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. 2006 Jun 15;573(Pt 3):697-709.
doi: 10.1113/jphysiol.2006.110031. Epub 2006 Apr 13.

Subthalamic stimulation evokes complex EPSCs in the rat substantia nigra pars reticulata in vitro

Ke-Zhong Shen  1 , Steven W Johnson
Affiliations

Subthalamic stimulation evokes complex EPSCs in the rat substantia nigra pars reticulata in vitro

Ke-Zhong Shen et al. J Physiol. .

Abstract

The subthalamic nucleus (STN) plays an important role in movement control by exerting its excitatory influence on the substantia nigra pars reticulata (SNR), a major output structure of the basal ganglia. Moreover, excessive burst firing of SNR neurons seen in Parkinson's disease has been attributed to excessive transmission in the subthalamonigral pathway. Using the 'blind' whole-cell patch clamp recording technique in rat brain slices, we found that focal electrical stimulation of the STN evoked complex, long-duration excitatory postsynaptic currents (EPSCs) in SNR neurons. Complex EPSCs lasted 200-500 ms and consisted of an initial monosynaptic EPSC followed by a series of late EPSCs superimposed on a slow inward shift in holding current. Focal stimulation of regions outside the STN failed to evoke complex EPSCs. The late component of complex EPSCs was markedly reduced by ionotropic glutamate receptor antagonists (2-amino-5-phosphonopentanoic acid and 6-cyano-7-nitro-quinoxalone) and by a GABAA receptor agonist (isoguvacine) when these agents were applied directly to the STN using a fast-flow microapplicator. Moreover, the complex EPSC was greatly enhanced by bath application of the GABAA receptor antagonists picrotoxin or bicuculline. These data suggest that recurrent glutamate synapses in the STN generate polysynaptic, complex EPSCs that are under tonic inhibition by GABA. Because complex EPSCs are expected to generate bursts of action potentials in SNR neurons, we suggest that complex EPSCs may contribute to the pathological burst firing that is associated with the symptoms of Parkinson's disease.

