch et al., 2001; Marcoli et al., 2003) which could alter JNK phosphorylation, we pre-treated slices with 1.2uM tetrodotoxin (TTX) for 20 min. CPA effectively increased phospho-JNK in the presence of T T X (177 ± 17.8% of control; n=10; p < 0.01) (Fig. AA). Slices were incubated in DPCPX (500nM; 20 min) or normal aCSF and then treated with CPA (500nM; 10 min). We observed that the CPA-induced increase in JNK phosphorylation was attenuated in the presence of DPCPX (Fig. AA) to 125 ± 21% (n=10; p > 0.20) of the control level (+TTX). These findings indicate that A i receptor stimulation selectively increases JNK phosphorylation in the rat hippocampus. 4.3.7. CPA-dependent increases in JNK phosphorylation are blocked by inhibition of JNK and p38 MAPK In a previous study, we found that p38 M A P K activity contributes to adenosine A\-receptor mediated synaptic depression (Brust et al., 2005). The p38 M A P K inhibitor 152 Figure 4.4. The increase in J N K phosphorylation induced by CPA was blocked by Ai receptor antagonism. Hippocampal slices were pre-incubated in normal aCSF or DPCPX (500nM; 20 min) and then exposed to CPA (500nM; 10 min). The membrane fraction was separated by centrifugation for Western blot analysis. Upper panel, Representative Western blots showing phosphorylated J N K (phospho-JNK) and P-actin immunoreactivity in response to CPA treatment in the presence and absence of DPCPX. Lower panel, Quantified phospho-JNK immunoreactivity (mean ± SEM) for control (n=10), CPA (n=10), and CPA + DPCPX (n=10). Data were normalized to p-actin immunoreactivity. Statistical significance compared with control was assessed using a Student Mest, where * denotes p < 0.05 and ** denotes p < 0.01. Phospho-JNK 2/3 P-actin O O mm <mm « a»48 48 Membrane (+TTX) P 200 Con C P A +DPCPX 153 SB203580 attenuated CPA-induced depression of fEPSPs, and CPA treatment increased the phosphorylation of p38 M A P K in membrane fractions. In the present study, we tested whether SP600125 and SB206393 (a second generation p38 M A P K inhibitor) affected CPA-induced increases in JNK phosphorylation. We also investigated whether the A] induced activation of p38 M A P K and JNK was through parallel or serial pathways. Slices were incubated in control aCSF (n=9), 20uM SP600125 (n=4), or 5uM SB206393 (n=4) for one hour prior to CPA treatment (500nM; 10 min). As expected, CPA increased phospho-JNK immunoreactivity in normal aCSF (in this case to 152 ± 14.0% of control; n=9; p < 0.01) (Fig 5B, lane 2). We found that CPA failed to increase phospho-JNK in the presence of SP600125 (Fig. 4B, lane 3). In slices pre-treated with SP600125, phospho-JNK levels were 93 ± 12.0% of baseline (n=4), which was significantly less than the increase to 152% observed in slices exposed to CPA in control conditions (p < 0.05). Surprisingly, the p38 M A P K inhibitor SB206393 also prevented increases in JNK phosphorylation due to CPA (Fig. 4B, lane 4). In slices pre-incubated in SB206393 and treated with CPA (500nM; 10 min), phospho-JNK immunoreactivity was reduced to 68 ± 13.0%) of baseline (n=5). The level of phospho-JNK immunoreactivity observed in slices pre-incubated with SB206393 was significantly less than both slices exposed to normal aCSF (p < 0.01) and slices exposed to normal aCSF plus CPA (p < 0.01). Together these data show that inhibiting JNK and/or p38 M A P K activity prevents CPA-induced increases in JNK phosphorylation. 154 4.3.8. CPA-dependent increases in p38 MAPK phosphorylation are blocked by p38 MAPK inhibition but not JNK inhibition The above data show that p38 M A P K and JNK activation are both activated by A l receptor stimulation, and suggest that p38 M A P K activity may be required for JNK activation. To gain further insight into whether p38 M A P K and JNK are activated in parallel or sequential pathways, the JNK inhibitor SP600125 was tested for its ability to affect CPA-induced p38 M A P K phosphorylation. Slices were incubated in control aCSF, 20pM SP600125, or 5pM SB206393 for one hour prior to CPA treatment (500nM; 10 min). As in our previous study (Brust et al., 2005), activation of A i receptors with CPA increased p38 M A P K phosphorylation in membrane fractions of rat hippocampal lysates (in this case to to 131 ± 11.8% of control; n=5; p < 0.05; Fig. 5A). Incubating slices in SP600125 did not prevent p38 M A P K phosporylation, which increased to 138 ± 18.5% of control (n=5; p < 0.05; Fig. 5B). However, SB206393 completely abolished the CPA-induced increase in p38 phosphorylation, and actually decreased phospho-p38 immunoreactivity to 71 ± 10.5% of baseline (n=4; p < 0.05). To summarize, JNK phosphorylation is prevented by both p38 M A P K inhibition (with SB206393) and JNK inhibition (with SP600125) (see above), whereas p38 M A P K phosphorylation is prevented by SB206393 but not SP600125. Therefore, p38 M A P K phosphorylation appears to be necessary for JNK phosphorylation. Our data supports a model where p38 M A P K and JNK are activated serially instead of in parallel following A i receptor stimulation. 155 Figure 4.5. Adenosine A i receptor stimulation sequentially activates p38 M A P K and J N K . Hippocampal slices were either pre-incubated in normal aCSF, SP600125 (20pM; 1 hour), or SB206393 (5pM; 1 hour) and then exposed to CPA (500nM; 10 min). The membrane fraction was separated by centrifugation for Western blot analysis. A (upper panel), Representative Western blots showing phosphorylated JNK (phospho-JNK) and p-actin immunoreactivity in response to CPA treatment in the presence and absence of SP600125. SP600125 and SB206393 prevent CPA-induced JNK phosphorylation. Lower panel, Quantified phospho-JNK immunoreactivity (mean ± SEM) for control (n=9), CPA (n=9), and CPA + SP600125 (n=4). Data were normalized to p-actin immunoreactivity. B (upper panel), Representative Western blots showing phosphorylated p38 M A P K (phospho-p38) and p-actin immunoreactivity in response to CPA treatment in the presence and absence of SB206393. SB206393 prevents CPA-induced p38 M A P K phosphorylation, but SP600125 does not. Lower panel, Quantified phospho-p38 immunoreactivity (mean ± SEM) for control (n=5), CPA (n=5), and CPA + SB206393 (n=4). Data were normalized to P-actin immunoreactivity. Statistical significance compared with control was assessed using a Student /-test, where * denotes p < 0.05 and ** denotesp < 0.01. 156 s i fj o * * Phospho-JNK 2/3 mmm* Membrane 48_ 48 B Phospho-p38 P-actin J f * # i i O O % % 48 48 Membrane =5 200! Con CPA +SP +SB Con CPA +SP +SB 157 4.4. Discussion In the present study, we provide evidence that A i receptor activation stimulates JNK activity in hippocampal slices, and that JNK activation is necessary for A i receptor-mediated synaptic depression. We demonstrated that adenosine, hypoxia, and CPA decreased fEPSP slopes in a pathway requiring JNK activity. Inhibition of JNK with both SP600125 and JNK inhibitor V , but not an inactive analogue, attenuated A] receptor-dependent synaptic depression. Western blot analysis confirmed that A i receptor stimulation phosphorylated membrane associated JNK in a time course consistent with CPA-induced attenuation of fEPSPs. In our western blot analysis, we observed that the phospho-JNK antibody recognized two bands: a 46 kDa band composed of all three JNK isoforms, and a band at 54 kDa band, which comprises only JNK2 and the neuronal specific JNK3 isoform (Kuan et al., 2003). The 54 kDa band contained much less total immunoreactivity than the 46 kDa band, probably because JNK1, which is responsible for the high level of basal JNK activity in the brain (Yang et al., 1997), exists primarily as p46JNK (Kuan et al., 2003). Both JNK2 and JNK3 exist as p54JNK and p46JNK (Kuan et al., 2003). Although we observed that CPA initially (within 2 min) increased the amount of phospho-JNK immunoreactivity in both bands, CPA-induced phosphorylation was proportionally greater in the JNK2/3 54 kDa band than the JNK1/2/3 46 kDa band. This suggests that there is a correlation between A l receptor activation and the specific phosphorylation of neuronal JNK3, although effects on JNK1 and JNK2 cannot be ruled out. Co-immunoprecipitation revealed that JNK, but not phospho-JNK, is physically associated with A] receptors. The differential binding of JNK and phospho-JNK supports 158 a model in which A i receptors and JNK exist in a signalling complex, and that A i receptor activation phosphorylates JNK causing it to dissociate from the complex. Such a model would imply that the A i receptor-JNK complex is in constant dynamic equilibrium, with JNK continually cycling into the complex to replace dissociated phospho-JNK. Presumably, the phosphorylated JNK then mediates inhibition of neurotransmission. Although the precise mechanism of JNK-induced inhibition of neurotransmission has yet to be determined, there is tantalizing evidence that JNK activation is intimately linked to decreased glutamate release in the hippocampus (Vereker et al., 2000b; Vereker et al., 2000a; Lynch and Lynch, 2002; Curran et al., 2003; Costello and Herron, 2004). JNK inhibition with SP600125 increases baseline synaptic transmission and reduces paired-pulse facilitation, which is consistent with enhanced neurotransmitter release (Costello and Herron, 2004). Moreover, glutamate release is inhibited in synaptosomes exhibiting elevated JNK phosphorylation (induced by applying a tetanus in slices obtained from interleukin-ip-treated rats), providing a further indication that endogenous JNK activity regulates neurotransmitter release (Vereker et al., 2000a). In agreement with the