Pulses Of ACh Research Articles

The quantal catecholamine release from mouse chromaffin cells challenged with repeated ACh pulses is regulated by the mitochondrial Na+ /Ca2+ exchanger.

Upon repeated application of short ACh pulses to C57BL6J mouse chromaffin cells, the amperometrically monitored secretory responses promptly decayed to a steady-state level of around 25% of the initial response. A subsequent K+ pulse, however, overcame such decay. These data suggest that mouse chromaffin cells have a ready release-vesicle pool that is selectively recruited by the physiological neurotransmitter ACh. The ACh-sensitive vesicle pool is refilled and maintained by the rate of Ca2+ delivery from mitochondria to the cytosol, through the mitochondrial Na+ /Ca2+ exchanger (mNCX). ITH12662, a novel blocker of the mNCX, prevented the decay of secretion elicited by ACh pulses and delayed the rate of [Ca2+ ]c clearance. This regulatory pathway may be physiologically relevant in situations of prolonged stressful conflicts where a sustained catecholamine release is regulated by mitochondrial Ca2+ circulation through the mNCX, which couples respiration and ATP synthesis to long-term stimulation of chromaffin cells by endogenously released ACh. Using caged-Ca2+ photorelease or paired depolarising pulses in voltage-clamped chromaffin cells (CCs), various pools of secretory vesicles with different readiness to undergo exocytosis have been identified. Whether these pools are present in unclamped CCs challenged with ACh, the physiological neurotransmitter at the splanchnic nerve-CC synapse, is unknown. We have explored here whether an ACh-sensitive ready-release vesicle pool (ASP) is present in C57BL6J mouse chromaffin cells (MCCs). Single cells were fast perfused with a Tyrode solution at 37°C, and challenged with 12 sequential ACh pulses (100μm, 2s, every 30s) plus a K+ pulse given at the end (75mm K+ ). After the first 2-3 ACh pulses the amperometrically monitored secretory responses promptly decayed to a steady-state level of around 25% of the initial response. The last K+ pulse, however, overcame such decay. Repeated ACh pulses to voltage-clamped cells elicited non-desensitising nicotinic currents. Also, the [Ca2+ ]c transients elicited by repeated ACh pulses that were superimposed on a stable baseline elevation did not undergo decay. The novel blocker of the mitochondrial Na+ /Ca2+ exchanger (mNCX) ITH12662 prevented the decay of secretion elicited by ACh pulses and delayed the rate of [Ca2+ ]c clearance. The experiments are compatible with the idea that C57BL6J MCCs have an ASP vesicle pool that is selectively recruited by the physiological neurotransmitter ACh and is regulated by the rate of Ca2+ delivery from mitochondria to the cytosol, through the mNCX.

A role of local VTA GABAergic neurons in mediating dopamine neuron response to nicotine

