Frequency-specific and D2 receptor-mediated inhibition of glutamate release by retrograde endocannabinoid signaling

Abstract

The mechanisms underlying modulation of corticostriatal synaptic transmission by D2-like receptors (D2Rs) have been controversial. A recent study suggested that D2Rs inhibit glutamate release at this synapse, but only during high-frequency synaptic activation. Because the release of postsynaptic endocannabinoids (eCBs), which act as retrograde messengers to inhibit presynaptic glutamate release, can be triggered by D2R activation and intense synaptic activation, such a mechanism could mediate dopaminergic modulation of corticostriatal transmission. Here, we show that D2R activation reduces excitatory transmission onto striatal medium spiny neurons at a stimulation frequency of 20 Hz but not at 1 Hz. This form of inhibition requires CB1 receptor activation, as evidenced by the fact that it is blocked by AM251 [N-(piperidin-1-yl)-1-(2,4-dichlorophenyl)-5-(4-chlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide], a CB1 antagonist, and is absent in CB1 knockout mice. It is also blocked by postsynaptic intracellular calcium chelation, by group I metabotropic glutamate receptor antagonism, and by inhibition of postsynaptic phospholipase C. These results demonstrate a previously unrecognized role for retrograde eCB signaling in reversible and frequency-specific inhibition of glutamate release by the activation of striatal D2Rs.

Fig. 1.

Activation of D₂Rs inhibits glutamate release at the corticostriatal synapse. (A) Schematic illustration of the corticostriatal slice preparation. Note that the stimulating electrode illustrated is placed in the cortex outside of the white matter. (B) Quinpirole inhibits EPSCs when a three-pulse train is used. (C) A two-pulse train is also effective in revealing D₂R-mediated inhibition of EPSCs. Inhibition is blocked by pretreatment with sulpiride, a D₂R antagonist.

Fig. 2.

Activation of D₂ receptors inhibits glutamate transmission only when paired with HF stimulation. (A) Depression is observed after bath application of quinpirole using the three-pulse protocol with 20-Hz afferent stimulation. Only the first EPSC is shown. (B) Depression is observed after bath application of quinpirole using the two-pulse protocol with 20-Hz afferent stimulation. (C) No depression is observed at 1 Hz. After recording EPSCs at 20 Hz during the predrug baseline period, the frequency of stimulation was reduced to 1 Hz during the first 5 min of quinpirole application. (D) When stimulation at 20 Hz was resumed in the presence of quinpirole, inhibition developed gradually, with a time course similar to that seen in A. (Scale bars: 100 pA, 25 ms.)

Fig. 3.

Group I mGluRs are necessary for quinpirole-induced inhibition. Inhibition is blocked by bath application of CPCCOEt, a group I mGluR antagonist (A), postsynaptic loading of BAPTA, a calcium chelator (B), postsynaptic loading of thapsigargin, an inhibitor of intracellular calcium pumps (C), and U73122, an inhibitor of phospholipase C (D). (Scale bars: 100 pA, 25 ms.)

Fig. 4.

Shifting the frequency dependence with inhibition of glutamate transport. (A) Quinpirole-induced synaptic depression is observed at 20 Hz with TBOA in the bath. (B) The effect in A is blocked by CPCCOEt. (C) Quinpirole-induced synaptic depression is also observed at 1 Hz with TBOA in the bath. (D) The effect in C is blocked by CPCCOEt. (Scale bars: 100 pA, 25 ms.)

Fig. 5.

Activation of CB₁ receptors is necessary for HF-specific inhibition. (A) Application of AM251 blocked synaptic depression produced by quinpirole. (B) Pretreatment with WIN552,12-2, a CB₁ receptor agonist, occluded the quinpirole-induced inhibition. (C and D) Quinpirole-induced synaptic depression was normal in wild-type mice (C) but absent in CB₁ knockout mice (D). (Scale bars: 100 pA, 25 ms.)

Fig. 6.

CB₁ activation is downstream of signaling mediated by D₂Rs and group I mGluRs. Pretreatment with sulpiride (A) or CPCCOEt (B) did not affect inhibition of glutamate release caused by bath application of WIN552,12-2. (Scale bars: 100 pA, 25 ms.)

Fig. 7.

Quinpirole-induced, frequency-dependent inhibition is observed under more physiological conditions. (A) No inhibition of EPSPs measured in current-clamp mode was observed with 1-Hz stimulation. (B) Significant inhibition of EPSPs was observed with 20-Hz stimulation. (C) Inhibition of EPSPs at 20 Hz was blocked by intracellular loading of the calcium chelator EGTA.