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Comparative Study
. 2009 Sep 2;29(35):11011-9.
doi: 10.1523/JNEUROSCI.2519-09.2009.

Robust pacemaking in substantia nigra dopaminergic neurons

Jaime N Guzman  1 , Javier Sánchez-Padilla, C Savio Chan, D James Surmeier
Affiliations
Comparative Study

Robust pacemaking in substantia nigra dopaminergic neurons

Jaime N Guzman et al. J Neurosci. .

Abstract

Dopaminergic neurons of the substantia nigra pars compacta are autonomous pacemakers. This activity is responsible for the sustained release of dopamine necessary for the proper functioning of target structures, such as the striatum. Somatodendritic L-type Ca2+ channels have long been viewed as important, if not necessary, for this activity. The studies reported here challenge this viewpoint. Using a combination of optical and electrophysiological approaches in brain slices, it was found that antagonism of L-type Ca2+ channel effectively stopped dendritic Ca2+ oscillations but left autonomous pacemaking unchanged. Moreover, damping intracellular Ca2+ oscillations with exogenous buffer had little effect on pacemaking rate. Although not necessary for pacemaking, L-type channels helped support pacemaking when challenged with cationic channel blockers. Simulations suggested that the insensitivity to antagonism of L-type channels reflected the multichannel nature of the pacemaking process. The robustness of pacemaking underscores its biological importance and provides a framework for understanding how therapeutics targeting L-type Ca2+ channels might protect dopaminergic neurons in Parkinson's disease without compromising their function.