The local circuitry of the tegmental area (VTA) consists primarily of dopamine (DA) and GABA neurons. Interactions between DA and GABA neurons are critical for regulating DA neuron activity, and thus DA efflux throughout the brain. One striking example that demonstrates the significance of local interactions between DA and GABA neurons is related to nicotine reinforcement. Experimentally, it was shown that activation of nicotinic acetylcholine (nAch) receptors on GABA neurons by Ach leads to an increase in both firing and bursting of the DA neuron, while nicotine produces an opposite effect [1]. In order to investigate the mechanism of this GABA-mediated effect, we created a biologically plausible model of local VTA microcircuitry. The model network consists of a population of GABA neurons innervating one DA neuron. DA neuron dynamics are described by a conductance based model; which includes intrinsic and synaptic currents conducted by NMDA, AMPA, GABA, and nAch receptors. GABA neurons that are described by Wang-Buszaki equations provide inhibitory drive to the DA neuron. Excitatory inputs (Glu and Ach) to DA and GABA neurons are modeled as Poisson-distributed spike trains. Ach pulses act as synchronizing inputs to a population of GABA neurons, due to a transient activation of GABA nAch receptors, while nicotine persistently activates nAch receptor, causing an increase in firing frequency of GABA neurons. Modeling revealed that synchronization of GABA neurons by cholinergic input could provide a mechanism for the elevation of DA neuron firing frequency and bursting. Synchronized GABA inputs act via phasic disinhibition that promotes rebound spikes, but is conditioned on the presence of depolarizing currents to the DA neuron. The opposite effects of nicotine applied to the GABA neuron only (decrease in firing and bursting of DA neuron), could be the result of desynchronization in population of GABA neurons, produced by tonic activation of nAch receptor. A desynchronized GABA population provides constant inhibition to the DA neuron that suppresses firing (see Figure ​Figure1).1). These data highlight the important and powerful role local circuit dynamics VTA. Furthermore, synchrony amongst GABA neurons seems to be a critical intermediary that regulates DA neuron activity and, by extension, DA release throughout the brain. Figure 1 A) Quantification of Ach mediated firing of DA neuron (firing rate and %SWB) B) Nicotine-elicited modifications in firing rate of DA neuron. Vertical black lines indicate nicotine injection. Horizontal dashed lines indicate the baseline firing rate. ...

Amino Acid Residues That Confer High Selectivity of the α6 Nicotinic Acetylcholine Receptor Subunit to α-Conotoxin MII[S4A,E11A,L15A

Nicotinic acetylcholine receptors (nAChRs) containing alpha3 and beta2 subunits are found in autonomic ganglia and mediate ganglionic transmission. The closely related alpha6 nAChR subtype is found in the central nervous system where changes in its level of expression are observed in Parkinson's disease. To obtain a ligand that discriminates between these two receptors, we designed and synthesized a novel analog ofalpha-conotoxin MII, MII[S4A,E11A,L15A], and tested it on nAChRs expressed in Xenopus oocytes. The peptide blocked chimeric alpha6/alpha3beta2beta3 nAChRs with an IC(50) of 1.2 nm; in contrast, its IC(50) on the closely related alpha3beta2 as well as non-alpha6 nAChRs was three orders of magnitude higher. We identified the residues in the receptors that are responsible for their differential sensitivity to the peptide. We constructed chimeras with increasingly longer fragments of the N-terminal ligand binding domain of the alpha3 subunit inserted into the homologous positions of the alpha6 subunit, and these were used to determine that the region downstream of the first 140 amino acids was involved. Further mutagenesis of this region revealed that the alpha6 subunit residues Glu-152, Asp-184, and Thr-195 were critical, and replacement of these three residues with their homologs from the alpha3 subunit increased the IC(50) of the peptide by >1000-fold. Conversely, when these key residues inalpha3 were replaced with those fromalpha6, the IC(50) decreased by almost 150-fold. Similar effects were seen with other alpha6-selective conotoxins, suggesting the general importance of thesealpha6 residues in conferring selective binding.

Acetylcholine and potassium elicit different patterns of exocytosis in chromaffin cells when the intracellular calcium handling is disturbed.

During fast superfusion of bovine chromaffin cells with normal Krebs-HEPES solution containing 2 mM Ca2+, pulses of 100 microM ACh or 100 mM K+ of increasing duration (1-5 s) caused similar exocytosis of about 3-4 microC catecholamine. Depletion of endoplasmic reticulum (ER) Ca2+ by pretreatment with 1 microM thapsigargin, 10 mM caffeine and 10 microM ryanodine more than halved the responses to ACh but did not affect the responses to K+ responses. In these ER Ca2+-depleted cells the protonophore carbonylcyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) (20 microM given during the 5 s preceding each pulse) augmented the responses to ACh responses fourfold for all pulse durations applied (1-5 s) whereas responses to K+ were potentiated twofold with 1 to 2 s pulses but were not affected with longer pulse durations. ACh pulses applied to fura-2-loaded cells evoked increases of bulk cytosolic [Ca2+] ([Ca2+](c)) that were substantially smaller than those elicited by K+ pulses. Confocal microscopy of fluo-3-loaded cells showed that ACh pulses elicited discrete and more localized [Ca2+](c) elevations, whereas K+ pulses produced higher [Ca2+](c) transients that spread out quickly throughout the cytosol. These results suggest that mitochondria sense the increase of local [Ca2+](c) elicited by ACh (that evokes firing of action potentials) much better than that induced by K+ (that produces sustained cell depolarisation). This implies that mitochondria are more sensitive to the local [Ca2+](c) changes resulting from the physiological triggering of action potentials by ACh.