above studies, we also found that inhibition of JNK increased excitability in hippocampal slices. Because ambient adenosine in the extracellular space is a tonic suppressor of glutamate release under basal conditions, the increase in synaptic transmission due to SP600125 or JNK Inhibitor V may occur because JNK inhibition removes tonic Ai-receptor-mediated depression of neurotransmission. We have previously demonstrated that p38 M A P K activation is necessary for adenosine A i receptor-mediated synaptic depression (Brust et al., 2005), as well as Bz-159 ATP-induced depression of mossy fiber-CA3 synaptic transmission (Armstrong et al., 2002). Inhibition of p38 M A P K activity almost completely blocks the depression of evoked CA1 fEPSPs induced by the A i receptor agonist CPA, adenosine, or brief hypoxia. Moreover, CPA phosphorylates p38 M A P K within minutes in hippocampal slices. The link between adenosine A i receptors and p38 M A P K is well established in other cells, as A] receptor stimulation activates p38 M A P K in transfected Chinese hamster ovary cells (Robinson and Dickenson, 2001). A] receptors also activate p38 M A P K in the perfused rat heart (Zhao et al., 2001b; Schulte et al., 2004), where A,-p38 M A P K signalling mediates ischemic preconditioning. Here, we show that CPA-induced JNK phosphorylation is blocked by p38 M A P K inhibition but CPA-induced p38 phosphorylation is not blocked by JNK inhibition, revealing a sequential activation of p38 M A P K then JNK upon A l receptor stimulation. Our previous results (Brust et al., 2005) also demonstrated that p38 M A P K inhibition with SB203580 greatly attenuated CPA-induced inhibition of synaptic transmission (i.e., in the presence of SB203580, CPA only produced <10% inhibition of synaptic transmission compared with -70% inhibition in control). In contrast, CPA was still able to depress synaptic transmission by about 30%) in slices pre-incubated with JNK inhibitors (SP600125 or JNK inhibitor V , present study). Our data thus indicate that p38 M A P K activation may be necessary for JNK activation following A i receptor stimulation, and that both p38 M A P K and JNK contribute to inhibition of synaptic transmission. The mechanism by which JNK is activated following p38 M A P K is unclear at present, and may involve multiple substrates. For example, adenosine A i receptor activation leads to protein-phosphatase 2a translocation in both cardiac tissue (Liu and 160 Hofmann, 2003) and hippocampal tissue (Brust et al., 2005) in a pathway requiring p38 M A P K activation. Inhibiting PP2a activity (with okadaic acid) also blocks JNK phosphorylation in response to TNF-a, suggesting that PP2a regulates activation of JNK (Ray et al., 2005). Therefore, p38 M A P K may activate JNK through PP2a translocation and activation. Alternatively CK2 (formerly casein kinase II) may function as a mediator of p38 M A P K - J N K signalling. CK2 is directly activated by p38 M A P kinase (Sayed et al., 2000), and also phosphorylates JNK (Min et al., 2003). However, determining whether JNK is a downstream target of p38 M A P K in the hippocampus will require further studies. The primary mechanism underlying A] receptor-mediated inhibition of neurotransmission is thought to reflect a G protein-coupled inhibition of calcium influx in nerve terminals (Dunwiddie and Masino, 2001). This is not the only possibility however, because adenosine also decreases excitability by activating G protein-gated inwardly rectifying potassium channels (GIRKs) in the postsynaptic membrane (Luscher et al., 1997). Although JNK is involved in the activation of voltage-gated K+ channels (Gao et al., 2004), whether JNK modulates GIRKs following A i receptor stimulation remains to be established. During trauma such as oxygen deprivation, adenosine increases to concentrations of lOpM or more in the extracellular space where it acts as an endogenous neuroprotectant by inhibiting potentially toxic glutamate release (Rudolphi et al., 1992; Von Lubitz, 1999; Latini and Pedata, 2001; Marcoli et al., 2003; Boeck et al., 2005). JNK phosphorylation rapidly increases (within minutes) following cerebral ischemia (Comerford et al., 2004; Gao et al., 2005), possibly as a result of adenosine release and 161 subsequent A] receptor-mediated JNK activation. However, whether JNK activation is neuroprotective or neurodegenerative is controversial. JNK mediates neurodegeneration by activating genetic programs through phosphorylation of the nuclear transcription factor c-Jun/AP-1, the release of cytochrome c or the pro-inflammatory actions of microglia (Manning and Davis, 2003). Indeed ischemic injury and excitoxicity is dramatically reduced in the brains of mice lacking the JNK3 gene (Kuan et al., 2003) or treated with a JNK-inhibitor (Borsello et al., 2003; Gao et al., 2005) in the days following an ischemic insult. Although these findings support role for JNK in neuronal death after ischemia/reperfusion injury, the long-term effects of JNK activation may differ significantly from the effect of JNK activation during an ischemic insult, when it may contribute to adenosine-mediated neuroprotection. In contrast to its role in apoptosis during ischemia, JNK activation promotes cell survival by phosphorylating Akt/PKB in ventricular myocytes after hypoxia-reoxygenation (Shao et al., 2006). The dichotomous actions of JNK could result from differential availability and preferential site-specific phosphorylation of a given JNK substrate depending on the context of JNK activation. Alternatively, analysis of the consequences of JNK activation at different time points during and following ischemic insults may yield different results. In the hours and days post-ischemia, JNK activation may indeed lead to apoptosis (Manning and Davis, 2003), whereas immediately during an ischemic insult JNK activation may confer protection from excitoxicity by inhibiting glutamate release in response to A i receptor stimulation. Because JNK inhibitors are potential therapeutics for stroke, it will be valuable to ensure that the timing of their use does not impede the neuroprotective power of endogenous adenosine. 162 The present study demonstrates that JNK activation is necessary for adenosine A i receptor-mediated depression of neurotransmission at CA3-CA1 synapses in the rat hippocampus. Adenosine Aj receptor mediated JNK signalling may represent a novel mechanism underlying neuroprotection and inhibition of neurotransmitter release in the central nervous system. 4.5. 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Zhu Y , Pak D, Qin Y , McCormack SG, Kim MJ , Baumgart JP, Velamoor V , Auberson Y P , Osten P, van Aelst L , Sheng M , Zhu JJ (2005) Rap2-JNK removes synaptic A M P A receptors during depotentiation. Neuron 46:905-916. 167 C h a p t e r F i v e : G e n e r a l D i s c u s s i o n 168 5.1. Introduction The research described here tests the hypothesis that M A P K s are a component of purinergic signalling in the central nervous system. A breadth of biochemical, pharmacological, and electrophysiological techniques provides evidence that the synaptic inhibition caused by purinoceptor stimulation requires the activation of p38 M A P K and/or JNK in the hippocampus. The data suggests that P2X 7 receptors, located in mossy fiber terminals, decrease glutamate release through activation of p38 M A P K . Further studies in the CA1 region suggest that sequential activation of p38 M A P K and JNK is required for A l receptor-mediated inhibition of neurotransmission. A working hypothesis that emerges from these observations is that A i receptors exist in a complex with p38 M A P K and JNK and that this complex functions to decrease C a 2 + influx through presynaptic neuronal calcium channels upon A i receptor-dependent phosphorylation of p38 M A P K and JNK. Although the evidence presented in the previous three chapters provides a compelling case for such a notion, some potential limitations are discussed below, and alternate interpretations of the data are identified. In addition, the findings presented here are integrated and evaluated in the context of current knowledge. Finally, implications to the field of study as well as ideas for future experiments are proposed. 5.2. P2X7 receptor-mediated synaptic depression In our study investigating the role of P2X 7 receptor-mediated synaptic depression of mossy fiber-CA3 synaptic transmission, we used an antibody directed towards the C-169 terminus of the P2X 7 receptor protein (Cterm-ab) to show P2X 7 receptor immunoreactivity in mossy fiber terminals (Armstrong et al., 2002). We observed labelling of small-terminal-like puncta throughout the hippocampus, a finding that has since been replicated in another study using the same antibody (Sim et al., 2004). P2X 7 immunoreactivity was particularly dense in the termination zones of hippocampal mossy fibers, where it was colocalized with the presynaptic marker syntaxin 1A/B but not the dendritic marker MAP-2 . Based on this immunohistochemical data, we concluded that P2X 7 are abundant in mossy fiber terminals. We used electrophysiology and pharmacology to confirm that selective P2X 7 receptor activation had functional consequences in area CA3 of the hippocampus. The P2X 7 receptor agonist Bz-ATP induced a rapid and long-lasting synaptic depression that was inhibited by the P2X 7 receptor antagonist oxidized periodate-ATP, but not the P2Y receptor antagonist reactive blue 2 or the P2Xi . 