Dopamine D1 receptors and group I metabotropic glutamate receptors contribute to the induction of long-term potentiation in the nucleus accumbens

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Neuropharmacology 54 (2008) 837e844 www.elsevier.com/locate/neuropharm

Dopamine D1 receptors and group I metabotropic glutamate receptors contribute to the induction of long-term potentiation in the nucleus accumbens Sietske M. Schotanus, Karima Chergui* Department of Physiology and Pharmacology, Section of Molecular Neurophysiology, The Karolinska Institute, Von Eulers va¨g 8, 171 77 Stockholm, Sweden Received 8 October 2007; received in revised form 15 December 2007; accepted 31 December 2007

Abstract Long-term changes in the efficacy of glutamatergic synaptic transmission in the striatal complex are proposed to underlie motor learning and neuroadaptations leading to addiction. Dopamine and glutamate play key roles in the induction of long-term potentiation (LTP) and long-term depression (LTD) in the dorsal striatum, but their contribution to synaptic plasticity in the ventral striatum (nucleus accumbens, NAc) has been less extensively studied. We have examined the role of dopamine, glutamate and GABA in the induction of LTP in mouse brain slices containing the NAc. High-frequency stimulation of glutamatergic inputs elicited LTP of field excitatory postsynaptic potentials/population spikes (fEPSP/ PSs) in the core region of the NAc. GABA did not seem to participate in LTP induction because LTP was not altered in the presence of either a GABAA- (bicuculline) or a GABAB- (CGP 55845) receptor antagonist. However, the dopamine D1 receptor antagonist SCH 23390, but not the dopamine D2 receptor antagonist sulpiride, impaired LTP. The dopamine reuptake blocker nomifensine also inhibited LTP induction. We found that group I metabotropic glutamate receptors (mGluRs) contribute to LTP induction because the mGluR1 antagonist LY 367385, or the mGluR5 antagonist MPEP, blocked LTP induction. Furthermore, the glutamate reuptake blocker DL-TBOA also impaired LTP. The present results demonstrate that dopamine and glutamate play critical roles in the mechanisms of induction of LTP in the NAc through the activation of dopamine D1 receptors and group I mGluRs. However, LTP is negatively regulated when endogenous levels of dopamine or glutamate are elevated. Ó 2008 Elsevier Ltd. All rights reserved. Keywords: Long-term potentiation; Nucleus accumbens; Field excitatory postsynaptic potential; Dopamine receptors; Metabotropic glutamate receptors; Mouse

1. Introduction The striatal complex is an important component of the basal ganglia, a group of several brain nuclei that controls numerous psychomotor behaviors. Based on neuroanatomical and behavioral studies, the striatal complex has been divided into the dorsal striatum (caudate-putamen), and the ventral striatum (nucleus accumbens, NAc). A dorsolateraleventromedial functional organization of the striatal complex has also been described. Although basic neurophysiological properties and neurochemical features of the medium spiny neurons that

* Corresponding author. Tel.: þ46 8 524 86882. E-mail address: [email protected] (K. Chergui). 0028-3908/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2007.12.012

compose the striatal complex are similar in the dorsal striatum and in the NAc, these two striatal subdivisions serve different functions. The dorsal striatum serves motor functions and habit learning whereas motivation, reward and attention are ascribed to the NAc (Gerfen, 1992; Nicola et al., 2000; Lovinger et al., 2003; Voorn et al., 2004). These functional differences may be due to differences in extrinsic connections because, for example, excitatory inputs to the NAc arise from limbic structures such as the hippocampus, amygdala and prefrontal cortex whereas the dorsal striatum receives glutamatergic inputs from the cortex and thalamus (McGeorge and Faull, 1989; Berendse and Groenewegen, 1990; O’Donnell and Grace, 1995). Long-lasting activity-dependent changes in glutamatergic synaptic efficacy constitute cellular candidates for information