Quantal endplate currents from newborn to adult mice and the switch from embryonic to adult channel type

Quantal endplate currents (qEPCs) were recorded extracellularly by a macropatch electrode from excised diaphragms of mice. During the first 3 days after birth, the mean rise time t r was 0.5 ms (0.1–0.9 peak, 20 °C). Double-exponential, amplitude-weighted fits of the decay discerned almost equally abundant components τ 1′≈6 ms and τ 2′≈9 ms. Beginning on day 3, on days 4 and 5 after birth both t r and the τ′ dropped. Further decreasing slowly, adult values were reached at day 8, with t r≈0.3 ms, τ 1′≈2 ms and a very weak τ 2′≈6 ms component. When compared to the kinetics of fetal channels, the τ 1′ and τ 2′ of up to 3 day qEPCs could correspond to the short and long splice variants of the fetal channel type. The τ 1′ of adult muscles of 2 ms agrees well with the burst durations of adult channels while a weak longer τ 2′ component may represent ‘extrasynaptic’ channels. The long t r of very young mice may correspond to the relatively slow rise of channel currents elicited by ACh pulses in mouse myoballs.

Facilitation of glutamatergic neurotransmission by presynaptic nicotinic acetylcholine receptors

The profiles of presynaptic facilitation of glutamate release as elicited by nicotine and acetylcholine were compared in two limbic pathways recapitulated in vitro. At synapses of medial habenula (MHN) and interpeduncular nucleus (IPN) neurons, application of nicotine increased the frequency of TTX-resistant, spontaneous postsynaptic currents (SSCs) by an average of 5-fold. In contrast, the average increase in SSC frequency elicited by nicotine was more than 120 fold at synapses of olfactory bulb (OB) and amygdala neurons. At both preparations, pulses of ACh caused presynaptic facilitation that lasted longer than that elicited by nicotine. The subunit composition of presynaptic nAChRs may contribute to the different profiles of facilitation observed. The large magnitude, fast kinetics, and α–bungarotoxin sensitivity of facilitation observed at OB–amygdala synapses is consistent with participation of α7-type nAChRs. As subunit-selective deletion of α5 or α7 altered the profile of nicotine-elicited facilitation at MHN–IPN synapses, presynaptic nAChRs at MHN–IPN synapses appear to be more complex. Such heteromeric combinations of nAChRs may contribute to the lower magnitude and slower kinetics of presynaptic facilitation at MHN–IPN synapses. Calcium influx through either voltage-gated calcium channels or directly through presynaptic α7-containing nAChRs is sufficient to support nicotine-elicited facilitation of glutamate release. Resultant increases in intracellular calcium may further modulate presynaptic nAChR activity in a subunit-composition dependent manner.

The novel Na(+)/Ca(2+) exchange inhibitor KB-R7943 also blocks native and expressed neuronal nicotinic receptors.

