Ionic currents and spontaneous firing in neurons isolated from the cerebellar nuclei

Abstract

Neurons of the cerebellar nuclei fire spontaneous action potentials both in vitro, with synaptic transmission blocked, and in vivo, in resting animals, despite ongoing inhibition from spontaneously active Purkinje neurons. We have studied the intrinsic currents of cerebellar nuclear neurons isolated from the mouse, with an interest in understanding how these currents generate spontaneous activity in the absence of synaptic input as well as how they allow firing to continue during basal levels of inhibition. Current-clamped isolated neurons fired regularly ( approximately 20 Hz), with shallow interspike hyperpolarizations (approximately -60 mV), much like neurons in more intact preparations. The spontaneous firing frequency lay in the middle of the dynamic range of the neurons and could be modulated up or down with small current injections. During step or action potential waveform voltage-clamp commands, the primary current active at interspike potentials was a tetrodotoxin-insensitive (TTX), cesium-insensitive, voltage-independent, cationic flux carried mainly by sodium ions. Although small, this cation current could depolarize neurons above threshold voltages. Voltage- and current-clamp recordings suggested a high level of inactivation of the TTX-sensitive transient sodium currents that supported action potentials. Blocking calcium currents terminated firing by preventing repolarization to normal interspike potentials, suggesting a significant role for K(Ca) currents. Potassium currents that flowed during action potential waveform voltage commands had high activation thresholds and were sensitive to 1 mm TEA. We propose that, after the decay of high-threshold potassium currents, the tonic cation current contributes strongly to the depolarization of neurons above threshold, thus maintaining the cycle of firing.

Fig. 1.

Spontaneous firing of neurons isolated from the cerebellar nuclei. A, Representative isolated cerebellar nuclear neurons. Scale bar, 20 μm. B, Spontaneous action potentials recorded from each of the six cells inA, all plotted on the same scale. Records are from cells in the corresponding positions in A. C, Histogram showing the distribution of firing frequencies in all of the cells that were tested (n = 50).

Fig. 2.

Firing patterns evoked by current injections.A, Top, Spontaneous firing and responses to steady injections of hyperpolarizing current, as labeled, of a single neuron. A, Bottom, Firing frequency versus injected current for the same neuron;R² = 0.9997. B, Responses of a different neuron to depolarizing and hyperpolarizing current steps. First panel, Depolarization block elicited by a 50 pA depolarizing step. Second panel, Increased firing rate with a 20 pA depolarizing step. Third panel, Input resistance (672 MΩ), as measured with hyperpolarizing current steps. For clarity, not all of the traces that were used to calculate input resistance are shown. Thearrow indicates depolarizing sag. Fourth panel, Current steps. C, Firing frequency versus input resistance for seven “silent” neurons (filled symbols) and 36 spontaneously firing cells (open symbols). Firing frequency of spontaneous cells did not correlate with input resistance (line,R² = 0.0008). D, Resting potential of a silent neuron (−35 mV) and regular firing induced by a 20 pA injection of steady hyperpolarizing current.

Fig. 3.

TTX-sensitive sodium currents elicited by step depolarizations. A, TTX-sensitive sodium currents evoked by step depolarizations from −90 mV in 2 mV increments. For clarity, only alternate traces are shown positive to −20 mV. B, Peak current versus voltage relation of traces in A.Solid line is a straight line fit to the linear portion of the curve from which the reversal potential,V_rev, was extrapolated.C, Conductance–voltage relation (open symbols) and steady-state availability curve (closed symbols) for the neuron for which the responses are illustrated in A. Activation data were fit as G= G_max/(1 + exp [− (V −V_half)/k]), whereG_max is the maximal conductance,V_half is the half-maximal activation voltage, and k is the slope factor. Inactivation (availability) data were fit as the % availability = 100/(1 + exp [(V −V_half)/k]), where the percentage of availability is the current evoked at 0 mV after a 200 msec conditioning step that is normalized by the maximal current evoked at 0 mV after conditioning at −90 mV, V_halfis the half-maximal inactivation voltage, and k is the slope factor. Parameters of the fits are indicated on the plot.D, Recovery from inactivation of TTX-sensitive sodium currents. The variable recovery interval at −65 mV, Δt, ranged from 2 to 68 msec, in 3 msec increments. In this and all other figures of currents, the horizontal dashed line indicates 0 pA. E, Time course and extent of recovery of transient current shown in D. The peak current evoked by the test step to 0 mV was normalized to the peak current evoked by the conditioning step to 0 mV and was plotted against the recovery interval. Data were fit with a single exponential that estimated a maximum of 48% recovery, with a time constant of recovery, τ, of 12.2 msec.