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storage in behavioral learning (Malenka and Bear, 2004). In the striatal complex these changes are believed to underlie motor learning, habit formation, and neuroadaptations leading to addiction (Calabresi et al., 2000a; Lovinger et al., 2003; Wolf et al., 2004; Hyman et al., 2006). Long-term potentiation (LTP) and long -term depression (LTD) of glutamatergic synaptic transmission have both been described in the dorsal striatum and in the NAc, in in vitro and in vivo preparations (Calabresi et al., 1992, 2000a; Boeijinga et al., 1993; Pennartz et al., 1993; Charpier and Deniau, 1997). Interestingly, the features of LTP and LTD and the experimental protocols used for their induction differ between these two striatal subdivisions. High-frequency stimulation (HFS) of glutamatergic inputs is often utilized to induce LTP in various brain regions (Malenka and Bear, 2004). In cortico-striatal brain slices, HFS induces an N-methyl-D-aspartate (NMDA)-receptor dependent form of LTP in the NAc (Pennartz et al., 1993; Kombian and Malenka, 1994) (unpublished observations), and in the striatum (Calabresi et al., 2000a; Partridge et al., 2000; Kerr and Wickens, 2001; Fino et al., 2005; Kung et al., 2007; Chen et al., 2008), but it also evokes an NMDA-receptor independent form of LTD in the dorsal striatum (Calabresi et al., 1992; Choi and Lovinger, 1997). The cellular mechanisms involved in HFS-induced LTD and LTP in the dorsal striatum have been extensively described. They involve a complex interplay between postsynaptic membrane depolarization and presynaptic events (Calabresi et al., 2000a, 2007; Lovinger et al., 2003; Wang et al., 2006). Glutamate and two other major neurotransmitters, dopamine and GABA, are released in the striatum following HFS (Calabresi et al., 1995; Partridge et al., 2002). The critical contribution of dopamine D1 and D2 receptors, and of group I metabotropic glutamate receptors (mGluRs), in LTD and LTP in the dorsal striatum has been demonstrated through the use of pharmacological tools, transgenic animals, and by lesion studies (Calabresi et al., 2000a,b; Kerr and Wickens, 2001; Sung et al., 2001; Gubellini et al., 2004; Kung et al., 2007). Dopamine, glutamate and GABA are abundant in the NAc, but only little information is available for their contribution in LTP induction in this striatal subdivision. The aim of the present study was therefore to examine the possible contribution of these neurotransmitters in HFS-induced LTP in the NAc, with particular emphasis on GABAA and GABAB receptors, dopamine D1 and D2 receptors, and group I mGluRs. We have also examined the ability of endogenous dopamine and glutamate to modulate LTP induction in the NAc. A part of the present study has been previously published in the form of an abstract (Schotanus and Chergui, 2006). 2. Methods 2.1. Brain slice preparation Experiments using mice were approved by our local ethical committee (Stockholms norra djurfo¨rso¨ksetiska na¨mnd) and were performed as described previously (Schotanus et al., 2006). Male C57Bl6 mice aged 4e7 weeks were anesthetized by inhalation of fluothane and underwent cervical dislocation followed by decapitation under anesthesia. Their brains were rapidly removed

and coronal brain slices (400 mm thick), containing the NAc were prepared with a microslicer (VT 1000S, Leica Microsystem, Heppenheim, Germany). Slices were incubated, for at least 1 h, at 32  C in oxygenated (95% O2 þ 5% CO2) artificial cerebrospinal fluid (aCSF) containing (in mM): 126 NaCl, 2.5 KCl, 1.2 NaH2PO4, 1.3 MgCl2, 2.4 CaCl2, 10 glucose and 26 NaHCO3, pH 7.4. Slices were transferred to a recording chamber (Warner Instruments, Hamden, CT) mounted on an upright microscope (Olympus, Solna, Sweden) and were continuously perfused with oxygenated aCSF at 28  C.

