Changes in cationic selectivity of the nicotinic channel at the rat ganglionic synapse: a role for chloride ions?

The permeability of the nicotinic channel (nAChR) at the ganglionic synapse has been examined, in the intact rat superior cervical ganglion in vitro, by fitting the Goldman current equation to the synaptic current (EPSC) I-V relationship. Subsynaptic nAChRs, activated by neurally-released acetylcholine (ACh), were thus analyzed in an intact environment as natively expressed by the mature sympathetic neuron. Postsynaptic neuron hyperpolarization (from -40 to -90 mV) resulted in a change of the synaptic potassium/sodium permeability ratio (P(K)/P(Na)) from 1.40 to 0.92, corresponding to a reversible shift of the apparent acetylcholine equilibrium potential, E(ACh), by about +10 mV. The effect was accompanied by a decrease of the peak synaptic conductance (g(syn)) and of the EPSC decay time constant. Reduction of [Cl(-)](o) to 18 mM resulted in a change of P(K)/P(Na) from 1.57 (control) to 2.26, associated with a reversible shift of E(ACh) by about -10 mV. Application of 200 nM αBgTx evoked P(K)/P(Na) and g(syn) modifications similar to those observed in reduced [Cl(-)](o). The two treatments were overlapping and complementary, as if the same site/mechanism were involved. The difference current before and after chloride reduction or toxin application exhibited a strongly positive equilibrium potential, which could not be explained by the block of a calcium component of the EPSC. Observations under current-clamp conditions suggest that the driving force modification of the EPSC due to P(K)/P(Na) changes represent an additional powerful integrative mechanism of neuron behavior. A possible role for chloride ions is suggested: the nAChR selectivity was actually reduced by increased chloride gradient (membrane hyperpolarization), while it was increased, moving towards a channel preferentially permeable for potassium, when the chloride gradient was reduced.

Regulation of the subthreshold chloride conductance in the rat sympathetic neuron.

The mechanisms that control chloride conductance (gCl) in the rat sympathetic neuron have been studied by the two-electrode voltage-clamp technique in mature, intact superior cervical ganglia in vitro. In addition to voltage dependence in the membrane potential range -120/-50 mV, gCl displays time- and activity-dependent regulation (sensitization). The resting membrane potential is governed by voltage-dependent gK and gCl, which determine values of cell input conductance ranging from 7 to 18 nS (full deactivation) to an upper value of about 130 nS (full activation and maximal gCl sensitization). The quiescent neuron, held at constant membrane potential, spontaneously and gradually moved from a low- to a high-conductance status. An increase (about 40 nS) in gCl accounted for this phenomenon, which could be prevented by imposing intermittent hyperpolarizing episodes. Following spike firing, gCl increased by 20-33 nS, independent of the cell conductance value preceding tetanization, and thereafter decayed to the pre-stimulus level within 5 min. Intracellular sodium depletion and its successive ionophoretic restoration moved the neuron from a stable low-conductance state to maximum gCl sensitization, pointing to a link between gCl sensitization and [Na+]i. The dependence of gCl build-up on [Na+]i and the time-course of such Na+-related modulation have been examined: gCl sensitization was absent at 0 [Na+]i, was well developed (20 nS) at 15 mM and tended towards a saturating value of 60 nS for higher [Na+]i. Sensitization was transient in response to neuron activity. In the silent neuron, sensitization of gCl shifted membrane potential over a range of about 15 mV.

Synaptic and somatic effects of axotomy in the intact, innervated rat sympathetic neuron.

A biophysical description of the axotomized rat sympathetic neuron is reported, obtained by the two-electrode voltage-clamp technique in mature, intact superior cervical ganglia in vitro. Multiple aspects of neuron functioning were tested. Synaptic conductance activated by the whole presynaptic input decreased to 29% of the control value (0.92 muS per neuron) 1 day after axotomy and to 18% after 3 days. Despite the decrease in amplitude of the macroscopic current, miniature excitatory postsynaptic current (mEPSC) mean conductance, acetylcholine (ACh) equilibrium potential, and EPSC decay time constant were unaffected. Synaptic efficacy was tested during paired-pulse or maintained stimulation (5, 10, and 15 Hz, 10-s duration). Quantal release in axotomized neurons was preserved during the tetanus despite the reduction of the initial EPSC amplitude, suggesting that ACh secretion depended on the number of surviving synapses; each of them exhibited dynamic behavior during trains similar to that of normal synapses. Facilitation of EPSC amplitude was noted in 2-day axotomized neurons during the first few impulses in the train. Voltage-dependent potassium currents (the delayed I(KD) and the transient I(A)) exhibited an early drastic decrease in peak amplitude; these effects persisted 7 days after axotomy. Marked changes in I(A) kinetics occurred after injury: the steady-state inactivation curve shifted by up to +17 mV toward positive potentials and the voltage sensitivity of inactivation removal became steeper. I(A) impairment was reflected in a reduced inward threshold charge for discharge and reduced spike repolarization rate. Synaptic and somatic data were applied in a mathematical model to describe the progressive decrease in the safety factor, and the eventual failure of ganglionic transmission after axotomy.

Voltage- and activity-dependent chloride conductance controls the resting status of the intact rat sympathetic neuron.

Remarkable activity dependence was uncovered in the chloride conductance that operates in the subthreshold region of membrane potential, by using the two-microelectrode voltage-clamp technique in the mature and intact rat sympathetic neuron. Both direct and synaptic neuron tetanization (15 Hz, 10-s duration to saturate the response) resulted in a long-lasting (not less than 15 min) increase of cell input conductance (+70-150% 10 min after tetanus), accompanied by the onset of an inward current with the same time course. Both processes developed with similar properties in the postganglionic neuron when presynaptic stimulation was performed under current- or voltage-clamp conditions and were unaffected by external calcium on direct stimulation. The posttetanic effects were sustained by gCl increase because both conductance and current modifications were blocked by 0.5 mM Anthracene-9-carboxylic acid (a chloride channel blocker) but were unaffected by TEACl or cesium chloride treatments. The chloride channel properties were modified by stimulation: their voltage sensitivity and rate of closure in response to hyperpolarization strongly increased. The voltage dependence of the three major conductances governing the cell subthreshold status (gCl, gK, and gL) was evaluated over the -40/-110 mV membrane potential range in unstimulated neurons and compared with previous results in stimulated neurons. A drastic difference between the voltage-conductance profiles was observed, exclusively sustained by gCl increase. The chloride channel thus hosts an intrinsic mechanism, a memory of previous neuron activity, which makes the chloride current a likely candidate for natural controller of the balance between opposite resting currents and thus of membrane potential level.