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Changes in extracellular dopamine levels in the nucleus accumbens induced by low frequency stimulation… Taepavarapruk, Pornnarin 1998

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C H A N G E S I N E X T R A C E L L U L A R D O P A M I N E L E V E L S I N T H E N U C L E U S A C C U M B E N S I N D U C E D B Y L O W F R E Q U E N C Y S T I M U L A T I O N OF T H E V E N T R A L S U B I C U L U M / C A 1 R E G I O N OF T H E H I P P O C A M P U S : A M I C R O D I A L Y S I S S T U D Y I N F R E E L Y M O V I N G R A T S by P O R N N A R I N T A E P A V A R A P R U K B.Sc, Silpakorn Un i v e r s i t y , Thailand, 1989 M. Sc., Chulalongkorn University, Thailand, 1993 A T H E S I S S U B M I T T E D I N P A R T I A L F U L F I L L M E N T OF 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 M A S T E R OF S C I E N C E i n 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 Department of Neuroscience We accept this thesis as conforming to the required standard 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 February 1998 © Pornnarin Taepavarapruk, 1998 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of WtVLrOSCiewce The University of British Columbia Vancouver, Canada DE-6 (2/88) 11 A B S T R A C T The present study util ized microdialysis in freely-moving rats to investigate 1) changes i n extracellular dopamine (DA) level i n the nucleus accumben (NAc) induced by electrical stimulation of the ventral s u b i c u l u m / C A l ( V s u b / C A l ) area of hippocampus, and 2) the role of metabotropic glutamate receptors (mGluRs) i n mediating D A release i n the N A c induced by low frequency stimulation of V s u b / C A l . It has been shown that electrical stimulation of V s u b / C A l at 2 H z for 100 sec induces a significant decrease (p < 0.05) i n basal D A levels i n the N A c which is long lasting throughout the 2 hr monitoring period. Reverse dialysis of the specific antagonist at m G l u R l / 2 , (+)-a-methyl-4-carboxyphenylglycine (MCPG) , at doses 10 LLM and 100 LuVL d id not prevent the suppressive effects on D A levels induced by low frequency stimulation of V s u b / C A l . However, reverse dialysis wi th the group 2 and 3 m G l u R antagonist, (+)-a-methyl-4-phosphonophenylglycine (MPPG), at dose of 100 u M and 1 m M , significantly blocked the prolonged suppressive effects of 2 H z V s u b / C A l stimulation on D A efflux. These results suggest that presynaptic group 2 and 3 mGluRs are likely to be involved i n a mechanism underlying the late phase of synaptic transmission at glutamatergic-dopaminergic synapses i n the N A c . However, the question w h i c h specific m G l u R subtypes are responsible for these init ial and long lasting suppressive mechanisms w i l l require further research. iii T A B L E OF C O N T E N T S Abstract i i Table of contents i i i List of Tables v List of Figures v i List of Abbreviations v i i i Acknowledgment xi I. Introduction 1 Anatomical and physiological role of the nucleus accumbens 1 Functional interaction of D A and G l u i n the N A c 5 Metabotropic glutamate receptors (mGluRs): their characteristics and anatomical distribution i n the C N S 6 Functional roles of mGluRs i n mediating D A release i n the N A c 10 II. Methods 16 Subjects 16 Surgery 16 Microdialysis Procedures 17 Analysis of Dialysates 20 Electrical Stimulation Procedure 21 Pharmacological Studies 21 Histology 22 Data Analysis 22 iv III. Results 24 Effect of V s u b / C A l stimulation on extracellular levels of D A in the N A c 24 Pharmacological studies: Effects of selective antagonists at mGluRs on basal D A level i n the N A c 26 Role of mGluRs in regulating D A release i n the N A c induced by 2 H z stimulation of V s u b / C A l 34 IV. Discussion 40 Effects of V s u b / C A l stimulation on extracellular levels of D A i n the N A c 40 Effects of the selective m G l u R antagonists on basal D A levels i n the N A c and on the suppressive effects in N A c D A release induced by 2 H z stimulation of V s u b / C A l 46 V . References 53 LIST OF T A B L E S Table 1 Dendrogram and pharmacological distinction between cloned m G l u R subtypes 8 Table 2 A summary of potency of several agonists of mGLuRs as tested on cell lines that express m G l u R subtypes 12 Table 3 Summary of potency of phenylglycine compounds as antagonists of m G l u R specific agonists i n different tissue preparations 13 VI L I S T OF F I G U R E S Fig. 1 The general scheme of the afferent and efferent circuits of the N A c that are involved i n regulating locomotor activity 3 Fig. 2 Schematic view of the possible roles of m G l u R l at the glutamatergic synapse 9 Fig. 3 Diagram of proposed mechanism that regulates the release of D A i n the N A c 15 Fig. 4 Illustration of a dialysis system 18 Fig. 5 Diagram of preparation used i n the study 19 Fig. 6 Time-courses of the effects of electrical stimulation of V s u b / C A l at three different frequencies (2 H z , 5 H z and 10 Hz) on extracellular D A levels i n the N A c 25 Fig. 7 Time-courses of the effects of 2 H z stimulation of V s u b / C A l on extracellular D A levels i n the N A c 27 Fig. 8 Time-courses of the effects of M C P G administration at doses of 10 u M , 100 | i M or 1 m M on basal levels of D A i n the N A c 28 Fig. 9 Time-courses of the effects of M P P G administration at doses of 100 ^ . M , 1 m M or 10 m M on basal levels of D A i n the N A c 31 Fig. 10 Time-courses of the effects of administration 10 m M M P P G or its vehicle on basal levels of D A i n the N A c 33 Fig. 11 Effects of M C P G (lOuM) in combination wi th 2 H z stimulation of V s u b / C A l on extracellular D A levels i n the N A c 36 vu Fig. 12 Effects of M C P G (IOOLIM) i n combination wi th 2 H z stimulation of V s u b / C A l on extracellular D A levels in the N A c 37 Fig. 13 Effects of M P P G (IOOLIM) i n combination wi th 2 H z stimulation of V s u b / C A l on extracellular D A levels in the N A c 38 Fig. 14 Effects of M P P G (1 m M ) i n combination wi th 2 H z stimulation of V s u b / C A l on extracellular D A levels in the N A c 39 Fig. 15 Diagram of proposed mechanism that involves the role mGluRs in regulating the release of D A i n the N A c 43 Fig. 16 Diagram of proposed mechanism that involves the effects of M P P G (100 LIM) on the release of D A i n the N A c 49 Fig. 17 Diagram of proposed mechanism that involves the effects of M P P G on the suppression of D A efflux induced by 2 H z stimulation 52 L I S T OF A B B R E V I A T I O N S (1S,3R)-ACPD = trans (1S,3R)- l-aminocyclopentane-l,3-dicarboxylic acid A N O V A = analyses of variance A M P A = a-amino-3-hydroxy-5-methylisoxazole-4-propionate A P = antero-posterior AP-5 = DL-2-amino-5-phosphopentanoic acid B K = big C a 2 + activated K + channel °C = degree celsius C a 2 + = calc ium C a C l 2 . 2 H 2 0 = calcium chloride dihydrate = concentration i n the medium C o u t = concentration i n the outflow (S) 4CPG = (S)-4-carboxy-phenylglycine (S) 4 C 3 H P G = (S)-4-carboxy-3-hydroxyphenylglycine (S)3C4HPG = (S)-3-carboxy-4-hydroxyphenylglycine cyclic A M P = adenosine 3'5'-cyclic monophosphate D A = dopamine D C G I V = (2S /2'R /3'R)-2-(2,3-dicarboxycyclopropyl) glycine D N Q X = 6,7-dinitroquinoxaline-2,3-dione E A A = excitatory amino acid ECD = electrochemical detector E D T A = ethylenediaminetetraacetic acid ix G l u = glutamate H P L C = high pressure l iquid chromatography i G l u R = ionotropic glutamate receptor IK = inward rectifier K + channel i.p. = intraperitoneal IP 3 = inositol 1,4,5-triphosphate K A = kainic acid KC1 = potassium chloride L-AP4 = L-2-amino-4-phosphonobutyrate L-CCG-I = (2S,l'S /2'S)-2-(2'-carboxycyclopropyl) glycine L-SOP = L-serine-O-phosphate L T D = long term depression L - V S C C = L-type voltage-sensitive calcium channel L Y 354730 = (±)-2-aminobicylclo [3.1.0] hexane-2,6-dicarboxylate M C C G = 2S /lS',2S'-2-methyl-2-(2'-carboxycyclopropyl) glycine M C P G = (±)-a -methyl-4-carboxyphenylglycine M 3 C M 4 H P G = (+)-a-methyl-3-carboxymethyl-4-hydroxyphenylglycine M g C l 2 . 6 H 2 0 = magnesium chloride m G l u R = metabotropic glutamate receptor M L = medial-lateral M P P G = (+)-a-methyl-4-phosphonophenyl glycine M S P G = (+)-a-methyl-4-sulphonophenylglycine X MTPG (±)-a-methyl-4-tetrazolylphenylglycine M . W = molecular weight NAc = nucleus accumbens NaCl = sodium chloride NaOH = sodium hydroxide N M D A = N-methyl-D-aspartate N-VSCC = N-type voltage-sensitive calcium channel 6-OHDA = 6-hydroxydopamine PKC = protein kinase C QA = quisqualic acid V T A = ventral tegmental area XI A C K N O W L E D G M E N T I wou ld like to express my sincere gratitude to my supervisor, Dr. A . G . Phil l ips, for his k ind guidance and generosity. Without his assistance, I would not have been able to complete this work. I wou ld also like to thank Dr. C D . Blaha, Dr. A . C . Coury and Dr. C R . Yang for their assistance and thoughtful suggestions. I wou ld like to take the opportunity to thank my chair and committee member of the supervisory committee, Dr. P. Finlayson and Dr. L . Raymond for their invaluable insight and discussion on this work. I wou ld like to thank Stan Floresco, Dennis Fiorino and Soyon A h n for their kindly suggestions and Fred Lapaine for his technical assistance. Finally, I w o u l d like to express my appreciation for all love and support I have received from my parents and my husband, Niwat . This research was supported by a grant (PG-12808) from the M R C to A . G . Phill ips, C D . Blaha, and C R . Yang. Pornnarin Taepavarapruk is a recipient of Royal Thai Government Scholarship. 1 I N T R O D U C T I O N Anatomical and physiological role of the nucleus accumbens. The nucleus accumbens of the ventral striatum (NAc) is a l imbic structure that has been implicated i n a number of functions including learning and adaptive behavior (Mogenson et al., 1980), reward (Fibiger and Phil l ips, 1988; Phil l ips et al., 1989; Koob, 1992), motivation (Mogenson et al., 1993), locomotor activity (Yang and Mogenson, 1987; Koob and Swerdlow, 1988; Gold et al., 1988; Dreher and Jackson, 1989; W o n g et al., 1991; W u et al., 1992; Brudzynski et al., 1993; W u and Brudzynski , 1995). It is also wel l established that dopamine terminals wi th in the N A c originating from cell bodies i n the ventral tegmental area (VTA) are a crucial substrate for the locomotor activating properties of psychostimulant drugs such as cocaine and amphetamine (Gold et al., 1988; Pulvirent i et al., 1991; Kelley and Throne, 1992; Steketee et al., 1992; Nestler, 1993; Smith et a l , 1995). Recently, it has been suggested that the N A c has comparatively h igh level of a dopamine (DA) receptor subtype of D2 family, known as the D3 receptor (Sokoloff and Schwartz, 1995) which is thought to play a role i n psychiatric disease such as schizophrenia or Gilles de la Tourette's syndrome (Gilbert et al., 1995). This nucleus can be divided into core and shell subdivisions due to the differences i n immunological characterization using calcium binding protein ca lb ind in -D 2 8 k D A (CaBP), substance P, and acetylcholinesterase as the markers (Jongen-Reio et al., 1994) and i n their organization of afferent and efferent projections (Groenewegen et al., 1989; Johnson et al., 1994). Anatomical studies have shown the N A c receives strong excitatory input from two major l imbic structures; hippocampus and amygdala, and these afferents are glutamatergic i n 2 nature (Yim and Mogenson, 1982; Yang and Mogenson, 1984; Groenewegen et al., 1987; Robinson and Beart, 1988). As previously observed by Mogenson and colleagues (1980), the N A c has been proposed to serve as an interface between the limbic and motor systems as it appears to play an important role i n relaying limbic information to the motor effector sites. This hypothesis was later supported by anatomical, electrophysiological and behavioral findings showing that this nucleus serves as a "limbic-motor interface", by integrating signals from limbic regions and then transferring them to the subpallidal region and then to the mesencephalic locomotor area (Yang and Mogenson, 1987; Y i m and Mogenson, 1988; Mogenson et al, 1993). The diagram of this circuit is shown i n Fig. 1. As noted, the N A c receives a large dopaminergic input from the V T A . Electrical stimulation of the V T A attenuates the excitatory inputs to the N A c from the parafascicular nucleus of the thalamus (Sasa et al., 1991), amygdala (Yim and Mogenson, 1982), or from the hippocampus (Yang and Mogenson, 1984). The G A B A e r g i c medium spiny neuron, the major neuron i n the N A c , has been shown to receive both dopaminergic input from the V T A and glutamatergic input from the hippocampus and accordingly many researchers have been trying to elucidate both anatomical and functional interactions between these two major inputs. W i t h respect to the anatomical evidence, Sesack and Pickel (1990) reported that the hippocampal glutamatergic and mesolimbic catecholaminergic terminals were found to converge either on the same spine head or on the different part of the same dendrites of the accumbens spiny neurons. As wel l , at the physiological level, early work revealed that the convergence of the inputs to accumbens neurons from the hippocampal glutamatergic and V T A dopaminergic neurons i n 3 Fig. 1 The general scheme of the circuits showing A) the projections of the ventral subiculum, the posterior prelimbic cortex (prelimbic Cx posterior), and the posterior part of the basolateral amygdala (amygdala BL posterior) to the caudomedial accumbens (Acb), and B) the connections of the Acb with the mesencephalic locomotor region (MLR) via the ventral pallidum (VP). The broken line represents the ventral pallidal back-projection to the Acb (from Pennartz et al., 1994). 