Oscar Sacchi

The slow Ca(2+)-activated K+ current, IAHP, in the rat sympathetic neurone.

1. Adult and intact sympathetic neurones of the rat superior cervical ganglion maintained in vitro at 37 degrees C were analysed using the two‐electrode voltage‐clamp technique in order to investigate the slow component of the Ca(2+)‐dependent K+ current, IAHP. 2. The relationship between the after‐hyperpolarization (AHP) conductance, gAHP, and estimated Ca2+ influx resulting from short‐duration calcium currents evoked at various voltages proved to be linear over a wide range of injected Ca2+ charge. An inflow of about 1.7 x 10(7) Ca2+ ions was required before significant activation of gAHP occurred. After priming, the gAHP sensitivity was about 0.3 nS pC‐1 of Ca2+ inward charge. 3. IAHP was repeatedly measured at different membrane potentials; its amplitude decreased linearly with membrane hyperpolarization and was mostly abolished close to the K+ reversal potential, EK (‐93 mV). The monoexponential decay rate of IAHP was a linear function of total Ca2+ entry and was not significantly altered by membrane potential in the ‐40 to ‐80 mV range. 4. Voltage‐clamp tracings of IAHP could be modelled as a difference between two exponentials with tau on approximately 5 ms and tau off = 50‐250 ms. 5. Sympathetic neurones discharged only once at the onset of a long‐lasting depolarizing step. If IAHP was selectively blocked by apamin or D‐tubocurarine treatments, accommodation was abolished and an unusual repetitive firing appeared. 6. Summation of IAHP was demonstrated under voltage‐clamp conditions when the depolarizing steps were repeated sufficiently close to one another. Under current‐clamp conditions the threshold depolarizing charge for action potential discharge significantly increased with progressive pulse numbers in the train, suggesting that an opposing conductance was accumulating with repetitive firing. This frequency‐dependent spike firing ability was eliminated by pharmacological inhibition of the slow IAHP. 7. The IAHP was significantly activated by a single action potential; it was turned on cumulatively by Ca2+ load during successive action potential discharge and acted to further limit cell excitability.

Storage and release of acetylcholine in the isolated superior cervical ganglion of the rat.

The storage and release of acetylcholine and choline were studied in the isolated superior cervical ganglion of the rat by a radioenzymic method. The acetylcholine and choline contents were 202.2 +/- 5.1 and 624.7 +/- 20.2 pmole/ganglion, respectively. The transmitter tissue store was unaffected during 1 h of superfusion in choline--Krebs solution, while a 20% decrease was exhibited after 2 h and then remained approximately stable. Conversely, choline content declined to 50% within 1 h and further to 37% of the original level by 4 h. About 24% of the choline assayed in the intact preparation is located in the connective sheath. Preganglionic nerve stimulation at 10--20/sec or potassium stimulation (40 mM KCl) invariably decreased the transmitter tissue stores by 25--45%; such a depletion is independent of the presence or absence of external choline. By contrast, the presence of choline proved to be a prerequisite for the efficient release of acetylcholine from eserinized ganglia during continuous 10/sex stimulation. A drastic depression in the acetylcholine release is described which is related to the time of preincubation of the ganglia with eserine prior to stimulation. Indeed, a 30 min exposure to eserine, compared with a 5 min period, resulted in a 4-fold decrease in the steady output rate. Under optimal conditions, the initial volley output at 10/sec was 1.3 X 10(-4) of the releasable transmitter pool and 1.9 X 10(-4) during the steady-state output. These results are discussed in the light of the electrophysiological knowledge of the quantal release process at the ganglionic synapse.

A model of signal processing at a mammalian sympathetic neurone.

A computational model has been developed for the action potential and, more generally, the electrical behaviour of the rat sympathetic neurone. The neurone is simulated as a complex system in which five voltage-dependent conductances (gNa, gCa, gKV, gA, gKCa), one Ca2+-dependent voltage-independent conductance (gAHP) and the activating synaptic conductance coexist. The individual currents are mathematically described, based on a systematic analysis obtained for the first time in a mature and intact mammalian neurone using two-electrode voltage-clamp experiments. The simulation initiates by setting the starting values of each variable and by evaluating the holding current required to maintain the imposed membrane potential level. It is then possible to simulate current injection to reproduce either the experimental direct stimulation of the neurone or the physiological activation by the synaptic current flow. The subthreshold behaviour and the spiking activity, even during long-lasting current application, can be analysed. At every time step, the program calculates the amplitude of the individual currents and the ensuing changes; it also takes into account the accompanying K+ accumulation process in the perineuronal space and changes in Ca2+ load. It is shown that the computed time course of membrane potential must be filtered, in order to reproduce the limited bandwidth of the recording instruments, if it is to be compared with experimental measurements under current-clamp conditions. The membrane potential trajectory and single current data are written in files readable by graphic software. Finally, a screen image is obtained which displays in separate graphs the membrane potential time course, the synaptic current and the six ionic current flows. The simulated action potentials are comparable to the experimental ones as concerns overshoot amplitude and rising and falling rates. Therefore, this program is potentially helpful in investigating many aspects of neurone behaviour.

Effectiveness of some anions in sustaining the efferent inhibition in the frog labyrinth

Efferent inhibition in the frog labyrinth is sustained by the release of acetylcholine (ACh) which opens a Cl(-)-channel in the hair cell membrane. To investigate more closely the nature of the permeability change underlying the ACh reaction, the external Cl(-) was replaced by anions of increasing hydrated size, and to test the possible role of a Cl(-)-pump in the sensory cells, drugs were applied which are known to block active cl(-) pumping in other systems. Experiments indicate that the ACh-operated inhibitory channel of the hair cell is larger than at other inhibitory synapses (or approximately 0.7 nm), while pharmacological treatments (DNP, NaN3, acetazolamide, ammonium acetate, DIDS) fail to demonstrate any active distribution of Cl(-) across the hair cell membrane.

Synaptic stimulation of nicotinic receptors in rat sympathetic ganglia is followed by slow activation of postsynaptic potassium or chloride conductances

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 ∼ 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 τ = 148.5 ms) in 18 neurons or according to a two‐exponential time course (τ = 51.8 and 364.9 ms, respectively) in 19 different neurons. The mean peak conductance activated was ∼ 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.

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 (PK/PNa) from 1.40 to 0.92, corresponding to a reversible shift of the apparent acetylcholine equilibrium potential, EACh, by about +10 mV. The effect was accompanied by a decrease of the peak synaptic conductance (gsyn) and of the EPSC decay time constant. Reduction of [Cl−]o to 18 mM resulted in a change of PK/PNa from 1.57 (control) to 2.26, associated with a reversible shift of EACh by about −10 mV. Application of 200 nM αBgTx evoked PK/PNa and gsyn 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 PK/PNa 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.