3 j 5 i 6 receptor antagonist PPADS. Together, our data indicated that P2X 7 receptors are present in mossy fiber presynaptic terminals where they are functionally important in depressing mossy fiber-CA3 synaptic transmission. P2X 7 receptors are only activated by high concentrations of ATP (North, 2002), such as would occur during a tetanus or seizure. Thus, P2X 7 receptor-dependent inhibition of neurotransmission may be an important neuroprotective mechanism in the brain because of its potential to dampen potentially toxic release of excitatory neurotransmitters (i.e. glutamate). The P2X 7 receptor is a particularly intriguing membrane receptor because it can not only function as a non-selective cation channel, but also as a mediator of cell membrane permeabilization (North, 2002). This unique property is due to a longer 170 intracellular C-terminus than other P2X members, conferring the ability to form a large permeabilizing pore. In addition, the P2X 7 receptor is C a 2 + permeable, which could help explain the diverse effects of P2X 7 receptor stimulation on processes relating to immune responses, cell survival, release of interleukin-lb, activation of NFkB and NFAT, and the induction of both necrotic and apoptotic cell death (North, 2002). The mechanism by which P2X 7 receptors initiate second messenger cascades is not known in nearly as much detail as prototypical PI or P2Y G protein-dependent signalling. 5.2.1. Possible limita Hons In addition to the P2X7 receptor Cterm-ab that we used in our study (Alamone Laboratories), two other antibodies have been used to examine P2X 7 localization in rat brain (EctoAb-1 and EctoAb-2). Unfortunately, each of these three antibodies show different patterns of P2X 7 immunoreactivity (Sim et al., 2004), making it difficult to determine which pattern reliably indicates P2X 7 receptor expression in rat brain. It should be noted that the Cterm-ab that we used in our P2X 7 receptor study is the most specific or the three antibodies, recognizing a single band at the appropriate molecular weight in H E K cells (Sim et al., 2004). In an attempt to determine which pattern of immunoreactivity is an accurate representation of P2X 7 protein expression, Annmarie Surprenant and colleagues (Sim et al., 2004) examined the patterns of immunoreactivity of each of these antibodies in two types of P2X 7 knock-out mice (Pfizer P2X 7 " A and Glaxo P2X 7" A). Surprisingly, there was no difference in the pattern of P2X 7 immunoreactivity between wild-type mice and either of the two knockout mice in the hippocampus (Sim et al., 2004). In contrast, the robust immunoreactivity detected by the Cterm-ab and the 171 EctoAb-1 in the lung and submandibular gland was completely absent in both Pfizer P2X 7" / _ and Glaxo P2X 7 " A mice. Moreover, Western blot analysis using these antibodies revealed the presence of a band at the appropriate weight to represent P2X 7 receptor protein (75 kDa) in the brain, lung, and submandibular gland of wild-type mice that was absent in the lung and submandibular gland, but not brain, of both P2X 7 knockout mice. Surprenant et al. (2004) argue that ourselves (Armstrong et al., 2002) and others who have described P2X 7 receptor expression and function in neurons (Deuchars et al., 2001; Atkinson et al., 2002; Lundy et al., 2002; Sperlagh et al., 2002; Ishii et al., 2003; Cavaliere et al., 2004) have been "fooled" by the presence of a brain-specific protein that happens to be the same molecular weight as the P2X 7 receptor and that happens to be recognized by all three P2X 7 receptor antibodies. The authors conclude that "the only reasonable explanation for all of these results is that P2X 7 receptor protein is either not expressed in neurons of the normal adult rodent brain, or that levels are too low to be detected by any method currently available." An alternate explanation is that a slightly different isoform of the P2X 7 receptor protein exists in the brain, and that this neuronal isoform is not recognized by the P2X 7 receptor antibodies in wild-type or knockout mice, whereas the isoform expressed in peripheral tissue is recognized by these antibodies. It may seem unlikely that a different isoform of the P2X 7 receptor protein that does not contain the appropriate epitopes is expressed in brain and not the periphery. However, seven additional variants of the human P2X 7 receptor resulting from alternative splicing have recently been reported, with differential expression patterns in different tissues (Cheewatrakoolpong et al., 2005). One variant lacked the entire cytoplasmic tail, yet C a 2 + flux was largely unaffected. This 172 finding supports the idea that different isoforms are expressed in different tissues. The Pfizer P2X7~/" knockout mouse was generated by deletion of the amino acids from C y s 5 0 6 to Pro 5 3 2 in the carboxyl-terminal domain of the P2X 7 receptor gene product (Solle et al., 2001). Therefore, the discovery of a functional P2X 7 variant lacking the cytoplasmic tail supports the notion that genetic deletion of the cytoplasmic tail (as in the Pfizer P2X 7~A knockout mice) may not actually functionally inhibit P2X 7 receptor channels. Real-time PCR revealed that the alternatively spliced (and presumably functional) P2X 7 variant lacking the cytoplasmic tail was abundantly expressed in brain tissue (Cheewatrakoolpong et al., 2005). Clearly, it will take further investigation to determine whether P2X 7 receptors truly are absent in hippocampal neurons. Another study that casts doubt on the hypothesis that P2X 7 receptors are localized in presynaptic nerve terminals and function to decrease neurotransmitter release was recently conducted by Dirk Dietrich and colleagues (Kukley et al., 2004). Although the authors confirmed that Bz-ATP produces a pronounced inhibition of mossy fiber-CA3 synaptic transmission, they suggest this is caused by activation of adenosine receptors following enzymatic conversion of Bz-ATP to Bz-adenosine and heteroexchange with cellular adenosine via nucleoside transporters. Bz-ATP mimics both the mossy fiber-CA3 synaptic depression and the activation of GIRK channels induced by A) receptor activation (Luscher et al., 1997). Furthermore, all effects of Bz-ATP could be reversed by the selective A] receptor antagonist DPCPX. Therefore, the authors conclude that Bz-ATP application onto hippocampal slices leads to A i receptor activation, and that Bz-ATP induced synaptic depression is actually A i receptor-mediated. 173 Because Bz-Adenosine, the hydrolyzed product of Bz-ATP, is unlikely to be an effective A i receptor agonist (Klotz, 2000; Kukley et al., 2004), the authors conducted further experiments to determine the mechanism by which Bz-ATP ultimately stimulates A i receptors. They found that inhibition of either ecto-nucleotidases (with A R L 67156, ITP, and concanavalin A) or nucleoside transporters (with excess inosine) blocks Bz-ATP-induced currents, suggesting that Bz-ATP is converted to adenosine by a combined action of ecto-nucleotidases and nucleoside transporters. In our P2X 7 receptor study, we found that Bz-ATP-induced depression of mossy fiber-CA3 synaptic transmission was blocked by p38 M A P K inhibition with SB203580, but adenosine-induced synaptic depression was not. The Dietrich group (2004) attempts to account for the difference in the effect of SB203580 on Bz-ATP- versus adenosine-induced synaptic depression by proposing that SB203580 is actually blocking nucleoside transporters and preventing the exchange of Bz-adenosine for adenosine. The net effect of SB203580 in hippocampal slices, they argue, is blockade of adenosine release into the extracellular space where it can act on A] receptors. The evidence they present in support of this hypothesis is that the activity of Bz-ATP was potently suppressed by treating slices with SB203580 (in agreement with our results), whereas the current evoked by ATP was not. Since ATP is rapidly degraded to adenosine by ecto-nucleotidases (Zimmermann and Braun, 1996; Cunha et al., 1998), the authors have in effect simply replicated our results without any new evidence to support their interpretation over ours. Furthermore, the net effect of nucleoside transporters is the uptake of adenosine from the extracellular space (Fredholm et al., 1994; Latini and Pedata, 2001). Therefore, i f SB203580 truly were blocking nucleoside transporters, then treating slices with 174 SB203580 would be expected to increase ambient adenosine in the extracellular space and cause a decrease in baseline synaptic transmission, as has been reported (Dunwiddie and Diao, 2000). This is clearly not the case, as we have demonstrated that perfusion of SB203580 onto slices actually increases the amplitude of fEPSPs (Fig. 3.4), which is not consistent with an increase in adenosine tone due to the inhibition of nucleoside transporters. Perhaps the main problem with the study Kukely et al. (2004) is that most of their experiments use postsynaptic K + currents as an assay for the effects of Bz-ATP and SB203580. It is difficult to extrapolate such data to presynaptic terminals, where the signalling pathway linking P2X 7 or A i receptors to decreased neurotransmission may be significantly different. One question that remains is why adenosine-induced synaptic depression was not attenuated by SB203580 in mossy fiber synapses (Armstrong et al., 2002), whereas in the CA1 region it was inhibited by ~50% (Figs. 3.4 & 3.5). Mossy fiber-CA3 synapses have distinct properties from other central synapses such as the CA3-CA1 synapse (Nicoll and Schmitz, 2005). For example, they can exhibit larger paired-pulse facilitation (with a ratio of > 3) and can undergo frequency facilitation (changing the frequency