Abstract
Understanding how striatal neurons integrate glutamatergic and GABAergic inputs is essential for understanding the control of movement and the formation of striatal-based memories. Here we show that GABAergic synapses on striatal medium spiny neurons (MSNs) are more sensitive than glutamatergic synapses on the same cells to endocannabinoid (eCB) signaling, and that protocols that induce short-lasting cannabinoid 1 receptor (CB 1 R)-dependent depression at glutamatergic synapses are sufficient to induce long-term depression (LTD) at GABAergic synapses. We also show that the frequency and duration of glutamatergic input are strong determinants of the net effect of eCB signaling, and key factors in determining if LTD has a net disinhibitory or inhibitory action in striatum. Plastic changes in net output from striatal MSNs are thus a complex function of disinhibitory and inhibitory LTD combined with other forms of synaptic plasticity such as long-term potentiation at excitatory synapses.
Introduction
The striatum is the largest nucleus of the basal ganglia and has important roles in planning and execution of controlled movements (Graybiel et al., 1994; Balleine et al., 2007) . The majority of synapses (ϳ80%) are asymmetric glutamatergic synapses originating from the cortex (associative areas of the frontal and parietal lobes and motor and somatosensory cortex) and thalamus (lateral parafascicular nucleus and the central lateral nucleus) (Wilson, 2007) , while the majority of neurons in the striatum (Ͼ90%) are GABAergic medium spiny neurons (MSNs) (Tepper et al., 2007) .
Introduction
The endocannabinoids (eCBs) are key neuromodulatory agents involved in basal ganglia function (eCBs) (Hohmann and Herkenham, 2000; Hilário et al., 2007) . eCBs are released postsynaptically and activate presynaptic cannabinoid 1 receptors (CB 1 R) that depress transmission at both glutamatergic and GABAergic synapses in the basal ganglia (Gerdeman and Lovinger, 2001; Freiman et al., 2006; Narushima et al., 2006; Adermark and Lovinger, 2007a; Lovinger, 2007) . The density of CB 1 Rs is higher at GABAergic than at glutamatergic synapses in striatum (Uchigashima et al., 2007) , and our previous research has indicated that eCB signaling is more easily activated at GABAergic synapses (Adermark and Lovinger, 2007b) . Understanding how striatal neurons integrate information from their various synaptic inputs is essential for understanding the control of movement and the formation of striatal-based memories, and the goal of this study was to further evaluate the requirements for eCB signaling and induction of long-term depression (LTD) at glutamatergic and GABAergic synapses in the striatum.
Materials and Methods
Brain slice preparation. Coronal brain slices containing the striatum were prepared from p16 -20 rats as previously described (Adermark and Lovinger, 2007a) . Slices were allowed to equilibrate for at least 1 h in artificial CSF (aCSF) containing (in mM): 124 NaCl, 4.5 KCl, 2 CaCl 2 , 1 MgCl 2 , 26 NaHCO 3 , 1.2 NaH 2 PO 4 , and 10 D-glucose, continuously bubbled with a mixture of 95% O 2 /5% CO 2 gas.
Materials and Methods
Whole-cell voltage clamp recordings. Electrophysiological recordings were performed as previously described (Adermark and Lovinger, 2007a) . Internal solutions consisted of (in mM): 120 CsMeSO 3 , 5 NaCl, 10 TEA-Cl, 10 HEPES, 5 QX-314, 1.1 EGTA, 4 Mg-ATP, 0.3 Na-GTP, for experiments examining EPSCs, and 150 CsCl, 10 HEPES, 2 MgCl 2 , 0.3 Na-GTP, 3 Mg-ATP, 0.2 BAPTA, for experiments examining IPSCs. For IPSC measurements, 5 M 1,2,3,4-tetrahydro-6-nitro-2,3-dioxobenzo[f]quinoxaline-7-sulfonamide disodium salt (NBQX) and 50 M 5 D,L-2-amino-5 phosphonovaleric acid (AP-5) was added to the aCSF. For EPSC recordings AP-5 and 50 M picrotoxin were added to the aCSF. Currents were measured in conventional ruptured-patch whole-cell mode in medium spiny neurons (MSNs) voltage clamped at Ϫ50 mV, unless stated. In a subset of recordings MSNs were loaded with the eCB arachidonoyl ethanolamide (anandamide, AEA, 50 M) (Ronesi et al., 2004; Adermark and Lovinger, 2007b) . EPSC/IPSC data from AEAloaded cells are shown starting from the point when a stable baseline response was observed which typically occurred within 3 min after establishing the whole cell configuration (Adermark and Lovinger, 2007b) .
