Expression and function of neuronal nicotinic ACh receptors in rat microvascular endothelial cells

The expression and function of nicotinic ACh receptors (nAChRs) in rat coronary microvascular endothelial cells (CMECs) were examined using RT-PCR and whole cell patch-clamp recording methods. RT-PCR revealed expression of mRNA encoding for the subunits alpha(2), alpha(3), alpha(4), alpha(5), alpha(7), beta(2), and beta(4) but not beta(3). Focal application of ACh evoked an inward current in isolated CMECs voltage clamped at negative membrane potentials. The current-voltage relationship of the ACh-induced current exhibited marked inward rectification and a reversal potential (E(rev)) close to 0 mV. The cholinergic agonists nicotine, epibatidine, and cytisine activated membrane currents similar to those evoked by ACh. The nicotine-induced current was abolished by the neuronal nAChR antagonist mecamylamine. The direction and magnitude of the shift in E(rev) of nicotine-induced current as a function of extracellular Na(+) concentration indicate that the nAChR channel is cation selective and follows that predicted by the Goldman-Hodgkin-Katz equation assuming K(+)/Na(+) permeability ratio of 1.11. In fura-2-loaded CMECs, application of ACh, but not of nicotine, elicited a transient increase in intracellular free Ca(2+) concentration. Taken together, these results demonstrate that neuronal nAChR activation by cholinergic agonists evokes an inward current in CMECs carried primarily by Na(+), which may contribute to the plasma nicotine-induced changes in microvascular permeability and reactivity induced by elevations in plasma nicotine.     

Modulatory action of acetylcholine on the Na-dependent action potentials in Kenyon cells isolated from the mushroom body of the cricket brain

Kenyon cells, intrinsic neurons of the insect mushroom body, have been assumed to be a site of conditioning stimulus (CS) and unconditioned stimulus (US) association in olfactory learning and memory. Acetylcholine (ACh) has been implicated to be a neurotransmitter mediating CS reception in Kenyon cells, causing rapid membrane depolarization via nicotinic ACh receptors. However, the long-term effects of ACh on the membrane excitability of Kenyon cells are not fully understood. In this study, we examined the effects of ACh on Na⁺ dependent action potentials (Na⁺ spikes) elicited by depolarizing current injection and on net membrane currents under the voltage clamp condition in Kenyon cells isolated from the mushroom body of the cricket Gryllus bimaculatus. Current-clamp studies using amphotericin B perforated-patch recordings showed that freshly dispersed cricket Kenyon cells could produce repetitive Na⁺ spikes in response to prolonged depolarizing current injection. Bath application of ACh increased both the instantaneous frequency and the amplitudes of Na⁺ spikes. This excitatory action of ACh on Kenyon cells is attenuated by the pre-treatment of the cells with the muscarinic receptor antagonists, atropine and scopolamine, but not by the nicotinic receptor antagonist mecamylamine. Voltage-clamp studies further showed that bath application of ACh caused an increase in net inward currents that are sensitive to TTX, whereas outward currents were decreased by this treatment. These results indicate that in order to mediate CS, ACh may modulate the firing properties of Na⁺ spikes of Kenyon cells through muscarinic receptor activation, thus increasing Na conductance and decreasing K conductance.     

Acetylcholine responses of identified neurons in Helix pomatia--II. Pharmacological properties of acetylcholine responses

A pharmacological separation of depolarizing and hyperpolarizing mechanisms involved in the generation of acetylcholine (ACh) depolarizations was attempted in the identified neurons B1 and B3 of the buccal ganglia of Helix pomatia. The selectivity of the drugs employed was assayed in non-identified buccal neurons in which ACh increased a hyperpolarizing Cl- conductance. Voltage clamp techniques were used. Under control conditions the depolarizing ACh currents increased non-linearly with more negative membrane potentials. The hyperpolarizing ACh currents showed a linear potential dependence. The buffer substance Tris (5 mmol/l) depressed the depolarizing ACh currents. The effect was accentuated with more negative membrane potentials. Tris failed to affect hyperpolarizing ACh responses. HEPES (5 mmol/l) did not change depolarizing or hyperpolarizing ACh responses. d-Tubocurarine (0.02-0.2 mmol/l), hexamethonium (0.5-5.0 mmol/l) and atropine (0.1 mmol/l) blocked the depolarizing and hyperpolarizing ACh responses. Arecoline (0.1 mmol/l) had neither an agonistic nor an antagonistic effect on the identified and on the non-identified neurons. It displayed an anticholinesterase activity. Anthracene-9-carbonic acid (0.5 mmol/l) depressed selectively the hyperpolarizing ACh responses. In the neurons B1 and B3 no pharmacologically separable hyperpolarizing ACh responses were detected to be superimposed on the ACh depolarizations.     