4 the N A c is similar i n detail to the converging inputs from amygdala and V T A (Yim and Mogenson, 1982). Subsequent work was conducted by Yang and Mogenson (1984) to investigate the influence of the mesolimbic dopamine pathway on signals reaching the N A c from the hippocampus. In short, results from this study indicated that the hippocampus sends strong excitatory inputs to both the silent and spontaneously active accumbens neurons. Condi t ion ing stimulation of the V T A (a train of 10 H z , 300 LLA, 0.15 ms duration), or iontophoretic application of D A , attenuated approximately 40-60% of the excitatory response of the accumbens neurons to hippocampal stimulation for a sustained period of time. Iontophoretic application of trifluoperazine, or the injection of haloperidol into the N A c could significantly block this prolonged suppressive effect. It is thus feasible that this suppressive action of the mesolimbic dopaminergic input is functionally important in modulating the extent to w h i c h the hippocampus can influence motor mechanisms, v ia the N A c , for the initiation of behavioral response. From electrophysiological and behavioral studies, there is considerable evidence that D A released from the mesolimbic terminals i n the N A c can exert a "gating" influence on the limbic input by selecting only particular signals to be transmitted through the N A c (Yang and Mogenson, 1986; Mogenson et al., 1988). The gated limbic signals are subsequently relayed from the N A c to basal forebrain motor effector sites to initiate adaptive behaviors (Yang and Mogenson, 1985). Functional interaction of D A and G l u i n the N A c i n the regulation of locomotion. A n early study reported that bilateral intra-accumbens injection of D A caused a dose-dependent locomotor hyperactivity i n reserpine-nialamide pretreated rats (Jackson et al., 1975). When a large dose of D A was administered, a 5 suppression of exploratory locomotor activity was produced i n the first 3 min, and then followed by stimulation of exploratory locomotor activity (Svensson and Ahlenius , 1983). DA-induced locomotor activity has been found to involve both D l and D2 receptor stimulation wi th in the N A c (Dreher and Jackson, 1989). O n the other hand, the neurotoxin 6-hydroxydopamine (6-OHDA) which specifically damages to dopaminergic neurons when injected into the N A c , has been shown to produce motor hypoactivity which can be ameliorated by administration of the D A agonist, apomorphine (Wolterink et al., 1990). Based on observations of the rotational behavior induced by the N M D A antagonist, DL-2-amino-5-phosphonopentanoic acid (AP-5) i n mice, it was found that glutamate (Glu) has a dual function and affects behavior i n different directions depending on the degree of D A D2 receptor stimulation (Svensson et al., 1992,1994a, 1995). This suggests that D A D2 receptors can modulate the behavioral effects of glutamatergic neurotransmission i n the N A c . Conversely, numerous studies have shown that excitatory amino acids (EAAs) also play a role i n regulation of motor behavior by influencing dopaminergic mechanism. Agonists selective for different types of G l u receptors, when injected into the N A c have been shown to induce hyperactivity i n rats. For example, intra-accumbens injection of kainate (KA) , quisqualate (QA), an agonist at A M P A receptor, and N-methyl-D-aspartate ( N M D A ) produced a pronounced hypermotility response which has been shown to be antagonized by reserpine, haloperidol or fluphenazine (Donzanti and Uretsky, 1983) or antagonists of E A A receptor (Hamill ton et al., 1986). Similarly, intra-accumbens injection of a-amino-3-hydroxy-5-methylisoxazole-4-propionate ( A M P A ) has been shown to produce a marked dose-dependent increase in locomotor activity which is not mediated by 6 the activation of N M D A receptors (Shreve and Uretsky, 1988). These findings exemplify the distinctive role of separate glutamatergic ionotropic receptors i.e., N M D A vs. A M P A i n regulating motor tonic activity i n mammals. In addition, local application of the glutamatergic receptor agonists, K A and Q A , by reverse dialysis increase the efflux of D A in the N A c (Imperato et al., 1990a) while the ionotropic glutamate receptor agonist, N M D A , failed to evoke D A release (Imperato et a l , 1990b). However, others have reported that D A release could be evoked by G l u acting at either N M D A or n o n - N M D A ionotropic receptors (Svensson et al., 1994b). Moreover, microinjection of G l u agonists, either N M D A or A M P A , into the N A c has been found to increase the locomotor activity. Prior administration of the D A D2 agonist, quinpirole, has been found to reduce this hyperkinetic effects i n dose dependent manner suggesting that D A mediated the locomotor activity induced by the G l u agonists (Wu et al., 1992). From the accumulated data, it appeared that glutamatergic afferents from the hippocampus and dopaminergic afferents from the V T A may interact w i t h each other i n the N A c to influence a variety of behavioral responses. Metabotropic glutamate receptors (mGluRs): Their characteristics and anatomical distr ibutions i n the C N S . The metabotropic excitatory amino acid receptor is a member of the glutamate receptor family that couples to G-protein to modulate mult iple second messenger systems. Presently, eight subtypes of mGluRs have been identified and classified into three groups according to their amino acid sequence identities, signal transduction mechanisms, and agonist selectivity (Table 1 and Fig. 2) (Tanabe et al., 1992; for reviews see Nakanishi , 1994; P i n and Bockaert, 1995; P i n and Duvoisin, 1995; Conn and P in , 1997). 7 The m G l u R group I ( m G l u R l and mGluR5) is coupled to phospholipase C (PLC) to stimulate phosphoinositide (PI) hydrolysis resulting i n an increase i n intracellular calcium (Ca 2 +) as studied i n transfected C H O cells (Aramoni and Nakanishi , 1992). By using immunocytochemical-ultrastructural approaches, Mar t in et al. (1992) reported the abundant presence of m G l u R l i n the olfactory bulb, hippocampus, globus pallidus, thalamus, substantia nigra, superior colliculus and cerebellum. O n a subcellular level, m G l u R l is localized postsynaptically to dendritic shafts, dendritic spines and also i n neuronal cell bodies. Similarly, a high density of mGluR5 is located postsynaptically i n the olfactory bulb, anterior olfactory nuclei, olfactory tubercle, cerebral cortex, hippocampus, lateral septum, striatum, inferior colliculus, spinal trigeminal nuclei as wel l as i n the nucleus accumbens (Shigemoto et al., 1993). The m G l u R group 2 (mGluR2 and mGluR3) and group 3 (mGluR4, mGluR6 mGluR7 , and mGluR8), however, are negatively l inked to adenylate cyclase activity resulting i n a reduction of adenosine 3'5'-cyclic monophosphate (cyclic A M P ) formation (Schaffhauser et al., 1997). The study of Petralia et al. (1996) has reported the location of mGluR2 and/or mGluR3 in glutamatergic presynaptic terminals, mostly at mossy fiber synapses i n the hippocampus, supporting the idea that presynaptic metabotropic mGluRs may play a role i n plastic changes at glutamatergic synapses. Immunocytochemical evidence has shown that m G l u R 2 is located both pre- and postsynaptically mainly i n the cerebellar cortex, hippocampus, neocortical and limbic cortical regions (Neki et al., 1996). Both m G l u R group 2 and m G l u R group 3 are also thought to act presynaptically as autoreceptor invo lved i n the induction of long term synaptic depression at various brain regions, i.e., locus coeruleus (Dube' and Marshal l , 1997), caudate Table 1 Dendrogram and pharmacological distinction between cloned mGluR subtypes, (adapted from Pin and Bockaert, 1995; Pin and Duvoisin, 1995) 30 40 50 60 70 80 90 % Identity I 1 1 1 1 I I mGluRl mGluR5 mGluR2 mGluR3 mGluR4 mGluR7 mGluR8 Group II in Transduction T PI hydrolysis IcAMP •I cAMP Rank order of agonist potency quisqualate > L-glutamate > ibotenate > L-homocysteine sulfinate >trans-ACPD DCG-IV>L-CCG-I> L-glutamate > (1S,3R) ACPD > 4C3HPG > ibotenate > quisqualate L-AP4 > L-SOP > glutamate > ibotenate > quisqualate > (1S,3R)-ACPD Antagonists MCPG 4C3HPG, 4CPG for mGluRl but not mGluR5 MCPG for mGluR2 MPPG, MSPG, MTPG M3CM4HPG, M4H3PMPG for both mGluR group 2 and group 3 mGluR6 00 9 Fig. 2 Schematic view of the possible roles of mGluRs at the glutamatergic synapse. The m G l u R l / 5 located mainly at post-synaptic neurons can either inhibit or potentiate L-Type voltage-sensitive calcium channel (L-VSCC). Act ivat ion of m G l u R l / 5 could stimulate the release process by st imulating protein kinase C (PKC) and inositol 1,4,5-triphosphate (IP3) hydrolysis w h i c h induce an increase of C a 2 + from intracellular storages. The m G l u R l / 5 can activate big C a 2 + activated K + channel (BK), inhibit the inward rectifier K + channels (IKs) and particularly modulate the activity of N M D A and A M P A channel at postsynaptic neurons. m G l u R group 2 and 3 are located mainly at presynaptic neurons responsible for inhibiting the release process possibly via N -type V S C C (from P in and Bockaert, 1995). 