Mechanism of spontaneous firing in dorsomedial suprachiasmatic nucleus neurons

Abstract

We studied acutely dissociated neurons from the dorsomedial (shell) region of the rat suprachiasmatic nucleus (SCN) with the aim of determining the ionic conductances that underlie spontaneous firing. Most isolated neurons were spontaneously active, firing rhythmically at an average frequency of 8 +/- 4 Hz. After application of TTX, oscillatory activity generally continued, but more slowly and at more depolarized voltages; these oscillations were usually blocked by 2 microm nimodipine. To quantify the ionic currents underlying normal spontaneous activity, we voltage clamped cells using a segment of the spontaneous activity of each cell as voltage command and then used ionic substitution and selective blockers to isolate individual currents. TTX-sensitive sodium current flowed throughout the interspike interval, averaging -3 pA at -60 mV and -11 pA at -55 mV. Calcium current during the interspike interval was, on average, fourfold smaller. Except immediately before spikes, calcium current was outweighed by calcium-activated potassium current, and in current clamp, nimodipine usually depolarized cells and slowed firing only slightly (average, approximately 8%). Thus, calcium current plays only a minor role in pacemaking of dissociated SCN neurons, although it can drive oscillatory activity with TTX present. During normal pacemaking, the early phase of spontaneous depolarization (-85 to -60 mV) is attributable mainly to background conductance; cells have relatively depolarized resting potentials (with firing stopped by TTX and nimodipine) of -55 to -50 mV, although input resistance is high (9.5 +/- 4.1 GOmega). During the later phase of pacemaking (positive to -60 mV), TTX-sensitive sodium current is dominant.

Figure 1.

Dissociation of dmSCN neurons staining for AVP. A, Localization of dmSCN based on staining for AVP. Rabbit anti-AVP antibodies were used at a dilution of 1:1000, with a secondary antibody of biotinylated goat anti-rabbit IgG, followed by avidin-biotinylated peroxidase complex and diaminobenzidine substrate to form a reaction product. As a positive control, staining of both the paraventricular nucleus and supraoptic nucleus, both known to contain AVP-positive neurons, was also prominent. Scale bar, 100 μm. B, Morphology of freshly dissociated SCN neurons. Scale bar, 20 μm. C, Staining of dissociated SCN neurons with anti-AVP antibodies. Left, Rabbit anti-AVP antibodies were used at a dilution of 1:1000, with a secondary antibody of biotinylated goat anti-rabbit IgG, followed by avidin-biotinylated peroxidase complex and diaminobenzidine substrate to form a reaction product. Right, Control using neurons treated identically but with nonimmunized rabbit IgG rather than anti-AVP. Scale bar, 50 μm. In another negative control, dissociated cortical neurons failed to be stained by the anti-AVP antibodies.

Figure 7.

Voltage-activated ionic current elicited by step depolarizations. A, Total ionic current evoked by voltage steps from a holding potential of -78 mV studied with physiological solutions. There was no correction for leak or background current. Capacity transients were electronically nulled using the amplifier circuitry, and remaining transients were corrected digitally by scaling and subtracting the capacity transient for a 10 mV hyperpolarization. B, Steady-state current measured at the end of 200 msec steps. Note low steady-state background conductance, inward inflection between -70 and -50, corresponding to persistent sodium current, and steep activation of apparent voltage-dependent potassium current positive to -50 mV. The solid line shows linear fit to the current-voltage relationship between -100 and -75 mV. C, Peak inward transient current during test step.

Figure 8.

Isolation of voltage-activated sodium and calcium currents elicited by step depolarizations. A, Ionic currents evoked in response to depolarizing pulses in control (left), with 10 mm TEA present in the external solution (middle), and with both TEA and 300 nm TTX present (right). B, TTX-sensitive sodium current obtained by subtraction of currents before and after TTX. C, Voltage dependence of peak transient sodium conductance, obtained by converting TTX-sensitive sodium current to a conductance (using the measured reversal potential of +45 mV). The solid line is plotted according to G_Na = G_max/[1 + exp(-(V - V_h)/k)], where G_max is the maximal conductance (36 nS), V is the test potential, V_h is the midpoint (-34 mV), and k is the slope factor (3.8 mV). D, Ionic currents evoked by a series of solutions enabling isolation of calcium current: a solution in which external sodium in Tyrode's solution was replaced by TEA (left) and the same solution but with cobalt replacing calcium (middle). Subtraction of currents in these solutions yields calcium current (right). E, Open symbols indicate depolarization-evoked calcium conductance, calculated by converting calcium current to conductance using a reversal potential of +57 mV. The solid curve is plotted according to G_Ca = 1.9 nS/[1 + exp(-(V + 20)/9.5)]; the closed symbols and fit: G_Na (from C) plotted on same scale to illustrate relative size of voltage-activated calcium and sodium conductances.