Anoxia reversibly suppresses neuronal calcium currents in rat hippocampal slices

Intracellular recording from CA1 neurons confirmed that short periods of anoxia (95% N2 + 5% CO2 for 2-4 min) have a hyperpolarizing action, caused by a rise in K conductance. After blockage of K channels with extracellular Cs+ and tetraethylammonium (or intracellular Cs+), large inward currents of Ca were evoked by depolarizing pulses: transient currents at a holding potential near -70 mV, and more sustained ones near -50 mV. Both types of Ca current were much reduced or fully suppressed after 1-3 min of anoxia, but they largely (or fully) recovered within 1-10 min of starting reoxygenation.     

Factors affecting slow regular firing in the suprachiasmatic nucleus in vitro

In isolated slices of hypothalamus, suprachiasmatic nucleus (SCN) neurons were recorded intracellularly. Blockade of Ca++ channels increased spike duration, eliminating an early component of the afterhyperpolarization (AHP) that followed evoked spikes. The duration and reversal potential of AHPs were, however, unaffected, suggesting that only an early, fast component of the AHP was Ca(++)-dependent. Unlike other central neurons that exhibit pacemaker activity, therefore, SCN neurons do not display a pronounced, long-lasting Ca(++)-dependent AHP. Extracellular Ba++ and intracellular Cs+ both revealed slow depolarizing potentials evoked either by depolarizing current injection, or by repolarization following large hyperpolarizations. They had different effects on the shape of spikes and the AHPs that followed them, however. Cs+, which blocks almost all K+ channels, dramatically reduced resting potential, greatly increased spike duration (to tens of milliseconds), and blocked AHPs completely. In contrast, Ba++ had little effect on resting potential and produced only a small increase in spike duration, depressing an early Ca(++)-dependent component and a later Ca(++)-independent component of the AHP. The relatively weak pacemaker activity of SCN neurons appears to involve voltage-dependent activation of at least one slowly inactivating inward current, which brings the cells to firing threshold and maintains tonic firing; both Ca(++)-dependent and Ca(++)-independent K+ channels, which repolarize cells after spikes and maintain interspike intervals; and Ca++ channels, which contribute to activation of Ca(++)-activated K+ currents and may also contribute to slow depolarizing potentials. In the absence of powerful synaptic inputs, SCN neurons express a pacemaker activity that is sufficient to maintain an impressively regular firing pattern. Slow, repetitive activation of optic input, however, increases local circuit activity to such an extent that the normal pacemaker potentials are overridden and firing patterns are altered. Since SCN neurons are very small and have large input resistances, they are particularly susceptible to synaptic input.     

Chemosensitive conductance and inositol 1,4,5-trisphosphate-induced conductance in snake vomeronasal receptor neurons