We studied the effects of the novel Na(+)/Ca(2+) exchange inhibitor KB-R7943, 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulphonate, on the native nicotinic receptors present at the bovine adrenal chromaffin cells, as well as on rat brain alpha(3)beta(4) and alpha(7) nicotinic acetylcholine receptors (AChRs) expressed in Xenopus oocytes. As expected, KB-R7943 blocked the Na(+)-gradient dependent (45)Ca(2+) uptake into chromaffin cells (IC(50) of 5.5 microM); but in addition, the compound also inhibited the (45)Ca(2+) entry and the increase of cytosolic Ca(2+) concentration, [Ca(2+)](c), stimulated by 5 s pulses of ACh (IC(50) of 6.5 and 1.7 microM, respectively). In oocytes expressing alpha(3)beta(4) and alpha(7) nicotinic AChRs, voltage-clamped at -60 mV, inward currents elicited by 1 s pulses of 100 microM ACh (I(ACh)) were blocked by KB-R7943 with an IC(50) of 0.4 microM and a Hill coefficient of 0.9. Blockade of alpha(3)beta(4) currents by KB-R7943 was noncompetitive; moreover, the blocker (0.3 microM) became more active as the ACh concentration increased (34 versus 66% blockade at 30 microM and 1 mM ACh, respectively). Inhibition of alpha(3)beta(4) currents by 0.3 microM KB-R7943 was more pronounced at hyperpolarized potentials. If given within the ACh pulse (10 microM), the inhibition amounted to 33, 64 and 80% in oocytes voltage-clamped at -40, -60 and -100 mV, respectively. The onset of blockade was faster and the recovery slower at -100 mV; the reverse was true at -40 mV. In conclusion, KB-R7943 is a potent blocker of nicotinic AChRs; moreover, it displays many features of an open-channel blocker at the rat brain alpha(3)beta(4) AChR. These results should be considered when KB-R7943 is to be used to study Ca(2+) homeostasis in cells expressing nicotinic AChRs and the Na(+)/Ca(2+) exchanger.

The relationship between acetylcholine-evoked Ca(2+)-dependent current and the Ca2+ concentrations in the cytosol and the lumen of the endoplasmic reticulum in pancreatic acinar cells.

In a study of isolated mouse pancreatic acinar cells, we used the patch-clamp whole-cell recording configuration to monitor the Ca(2+)-dependent inward ionic current and simultaneously measured the Ca2+ concentration in either the cytosol ([Ca2+]i) or the lumen of the endoplasmic reticulum ([Ca2+]Lu), using appropriate Ca(2+)-sensitive fluorescent probes. A high concentration of acetylcholine (ACh, 10 microM) evoked an increase in [Ca2+]i, which resulted in the activation of Ca(2+)-dependent inward current. Continued ACh application for several minutes led to a marked reduction in both the current and the [Ca2+]i response and after about 4-10 min of sustained ACh stimulation, the inward current response had disappeared and [Ca2+]i was back to the pre-stimulation level. Repeated stimulation with shorter pulses of ACh (10 microM) resulted in responses of declining magnitude both in terms of inward current and [Ca2+]i rises. The ACh-activated inward current was entirely dependent on the elevation of [Ca2+]i, but at a relatively high [Ca2+]i the current was saturated. ACh caused a rapid release of Ca2+ from the lumen of the endoplasmic reticulum and after discontinuation of stimulation, [Ca2+]Lu was only very slowly (10-15 min) fully restored to the pre-stimulation level. Repeated applications of ACh did not change the relationships between the Ca(2+)-dependent current and [Ca2+]i or the current and [Ca2+]Lu. When [Ca2+]Lu was greater than 100 microM, the ACh-evoked Ca2+ release from the store was so large that the current response was initially saturated. We conclude that the ACh-evoked current response essentially depends on the release of stored Ca2+. Desensitization is mainly due to the relatively slow reloading of the intracellular stores with Ca2+.

Two mutations linked to nocturnal frontal lobe epilepsy cause use-dependent potentiation of the nicotinic ACh response.