2.2. Electrophysiology Extracellular field potentials were recorded using a glass micropipette filled with aCSF positioned on the slice surface. Signals were amplified 500 times via an Axopatch 200B amplifier (Axon Instruments, Foster City, CA), acquired at 10 kHz, filtered at 2 kHz and recorded on a Dell Computer using acquisition and data analysis software from Axon Instruments (pClamp9). Synaptic responses were evoked with a concentric bipolar stimulating electrode (FHC, Bowdoinham, ME) placed near the recording electrode on the surface of the slice. Single stimuli (0.1 ms duration) were applied every 15 s at an intensity yielding 50% maximal response as assessed by a stimulus/response curve established, for each slice, at the beginning of the recording session, by measuring the amplitude of the field potential evoked by increasing stimulation intensities. As described earlier (Schotanus et al., 2006), brief electrical stimulation of cortico-accumbens slices elicited a characteristic biphasic response, with two negative components. The first negative component, termed N1, is a non-synaptic compound action potential which is independent of glutamate release and activation of glutamate receptors. The second negative component, termed ‘‘field excitatory postsynaptic potential/population spike’’ (fEPSP/PS), reflects excitatory monosynaptic transmission in a population of neurons. This component is mediated by endogenous glutamate released upon electrical stimulation of glutamate-containing fibers present in the slice and is mediated by glutamate receptors of the AMPA type. Numerical values are expressed as mean  S.E.M., with n indicating the number of slices tested. Data are expressed as percent of the baseline measured for each slice during the 5 min preceding application of high frequency stimulation. Statistical significance of the results was assessed by using the twotailed Student t-test for unpaired observations. All chemicals and drugs were purchased from Sigma except for DLTBOA, LY 367385, MPEP hydrochloride and CGP 55845, which were purchased from Tocris Bioscience (Bristol, UK). Drugs were applied in known concentrations in the perfusion solution by switching a three-way tap.

3. Results 3.1. Induction of LTP in the nucleus accumbens We have examined the properties of glutamatergic synaptic plasticity in the core region of the NAc by measuring the amplitude of the fEPSP/PS evoked by brief electrical stimulation of glutamatergic fibers present in the brain slice. We recorded a 15-min baseline fEPSP/PS evoked by single pulse stimulation before high-frequency stimulation (HFS) was applied. Previous studies demonstrated that 100-Hz trains applied up to four times induced LTP in the NAc (Pennartz et al., 1993; Kombian and Malenka, 1994; d’Alcantara et al., 2001; Schramm et al., 2002; Li and Kauer, 2004). We tested this induction protocol in our experimental conditions. We found that the amplitude of the fEPSP/PS measured 1 h following HFS (three trains at 100 Hz, 1 s duration, 10 s inter-train intervals, same stimulation intensity as for baseline) was increased to 139.3  9.0% (n ¼ 15) from baseline fEPSP/PS amplitude (Fig. 1). This potentiation was significant ( p < 0.01), stable, and lasted for >1 h.

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We also tested the involvement of GABAB receptors by applying the GABAB receptor antagonist CGP 55845 (1 mM). This antagonist did not affect the amplitude of the fEPSP/PS when applied alone (percentage of baseline: 97.8  10.4%, n ¼ 3). Moreover, in the presence of CGP 55845, LTP was induced to a similar extent as in control experiments (percentage of baseline: 148.1  19.1%, n ¼ 9, not statistically different from control experiments without CGP 55845, p > 0.05, Fig. 2B). These results show that neither GABAA nor GABAB receptors contribute to LTP induction in the mouse NAc.

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Fig. 1. LTP induction in the nucleus accumbens. Glutamatergic synaptic transmission was examined in the NAc by recording extracellular field excitatory postsynaptic potentials/population spikes (fEPSP/PSs) evoked by electrical stimulation of glutamatergic fibers in mouse brain slices. Graph shows the mean (S.E.M.) amplitude of the fEPSP/PS, expressed as a percentage of baseline in n ¼ 15 slices, before and after high frequency stimulation (HFS), applied at the time indicated by the arrow. Lower panel shows records of fEPSP/PS measured in one slice, before (left trace) and after (right trace) HFS, at the time points indicated on the graph.

