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. 2009 Jan;29(1):32-41.
doi: 10.1111/j.1460-9568.2008.06551.x.

Endocannabinoid-dependent plasticity at GABAergic and glutamatergic synapses in the striatum is regulated by synaptic activity

Louise Adermark  1 , Giuseppe Talani, David M Lovinger
Affiliations

Endocannabinoid-dependent plasticity at GABAergic and glutamatergic synapses in the striatum is regulated by synaptic activity

Louise Adermark et al. Eur J Neurosci. 2009 Jan.

Abstract

Long-term depression (LTD) at striatal synapses is mediated by postsynaptic endocannabinoid (eCB) release and presynaptic cannabinoid 1 receptor (CB(1)R) activation. Previous studies have indicated that eCB mobilization at excitatory synapses might be regulated by afferent activation. To further address the role of neuronal activity in synaptic plasticity we examined changes in synaptic strength induced by the L-type calcium channel activator 2,5-dimethyl-4-[2-(phenylmethyl)benzoyl]-1H-pyrrole-3-carboxylic acid methyl ester (FPL 64176, FPL) at glutamatergic and gamma-aminobutyric acid (GABA)ergic synapses in the striatum. We found that the basic mechanisms for FPL-mediated eCB signaling are the same at glutamatergic and GABAergic synapses. FPL-induced LTD (FPL-LTD) was blocked in slices treated with the CB(1)R antagonist AM251 (2 microm), but established depression was not reversed by AM251. FPL-LTD was temperature dependent, blocked by protein translation inhibitors and prevented by intracellular loading of the anandamide transporter inhibitor VDM11 (10 microm) at both glutamatergic and GABAergic synapses. FPL-LTD at glutamatergic synapses required paired-pulse afferent stimulation, while FPL-LTD at GABAergic synapses could be induced even in the absence of explicit afferent activation. By evaluating tetrodotoxin-insensitive spontaneous inhibitory postsynaptic currents we found that neuronal firing is vital for eCB release and LTD induction at GABAergic synapses, but not for short-term depression induced by CB(1)R agonist. The data presented here suggest that the level of neuronal firing regulates eCB signaling by modulating release from the postsynaptic cell, as well as interacting with presynaptic mechanisms to induce LTD at both glutamatergic and GABAergic synapses in the striatum.