10 nucleus (Cozzi et al., 1997), and hippocampus (Kamiya et al., 1996; Manahan-Vaughan, 1997). Functional roles of m G l u R s i n mediating D A release i n the N A c . As previously mentioned, numerous studies have addressed the role of ionotropic glutamate receptors (iGluRs) i n mediating D A release i n the N A c as wel l as their effects on motor behaviors. Recent evidence also indicates a functional interaction between mGluRs and D A i n limbic striatum, particularly i n the N A c . Taber and Fibiger (1995) employing in vivo microdialysis revealed an inhibitory effect of mGluRs on D A release evoked by prefrontal cortex (PFC) stimulation. Furthermore, a selective m G l u R agonist, trans (1S,3R)-1-aminocyclopentane-l,3-dicarboxylic acid (r-ACPD), when applied locally to the N A c by reverse dialysis caused a dose-dependent effect on D A release i n which a low dose (100 LIM) induced a decrease whereas a higher dose (1 m M ) induced an increase i n extracellular D A levels i n the N A c . The later effect w i th doses i n the m M range is consistent wi th the finding of Ohno and Watanabe (1995) w h o reported a persistent increase in D A release i n the N A c following local perfusion of A C P D (1 m M ) and this facilitatory effect could be antagonized by co-perfusion wi th (+)-a-methy-4-carboxyphenylglycine (MCPG), a specific antagonist of m G l u R 1/2. In a behavioral study, Attarian and Amalr ic (1997) found that activation of mGluRs by perfusion of A C P D to the N A c caused a dose-dependent increase i n locomotor activity i n rats and this effect appeared to be mediated through the D A system i n the N A c . Taken together, these studies suggest that low doses of m G l u R agonists may exert inhibitory effects on D A levels i n the N A c whereas higher less specific doses may facilitate D A neurotransmission. Table 2 and 3 show the data 11 from the recent studies demonstrating the potency of several agonists and antagonists at mGluRs. A recent study by Blaha et al. (1997) using in vivo chronoamperometry w i t h stearate-graphite paste electrodes i n urethane-anaesthetized rats has shown that stimulation of the ventral s u b i c u l u m / C A l ( V s u b / C A l ) region of the hippocampus wi th burst patterned monophasic pulses (10-100 Hz/burs t delivered at 0.8-4 Hz) could evoke a three component change i n the D A signal i n the N A c . In brief, the first and the third component, i n which D A signals are significantly increased above baseline following burst-stimulation of V s u b / C A l could be antagonized by microfusion of the i G l u R antagonists, AP-5 , 6,7-dinitroquinoxaline-2,3-dione (DNQX), and kynurenate. In contrast, the second component i n which the D A signal is significantly decreased below the baseline was blocked selectively by microinfusion of the m G l u R antagonist, M C P G . These results suggested that G l u released physiologically from V s u b / C A l inputs to the N A c selectively activates i G l u R and m G l u R wi th in the N A c resulting in an increase or an decrease i n extracellular D A levels, respectively. From available data, the mGluRs appear to play a functional role i n inhibit ing the release of D A from the dopaminergic neurons i n the N A c as suggested by several lines of evidence. Firstly, anatomical evidence from a m R N A in situ hybridization study has revealed the expression of mGluRs ( m G l u R l -mGluR5) i n the N A c (Testa et al., 1994). Secondly, an in vivo study has demonstrated that reverse dialysis of A C P D at low concentration (100 uM) into the N A c produces a marked decrease i n extracellular D A level i n the N A c (Taber and Fibiger, 1995). Thirdly, the recent in vitro study in the rat N A c slice preparation has shown that m G l u R group 2/3 but not group 1 is implicated i n the inhibi t ion of 12 Table 2 A summary of potency of several agonists and antagonists of mGluRs as tested on cell lines that express m G l u R subtype (from Conn and Pin , 1997) mGluRla mGluRSa mGIuR2 mGluR3 mGluR4a mG!uR6 mGluR7 mGluR8 Agonists Glutamate 9-13 3-10 4-20 4-5 3-20 16 1000 0.02 Quisqualate 0.2-3.0 0.03-0.3 >1000 40 100-1000 >300 — — Ibotenate 10-60 2-10 35-250 10-15 100-1000 >300 — — IS, 3R-ACPD 10-80 5-7 18 8 »300 300 — — IS, 3S-ACPD >300 >300 13 30 50 — — — L-CCG-1 50 — 0.3-0.4 1 9-50 — — — DCG-IV n.e. n.e. 0.3 0.2 >1000 — n.e. — 2R, 4R-APDC — — 3* — — — — — L-AP4 n.e. n.e. n.e. n.e. 0.4-1,2 . 0.9 160-500 0.4 L-SOP n.e. — — — 2-5 2.7 >160 — L-AP3 Ant. Ant. — — — — n.e. — 1-HPG 68-100 14-35 n.e. — n.e. — — — 3, 5-DHPG 6.6 2 n.e. — n.e. — n.e. — 4C3HPG Ant. >300 20-50 — n.e. — — — i-ADA 190 30 >1000 — n.e. — — — CPAP4 — — — — 0.6 — — — ntagonists MCPG 40-200 >200 100-1000 >1000 n.e. — n.e. — 4C3HPG 10-40 P. Ag. (?) Ag. — n.e. — — — 3C4HPG 300-400 — Ag- — n.e. — — — 4CPG 15-65 >500 Ag. (?) — n.e. — — — MPPG >1000 n.e. 100 — 54 — — — MSPG n.e. — 250 — »1000 — — — MTPG >1000 n.e. 450 — n.e. — — — MCCG-I n.e. — 84 — n.e. — — — MAP4 n.e. — 500 — 90-190 — — — L-AP3 >1000 >1000 — — — — — ABHD-I 300 — — — — — AIDC 7 — n.e. — n.e. — — PCCG-IV n.e. n.e. 8 — Ag. — — 7HCCMA 2 — n.e. — — — — ADPD n.e. n.e. 18,1 — — — — — Noted: potency is shown in EC 5 0 , IC 5 0 , or K b values QiM); n.e., no effect; —, not determined. Table 3 Summary of potency of phenylglycine compounds (in rank orders) as antagonists of mGluR specific agonists in different tissue preparations (from Bedingfield et al., 1996). Coupling Tissue Rank Adenyly) cyclase Phosholipase C Coupling Tissue Rank Adult rat cortex Neonatal spinal cord Neonatal cortex Granule cells Coupling Tissue Rank L-CCG-1 L-AP4 (1S,3S)-ACPD L-AP4 (15,3/?)-ACPD L-Quisqualate 1 MPPG MPPG MTPG b MPPG b (+ VM4CPG 8 (SMCPG 2 MSPG MSPG MPPG b MSPG b 4C2IPG 4C2IPG 3 M3CM4HPG M3CM4HPG MSPG b MTPG b (5)-4CPG • (+ )-M4CPG 4 M3CMPG " M3CMPG * ( + VM4CPG a (+ )-M4CPG a MPPG 5 M3C4HPG ' M4H3PMPG M4C3HPG a 6 ( + )-M4CPG a. M3C4HPG " MTPG 7 E4CPG " (+ VM4CPG * M4CMPG 8 8 M3CPG " B4CPG 8 MSPG 9 M4H3PMPG M3CPG " M3CM4HPG 10 M4CMPG '" MTPG M4C3C1PG 8 11 (- )M4CPG a M4C3C1PG " M3CMPG 8 12 MTPG (- )M4CPG a M3CPG a 13 M4C3C1PG 8 M4CMPG 8 M3C4HPG 8 Abbreviations: (#S)-a-rnetriyl-4-phosprionophenylglycine (MPPG; (/?S)-a-methyl-4-sulphonophenylglycine (MSPG); (/?5)-Q-meihyl-3-carboxymethyl-4-hydroxyphenylglycine (M3CM4HPG); (/?5)-a-methyl-3-carboxymethylphenylglycine (M3CMPG); (/?5)-a-methyl-3-carboxy-4-hydroxyphenylglycine (M3C4HPG); ( + )-a-methyl-4-carboxyphenylglycine (M4CPG); (/?5)-a-ethyl-4-carboxyphenylglycine (E4CPG); (/?5)-Q-methyl-3-carboxyphenylglycine (M3CPG); (/?5)-a-methyl-4-hydroxy-3-phosphonomethylphcnylglycine (M4H3PMPG); (/?5)-a-methyl-4-carboxymethylphenylglycine (( + VM4CMPG); (/?5)-Q-methyl-4-ietrazolylphenylglvcine (MTPG); (/?5)-a-methyl-4-carboxy-3-chlorophenylglycine (M4C3CIPG); (S)-4-carboxyphenylg)ycine ((5)-4CPG): (A?5)-4-carboxy-2-iodophenylglycine (4C2IPG). Previously published data from (a) Bedingfield et al. (1995); (b) Jane et al. (1995). 14 transmission at the prefrontal-accumbens synapses v ia a presynaptic mechanism (Manzoni et al., 1997). Finally, in vivo experiments i n urethane anaesthetized rats have shown that electrical stimulation of V s u b / C A l at 2 H z frequency induces a marked decrease of D A efflux below the baseline level which could be blocked by co-application wi th M C P G (Blaha et al., unpublished data). Therefore, it is hypothesized that 1) 2 H z stimulation of V s u b / C A l i n the freely moving rats w i l l modulate the release of G l u which subsequently induce an inhibi t ion of D A release from the dopaminergic terminals i n the N A c , and 2) these suppressive effects of V s u b / C A l stimulation are mediated by m G l u R s located on presyaptic G l u terminals. A diagram of the proposed role of mGluRs i n regulating D A release i n the N A c is shown in Fig. 3. The present study employed in vivo microdialysis techniques wi th high performance l iquid chromatography (HPLC) analysis of the dialysate obtained from the N A c of the unanaesthetized, freely moving rats. This procedure provided the unequivocal identification of extracellular D A and when combined wi th freely moving subjects removed any confounding effects of anaesthetic agents. This procedure has the added advantage that drugs can be applied locally to the N A c by reverse dialysis while collecting the dialysate for analysis from the same area. The main aims of the study were: 1) to investigate the effects of low frequency electrical stimulation of V s u b / C A l o n extracellular D A levels i n the N A c , and 2) the effects of M C PG, a specific antagonist of m G l u R at m G l u R l / 2 , and M P P G , an antagonist of m G l u R group 2 and 3 , o n basal levels of D A i n the N A c , as wel l as on changes i n levels of D A induced by V s u b / C A l 2 H z frequency stimulation. In order to determine whether any observed inhibi t ion is of short or long duration, D A was measured for 120 m i n following 2 H z stimulation. 15 Fig. 3 Diagram of a proposed mechanism that regulates the release of D A i n the N A c . A) Under normal condition: the spontaneous release of G l u f rom glutamatergic terminals of the V s u b / C A l induces the release of D A from the dopaminergic terminals i n the N A c via iGluRs. B) 2 H z stimulation: the release of G l u from the V s u b / C A l is reduced by the mechanism of presynaptic m G l u R group 2/3 following 2 H z stimulation wh ich eventually attenuates the release of D A from the terminals of V T A afferents i n the N A c . 16 M E T H O D S Subjects The subjects i n all experiments were male Long-Evans rats (Charles R ive r ; St. Constant, Quebec, Canada), weighing between 350-400 g at the time of surgery. They were indiv idual ly housed i n plastic cages i n a colony room wi th an ambient temperature of 25 °C and a 12:12 hr l ightdark cycle (light on at 7 am). Food (Purina Rat Chow) and water were available ad libitum. Surgery Prior to cannula implantation, rats were anesthetized wi th ketamine hydrochloride (100 mg/kg , i.p.) and xylazine (10 mg/kg , i.p.) and placed in a stereotaxic apparatus. The dorsal skull surface was exposed and holes were drilled. A microdialysis probe guide cannula (19 ga.) was implanted over the N A c (co-ordinates: A P = +1.7 m m from bregma, M L = +1.2 m m from mid l ine , and D V = -1.0 m m from dura; according to the atlas of Paxinos and Wat son (1986)). A bipolar stimulating electrode was implanted ipsilateral to the microdialysis probe guide cannula wi th the tip centered in the V s u b / C A l region of the hippocampus (co-ordinates from bregma: A P = -5.8 m m from bregma, M L = ±5.6 m m from midline, and D V = -6.5 m m from dura). The guide cannula and the stimulating electrode were secured chronically to the skul l wi th four set screws and dental acrylic. The animals were allowed to recover fully for at least 2 days before implanting a microdialysis probe. 17 Microd ia lys i s Procedure Microdialysis probes (Fig. 4 A ) used i n all experiments were constructed i n the laboratory. The probes were of a concentric-style consisting of a 24-ga stainless steel cannula (34 mm), fused silica tubing ( 75 p i i.d. x 150 um o.d.), polyethylene (PE 50) tubing and a semipermeable hollow-fiber membrane (2 m m of exposed membrane, 340 um o.d., 65,000 M . W . cut off, Fil tral 12, Hospal-gambro). Epoxy glue was used to seal joints and plug the dialysis fiber tip. The inlet tubing was connected to a swivel which was mounted above the testing chamber. A l iquid switch ( C M A / 1 1 0 , Carnegie Medicin), located between the swivel and the infusion pumps, was used manually to exchange the standard perfusate solution to one containing drug. Figure 5 shows a diagram of the preparation used i n the study. The relative recovery for D A was obtained pre-m vivo by immersing the tip of the probe i n a medium containing D A at known concentration and perfusing wi th perfusate at a constant rate of 1 | i l / m i n for at least 30 m i n before collecting samples at a fixed interval of 10 min . D A concentration i n the outflow was determined by H P L C - E C D . The relative recovery was calculated as: Recovery in vitro = C o u t / C i n Where C o u t is the D A concentration i n the outflow and C i n is the m e d i u m . Typical in vitro recovery for D A at ambient temperature and 1 u l / m i n was 22%. The day before conducting an experiment, the microdialysis probe was continuously perfused wi th perfusate solution (147 m M N a C l , 3.0 m M KC1, 1.3 m M C a C l 2 . H 2 0 , 1 . 0 m M M g C l 2 . 6 H 2 0 , 0.01 M Sodium phosphate buffer; p H 7.3-7.4) at a constant rate of 1 u l / m i n using a syringe pump (Harvard model 22). The probe was secured i n the microdialysis probe collar (Fig. 4B) by the top screw. 18 A) Microdialysis Probe Outlet Tubing yfc (Silica) Inlet Tubing (PE 50; Silica) P E 50 Tubing Stainless Steel Tubing (24 ga.) Epoxy Microdialysis Probe Collar Thread for Protective C o i l Attachment 15 m m 34 mm Setting Screws C) Guide Cannula . Stainless Steel Tubing (19 ga.) Dialysis _ Membrane -»'s m m - E p o x y Fig. 4 A graphic illustration of a dialysis system. The system consists of a concentric design microdialysis probe (A) wi th a tip of 2 m m for collecting dialysate from the N A c , a microdialysis probe collar (B) for securing the probe and an implanted guide cannula, and a guide cannula (C) which is used to implant chronically and stereotaxically over the area of the N A c . (adapted from Fiorino et al., 1993) Fig. 5 Diagrammatic illustration of the set up (A) which consists of a testing chamber equipped wi th two swivels, an electrical stimulator, an isolator unit, two syringe pumps and a l iquid switch. The sagittal view of the rat's brain (B) shows the placement of the stimulating electrode and the microdialysis probe. 