3.2. Involvement of GABA in the induction of LTP GABA is the main neurotransmitter utilized by medium spiny projection neurons in the striatum, and by several types of striatal interneurons. HFS produces the release of GABA in the striatum (Calabresi et al., 1995). However, electrophysiological studies that investigate the properties and regulation of glutamatergic synaptic transmission and plasticity frequently use GABAA receptor antagonists to pharmacologically isolate glutamate-mediated responses. We therefore examined whether this treatment affected the properties of LTP in the NAc, and whether GABA was involved in the induction of LTP. We used the GABAA receptor antagonist bicuculline methiodide (10 mM), which, when applied alone, increased the fEPSP/PS amplitude to 155.9  11.8% (n ¼ 4) from baseline values. This effect was previously reported in the NAc and was shown to be due to disinhibition of circuits intrinsic to the cortex (Buckby and Lacey, 2001). In slices pre-incubated with bicuculline, we found that LTP was induced following HFS, although the time course of the initial phase of LTP seemed to be altered by this treatment. The amplitude of the fEPSP/PS measured 1 h following HFS was increased to 120.1  11.3% (n ¼ 9) from baseline fEPSP/PS amplitude (Fig. 2A). This potentiation was not significantly different from that obtained in control conditions, without bicuculline ( p > 0.05). The lack of involvement of GABAA receptors in LTP is also supported by the absence of effect of bicuculline on cumulative membrane depolarization of single projection neurons induced by 100 Hz train stimulations (unpublished observations).

Dopamine in the dorsal striatum and NAc arises from midbrain dopaminergic neurons located in the substantia nigra and ventral tegmental area. Dopamine is massively released in the dorsal striatum following HFS (Calabresi et al., 1995; Partridge et al., 2002). In the dorsal striatum, induction of LTD by HFS is dependent upon the activation of the two major types of dopamine receptors, the D1 and the D2 receptor, whereas HFS-induced LTP requires the activation of D1 receptors only (Calabresi et al., 1992, 2000b; Kerr and Wickens, 2001). We first evaluated the contribution of dopamine D1 receptors in LTP induction in the NAc, by applying the dopamine D1 receptor antagonist SCH 23390 (10 mM). This antagonist did not affect the amplitude of the fEPSP/PS when applied alone (percentage of baseline: 100.4  14.7%, n ¼ 3). We found that HFS did not induce LTP in the presence of SCH 23390 (10 mM) in the perfusion solution (percentage of baseline: 102.1  5.2%, n ¼ 8, p < 0.01 compared to control experiments without SCH 23390, Fig. 3A). An earlier study found that a low concentration (1 mM) of SCH 23390 did not impair LTP in the NAc (Pennartz et al., 1993). We tested the possibility that this concentration was not sufficient to completely block dopamine D1 receptors, by reducing the concentration of SCH 23390. We found that LTP was induced in the presence of 1 mM SCH 23390 (percentage of baseline: 128.4  7.3%, n ¼ 5, p < 0.05 compared to baseline), which confirms the finding by Pennartz et al. (1993). These results demonstrate that 10 mMdbut not 1 mMdSCH 23390 blocks LTP in the NAc. We also examined the involvement of dopamine D2 receptors by applying the D2 receptor antagonist sulpiride (10 mM), which, when applied alone, did not affect the amplitude of the fEPSP/PS (percentage of baseline: 106.8  3.6%, n ¼ 3). In slices pre-incubated with sulpiride, HFS increased the amplitude of the fEPSP/PS to a comparable level as that seen in control experiments (percentage of baseline: 132.6  11.2%, n ¼ 9, p > 0.05 compared to control experiments without sulpiride, Fig. 3B). Because dopamine D1 and D2 receptors cooperate to induce LTD in the dorsal striatum (Centonze et al., 2001), we also tested whether antagonism of both D1 and D2 receptors would affect LTP induction in the NAc. In the presence of SCH 23390 (10 mM) and sulpiride (10 mM), the amplitude of the fEPSP/PS measured 1 h following HFS was not significantly different from baseline values

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Fig. 2. Effect of GABAA- and GABAB-selective antagonists on LTP induction in the nucleus accumbens. Graphs show the mean (S.E.M.) amplitude of the fEPSP/PS, expressed as a percentage of baseline, before and after HFS in the presence of the GABAA receptor antagonist bicuculline (10 mM, n ¼ 9, graph A) and in the presence of the GABAB receptor antagonist CGP 55845 (1 mM, n ¼ 9, graph B). Representative fEPSP/PS before and after HFS, at the time points indicated on the graphs, are shown above the graphs. Bicuculline and CGP 55845 were applied in the perfusion solution during the time indicated by the open bars. HFS was applied at the time indicated by the arrows.

