Role of p/q-Ca2+ channels in metabotropic glutamate receptor 2/3-dependent presynaptic long-term depression at nucleus accumbens synapses

Abstract

The nucleus accumbens (NAc) is an important cerebral area involved in reward and spatial memory (Pennartz et al., 1994), but little is known about synaptic plasticity in this region. Here, electron microscopy revealed that, in the NAc, metabotropic glutamate receptors 2/3 (mGlu2/3) immunostaining was essentially associated with axonal terminals and glial processes, whereas postsynaptic dendrites and neuronal cell bodies were unstained. Electrophysiological techniques in the NAc slice preparation demonstrated that activation of mGlu2/3 with synaptically released glutamate or specific exogenous agonist, such as LY354740 (200 nm, 10 min), induced long-term depression of excitatory synaptic transmission (mGlu2/3-LTD). Tetanic-LTD and pharmacological mGlu2/3-LTD occluded each other, suggesting common mechanisms. The mGlu2/3-LTD did not require synaptic activity but depended on the cAMP-protein kinase A cascade. Selective inhibition of P/Q-type Ca(2+) channels with omega-agatoxin-IVA occluded the expression of mGlu2/3-LTD, and, conversely, the inhibitory effects of omega-agatoxin-IVA were abolished during mGlu2/3-LTD. Thus, mGlu2/3 play an important role in the control of use-dependent synaptic plasticity at prelimbic cortex-NAc synapses: their activation causes a form of LTD mediated by the long-lasting reduction of P/Q-type Ca(2+)channels contribution to transmitter release.

Fig. 1.

Immunolocalization of mGlu2/3 in the nucleus accumbens. A, At the light microscope level, immunostaining is associated with elongated processes (arrows) and dot structures dispersed between unlabeled neuronal cell bodies (N). B–D, At the electron microscope level, immunostaining is essentially associated with axon terminals containing numerous synaptic-like vesicles (ax) and glial processes (gl), whereas dendritic profiles (D) always appear unstained. Note that labeled axon terminals frequently form typical synaptic contacts with unlabeled dendrites (arrowheads in B andC), whereas labeled glial processes frequently enwrap either labeled (C) or unlabeled (D) synaptic axon terminals.Asterisks in B–D indicate the unlabeled axon terminals. Scale bars: A, 25 μm;B–D, 1 μm.

Fig. 2.

Characterization of pharmacological mGlu2/3-induced LTD. A, Summarized data showing that application of LY354740 (200 nm) resulted in an initial depression, which was followed during washout by an LTD.Inset shows traces (average of 10 consecutive fEPSPs) taken at the points indicated in a typical experiment. Calibration: 0.3 mV, 10 msec. B, Typical whole-cell experiment showing LTD induced by LY354740. Inset shows traces (average of 10 consecutive eEPSCs) taken at points indicated on the graph. Calibration: 100 pA, 25 msec. C, Inhibition of eEPSC 30 min after LY354740 is accompanied by an increase in the paired-pulse ratio. D, In presence of the group 2 mGlu antagonist LY341495 (200 nm), the initial depression induced by LY354740 was reduced and the LTD was prevented. E, Application of LY341495 (200 nm) 60 min after LTD induced by LY354740 did not reverse the LTD. F, Dose–response curves for the LY354740-induced initial inhibition of the fEPSP and for LTD.

Fig. 3.

Induction of synaptic mGlu2/3-LTD and mutual occlusion with pharmacological LTD. A, High-frequency tetanus (3 times for 1 sec at 100 Hz, 20 sec interval) can induce LTD, LTP, or no plasticity in the NAc. Summarized histogram showing the percentage of slices exhibiting LTD (defined as a inhibition of at least 10% of baseline fEPSP, 30 min after tetanus), LTP (defined as an enhancement of at least 10% of baseline fEPSP, 30 min after tetanus), or no plasticity in control conditions or after perfusion with the highly selective mGlu2/3 antagonist LY341495 (200 nm, 15 min). Blocking mGlu2/3 receptors during the tetanus clearly reduced the number of slices expressing LTD and increased the number of slices exhibiting either LTP or no change in synaptic efficacy.B, Typical experiment showing tetanus-induced LTD in control medium. The inset shows traces (average of 10 consecutive fEPSPs) taken at the points indicated. Calibration: 0.4 mV, 5 msec. C, Summary of all of the experiments comparing the effects of high-frequency stimulation of prelimbic afferents in control conditions and after blockade of mGlu2/3. D, Typical experiment showing that, after inducing the mGlu2/3-LTD with LY354740 (200 nm), the tetanus induces no more LTD (here it induces LTP). E, Summary of all of the experiments showing the occlusion of tetanus-induced LTD after induction of mGlu2/3-LTD with LY354740. F, Typical experiment showing that, after saturation of tetanus-induced LTD, LY354740 (200 nm) did not induce LTD. G, Summary of all of the experiments showing the occlusion of mGlu2/3-LTD induced with LY354740 (200 nm) after induction of tetanus-induced LTD. Tet, Tetanus (three times at 1 sec at 100 Hz). *p < 0.05; **p< 0.01.