1. We constructed rat homologues (S252F and +L264) of two human alpha4 nicotinic mutations - alpha4(S248F) and alpha4(777ins3) - that have been linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) and co-expressed them with wild-type rat beta2 subunits in Xenopus oocytes. 2. The S252F and +L264 mutations had three common effects on the ACh response. First, they caused use-dependent potentiation of the response during a train of brief 100 nM ACh pulses. Second, they delayed the rise times of the 5-15 nM (+L264) and 30 nM (S252F) ACh responses. Third, they reduced extracellular Ca2+-induced increases in the 30 microM ACh response. 3. Beside these shared effects, the S252F mutation also reduced the channel burst duration measured from voltage-jump relaxations, enhanced steady-state desensitization and reduced the single-channel conductance. In contrast, the +L264 mutation prolonged the channel burst duration, did not affect desensitization and slightly increased single-channel conductance. Neither mutation affected the number of surface receptors measured by antibody binding but the S252F mutation reduced the maximum ACh response. 4. The ACh concentration dependence of use-dependent potentiation and the delay in the rising phase of the mutant ACh response suggest that these effects are caused by a slow unblocking of the closed mutant receptors. Use-dependent potentiation of the mutant response during a series of high-frequency cholinergic inputs to the presynaptic terminal could trigger ADNFLE seizures by suddenly increasing nicotinic-mediated transmitter release.

Purified IgG from seropositive and seronegative patients with mysasthenia gravis reversibly blocks currents through nicotinic acetylcholine receptor channels.

The mechanism of block of nicotinic acetylcholine receptor (nAChR) channels by purified antibodies from patients with myasthenia gravis (MG) was investigated by using an ultrafast system for solution exchange at outside-out patches. IgG of MG patients and controls was purified by using protein A-Sepharose columns. Probes from 9 seropositive MG patients and 3 seronegative MG patients were tested. As a preparation, cultured mouse myotubes expressing the embryonic-type nAChR channels were used. Twenty-millisecond pulses of 1.0 mM ACh were applied repetitively to outside-out patches. Outside-out patches were preexposed with IgG in concentrations between 0.1 and 200 mg/L during application of ACh pulses. The peak current amplitude was reduced to values between 6% and 71% of control for the 9 seropositive and 3 seronegative MG patients. The block was concentration dependent and fully reversible after washout of antibodies. Incubation with IgG from different control patients did not reduce the peak current amplitude. In addition, our findings with purified IgG from seronegative MG patients support the idea of the immunopathogenesis of this disorder and may allow the development of a diagnostic test for seronegative MG patients.

Role of phosphorylation in desensitization of acetylcholine receptors expressed in Xenopus oocytes

The nicotinic acetylcholine receptor (AChR) is a pentameric complex made up of four types of subunits in the stoichiometry alpha 2 beta gamma delta. These subunits have been shown to be differentially phosphorylated by cAMP-dependent protein kinase (PKA) protein kinase C, and a protein tyrosine kinase. A variety of studies have suggested that phosphorylation of the AChR in vitro and in vivo regulates the rate of desensitization of the receptor. In this study we have used site-specific mutagenesis and patch-clamp techniques to examine the role of phosphorylation in the regulation of desensitization of the AChR expressed in Xenopus oocytes Expression of wild-type AChR in Xenopus oocytes results in the constitutive phosphorylation of the AChR on the gamma and delta subunits. This phosphorylation is apparently due to the high basal level of PKA in oocytes since a specific peptide inhibitor of PKA completely eliminated phosphorylation of the AChR by oocyte extracts in vitro. The phosphorylation of the AChR in oocytes was not significantly enhanced by forskolin or cAMP analogs or by coexpression with the catalytic subunit of PKA, suggesting that the basal activity of PKA in oocytes is sufficient to phosphorylate the receptor to a high stoichiometry. Using site-specific mutagenesis, the sites of phosphorylation were determined to be serines 353 and 354 on the gamma subunit and serines 361 and 362 on the delta subunit. To examine the functional properties of wild-type and mutant receptors lacking phosphorylation sites, we used patch-clamp techniques to measure the responses of out-side-out patches to repetitive pulses of ACh using a rapid perfusion system. Wild-type and mutant receptors showed rapid concentration-dependent activation and desensitization to applied agonist. The time constant of desensitization of ensemble mean currents ranged from several hundred milliseconds at low ACh concentrations to 100-200 msec at saturating concentrations. The desensitization time constants for mutant receptors lacking all phosphorylation sites were significantly slower than wild-type phosphorylated receptors at all concentrations of ACh tested. In addition, mutant receptors that had the serine residues changed to glutamate residues in order to mimic the negative charge of the phosphorylated serine residue produced receptors that had desensitization rates approaching those of the wild-type phosphorylated receptor. These results provide further support that phosphorylation of the nicotinic ACh receptor regulates rate of desensitization.