of stimulation from low to high dramatically increases synaptic strength). In addition, the induction of LTP is independent of N M D A receptor activation. Given the distinct functional properties of mossy fiber synapses, it is perhaps not surprising that mossy fiber terminals also have markedly different expression of voltage-dependent calcium channels, exhibiting predominantly P-type calcium-dependent evoked release of neurotransmitter (Castillo et al., 1994). Therefore, a possible explanation for the 175 differential effect of p3 8 M A P K on adenosine-mediated inhibition of neurotransmitter release could be that Ai-p38 M A P K signalling specifically inhibits the N-type calcium channel subtype. The role of P2X 7 receptors in inhibiting presynaptic glutamate release was further challenged by studies showing that Bz-ATP actually facilitated glutamate release in the rat hippocampus (Sperlagh et al., 2002) and spinal cord (Deuchars et al., 2001). However, the question of whether P2X 7 receptor activation mediates presynaptic inhibition or facilitation may be moot i f P2X 7 receptors are not present in the brain at all. In support of this notion, Rodrigo Cunha and colleagues (2005) confirmed that Bz-ATP ' evoked release of glutamate in purified nerve terminals, but they showed that the facilitation of glutamate release was blocked by suramin and not by the P2X 7 receptor antagonist brilliant blue G. This pharmacological profile indicates the involvement of P2Xi receptors and not P2X 7 receptors because P2X 7 receptors, particularly in the rat, are sensitive to brilliant blue G and not suramin (North, 2002). 5.2.2. Future Directions Clearly more studies will be required to determine whether P2X 7 receptors are expressed in neurons, glia, or both in the central nervous system. Approaches using techniques in molecular biology are probably the most appropriate. One such approach may be to identify the protein from SDS-PAGE gels prepared from hippocampal lysates or mossy fiber synaptosomes using mass spectrometry. In such an experiment, the excised gel slice containing the protein of interest is subjected to proteolytic digestion followed by mass spectrometric analysis of the resulting peptides. Until there is a definitive answer as 176 to whether P2X 7 receptors are expressed in neurons, it is hard to justify further functional studies. 5.3. Adenosine A<\ receptor signalling through p38 MAPK This study is an overdue investigation into the molecular mechanisms underlying the inhibition of glutamate release by adenosine, a powerful and ubiquitous inhibitory modulator of excitatory synaptic transmission in the mammalian central nervous system. Although the inhibitory actions of adenosine have been known for some time, the signalling by which this occurs has not been studied in detail. A number of independent lines of evidence suggest that p38 mitogen-activated protein kinase (MAPK) may play an important role in Ai-mediated modulation of the CA3-CA1 synapse. First, there is a precedent for A i receptor activation of p38 M A P K in a smooth muscle cell line (Robinson & Dickenson, 2001). Second, there is a growing body of evidence supporting a role for p38 M A P K in synaptic plasticity in the hippocampus (see Introduction). Third, there is evidence, from studies of NG108-15 cells, that N-type current can be modulated by a pathway that involves p38 M A P K (Wilk-Blaszczak et al., 1998). A n obvious hypothesis that emerges from this is that A i receptor presynaptic inhibition at the CA3-CA1 synapse is due to a p38 M A P K -dependent suppression glutamate release. This study uses a variety of techniques to put the above hypothesis to the test. First, adenosine, acting via the inhibitory A i receptor, activates p38 M A P K and this activation is responsible for the inhibition of glutamate release, perhaps ultimately via an action on voltage-gated C a 2 + channels. Second, the A i agonist CPA phosphorylates p38 177 M A P K , and this is blocked by an A i antagonist and a p38 M A P K inhibitor. Third, the A i receptor and p38 M A P K co-immunoprecipitate. Fourth, a downstream substrate of p38 M A P K , PP2a, is also activated by adenosine A] receptors. Finally, the synaptic depression mediated by exogenous adenosine, endogenous adenosine released during hypoxia, or the agonist CPA is blocked by p38 M A P K inhibitors, providing an important physiological context for the above observations. A i receptor signalling through p38 M A P K could represent a novel mechanism underlying neuroprotection and presynaptic inhibition in the mammalian brain. This study extends the findings of our previous work showing that Bz-ATP-mediated depression of mossy fiber-CA3 synaptic transmission required activation of p38 M A P K . We have now established that p38 M A P K functions in synaptic depression at two distinct synapses following activation of two distinct purinoceptors. 5.3.1. Possible limitations One potential limitation of the study is that it depends entirely on the specificity of the p38 M A P K inhibitors. Although it is reassuring that consistent results were obtained with two different inhibitors and not when an inactive analogue was used, a critic could easily propose that the p38 M A P K inhibitors interfere directly with Aj receptor activation, especially given the demonstrated constitutive association of phospho-p38 M A P K and A i receptors. In support of this view, there is evidence that, in addition to classical adenosine receptor ligands, enzyme inhibitors commonly used in transduction research also bind adenosine receptors. For example, the protein kinase C inhibitor chelerythrine and the tyrosine kinase inhibitor genistein bind adenosine receptors and compete with adenosine receptor ligands for binding (Okajima et al., 1994; Ji et al., 1996; Schulte and 178 I Fredholm, 2002). Appropriate control experiments must be used in studies employing such substances, as both chelerythrine and genistein bind adenosine A i receptors at concentrations in which they are often used for inhibiting intracellular signalling pathways. However this is not the case for the p38 M A P K inhibitor SB203580. A binding assay revealed that 20pM SB203580 (which is the concentration used in our study) does not compete with A i receptor antagonist DPCPX binding in a rat membrane preparation (Schulte and Fredholm, 2003; see Figure 1). 5.3.2. Future Directions An obvious hypothesis that emerges from the research presented here is that p38 M A P K modulates neuronal calcium channels. It would be interesting to test whether calcium transients in central nerve terminals are sensitive to p38 M A P K inhibition. Simulataneously recording from presynaptic and postsynaptic neurons is possible in the Calyx of Held. It would thus be possible to test whether SB203580 prevents adenosine-induced decreases in C a 2 + influx in Calyx terminals as well the effects pf SB203580 on transmitter release. Regulation of calcium channels by p38 M A P K could be either direct or indirect. One avenue of future research that may be worth pusuing is determining whether p38 M A P K directly binds to either the a l or p subunit of Cav2.2 (N-type calcium channel), and what functional consequence on calcium currents such an interaction may have. Such experiments could be conducted by expressing channels in a heterologous expression system such as COS-7 or H E K 293 cells. If p38 M A P K does directly interact with Cav2.2 a l or P-subunits, further experiments using mutant channels could help 179 define the structural components of the channel that are functionally important for this interaction. 5.4. Adenosine A 1 receptor signalling through JNK Having already shown that p38 M A P K , but not ERK, is activated by A i receptors to depress synaptic transmission, and knowing that p38 M A P K and JNK are often activated by similar stimuli, we next tested whether A i receptors activated JNK and whether A i -JNK signalling played a role in synaptic depression. We found that JNK and p38 M A P K were similarly activated by the A i agonist CPA. Like CPA-induced p38 M A P K activation, CPA-induced JNK activation was blocked by the A i receptor antagonist DPCPX. Moreover, like p38 M A P K , JNK activity was required for adenosine and CPA depression of fEPSPs in the CA1 region. Unlike phospho-p38, phospho-JNK did not co-immunoprecipitate with the A\ receptor. However, using a pan-specific JNK antibody, we found that JNK did co-immunoprecipitate with the A , receptor. Based on the above evidence, we proposed that A i receptors and JNK exist in a membrane-associated complex. Stimulation of A i receptors within this complex leads to phosphorylation of JNK and dissociation of phospho-JNK, which leads to a decrease in glutamate release. We also proposed that p38 M A P K activation is upstream of JNK activation in the signalling cascade initiated by Aj receptor simulation. This idea is based on pharmacological evidence showing that inhibition of p38 M A P K blocked CPA-induced JNK phosphorylation whereas inhibition of JNK had no effect on CPA-induced p38 M A P K activation. With this study we have extended the findings of our previous work by describing a potential mechanism underlying presynaptic inhibition, whereby 180 activation of both p38 M A P K and JNK following A i receptor stimulation is a major component of adenosine mediated decreases in neurotransmitter release in the hippocampus, possibly by regulating N-type calcium channel phosphorylation through PP2a (Fig. 5.1). The evidence presented in this study suggests that JNK is a downstream target of p38 M A P K activation, and that both p38 M A P K and JNK are mediators of A i-receptor-dependent synaptic depression. 