(percentage of baseline: 103.3  5.6%, n ¼ 5, p < 0.01 compared to control experiments, Fig. 3C). We also examined the ability of endogenous dopamine to modulate LTP induction, as suggested in an earlier study (Li and Kauer, 2004). We applied the dopamine reuptake blocker nomifensine (10 mM), which delays the clearance from the extracellular space of dopamine released after each stimulation pulse during HFS. Nomifensine did not affect the amplitude of the fEPSP/PS (percentage of baseline: 109.5  2.1%, n ¼ 4) when applied alone, but it prevented the induction of LTP in the NAc (percentage of baseline: 107.2  6.1%, n ¼ 10, p < 0.01 compared to control experiments, Fig. 3D). Given that a dopamine D2 receptor agonist impairs LTP in the NAc (Li and Kauer, 2004), we tested the possibility that nomifensine could inhibit LTP through the activation of dopamine D2 receptors that counteracted the mechanisms of LTP induction. We found that LTP was still impaired when nomifensine and sulpiride were co-applied in the perfusion solution (percentage of baseline: 117.4  8.3%, n ¼ 5, p > 0.05 compared to control experiments, Fig. 3D). This result demonstrates that nomifensine does not inhibit LTP by promoting a D2 receptor-mediated inhibition of mechanisms of induction of LTP. Thus, LTP in the NAc is impaired when dopamine D1, but not D2, receptors are blocked, but also in the presence of excess dopamine.

of the fEPSP/PS measured 1 h following HFS was not significantly different from baseline values (percentage of baseline: 108.1  9.6%, n ¼ 7, p < 0.05 compared to control experiments, Fig. 4A). LTP was also impaired when slices were perfused with the mGluR5 antagonist MPEP (20 mM) (percentage of baseline: 105.2  7.2%, n ¼ 12, p < 0.01 compared to control experiments, Fig. 4B). When applied alone, these two antagonists did not affect the amplitude of the fEPSP/PS (percentage of baseline: 99.2  7.6, n ¼ 4 for LY 367385; 101.4  4.7%, n ¼ 6 for MPEP). Thus, LTP in the NAc requires the activation of mGluR1 and mGluR5. We also examined the possibility that excess glutamate could modulate LTP induction by applying the glutamate reuptake blocker DL-TBOA (20 mM) in the perfusion solution in order to further promote glutamate spillover outside the synaptic cleft. In the presence of DL-TBOA, HFS did not induce LTP (percentage of baseline: 107.1  5.7%, n ¼ 8, p < 0.01 compared to control experiments, Fig. 4C). We examined the effect of DL-TBOA on baseline fEPSP/PS amplitude to test the possibility that DL-TBOA occluded LTP. We found that, when applied alone, DL-TBOA increased the fEPSP/PS amplitude to a degree similar to that observed following HFS in control slices (percentage of baseline: 142.0  8.7%, n ¼ 4, Fig. 4C). 4. Discussion

3.4. Involvement of glutamate in the induction of LTP We tested the hypothesis that glutamate might contribute to LTP induction in the NAc by examining the involvement of group I mGluRs, i.e. mGluR1 and mGluR5. These receptors were shown to contribute to HFS-induced LTD and LTP in the dorsal striatum (Sung et al., 2001; Gubellini et al., 2004). When slices were perfused with the mGluR1 antagonist LY 367385 (100 mM), HFS did not induce LTP. The amplitude

The present study evaluated the role of three major neurotransmitters, GABA, dopamine and glutamate, in the induction of LTP in the NAc. We found that the activation of GABAA or GABAB receptors is not required for LTP induction, suggesting that GABA does not contribute to LTP in the NAc. However, our results show that activation of dopamine D1, but not D2 receptors, and of group I mGluRs, is required for HFS-induced LTP in the NAc. These observations clearly indicate