Materials and Methods
Unless otherwise stated, baseline synaptic currents were evoked by paired (50 ms interpulse interval) stimuli delivered every 20 s through a bipolar electrode placed at the border of the dorsolateral striatum and the overlying white matter. Stimulus parameters were adjusted to elicit baseline EPSC or IPSC amplitudes between 200 and 400 pA. Experiments were performed at 29Ϫ32°C, with the temperature not varying more than 1°C throughout a given experiment. The amplitude of EPSCs and IPSCs was measured using Clampfit 9.2 as previously described (Adermark and Lovinger, 2007b) . Experiments were discontinued if the series resistance varied by Ͼ20% or increased Ͼ30 M⍀.
Materials and Methods
Field potential recordings. Extracellular field recordings were conducted as previously described . Test stimuli were delivered at a frequency of 0.05 Hz through a bipolar twisted tungsten wire placed at the border of the dorsolateral striatum and the overlying white matter. Slices were kellyryan@example.org ml/min with regular aCSF, or aCSF containing picrotoxin (50 M), AP-5 (50 M), or AM251 (3 M). Experiments were performed at 30°C. The population spike amplitude was measured using Clampfit 9.2, as previously described .
Materials and Methods
Statistical analysis. The amplitude of the EPSC, IPSC, or PS, once a stable baseline was achieved, was compared with amplitudes 20 -25 min after stabilization of the baseline and presented as mean value Ϯ 95% confidence interval (CI), unless otherwise stated. Clampex 9.2 was used for data acquisition (Molecular Devices), and graphs were assembled in GraphPad Prism (GraphPad Software), and Photoshop. Time course figures are plotted as mean amplitude compared with baseline, with SEM, and two-tailed paired t test was used for statistical analysis.
Materials and Methods
Drugs. Chemicals were purchased from Sigma or Tocris. AEA was dissolved in ethanol to 25 mM and used at 50 M in the intracellular solution. NBQX was dissolved in H 2 O to 50 mM and used at 5 M, and APV and picrotoxin was dissolved in aCSF (50 M). The CB 1 receptor blocker AM251 and the CB 1 agonist WIN 55,212-2 were dissolved in DMSO to 20 and 10 mM respectively, stock solutions were diluted in aCSF containing bovine serum albumin (BSA) (0.5 g/L) and used at 3 M (AM251) and 1 M or 50 nM (WIN 55, . Cycloheximide was dissolved in aCSF to 80 M. Metabotropic glutamate receptor antagonists MPEP and CPCCOEt were dissolved in DMSO to 100 mM and used at 40 M (MPEP) and 80 M (CPCCOEt).
Results

Low frequency stimulus-induced LTD at GABAergic synapses
To evaluate eCB signaling and LTD in response to stimulation of intrinsic afferents, we applied a single stimulus train of 60 pulses delivered at 1 Hz at the border to the white matter overlying striatum. This protocol induced a robust depression of IPSC amplitude in MSNs voltage clamped at Ϫ50 mV (IPSC amplitude ϭ 39 Ϯ 11% of baseline, n ϭ 6, p Ͻ 0.001), while, as expected, only a small depression was seen at glutamatergic synapses (EPSC amplitude ϭ 87 Ϯ 8.5%, n ϭ 6, p Ͻ 0.01, IPSC vs EPSC p Ͻ 0.001) (Fig. 1a) , indicating that this protocol might induce long-lasting disinhibition (DLL) of striatal output. Inhibition of group I mGluRs with combined application of CPCCOEt (80 M) and MPEP (40 M) prevented stimulation-induced depression at GABAergic synapses suggesting that glutamate is vital for this form of depression (IPSC amplitude ϭ 87 Ϯ 8.7% of baseline, n ϭ 7, p Ͼ 0.05) (Fig. 1b) . Stimulation-induced depression at GABAergic synapses was abolished in slices treated with AM251 throughout the experiment (IPSC amplitude ϭ 98 Ϯ 10%, n ϭ 6, p Ͼ 0.05) (Fig. 1c) . However, AM251 treatment beginning 5 min after the end of 1 Hz stimulation and continuing for up to 60 min was unable to reverse established depression (IPSC amplitude after 40 -45 min of AM251 treatment ϭ 41 Ϯ 5.7%, n ϭ 5) (Fig.  1c) , suggesting that this stimulation protocol induces LTD at GABAergic synapses in the striatum. The depression in IPSC amplitude induced by a train of 60 pulses at 1 Hz was not significantly different if the stimulating electrode was placed in the dorsolateral striatum itself (IPSC amplitude (local stimulation) ϭ 53 Ϯ 13%, n ϭ 5, p Ͻ 0.01: local vs white matter stimulation, p Ͼ 0.05).
