D5 (not D1) dopamine receptors potentiate burst-firing in neurons of the subthalamic nucleus by modulating an L-type calcium conductance

Abstract

Dopamine is a crucial factor in basal ganglia functioning. In current models of basal ganglia, dopamine is postulated to act on striatal neurons. However, it may also act on the subthalamic nucleus (STN), a key nucleus in the basal ganglia circuit. The data presented here were obtained in brain slices using whole-cell patch clamp. They reveal that D5 dopamine receptors strengthen electrical activity in the subset of subthalamic neurons endowed with burst-firing capacity, resulting in longer discharges of spontaneous or evoked bursts. To distinguish between D1 and D5 subtypes, the action of agonists in the D1/D5 receptor family was first investigated on rat subthalamic neurons. Single-cell reverse transcription-PCR profiling showed that burst-competent neurons only expressed D5 receptors. Accordingly, receptors localized in postsynaptic membranes within the STN were labeled by a D5-specific antibody. Second, agonists in the D1/D5 family were tested in mouse brain slices. It was found that these agonists were active in D1 receptor knock-out mice in a similar way to wild-type mice or rats. This proved that D5 rather than D1 receptors were involved. Pharmacological tools (dihydropyridines, omega-conotoxins, and calciseptine) were used to identify the target of D5 receptors as an L-type channel. This was reached via G-protein and protein kinase A. The action of dopamine on D5 receptors therefore shapes neuronal activity. It contributes to normal information processing in basal ganglia outside striatum. This finding may be useful in drug therapy for various disorders involving changes in STN activity, such as Parkinson's disease and related disorders.

Fig. 1.

Activation of receptors in the D1 family strengthens spontaneous burst-firing. A, Representative examples of spontaneous burst-firing in a subthalamic neuron. The recording was made in the presence of synaptic transmission blockers (APV, 40 μm; CNQX, 10 μm; bicuculline, 10 μm) for this experiment as with all others, and at zero current level. Burst duration was notably increased during perfusion of SKF 82958 (5 μm). B, Top, The duration of spontaneous bursts recorded in the cell-attached configuration is made longer by SKF 81297 (5 μm).Calibration: 10 pA, 3 sec. Bottom, Representative bursts on an expanded time scale. C, A box plotsummary of the changes in typical burst features with SKF 82958 or SKF 81297 (5 μm). The central line in thebox shows the distribution median. Theedges of the box are the interquartiles. The lines running from the edge of thebox show the distribution extremes. Thesquare displays the mean. n, Number of experiments.

Fig. 2.

Action of agonists of receptors in the D1 family on burst-competent neurons. A, Burst-competent neurons switch from regular, single-spike firing mode at zero current level to burst-firing with negative current injection. B,C, Depolarizing or hyperpolarizing stimuli trigger plateau potentials. The two regenerative discharges markedly outlast the stimulus. They are potentiated by SKF 81297 (3 μm). Potentiation is reversed by the D1-like receptor-specific antagonist, SCH 23390 (10 μm).

Fig. 3.

Agonists of receptors in the D1 family increase the duration of the regenerative depolarization.A, Superimposed records of plateau potentials in the presence of TTX (1 μm) and with Ba²⁺replacing Ca²⁺. Washing off of SKF 82958 partially restored the surface of the plateau. The inset shows a plateau potential recorded in normal Ringer's solution, in addition to that recorded during bath perfusion of TTX, with Ba²⁺ instead of Ca²⁺.B, Time course of the action of D1-like agonists (SKF 81297 and 82958; 3–5 μm). The bar shows the addition of agonists to bath perfusion. *Significantly different from pretest values. C, The D1 receptor antagonist, SCH 23390, reversed the action of SKF 81297, whereas the selective D2 receptor antagonist, raclopride, had no effect, as illustrated by representative traces of plateau potentials. Box plotsof the changes in the plateau potential surface induced by D1-like agonists (3–5 μm) together with SCH 23390 (10 μm) or raclopride (5 μm) substantiate this finding.

Fig. 4.

Calcium-activated channels are not involved in potentiation of plateau potential. A, Activation of receptors in the D1 family still increases plateau potential when free cytosolic Ca²⁺ is heavily buffered by BAPTA (10 μm) in the intrapipette solution. TEA (20 mm) and TTX (1 μm) were added to the recording solution.B, Box plots present the changes in plateau potential surface induced by D1 agonists (3–5 μm) in three experimental conditions designed to inhibit Ca²⁺-activated channels: top, with 20 mm TEA in the perfusion solution, to block calcium-activated potassium current; middle, with 10 mm BAPTA in the intrapipette medium; bottom, with choline replacing Na⁺, to block Ca²⁺-activated nonspecific channels, in addition to TEA (20 mm).

