Activation of group II metabotropic glutamate receptors inhibits synaptic excitation of the substantia Nigra pars reticulata

Abstract

Loss of nigrostriatal dopaminergic neurons in Parkinson's disease (PD) leads to increased activity of glutamatergic neurons in the subthalamic nucleus (STN). Recent studies reveal that the resultant increase in STN-induced excitation of basal ganglia output nuclei is responsible for the disabling motor impairment characteristic of PD. On the basis of this, it is possible that any manipulation that reduces activity at excitatory STN synapses onto basal ganglia output nuclei could be useful in the treatment of PD. We now report that group II metabotropic glutamate receptors (mGluRs) are presynaptically localized on STN terminals and that activation of these receptors inhibits excitatory transmission at STN synapses. In agreement with the hypothesis that this could provide a therapeutic benefit in PD, a selective agonist of group II mGluRs induces a dramatic reversal of catalepsy in a rat model of PD. These results raise the exciting possibility that selective agonists of group II mGluRs could provide an entirely new approach to the treatment of PD. These novel therapeutic agents would provide a noninvasive pharmacological treatment that does not involve the manipulation of dopaminergic systems, thus avoiding the problems associated with current therapies.

Fig. 1.

Activation of group II mGluRs reduces EPSCs at the STN–SNr synapse. A, Evoked EPSCs before (Control), during (LY354740), and after (Wash Out) a brief local application of LY354740. Applications of LY354740 dramatically reduce EPSCs, and this effect is reversible. B, Average time course of the effect of 100 nm LY354740 (application indicated byhorizontalbar). Eachpoint represents the mean (± SEM) of data from five cells. C, Dose–response relationship of LY354740-induced inhibition of EPSCs. The effect of inhibition of EPSCs is maximal at 100 nm. Each point represents the mean of three experiments. D, Effects of specific group II mGluR agonists on EPSCs at the STN–SNr synapse and block of the LY354740-induced inhibition of EPSCs by application of group II mGluR antagonists before application of the agonist. Agonists include LY354740 (100 nm), APDC (100 μm), and DCG-IV (3 μm). Antagonists include LY341495 (100 nm) and CPPG (500 μm). Each verticalbar represents the mean (± SEM) of data collected from five cells (*p < 0.01).

Fig. 2.

Group II mGluRs are presynaptically localized at asymmetric terminals in the SNr. A–D, Electron micrographs demonstrating presynaptic mGluR2/3 immunoreactivity at asymmetric terminals in the SNr. Labeled (*) axon terminals (t) are shown synapsing on unlabeled dendrites (d) and dendritic spines (s). E, An example of a labeled terminal forming a symmetric synapse. Synapses are indicated byarrows. Scale bar: A, 301 nm;B, 203 nm; C, 315 nm; D, 263 nm; E, 207 nm.

Fig. 3.

Activation of group II mGluRs has no effect on the response to exogenously applied kainate. A,Representative traces of kainate-evoked currents in the SNr projection neurons before (Control; left) and during application of 100 nm LY354740 (right).B, Time course of the effect of LY354740 on the amplitude of kainate-evoked currents. C, Mean data demonstrating the lack of effect of group II mGluR activation on kainate-evoked currents (mean ± SEM; p > 0.05; n = 5).

Fig. 4.

Inhibition of EPSCs at the STN–SNr synapse is mediated by a presynaptic mechanism. A,Examples of mEPSCs before (Pre-Drug; left) and during application of 100 nm LY354740 (right).B, Overlayed averages of all mEPSCs recorded before and during LY354740 application, demonstrating the lack of effect on the amplitude and kinetics of mEPSCs. C, Amplitude histograms of mEPSCs before (left) and during application of 100 nm LY354740 (right).D, Cumulative frequency plots illustrating the lack of effect of LY354740 on mEPSC amplitude (left) and inter-event interval (right) (Kolmogorov–Smirnov test;p = 0.99). The data shown are representative of five separate experiments.

Fig. 5.

Activation of group II mGluRs reduces the frequency of EPSCs evoked by glutamate application to the STN.A, A demonstration of the experimental paradigm used. Direct application of glutamate (100 μm; 1 ml/min; 30 sec) to the STN produces approximately a threefold increase in EPSC frequency without affecting EPSC amplitude. Moving the microapplicator to a position above the cerebral peduncle (cp) produced no change in the frequency of EPSCs, indicating that the glutamate effect is caused by selective activation of STN neurons and not by fibers of passage. B, Examples of glutamate-evoked EPSCs both before (left) and during the application of 100 nm LY354740 (right). C, Overlayedtraces of average glutamate-evoked EPSCs before and during 100 nm LY354740 application indicating no change in the amplitude or kinetics of the responses. D,Cumulative frequency plots illustrating a lack of effect of LY354740 on amplitude (left; Kolmogorov–Smirnov test;p > 0.05) and a significant increase in interevent interval (right; Kolmogorov–Smirnov test;p < 0.01), indicating that LY345740 selectively reduces the frequency of glutamate-evoked EPSCs. E,Frequency–amplitude histograms demonstrating a decrease in the frequency but no change in the mean amplitude of glutamate-evoked EPSCs. F, Mean (± SEM) data demonstrating that glutamate induces approximately a threefold increase in frequency over basal values without altering amplitude. This glutamate-evoked increase is significantly reduced by LY345740. Each verticalbar represents the mean (± SEM) of data collected from five cells (*p < 0.05).

Fig. 6.

Activation of group II mGluRs does not effect the excitability of STN neurons. A,Representative current-clamp recording demonstrating that application of 100 nm LY345740 does not alter membrane potential.B, Overlayed traces of responses to the injection of hyperpolarizing current demonstrating that LY354740 has no effect on input resistance. C, Representativetraces of experiments in which small depolarizing current injections were used to determine the lowest potential at which an STN neuron would produce an action potential. Application of 100 nm LY354740 has no effect on this potential. D, Mean (± SEM) of data demonstrating the lack of effect of group II mGluR activation on membrane potential, input resistance, or lowest spike potential. Data are from three to seven cells per condition. E, F, Representativetraces (E) and time course (F) demonstrating that LY354740 does not alter presynaptic fiber volleys evoked by stimulation of the STN.G, Mean (± SEM) of data from four independent experiments demonstrating that activation of group II mGluRs has no effect on presynaptic fiber volleys. The fiber volley is blocked by the application of 500 nm TTX indicating that the volley is a measurement of presynaptic axonal action potential.

Fig. 7.

Activation of group II mGluRs has no effect on inhibitory transmission in the SNr. A, Representativetraces of evoked IPSCs before (Pre-Drug; left) and during the application of 100 nm LY354740 (right). B, Time course of the effect of LY354740 on IPSC amplitude. C, Mean data demonstrating the lack of effect of group II mGluR activation on IPSC amplitude. Data represent the mean (± SEM) of seven separate experiments (p > 0.05).

Fig. 8.

Activation of group II mGluRs reverses catalepsy in an animal model of Parkinson's disease. The degree of haloperidol-induced catalepsy was measured as either latency to the first paw movement when the animal was placed on a vertical grid (A) or latency to remove a paw from a bar (B). Haloperidol (1.5 mg/kg, i.p.) induces a pronounced catalepsy that was reversed in a dose-dependent manner by LY354740 (3–30 mg/kg, i.p.) (*p < 0.05). LY354740 alone had no effect on either measure of catalepsy. Data shown represent the mean (± SEM) of data collected from eight animals.