Low frequency stimulus-induced LTD at GABAergic synapses
GABAergic synapses are highly sensitive to CB 1 R activation Bath application of a low concentration of the CB 1 R agonist WIN 55,212-2 (50 nM) was unable to significantly affect postsynaptic currents at glutamatergic synapses (EPSC amplitude ϭ 87 Ϯ 10% of baseline, n ϭ 6, p Ͼ 0.05). The same treatment induced a robust depression of GABAergic IPSC amplitude (IPSC amplitude ϭ 51 Ϯ 21% of baseline, n ϭ 8, p Ͻ 0.01), suggesting that GABAergic synapses are more sensitive to CB 1 R activation (Fig. 2a) .
Low frequency stimulus-induced LTD at GABAergic synapses
Treatment with a higher concentration of the CB 1 R agonist WIN55,212-2 (1 M) induced a robust depression at both glutamatergic and GABAergic synapses (EPSC amplitude at t ϭ 15-20 min ϭ 62 Ϯ 18% of baseline, n ϭ 5, p Ͻ 0.01; IPSC amplitude ϭ 64 Ϯ 9.2% of baseline, n ϭ 12, p Ͻ 0.001), that was reversed by subsequent application of the CB 1 R antagonist AM251 (3 M) within 15 min of the onset of antagonist treatment at glutamatergic synapes (EPSC amplitude after 15-20 min AM251 treatment ϭ 87 Ϯ 10% of baseline, n ϭ 7, p Ͼ 0.05), while no recovery was detected at GABAergic synapses (IPSC amplitude after 15-20 min AM251 treatment ϭ 50 Ϯ 14% of baseline n ϭ 12, p Ͻ 0.001) (Fig. 2b) . To simulate a condition in which eCBs have been produced but are not yet released from the postsynaptic cell we loaded MSNs with the eCB arachidonoylethanolamide (anandamide or AEA) (50 M), which, when combined with paired pulse stimulation induces a robust depression at both excitatory and inhibitory synapses (Ronesi et al., 2004; Adermark and Lovinger, 2007b) . Similar to agonist-mediated CB 1 R activation, AEAinduced depression was reversed by subsequent AM251 treatment at glutamatergic synapses, but not at GABAergic synapses (EPSC amplitude at t ϭ 15-20 min ϭ 64 Ϯ 5.1% of baseline, n ϭ 5, EPSC amplitude at t ϭ 25-30 min ϭ 114 Ϯ 9.3% of baseline, n ϭ 6, p Ͼ 0.05; IPSC amplitude at t ϭ 15-20 min ϭ 62 Ϯ 11% of baseline, n ϭ 12, p Ͻ 0.001, IPSC amplitude at t ϭ 25-30 min ϭ 59 Ϯ 19% of baseline, n ϭ 12, p Ͻ 0.001) (Fig. 3a) . Furthermore, AEA loading combined with single pulse stimulation, which is insufficient to induce depression at glutamatergic synapses (Adermark and Lovinger, 2007b), induced depression at GABAergic synapses that was not reversed by subsequent CB 1 R antagonist treatment. This depression was not different in cells clamped at Ϫ50 mV compared with MSNs clamped at Ϫ70 mV (IPSC amplitude ϭ 47 Ϯ 13% of baseline, n ϭ 9, p Ͻ 0.001 (Hp ϭ Ϫ50 mV), and 40 Ϯ 8.5% of baseline, n ϭ 7, p Ͻ 0.001 (Hp ϭ Ϫ70 mV) (Fig. 3a) .