Fig. 5.

Potentiation of plateau potential involves L-type calcium channels. A, Time course of the action of the dihydropyridine, L-type channel antagonist, nifedipine. Nifedipine prevented the action of SKF 81297. The inset displays superimposed sample traces taken from positions1-3. Calibration: 10 mV, 500 msec. TEA (20 mm) and TTX (1 μm) were present.B, The increase in the surface of plateau potential by SKF 81297 (3–5 μm) is reversed by perfusion of nifedipine. Note that nifedipine (6 μm) abolishes the plateau potential. Inset, Superimposed traces taken from positions 1-3. Calibration: 10 mV, 500 msec. C, The dihydropyridine, L-type channel agonist, BayK 8644, occluded the effect of SKF 81297, as illustrated by representative traces of plateau potentials obtained successively in control, during perfusion of BayK 8644 alone, and during perfusion of BayK 8644 with SKF 81297.Box plots summarize the changes in the surface of plateau potential induced by BayK 8644 and by nifedipine (data fromA and B). D, Coapplication of N- and P/Q-type calcium channel blockers, ω-conotoxin MVIIC (250 nm) and ω-conotoxin GVIA (500 nm), reduced plateau potential. However, addition of 3 μm SKF 81297 in presence of both toxins significantly increased plateau potential (trace 3), indicating that N- and P/Q-type Ca²⁺ channels were not involved. Perfusion of calciseptine (200 nm), a specific L-type channel antagonist, occluded the action of SKF 81297 and abolished the plateau potential (trace 4). Box chartssummarize the results from several tests (*significant atp < 0.05).

Fig. 6.

Expression of D5 receptor in subthalamic neurons.A, Left, Sample records of plateau potentials and PCR amplicons from an SKF 81297-sensitive, burst-competent neuron. Right, RT-PCR products obtained from whole-brain RNA extract. D5 receptor mRNA was only detected by single cell-RT PCR amplification, whereas both D1 and D5 receptor mRNAs were detected from whole-brain tissue. Ethidium bromide-stained products of RT-PCR amplifications were resolved by electrophoresis. The positions of standard nucleotide bands of molecular weight markers (M) are indicated to theright of the gels. B, Dopamine D5 receptor immunoperoxidase staining of a coronal section of rat brain shows immunoreactivity in many neuronal cell bodies within the STN.C, Electron micrograph revealing labeling of D5 receptors (top arrow) in a dendrite (d) making asymmetric synapse (arrowheads) with an axon (a). Labeling (bottom arrows) is also associated with microtubules and endoplasmic reticulum. Scale bars: B, 200 μm; C, 500 nm.

Fig. 7.

Potentiation of plateau potential is caused by D5, not D1, receptors. A, Mouse subthalamic burst-competent neurons display firing properties similar to that of rat neurons. Burst-firing is induced by injecting a negative current. Depolarizing (+80 pA; 200 msec) and hyperpolarizing (−80 pA, 1 sec) stimuli produce long-lasting plateau potential and postinhibitory rebound burst, respectively. B, Sample records of plateau potentials from mouse neurons in the presence of TTX (1 μm) and TEA (20 mm). Wild-type and D1 receptor null mutant (D1 −/−) mice displayed strong plateau potentials when stimulated by short current pulses (+80 pA, 200 msec). The agonist of receptors in the D1 family, SKF 81297 (3–5 μm), was active in neurons from the D1 −/− mice as well as in neurons from wild-type mice. The increase in surface of the plateau potential measured in D1 −/− mice was not significantly different (p = 0.99; Mann–Whitney U test) from that measured in their wild-type counterpart, as summarized by the box plots.

Fig. 8.

G-protein and protein kinase A are involved in the action of D1-like, presumably D5, receptor. A, Time course of changes in plateau potential surface during recording with a pipette medium containing GTP-γ-S and GDP-β-S. The sample records1, 2, and 3 were taken at the beginning and after 6 and 11 min of perfusion, respectively.B, Representative examples and time course of changes in plateau potential during perfusion of a membrane-permeant antagonist of protein kinase A, H-89 (1–3 μm). Addition of SKF 81297 (3 μm) failed to increase plateau potential. Perfusion of the membrane-permeant cAMP analog, 8-bromo-cAMP (10 μm), per se potentiated plateau potentials. Box plotssummarize the results of the trials with H-89 and 8-bromo-cAMP.