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Figures

Figure 1.
Figure 1.
Low concentrations of DHPs suppress dendritic Ca2+ oscillations but do not slow pacemaking. a, Digitized cell-attached patch recordings from an SNc DA neuron before and after application of isradipine (5 μm). The median discharge rate before isradipine application was 2.2 and 2.4 Hz after (p > 0.05; n = 10). b, Whole-cell recording from the cell shown to the left (projection image) before and after isradipine (5 μm) application; there was no significant change in discharge rate in this cell or in 10 others. At the bottom, two-photon laser scanning microscopy measurements of Fluo-4 fluorescence (G) at a proximal dendritic location (∼40 μm from the soma) normalized by the fluorescence of the red Alexa dye used to image the cell. c, Somatic recording during imaging at a more distal dendritic location (∼120–200 μm from the soma). Note the complete elimination of the spike-associated dendritic Ca2+ transient at the distal imaging site. Similar results were obtained in six other neurons. d, All points histogram of fluorescence at the distal location of the cell in c showing that the median fluorescence was reduced by isradipine. Similar results were obtained in six cells. AU, Arbitrary units. e, Summary showing that, in whole-cell recordings in which imaging was done, isradipine did not change the discharge rate (p > 0.05; n = 10). f, Spectral analysis of the fluorescence signal readily detected oscillations at the pacemaking frequency; power at this frequency was eliminated by isradipine. Similar results were obtained in six cells.
Figure 2.
Figure 2.
Submicromolar levels of DHPs are sufficient to block SOPs without compromising pacemaking activity. a, Representative SOP traces (after blockade of sodium channels with 1 μm TTX) in the presence or absence of isradipine (5 μm). Micromolar concentrations of isradipine (5 μm) blocked the TTX-insensitive SOPs (n = 10 cells). b, Submicromolar concentrations of DHPs (200 nm isradipine) had no effect in pacemaking firing rate (p > 0.05; n = 5 cells). c, Representative current-clamp trace showing no SOPs of a DA neuron pretreated with 200 nm isradipine for 1 h (n = 5 cells). d, Summary box plots showing that 200 nm isradipine had no effect in spike rate. e, Spectral analysis of the fluorescence showing a significant reduction in the power of the frequency detected in control, highly reduced power in the presence of 200 nm isradipine and complete elimination of frequency by 5 μm isradipine.
Figure 3.
Figure 3.
SOPs are absent after chelation of intracellular calcium. a, Recording from an SNc DA neuron with an electrode containing 5 mm BAPTA. Before break-in, the pacemaking rate could be seen; after break-in, the rate slowly increased and the afterhyperpolarization became less pronounced, as shown in d. Similar results were obtained in six cells. b, Application of TTX demonstrated the absence of SOPs in the presence of BAPTA. (n = 4). c, Voltage traces from b early (black trace) and later (red trace) in the dialysis with BAPTA showing the change in the afterhyperpolarization. d, The discharge rate before application of TTX is plotted against the frequency of SOP oscillation. Note the scatter around the line with a slope of 1 (n = 19). e, Plots of successive spike intervals or successive SOP intervals (measured from the point at which the voltage reached the median voltage of the oscillation). A sample of nine cells is plotted. Note the tight clustering of the spike data and the dispersion of the SOP data.
Figure 4.
Figure 4.
Dendritic Ca2+ oscillations are phase locked to somatic spiking. a, Projection image of an SNc DA neuron recorded from with a somatic patch electrode containing Alexa 594 (50 μm) and the Ca2+ dye Fluo-4 (200 μm). To the right are somatic voltage recordings during pacemaking. At the bottom are averages of the fluorescence changes (G/R) at two dendritic locations shown by blue (proximal) and red (distal) dots. The averages were computed by aligning segments of the fluorescence records of 10 successive spikes; because the timing of the next spike varied, the timing of the fluorescence change after the initial spike in the average is only approximate and was used to gauge the phase relationship of the Ca2+ oscillation. b, A simulation using a single compartment model showing the relationship between the voltage (black trace) and the rise in intracellular Ca2+ concentration (red) before and after block of Na+ channels; note the similarity between these records and the data in a where Ca2+ concentration rises just before the spike and peaks after the spike, falling during the afterhyperpolarization. c, Recording from an SNc DA neuron in which ectopic spikes were introduced by brief (2 ms) current pulses; the green bar shows the timing of the ectopic spikes. d, Histogram of the intervals during normal pacemaking (black filled) and the intervals after an ectopic spike (green filled) showing that it generated an interval typical of normal pacemaking, regardless of its phase relationship to the ongoing spiking. Similar behavior was seen in all four cells examined in this way. e, At the top, somatic voltage records from one of these experiments showing the ectopic spike; at the bottom, fluorescence records (G/R) at the same locations shown in a, but now aligned with the ectopic spike. Note that, at the proximal location, the ectopic spike evoked a Ca2+ transient but not at the distal location; nevertheless, the oscillation in the Ca2+ fluorescence was reset to coincide with the next spike in the series.
Figure 5.
Figure 5.
Simulations of pacemaking behavior and sensitivity to channel antagonism. a, Voltage measurements from a single compartment simulation of an SNc DA neuron before and after removal of Na+ channels, showing SOP-like behavior, and then after the additional removal of Cav1.3 Ca2+ channels, showing cessation of the SOP. Restoring the channels led to restoration of pacemaking. Inset is intracellular Ca2+ concentrations in the model before and after removal of Na+ channels. Timing of the changes is shown in the bars at the top. b, Voltage measurements of a simulation in which there was successive removal of first Cav1.3 and then combined removal of HCN and Cav1.3 channels. Note that pacemaking stopped only with the removal of both channels. c, Digitized cell-attached patch recordings before and after application of ZD 7288 (10 μm) showing no significant change in discharge rate (see summary in d). At the bottom, digitized cell-attached patch recordings before and after application of ZD 7288 (10 μm) and isradipine (5 μm) showing silencing of the cell. d, Summary of experiments as in c. The fall in discharge rates with coapplication of ZD 7288 (10 μm) and isradipine (5 μm) or with application of high concentrations of ZD 7288 (100 μm) alone was significant (**p < 0.01). Sample sizes were control (n = 6), isradipine (5 μm; n = 7), low ZD 7288 (10 μm; n = 8), low isradipine plus low ZD 7288 (n = 5), and high ZD 7288 (100 μm; n = 4).
Figure 6.
Figure 6.
Pacemaking in DA neurons silenced with high DHP is restored by blocking Kv1 channels. a, NEURON simulation model of pacemaking firing previous and after blockade of Cav1.3, 20% reduction in sodium current, and 20% reduction in leak currents. Combined partial blockade of these inward currents silenced pacemaking as in the experimental conditions described below (b, c). Spiking activity was restored on blockade of Kv1 channels. b, Cell-attached patch recording from an SNc DA neuron that had been silenced by isradipine (20 μm) and then restarted by bath application of α-dendrotoxin (DTX; 100 nm). c, Summary of similar experiments showing discharge rates in control (n = 8), isradipine exposed (n = 6), and then cells exposed to either α-dendrotoxin (100 nm; n = 6) or 4-aminopyridine (100 μm; n = 6); the change in discharge rate after exposure to either toxin was significant (**p < 0.01) when compared with the isradipine-induced silenced state.
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