AEA-Induced LTD at GABAergic synapses is sensitive to protein synthesis inhibition
The finding that antagonist treatment cannot reverse established synaptic depression suggests that LTD has been induced under many of the experimental conditions mentioned above. To confirm this finding we pretreated slices with the protein translation inhibitor cycloheximide (80 M) that prevents LTD without disrupting CB 1 R-dependent short-term depression (Yin et al., 2006) . Extracellular perfusion of cycloheximide did not prevent depression of EPSC amplitude induced by postsynaptic AEA, indicating that protein synthesis is not required for eCB release or CB 1 R activation (EPSC amplitude at t ϭ 20 -25 min in cycloheximide treated slices ϭ 70 Ϯ 7.5%, n ϭ 7, p Ͻ 0.001; EPSC amplitude in control slices ϭ 65 Ϯ 8.9%, n ϭ 9, p Ͻ 0.01; cycloheximide treated vs aCSF, p Ͼ 0.5) (Fig. 3b) . In slices perfused with cycloheximide, AEAinduced depression was reversed by AM251 (IPSC amplitude after 15-20 min AM251 treatment ϭ 94 Ϯ 14%, n ϭ 11, p Ͼ 0.65), while no antagonist reversal was observed in the absence of cycloheximide (Fig. 3c) . These findings indicate that AEA-loading is sufficient to induce protein synthesis inhibitor-sensitive LTD at GABAergic synapses, which is not the case for glutamatergic synapses.
AEA-Induced LTD at GABAergic synapses is sensitive to protein synthesis inhibition
Previously published data suggest that induction of eCB-dependent LTD requires both CB 1 R signaling and another coincident presynaptic signal (Sjöström et al., 2003; Singla et al., 2007; Adermark et al., 2009) . It is thus possible that at GABAergic synapses that are rich in CB 1 R, LTD may be easily induced even with moderate stimulus trains, while at relatively CB 1 R-poor glutamatergic synapses both strong CB 1 R activation and afferent stimulation may be needed to induce LTD. When we combined AEA-loading with one train of 60 pulses delivered at 1 Hz we observed an inhibition of EPSC amplitude comparable to that observed with AEA loading alone. However, AM251 treatment after combined AEA loading and 60 s, 1 Hz stimulation did not reverse the depression in EPSC amplitude, indicating that AEA-loading combined with moderate afferent activation converts eCB-induced short-term de- pression to LTD (EPSC amplitude at t ϭ 30 -35 min ϭ 46 Ϯ 9.8%, n ϭ 5, p Ͻ 0.001) (Fig. 3d) .
Frequency dependent inversion of striatal output
Our findings indicate that eCB signaling and LTD are induced at different afferent stimulus frequencies at GABAergic synapses compared with glutamatergic synapses. Thus it is possible that eCB release produced by sustained low frequency synaptic activation produces a long-lasting increase in MSN output, while higher frequency activation produces a net decrease in output in the same set of cells. To test this hypothesis we examined stimulation-induced changes in population spike (PS) amplitude using field potential recording in the dorsolateral striatum. To establish the impact of GABAergic innervation on MSN output, we perfused slices with picrotoxin (50 M), which significantly enhanced PS amplitude (PS amplitude at t ϭ 30 -35 min ϭ 135 Ϯ 11% of baseline, n ϭ 16, p Ͻ 0.001) (Fig. 4a) , showing that GABAergic inhibition is important for the modulation of striatal output. PS amplitude was not significantly affected by 60 s, 1 Hz stimulation in slices perfused with picrotoxin ( p Ͼ 0.05) (PS amplitude at t ϭ 55-60 min ϭ 124 Ϯ 9.5% of baseline, n ϭ 16, p Ͻ 0.01) (Fig. 4a) .