Fig. 4.

TTX-sensitive sodium currents evoked with spike-train command waveforms. A, Top, Voltage-clamp command waveform, consisting of a prerecorded train of spontaneous action potentials with average frequency, peak potential, and trough potential. A, Bottom, Mean TTX-sensitive current elicited by five spike-train commands. The intersweep holding potential was −68 mV for 2 sec. B,Top, Resumption of spontaneous firing after release from voltage clamp at −68 mV. B, Bottom, Differentiation of the current-clamp record, showing the decrease in peak dV/dt of each spike over the first 10–15 action potentials. C, Top, The ninth action potential waveform of the spike-train command (A), with (thin trace) and without (bold trace) a 10 mV DC hyperpolarization.C, Bottom, TTX-sensitive current evoked by the control (thin trace) and DC-shifted (bold trace) voltage command. This is a different cell fromA.

Fig. 5.

Long-lasting TTX-sensitive sodium currents.A, Top, TTX-sensitive sodium currents evoked by step depolarizations from −90 mV in 10 mV increments.A, Bottom, Current–voltage relation of the mean steady-state current measured between 90 and 100 msec (within the dashed box of the top panel). This is the same record as Figure 3A. B, Mean of the last three traces in Figure 3D, with recovery intervals of 62, 65, and 68 msec, expanded to illustrate the TTX-sensitive current on repolarization to −65 mV. The small resurgent sodium current decayed with an exponential time constant of 12 msec to −4 pA. C, Expansion of eighth and ninth spike command and evoked currents of Figure 4A, illustrating the interspike TTX-sensitive current. D, Current-clamp records of spontaneous firing in control Tyrode's and subsequent silencing on exposure to TTX, as labeled, in one neuron. In TTX the cell rested at −42 mV.

Fig. 6.

Firing patterns of neurons during blockade of calcium currents and calcium-activated currents. A, Spontaneous firing in control Tyrode's and subsequent silencing on exposure to Co Tyrode's. In Co Tyrode's the neuron rested at −37 mV.B, Responses in control Tyrode's (dotted line) and Co Tyrode's (solid line) to depolarizing current steps superimposed on a steady hyperpolarizing current. The peak amplitude of the first action potential depolarized by 9 mV in Co Tyrode's, and the trough depolarized by 7 mV.C, Spontaneous firing of a single neuron in control Tyrode's and in Tyrode's with 100 μm bicuculline methiodide (BMI). D, Firing frequencies of seven cells in control solutions and on exposure to 100 μm BMI (open symbols). Closed symbols show the mean data.

Fig. 7.

Total TTX-insensitive currents evoked by step and spike-train voltage-clamp commands in physiological solutions.A, Top, Middle, Raw currents evoked by step depolarizations in control Tyrode's with TTX (Ca Tyr +TTX) and Co Tyrode's with TTX (Co Tyr + TTX). A,Bottom, Cobalt-sensitive currents, obtained by subtraction of the currents in the top andmiddle panels. B, Current–voltage relation of peak currents in A. C, Raw current elicited by the spike-train command in control Tyrode's with TTX. Intersweep holding potential was −68 mV for 2 sec; mean of five sweeps. D, Top, Expansion of current within dashed lines in C to illustrate TTX-insensitive inward current at interspike potentials.D, Middle, Currents evoked in Co Tyrode's by the spike-train command. The illustrated portion of the record corresponds to that shown in the top panel.D, Bottom, Cobalt-sensitive currents, obtained by subtraction of currents in the top andmiddle panels.