5.4.1. Possible limitations Like the App38 M A P K study, the A p J N K study depends heavily on the specificity of the JNK inhibitors. In an attempt to ensure that the observed effects were due to specific inhibition of JNK, we used two distinct JNK inhibitors as well as an inactive analogue. Moreover, we found that SP600125 attenuated CPA-induced synaptic depression at doses of 20uM and 5pM, the latter concentration being one quarter of the concentration commonly used to inhibit JNK in hippocampal slices (Curran et al., 2003; Costello and Herron, 2004). It would be difficult to determine the concentration of SP600125 within the neurons of hippocampal slices incubated with SP600125. In any case, the intracellular concentration of SP600125 is almost certainly less than the concentration of SP600125 in the bath solution. There was a dose dependent effect of SP600125 on the magnitude of CPA-induced fEPSP depression. In the presence of 20pM SP600125 CPA depressed fEPSPs by 29.0 ± 7.8% (n=6), whereas in 5pM SP600125 CPA was able to depress fEPSPs by 41.4 ± 7.7% (n=8; p < 0.05). (CPA depression of fEPSPs in control conditions is -70%). Although it is possible that the increased potency of SP600125 at the higher dose reflects an increase in non-specific inhibition of another kinase, this is unlikely considering that 181 Figure 5 .1 . Model of adenosine A ( receptor-MAPK signalling in the nerve terminal. Adenosine A i receptors and p38 M A P K are physically associated in presynaptic terminals. Stimulation of A i receptors leads to p38 M A P K phosphorylation, activation of JNK, and translocation of PP2a to the membrane. PP2a activation could then modulate neuronal N-type calcium channel function, leading to a decrease in neurotransmitter release. 182 we obtained the same results for SP600125 at 20uM (29.0 ± 7.8% CPA-depression; n=6) as we did for JNK Inhibitor V at 5uM (28.6 ± 1.0%; n=8), whilst preincubation in 5uM of the inactive analogue had no effect on CPA-induced depression. Rather, the increased ability of SP600125 to block JNK activity at higher doses is probably simply a reflection of increased specific inhibition of the JNK signalling pathway. JNK inhibition due to SP600125 is thus most likely submaximal at a concentration of 5uM. 5.4.2. Future Directions The results presented herein indirectly indicate that Aj receptor stimulation is selectively activating the JNK3 isoform, as CPA-induced phosphorylation of p54-JNK (which consists of JNK2/3) is proportionally greater than that of p46-JNK (consisting of JNK1/2/3). The high baseline level of JNK phosphorylation in the brain is due to JNK1. The greater increase in p54-JNK vs p46-JNK phosphorylation upon CPA treatment is suggestive that A i receptors specifically activate the neuronal-specific JNK3 isoform. A powerful test of this hypothesis could be accomplished using transgenic mice in which the JNK3 gene has been deleted (Kuan et al., 2003). It would be expected that both CPA-induced synaptic depression and JNK phosphorylation would be attenuated in JNK3 knockout mice. Another possibility would be to interfere with the translation of JNK3 protein using R N A interference. Another aspect of the current research that lends itself to further study is whether A i receptors mediate some of their effects by directing the recruitment, activation, and scaffolding of cytoplasmic signalling complexes via |3-arrestins. The classic role of P-arrestins, based on studies of their interaction with B2-adrenergic receptors, was to 183 desensitize seven transmembrane receptors. The mechanism of p-arrestin desensitization is as follows: the activated (agonist-bound) form of the receptor is rapidly phosphorylated by G protein-coupled receptor kinases, which leads to recruitment of P-arrestins and results in receptor desensitization (Lefkowitz and Shenoy, 2005). However, in addition to desensitization of G protein-signalling, recent evidence indicates that P-arrestins can also act as signal transducers themselves. For example, P-arrestins can function as endocytic adaptors, linking receptors to the clathrin-coated pit machinery, and thus facilitate endocytosis of G protein-coupled receptors (Lefkowitz and Shenoy, 2005). Of particular interest is the finding that JNK3, as well as its upstream activators ASK1 and M K K 4 , exist in a complex with P-arrestin 2 in the mouse brain, where it acts as a scaffold to bind the components of the JNK module (McDonald et al., 2000). The presence of P-arrestin 2 causes cytosolic retention of JNK3 and enhances A S K 1 -dependent JNK3 phosphorylation. In addition, stimulation of the angiotensin II type l a receptor activates JNK3 and induces colocalization of p-arrestin 2 and active JNK3 to intracellular vesicles. Through its actions as a scaffold, P-arrestin is able to couple a specific M A P K pathway to a particular G protein-coupled receptor. Does P-arrestin signalling mediate the effects of A i receptor stimulation in the hippocampus? We tested this hypothesis by attempting to co-immunoprecipitate p-arrestin 2 and the Aj receptor. We found that p-arrestin 2 and the A i receptor did not co-immunoprecipate (data not shown). However, two additional experiments suggest that the Aj receptor may by associated with p-arrestin after all: 1) the A i receptor and the endocytic protein endophilin co-immunoprecipitate; and 2) endophilin and p-arrestin 2 co-immunoprecipitate (data not shown). We thus have indirect evidence that the A] 184 receptor and {3-arrestin can exist in a complex, as the A i receptor is associated with endophilin which is in turn associated with (3-arrestin 2. However, further experiments will be needed to determine whether (3-arrestin plays a functional role in A i - J N K signalling in the hippocampus. Finally, we also have evidence that selective Aj receptor stimulation leads to a long lasting (> 4 hours) synaptic depression that, in contrast to short-term (i.e. 10 min) depression, is independent of JNK inhibition (Fig. 5.2). One explanation is that the short-term depression caused by A i receptor stimulation is due to JNK-dependent changes in release probability, whereas A i receptor-mediated long-term depression (LTD) is caused by internalization of postsynaptic A M P A receptors in a mechanism similar to classic frequency-dependent LTD. Two major predictions arise from this hypothesis: 1) Prolonged exposure to CPA will decrease GluR2 surface expression; and 2) Preventing A M P A receptor internalization will block CPA-LTD. The first prediction could be tested using a biotinylation assay of GluR2 surface expression. A test of the second prediction could be accomplished using a peptide interference strategy. For example, LTD can be abolished by infusion of a peptide through the recording pipette that specifically binds to AP2, and thus prevents recruitment of AP2 to A M P A receptors, assembly of the clathrin coat, and endocytosis of A M P A receptors (Lee et al., 2002). We have results from biotinylation experiments showing that prolonged stimulation of A i receptors (10-30 min) leads to a decrease in receptor surface 185 Figure 5.2. The A i receptor agonist C P A induces a form of long-term depression that is insensitive to J N K inhibition. Plot of the normalized fEPSP slope over time. Slices were either preincubated in 5uM SP600125 (JNK Inhibitor II) or normal aCSF. CPA was applied for 10 min. 120i CD C 1 100-CO . Q - 80H CD Q . O CO CL co 60-Q_ LU M— T 3 CD N 1 4 0 i E 40nM CPA n=6 20-~20 40 60 80 100 120 140 Time (minutes) • 40 nM CPA O 5nM JNK Inhibitor II + 40nM CPA 186 expression. This is consistent with agonist-induced internalization of postsynaptic A i receptors. A i receptors are known to be physically associated with N M D A receptors. Whether A i receptor stimulation causes LTD by internalizing glutamate receptors is an open question. Adenosine A ! receptor-dependent internalization of glutamate receptors would be a novel mechanism underlying G protein-dependent modulation of synaptic plasticity in the brain and would be expected to have a wide array of functional consequences. 