Combined activation of L-type Ca2+ channels and synaptic transmission is sufficient to induce striatal long-term depression

Abstract

Changes in synaptic strength at striatal synapses, such as long-term depression (LTD), may be involved in striatal-based learning and memory. Several molecular mechanisms have been implicated in striatal LTD, but it is not clear which mechanisms are crucial for LTD induction. We found that the activation of L-type calcium channels by 2,5-dimethyl-4-[2-(phenylmethyl)benzoyl]-1H-pyrrole-3-carboxylic acid methylester (FPL64176), combined with modest postsynaptic depolarization and synaptic activation, is sufficient to induce robust LTD (FPL-LTD). The L-channel activator 1,4-dihydro-2,6-dimethyl-5-nitro-4-[2(trifluoromethyl)phenyl]pyridine-3-carboxylic acid methyl ester (Bay K 8644) has a similar action. FPL-LTD occludes LTD induced by high-frequency stimulation (HFS-LTD) and requires elevated postsynaptic calcium and retrograde endocannabinoid signaling, properties similar to those of HFS-LTD. In contrast, FPL-LTD does not require the activation of metabotropic glutamate receptors (mGluRs), phospholipase C, or dopamine D2 receptors. FPL-LTD induction also requires afferent stimulation. These findings suggest a scenario in which L-type calcium channel activation is a crucial switch for LTD induction, and mGluRs and D2 receptors can be bypassed if this channel is activated.

Figure 1.

Activation of L-type calcium channels induces LTD in a concentration-dependent manner at the corticostriatal synapse. a–c, Data show EPSC amplitude in MSNs clamped at–50 mV and stimulated every 20 s, with paired pulse stimuli delivered with a 50 ms interpulse interval (50 μm AP-5 and 50 μm picrotoxin present in the bath solution). a, EPSC amplitude in a single-cell example experiment during treatment with 500 nm FPL. FPL–LTD typically was induced after 5 min of drug perfusion. These traces also include the response to the poststimulus hyperpolarizing step used to calculate the relative input resistance. b, Averaged data for slices treated with 500, 50, or 10 nm FPL. c, Averaged data for seven slices treated with 1 μm Bay K 8644. The PPR (EPSC 2/EPSC 1) was increased significantly after FPL treatment. d, PPR expressed as a percentage of pre-drug baseline values for six experiments with a concentration of 500 nm FPL. e, Graph shows PPR values 20–25 min after the onset of the recording. Data are the mean values with 95% CI. ** p < 0.01; *** p < 0.001. f, FPL–LTD occludes HFS–LTD. HFS did not induce LTD when given after the onset of FPL–LTD (n = 6; filled circles), and HFS was similarly ineffective in cells in which FPL did not induce LTD (n = 8; empty circles). EPSC values are the mean ± SEM from responding MSNs. Traces are EPSCs evoked by paired pulses before (black line) and after (gray line) LTD induction. Data are presented as the mean ± SEM; n.s., not significant. Calibration: 100 pA, 50 ms.

Figure 2.

FPL–LTD is dependent on increased postsynaptic [Ca²⁺]_i. a, LTD was not induced in cells held at–70 mV. b, No change in synaptic strength was detected during chelation of postsynaptic intracellular calcium with 20 mm BAPTA. c, FPL–LTD was inhibited during incubation with the calcium channel blocker nifedipine (20 μm) (n = 6). EPSC values are the mean ± SEM.

Figure 3.

FPL–LTD induction requires CB₁ receptor activation and protein translation. a, LTD was blocked by the CB₁ receptor antagonist AM251 (3 μm), suggesting that FPL perfusion induces endocannabinoid release (n = 7). b, Established FPL–LTD was not reversed by blockade with AM251, which is in line with studies from LTD induced by HFS (n = 5). EPSC values are the mean ± SEM. Traces are EPSCs evoked by paired pulses before (black line) and after (gray line) LTD induction. Calibration: 100 pA, 50 ms.

Figure 4.

FPL–LTD does not require activation of group I mGluRs or D₂Rs. a, FPL induces LTD in the combined presence of the group I mGluR antagonists MPEP (40 μm) and CPCCOEt (80 μm) (n = 6). b, FPL–LTD remained during intracellular loading with the PLC inhibitor U73122 (5 μm). c, The D₂R antagonist sulpiride (5 μm) also did not alter LTD (n = 6). d, FPL–LTD could be induced in both D₁-GFP and D₂-GFP mice. EPSC values are the mean ± SEM from responding MSNs. Traces are EPSCs evoked by paired pulses before (black line) and after (gray line) LTD induction. Calibration: 100 pA, 50 ms.

Figure 5.

FPL–LTD requires afferent stimulation. a, EPSC amplitude was at control level when afferent activation was stopped during the 10 min FPL treatment but rapidly decreased as stimulation continued (n = 6). Stars mark the time points at which sEPSCs were sampled. EPSC amplitude values are the mean ± SEM. b, Traces are EPSCs evoked by paired pulses at baseline (black line) and after 15 min of afferent activation (gray line). Calibration: 100 pA, 50 ms. c, Representative traces showing sEPSCs before FPL treatment, just after treatment but before afferent stimulation, and after a subsequent 10 min period of afferent stimulation. sEPSCs were not altered after a 10 min application of FPL, but after a subsequent 10 min presynaptic stimulation period the frequency of events was reduced significantly (c, e). e, sEPSC rise time was unaffected by FPL, and the sEPSC amplitude assessed by mean amplitude and cumulative probability distribution analyses also was not altered by FPL (d, e). Graphs show the mean values with 95% CI. Data are based on seven recordings and are compared with a paired Student's t test; *p < 0.05. n.s., Not significant.