Fig. 8.

Potassium currents pharmacologically isolated with cobalt substitution and 1 mm TEA. A,B, Currents evoked by step and spike-train voltage-clamp commands (top panels). Time scale bars inA and B apply to all panels inA and B, respectively. Vertical scale bars in B apply to the corresponding panels inA. Currents evoked by spike-train commands are the mean of five traces. Intersweep holding potential was −68 mV for 2 sec. This is a different cell from Figure 6. Top trace, Calcium-independent current, sensitive to 1 mm TEA, also referred to as K(V)_TEA. Middle traces, Calcium-dependent current, sensitive to 1 mm TEA, also referred to as K(Ca)_TEA. Bottom trace, Calcium-dependent current, resistant to 1 mm TEA (including calcium and calcium-activated potassium currents). Note that little outward current is measured between spike commands in response to the spike-train protocol. C, Conductance–voltage relation for peak K(Ca)_TEA currents (filled symbols) and peak K(V)_TEA currents (open symbols). Data were fit with Boltzmann equations as in Figure3B, and the estimated parameters are indicated in the plot.

Fig. 9.

I_h in cerebellar nuclear neurons and in bushy cells of the ventral cochlear nucleus.A, Currents were elicited by 500 msec hyperpolarizing steps from −48 mV in −10 mV increments. I_hwas isolated by subtraction of the records in Tyrode's with 2 mm CsCl from those in control Tyrode's. A,Top traces, Cesium-sensitive current recorded from a cerebellar nuclear neuron (CbN).A, Bottom trace, Cesium-sensitive current recorded in a bushy cell from the ventral cochlear nucleus (VCN). B, Mean current between 400 and 500 msec versus voltage for cerebellar nuclear neurons (filled circles; n = 6) and ventral cochlear nuclear neurons (open triangles;n = 5). Five times the mean current for cerebellar nuclear neurons is plotted for comparison (5×CbN, open circles).

Fig. 10.

Reduction of TTX-insensitive steady inward current in low-sodium solutions. A, Raw currents evoked by 250 msec step hyperpolarizations. Holding potential is −58 mV.A, Left traces, Currents elicited in control Tyrode's with TTX. A, Right traces, Currents elicited in NMDG-Tyrode's with TTX. Scale bars apply to both panels. Shown are data from one cell.B, Current–voltage relations of mean currents in each panel of A (circles) as well as currents evoked by the same protocol in the same cell, recorded in solutions containing 2 mm CsCl (triangles). Thelines indicate linear regression over the points, with (dashed lines) and without (solid lines) cesium present. C, Mean extrapolated reversal potential of currents analyzed as in B, in high (155 mm) and low (6 mm) sodium solutions (n = 8). D, Raw currents elicited by the spike-train command in control Tyrode's with TTX (top trace) and in NMDG-Tyrode's with TTX (bottom trace). Intersweep holding potential was −68 mV for 2 sec; mean of five sweeps.

Fig. 11.

Dependence of the resting potential on extracellular sodium concentration. A, Spontaneous firing of a neuron in control Tyrode's (control) and resting potentials of the same cell on exposure to control Tyrode's with TTX (TTX), Co Tyrode's with TTX (Co + TTX), and NMDG-Tyrode's with TTX (lowNa + TTX). B, Within-cell comparisons of resting membrane potentials (RMP) measured in Tyrode's with TTX and in one or more of the following solutions: Co plus TTX, low Na plus TTX, or control Tyrode's with TTX and 2 mm CsCl.