5.5. The physiological relevance of ambient adenosine Basal transmitter release in the hippocampus is depressed by ambient adenosine (adenosine tone). Removing the inhibitory action of adenosine using an A i receptor antagonist (e.g. DPCPX) typically increases baseline synaptic transmission at Schaffer collateral-CAl synapses by 20-30% (Dunwiddie and Diao, 1994; Brundege and Dunwiddie, 1996; de Mendonca et al., 1997; Latini et al., 1999a; Bon and Garthwaite, 2002). The role of ambient adenosine in modulating baseline synaptic transmission and synaptic plasticity is controversial. It was recently reported that a number of forms of plasticity unique to mossy fiber synapses (i.e. large paired-pulse ratio, capacity to undergo frequency facilitation, large posttetanic potentiation, and presynaptic expression of LTP) were specifically due to the tonic presynaptic inhibition by a local, high concentration of ambient adenosine (Moore et al., 2003). These forms of synaptic plasticity are distinct to mossy fiber synapses because of the extremely low basal release probability of mossy fiber terminals. Previously, the low-release probability of mossy fibers was attributed to intrinsic properties of the synapse, such as high endogenous C a 2 + 187 buffering in terminals, release-incompetent presynaptic calcium channels, and strongly inactivating presynaptic K + channels. However, Moore et al. (2003) suggest the low-release probability is in fact imposed on mossy fiber synapses by tonic activation of presynaptic A i receptors. The authors came to this conclusion based on their findings that blockade of A i receptors (with DPCPX), enzymatic degradation of extracellular adenosine, and the genetic deletion of the Aj receptor all caused a dramatic (~ 5-fold) increase of baseline mossy fiber-CA3 synaptic transmission, whilst both short-term plasticity (paired-pulse facilitation and frequency facilitation) and long-term potentiation were almost absent under these conditions. The results of the study conducted by Moore et al. (2003) were recently called into question by the Dietrich group (Kukley et al., 2005), who found no evidence that ambient adenosine regulates plasticity at mossy fiber synapses. In stark contrast with the -500% increase in baseline synaptic transmission caused by DPCPX reported by Moore et al. (2003), Kukley et al. (2005) found that blockade of A i receptors caused only a modest 20-30% increase in mossy fiber responses. Furthermore, the magnitude of posttetanic potentiation, paired-pulse facilitation, and frequency facilitation were all unchanged when A i receptors were either genetically deleted or antagonized by DPCPX. The authors suggest that release probability of mossy fiber terminals is low enough to spawn these forms of synaptic plasticity without the requirement for potent inhibitory adenosine tone. According to Kukley et al. (2005), the most likely reason for the differences in adenosine tone observed in their study versus that of Moore et al. (2003) is that Moore et al. (2003) used a submerged recording chamber that led to hypoxia in their slices. 188 However, a difference in the extracellular adenosine concentration does not explain why Kukely et al. (2005) and Moore et al. (2003) achieved vastly different results for frequency facilitation (-180% vs -400%) and posttetanic stimulation (-280% vs -520%) when A l receptors were antagonized by D P C P X or genetically deleted. In the hippocampal slice preparation, the concentration of extracellular adenosine is dependent upon the temperature of slice incubation. The concentration of extracellular adenosine at 21°C is approximately double that of slices incubated at 32°C (Dunwiddie and Diao, 2000). The increase in extracellular adenosine at lower temperatures is due to diminished activity of dipyridamole-sensitive (ei) nucleoside transporter. Given the effect of temperature on adenosine tone, it is surprising that Kukley et al. (2005) did not discuss, let alone test, whether temperature differences could account for some of the discrepancies between their results and those obtained by Moore et al. (2003); especially considering Moore et al. (2003) conducted all experiments at room temperature and Kukley et al. (2005) conducted their experiments at 30°C. The larger effects of DPCPX observed by Moore et al. (2003) may be attributable to increased adenosine tone resulting from decreased adenosine uptake by ei transporters. In support of a physiological role for adenosine tone in regulating baseline synaptic transmission and plasticity, Pascual et al. (2005) recently reported that purinergic release from astrocytes enhances adenosine tone and modulates synaptic plasticity. Blockade of astrocytic ATP release (and thus accumulation of extracellular adenosine) was accomplished using inducible transgenic mice expressing a dominant-negative SNARE domain selectively in astrocytes. Mice with impaired astrocytic purinergic signalling exhibited increased baseline synaptic transmission and reduced 189 LTP. Moreover, activity-dependent heterosynaptic depression was completely abolished. Therefore, tonic suppression of excitatory transmission mediated by astrocytes regulates the degree to which a synapse may be plastic. In addition, activation of an astrocyte to release ATP leads to widespread coordination of synaptic networks, as astrocytes-derived adenosine can depress distant synapses. A n interesting observation is that activation of metabotropic glutamate receptors with DHPG, which leads to a form of LTD, appears to abolish adenosine tone in the slice (data not shown). Perfusing the A i receptor agonist DPCPX onto slices after induction of DHPG-LTD does not increase the slope of fEPSPs. This is in contrast to CPA-LTD, which is still sensitive to DPCPX administration. Manipulation of adenosine tone by receptor-dependent processes could be an important mechanism underlying metaplasticity (i.e. the phenomenon whereby the history of synaptic activity influences the capacity of the synapse to undergo future plasticity). Another fascinating observation is that the inhibition of CK2 (formerly casein kinase II) with 4,5,6,7-Tetrabromo-2-azabenzimidazole (TBB) (Sarno et al., 2005) completely abolishes Schaffer-collateral fEPSPs, and this is reversible by D P C P X (data not shown), suggesting that inhibiting CK2 activity leads to a huge increase in adenosine tone. TBB is likely exerting its effects by interfering with CK2-dependent regulation of equilibrative nucleoside transporters (Stolk et al., 2005), although this has not yet been demonstrated in nervous tissue. Nonetheless, it would appear that CK2-dependent modulation of nucleoside transporters is a novel mechanism underlying regulation of adenosine tone in the central nervous system. Such a mechanism could have important clinical implications in anaesthesia and neuroprotection. 190 5.6. A revised role for p38 MAPK and JNK in neuroprotection One of the major contributions of the research presented in this thesis to the field of neuroscience may be a greater understanding of the mechanisms underlying neuroprotection. There is a great deal of controversy over whether or not p38 M A P K and JNK exert neuroprotective or neurodegenerative influences when the brain undergoes trauma such as an ischemic insult. I believe that both scenarios are likely true under specific circumstances, and that the research presented here provides a framework for determining when p38 M A P K and JNK activation is likely to be neuroprotective and when it is likely to be a harbinger of cell death. 5.6.1. Whether p38 MAPK and JNK activation is neuroprotective or neurodegenerative may be a matter of timing. There is extensive evidence that adenosine, acting at A] receptors, attenuates ischemic injury in the heart and brain (see introduction). The data presented in this thesis establish that p38 M A P K and JNK are activated by A i receptor stimulation, and are necessary for A] receptor-dependent inhibition of neurotransmission, which is the primary means by which adenosine protects cells from excitotoxicity. Therefore one would predict that, like adenosine, p38 M A P K and JNK are also involved in protecting cardiac and/or neuronal tissue during an ischemic insult. Previous studies have found that the p38 M A P K pathway (Irving et al., 2000; Barone et al., 2001) and the JNK pathway (Comerford et al., 2004; Gao et al., 2005) are activated within minutes following hypoxia/ischemia in brain tissue. It is reasonable to speculate that the increase in p38 M A P K and JNK activity following hypoxia/ischemia is attributable to increased 191 activation of A i receptors resulting from increased release of endogenous adenosine during the ischemic insult. However, whether p38 M A P K and JNK activation is neuroprotective is controversial. There is a body of evidence that would seem to disprove the hypothesis that JNK and p38 M A P K activation underlie the neuroprotective effects of adenosine in the hippocampus. JNK and p38 M A P K are well-established mediators of cell death in the brain, where they are activated to execute apoptotic cell death in vulnerable brain areas after hypoxic/ischemic injury (Herdegen et al., 1998; Ozawa et al., 1999; Irving et al., 2000; Legos et al., 2002; Zhu et al., 2002b). JNK mediates neurodegeneration by activating genetic programs through phosphorylation of the nuclear transcription factor c-Jun/AP-1, the release of cytochrome c or the pro-inflammatory actions of microglia (Manning and Davis, 2003). Similarly, p38 M A P K is activated by a number of stimuli that cause apoptosis (Xia et al., 1995; Juo et al., 1997), and is thought to function downstream of caspase activation in mediating apoptotic cell death (Fernandes-Alnemri et al., 1996; Huang et al., 1997). In support of a deleterious role for JNK in cerebral ischemia, systematic administration of the JNK inhibitor SP600125 (Gao et al., 2005) or a peptide inhibitor of JNK (Borsello et al., 2003) protects against ischemic injury. SP600125 attenuates the mitochondrial apoptosis signaling pathway by preventing ischemia-induced translocation of Bax and Bim, release of cytochrome C and Smac, and activation of caspase-9 and caspase-3. The JNK inhibiting peptide, even when injected 6 hours post-reperfusion, reduces lesion volumes by more than 90% and confers significant behavioural improvement on a task measuring locomotor performance as long as 14 days post-192 ischemia. In addition, disruption of the gene encoding specifically the neuronal JNK3 isoform in mice prevents apoptosis of hippocampal neurons and decreases seizure activity in response to the excitotoxic glutamate-receptor agonist kainic acid (Yang et al., 1997). Moreover, stress-induced JNK activity and brain injury is reduced in JNK3 knockout mice after cerebral ischemia/hypoxia (Kuan et al., 2003). These data indicate that extinction of the JNK3 signalling pathway leads to neuroprotection and that JNK3 activation is an important component in the pathogenesis of