SK2 and SK3 expression differentially affect firing frequency and precision in dopamine neurons

Abstract

The firing properties of dopamine (DA) neurons in the substantia nigra (SN) pars compacta are strongly influenced by the activity of apamin-sensitive small conductance Ca(2+)-activated K(+) (SK) channels. Of the three SK channel genes expressed in central neurons, only SK3 expression has been identified in DA neurons. The present findings show that SK2 was also expressed in DA neurons. Immuno-electron microscopy (iEM) showed that SK2 was primarily expressed in the distal dendrites, while SK3 was heavily expressed in the soma and, to a lesser extent, throughout the dendritic arbor. Electrophysiological recordings of the effects of the SK channel blocker apamin on DA neurons from wild type and SK(-/-) mice show that SK2-containing channels contributed to the precision of action potential (AP) timing, while SK3-containing channels influenced AP frequency. The expression of SK2 in DA neurons may endow distinct signaling and subcellular localization to SK2-containing channels.

Figure 1

Apamin-sensitive currents recorded from wild type, SK2^−/− and SK3^−/− DA neurons. Pharmacological isolation of the apamin-sensitive SK current in wild type (A), SK2^−/− (B) and SK3^−/− (C) DA neurons. The currents were evoked by a 50 ms depolarizing pulse to 0 mV from −70 mV holding potential, followed by a return to −50 mV. The voltage protocol is schematically presented above the data trace in panel A. The black traces are currents recorded in control solution, while the red traces are currents recorded after apamin (200 nM) application. To obtain the subtracted, apamin sensitive current (inset), each record was zeroed to the mean, steady state current following repolarization to −50 mV. Each trace is the average of four sequential trials. (D) Averages ± S.E.M. for the apamin-sensitive currents for each genotype. Scale bars: current, 0.2 nA, time, 0.2 s.

Figure 2

SK2 and SK3 are expressed in wild type DA neurons. Electron micrographs from SN showing immunoparticles for SK channel subunits and TH immunoreactivity as detected using double labeling immunogold-HRP. (A, B) Immunoparticles for SK2 protein (arrows) were found along the plasma membrane in dendrites (Den) of TH-positive neurons. (D) Immunoparticles for SK3 (arrows) were detected along the plasma membrane of the soma (Cyt, cytoplasm; N, nucleus) and (D) the dendrites (Den) of TH-positive neurons labeled with the HRP reaction product. (C,F) Summary graphs showing the sub-cellular distribution of SK2 and SK3 immunoparticles, respectively. Scale bars: 500 nm.

Figure 3

SK channel activity regulates the timing and frequency of APs in wild type DA neurons. Panels A,B,Ci, and Di are derived from the same representative wild type neuron. (A) Top traces show examples of spontaneous APs recorded in the loose patch configuration from a wild type DA neuron in control solution (black) and after apamin application (red). Below is a diary plot of the mean ISIs binned over x s for the same cell. Apamin was added at time 0. The shaded areas denote the control (black) and apamin (red) ISIs used for graphs and statistical comparisons. (B) Histogram of ISIs recorded in control solution (black) and after apamin application (red) for the indicated period in Panel A. (C) Cumulative probability histograms of ISIs from a representative cell (C_i) and for all wild type cells (C_ii) in control solution (black) after apamin application (red). The black and red points denote the mean ISIs. (D_i) Diary plot of the effect of apamin (added at time 0) on the CV of ISIs. Each point represents the CV calculated from 30 s epochs of ISIs. Error bars are mean ± S.E.M.

Figure 4

Effects of SK channel activity in SK2^−/− DA neurons. Panels A,B,Ci, and Di are derived from the same representative SK2^−/− neuron. (A) Top traces show examples of spontaneous APs recorded in the loose patch configuration from an SK2^−/− DA neuron in control solution (black) and after apamin application (red). Below is a diary plot of the ISIs for the same cell. Apamin was added at time 0. The shaded areas denote the control (black) and apamin (red) ISIs used for graphs and statistical comparisons. (B) Histogram of ISIs recorded in control solution (black) and after apamin application (red). (C) Cumulative probability histograms of ISIs from panel A (C_i) and for all SK2^−/− DA neurons (C_ii) in control solution (black) after apamin application (red). The black and red points denote the mean ISIs. (D) Diary plot of the effect of apamin (added at time 0) on the CV of ISIs. Each point represents the CV calculated from 30 s epochs of ISIs. Error bars are mean ± S.E.M.

Figure 5

SK2-containing channels influence AP timing in SK3^−/− DA neurons. Panels A,B,Ci, and Di are derived from the same representative SK3^−/− neuron. (A) Top traces show example traces of spontaneous APs recorded in the loose patch configuration from an SK3^−/− DA neuron in control solution (black) and after apamin application (red). Below is a diary plot of the ISIs for the same cell. Apamin was added at time 0. The shaded areas denote the control (black) and apamin (red) ISIs used for graphs and statistical comparisons. (B) Histogram of ISIs recorded in control solution (black) and after apamin application (red). (C) Cumulative probability histograms of ISIs from panel B (C_i), and for all SK3^−/− DA neurons (C_ii) shown in control solution (black) after apamin application (red). The black and red points denote the mean ISIs. (D) Diary plot of the effect of apamin (added at time 0) on the CV of ISIs. Each point represents the CV calculated from 30 s epochs of ISIs. Error bars are mean ± S.E.M.

Figure 6

Summary bar graphs showing the effects of apamin (200 nM) on the mean ISI (top) and ISI-CV (bottom) in the different genotypes examined.

Figure 7

AP timing is selectively affected by blocking SK2-containing channels. The effects of 300 pM apamin on wild type DA neurons are shown for individual cells (grey) and population means ± S.E.M. (black). (A,B) Effects of apamin on CV and ISI, respectively. (C) The fold change relative to control for CV and ISI.