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Abstract: Nicotinic responses to endogenous acetylcholine and to exogenously applied agonists have been studied in the intact or denervated rat sympathetic neuron in vitro, by using the two-microelectrode voltage-clamp technique. Preganglionic denervation resulted in progressive decrease of the synaptic current (excitatory postsynaptic current, EPSC) amplitude, which disappeared within 24 h. These effects were accompanied by changes in ion selectivity of the nicotinic channel (nAChR). The extrapolated EPSC null potential (equilibrium potential for acetylcholine action, ESyn) shifted from a mean value of −15.9±0.7 mV, in control, to −7.4±1.6 mV, in denervated neurons, indicating a decrease of the permeability ratio for the main components of the synaptic current (PK/PNa) from 1.56 to 1.07. The overall properties of AChRs were investigated by applying dimethylphenylpiperazinium or cytisine and by examining the effects of endogenous ACh, diffusing within the ganglion after preganglionic tetanization in the presence of neostigmine. The null potentials of these macrocurrents (equilibrium potential for dimethylphenylpiperazinium action, EDMPP; and equilibrium potential for diffusing acetylcholine, EACh, respectively) were evaluated by applying voltage ramps and from current–voltage plots. In normal neurons, ESyn (−15.9±0.7 mV) was significantly different from EDMPP (−26.1±1.0) and EACh (−31.1±3.3); following denervation, nerve-evoked currents displayed marked shifts in their null potentials (ESyn=−7.4±1.6 mV), whereas the amplitude and null potential of the agonist-evoked macrocurrents were unaffected by denervation and its duration (EDMPP=−26.6±1.2 mV). It is suggested that two populations of nicotinic receptors, synaptic and extrasynaptic, are present on the neuron surface, and that only the synaptic type displays sensitivity to denervation. [Copyright &y& Elsevier]

The blue shark Prionace glauca , among the most common and widely studied pelagic sharks, is a top predator, exhibiting the widest distribution range. However, little is known about its population structure and spatial dynamics. With an estimated removal of 10-20 million individuals per year by fisheries, the species is classified as "Near Threatened" by International Union for Conservation of Nature. We lack the knowledge to forecast the long-term consequences of such a huge removal on this top predator itself and on its trophic network. The genetic analysis of more than 200 samples collected at broad scale (from Mediterranean Sea, North Atlantic and Pacific Oceans) using mtDNA and nine microsatellite markers allowed to detect signatures of genetic bottlenecks but a nearly complete genetic homogeneity across the entire studied range. This apparent panmixia could be explained by a genetic lag-time effect illustrated by simulations of demographic changes that were not detectable through standard genetic analysis before a long transitional phase here introduced as the "population grey zone ." The results presented here can thus encompass distinct explanatory scenarios spanning from a single demographic population to several independent populations. This limitation prevents the genetic-based delineation of stocks and thus the ability to anticipate the consequences of severe depletions at all scales. More information is required for the conservation of population(s) and management of stocks, which may be provided by large-scale sampling not only of individuals worldwide, but also of loci genomewide.

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.

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.

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.

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.

Two slow currents have been described in rat sympathetic neurons during and after tetanization of the whole preganglionic input. Both effects are mediated by nicotinic receptors activated by native acetylcholine (ACh). A first current, indicated as IAHPsyn, is calcium dependent and voltage independent, and is consistent with an IAHP-type potassium current sustained by calcium ions accompanying the nicotinic synaptic current. The conductance activated by a standard synaptic train was approximately 3.6 nS per neuron; it was detected in isolation in 14 out of a 52-neuron sample. A novel current, IADPsyn, was described in 42/52 of the sample as a post-tetanic inward current, which increased in amplitude with increasing membrane potential negativity and exhibited a null-point close to the holding potential and the cell momentary chloride equilibrium potential. IADPsyn developed during synaptic stimulation and decayed thereafter according to a single exponential (mean tau = 148.5 ms) in 18 neurons or according to a two-exponential time course (tau = 51.8 and 364.9 ms, respectively) in 19 different neurons. The mean peak conductance activated was approximately 20 nS per neuron. IADPsyn was calcium independent, it was affected by internal and external chloride concentration, but was insensitive to specific blockers (anthracene-9-carboxylic acid, 9AC) of the chloride channels open in the resting neuron. It is suggested that gADPsyn represents a specific chloride conductance activatable by intense nicotinic stimulation; in some neurons it is even associated with single excitatory postsynaptic potentials (EPSCs). Both IAHP and IADPsyn are apparently devoted to reduce neuronal excitability during and after intense synaptic stimulation.

The involvement of the postsynaptic membrane potential level in controlling synaptic strength at the ganglionic synapse was studied by recording nicotinic fast synaptic currents (EPSCs) from neurons in the intact, mature rat superior cervical ganglion, using the two-electrode voltage-clamp technique. EPSCs were evoked by 0.05-Hz supramaximal stimulation of the preganglionic sympathetic trunk over long periods; their peak amplitude (or synaptic charge transfer) over time appeared to depend on the potential level of the neuronal membrane where the nicotinic receptors are embedded. EPSC amplitude remained constant (n = 6) only if ACh was released within repeated depolarizing steps of the postganglionic neuron, which constantly varied between -50 and -20 mV in consecutive 10-mV steps, whereas it decreased progressively by 45% (n = 9) within 14 min when the sympathetic neuron was held at constant membrane potential. Synaptic channel activation, channel ionic permeation and depolarization of the membrane in which the nicotinic receptor is localized must occur simultaneously to maintain constant synaptic strength at the ganglionic synapse during low-rate stimulation (0.03-1 Hz). Different posttetanic (20 Hz for 10 s) behaviors were observed depending on the mode of previous stimulation. In the neuron maintained at constant holding potential during low-rate stimulation, the depressed EPSC showed posttetanic potentiation, recovering approximately 23% of the mean pretetanic values (n = 10). The maximum effect was immediate in 40% of the neurons tested and developed over a 3- to 6-min period in the others; thereafter potentiation vanished within 40 min of 0.05-Hz stimulation. In contrast, no statistically significant synaptic potentiation was observed when EPSC amplitudes were kept constant by repeated -50/-20-mV command cycles (n = 12). It is suggested that, under these conditions, posttetanic potentiation could represent an attempt at recovering the synaptic strength lost during inappropriate functioning of the ganglionic synapse.