20 After checking the volume of collected sample, the probe was inserted into the guide cannula (Fig. 4C) which had already been implanted over the N A c area of the animal. The bottom screw of the probe collar was then secured. The microdialysis probe was continuously perfused overnight at a flow rate of 1 \iM/min approximately 15-18 hr prior to experimental testing. Analys i s of Dialysates The dialysis membrane used in these studies does not allow proteins and other large molecules above a M . W . of 65 K Daltons into the perfusate, therefore the sample is relatively cleaned up and can be injected directly into the H P L C system without further purification (Benveniste and Huttemeier, 1990). The FfPLC-ECD system used i n the experiments consisted of a Biorad p u m p (Richmond, C A ) delivering 0.8 m l / m i n at a pressure of 123 K g / c m 2 , an EC10W two-position injector, an Ultrasphere column (Beckmann, Fullerton, C A , ODS 5 u.m, 15 cm x 4.6 mm), an E S A (Bedford, M A ) , and a Coulochem II EC detector. The working potentials were: +450 m V (electrode 1), -300 m V (electrode 2) and +450 m V (guard cell). The mobile phase consisted of 6 g/1 sodium acetate, 70 mg/1 octyl sulfate (adjustable), 10 mg/1 ethylenediaminetetraacetic acid (EDTA), 35 ml/1 glacial acetic acid and 865 m l M i l l i Q purified water. The mobile phase was adjusted to p H 3.5 wi th glacial acetic and filtered through 0.22 |a.m sterile nylon filter unit (Millipore). Filtered methanol (10% per volume) was added to the mobile phase solution and degassed prior to use. Chromatograms were registered on a dual pen chart recorder (Kipp and Zonen, Bohemia, N Y ) . D A was quantified from each sample by comparing sample peak heights to peak heights from a calibration curve of the standard solution containing D A at three different concentrations. 21 Electrical S t imula t ion O n the day of experiment, the dialysate samples were collected every 10 m i n and immediately analyzed by H P L C - E C D . Electrical stimulation was performed after establishing a stable D A baseline defined as three consecutive samples wi th less than 10% variation i n peak height. Cathodal constant current pulses were delivered to the V s u b / C A l through an isolator (Iso-flex, A.M.P.I) v i a a master stimulator (a dual channel stimulator model 4710, Ortec, E G & G company). Parameters for electrical stimulation were a total of 200 pulses, 300 LIA delivered at 2 H z , 5 H z or 10 H z for 100 sec, 40 sec or 20 sec, respectively. For most of the experiments reported i n this thesis, 2 H z stimulation frequency was used to produce a reliable inhibition of D A release. Pharmacological Studies After establishment of baseline D A levels, specific m G l u R antagonists, either M C P G (at doses of 10 u M , 100 u M or 1 mM) or M P P G (at doses of 100 u M , 1 m M or 10 m M ) , were administered locally for 20 m i n via reverse dialysis (i.e., through the same microdialysis probe used to measure D A ) . Both drugs were purchased from Precision Biochemical Inc. (Vancouver, Canada). A 10 m M M C P G or M P P G stock solution was prepared by dissolving wi th 50-100 Lil of 0.1 M N a O H solution which was then topped up to 1 m l wi th normal perfusion medium and kept frozen as aliquots at -20 ° C Drug was diluted from frozen aliquots wi th normal perfusate solution immediately prior to use. The p H of M C P G and M P P G solutions used i n the present study are shown as follows: 22 D r u g Concent ra t ion p H value M C P G 10 u M 7.3 100 u M 7.4 1 m M 7.6 M P P G 100 u M 7.6 1 m M 7.6 10 m M 8.5-8.7 In subsequent experiments, M C P G at dose of 10 [iM and 100 \xM or M P P G at doses of 100 ( iM and 1 m M was administered i n combination wi th electrical stimulation of V s u b / C A l . H i s to logy After completion of each experiment, DC current (100 u A for 10 sec) was passed through the bipolar stimulating electrode. Animals were perfused transcardially wi th 0.9% saline followed by 4% formaldehyde solution. The brains were then removed and placed i n 10% sucrose i n 4% formaldehyde. Serial 50 am coronal sections were cut on a freezing microtome and stained for Niss l substance wi th cresyl violet. Placements of the dialysis probe and the bipolar stimulating electrode were determined under a light microscope. Data Ana lys i s The data are presented as the percentage of the mean of three samples before the first electrical stimulation or before drug perfusion. Data f rom implanted control group, electrical stimulated group, drug-treated group or drug-treated i n combination wi th electrical stimulation groups, were analyzed for statistical significance using two-way, between/within subjects mixed analysis of 23 variance ( A N O V A ) design, wi th treatment as the between subjects factor, and sample as the wi th in subjects factor, unless stated otherwise. Whenever a significant treatment x sample interaction was observed, subsequent simple main effects analyses were conducted using one-way between subjects A N O V A with Dunnet's or Tukey's post hoc tests. These analyses assessed differences between groups at each sample. 24 R E S U L T S 1) Effect of V s u b / C A l st imulation on extracellular levels of D A i n the N A c . The baseline value of the extracellular D A concentration i n dialysates collected from the N A c was 2.42 + 0.08 n M (mean ± S.E.M., n= 67). These values were uncorrected for probe recovery which were i n the range of 20-22%. The init ial experiment was designed to examine the effect of electrical stimulation of the V s u b / C A l at three different sequential frequencies o n extracellular levels of D A in the N A c . A stimulation current intensity of 300 u A and a total of 200 pulses were used as a fixed parameter for all stimulation. A set of three consecutive electrical stimulation of V s u b / C A l wi th a period of seventy minutes between each stimulation at 2 H z , 5 H z , and 10 H z for 100 sec, 40 sec, and 20 sec, respectively, was found to induce changes in extracellular level of D A in a frequency-dependent manner (Fig. 6). Stimulation of V s u b / C A l at lowest frequency (2 Hz) tested caused a decrease i n extracellular D A levels in the N A c which reached statistical significant p < 0.05 at 30 min and p < 0.01 at 50, and 70 m i n following electrical stimulation. In contrast, subsequent stimulation 5 H z caused no change i n the depressed baseline induced by 2 H z s t imulat ion. Subsequent st imulation at 10 H z resulted i n an increase i n extracellular D A levels back to the pre-2 H z stimulation baseline. It should be noted that these experiments were not designed to test systematically the effects of different stimulation frequencies on D A release. A l l subsequent experiments focused o n the effects of 2 H z stimulation on D A efflux in the N A c . In further experiments (n=9), electrical stimulation of V s u b / C A l at 2 H z for 100 sec alone was shown to produce a significant and long lasting decrease i n 25 Fig. 6 Effects of electrical stimulation of V s u b / C A l on extracellular concentration of D A i n ipsilateral N A c . Each stimulus consisted of 200 pulses of constant current (300 uA) single pulses delivered at 2 H z (SI), 5 H z (S2), and 10 H z (S3) for 100 sec, 40 sec, and 20 sec, respectively. The data are expressed as a percentage of the mean of three basal samples before the first stimulation (SI). Each value is the mean + S E M (n=5). * significantly different from prestimulation baseline p < 0.05, ** p < 0.01, one way repeated measure A N O V A with Dunnet's post hoc test. 26 extracellular D A levels i n the N A c that lasted throughout the 120 m i n period of measurement (p < 0.01) as compared to non-stimulated animals (control group) (Fig- 7). 2) Pharmacological studies. According to available data, mGluRs i n the N A c have been shown to modulate D A release from terminals of dopaminergic neurons and to inhibi t transmitter release i n several other brain regions (Kamiya et al., 1996; Cozz i et al., 1997; Manahan-Vaugnan, 1997). Therefore, the following experiments examined the role of mGluRs in modulating the basal release of D A as wel l as i n mediating the suppressive effect on extracellular D A levels i n the N A c observed i n the previous study following 2 H z stimulation of V s u b / C A l . 2.1 Effects of selective m G l u R s antagonists on basal D A levels i n the N A c . M C P G , a specific antagonists at m G l u R l / 2 (Sekiyama et al., 1996), and M P P G , a potent L-AP4-sensitive presynaptic mGluR antagonists (Jane et al., 1995; Robert, 1995), were tested for their local effects on basal extracellular D A levels i n the N A c . A s shown i n Fig. 8, reverse dialysis of M C P G at concentrations of 10 u.M (n=5) and 100 u M (n=6) for 20 min caused no significant change i n D A levels after drug perfusion was discontinued. Reverse dialysis of M C P G at dose 1 m M (n=6) also was without significant effect on baseline D A levels but again a trend for a delayed decrease was more marked and persisted throughout the period of sample collection. Moreover, the variability of the data at the dose of 1 m M was higher than those of 10 u M and 100 u M . The effects of M C P G on basal levels of D A release i n the N A c are summarized i n Fig 8. In contrast, reverse dialysis of M P P G at dose of 100 u M for 20 m i n (n= 5) caused a decrease i n D A level in the N A c which achieved significance (p < 0.05) 27 Fig. 7 Effects of electrical stimulation of V s u b / C A l on extracellular concentration of D A in ipsilateral N A c . The stimulus consisted of 200 pulses of constant current (300 uA) single pulses delivered at 2 H z for 100 sec. Dialysate samples were collected every 10 min , and stimulation was delivered after stable baselines were established. Each value is expressed as a percentage of the mean of four basal samples before stimulation (S) i n the 2 H z frequency stimulation group (n=9). Each value is the mean +. S E M . There was a significant group x sample interaction (F(14, 182) = 5.563, p < 0.001) * significantly different from the control group, p < 0.01, simple m a i n effects analysis wi th Dunnet's post hoc test. 28 Fig.8 Time-course of the effects of local administration of M C P G at doses of 10 U .M (n=5), 100 LLM (n=6) or I m M (n=6) on basal levels of D A i n the N A c . Dialysate samples were collected every 10 min and each dose of M C P G was perfused for 20 m i n as indicated by horizontal bar. Each value is expressed as a percentage of the mean + S E M of three basal samples before drug administration. A n overal l A N O V A analysis revealed no significant different between the drug-treated groups and the control group. DAoutput(% baseline) DA output (% baseline) DA output (% baseline) 30 at 80, 90, 100, and 120 min time points. Perfusion of M P P G at a dose of 1 m M , however, resulted i n no significant changes i n D A levels i n the N A c . Appl icat ion of M P P G at dose of 10 m M caused a biphasic effect i n which a significant decrease i n D A levels (p < 0.05) was observed at 20 and 40 min time points, followed by a return to baseline. A transient increase wi th large variance was observed at 70 min. Dose-related effects of M P P G on basal levels of D A release i n the N A c are shown i n Fig. 9. As previously mentioned i n the methods section, a diluted N a O H solution was used to dissolve both drugs, and all of the 5 solutions were found to be in the range of 7.3-7.6 p H . However, the p H of a 10 m M M P P G solution was i n the range of 8.6-8.7. To determine whether p H may account for the biphasic effects on D A efflux observed following the application of 10 m M M P P G , an additional experiment was conducted consisting of perfusion wi th a vehicle solution (diluted N a O H , p H 8.7) into the N A c (n=4). As shown i n Fig. 10, a vehicle solution wi th a p H of 8.7 induced a delayed decrease in basal D A levels i n the N A c which reached statistical significance (p < 0.05) at 40 and 50 min time points as compared to the control (normal p H perfusion medium) group. W h e n the data from the control (normal perfusion medium) group, the vehicle-d i l u t ed N a O H , p H 8.7) treated group, and the M P P G (dose 10 m M ) treated group were compared and analyzed for statistical significance, it was found that both the MPPG-treated and the vehicle-treated groups showed a significant decrease in D A levels at 20, 30, and 40 m i n time points and at 40 and 50 m i n time points, respectively. In addition, there was a significant increase: i n D A levels i n the MPPG-treated group at the 70 min time point compared to the vehicle-treated group. 