Chaotic activity in a mathematical model of the vagally driven sinoatrial node.

Phase-locking behavior and irregular dynamics were studied in a mathematical model of the sinus node driven with repetitive vagal input. The central region of the sinus node was simulated as a 15 x 15 array of resistively coupled pacemakers with each cell randomly assigned one of 10 intrinsic cycle lengths (range 290-390 msec). Coupling of the pacemakers resulted in their mutual entrainment to a common frequency and the emergence of a dominant pacemaker region. Repetitive acetylcholine (ACh; vagal) pulses were applied to a randomly selected 60% of the cells. Over a wide range of stimulus intensities and basic cycle lengths, such perturbations resulted in a large variety of stimulus/response patterns, including phase locking (1:1, 3:2, 2:1, etc.) and irregular (i.e., chaotic) dynamics. At a low ACh concentration (1 microM), the patterns followed the typical Farey sequence of phase-locked behavior. At a higher concentration (5 microM), period doubling and aperiodic patterns were found. When a single pacemaker cell was perturbed with repetitive ACh pulses, qualitatively similar results were obtained. In both types of simulation, chaotic behavior was investigated using phase-plane (orbital) plots, Poincaré mapping, and return mapping. Period-doubling bifurcations (2:2, 4:4, and 8:8) were found temporally and spatially within the array. Under certain conditions of stimulation, the attractor in the return map during chaotic activity of the single cell resembled the Lorenz tent map. However, when electrical coupling between cells was allowed, the interactions with neighboring cells exhibiting chaotic dynamics resulted in characteristic alterations of the attractor geometry. Our results suggest that irregular dynamics obeying the rules derived from other chaotic systems are present during vagal stimulation of the sinus node. In addition, application of the same analytical tools to the analysis of simulation of reflex vagal control of sinus rate suggests that chaotic dynamics can be obtained in the physiologically relevant case of the baroreceptor reflex loop. These results may provide insight into the mechanisms of dynamic vagal control of heart rate and may help to provide insights into clinically relevant disturbances of cardiac rate and rhythm.

Effects of Mytilus inhibitory peptides on mechanical responses of various molluscan muscles

1. Effects of the Mytilus inhibitory peptides (MIPs), Ala 2-MIP and Ser 2-MIP, on mechanical responses of various molluscan muscles were examined. 2. In the ABRM of Mytilus edulis, all contractions in response to repetitive electrical pulses of stimulation, ACh, FMRFamide and caffeine were inhibited by MIPs. Contraction by 400 mM K + ASW was invariably not affected, but that by 100 mM K + ASW was slightly inhibited in some muscles. 3. In the pedal retractor muscle of Mytilus edulis, contractions in response to repetitive electrical pulses of stimulation and ACh were also inhibited by MIPs. 4. In the heart of Meretrix lusoria, its activity was inhibited by MIPs. Ala 2-MIP was found to be more potent than Ser 2-MIP. The ACh-antagonist benzoquinonium did not change the inhibitory effects of the peptides. In the heart of Tapes japonica, both of the peptides showed weak inhibitory effect at 100 nM. At 10 μM, however, Ala 2-MIP showed inhibitory effect followed by excitatory effect, and Ser 2-MIP showed excitatory effect and then brought about a transient cystolic arrest. 5. Twitch and tetanic contractions of the penis retractor muscle of Achatina fulica were inhibited by MIPs. Ala 2-MIP was found to be more potent than Ser 2-MIP. 6. MIPs seem to have an inhibitory effect on many molluscan muscles. There may exist a family of MIP-related peptides in molluscs.