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Time (min) Fig. 3. Effect of D1- and D2-selective antagonists and of a dopamine reuptake blocker on LTP induction in the nucleus accumbens. Graphs show the mean (S.E.M.) amplitude of the fEPSP/PS, expressed as a percentage of baseline, before and after HFS in the presence of the D1 receptor antagonist SCH 23390 (10 mM, n ¼ 8, graph A); in the presence of the D2 receptor antagonist sulpiride (10 mM, n ¼ 9, graph B); in the presence of SCH 23390 (10 mM) and sulpiride (10 mM, n ¼ 5, graph C); in the presence of the dopamine reuptake blocker nomifensine (10 mM, n ¼ 10, graph D, open symbols); and in the presence of nomifensine (10 mM) and sulpiride (10 mM) (n ¼ 5, graph D, filled symbols). Representative fEPSP/PS before and after HFS, at the time points indicated on the graphs, are shown above the graphs. Drugs were applied in the perfusion solution during the time indicated by the open and filled bars. HFS was applied at the time indicated by the arrows.

that dopamine and glutamate play key roles in the induction of LTP in the NAc. Interestingly, we also found that dopamine and glutamate reuptake blockers impair LTP, suggesting that excess dopamine and glutamate negatively regulates LTP in the NAc. This study identifies mechanisms of induction and modulation of LTP in the ventral striatum and demonstrates similarities and differences between the properties of longterm changes in synaptic strength in the NAc and in the dorsal striatum. Additional experiments are however needed to identify the intracellular signaling cascades activated by dopamine and glutamate in LTP in the NAc. Further studies are also required to determine if these neurotransmitters interfere with mechanisms intrinsic to LTP or if they modulate membrane depolarization and NMDA receptor activity, as shown previously by Li and Kauer (2004).

4.1. Involvement of dopamine in LTP induction in the nucleus accumbens Dopamine plays a critical role in reward-directed behaviors and in motor function. Modulation by dopamine of plasticity at glutamatergic synapses in the NAc might promote rewardseeking behaviors and underlie neuroadaptations leading to addiction (Wolf et al., 2004; Hyman et al., 2006). Our results demonstrate a critical involvement of dopamine, acting through D1 but not D2 receptors, in LTP induction in the NAc. This requirement is identical to that observed in the dorsal striatum for HFS-induced LTP, but not for HFS-induced LTD, which depends upon both dopamine D1 and D2 receptors (Calabresi et al., 2000b; Kerr and Wickens, 2001). In particular, HFS-induced LTD in the dorsal striatum was recently

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Time (min) Fig. 4. Effect of mGluR1- and mGluR5-selective antagonists and of a glutamate reuptake blocker on LTP induction in the nucleus accumbens. Graphs show the mean (S.E.M.) amplitude of the fEPSP/PS, expressed as a percentage of baseline, before and after HFS in the presence of the mGluR1 receptor antagonist LY 367385 (100 mM, n ¼ 7, graph A); in the presence of the mGluR5 receptor antagonist MPEP (20 mM, n ¼ 12, graph B); and in the presence of the glutamate reuptake blocker DL-TBOA (20 mM, n ¼ 8, graph C). Inset in graph C shows the time course of the effect of DL-TBOA (20 mM, n ¼ 4) on the amplitude (mean  S.E.M.) of the fEPSP/PS, expressed as a percentage of baseline. Superimposed fEPSP/PS before and after HFS, at the time points indicated on the graphs, are shown in graphs A and B. Drugs were applied in the perfusion solution during the time indicated by the open bars. HFS was applied at the time indicated by the arrows.