Figure 2.

Spontaneous firing in a dissociated SCN neuron recorded first in cell-attached patch mode and then in whole-cell current-clamp mode. A, Action currents from spontaneous firing recorded in cell-attached mode. B, Spontaneous firing recorded in whole-cell mode 7 min after breaking through into whole-cell mode. The pipette solutions consisted of (in mm) 123 K-methanesulfonate, 9 NaCl, 1.8 MgCl₂, 0.9 EGTA, 9 HEPES, 14 creatine phosphate, 4 MgATP, and 0.3 GTP, pH 7.3. The external solution included (in mm) 1.2 CaCl₂, 3.5 KCl, 150 NaCl, 1 MgCl₂, 10 glucose, and 10 HEPES, pH 7.4.

Figure 3.

Oscillations of membrane potential in the presence of TTX. A, Effect of cumulative application of 300 nm TTX and 2 μm nimodipine to an SCN neuron firing spontaneously at 4.9 Hz. B, Two-second periods shown in each condition at an expanded time scale.

Figure 4.

Action potential clamp technique using spontaneous action potentials. A period of spontaneous firing (no stimulation or steady injected current) was recorded in fast current-clamp mode (first row; left). The right column shows a single spike on an expanded time scale. Then the amplifier was switched to voltage-clamp mode, with compensation for whole-cell capacitance and series resistance; this tuning was done before the current-clamp recording, using a 10 mV hyperpolarizing step from -70 mV. The segment of spontaneous firing was used as voltage command. Second row, The ionic current elicited by this command (with capacitative current removed using electronic capacitance compensation in the patch clamp circuitry) in control Tyrode's solution. Third row, Ionic current after moving cell to Tyrode's solution plus 10 mm TEA chloride. Fourth row, Current after moving cell to the same solution but with the addition of 300 nm TTX. Fifth row, Current after moving cell to solution in which Na⁺ was replaced by TEA⁺. Sixth row, With cell in similar solution, except with Ca²⁺ replaced by Mg²⁺. The current remaining during the spike waveform in this solution is consistent with being capacitative transients attributable to incomplete compensation of whole-cell capacitance.

Figure 5.

Interspike sodium and calcium currents determined using the action potential clamp technique. A, Voltage during the cycle of spontaneous firing was signal averaged by aligning action potentials from a 5 sec period of recording at their peaks and signal averaging. A unit cycle of firing resulting from this procedure was concatenated so as to illustrate slightly more than one complete cycle. B, Sodium current (red) in response to the 5 sec action potential waveform was calculated by subtraction of currents before and TTX as in Figure 4. Current was signal averaged in an identical manner as the voltage in A (using the times of the action potential peaks to perform the alignment). Calcium current (blue) was signal averaged in an identical manner based on the subtraction of currents recorded with and without Ca²⁺ (replaced by Mg²⁺), both in the presence of 155 mm TEA⁺ to block calcium-activated potassium currents. Inset, Sodium and calcium currents during the spike, shown on an expanded time base. C, Same currents as in B shown on an expanded current scale to resolve currents during the interspike interval.

Figure 6.

Comparison of current from calcium channels and TTX-sensitive sodium channels during the interspike interval of 13 SCN neurons. In each cell, sodium and calcium currents flowing during the cycle of spontaneous firing were determined as in Figure 5. To allow comparisons among cells, current as a function of time was converted to current as a function of interspike voltage for each cell. A, Sodium (red) and calcium (blue) currents during the interspike interval, averaged over 13 cells. The error bars show SD. B, Cell-by-cell comparison of sodium and calcium currents integrated from the trough after a spike to the time at which the voltage reached -55 mV. Cells are arranged by their firing rate; there was no systematic dependence of relative contribution of sodium versus calcium current on firing rate.

Figure 9.

Voltage dependence of nimodipine-sensitive calcium current. A, Top, Total calcium current with physiological calcium (1.2 mm) as charge carrier, obtained by subtraction of currents after replacing calcium with cobalt (with sodium replaced with 155 mm TEA to block potassium currents) as in Figure 8. Middle, Same, but with both calcium- and cobalt-containing solutions containing 2 μm nimodipine. Bottom, Nimodipine-sensitive calcium current. B, Normalized total calcium conductance (open symbols) and nimodipine-sensitive calcium conductance (closed symbols) as a function of voltage. Conductance was calculated from peak current using a reversal potential of +57 mV. The solid lines indicate Boltzmann functions plotted according to G_max/[1 + exp(-(V - V_h)/k)], where G_max is the maximal conductance, V is the test potential, V_h is the midpoint, and k is the slope factor, with the indicated values.