Snake vomeronasal receptor neurons in slice preparations were studied using the patch-clamp technique in the conventional and nystatin-perforated whole-cell configurations. The mean resting potential was approximately -70 mV; the average input resistance was 3 GOmega. Neurons required current injection of only 1-10 pA to display a variety of spiking patterns. Intracellular dialysis of 100 microM inositol 1,4,5-trisphosphate (IP(3)) evoked an inward current in 38% of neurons, with an average peak amplitude of 16.4 +/- 2.8 pA at a holding potential of -70mV. Application of 100 microM 3-deoxy-3-fluoro-D-myo-inositol 1,4,5-trisphosphate (F-IP(3)), a derivative of IP(3), also evoked an inward current in 4/8 (50%) neurons (32.6 +/- 58 pA at -70 mV, n = 4). The reversal potentials of the induced components were estimated to be -14 +/- 5 mV for IP(3) and -17 +/- 3 mV for F-IP(3). Bathing the neurons in 10 microM ruthenium red solution greatly reduced the IP(3)-evoked inward current to 1.6 +/- 1.1 pA at -70 mV (n = 6). With Cs(+)-containing internal solution, neither the Ca(2+)-ATPase inhibitor thapsigargin (1-50 microM) nor the Ca(2+)-ionophore ionomycin (10 microM) evoked a significant current response, suggesting that IP(3) can elicit current response in the neurons without mediation by intracellular Ca(2+) stores. Intracellular application of 1 mM cAMP evoked no detectable current response. Extracellular application of chemoattractant for snakes evoked a very large inward current. The reversal potential of the chemoattractant-induced current was similar to that of the IP(3)-induced current. The present results suggest that IP(3) may act as a second messenger in the transduction of chemoattractants in the garter snake vomeronasal organ.     

The inhibitory effects of dantrolene on action potential-induced calcium transients in cultured rat dorsal root ganglion neurons

We investigated the actions of dantrolene Ca(2+)-induced on Ca(2+)-release (CICR) evoked by action potentials in cultured rat sensory neurons. The effect of dantrolene on action potential after-depolarization and voltage-activated calcium currents was studied in cultured neonatal rat dorsal root ganglion cells (DRG) using the whole-cell patch-clamp technique. Depolarizing current injection evoked action potentials and depolarizing after-potentials, which are activated as a result of CICR following a single action potential in some cells. The type of after-potentials was determined by inducing action potentials from the resting membrane potential. Extracellular application of dantrolene (10 microM) abolished after-depolarizations without affecting action potential properties. Furthermore, dantrolene significantly reduced repetitive action potentials after depolarizing current injection into these neurons, but had no significant effect on the steady-state current voltage relationship of calcium currents in these neurons. We conclude that dantrolene inhibits the induction of action potential after depolarizations by inhibiting CICR in cultured rat sensory neurons.     

Acetylcholine responses of identified neurons in Helix pomatia--I. Interactions between acetylcholine-induced and potential-dependent membrane conductances

The influence of potential-dependent membrane conductances on amplitude and time course of acetylcholine (ACh) responses was studied. The investigations were performed on the identified neurons B1 and B3 of the buccal ganglion of Helix pomatia. The neurons B1 and B3 were depolarized by ACh. The depolarization was accompanied by a decrease of membrane resistance. An inward rectification occurring negative to the resting membrane potential (RMP) reduced the amplitude of the ACh depolarizations. An outward rectification occurring positive to the RMP consisted of two parts and ceiled the ACh responses. The early outward current reduced the amplitude and modified the time course of ACh responses. Local responses or axonal action potentials increased the amplitude of the ACh depolarizations.     

Halothane suppresses slow inward currents in hippocampal slices

Single-electrode voltage-clamp experiments were made on CA1 neurons in the presence of tetrodotoxin and K channel blockers. Applications of halothane (1-3% v/v) for 3-10 min caused a similar marked and reversible depression of slow inward currents (probably Ca currents) evoked by depolarizing pulses from a holding potential near -80 or near -40 mV. The peak amplitudes of the inward currents were much reduced, in a concentration-dependent manner, and they decayed more rapidly (half-decay time was shortened by a quarter). In most cases, leak conductances were diminished by halothane, making it unlikely that the suppression of inward currents was primarily caused by enhancement of outward currents. A similar inactivation of Ca currents in presynaptic terminals would explain why halothane depresses synaptic transmission.     

