L-Type calcium channels mediate a slow excitatory synaptic transmission in rat midbrain dopaminergic neurons

Abstract

Patch pipettes were used to record whole-cell synaptic currents under voltage-clamp in dopaminergic neurons in slices of rat substantia nigra pars compacta and ventral tegmental area. We report that dihydropyridines (DHPs), L-type Ca2+ channel antagonists, depressed a slow EPSC (EPSCslow) evoked by a train of focally delivered electrical stimuli. In fact, the amplitude of the EPSCslow was reduced by the DHP antagonists nifedipine (1-100 microM), nimodipine (1-100 microM), and isradipine (30 nM-100 microM) in a concentration-dependent and reversible manner. On the other hand, Bay-K 8644 (1 microM), an L-type Ca2+ channel agonist, increased the EPSCslow. The DHPs depressed the EPSCslow only when the high-frequency stimulation that was used to evoke this synaptic current lasted >70 msec. On the other hand, Bay-K 8644 increased the amplitude of the EPSCslow only when it was evoked by a train <70 msec. Moreover, the DHPs did not affect the EPSCfast, the IPSCfast, and the IPSCslow. The inhibition of the EPSCslow caused by the DHPs is attributed to presynaptic mechanisms because (1) the inward current generated by exogenously administered glutamate was not affected and (2) the EPSCslow was reduced to a similar degree even when the activation state of postsynaptic L-type Ca2+ channels was changed by holding the neurons at -100, -60, and +30 mV. Finally, a DHP-sensitive component of the EPSCslow could even be detected after the blockade of N-, Q-, and P-type Ca2+ channels by the combination of omega-conotoxin GVIA, omega-agatoxin IVA, and omega-conotoxin MVIIC. Taken together, these results indicate that under certain patterns of synaptic activity, L-type Ca2+ channels regulate the synaptic release of excitatory amino acids on the dopaminergic neurons of the ventral mesencephalon.

Fig. 1.

Properties of the EPSC_slow.A, The graph indicates that the amplitude of the EPSC_slow is dependent on the duration of the train. The amplitude of the synaptic current was measured after the stimulation. Each point represents at least three experiments. Ba, The NMDA antagonist APV depresses the EPSC_slow in a reversible manner, whereas the AMPA/kainate antagonist CNQX (10 μm) did not produce any effect on the EPSC_slow. The stimulus artifacts were blanked (records are average of 4 sweeps). Bb, The metabotropic antagonist (+)-MCPG (300 μm) also depresses the EPSC_slowin a reversible manner.

Fig. 2.

Effects of dihydropyridines on the slow excitatory synaptic transmission. The traces represent the EPSC_slow in control, during and after (wash) the effect of nifedipine (3 μm), nimodipine (10 μm), and isradipine (1 μm). The bottom traces inAa, Ab, and Ac are superimposed (control vs the effect of the L-type Ca²⁺ antagonists). B, Dose–response plots of the inhibition of the EPSC_slow caused by nifedipine (left), nimodipine (middle), and isradipine (right). Each point is an average of at least four different experiments. Only one experiment per slice was performed. To calculate the percentage inhibition of the EPSC_slow, each cell was taken as its own control.

Fig. 3.

A, The percentage of inhibition of the EPSC_slow caused by nifedipine is dependent on the duration of the electrical stimulation. Aa, Superimposed EPSC_fast in control condition and after the application of nifedipine. Ab, Ac, A 50 msec and a 200 msec train of stimuli were delivered to evoke EPSCs_slow. In the presence of nifedipine, the EPSC_slow evoked by the longer train (c) was depressed, but the EPSC evoked by the short train (b) was not affected. The time and current bars in c are also valid for b. Note that the EPSC_fast in a and the EPSC_slow in c were elicited alternately in the same neuron. B, The graph shows that the depression of the EPSC_slow caused by nifedipine (10 μm) is dependent on the duration of the electrical stimulus.

Fig. 4.

A, Nifedipine (10 μm) had no effect on the amplitude of the GABA_A(a) and GABA_B(b) IPSCs. The GABA_A current is inward because the recording pipette was filled with a solution containing potassium chloride (see Materials and Methods). Ba, Bb, Superimposed traces showing that the L-type Ca²⁺ channel agonist Bay-K 8644 (1 μm) increased the EPSC_slow evoked by a short train (a) but did not augment the EPSC_fast(b). The EPSC_fast and the EPSC_slow were elicited alternatively in the same cell. c, Plot taken from the same dopamine neuron showing the time course of the enhancing effect of Bay-K 8644 on the EPSC_slow.

Fig. 5.

Presynaptic effects of nifedipine.a, Changes in the holding potential from −60 mV to 30 mV or −100 mV did not affect the depression of the EPSC produced by nifedipine. Typical traces taken form experiments showing the similar degree of inhibition produced by nifedipine (10 μm).b, The horizontal columns show that the percentage of inhibition caused by nifedipine (10 μm) at 30 mV, −60 mV, and −100 mV is not statistically different among the three groups of neurons tested (p > 0.05). Each column represents an average of at least five cells. To improve space-clamp between proximal somatic and distal dendritic regions, theI_h current was also reduced by extracellular CsCl (1 mm) during the hyperpolarization.

Fig. 6.

a, Traces from an experiment in which a 200 msec train was given alternately with a 50 msec extracellular (puff) application of glutamate (1 mm). Nifedipine (10 μm) decreased the amplitude of the EPSC_slow but not the inward current evoked by the local application of glutamate. b, The plot represents the time course of the experiment shown ina.

Fig. 7.

A, The graph shows the depressant effects of ω-conotoxin GVIA (1 μm), ω-AGA-IVA (200 nm), and ω-conotoxin MVIIC (5 μm) on the EPSC_slow evoked by a short train of stimuli. Note that nifedipine (30 μm) added to the three toxins did not depress the synaptic current. A, B, In the presence of ω-conotoxin GVIA (1 μm), ω-AGA-IVA (200 nm), and ω-conotoxin MVIIC (5 μm), nifedipine (30 μm) reversibly depressed the residual EPSC_slow. The residual synaptic current recorded after the treatment with the toxins was blocked by cadmium (300 μm). Ba, Sample records of a cell obtained at the times indicated by the numbers 1–6 in the graph.Bb, Graph of the amplitude of the EPSC _slowduring the application of the natural toxins nifedipine (30 μm) and cadmium (300 μm). Each point is an average of four experiments performed on four different slices.