shown to be indirectly mediated by dopamine D2 receptors located on cholinergic interneurons (Wang et al., 2006), a cellular mechanism that may therefore not be involved in HFS-induced LTP in the NAc given the lack of contribution of D2 receptors. The requirement of dopamine D1 receptors in LTP induction in the NAc is in agreement with previous studies in primary cultures prepared from NAc showing that D1 receptor stimulation leads to increased PKA-mediated phosphorylation and externalization onto the cell membrane of AMPA receptors, which should promote LTP (Wolf et al., 2004). The lack of involvement of dopamine receptors in LTP induction in the NAc observed in an earlier study (Pennartz et al., 1993) might be due to differences in the protocols used for LTP induction. However, an incomplete blockade of dopamine receptors with the low concentration of the antagonist used is a more likely explanation, because we found that a high, but not a low, concentration of a D1 receptor antagonist inhibits LTP. We also found that blockade of dopamine reuptake with nomifensine inhibits LTP induction. In the dorsal striatum, dopamine transport blockers such as nomifensine or cocaine do not affect the induction or expression of LTD (Partridge et al., 2002). Our results in the NAc are in accordance with the observations that LTP in the NAc is impaired by the psychomotor stimulant drug amphetamine, or by dopamine D1 or D2 receptor agonists (Li and Kauer, 2004). Our results suggest that the inhibitory effect of nomifensine on LTP is not due to D2 receptor-mediated activation of cellular mechanisms that counteract LTP; rather this effect is likely due to a decreased excitability of medium spiny neurons during HFS (Li and Kauer, 2004). Taken together, these results show that the effect of dopamine reuptake blockers, and of drugs of abuse, on synaptic plasticity in the striatal complex is restricted to the NAc, the striatal subdivision that plays a key role in drug addiction. Our findings also strengthen the idea that a strict spatial and temporal pattern of dopamine release is required for LTP induction. Indeed, as proposed earlier, induction of LTP might depend on spatially restricted activation of dopamine receptors near dopamine release sites, time-locked with HFS (Partridge et al., 2002). Taken together, these results further support the suggestion that phasic and tonic dopamine release have opposite effects on LTP (Arbuthnott et al., 2000; Centonze et al., 2001). Thus, phasic activation of dopamine receptors would promote LTP in the NAc while tonic release of dopamine, such as that obtained in the presence of a reuptake blocker, would impair LTP. 4.2. Involvement of glutamate in LTP induction in the nucleus accumbens In the dorsal striatum, both mGluR1 and mGluR5 receptors are localized at postsynaptic sites, in medium spiny neurons, and contribute to induction of LTD and LTP, because HFS-induced LTD and LTP are blocked in the presence of group I mGluR selective antagonists (Sung et al., 2001; Gubellini et al., 2004). We found that in the NAc, both mGluR1 and mGluR5 contribute to LTP induction. The mechanism

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involved may include regulation of intracellular Ca2þ levels and of NMDA receptor activity. Indeed, HFS-induced LTD in the dorsal striatum and HFS-induced LTP in the NAc are both dependent on an increase in intracellular concentration of Ca2þ, which may be achieved by activation of the group I mGluRs/PLC and IP3/DAG pathway by glutamate released during HFS (Calabresi et al., 1994; Kombian and Malenka, 1994; Gubellini et al., 2004). In addition, LTP in the NAc is dependent upon the activation of NMDA receptors and, in the dorsal striatum, postsynaptic mGluR1 and mGluR5 were shown to modulate the activity of synaptic and extrasynaptic NMDA receptors (Kombian and Malenka, 1994; Calabresi et al., 2000a; Pisani et al., 2001; Chergui et al., 2005; Schotanus and Chergui, 2006). Thus, the possibility exists that Group I mGluR antagonists interfere with cellular mechanisms that lead to the induction of LTP in the NAc. As for dopamine reuptake blockade, we found that a glutamate reuptake blocker, DL-TBOA, impaired LTP. The precise locus of action of glutamate overflow during HFS in the presence of DL-TBOA is however unclear. Our results indicate that this excess glutamate might occlude HFS-induced LTP in the NAc as suggested earlier for LTD in the dorsal striatum (Calabresi et al., 1999b). It is also probable that excess glutamate impairs LTP induction through the inhibition of dopamine release during HFS. Indeed, a previous study showed that glutamate reuptake blockers depress the evoked dopamine release in the dorsal striatum through the activation of mGluRs located on dopaminergic presynaptic terminals (Zhang and Sulzer, 2003). Thus, glutamate released during HFS contributes to the induction of LTP in the NAc via the activation of mGluR1 and mGluR5; however, excess glutamate inhibits induction of LTP. 4.3. Other neurotransmitters GABA is the main neurotransmitter used by medium spiny striatal neurons and by several classes of interneurons. Our observation that GABAA receptors do not contribute to LTP induction in the NAc is in accord with the lack of involvement of these receptors in HFS-induced LTD in the dorsal striatum (Calabresi et al., 1992). We additionally found that GABAB receptors do not contribute to LTP induction in the NAc, further supporting the lack of involvement of GABA in this form of LTP. The observation that the release of GABA in the striatum following HFS is lower and shorter lasting than that of glutamate (Calabresi et al., 1995) provides a probable explanation for the lack of involvement of this neurotransmitter in LTP in the NAc. Other neurotransmitters and retrograde messengers could potentially contribute to LTP induction in the NAc, as shown in the dorsal striatum for HFS-induced LTD and LTP. Among these neurotransmitters and molecules, acetylcholine (supplied by large-sized cholinergic interneurons) was shown to play significant roles in LTD and LTP in the dorsal striatum. Thus, a decreased activity of muscarinic M1 receptors following activation of dopamine D2 receptors located on cholinergic interneurons and subsequent decrease in acetylcholine