glutamate neurotoxicity. There is also evidence that p38 M A P K activation is deleterious during cerebral ischemia. The p38 M A P K inhibitor SB203580 protects against neuronal death after N M D A receptor overstimulation (Kawasaki et al., 1997) or hypoxia (Zhu et al., 2002b). A second generation p38 M A P K inhibitor, SB206393, also reduces brain injury (Legos et al., 2002) and neurological deficits (Barone et al., 2001) due to cerebral ischemia. It is important to note that most of the neuroprotective effects of p38 M A P K and JNK inhibition have been reported in studies that examined ischemic injury or neurological deficits many hours, and often days, post-ischemia. Moreover, the most dramatic neuroprotective effects have often been described in studies where the p38 M A P K or JNK inhibitor was administered for many hours beginning at some time point after reperfusion. For example, Barone et al. (2001) administered SB239063 continuously for 6 hours post-middle cerebral artery occlusion (MCAO) starting 15 min following the onset of ischemia. Borsello et al. (2003) achieved their greatest reduction in infarct size by injecting a peptide inhibitor of JNK 6 hours post-reperfusion. Because JNK and p38 activity increases within minutes of the onset of ischemia (Sugino et al., 193 2000), early activation of JNK and p38 is clearly not responsible for the ischemic injury observed in these studies. Early activation of p38 M A P K and JNK due to ischemia is almost certainly mediating different physiological processes than activation of these kinases hours or days post-ischemia. In support of this notion an experiment was conducted where rats were given SB203580 or vehicle prior to transient middle cerebral artery occlusion and serial magnetic resonance imaging was used to evaluate the extent of the resulting lesion (Lennmyr et al., 2003). After 1 day, lesions were significantly larger in the SB203580 group compared with vehicle, suggesting that p38 M A P K may aggravate ischemic brain injury when present during the ischemic insult, which is remarkably similar to the deleterious effect of A i receptor antagonism (Rudolphi et al., 1992; Zhou et al., 1994). These data make it clear that not only is inhibition of p38 M A P K or JNK during transient ischemia unnecessary to achieve neuroprotection, but inhibiting these kinases during an ischemic insult may actually exacerbate ischemic injury. The results of the research presented in this thesis may offer an explanation for why late activation of p38 M A P K and JNK is helpful whereas early activation may be harmful. Based on our discovery that adenosine inhibits neurotransmission by activating p38 M A P K and JNK through A i receptor stimulation, early activation is likely mediating the neuroprotective effects of endogenous adenosine acting at A i receptors, whereas late activation is involved in completely separate processes that mediate cell death. Evidence that early activation of p38 M A P K and JNK is beneficial has long been supported by studies of ischemic injury in the heart. In heart tissue, adenosine activates both p38 M A P K and JNK (Haq et al., 1998), and both p38 M A P K and JNK mediate 194 cardioprotection in response to ischemia (Weinbrenner et al., 1997; Baines et al., 1998; Baines et al., 1999; Barancik et al., 1999; Fryer et al., 2001c). Ischemic preconditioning (or ischemic tolerance) is a phenomenon in which a brief sublethal ischemic insult induces long-term resistance to the effects of a subsequent more severe ischemic insult that depends on Ai-receptor activation (Thornton et al., 1992; Tsuchida et al., 1993) in a pathway requiring p38 M A P K (Zhao et al., 2001b; Schulte et al., 2004). Recent studies have confirmed that A i receptor-mediated delayed preconditioning against myocardial infarction is dependent on p38 M A P K in vivo (Lasley et al., 2005). The role of p38 M A P K and JNK in mediating ischemic preconditioning has not been studied as extensively in the brain as it has in the heart. Nonetheless, there is a growing body of evidence, albeit indirect, that supports the hypothesis that p38 M A P K and JNK mediate A] receptor-dependent ischemic tolerance in the brain. As in the heart, the adenosine A] receptor is thought to trigger ischemic tolerance in the gerbil hippocampus (Kawahara et al., 1998). Evidence that p38 M A P K contributes to ischemic / / tolerance in the same system came from the finding that SB203580, administered 30 min before a 2-minute sublethal ischemic insult, increases neuronal cell death in CA1 neurons in response to a second 5 minute ischemic insult 48 hours after reperfusion (Nishimura et al., 2003). Perhaps the most compelling evidence that A i receptors can activate p38 M A P K in the brain with functional consequences, aside from the research presented in this thesis, comes from integrating the findings of a number of studies on isoflurane-induced tolerance for focal cerebral ischemia. Isoflurane preconditions neurons to improve tolerance for subsequent ischemic insults in both in vitro and in vivo models (Kapinya et 195 al., 2002; Zhao and Zuo, 2004). In the heart, isoflurane-induced tolerance shares a number of cellular mechanisms with ischemic preconditioning (Aizawa et al., 2004), including the requirement for an A]-receptor mediated pathway (Roscoe et al., 2000). Recently, it was reported that isoflurane tolerance against focal cerebral ischemia is dependent on an A i receptor-mediated pathway as well (Liu et al., 2006). This is a particularly interesting finding because isoflurane tolerance against cerebral ischemia requires the activation of p38 M A P K in vivo (Zheng and Zuo, 2004). The above data demonstrate that a physiological process (isoflurane tolerance) in the brain depends on both A i receptor stimulation and p38 M A P K activation. It would be interesting to directly test whether A i receptor antagonism prevents isoflurane-mediated increases in p38 M A P K phosphorylation. A l receptor-dependent activation of p38 M A P K in response to isoflurane may represent a novel neuroprotective mechanism in the mammalian brain. The apparent dichotomy between the neuroprotective and neurodegenerative roles of p38 M A P K and JNK in ischemia will likely disappear with more precise knowledge of the timing and context of their activation. Obviously, p38 M A P K and JNK activation will have a different function in presynaptic nerve terminals during an ischemic insult than in microglia surrounding an infarct a week after a stroke. Because both p38 M A P K and JNK inhibitors have been put forward as potential therapeutics for stroke, it will be valuable to test the hypothesis that inhibition of p38 M A P K and JNK is harmful during an ischemic insult but beneficial after reperfusion. Ultimately, ischemic injury might best be decreased by enhancing p38 M A P K and JNK activity during hypoxia/ischemia and decreasing p38 M A P K and JNK activity beginning in the hours post-ischemia. 196' 5.7. Presynaptic inhibition revisited Another major contribution of the research presented in this thesis is that it proposes a novel mechanism underlying presynaptic inhibition, namely p38 MAPK/JNK-dependent inhibition of neurotransmitter release following A i receptor stimulation. Regulation of neurotransmitter release can occur via direct interaction with proteins associated with vesicular release, or by modulation of presynaptic neuronal calcium channels. Modulation of neuronal calcium channels by G protein-coupled receptors has been studied extensively, and a number of canonical mechanisms by which activated G proteins regulate calcium channel function in presynaptic terminals have been described. A discussion of these classical pathways is presented here in an attempt to reconcile what is known about G protein-dependent modulation of neuronal calcium channels and the somewhat radical idea proposed here that p38 M A P K and JNK play a role in G protein-dependent presynaptic inhibition. 5.7.1. Classic G protein modulation of neuronal calcium channels Calcium channels are divided into low-threshold (T-types) and high threshold (L-, N - , P/Q-, and R-types) and have been classified according to both their electrophysiological and pharmacological properties (Catterall, 2000). A prototypical high-threshold neuronal calcium channel is a heterotrimeric complex that consists of a pore-forming al-subunit, a P-subunit, and an a2o~-subunit. The a l subunit contains four conserved structural domains (domain I-IV) linked by cytoplasmic hydrophobic linkers. The diversity of a l -subunits accounts for all known voltage-gated calcium currents. Most neurons express multiple types of a l - and P-subunits, although the subcellular localization of individual 197 subtypes is more prescribed. For example, the C C I E (Cav2.3) R-type and a l e (Cavl.2) and a l D (Cavl.4) L-types are localized to cell bodies and dendrites, whereas the a l B (Cav2.2) N type and a l A (Cav2.1) P/Q type subunits are concentrated in large numbers at presynaptic terminals, where they trigger neurotransmitter release (Catterall, 2000; Elmslie, 2003). Activation of G protein-coupled receptors, including opioid, cannabinoid, neuropeptide Y , and adenosine receptors, inhibits N-type current by releasing GBy from the trimeric G-aPy complex allowing it to directly bind to presynaptic N-type calcium channels in a 1:1 stoichiometry (Elmslie, 2003). Binding of GPy to the I-II linker of the N-type calcium channel stabilizes the channel in the closed state, and thus decreases neurotransmitter release in response to subsequent action potentials arriving at the terminal. This GPy-mediated inhibition is strongly voltage dependent, is relieved by action potential trains, and is affected by the type of p-subunit (Cavpi vs Cavp2a), associated with the N-type channel complex (Snutch, 2005). Moreover, P K C phosphorylation of the N-type channel can also inhibit the Gpy-N-type channel interaction (Zamponi and Snutch, 1998). There is evidence that in addition to GPy-dependent inhibition, G protein-coupled receptors modulate neuronal calcium channels via parallel GPy-independent pathways as well. For example, retinal ganglion cells possess baclofen-sensitive G A B A receptors that, when activated, can either directly inhibit voltage-dependent calcium channels via GPy binding, or indirectly inhibit these channels through an independent parallel P K A -dependent pathway (Zhang et al., 1997). 