31 Fig. 9 Time courses of the effects of local administration of M P P G at doses of 100 (n=5), 1 m M (n=5) and 10 m M (n=5) on basal levels of D A i n the N A c . Dialysate samples were collected every 10 min and each dose of M P P G was perfused for 20 min (horizontal bar) after establishing a stable baseline. Each value is expressed as a percentage of the mean + S E M of three basal samples before the drug administration. There was a significant group x sample interaction (F (14, 434) = 1.843, p < 0.001), * significantly different from the control group, p < 0.05, simple main effects analysis wi th Dunnet's post hoc test. DA output (% baseline) DA output (% baseline) DA output (% baseline) 33 60 H 1 1 1 1 1 1 1 1 1 1 1 1 1 1 -20 -10 0 10 20 30 40 50 60 70 80 90 100 110 120 time (min) Fig. 10 Time-course of the effects of local administration of MPPG at a dose of 10 mM (n=5) and its pH adjusted vehicle (diluted NaOH, pH 8.7, n=4) on basal levels of DA in the NAc. Dialysate samples were collected every 10 min and MPPG or its pH adjusted vehicle were perfused for 20 min (horizontal bar) after establishing a stable baseline. Each value is expressed as a percentage of the mean + SEM of three basal samples before the drug administration. There was a significant group x sample interaction (F (14, 168) = 2.071, p < 0.005). * significantly different from the control group (normal pH perfusion medium) p < 0.05, ** p < 0.001, # significantly different from the vehicle group (diluted NaOH, pH 8.7) p < 0.005, simple main effects analysis with Tukey's post hoc test. 34 2.2 Role of mGluR in the inhibition of D A release in the NAc induced by 2 Hz stimulation of Vsub/CAl . The effects of M P P G at doses of 10 u M and 100 u M and M P P G at doses of 100 u M and 1 m M on the decrease i n D A levels i n the N A c induced by 2 H z stimulation of V s u b / C A l were investigated. It was found that M C P G at doses of 10 u.M (n=5) and 100 u M (n=6) applied 10 m i n prior to electrical st imulation did not antagonize the suppressive effect on D A efflux i n the N A c induced by 2 H z stimulation of V s u b / C A l as shown i n Fig. 11 and 12, respectively. Statistical analysis revealed no significant difference between the 2 H z stimulation (control) group and the 2 H z stimulation plus M C P G group. Local administration of M P P G at a dose of 100 u M (n=6) into the N A c 10 m i n prior to V s u b / C A l stimulation (Fig. 13) showed a trend of attenuation of the 2 H z stimulation-induced decrease i n D A levels compared to the control (2 H z stimulation) group during the first 20 min post-stimulation period, however, these values d id not differ significantly. These D A levels i n the N A c gradually returned to basal values 80 min after applying electrical stimulation. There was a significant group x sample interaction and simple main effects analysis w i t h Dunnet's post hoc test showed significant differences between this MPPG-treated group and the control (2 H z stimulation) group at 100 (p < 0.01), 110, and 120 (p < 0.005) m i n time points following electrical stimulation. A t a higher dose of M P P G ( ImM), the levels of D A i n the N A c returned to basal values sooner compared to the effects of 100 u M M P P G (Fig. 14). As wi th the lower dose of M P P G , the magnitude and time course of the init ial phase of suppressive effects of 2 H z stimulation on D A efflux was not altered by 1 m M M P P G . There was a significant group x sample interaction and simple main effects analysis w i t h 35 Dunnet's post hoc tests showed significant differences between this group and the control (2 H z stimulation) group at 70 (p < 0.05), 80 (p < 0.001), 90 (p < 0.01), 100 (p < 0.001), 110 (p < 0.05) and 120 (p < 0.001) m i n time points fol lowing electrical stimulation of V s u b / C A l at 2 H z . 36 120 110--TJ 2Hz (n=9) 2Hz+10uM-MCPG (n=5) -20 -10 0 10 20 30 40 50 60 70 80 90 100 110 120 time (min) Fig. 11 Effects of M C P G at dose of 10 LIM (n=5) i n combination wi th V s u b / C A l stimulation on extracellular concentration of D A i n the N A c . The st imulus consisted of 200 pulses of constant current (300 | iA) single pulses delivered at 2 H z for 100 sec. Dialysate samples were collected every 10 m i n and M C P G was perfused for 20 m i n (horizontal bar) after establishing a stable baseline. Stimulation was performed after 10 min of drug perfusion. Each value is expressed as a percentage of the mean + S E M of three basal samples before stimulation (S). A n overall A N O V A analysis revealed no significant different between these two groups. 37 Fig. 12 Effects of M C P G at dose of 100 u M (n=6) i n combination wi th V s u b / C A l stimulation on extracellular concentration of D A i n i:he N A c . The s t imulus consisted of 200 pulses of constant current (300 (iA) single pulses delivered at 2 H z for 100 sec. Dialysate samples were collected every 10 m i n and M C P G was perfused for 20 m i n (horizontal bar) after establishing a stable baseline. Stimulation was performed after 10 min of drug perfusion. Each value is expressed as a percentage of the mean + S E M of three basal samples before stimulation (S). A n overall A N O V A analysis revealed no significant different between these two groups. 38 2Hz (n=10) 2Hz+100uM-MPPG (n=6) ~i 1 i r -20 -10 0 10 20 30 40 50 60 70 80 90 100 110 120 time (min) Fig. 13 Effects of M P P G at dose of lOOuM (n=6) i n combination wi th V s u b / C A l stimulation on extracellular concentration of D A i n the N A c . The st imulus consisted of 200 pulses of constant current (300 uA) single pulses delivered at 2 H z for 100 sec. Dialysate samples were collected every 10 m i n and M P P G was perfused for 20 m i n (horizontal bar) after establishing a stable baseline. Stimulation was performed after 10 min of drug perfusion. Each value is expressed as a percentage of the mean + S E M of three basal samples before stimulation (S). There was a significant group x sample interaction (F (14, 378) = 2.136, v < 0.001), * significantly different from control inhibition, p < 0.01, ** p < 0.005, simple main effects analysis wi th Dunnet's post hoc test. 39 120 n 2Hz (n=9) 2H.z+lmM-MPPG (n=6) 110-< D 70-D R U G 60 H 1 1 1 1 1 1 1 1 1 r 1 1 1 1 -20 -10 0 10 20 30 40 50 60 70 80 90 100 110 120 time (min) Fig. 14 Effects of M P P G at dose of 1 m M (n=6) i n combination wi th V s u b / C A l stimulation on extracellular concentration of D A i n the N A c . The st imulus consisted of 200 pulses of constant current (300 LLA) single pulses delivered at 2 H z for 100 sec. Dialysate samples were collected every 10 m i n and M P P G was perfused for 20 m i n (horizontal bar) after establishing a stable baseline. Stimulation was performed after 10 min of drug perfusion. Each value is expressed as a percentage of the mean + S E M of three basal samples before stimulation (S). There was a significant group x sample interaction (F (14, 378) = 2.136, p< 0.001), * significantly different from control inhibi t ion, p < 0.05, ** p < 0.01, *** p < 0.001, simple m a i n effects analysis w i th Dunnet's post hoc test. 40 D I S C U S S I O N Effect of V s u b / C A l st imulation on basal extracellular levels of D A i n the N A c . The results from the present study indicate that electrical stimulation of the V s u b / C A l at three consecutive frequencies (2 H z , 5 H z , and 10 H z for 100 sec, 40 sec, and 20 sec, respectively) induces frequency-dependent changes i n extracellular D A levels i n the N A c . A significant decrease i n N A c D A levels was observed when the V s u b / C A l was stimulated at 2 H z frequency. 10 H z stimulation, but not 5 H z stimulation, caused a marked increase i n D A levels which reversed the inhibitory effect induced previously by 2 H z stimulation. As previously noted, these experiments were not designed to test systematically the effects of each individual stimulation frequency on D A release i n the N A c . The issue concerning the effects of each individual stimulation frequency on N A c D A levels w i l l be the subject of further investigations. The main body of experiments focused on the effects of 2 H z stimulation o n N A c D A levels. The results from the experiment applying electrical stimulation at 2 H z alone confirmed that 2 H z stimulation reliably induced a suppressive effect on N A c D A levels wi th lasted throughout the two hr monitoring period. The suppressive effect on N A c D A levels observed i n this dialysis study is consistent wi th previous findings of a study employing in v ivo chronoamperometry (unpublished data, Blaha et al.). The later study showed that V s u b / C A l stimulation at 2 H z for 10 sec could induce a relatively brief decrease i n basal D A efflux i n the N A c . However, the suppressive effect observed i n the present study persisted throughout the 2 hr period of measurement compared to the previous chronoamperometric study i n which D A oxidation currents were decreased for 41 only 10 m i n after application of electrical stimulation. The difference in the duration of suppression may be the result of several procedural differences, including a) type and tip size of stimulating electrode) which may have an- impact on the area of current spread, b) the duration of stimulation (100 sec and 10 sec), and most importantly c) the condition of experimental animals (unanaesthetized vs. anaesthetized) which has been reported to influence the basal concentrations of extracellular D A and particularly G l u levels in the rat striatum (Shiraishi et al., 1997). A proposed mechanism under ly ing the suppressive effects of 2 H z s t imula t ion on N A c D A levels. As noted, mGluRs have been classified into three groups based o n differences i n their amino acid sequences, pharmacological characteristics, and related second messenger systems (Nakanishi, 1994; P i n and Bockaert, 1995; P i n and Duvoisin, 1995; Conn and Pin , 1997). Recently, light and electron microscopy have revealed the predominant localization of mGluRs group 2 (mGluR2) and 3 (mGluR4a/7a/7b/8) i n presynaptic elements, whereas group 1 m G l u R ( m G l u R l / 5 ) are localized i n postsynaptic elements i n the rat hippocampus (Shigemoto et al., 1997). These findings are consistent wi th those of Bradley et al., (1996) showing that group 3 mGluRs (mGluR4a/7) are located presynaptically i n the hippocampus. In addition, mGluR4a has been found to be located on presynaptic elements i n the cerebellar cortex (Kinoshita et al., 1996). Several studies have suggested the role of group 2 and 3 mGluRs i n suppressing glutamatergic excitatory transmission i n some brain regions, most likely via a negative feedback presyaptic G l u autoreceptor mechanism (Kamiya et 42 al., 1996; C o z z i et al., 1997; Dube' and Marshal l , 1997). In contrast, group 1 mGLuRs , appear to be responsible for stimulating the release of neurotransmitter (Pin and Bockaert, 1995) and have been reported to facilitate N M D A - i n d u c e d responses i n a striatal slice preparation via a G l u postsynaptic mechanism (Pisani et al., 1997b). Therefore, it is reasonable to assume that, i n the N A c , group 2 and 3 mGluRs are located mainly on presynaptic glutamatergic terminals and are responsible for the inhibi t ion of G l u synaptic transmission by a presynaptic autoreceptor mechanism. Conversely, group 1 mGluR, as wel l as iGluRs , may be located mainly on postsynaptic dopaminergic terminals which may play a role i n modulating D A release. A t basal (non-electrically stimulated) conditions (Fig. 15A), it has been proposed that presynaptic mGluRs are unoccupied by basal levels of G l u (Scanziani et al., 1997). Basal levels of G l u are also assumed to modulate the basal levels of D A via iGluRs on dopaminergic terminals. This latter hypothesis is supported by several dialysis studies showing that E A A i.e., G l u and Asp play a modulatory role i n the release of D A i n the N A c through the iGluRs (e.g., N M D A or n o n - N M D A ) (Imperato et al., 1990a; 1009b; Youngren et a l , 1993). In addition, it has been suggested recently that mGluRs (hypothetically group 1 mGluRs) located on D A nerve terminals may also serve to enhance D A release i n the N A c (Ohno and Watanabe, 1995). A proposed series of mechanisms which may underlie the init ial and long term suppressive effects of 2 H z V s u b / C A l stimulation on D A efflux (Fig. 15B) may be that 1) stimulation-evoked release of G l u , over and above basal values, preferentially activates particular subtypes of presynaptic mGluRs . Al though we have no direct evidence for this mechanism, activation of these presumed group 2 43 Fig. 15 A proposed mechanism invo lv ing the role of mGluRs i n regulating D A release i n the N A c . A) A t non-stimulated basal condition, presynaptic mGluRs are assumed to be unactivated by spontaneous basal release of G l u from presynaptic terminals. The basal level of G l u is hypothesized to modulate the basal release of D A from the postsynaptic dopaminergic terminals. B) 2 H z stimulation: release of G l u from the presynaptic terminals induced by 2 H z stimulation of V s u b / C A l (1) activates presynaptic m G l u R s (2), resulting i n the inhibition of subsequent G l u release (3). Less G l u released from presynaptic terminals (4) and consequently a decrease i n D A release (5). Act ivat ion of an LTD-l ike mechanism (6) by evoked G l u release fol lowing 2 H z stimulation w o u l d result i n sustained inhibi t ion of G l u release, which i n turn wou ld result i n the prolonged reduction of basal D A levels. 44 and 3 presynaptic mGluRs by this stimulation-evoked transient elevation of G l u may serve to inhibit further G l u release from glutamatergic terminals via an mGluR-mediated autoreceptor inhibitory mechanism. The decreased amount of G l u released from glutamatergic terminals may, i n turn, lead to a decrease in the activation of postsynapitc iGluRs, that presumably regulate the release of D A from dopaminergic terminals. Consequently, this mechanism may account for the observed init ial decrease in D A efflux from dopaminergic terminals i n the N A c . In addition to m G l u R autoreceptor induced-inhibition, ii: is possible that 2) the excessive and transient elevation i n G l u release following 2 H z stimulation may lead to the activation of second messenger systems i n glutamatergic terminals. This second messenger mechanism may account for a long-lasting decrease i n subsequent G l u release. Thus, a mechanism resembling long-term depression (LTD) may play a role i n the long-term suppression i n D A efflux we have observed following 2 H z stimulation. The mechanism (#1) proposed above, accounting for the init ial suppression in D A efflux induced by 2 H z stimulation is supported by several lines of evidence. For example, the study of Testa et al. (1994) revealed m R N A expression of at least five subtypes of mGluRs i n the N A c and the prominent expression of m G l u R l i n dopaminergic neurons of the substantia nigra. In addition, several studies have suggested the involvement of group 2 and 3 presyaptic mGluRs i n presynaptic inhibi t ion mechanism of synaptic transmission i n various brain regions (Vignes et al., 1995; Kamiya et al., 1996; Cozz i et a l , 1997; Pisani et al., 1997a). Activat ion of two pharmacologically distinct receptors by application of selective agonists of group 2 mGluRs , (1S,1S)-ACPD and L-CCG-1, or the specific agonist of group 3 mGluRs , L-AP4, have been found to induce depression of excitatory synaptic 45 transmission i n the rat N A c slice preparation (Manzoni et al., 1997). This proposed mechanism is also supported by studies showing that L-AP4 can induce a reversible and dose-dependent depression of evoked EPSPs i n the locus coeruleus (Dube' and Marshal l , 1997), depression of monosynaptic excitation of motoneurons i n neonatal rat spinal cord (Jane et al., 1995), and suppression of basal synaptic transmission i n the lateral perforant path of the rat hippocampus (Bushell et al., 1996). In addition, application of f -ACPD i n rat striatal slice preparation has been found to induce a synaptic depressant effect on glutamatergic transmission at corticostriate synapses (Lovinger et al., 1993). Moreover, Taber and Fibiger (1995) have reported an inhibition i n basal D A release induced by relatively low concentrations of the group 2 m G l u R agonist, ACPE>, applied locally i n the N A c by reverse dialysis inn freely moving rats, strongly supporting a role for mGluRs i n regulating D A release i n the N A c . Evidence suggesting a role of mGluRs i n the expression of L T D (proposed mechanism #2) is the following. Kobayashi et al. (1996) have demonstrated that low frequency (1 Hz) stimulation together wi th activation of mGluRs , but not of iGluRs, induced presynaptic L T D at mossy fiber-CA3 synapses i n the hippocampus. More convincing evidence has been presented from a study wi th knockout mice deficient i n m G L u R 2 . This latter study showed an impairment of L T D at mossy fiber-CA3 synapses normally induced by low frequency stimulation (Yokoi et al., 1996). In addition, application of M C C G , a specific antagonist of group 2 m G L u R s , was found to inhibit the induction of L T D i n the dentate gyrus of the rat hippocampus (Huang et al., 1997). A study i n freely moving rats has also suggested the critical involvement of group 2 mGluRs in L T D induction (Manahan-Vaughan, 1997). Taken together, these results strongly sugg;est a role of presynaptic 46 m G l u R i n inducing the prolonged suppressive " L T D - l i k e " effects i n N A c D A levels induced by low frequency stimulation of V s u b / C A l . Effects of selective m G l u R antagonists on basal N A c D A levels and on the suppressive effects i n N A c D A release induced by 2 H z st imulat ion of V s u b / C A l . In the present study, two types of specific antagonists at mGluRs , M C P G and M P P G , were reverse dialyzed into the N A c i n order to test whether mGluRs are involved i n the tonic regulation of basal D A efflux, as wel l as the suppressive "LTD- l ike" effects i n N A c D A levels following 2 H z stimulation. M C P G and basal D A efflux. Appl icat ion of M C P G , a specific antagonist of m G l u R 1/2 (Sekiyama et al., 1996), at doses of 10 | i M , 100 u M , or 1 m M into the N A c caused a slight but n o n -significant decrease i n the basal levels of N A c D A . Therefore, it is unlikely that m G l u R l and/or mGluR2 are involved i n tonic regulation of D A release as wel l as i n the proposed mechanism #1. The density of m G l u R l expression i n the N A c is relatively higher than the mGluR2 expression (Testa et al., 1994), this fact along wi th the ability of M C P G to block both m G l u R l and mGluR2 (Bond and Lodge, 1995), raises the possibility that the small decrease in D A efflux observed fo l lowing application of M C P G may have resulted from the blockade of m G l u R l o n dopaminergic terminals i n the N A c . Overall , the present results are consistent wi th the study of Ohno and Watanabe (1995) which suggested that m G l u R s , particularly m G l u R l and mGluR2, i n the N A c are not involved i n the tonic regulation of D A since application of M C P G at a dose of 5 m M by itself d id not 47 cause any significant change i n accumbens D A levels. It is important to note, however, that this dose of M C P G attenuated the ability of locally applied A C P D to evoke D A overflow i n the N A c . M C P G and stimulated D A efflux. In the present study, reverse dialysis of M C P G at doses of 10 | i M or 100 u M , 10 min prior to the stimulation of V s u b / C A l at 2 H z d id not prevent the init ial or long-term suppressive effect on D A release, suggesting that m G l u R l and/or mGluR2 are not likely to be the major subtypes of mGluRs involved in these suppressive mechanisms. These results (for mechanism #2) are consistent w i t h the study of Selig et al. (1995) showing that m G l u R l / 2 do not play a significant role i n hippocampal L T D since application of M C P G did not block the L T D of EPSPs i n C A 1 hippocampal neurons induced by low frequency stimulation of Schaffer collaterals. However , it should be noted that application of M C P G did inhibit t-A C P D induced-LTD of EPSPs at Schaffer col la tera l -CAl synapse i n the hippocampal region of immature rats (Overstreets et al., 1997) and also inhibited both the A C P D - and the low frequency-induced L T D i n the dentate gyrus of the adult rats (O'Mara et al., 1995). The differences i n the experimental conditions i.e., concentration of drugs used, L T D induction protocols tested, area of the brain, and ages of animals examined between these studies may account for these conflicting results. M P P G and basal D A efflux. In the present study, it was found that reverse dialysis of M P P G at a dose of 100 LiM significantly decreased D A efflux i n the N A c after a delay, whereas a dose of 1 m M produced no significant change i n N A c D A levels. The highest dose of 48 M P P G resulted i n non-specific inhibitory effects. As noted i n the results, the f inal p H of the 10 m M solution of M P P F was i n the range of 8.5-8.7. Therefore, the transient decrease i n N A c D A levels observed at this dose is likely due to p H , rather than the drug itself. This was confirmed i n the additional study showing that the drug vehicle adjusted to a p H of 8.7 was able to decrease D A levels i n a manner similar to the drug. As previously noted, M P P G blocks the inhibi t ion of forskolin-stimulated c A M P production induced by L-AP4 i n rat striatal slices (Schaffhauser et al., 1997). Jane et al. (1995) reported the ability of M P P G to block the depression of the monosynaptic excitation of neonatal rat motoneurons induced by L-AP4, a selective agonist of group 3 mGluRs . However, M P P G has also been reported to block mGluR2 wi th 1/2 lower potency as shown i n Table 2. One possible explanation for the delayed suppressive effects on N A c D A release fol lowing washout of the blockade of mGluRs by M P P G (100 uM) may be that, during the application of M P P G , basal concentrations of G l u remain unaffected. Therefore, under these conditions no observable change i n N A c D A basal levels was observed. Fol lowing drug washout, these group 2 and 3 m G L u R s may have become supersensitive to the resting levels of G l u . If this were the case, activation of group 2 and 3 mGluRs by basal concentration of G l u may have resulted i n the inhibi t ion of subsequent G l u release from glutamatergic terminals. In turn, a decrease i n G l u release may have lead to the observed delayed decrease i n D A efflux from dopaminergic terminals. A diagram of this proposed mechanism is shown i n Fig. 16. Al though, there is no direct evidence for this mechanism, it could be tested by prolonging the perfusion of M P P G into the N A c . If the delayed decrease i n D A is mediated by an effect of basal levels of G l u acting o n 49 Fig. 16 The proposed mechanism invo lv ing the effects of M P P G (100 LIM) o n N A c D A levels. A) A blockade of group 2 and 3 mGluRs by M P P G failed to alter basal D A levels, suggesting that basal G l u release was unaffected by M P P G . B) After blockade, group 2 and 3 mGluRs , which are not activated by basal levels of G l u (1), become supersensitive to G lu . Act ivat ion of supersensitive mGluRs induces the inhibi t ion of G l u release. Less G l u (2) then cause less D A release from postsynaptic terminals. 