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release, contributes to the mechanisms of LTD (Wang et al., 2006). However, M1 receptors were also shown to contribute to HFS-induced LTP in the dorsal striatum (Calabresi et al., 1999a). Thus, the contribution of cholinergic interneurons to LTP in the NAc cannot be excluded. Endocannabinoids have been shown to mediate LTD induced by HFS in the dorsal striatum and by moderate frequency stimulation in the NAc (Robbe et al., 2002; Gerdeman and Lovinger, 2003). Nitric oxide (NO), which is released by striatal interneurons, is another molecule known to contribute to striatal LTD (Calabresi et al., 1999c, 2000b). We cannot exclude the involvement of endocannabinoids and of NO in HFS-induced LTP in the NAc; however, these molecules are most likely to play key roles in LTD rather than LTP in the NAc. Nonetheless, the contribution of other molecules and transmitters, such as adenosine, in LTP induction cannot be excluded given that inactivation of adenosine A2A receptors impairs LTP in the NAc (d’Alcantara et al., 2001). 5. Conclusions This study shows that dopamine and glutamate operate in conjunction to induce LTP in the NAc through the activation of dopamine D1 receptors and mGluR1 and mGluR5 receptors. Our findings also demonstrate that an excess of dopamine or of glutamate disrupts the mechanisms of LTP induction and suggest that functional differences between the dorsal and the ventral striatum could be attributable to specific features of synaptic plasticity in these two striatal subdivisions. Given that the NAc plays a prominent role in the early stages of drug use, the mechanisms involved in LTP that we describe in the present study, or the modulatory effect of dopamine and glutamate reuptake blockers on LTP, may contribute to the behavioral responses to drugs of abuse. Acknowledgment This work was supported by the Swedish Research Council (Vetenskapsra˚det K2006-33X-14517-04-3). References Arbuthnott, G.W., Ingham, C.A., Wickens, J.R., 2000. Dopamine and synaptic plasticity in the neostriatum. Journal of Anatomy 196, 587e596. Berendse, H.W., Groenewegen, H.J., 1990. Organization of the thalamostriatal projections in the rat, with special emphasis on the ventral striatum. Journal of Comparative Neurology 299, 187e228. Boeijinga, P.H., Mulder, A.B., Pennartz, C.M., Manshanden, I., Lopes da Silva, F.H., 1993. Responses of the nucleus accumbens following fornix/ fimbria stimulation in the rat. Identification and long-term potentiation of mono- and polysynaptic pathways. Neuroscience 53, 1049e1058. Buckby, L.E., Lacey, M.G., 2001. Epileptiform activity in the nucleus accumbens induced by GABA(A) receptor antagonists in rat forebrain slices is of cortical origin. Experimental Brain Research 141, 146e152. Calabresi, P., Maj, R., Pisani, A., Mercuri, N.B., Bernardi, G., 1992. Long-term synaptic depression in the striatumdphysiological and pharmacological characterization. Journal of Neuroscience 12, 4224e4233.

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