Axonal contribution to subthreshold currents inaplysia bursting pacemaker neurons

The contribution of axonal activity to the ionic currents which generate bursting pacemaker activity was studied by using the two-electrode voltage-clamp technique in Aplysia bursting neuron somata in conjunction with intraaxonal voltage recordings. Depolarizing voltage-clamp pulses applied to bursting cell somata triggered axonal action potentials. The voltage-clamp current recording exhibited transient inward current "notches" corresponding to each of the axonal spikes. The addition of 50 microM tetrodotoxin (TTX) to the bathing medium blocked the fast axonal spikes and current notches, revealing a slower axonal spike which was blocked by the replacement of external Ca2+ with Co2+. The inward current evoked by applying a depolarizing voltage-clamp pulse in the soma is distorted by the occurrence of the axonal Ca2+ spike. Elimination of the axonal spike, by injecting hyperpolarizing current into the axon, changes both the time course and the magnitude of the inward current. The axonal Ca2+ spikes are followed by a series of Ca2+-dependent afterpotentials: a rapid postspike hyperpolarization, a depolarizing afterpotential (DAP) and, finally, a long-lasting postburst hyperpolarization. The long-lasting hyperpolarization is not blocked by 50 mM external tetraethyl ammonium, an effective blocker of Ca2+-activated K+ current [IK(Ca)], and does not appear to reverse at EK. Hence, the axonal long-lasting hyperpolarization may not be due to IK(Ca). Somatic voltage-clamp pulses in bursting neurons are followed by a slow inward tail current, which is sometimes coincident with a DAP in the axon. In some cells, the amplitude of the slow inward tail current is greatly reduced if axonal spikes and DAPs are prevented by hyperpolarization of the axon, while, in other cells, elimination of axonal activity has little effect. Therefore, the slow inward tail current is not necessarily an artifact of poor voltage-clamp control over the axonal membrane potential but probably results from the activation of an ionic conductance mechanism located partly in the axon and partly in the soma.     

Excitatory responses of trigeminal neurons to substance P suggest involvement in sensory transmission

Responses to substance P application were studied with intracellular recording techniques in in vitro preparations of trigeminal root ganglion neurons of guinea pigs. Perfusion of substance P in micromolar concentrations markedly depolarized neurons and reduced their input conductances. Also, the threshold for spikes evoked by injections of depolarizing current pulses was decreased. Single electrode voltage-clamp recordings showed that substance P increased inward, and decreased outward currents evoked by hyperpolarizing voltage steps from holding potentials near rest. Depolarizing responses to substance P were attenuated in Na+-deficient solutions. The excitatory actions of this endogenous peptide on the perikarya of primary sensory neurons give rise to the possibility of physiological actions of substance P at multiple sites in the trigeminal system.     

Membrane and firing properties of avian medial vestibular nucleus neuronsin vitro

The intrinsic membrane and firing properties of medial vestibular nucleus (MVN) neurons were investigated in slices of the chick brainstem using intracellular recording and current injection. Avian MVN neurons fired spontaneous action potentials with very regular interspike intervals. The rapid repolarization of all action potentials was followed by an after-hyperpolarization. Intracellular injection of steps of hyperpolarizing current revealed both an inward rectification of the membrane potential during the step and a rebound depolarization following the offset of the step. In some neurons, the rebound depolarization resulted in bursts of action potentials. Steps of depolarizing current applied to spontaneously active neurons evoked increases in firing rate that were higher at the onset of the step than during the steady-state response. The relationship between current and firing rate was linear. The membrane and firing properties of avian MVN neurons were distributed continuously across the population of recorded neurons. These properties appear identical to those of rodent MVN neurons, suggesting that the composition and distribution of ion channels in the MVN neuronal membrane has been highly conserved across vertebrate species.Abbreviations MVN medial vestibular nucleus - VOR vestibulo-ocular reflex - AHP after-hyperpolarization     

Evoked Acetylcholine Release by Immortalized Brain Endothelial Cells Genetically Modified to Express Choline Acetyltransferase and/or the Vesicular Acetylcholine Transporter