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Figures

Fig. 1
Fig. 1
Basic properties of 2,5-dimethyl-4-[2-(phenylmethyl)benzoyl]-1H-pyrrole-3-carboxylic acid methyl ester (FPL)-LTD are similar at glutamatergic (FPL-eLTD) and GABAergic synapses (FPL-iLTD). (A) FPL (500 nm) induced a robust depression in MSNs clamped at −50 mV that was prevented, but not reversed, by CB1R antagonist (AM251, 2 µm). (B) FPL-LTD was blocked by intracellular loading of the AMT inhibitor VDM11 (10 µm), indicating that eCB signaling involves a postsynaptic release step at both glutamatergic and GABAergic synapses. Example traces show excitatory postsynaptic currents (EPSCs) in a VDM11-loaded MSN at baseline (black) and post FPL treatment at t = 20–25 min (gray). (C) FPL-eLTD was significantly reduced in slices perfused with the protein translation inhibitor cycloheximide (80 µm). Bath application (filled circles) was more successful in inhibiting FPL-eLTD compared with intracellular loading (open triangles), suggesting that protein synthesis is required outside the postsynaptic cell. Example traces show EPSCs in a cycloheximide-loaded MSN clamped at baseline (black) and post FPL treatment at t = 20–25 min (gray). FPL-iLTD was also dependent on protein translation, and completely prevented in slices pretreated with cycloheximide (80 µm). The graph shows the mean inhibitory postsynaptic current (IPSC) amplitude with SEM in MSNs after 10 min treatment with 500 nm FPL (*P < 0.05). (D) Treatment with another blocker of protein synthesis, anisomycin (20 µm; filled circles) also successfully inhibited FPL-eLTD. EPSC / IPSC amplitude data are mean ± SEM. Scale bars: 100 pA and 25 ms for all traces. aCSF, artificial cerebrospinal fluid.
Fig. 2
Fig. 2
Different requirement for afferent activation in 2,5-dimethyl-4-[2-(phenylmethyl)benzoyl]-1H-pyrrole-3-carboxylic acid methyl ester (FPL)-eLTD and FPL-iLTD. (A) FPL treatment combined with paired-pulse stimulation induced a robust depression with a similar time course and amplitude at glutamatergic and GABAergic synapses in the striatum. The depression in excitatory postsynaptic current (EPSC) amplitude was significantly reduced when the interpulse interval was increased from 50 ms to 1 s. (B) When afferents were activated by a single pulse delivered every 20 s FPL treatment was sufficient to induce FPL-iLTD, while the EPSC amplitude remained unaffected. (C) Synaptic depression of GABAergic transmission that was resistant to AM251 reversal (FPL-iLTD) could be induced even when afferent activation was suspended, but this depression was significantly smaller compared with FPL-iLTD induced during continuous afferent activation. EPSC / inhibitory postsynaptic current (IPSC) amplitude data are mean ± SEM. Example traces in (A) and (B) show IPSCs at baseline (black) and at t = 20–25 min (gray). Scale bar: 100 pA and 25 ms. aCSF, artificial cerebrospinal fluid.
Fig. 3
Fig. 3
2,5-Dimethyl-4-[2-(phenylmethyl)benzoyl]-1H-pyrrole-3-carboxylic acid methyl ester (FPL)-induced depression requires presynaptic activity. (A) FPL (10 min) treatment reduced the event frequency of spontaneous inhibitory postsynaptic currents (sIPSCs), but not miniature inhibitory postsynaptic currents (mIPSCs), showing that neuronal firing is vital for FPL-induced depression. sIPSC / mIPSC amplitude was not affected by FPL treatment. A small inward current was induced by FPL in both control and TTX-treated slices. Data are based on six cells in each group and presented as mean values compared with baseline with SEM. Statistics are paired t-test. ***P < 0.001. (B) Example traces shows mIPSCs and sIPSCs at baseline and following 10 min of FPL treatment. Note the slight inward current in the presence of FPL. Scale bar: 50 pA and 5 s. (C) Example trace showing mIPSCs at baseline, during FPL treatment, and washout. Note that the inward current reverses upon FPL washout. Scale bar: 25 pA and 1 min. (D) sIPSC frequency and amplitude were not affected by FPL treatment if the postsynaptic cell was clamped at −70 mV. The FPL-induced change in holding current remained in MSNs voltage-clamped at −70 mV, but was prevented by the L-type calcium channel blocker nifedipine (20 µm). *P < 0.05.
Fig. 4
Fig. 4
eCB release and LTD induction at GABAergic synapses require presynaptic activity. (A) Postsynaptic loading of the eCB AEA (50 µm), which previously has been shown to decrease the event frequency of recorded sIPSCs (Adermark & Lovinger, 2007b), was insufficient to affect mIPSC event frequency, amplitude, rise time or decay time within a 20-min cell-loading period. (B) Treatment with the cannabinoid 1 receptor (CB1R) agonist WIN 55,212-2 (1 µm) significantly reduced mIPSC frequency, suggesting that neuronal firing is not required for CB1R activation of downstream events involved in transient synaptic depression. However, the WIN-induced decrease in mIPSC frequency recovered after post-agonist AM251 treatment, suggesting that iLTD is not induced by CB1R activation when neuronal firing is prevented. Example traces show mIPSCs at baseline, after 10 min WIN 55,212-2 treatment and after 20 min washout with AM251. Data are based on eight cells in each group and presented as mean values compared with baseline with 95% confidence intervals. Statistics are paired t-test. *P < 0.05, # #P < 0.01. Scale bar: 40 pA and 1.3 s. MSN, medium spiny neuron.
Fig. 5
Fig. 5
2,5-Dimethyl-4-[2-(phenylmethyl)benzoyl]-1H-pyrrole-3-carboxylic acid methyl ester (FPL)-LTD is not sensitive to altered external potassium. (A and B) Increasing the extracellular level of KCl to 10 mm increased the spontaneous excitatory postsynaptic current (sEPSC) frequency, but did not facilitate FPL-eLTD. (C) Reducing extracellular KCl to 1 mm did not prevent FPL-iLTD. Data show spontaneous inhibitory postsynaptic current (sIPSC) values compared with baseline in 10-s bins before and after 10 min FPL treatment. FPL-iLTD in TTX-treated slices was also not facilitated by 10 mm KCl. Graph showing miniature inhibitory postsynaptic current (mIPSC) amplitude and frequency compared with baseline level after 10 min FPL application, note the absence of any depression of mIPSC frequency. (D) The amplitude and frequency of postsynaptic currents were not affected when external potassium was reduced to 1 mm, but were significantly depressed in slices treated with TTX (1 µm). Increasing the extracellular level of KCl to 10 mm increased the mIPSC amplitude and frequency (*P < 0.05, **P < 0.01).
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