50 supersensitive receptor thereby blocking them continuously wi th M P P G , one would expect to see no significant change i n the N A c D A levels until the drug was washed out. MPPG and stimulated D A efflux. Applicat ion of M P P G at doses of 100 u M or 1 m M 10 min prior to V s u b / C A l stimulation was found to block the presumed "LTD- l ike" effects (mechanism #2) but not the autoreceptor-mediated init ial suppressive effects (mechanism #1) o n D A levels, resulting i n the observed delayed return of D A levels to pre-stimulation baseline values (see figure 13 and 14). A proposed mechanism involving the effects of MPPG on the suppression of D A efflux induced by 2 Hz stimulation. As shown i n Fig. 13 and 14, after reverse dialysis of M P P G , the decrease i n N A c D A efflux was observed only i n the first 30-40 m i n following 2 H z stimulation after which D A levels returned to prestimulated values. One possible explanation for this relatively short lasting period of suppression i n D A levels may be that M P P G applied to the N A c by reverse dialysis 10 m i n prior to stimulation only partially blocks group 2 and 3 mGluRs on presynaptic terminals. M P P G is a competitive receptor antagonist, and therefore, the transient enhancement of synaptic G l u concentrations following 2 H z stimulation may be sufficient to displace M P P G from mGluRs, thereby resulting i n only partial mGluR blockade. Thus, sufficient G l u concentrations induced by 2 H z stimulation may activate some presyaptic mGluRs resulting in a subsequent decrease i n G l u release from presynaptic terminals by mechanism #1, an mGluR-mediated autoreceptor mechanism. Consequently, less G l u release from presynaptic terminals may cause 51 less activation of iGluRs on postsynaptic terminals resulting i n init ial decrease i n D A efflux i n the N A c . Furthermore, this lower level of G l u may be insufficient to induce " L T D - l i k e " effects (mechanism #2). As a consequence, the G l u and the antagonist (MPPG) competitive interactions at the presynaptic m G l u R may thus account for the observed blockade of the delayed suppression i n D A efflux. A diagram of this proposed mechanism is shown i n Fig. 17. General conclusion. In summary, the present study confirmed the hypothesis that 2 H z stimulation of V s u b / C A l wou ld result i n a rapid and prolonged suppression i n D A efflux i n the N A c . This study demonstrated that mGluRs , presumably located on presynaptic glutamatergic terminals, may be involved in the regulation of basal D A release at dopaminergic terminals i n the N A c via modulat ion of G l u release by an autoreceptor mGluR-mediated mechanism. Secondly, the prolonged suppressive effect on D A efflux may be related to an LTD-l ike phenomenon. Moreover, this late-phase suppression i n basal D A release induced by 2 H z stimulation of V s u b / C A l can be antagonized by selective blockade of group 2 and 3 mGluRs i n the N A c . Given the complexity i n the differential distribution of m G l u R subtypes i n the N A c and that only the effects of two specific mGluRs antagonists were examined, this thesis can not provide a definitive conclusion as to the subtypes of mGluRs that are responsible for the mechanism underlying the init ial and long lasting suppressive effects of V s u b / C A l on extracellular D A levels i n the N A c . Future studies are needed to examine the potential mechanisms underlying these Glu-induced actions on D A levels i n the N A c , as wel l as to fully characterize the role and identity of individual subtypes of mGluRs. 52 2 Hz stimulation + MPPG Fig. 17 The proposed diagram invo lv ing the effects of M P P G on the suppression of D A efflux induced by 2 H z stimulation. 2 H z s t imula t ion causes the release of G l u over the basal levels (1) and as a consequence activate group 2 and 3 mGluRs , which are partially blocked by M P P G . Less G l u (2) release, as a result of presynaptic autoreceptor activation, then cause less D A release from postsynaptic D A terminals. The amount of G l u release (2) is assumed to be insufficient to induce LTD-l ike effects, therefore the levels of D A i n the N A c return to prestimulated values. 53 R E F E R E N C E S Attarian S. and Amal r ic M . (1997) Microinfusion of the metabotropic glutamate receptor agonist lS,3R-l-aminocyclopentane-l,3-dicarboxylic acid into the nucleus accumbens induces dopamine-dependent locomotor activation i n the rat. Eur. J. Neurosci., 9, 809-816. Aramor i I., and Nakanishi S. (1992) Signal transduction and pharmacological characteristics of a metabotropic glutamate receptor, m G l u R l , i n transfected C H O cells. Neuron, 8, 757-765. Bedingfield J.S., Jane D.E., Kemp M . C , Toms N.J . , and Roberts P.T. (1996) N o v e l potent selective phenylglycine antagonists of metabotropic glutamate receptors. Eur. }. Neurosci., 309, 71-78. Benveniste H . and Huttemeier, C. (1990) Microdialysis- theory and application. Prog. Neurobiol, 35,195-215. Blaha C D . , Yang C.R., Floresco S.B., Barr A . M . , and Phil l ips A . G . (1997) Stimulation of the ventral subiculum of the hippocampus evokes glutamate receptor-mediated changes i n dopamine efflux i n the rat nucleus accumbens. Eur. }. Neurosci., 9, 902-911. Bond A . , and Lodge D. (1995) Pharmacology of metabotropic glutamate receptor-mediated enhancement of responses to excitatory and inhibitory amino acids on rat spinal neurons in vivo. Neuropharmacol, 34, 1015-1023. Bradley S.R., Levey A.I . , Hersch S .M. , and Con P.J. (1996) Immunocytochemical localization of group III metabotropic glutamate receptors i n the hippocampus wi th subtype-specific antibodies. /. Neurosci., 16, 2044-2056. 54 Brudzynsky S .M. , W u M . , and Mogenson G.J. (1993) Decrease i n rat locomotor activity as a result of changes in synaptic transmission to neurons wi th in the mesencephalic locomotor region. Can. f. Physiol. Pharmacol, 71, 394-406. Bushell T.J., Jane D.E. , Tse H - W . , Watkins J.C., Garthwaite J., and Collingridge G.L. (1996) Pharmacological antagonism of the action of group II and III m G l u R agonists i n the lateral perforant path of the rat hippocampal slices. Brit. J. Pharmacol, 117, 1475-1462. C o n n P.J., and P in J-P. (1997) Pharmacology and functions of metabotropic glutamate receptors. Annu. Rev. Pharmacol. Toxicol, 37, 205-237. Cozz i A . , Attucci S., Peruginelli F., Mar inozz i M . , Luneia R., Pellicciari R., and M o r o n i R. (1997) Type 2 metabotropic glutamate (mGlu) receptors tonically inhibit transmitter release in rat caudate nucleus: In vivo studies wi th (2S,1'S, 2'S,3'R)-2-(2'-carboxy-3'-phynylcyclopropyl) glycine,, a new potent and selective antagonist. Eur. J. Neurosci, 9, 1350-1355. Donzanti B .A . and Uretsky N.J . (1983) Effects of excitatory amino acids o n locomotor activity after bilateral microinjection into the rat nucleus accumbens: possible dependence on dopaminergic mechanisms. NeuropharmacoL, 22(8), 971-981. Dreher J.K. and Jackson D . M . (1989) Role of D l and D2 dopamine receptors i n mediating locomotor activity elicited from the nucleus accumbens of rats. Brain Res., 487, 267-277. Dube G.R. and Marshall K . C . (1997) Modula t ion of excitatory synaptic transmission i n locus coeruleus by multiple presynaptic metabotropic glutamate receptors. Neurosci., 80(2), 511-521. Fibiger H . C . and Phil l ips A . G . (1988) Mesocorticolimbic dopamine systems and reward. Ann. NY Acad. Sci., 537, 206-215. 55 Fiorino D.F., Coury A . , Fibiger H.C. , and Phil l ips A . G . (1993) Electrical s t imula t ion of reward sites i n the ventral tegmental area increases dopamine transmission i n the nucleus accumbens of the rat. Behav. Brain Res., 55, 131-141. Gilbert D.B., Mi l l a r J., and Cooper S.T. (1995) The putative dopamine D3 agonist,7-O H - D P A T , reduces dopamine release in the nucleus accumbens and electrical self stimulation to the ventral tegmentum. Brain Res., 681, 1-7. Go ld L . H . , Swardlow N.R., and Koop G.F. (1988) The role of mesolimbic dopamine i n conditioned locomotion produced by amphetamine. Behav. Neurosci., 102(4), 544-552. Groenewegen H.J. , Vermeulen-Van der Zee E. , Kortschot A . , and Witter M . P . (1987) Organization of the projections from the subiculum to the ventral striatum i n the rat. A study using anterograde transport of Phaseolus vulgalis leucoagglutinin. Neurosci., 23(1), 103-120. Hami l ton M . H . , Belleroche J.S., Gardiner I .M. , and Herberg L.J. (1986) Stimulatory effect of N-methy l aspartate on locomotor activity and transmitter release from rat nucleus accumbens. Pharmacol. Biochem. & Behav., 25, 943-948. Huang L.Q. , Rowan M.J . , and A n w y l R. (1997) m G l u R II agonist inhibi t ion of LTP induction, and m G l u R II antagonist inhibi t ion of L T D induction, i n the dentate gyrus in vitro. Neuroreport, 8, 687-693. Imperato A . , Honore T., and Jensen L . H . (1990a) Dopamine release i n the nucleus caudatus and i n the nucleus accumbens i n under glutamatergic control through n o n - N M D A receptors: a study i n freely-moving rats. Brain Res., 530, 223-228. 56 Imperato A . , Scrocco M . G . , Bacchi S., and Angelucci L . (1990b) N M D A receptors and i n v ivo dopamine release in the nucleus accumbens and caudatus. Eur. }. Pharmacol, 187, 555-556. Jackson D . M . , Ande N . , and Dahlstrom A . (1975) A functional effect of dopamine i n the nucleus accumbens and in some other dopamine-rich parts of the rat brain. Pschopharmacologia (Berl), 45, 139-149. Jane D.E., Pittaway K. , Sunter D . C , Thomas N . K . , and Watk in J.C. (1995) N e w phenylglycine derivative wi th potent and selective antagonist activity at presynaptic glutamate receptors i n neonatal rat spinal cord. Neuropharmacology, 34 851-856. Johnson L.R. , A y l w a r d R . L . M . , Hussain Z. , and Totterdell S. (1994) Input from the amygdala to the rat nucleus accumbens: its relationship wi th tyrosine hydroxylase immunoreactivity and identified neurons. Neuroscience, 61, 851-865. Jongen-Reio A . L . , Voorn P., and Groenewegen H.J. (1994) Immunohis tochemical characterization of the shell and core territories of the nucleus accumbens i n the rat. Eur. J. Neurosci. 6,1255-1264. Kamiya H . , Shinozaki H . , and Yamamoto C. (1996) Act ivat ion of metabotropic glutamate receptor type 2/3 suppresses transmission at rat hippocampal mossy fibre synapses. J. Physiol., 493,447-455. Kelley A . E . and Throne L .C . (1992) N M D A receptors mediate the behavioral effects of amphetamine infused into the nucleus accumbens. Brain Res. Bull, 20, 247-254. Kinoshita A . , Ohishi H . , Namura S., Shigemoto R., Nakanishi S. (1996) Presynaptic localization of a metabotropic glutamate receptor, mGluR4a, i n the cerebellar 57 cortex: A light and electron microscope study i n the rat. Neurosci. Lett., 207, 199-202. Kobayashi K . , Manabe T., and Takahashi T. (1996) Presynaptic long-term depression at the hippocampal mossy fiber-CA3 synapse. Science, 273, 648-650. Koob G.F. and Swerdlow N . R . (1988) The functional output of the mesolimbic dopamine system. Ann. NY Acad. Set, 537, 216-227. Koob G.F. (1992) Drugs of abuse: anatomy, pharmacology and function of reward pathways. Trends Pharmac. Set, 13,177-184. Lovinger D . M . , Tyler E., Fidler S., and Merri t A . (1993) Properties of a synaptic metabotropic glutamate receptor i n rat neostriatal slices. / . Neurophysiol., 69, 1236-1244. Lovinger D . M . and M c C o o l B .A. (1995) Metabotropic glutamate receptor-mediated presynaptic depression at corticostriatal synapses involves mGluR2 or 3. / . Neurophysiol, 73,1076-1083. Manahan-Vaughan D. (1997) Group 1 and 2 metabotropic glutamate receptors play differential roles i n hippocampal long-term depression and long term potentiation i n freely moving rats. /. Neurosci., 17, 3303-3311. Manzoni O., Michel J-P., and Bockaert J. (1997) Metabotropic glutamate receptors i n the rat nucleus accumbens. Eur. J. Neurosci., 9, 1514-1523. Mar t in L.J., Blackstone C.D., Huganir R.L. , and Price D.L. (1992) Cellular localization of a metabotropic glutamate receptor i n rat brain. Neuron, 9, 259-270. Mogenson G.J., Jone, D.L., and Y i m C.Y. (1980) From motivat ion to action: functional interface between the limbic system and motor system. Prog. Neurobiol, 14, 69-97. 