198 Protein kinases play an important role in the regulation of neuronal calcium channels, and the role of P K A , PKC, and CaMKII as N-type calcium channel substrates is particularly well documented (Nastainczyk et al., 1987; Hell et al., 1994; Zamponi et al., 1997). 5.7.2. MAPK modulation of neuronal calcium channels There is a growing body of evidence that N-type calcium channels are modulated by members of the M A P K family in addition to P K A , P K C , and CaMKII. For example, the inhibition of N-type calcium current by bradykinin, which requires the sequential activation of two G-proteins, heterotrimeric G13 and monomeric Racl/Cdc42, also requires the activation of p38 M A P K in NG108-15 cells (Wilk-Blaszczak et al., 1998). However, the mechanism by which p38 M A P K inhibits N-type channels is not well understood. In contrast, E R K modulation of N-type calcium channels has been studied in greater detail (Fitzgerald, 2000, 2002; Martin et al., 2006). The Ras/ERK signaling is responsible for tonic up-regulation of sensory neuronal N-type calcium channels (Collin et al., 1990; Fitzgerald and Dolphin, 1997; Lei et al., 1998). E R K modulation of N-type calcium channels is thought to reflect a direct phosphorylation of consensus sites within the intracellular linker between transmembrane domains I and II (I-II linker) (Martin et al., 2006). The CavP subunit binds to the Cav2.2-subunit within a conserved motif in the I-II linker called the al-interaction domain (Pragnell et al., 1994). This binding readily occurs because all CavP subunits have a complimentary a 1-interaction domain binding pocket (De Waard et al., 1994; Opatowsky et al., 2004). The presence of the CavP subunit is required for ERK-dependent modulation of Cav2.2 (Fitzgerald, 2002). 199 Because ERK-dependent modulation of Cav2.2 occurs regardless of which of the four neuronal CavB isoforms is co-expressed (Fitzgerald, 2002), it is likely that a universal mechanism of interaction exists at the channel level. The CavB subunit also has two putative E R K consensus sites that that fulfill the minimum requirement for E R K phosphorylation (Ser-Pro) that are conserved on all four CavP subtypes and these sites are functionally important for ERK-mediated Cav2.2 modulation (Martin et al., 2006). These two Cavp sites, plus phosphorylation of Ser-447 on the Cav2.2 I-II linker, are both necessary and sufficient for ERK-dependent modulation of Cav2.2 channels (Martin et al., 2006). The biophysical mechanism by which ERK-mediated phosphorylation modulates channel activity probably reflects altered channel gating mediated by conformational or electrostatic changes caused by phosphorylation of the I-II linker. Binding of the CavP subunit, which is required for ERK-dependent modulation of Cav2.2, may allow previously inaccessible sites on the Cav2.2 I-II linker to become targets for E R K phosphorylation (Martin et al., 2006). Phosphorylation of the conserved Ser-Pro consensus sites on CavP subunits may also influence channel function, as they are present within the functional core of the Cavp protein. Indeed, one of these sites (Ser-448) is found within the a l domain interaction binding pocket, and therefore even modest phosphorylation would be expected influence CavP-Cav2.2 interaction. E R K phosphorylation of CavP subunits could potentially regulate C a 2 + influx in a variety of ways depending on which Cavp is co-expressed with Cav2.2. For example, all Cavb subunits alter channel conductance, Cavp2a and Cavb4 alter activation/inactivation kinetics, and Cavp2a shifts voltage dependence (Wittemann et al., 2000). Therefore, 200 depending on which Cavp subunit is expressed with Cav2.2, E R K signaling could potentially allow channels expressed in different regions or cell types to be differentially modulated by the same neurotransmitter. Whether p38 M A P K and/or JNK directly interact with Cav2.2 in a similar fashion as E R K remains to be determined. The E R K signaling pathway often opposes the p38 M A P K and JNK signaling pathways. Therefore, it would not be surprising i f p38 M A P K or JNK activity suppressed C a 2 + influx through Cav2.2 and thus functioned in opposition to Ras/ERK-dependent enhancement of C a 2 + influx. One possible mechanism for p38 M A P K and/or JNK to oppose E R K phosphorylation of Cav2.2 is through activation of a serine/threonine protein phosphatase. The PP1, PP2a, PP2b, and PP2c families of Ser/Thr protein phosphatases are expressed in the brain, and play important roles in regulating the activity of neuronal calcium channels (Price and Mumby, 1999). For example, all four protein phosphatases dephosphorylate sites on both the I-II linker and the II-III linker the of the a l subunit after phosphorylation by PKCe, although PP2c was the most effective (Li et al., 2005). Moreover, PP2c and Cav2.2 channels form a functional signaling complex in neurons that is responsible for the rapid dephosphorylation of the channel after its phosphorylation by PKC. The results presented in this thesis show that A i receptor stimulation leads to translocation (and presumably activation) of PP2a. As has previously been reported in cardiac tissue (Liu and Hofmann, 2003), the results presented here show that translocation of PP2a to the plasma membrane due to A] receptor activation is dependent on p38 M A P K activity in the hippocampus (Fig. 3.3). p38 MAPK-dependent activation 201 of PP2a is a potential mechanism by which A i receptor activation could modulate neuronal calcium channel function. In support of this hypothesis, PP2a is known to be functionally associated with Cavl.2, and this interaction is responsible for reversing P K A phosphorylation of serine 1928 (Hall et al., 2006). It would be interesting to test whether PP2a is also associated with Cav2.2 and whether Ai-p38 MAPK-PP2a signaling is in fact a viable pathway mediating inhibition of neuronal calcium channels. It should be noted that prolonged exposure (> 2 hours) to the PP2a inhibitor Okadaic acid results in a complete run down of A M P A and N M D A responses in hippocampal slices (data not shown). Even a dramatic increase in stimulus intensity fails to elicit fEPSPs after prolonged Okadaic acid treatment. This observation suggests that Okadaic acid results in the death of the slice. Therefore, it is difficult to pharmacologically test whether PP2a activation contributes to synaptic depression. M A P K pathways are extremely complex and subject to regulation by numerous upstream activators. In contrast, direct inhibition of calcium channels through GPy is an all-or-none process with significantly less opportunity for subtle modulation. It is therefore not surprising that parallel protein kinase-dependent mechanisms have evolved for modulating neuronal calcium channels given their enormous structural and functional diversity. Indeed, modulation of neuronal calcium channels by M A P K has virtually unlimited potential for fine-tuning neurotransmitter release and thus synaptic transmission in the central nervous system. 5.8. Conclusion The body of work presented here gives insight into the mechanism by which purinergic signalling modulates neurotransmission in the central nervous system. Stimulation of 202 purinoceptors at both mossy fiber and Schaffer collateral synapses leads to decreased transmitter release in a pathway requiring M A P K activation (Fig. 5.3). Whether P2X 7 receptors are in fact expressed in mossy fiber terminals awaits confirmation using molecular biology. However, it is reassuring that the results of this study have been reliably replicated by other labs and it is only the interpretation of the results that is still an open question. Future experiments will also be required to determine the precise molecular mechanism underlying M A P K modulation of neuronal calcium channels. Nonetheless, a working model has emerged from this work whereby p38 M A P K and JNK exist in a complex with A i receptors in the CA1 region of the hippocampus where they are activated sequentially following A i receptor stimulation (see Fig. 5.1, page 182). Such a signalling complex could modulate neuronal calcium channels by regulating the activity of the. serine/threonine phosphatase PP2a. A]-p38-JNK signalling is a novel pathway mediating presynaptic inhibition. Because purinergic signalling is of such importance to the functioning of the nervous system under both normal and pathophysiological conditions, the results herein are expected to be of general interest to the field of neuroscience. 203 Figure 5.3. Summary of purinergic signalling through MAPKs in the hippocampus. In area CA1, p38 M A P K and JNK are activated sequentially following A\ receptor stimulation. 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