Immortalized rat brain endothelial RBE4 cells do not express choline acetyltransferase (ChAT), but they do express an endogenous machinery that enables them to release specifically acetylcholine (ACh) on calcium entry when they have been passively loaded with the neurotransmitter. Indeed, we have previously reported that these cells do not release glutamate or GABA after loading with these transmitters. The present study was set up to engineer stable cell lines producing ACh by transfecting them with an expression vector construct containing the rat ChAT. ChAT transfectants expressed a high level of ChAT activity and accumulated endogenous ACh. We examined evoked ACh release from RBE4 cells using two parallel approaches. First, Ca2+-dependent ACh release induced by a calcium ionophore was followed with a chemiluminescent procedure. We showed that ChAT-transfected cells released the transmitter they had synthesized and accumulated in the presence of an esterase inhibitor. Second, ACh released on an electrical depolarization was detected in real time by a whole-cell voltage-clamped Xenopus myocyte in contact with the cell. Whether cells synthesized ACh or whether they were passively loaded with ACh, electrical stimulation elicited the release of ACh quanta detected as inward synaptic-like currents in the myocyte. Repetitive stimulation elicited a continuous train of responses of decreasing amplitudes, with rare failures. Amplitude analysis showed that the currents peaked at preferential levels, as if they were multiples of an elementary component. Furthermore, we selected an RBE4 transgenic clone exhibiting a high level of ChAT activity to introduce the Torpedo vesicular ACh transporter (VAChT) gene. However, as the expression of ChAT was inactivated in stable VAChT transfectants, the potential influence of VAChT on evoked ACh release could only be studied on cells passively loaded with ACh. VAChT expression modified the pattern of ACh delivery on repetitive electrical stimulation. Stimulation trains evoked several groups of responses interrupted by many failures. The total amount of released ACh and the mean quantal size were not modified. As brain endothelial cells are known as suitable cellular vectors for delivering gene products to the brain, the present results suggest that RBE4 cells genetically modified to produce ACh and intrinsically able to support evoked ACh release may provide a useful tool for improving altered cholinergic function in the CNS.     

Pre- and postsynaptic effects of eugenol and related compounds on Helix pomatia L. neurons

We showed how eugenol blocks the synaptic transmission and gave a possible interpretation how it inhibits the excitation-contraction coupling that several authors described previously. Eugenol acts both in the pre- and postsynaptic side of the neurons. It blocks the Ca2+-currents, decreases the membrane potential of the neurons, increases the inward resistance and decreases the GABA, ACh and glutamate evoked excitatory responses in submillimolar concentration.     

Generalized posttetanic changes in the excitatory postsynaptic and acetylcholine-evoked currents in snail neurons

Heteroreceptor posttetanic changes in excitatory postsynaptic currents (EPSC) and inward currents evoked by the local iontophoretic application of acetylcholine (ACh) on the dorsal surface of PLa3 and PRa3 Helix lucorum neurons were studied. The following changes in the currents were revealed over the course of 1-1.5 h after tetanization. The rhythmical ACh application (0.5-1.0 cps, 10-40 s) evokes potentiation of the orthodromic EPSC. The tetanic orthodromic stimulation of one of the nerves (n. intestinalis, n. pallialis dexter, or n. pallialis sinister; 1-5 cps, 1-2 min) causes the potentiation of the ACh current and also heterosynaptic depression of the EPSC. It is concluded that activation of subsynaptic and nonsynaptic neurotransmitter chemoreceptors evokes the development of generalized posttetanic changes in neuronal responses.     

Electrophysiological properties of neurons of the red nucleus in rat brain slices

In experiments on rat brain slices, we carried out intracellular recording from neurons of the red nucleus (RN). Passive electrical properties of these neurons (input resistance, membrane time constant) were evaluated. We detected voltage-dependent rebound depolarization and time dependent inward rectification when passing hyperpolarizing pulses of current through the cell. Injections of depolarizing currents caused rhythmical firing of the neurons; the frequency of these firings depends upon the strength of injected current. Rhythmical firings were also characterized by rapid frequency adaptation when currents of different frequency were injected. Stimulation of regions of slices presumably corresponding to the decussion of the brachium conjunctivum mainly evoked EPSPs with a "fast" rise time in RN neurons. This suggests activation of synaptic input from the cerebellar nucleus interpositus. Stimulation of this same region sometimes evoked EPSP-IPSP mixtures and "pure" IPSPs in RN neurons.L. A. Orbeli Institute of Physiology, Armenian Academy of Sciences, Erevan. Translated from Neirofiziologiya, Vol. 23, No. 5, pp. 607–616, September–October, 1991.     

Voltage control during inward current flow in rat ventricular muscle using a double sucrose gap technique.