Frequency dependent inversion of striatal output
In normal ACSF, neither 60 s, 1 Hz stimulation nor HFS induced a significant change in PS amplitude (PS amplitude at t ϭ 25-30 min ϭ 92 Ϯ 16% of baseline, n ϭ 12, p Ͼ 0.05; t ϭ 45-50 min ϭ 119 Ϯ 20% of baseline, n ϭ 11, p Ͼ 0.05). It is possible that this result reflects combined activation of long-term potentiation (LTP) (Calabresi et al., 1992) , and LTD. We thus examined effects of the two stimulation patterns in slices treated with AP-5, which blocks NMDARs and should prevent LTP. Under this condition, 60 s, 1 Hz stimulation induced a small but consistent increase in PS amplitude (PS amplitude at t ϭ 25-30 min ϭ 115 Ϯ 7.3% of baseline, n ϭ 14, p Ͻ 0.01) indicating that a DLL has occurred, while HFS induced LTD (PS amplitude at t ϭ 45-50 min ϭ 85 Ϯ 9.5% of baseline, n ϭ 13, p Ͻ 0.01) (Fig. 4b) . PS amplitude was not enhanced by the 60 s, 1 Hz stimulation protocol in slices treated with AP-5 and picrotoxin (PS amplitude at t ϭ 25-30 min ϭ 97 Ϯ 6.1% of baseline, n ϭ 9, p Ͼ 0.05; PS amplitude after HFS at t ϭ 45-50 min ϭ 74 Ϯ 8.3% of baseline, n ϭ 9, p Ͻ 0.001) (Fig. 4c) , and perfusion with AM251 and AP-5 prevented the changes produced by either 60 s, 1 Hz stimulation or HFS (PS amplitude after 1 Hz stimulation at t ϭ 25-30 min ϭ 100 Ϯ 4.0% of baseline, n ϭ 9, p Ͼ 0.05; PS amplitude after HFS at t ϭ 45-50 min ϭ 92 Ϯ 9.6% of baseline, n ϭ 9, p Ͼ 0.05) (Fig.  4d) . Delivery of HFS before 60 s, 1 Hz stimulation induced a depression in PS amplitude and occluded DLL (PS amplitude at t ϭ 25-30 min ϭ 76 Ϯ 12% of baseline, n ϭ 12, p Ͻ 0.01; t ϭ 45-50 min ϭ 79 Ϯ 12% of baseline, n ϭ 9, p Ͻ 0.05).
Frequency dependent inversion of striatal output
To determine which stimulus parameters induce DLL, and which produce a switch from DLL to LTD, we applied single stimulus trains delivered at different frequencies and durations. Stimulation with a 60 s, 5 Hz train induced robust DLL (PS amplitude at t ϭ 35-40 min ϭ 139 Ϯ 19% of baseline, n ϭ 9, p Ͻ 0.01), while no change in PS amplitude was seen after 60 s, 10 Hz stimulation (PS amplitude at t ϭ 35-40 min ϭ 92 Ϯ 8.6% of baseline, n ϭ 9, p Ͼ 0.05; 5 Hz vs 10 Hz, p Ͻ 0.001) (Fig. 4e) . Pretreatment with AP-5 was not required for 60 s, 5 Hz-induced DLL (PS amplitude at t ϭ 35-40 min ϭ 118 Ϯ 13% of baseline, n ϭ 8, p Ͻ 0.05), indicating that DLL can occur under more "natural" conditions. Interestingly, 120 s, 5 Hz stimulation did not affect PS amplitude (PS amplitude at t ϭ 35-40 min ϭ 93 Ϯ 8.7% of baseline, n ϭ 9, p Ͼ 0.05), while 30 s, 10 Hz induced DLL (PS amplitude at t ϭ 35-40 min ϭ 132 Ϯ 22% of baseline, n ϭ 9, p Ͻ 0.05) (Fig. 4e) , indicating that when afferents are activated at frequencies well within the range exhibited by cortical inputs the experimental outcome is dependent on the duration of the stimulation. We also found that 60 s-0.5 Hz stimulation did not affect PS amplitude (PS amplitude at t ϭ 35-40 min ϭ 98 Ϯ 4.3% of baseline, n ϭ 9, p Ͼ 0.05), while 120 s-0.5 Hz induced a DLL that was not significantly different in magnitude from DLL induced by 60 s, 1 Hz stimulation (PS amplitude at t ϭ 35-40 min ϭ 112 Ϯ 9.4% of baseline, n ϭ 13, p Ͻ 0.05; 60 s, 1 Hz vs 120 s-0.5 Hz, p Ͼ 0.05) (Fig. 4f ) . Our observations suggest that plastic changes in net striatal output involve LTD, LTP and DLL at both GABAergic and glutamatergic synapses, and that the net balance is regulated by the frequency and duration of glutamatergic afferent input.
Discussion
The data presented here suggest that GABAergic, presumed inhibitory, synapses are more sensitive to eCB signaling and LTD induction compared with glutamatergic excitatory synapses in the striatum. This might involve different presynaptic activity requirements for LTD induction at the two types of synapses (Singla et al., 2007; Adermark et al., 2009 ), but could also be connected to the high expression of CB 1 R at inhibitory synapses (Uchigashima et al., 2007) . AEA-induced depression at GABAergic synapses was reversed by AM251 in the presence of cycloheximide, which provides independent support for the idea that the AEA effect at GABAergic synapses is truly LTD. Our findings also indicate that protocols which induce short-lasting eCB-mediated depression at glutamatergic synapses are sufficient to induce LTD at GABAergic synapses in the dorsolateral striatum.
