Heterogeneity of membrane properties in sympathetic preganglionic neurons of neonatal mice: evidence of four subpopulations in the intermediolateral nucleus

Abstract

Spinal cord sympathetic preganglionic neurons (SPNs) integrate activity from descending and sensory systems to determine the final central output of the sympathetic nervous system. The intermediolateral column (IML) has the highest number and density of SPNs and, within this region, SPN somas are found in distinct clusters within thoracic and upper lumbar spinal segments. Whereas SPNs exhibit a rostrocaudal gradient of end-target projections, individual clusters contain SPNs with diverse functional roles. Here we explored diversity in the electrophysiological properties observed in Hb9-eGFP-identified SPNs in the IML of neonatal mice. Overall, mouse SPN intrinsic membrane properties were comparable with those seen in other species. A wide range of values was obtained for all measured properties (up to a 10-fold difference), suggesting that IML neurons are highly differentiated. Using linear regression we found strong correlations between many cellular properties, including input resistance, rheobase, time constant, action potential shape, and degree of spike accommodation. The best predictor of cell function was rheobase, which correlated well with firing frequency-injected current (f-I) slopes as well as other passive and active membrane properties. The range in rheobase suggests that IML neurons have a recruitment order with stronger synaptic drives required for maximal recruitment. Using cluster analysis, we identified at least four subpopulations of SPNs, including one with a long time constant, low rheobase, and high f-I gain. We thus propose that the IML contains populations of neurons that are differentiable by their membrane properties and hypothesize they represent diverse functional classes.

Fig. 1.

Membrane property correlations. Figure shows correlation coefficients between membrane properties, with ρ values close to 1 showing strong positive relationships and ρ values close to −1 showing strong negative relationships. The firing frequency–injected current (f–I) slope refers to instantaneous firing frequency. Asterisks (*) denote statistically significant correlations (P < 0.05) and “?” denotes correlations with 0.05 < P < 0.1.

Fig. 2.

Transient outward and anomalous rectification. Ai: sample membrane response to a series of current steps (1-s duration, −15 to 10 pA, 5-pA steps), holding current −12.6 pA. Asterisk (*) indicates anomalous inward rectification; □ indicates transient outward rectification, seen as a much longer repolarization time to hyperpolarizing current steps. Aii: sample voltage response to a series of injected current pulses (1-s duration,10-pA steps) from a hyperpolarized holding potential. Note delay to first spike, due to transient outward conductance. B: sample current response to a series of voltage-clamp steps (−30 to +40 mV, 10-mV steps) from a hyperpolarized holding potential of −90 mV. Asterisk (*) indicates instantaneous increased conductance at hyperpolarized membrane potentials; □ indicates transient outward conductance, here activated at −70 mV and more pronounced at −60 and −50 mV. Next voltage step (−40 mV) produced an inward action current (not shown). Inset shows sample current response of a different cell to a −50-mV voltage step, when Cs⁺; replaced intracellular K⁺. Note the presence of the transient outward conductance. C: sample current–voltage plot of steady-state currents obtained during voltage-clamp recordings, revealing inward rectification at potentials less than about −80 mV.

Fig. 3.

Repetitive firing properties. A: sample response to 20-pA current injection (1-s duration). Note the slowing of the firing rate with each spike. B: frequency response of a typical sympathetic preganglionic neuron (SPN) showing spike-frequency adaptation (SFA) to multiple current injections, logarithmic scale. m was the natural log of the slopes of the lines shown. C: correlation between input resistance (R_in) and SFA slope m, averaged for each cell. Only cells with statistically significant SFA are shown.

Fig. 4.

Persistent inward currents (PICs). A: average steady-state current response to a series of 500-ms voltage steps with a CsF- and K-gluconate–based intracellular solution. Arrows denote the absence of anomalous rectifier and onset of negative slope conductance in CsF. B: sample neuron with a PIC resulting in negative conductance value, CsF-based intracellular solution. Arrows denote PIC onset and peak magnitude. C: PICs were largely masked by K⁺ conductances in K-gluconate–containing patch electrodes, but could be seen as small deviations from linear conductance during a slow voltage ramp (8 mV/s).

Fig. 5.

Cluster analysis. A: analysis of cluster validity, using 2 different indices. Both indices peak at 4 clusters, signifying best fit for the data set. B: distribution of cluster membrane properties as a function of rheobase. Bi: groups 2 and 3 have low rheobase values, with group 3 having larger f–I gains. Groups 1 and 4 are sequentially recruited and can be largely distinguished by rheobase values. Bii: group 3 neurons have statistically larger τ_m values than those of group 1 neurons. Biii: group 1 neurons display statistically larger I_peak values than those of both group 2 and group 3 SPNs and all have larger values than those of group 4 SPNs.