In rat ventricular muscle, measurements of the membrane potential with microelectrodes during depolarizing voltage steps showed that deviation of the membrane potential from the command signal were never larger than 15 mV during flow of the fast inward current and that voltage control was regained within 15 ms after the beginning of the voltage step. During the flow of the slow inward current, tail currents elicited by interrupting the time course of the slow current at different time intervals returned exponentially to the steady-state level, thus indicating acceptable voltage control. It is concluded that rat ventricular muscle is a rather favorable preparation for voltage-clapm experiments and this is attributed mainly to the geometry of the preparation.     

Relaxation studies on the interaction of hexamethonium with acetylcholine-receptor channels inAplysia neurons

The mode of action of the cholinergic antagonist hexamethonium on the excitatory responses of voltage-clamped Aplysia neurons to acetylcholine (ACh) has been examined by voltage- and concentration-jump relaxation analysis. At steady-state concentrations of ACh hyperpolarizing command steps induced inward current relaxations to a new steady-state level (Iss). The time constants of these inward relaxations, tau f, which approximate the mean single-channel lifetime, were increased both by increasing the membrane potential and by lowering the bath temperature (Q10 = 3) but were not affected by increasing the ACh concentration over the dose range employed. In the presence of hexamethonium hyperpolarizing command steps produced biphasic relaxations of the agonist-induced current. tau f was reduced in a voltage-dependent manner, the degree of reduction increasing with hyperpolarization. Slow, inverse relaxations were also triggered in the presence of hexamethonium. The time constant of this relaxation was reduced by increasing membrane potential and hexamethonium concentration. Both the estimated association (kf = 5 X 10(4) M-1 . sec-1) and the estimated dissociation (kb = 0.24-0.29 sec-1) rate constants derived from a three-state sequential model for block by hexamethonium were independent of the membrane potential. Similar rate constants were estimated from experiments with the concentration-jump technique, which were also independent of the membrane potential over the range -50 to -110 mV. It is suggested that the voltage-dependent actions of hexamethonium may originate either from an alteration of the channel opening and closing rate constants through an allosteric interaction with the ACh receptor, rather than through an influence of the transmembrane electric field on the rate of drug binding, or through a fast reaction which is rate-limited by voltage-independent diffusion.     

"Creep currents" in single frog atrial cells may be generated by electrogenic Na/Ca exchange

The objective of these experiments was to test the hypothesis that the "creep currents" induced by Na loading of single frog atrial cells (Hume, J. R., and A. Uehara. 1986. Journal of General Physiology. 87:833) may be generated by an electrogenic Na/Ca exchanger. Creep currents induced by Na loading were examined over a wide range of membrane potentials. During depolarizing voltage-clamp pulses, outward creep currents were observed, followed by inward creep currents upon the return to the holding potential. During hyperpolarizing voltage-clamp pulses, creep currents of the opposite polarity were observed: inward creep currents were observed during the pulses, followed by outward creep currents upon the return to the holding potential. The current-voltage relations for inward and outward creep currents in response to depolarizing or hyperpolarizing voltage displacements away from the holding potential all intersect the voltage axis at a common potential, which indicates that inward and outward creep currents may have a common reversal potential under equilibrium conditions and may therefore be generated by a common mechanism. Measurements of inward creep currents confirm that voltage displacements away from the holding potential rapidly alter equilibrium conditions. Current-voltage relationships of inward creep currents after depolarizing voltage-clamp pulses are extremely labile and depend critically upon the amplitude and duration of outward creep currents elicited during preceding voltage-clamp pulses. An optical monitor of mechanical activity in single cells revealed (a) a similar voltage dependence for the outward creep currents induced by Na loading and tonic contraction, and (b) a close correlation between the time course of the decay of the inward creep current and the time course of mechanical relaxation. A mathematical model of electrogenic Na/Ca exchange (Mullins, L.J. 1979. Federation Proceedings. 35:2583; Noble, D. 1986. Cardiac Muscle. 171-200) can adequately account for many of the properties of creep currents. It is concluded that creep currents in single frog atrial cells may be attributed to the operation of an electrogenic Na/Ca exchange mechanism.