Postsynaptic potentiation and desensitization in frog neuromuscular junction

The fractional increase in ACh responses that occurs at the beginning of each train of iontophoretically applied ACh pulses has been examined at the frog neuromuscular junction at room temperature, in the presence of active cholinesterase, during desensitization produced by a rapid sequence (every 20 s) of short (5 Hz, 5 s) iontophoretic trains of ACh. The fractional increase in ACh responses, which is used as an indicator of postsynaptic potentiation, becomes progressively greater with ACh application, often markedly (greater than 100%), although ACh responses are greatly reduced (as much as 90%) owing to desensitization. Clearly postsynaptic potentiation can exist concomitantly with desensitization. In addition, the dose-response curve is shifted to the right and its maximal response is diminished. The shift in the dose-response curve to the right, which can explain greater postsynaptic potentiation, is unlikely to be caused by accumulation of "monoligand-bound ACh receptor complexes," since experiments were done with active cholinesterase. The shift probably results from a greater number of desensitized receptors which, because of their large affinity for ACh molecules, serve as "high affinity traps." A small decrease of the maximal dose-response suggests only a small fractional decrease in the number of activable receptors, whereas a large shift to the right indicates a large fractional increase in the number of desensitized receptors. It appears that prior to ACh application only a small fraction of all receptors are desensitized. Alternatively, the shift to the right occurs because the cooperative action of ACh on receptors increases during desensitization.

Serotonin enhances the excitatory acetylcholine response in the RB cell cluster of Aplysia californica

The RB cells, an identified cluster of serotonergic neurons in the abdominal ganglion of Aplysia californica, are excited by ACh. We have found that serotonin (5-HT) enhances the ACh responses recorded from the RB cells. The locus and specificity of the serotonergic interaction with the ACh response were analyzed using the conventional two-electrode voltage-clamp technique. We compared the current responses evoked in RB cells by constant-current iontophoretic pulses of ACh in control solutions and in solutions containing various concentrations of 5-HT. Serotonin consistently enhances the ACh evoked response in a dose-dependent manner while simultaneously directly activating the RB cells by turning on a small steady-state inward current. The dose-response characteristics for the 5-HT-mediated steady-state current differ, however, from those of the 5-HT-induced ACh response facilitation. The ACh response enhancement appears to be an effect of 5-HT upon RB cell ACh receptors since the enhancement is present under conditions that eliminate presynaptic input, and 5-HT is still capable of enhancing the iontophoretically evoked response when the AChE-resistant agonist carbachol is substituted for ACh. We tested other neurons in the Aplysia CNS exhibiting ACh evoked responses qualitatively similar to the RB cell ACh responses; 5-HT does not enhance the iontophoretically evoked ACh responses of these cells. Also, although the RB cells are responsive to dopamine and histamine, these putative neurotransmitters do not enhance the ACh response.

Effects of divalent cations on acetylcholine-evoked membrane potential in the ionophore A23187 treated mouse pancreas.

Membrane potential and resistance were measured in isolated segments of mouse pancreas superfused in vitro using two intracellular microelectrodes. The acinar cells were stimulated by microionophoretic ACh application from an extracellular AChCl filled micropipette. The membrane depolarization and resistance reduction evoked by short pulses of ACh stimulation were abolished by treatment with A23187 containing Ca-free solution. The subsequent exposure of the A23187-treated tissue to a Ca or Sr containing solution restored the responses to short pulses of ACh. Addition of Ba, Co, or Mn instead of Ca had no such restorative effects. Mn or Co markedly suppressed the restorative effects of Ca or Sr. It is concluded that A23187 treatment causes depletion of intracellular Ca stores. The restorative effect of Ca or Sr indicates that short pulses of ACh stimulation evoke an increase in intracellular Ca or Sr concentration. Short pulses of ACh seem to release Ca from the stimulant-sensitive Ca pool and, in addition, to increase the membrane Ca permeability.