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Figures

Figure 1
Figure 1. Two types of neuron in the SNR
A, B and C show characteristics of presumed GABA-containing neurons. D, E and F show characteristics of presumed dopamine-containing neurons. A and D, photomicrographs of biocytin-filled neurons in 200 μm thick brain slices. B and E, current-clamp recordings from the two types of SNR neuron (0 pA holding current). Note that both cells fire action potentials spontaneously and regularly (12 Hz versus 2.5 Hz). C and F, voltage-clamp recordings showing responses to voltage steps (400 ms duration) from −70 mV to −140 mV (10 mV increments). Note that the presumed GABA neuron had a small Ih, whereas the presumed dopamine neuron had a pronounced Ih. All subsequent data were obtained from presumed GABA-containing neurons as identified by the electrophysiological characteristics illustrated in B and C.
Figure 2
Figure 2. Monosynaptic and polysynaptic components of complex EPSCs
A, a single electrical stimulus delivered to the STN evoked a complex EPSC with early (indicated by an arrow) and late components. Note the notches and inflections on the rising and decay phases in the late component. B, a complex EPSC evoked in the same neuron shown in A with lower stimulus intensity. Note the different time scale for this trace. C, three consecutive traces are superimposed that illustrate early and late components of complex EPSCs. The early component (indicated by arrow) had a constant latency and amplitude, suggesting that it was monosynaptic. EPSCs in the late component had variable latencies, which suggests a polysynaptic aetiology. Note that the late component EPSC amplitudes summate, which contributes to a long-lasting inward current that contains many notches and inflections. Numbers above traces are stimulation strengths 0.04 and 0.02 mA.
Figure 3
Figure 3. Stimulus-dependent characteristics of complex EPSCs
A, electrical stimulation of the slice evoked complex EPSCs only when the stimulation electrodes were placed in the STN (a). Electrodes placed rostral (b) or lateral (c) to the STN only evoked monosynaptic IPSCs. Moving electrodes medial (d) or caudal (e) to the STN failed to evoke complex EPSCs. Returning electrodes to the STN (f) evoked the complex EPSC. All traces in A are from the same neuron. Ba, effects of increasing stimulus intensities on EPSCs evoked by stimulation of the STN. Two responses are superimposed for each stimulation intensity. Note that the amplitude and latency of the early component (indicated by arrows) are relatively constant despite increasing stimulation strengths. However, the amplitudes of EPSCs in the late, polysynaptic component markedly increased with higher stimulation strengths. Recordings are from the same SNR neuron; stimulus intensity was 0.1 mA. Bb, local stimulation of the SNR near the recording site evoked only short latency, monosynaptic EPSCs in an SNR neuron. Note that the EPSC amplitude increased markedly at higher stimulation strengths. C, the amplitude of the complex EPSC increased with increasing number of electrical stimuli delivered to the STN. Numbers in parentheses indicate number of stimuli delivered. D, the complex EPSC is voltage dependent. The amplitude of the complex EPSC was larger at less hyperpolarized test potentials.
Figure 4
Figure 4. Polysynaptic characteristics of complex EPSCs
A, complex EPSC evoked in a SNR neuron under control conditions. B, EPSCs recorded during a 2 s train of stimuli delivered to the STN at 20 Hz. C, final EPSC evoked at the end of the 20 Hz train of stimuli. Note that the initial EPSC was relatively intact, whereas the late component of the complex EPSC was much reduced. Recordings in A, B and C are from the same neuron. D, superfusate containing high concentrations of divalent cations (4.2 mm Mg2+ and 7.0 mm Ca2+) reversibly reduced the late component of the complex EPSC. Note that the early, monosynaptic component, which is indicated by arrows, was relatively resistant to inhibition by the high divalent solution.
Figure 5
Figure 5. Local application of glutamate to the STN evokes EPSCs in SNR neurons
A, local fast-flow application of glutamate (100 μm) to the STN caused an inward shift in holding current and a significant increase in EPSC frequency in an SNR neuron. B, local application of glutamate near the recording site (SNR) produced an inward shift in holding current but failed to evoke spontaneous EPSCs. C, local application of glutamate to the internal capsule evoked no change in holding current or EPSC frequency. All recordings were made in the same SNR neuron.
Figure 6
Figure 6. Pharmacology of complex EPSCs
A, bath application of AP5 (50 μm) and CNQX (10 μm) blocked complex EPSCs. AP5, applied alone, markedly attenuated the complex EPSC (b). Although CNQX nearly abolished the complex EPSC (c), increasing the stimulus number to two (10 ms interval) caused an increase in amplitude of the complex EPSC despite continued application of CNQX (d). However, the combined application of AP5 and CNQX completely abolished the complex EPSC despite double stimuli. B, local, fast-flow application of AP5 (50 μm) and CNQX (10 μm) to the STN significantly reduced the late, polysynaptic component of the complex EPSC, whereas the early, monosynaptic component (indicated by arrows) was much less affected. Note that pairs of stimuli were used to evoke complex EPSCs. C, local application of AP5 and CNQX to the internal capsule did not affect the early or late component of the complex EPSC.
Figure 7
Figure 7. GABAA receptor blockade augments complex EPSCs
A, bath application of picrotoxin (100 μm) greatly increased the amplitude of the complex EPSCs evoked by STN stimulation. Arrows below traces indicate the early, monosynaptic component of the complex EPSC. Note that picrotoxin affects the late component much more than the initial EPSC. B, summary graph showing the time course for potentiation of the integrated area of the complex EPSC by picrotoxin (100 μm). Each data point is the mean ± s.e.m. of 5 SNR neurons.
Figure 8
Figure 8. Stimulation of GABAA receptors in the STN suppresses complex EPSCs
A, local, fast-flow application of the GABAA agonist isoguvacine (100 μm) to the STN completely abolished the complex EPSC recorded in an SNR neuron. Note that isoguvacine did not significantly shift holding current. B, local application of isoguvacine to the internal capsule had no effect on complex EPSCs or holding current recorded in the same SNR neuron. C, local application of isoguvacine to the SNR near the recording site caused an outward shift in holding current while only partially reducing the complex EPSC amplitude. D, summary graph showing the time course for suppression of the complex EPSC by isoguvacine. The complex EPSC is abolished when isoguvacine (100 μm) was applied directly to the STN (□), whereas it was only partially reduced when isoguvacine was applied near the recording site (▪). Each data point is the mean ± s.e.m. of 3–4 SNR neurons.
Figure 9
Figure 9. Polysynaptic currents in STN and burst firing in SNR neurons
A, low intensity stimulation of the STN near the recording site evoked multiple EPSCs with variable latencies in this STN neuron. Three superimposed responses are shown for each stimulation intensity. B, electrical stimulation of the STN evoked bursts of action potentials in this SNR neuron recorded under current clamp. A burst of 7–12 spikes was evoked by each stimulus. C, a burst of spikes evoked by STN stimulation is shown on an expanded time base. This recording is from the same SNR neuron as shown in ‘B’. The arrow indicates stimulus artifact.
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