Discussion
Interestingly, by pairing postsynaptic eCB-loading with 60 s, 1 Hz stimulation, the short-term depression of glutamatergic transmission normally induced by AEA could be converted to LTD (Fig. 3) . Since postsynaptic cells are loaded with 50 M AEA there should be an excess of eCBs at both GABAergic and glutamatergic synapses. Increased stimulation might therefore be required to synergize CB 1 R activation with some presynaptic mechanism for LTD-induction (Sjöström et al., 2003; Singla et al., 2007) . It should be noted that WIN55,212-2-induced depression at many synapses, including GABAergic striatonigral synapses, is fully reversed by subsequent AM251 treatment even when afferent activation is given at comparable levels to those used with our single-pulse stimulus protocol (Kreitzer et al., 2002; Wallmichrath and Szabo, 2002) . Thus, the conditions necessary to convert CB 1 R-mediated short-term synaptic depression to LTD likely vary in a synapse-specific manner.
Discussion
The necessity for mGluR activation in 1 Hz-LTD at GABAergic synapses indicates an important role for glutamatergic inputs in this form of DLL. This finding is in line with data from the hippocampus and basolateral amygdala indicating that glutamatergic synaptic activation of group I mGluRs is critical for afferent stimulus-induced LTD at inhibitory synapses (Chevaleyre and Castillo, 2003; Azad et al., 2004) . The role of glutamatergic transmission in this form of LTD indicates that the sustained synaptic depression most likely occurs at GABAergic synapses located on the distal dendrites and dendritic spines of MSNs, since only these synapses would be close to the glutamatergic synapses that are present on the outer two-thirds of the dendritic arbor. These observations suggest the interesting possibility that "feedback" inhibitory inputs from other MSNs are among the GABAergic synapses that exhibit 1 Hz-LTD.
Discussion
Using different stimulation frequencies (0.5, 1, 5, 10 Hz) and durations (30 s, 1 min, 2 min), we showed that when frequencies within the presumed physiological range are used, the number of pulses used, rather than just the frequency, is what determines whether the stimulation will enhance striatal output through DLL, or whether LTD occurs (Fig. 4e,f ) . During low frequency, short duration repetitive activation of glutamatergic inputs LTD of GABAergic synapses predominates; giving rise to DLL of striatal output. At higher frequencies, or longer stimulus durations, the glutamatergic synapses themselves are depressed, which would decrease output from projection neurons. The frequency/ duration of glutamatergic input is thus a strong determinant of the net effect of eCB signaling, and a key factor in determining whether LTD has a net disinhibitory or inhibitory action in striatum. Perfusion of picrotoxin amplified PS amplitude showing that GABAergic inhibition regulates striatal output. Furthermore, the increase in PS amplitude in AP-5 treated slices after 60 s, 1 Hz stimulation was blocked by picrotoxin or AM251, which is consistent with the idea that this DLL involves a change in GABAergic transmission mediated through CB 1 R activation. Thus, the striatal MSN shows unique patterns of glutamateactivated eCB-dependent plasticity that might cause frequencydependent inversion of net striatal output.
Discussion
In vivo, moderate frequency membrane potential oscillations (5 Hz) were recorded in MSNs that were caused by the synchronous firing of cortical inputs (Charpier et al., 1999) . In addition, in vitro recordings from layer 5 rat prefrontal cortical neurons demonstrated firing at irregular frequencies with an average of 5-10 Hz (Fellous et al., 2003) . In vivo recordings from the striatum have shown that MSNs fire at frequencies of 1-10 Hz, with occasional bursts reaching at least 30 Hz, while cholinergic neurons fire at 1-3 Hz, and that GABAergic interneurons can fire at 20 Hz (Burkhardt and Costa, unpublished observations). Since the frequencies used in this study are within the normal physiological firing range of striatal afferents and intrinsic neurons, it thus is likely that DLL is of physiological importance, suggesting that plastic changes in net output from striatal MSNs are a complex function of DLL and LTD combined with other forms of synaptic plasticity such as LTP at excitatory synapses (Calabresi et al., 1992; Partridge et al., 2000) . eCB-dependent plasticity at GABAergic and glutamatergic transmission may participate in striatal-based learning and memory by sculpting the net output of individual MSNs.

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