58 Mogenson, G.J., Yang, C.R., and Y i m , C.Y.(1988) Influence of dopamine on l imbic inputs to the nucleus accumbens. Ann. NY Acad. Sci., 537, 86-100. Mogenson G.J., Brudzynski S .M. , W u M . , Yang C.R., and Y i m C.Y. (1993) F r o m motivat ion to action: a review of dopaminergic regulation of limbic—> nucleus accumbens—> ventral pallidum—>pedunculopontine nucleus circuitries involved i n limbic-motor integration. In: Limbic Motor Circuits and Neuropsychiatry, (Kalivas, P .W. and Barnes, C D . , Eds.), pp 193-236. Boca Raton, Florida: C R C Press. Nek i A . , Ohishi H . , Kaneko T., Shigemoto R., Nakanishi S., and M i z u n o N . (1996) Pre- and postsynaptic localization of a metabotropic glutamate receptor, mGluR2 , i n the rat brain: an immunohistochemical study wi th a monoclonal antibody. Neurosci. Lett. 202, 197-200. Nestler E.J. (1993) Molecular mechanisms of drug addiction i n the mesolimbic dopamine pathway. Sent, in Neurosci., 5, 369-376. Nakanishi S. (1994) Metabotropic glutamate receptors: synaptic transmission, modulation, and plasticity. Neuron, 13,1031-1037. Ohno M . and Watanabe S. (1995) Persistent increase i n dopamine release fo l lowing activation of metabotropic glutamate receptors i n the rat nucleus accumbens. Neurosci. Lett., 200,113-116. O'Mara S .M. , Rowan M.J. , and A n w y l R. (1995) Metabotropic glutamate receptor-induced homosynaptic long-term depression and depotentiation in the dentate gyrus of the rat hippocampus in vitro. Neuropharmacol, 34, 983-989. Overstreet L.S., Pasternak J.F., Colley P.A. , Slater N.T. , and Trommer B.L. (1997) Metabotropic glutamate receptor mediated long-term depression i n developing hippocampus. Neuropharmacol., 36, 831-844. 59 Paxinos G . and Watson, C. (1986) The rat brain in stereotaxic coordinates, 2nd Ed., N e w York: Academic Press. Pennartz C . M . A . , Ameerun R.F., Groenewegen H.J., and Lopes Da Silva F . H . (1993) Synaptic plasticity in an in vitro slice preparation of the rat nucleus accumbens. Eur. f. Neurosci., 5,107-117. Pennartz C . M . A . , Groenewegen H.J., and Lopes Da Silva F . H . (1994) The nucleus accumbens as a complex of functionally distinct neuronal ensembles: an integration of behavioral, electrophysiology and anatomical data. Prog. Neurobiol., 42, 719-761. Petralia R.S., Wang Y . X . , Niedzie lshi A.S . , and Wenthold R.J. (1996) The metabotropic glutamate receptors, mGluR2 and mGluR3 , show unique postsynaptic, presynaptic and glial localizations. Neurosci. 71, 949-976. Phill ips A . G . , Blaha C.D. , and Fibiger H . C . (1989) Neurochemical correlates of brain-stimulation reward measured by ex vivo and in vivo analyses. Neurosci. Biobehav. Rev., 13, 99-104. P i n J-P. and Bockaert J. (1995) Get receptive to metabotropic glutamate receptors. Cur. Op in. in Neurobiol., 5, 342-349. P i n J.P. and Duvo i s in R. (1995) Review: Neurotransmitter receptors I. The metabotropic glutamate receptors: structure and functions. Neuropharmacol, 34,1-26. Pisani A . , Calabresi P., Centonze D. , and Bernardi G. (1997a) Act ivat ion of group III metabotropic glutamate receptors depresses glutamatergic transmission at corticostriatal synapse. Neuropharmacology, 36, 845-851. Pisani A . , Calabresi P., Centonze D., and Bernardi G. (1997b) Enhancement of N M D A responses by group I metabotropic glutamate receptor activation i n striatal neurones. Brit. }. Pharmacol, 120, 1007-1014. 60 Pulvirent i L . , Swerdlow N.R. , and Koob G.F. (1991) Nucleus accumbens N M D A antagonist decreases locomotor activity produced by cocaine, heroin or accumbens dopamine, but not caffeine. Pharmacol. Biochem. & Behav., 40, 841-845. Robinson T.G. and Bert P . M . (1988) Excitant amino acid projections from rat amygdala and thalamus to nucleus accumbens. Brain Res. Bull, 20, 467-471. Sasa M . , Hara M . , and Takaori S. (1991) Dopamine D - l receptor-mediated inh ib i t i on of nucleus accumbens neurons from the ventral tegmental area. Prog. Neuro-psychopharmacol. & Biol. Psychiat., 15, 119-128. Scanziani M . , Salin P .A. , Vogt K .E . , Malenka R.C. , and N i c o l l R . A . (1997) Use-dependent increases i n glutamate concentration activate presynaptic metabotropic glutamate receptors. Nature, 385, 630-633. Schaffhauser H . , Jakob-Rotne R., and Mute l V . (1997) Pharmacological characterization of metabotropic glutamate receptors l inked to the inh ib i t ion of adenylate cyclase activity i n rat striatal slices. Neuropharmacol, 36, 933-940. Sharp T., Zetterstrom T., and Ungerstedt U . (1986) A n i n v ivo study of dopamine release and metabolism i n rat brain regions using intracerebral dialysis. / . Neurochem., 47, 113-122. Selig D.K. , Lee H - K . , Bear M.F . , and Malenka R.C. (1995) Reexamination of the effects of M C P G on hippocampal LTP, L T D , and depotentiation. / . Neurophysiol, 74,1075-1082. Sekiyama N . , Hayashi Y . , Nakanishi S., Jane D.E. , Tse H - W . , Birse E.F., and Watk ins J.C. (1996) Structure-activity relationships of new agonists and antagonists of different metabotropic glutamate receptor subtypes. Brit. J. Pharmacol, 117, 1493-1503. 61 Sesack S.R., and Pickel V . M . (1990) In the rat medial accumbens, hippocampal and catecholaminergic terminals converge on spiny neurons and are i n apposition to each other. Brain Res. 527, 266-278. Shigemoto R., Nomura S., Ohishi H . , Sukihara H . , Nakanishi S., and M i z u n o N . (1993) Immunohistochemical localization of a metabotropic glutamate receptor, mGluR5, i n the rat brain. Neurosci. Lett., 163, 53-57. Shigemoto R., Kinoshi ta A . , Wada E., Nomura S., Ohishi H . , Takada M . , Flor P.J., N e k i A . , Abe T., Nakanishi S., and M i z u n o N ; (1997) Differential presynaptic localization of metabotropic glutamate receptor subtypes i n the rat hippocampus. /. Neurosci., 17, 7503-7522. Shiraishi M . , Kamiyama Y. , Huttemeier P.C. , and Benveniste H . (1997) Extracellular glutamate and dopamine measured by microdialysis i n the rat striatum during blockade of synaptic transmission i n anesthetized and awake rats. Brain Res., 759,221-227. Shreve P.E. and Uretsky N.J . (1988) Role of quisqualic: acid receptors i n the hypermotility response produced by the injection of A M P A into the nucleus accumbens. Pharmacol. Biochem. & Behav., 30, 379-384. Smith J. A . , M o Q., Guo H . , Kunko P . M . , and Robinson S.E. (1995) Cocaine increases extraneuronal levels of aspartate and glutamate i n the nucleus accumbens. Brain Res., 683, 264-269. Sokoloff P., and Scwartz J. (1995) N o v e l dopamine receptors half a decade later. Trends Pharmac. Set, 16, 270-274. Steketee J.D., Sorg B.A. , and Kalivas P.W. (1992) The role of the nucleus accumbens i n sensitization to drugs abuse. Prog. Neuro-psychopkarmacol. & Biol. Psychiat., 16,237-246. 62 Svensson L . and Ahlenius S. (1983) Suppression of exploratory locomotor activity by the local application of dopamine or /-noradrenaline to the nucleus accumbens of the rat. Pharmacol. Biochem. & Behav., 19, 693-699. Svensson L . , Carlsson M . L . , and Carlsson A . (1992) Interaction between glutamatergic and dopaminergic tone i n the nucleus accumbens of mice: evidence for a dual glutamatergic function wi th respect to psychomotor control. /. Neural. Transm., 88, 235-40. Svensson L. , Carlsson M . L . , and Carlsson A . (1994a) Glutamatergic neurons projecting to the nucleus accumbens can affect motor functions i n opposite directions depending on the dopaminergic tone. Prog. Neuro-Psychopharmacol. & Biol. Psychiat., 18, 1203-1218. Svensson L . , Zhang J., Johannessen K . , and Engel J. A . (1994b) Effect of local infusion of glutamate analogues into the nucleus accumbens of rats: an electrochemical and behavioral study. Brain Res., 643, 155-161. Svensson L . , Carlsson M . L . , and Carlsson A . (1995) Interactions between glutamate and dopamine i n the ventral striatum: evidence for dual glutamatergic function wi th respect to motor control. In: Age-related dopamine-dependent disorders (Segawa, M . and Nomura , Y . , Eds.) Monogr. Neural . Sci. Basel, Karger., 14,160-167. Taber M . T . and Fibiger H . C . (1995) Electrical stimulation of the prefrontal cortex increases dopamine release i n the nucleus accumbens of the rat: modula t ion by metabotropic glutamate receptors. /. Neurosci., 15, 3896-3904. Tanabe Y. , M a s u M . , Ishii T., Shigemoto R., and Nakanishi S. (1992) A family of metabotropic glutamate receptors. Neuron, 8, 169-179. 63 Testa C M . , Standaert D.G. , Yong A . B . , and Penney J.R (1994) Metabotropic glutamate receptor m R N A expression i n the basal ganglia of the rat. / . Neurosci. 14,3005-3018. Tombaugh G . C and Somjen G . C (1996) Effects of extracellular p H on voltage-gated N a + , K + , and C a 2 + currents i n isolated rat C A 1 neurons. /. Physiol, 493, 719-732. Vignes M . , Clarke V.R.J . , Davies C . H . , Chambers A . , Jane D.E., Watkins J .C , and Collingridge G.L. (1995) Pharmacological evidence for an involvement of group II and group III mGluRs i n the presynaptic regulation of excitatory synaptic response i n the C A 1 region of rat hippocampal slices. Neuropharmacology, 34, 973-982. Wolter ink G. , V a n Zanten E., Kamsteeg H . , Radhakishun FS., and V a n Ree J M . (1990) Functional recovery after destruction of dopamine systems in the nucleus accumbens of rats. I. Behavioral and biochemical studies. Brain Res., 507,92-100. Wong L.S., Eshel G. , Dreher J., Ong J., and Jackson D . M . (1991) Role of dopamine and G A B A i n the control of motor activity elicited from the rat nucleus accumbens. Pharmacol. Biochem. & Behav., 38, 829-835. W u M . , Brudzynski S .M. , and Mogenson G.J. (1992) Functional interaction of dopamine and glutamate i n the nucleus accumbens i n the regulation of locomotion. Can. J. Physiol. Pharmacol, 71, 407-413. W u M . and Brudzyski S .M. (1995) Mesolimbic dopamine terminals and locomotor activity induced from the subiculum. Neuroreport, 6, 1601-1604. Yang C R . and Mogenson, G.J. (1984) Electrophysiological responses of neurones i n the nucleus accumbens to hippocampal stimulation and attenuation of the excitatory responses by the mesolimbic dopaminergic system. Brain Res., 324, 69-84. 64 Yang C R . and Mogenson G.J. (1985) A n electrophysiological study of the neural projections from the hippocampus to the ventral pa l l idum and the subpallidal areas by way of the nucleus accumbens. Neuroscience, 15, 1015-1024. Yang C R . and Mogenson G.J. (1986) Dopamine enhances terminal excitability of hippocampal-accumbens neurons via D2 receptor: Role of dopamine i n presynaptic inhibition. / . Neurosci., 6, 2470-2478. Yang C R . and Mogenson G.J. (1987) Hippocampal signal transmission to the pedunculopontine nucleus and its regulation by dopamine D2 receptors i n the nucleus accumbens: an electrophysiological and behavioral study. Neuroscience, 23,1041-1055. Y i m C Y . and Mogenson G.J. (1982) Response of nucleus accumbens neurons to amygdala stimulation and its modulation and its modification by dopamine. Brain Res., 239, 401-415. Y i m C Y . and Mogenson G.J. (1988) Neuromodulatory action of dopamine i n the nucleus accumbens: A n in vivo intracellular study. Niiuroscience, 26,403-415. Yokoi M . , Kobayashi K. , Manabe T., Takahashi T., Sakaguchi I., Katsuura G., Shigemoto R., Ohishi H . , Nomura S., Nakamura K . , Kakao K . , Katsuki M . , and Nakanishi S. (1996) Impairment of hippocampal mossy fiber L T D i n mice lacking mGluR2. Science, 273, 645-647. Youngren K . D . , Daly D .A. , and Moghaddam B. (1993) Distinct actions of endogenous excitatory amino acids on the outflow of dopamine i n the nucleus accumbens. / . Phar. Exp. Ther., 264, 289-293. 

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