Figure 10.

Preferential activation of nimodipine-sensitive calcium current before spontaneous action potentials. A 5 sec segment of spontaneous activity was recorded in current clamp and used as command waveform in voltage clamp. A, Voltage trajectory before and during the spike, signal averaged over 48 spikes (aligned at spike peaks). B, Sodium current (red) was determined as the current sensitive to 300 nm TTX. Total calcium current (blue) was determined by recording current in a solution containing (in mm) 1.2 CaCl₂, 1 MgCl₂, 150 TEA-Cl, 3.5 KCl, 10 HEPES, and 10 glucose, pH to 7.4 with TEAOH, and subtracting current recorded in a solution that was identical, except with CoCl₂ replacing CaCl₂. Nimodipine-sensitive calcium current (black) was recorded as the current sensitive to 2 μm nimodipine applied in the solution containing (in mm) 1.2 CaCl₂, 1 MgCl₂, and 150 TEA-Cl. Current in each solution was signal averaged over 96-192 cycles of firing. C, Currents before spike shown on an expanded current and time scale (box in B).

Figure 11.

Effect of nimodipine on spontaneous firing. A, Three-second segments of spontaneous activity recorded in control (left), after 80 sec exposure to 2 μm nimodipine (middle), and after 3 min of washing with control solution (right). B, Voltage trajectory in control (red) and with nimodipine (blue), signal averaged over 1 sec (aligned at spike peaks).

Figure 12.

Calcium current and calcium-activated potassium current during spontaneous action potentials. Total calcium current and nimodipine-sensitive calcium current were determined as in Figure 10. In addition, calcium-activated potassium current was determined by first determining total potassium current as current blocked by 155 mm TEA (replacing 155 mm Na) starting with a background of Tyrode's solution containing 300 nm TTX, then determining non-Ca-dependent potassium current (performing the same replacement of Na by 155 mm TEA but using a pair of solutions in which 1.2 mm CaCl₂ was replaced by 1.2 mm CoCl₂) and then subtracting the two to yield calcium-activated potassium current. The sum of total calcium current and calcium-activated potassium current (gray trace; left) was net inward immediately before the spike but much larger and net outward during the falling phase of the spike. The sum of calcium current and calcium-activated potassium current could alternatively be determined as the net current sensitive to replacing 1.2 mm CaCl₂ with 1.2 mm CoCl₂ with a background of Tyrode's solution with 300 nm TTX; this gave a nearly identical current. The calcium-activated potassium current sensitive to block by nimodipine (i.e., that activated by nimodipine-sensitive calcium current) was determined by subtracting non-nimodipine-sensitive potassium current (determined by replacing 155 Na with 155 mm TEA using a pair of solutions both containing 2 μm nimodipine) from total potassium current determined as above. The sum of nimodipine-sensitive calcium current and nimodipine-sensitive calcium-activated potassium current (gray trace; right) was inward before the spike but net outward when integrated.

Figure 13.

Background currents in the interspike voltage range. Measured steady-state currents in an SCN neuron. Current was measured in response to a ramp of voltage from -98 to +12 mV delivered at 20 mV/sec, in control condition Tyrode's solution (red), after the addition of 300 mm TTX (blue) and after replacement of external Na ions by NMDG ions (with 300 nm TTX present in both solutions; green). The data shown were signal averaged over four to eight presentations of command. The solid line indicates linear fit to steady-state current in the presence of TTX between -90 and -78 mV, extrapolated to other voltages.

Figure 14.

Summary of inward currents driving spontaneous depolarization. Top, Voltage during the cycle of spontaneous firing in a cell with typical parameters (8 Hz; peak, +24 mV; trough, -79 mV), averaged over 80 cycles. Bottom, Population-averaged interspike sodium current (red), interspike calcium current (blue), and predicted background current (green). For each voltage during the interspike depolarization, the value of mean sodium current and mean calcium current was read off from smooth fits (cubic spline) to the mean interspike sodium and calcium currents plotted in Figure 6. The background current is based on an ohmic conductance with the slope corresponding to 9.5 GΩ (the mean measured in 70 cells) and the mean reversal potential of -29 mV estimated from linear fits to background current in the range -95 to -75 mV.