Fig. 4.

Transduction pathways of mGlu2/3-LTD.A, mGlu2/3-LTD was not blocked in the absence of presynaptic activity. Summary of all of the experiments in which evoked synaptic stimulation was stopped during bath perfusion of LY354740 (200 nm) and 30 min after the washout. B, Summarized data showing that treatment with the adenylate cyclase activator forskolin (10 μm) inhibited the initial depression attributable to LY354740 and completely abolished mGlu-LTD.C, Summary of the effects the PKA inhibitor KT 5720 (1 μm) on basal synaptic transmission. D, Summary of all of the experiments in which the slices were preincubated at least 2 hr with the PKA inhibitor KT 5720 at 1 μm. In this condition, the initial depression of fEPSP by LY354740 was slightly decreased, and LTD at 60 min was totally prevented. E, Summary of the effects the PKA inhibitor H 89 (10 μm) on basal synaptic transmission.F, Summary of the experiments in which slices have been treated 20 min before, during, and 10 min after LY354740 application with the PKA inhibitor H 89 (10 μm). In this condition, the LTD induced by LY354740 was completely prevented at 60 min. *p < 0.05; **p < 0.01.

Fig. 5.

mGlu2/3-LTD is attributable to the reduction of P/Q-type Ca²⁺ channels to evoked synaptic transmission. A–D, The mGlu2/3-LTD requires P/Q-type Ca²⁺ channels. A, Typical experiment in which the slice was perfused with ω-conotoxin-GVIA (1 μm). The fraction of synaptic transmission insensitive to ω-conotoxin-GVIA displayed a normal form of mGlu2/3-LTD.B, Typical experiment in which the slice was perfused with ω-agatoxin-IVA (200 nm). The fraction of synaptic transmission insensitive to ω-agatoxin-IVA did not display mGlu2/3-LTD. C, Typical experiment in which the slice was perfused with nimodipine (1 μm). This treatment had no effect on the mGlu2/3-LTD. D, Summary of all of the experiments performed as above. The histogram of the mGlu2/3-LTD at 60 min reveals that blocking P/Q-type HVA Ca²⁺ channels suppressed mGlu2/3-LTD. E–H, P/Q-type Ca²⁺ channels do not contribute to synaptic transmission when mGlu2/3-LTD is induced. E–G, Typical experiments in which, after induction of mGlu2/3-LTD with LY354740 (200 nm), the slices were perfused with ω-conotoxin-GVIA (1 μm), ω-agatoxin-IVA (200 nm), and nimodipine (1 μm), respectively.H, Summary of all of the experiments performed as above. The histogram represents mean fEPSP inhibition (i.e., fEPSP contribution) induced by ω-conotoxin-GVIA, ω-agatoxin-IVA, and nimodipine and taken after 15–20 min of drug application. It reveals that P/Q-type Ca²⁺ channels do not contribute to synaptic transmission once mGlu2/3-LTD is induced. Conversely, N-type contribution is augmented. *p < 0.05; **p < 0.01.

Fig. 6.

mGlu2/3-LTD is reduced on Ca²⁺-independent spontaneous EPSCs.A, B, Spontaneous EPSCs recorded in the absence of TTX express both acute and long-term effects of mGlu2/3 activation. A, Typical experiment. Representative consecutive 1 sec current sweeps from a cell (holding potential of −70 mV) in which sEPSCs were recorded in the absence, during, or 45 min after LY354740 (200 nm). Calibration: 30 pA, 200 msec. The distribution of sEPSC inter-event intervals was altered after bath-perfusion of LY354740. B, Mean frequency of sEPSCs was equally reduced during and 45 min after LY354740 perfusion. **p < 0.01.n.s., Not significant. C,D, Action potential-independent miniature EPSCs recorded in the presence of TTX (300 nm) express acute but reduced long-term effects of mGlu2/3 activation. C, Typical experiment. Representative consecutive 1 sec current sweeps from a cell (holding potential of −70 mV) in which mEPSCs were recorded in the absence, during, or 45 min after LY354740 (200 nm). Calibration: 30 pA, 200 msec. The distribution of mEPSC inter-event intervals was altered during application of the mGlu2/3 agonist but was back to normal after 45 min. The distribution of mEPSC amplitude was unchanged after bath perfusion of LY354740.D, Mean frequency of sEPSCs was significantly more reduced during than 45 min after LY354740 perfusion. *p < 0.05; **p < 0.01.