Diffusional/kinetic analysis of the neurotransmission process at the nerve-muscle junction.

Transmission at the neuromuscular junction is known to comprise the following steps: vesicular release of Ach pulses; transport across the synaptic cleft; partial hydrolysis by Ach-ase; binding to Ach receptors; ion channel opening and closure, and, thereby, alteration of sodium and potassium conductances. Combining these elements, a unified model has been constructed that is effective in describing the relation between neurotransmitter arrival at receptor sites and channel openings, leading to simple relationships for sodium and potassium conduction at the neuromuscular junction. Application of the model to mepc and epp data has been successful.

Membrane transport/reaction model of the neurotransmission process at the nerve-muscle junction

Transmission at the neuromuscular junction is known to comprise the following steps: synchronous and asynchronous vesicular release of Ach pulses (Ach = acetylcholine); transport across the synaptic cleft; partial hydrolysis by Ach-ase; binding to Ach receptors; ion channel opening and closure, and, thereby, alteration of sodium and potassium conductances. Combining these elements, a unified model has been constructed which is effective in describing the relation between neurotransmitter arrival/departure at receptor sites and channel opening/closure, leading to simple relationships for sodium and potassium conduction at the neuromuscular junction. Application of the model to miniature endplate current (mepc) and endplate potential (epp) data has been successful.

Further studies on the action of 5-hydroxytryptamine in the dorsal lateral geniculate nucleus of the cat

The possibility that 5-HT has a presynaptic inhibitory influence on the optic nerve input to the LGNd has been examined. We have compared the effect of iontophoretic application of 5-HT on the excitatory responses of LGNd neurons to visual stimulation and iontophoretic pulses of ACh and DLH. In contrast to previous studies the results suggest that 5-HT has only postsynaptic depressant effects and we found no evidence for a selective inhibition of the optic nerve input. It is proposed that the difference between the present results and those previously reported may reflect the method of stimulation of the optic nerve input. The weaker excitatory responses produced with electrical stimulation and diffuse light flashes of the earlier studies may be more susceptible to the postsynaptic depressant actions of 5-HT than the powerful visually evoked responses of the present study. Our data are only consistent with a postsynaptic depressant role for 5-HT in the LGNd.

Control of junctional acetylcholinesterase by neural and muscular influences in the rat.

1. The development of AChE at ectopic neuromuscular junctions forming between a transplanted foreign nerve (the superficial fibular nerve) and the denervated soleus muscle has been studied in adult rats. 2. Junctional AChE activity began to appear in the vicinity of the fibular nerve sprouts 6-7 days after section of the soleus nerve and 3-4 days after the onset of transmission. 3. No histochemically detectable AChE appeared when the fibular nerve was cut 0-4 days after the soleus nerve had been cut. 4. Direct electrical stimulation of the denervated soleus muscle caused plaques of true AChE, as determined by inhibitor studies, to appear in muscles where the fibular nerve had been cut 2-4 days after the soleus nerve but not in muscles where the two nerves had been cut at the same time. The plaques appeared only in the vicinity of fibular nerve sprouts and coincided with newly formed but stable peaks of ACh sensitivity. Local application of Neostigmine prolonged and increased the depolarising response evoked by pulses of ACh at these sites. 5. In muscles where the fibular nerve was intact the AChE plaques changed gradually over a few weeks from an immature appearance to a mature appearance characteristic of normal end-plates. In stimulated muscles where the fibular nerve had been cut the plaques stained intensely but remained morphologically immature. 6. We conclude (1) that muscle activity is important for the appearance of AChE at developing neuromuscular junctions and (2) that AChE accumulates only at sites on the muscle surface where the nerve fibres have left a 'trace' upon contact with the muscle fibres. These traces form quickly and persist after nerve-muscle interaction of as little as 2 days. The muscle appears as a major source of junctional AChE since stimulation of the muscle induces intense AChE activity in muscles where the nerve has degenerated.