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.     

Acetylcholine responses of identified neurons in Helix pomatia--III. Ionic composition of the depolarizing currents induced by acetylcholine

The ionic composition of the currents underlying the acetylcholine (ACh) depolarizations in the identified neurons B1 and B3 of the buccal ganglia of Helix pomatia was analysed. The equilibrium potential of the ACh responses was -2.8 +/- 0.6 mV (N = 49) and -4.0 +/- 0.7 mV (N = 79; mean +/- SEM) in the neurons B1 and B3, respectively. Replacement of NaCl in the bath solution by sucrose shifted the ACh equilibrium potential into the negative direction. A similar but less pronounced shift occurred when Ca2+ was substituted for Na+. Substitution of Cl- in the bath solution by propionate or an increase of the intracellular Cl- concentration did not affect the ACh equilibrium potential. Changes of K+ concentration in the bath between 1 and 50 mmol/l left the ACh equilibrium potential nearly unaffected when the Na+ concentration was at the control level. With a simultaneous reduction of extracellular Na+ an increase of K+ concentration shifted the ACh equilibrium potential towards more positive potentials. The findings are compatible with calculated K+ permeabilities if a K+ redistribution across the cell membrane is considered. In the neurons B1 and B3, channels operated by ACh are permeable for K+, Na+ and Ca2+, with the relative permeabilities 1.6:1.0:0.1.     

Outward currents in olfactory receptor neurons activated by odorants and by elevation of cyclic AMP

We studied the outward currents elicited by an odorous compound, isoamyl acetate, in isolated olfactory receptor neurons of the grass frog under whole-cell perforated-patch voltage-clamp recording. Odorant-induced outward currents were relatively rare, occurring in about 16% of the responding cells. Responses had smaller amplitudes and shorter time courses when compared to the more commonly found odorant-induced inward currents. There was a high correlation between odorant-induced outward current and responses evoked by either 8-(4-chlorophenylthio) adenosine 3':5'-cyclic monophosphate, a membrane-permeant cyclic adenosine monophosphate analog, or 3-isobutyl-1-methylxanthine, a phosphodiesterase inhibitor. The outward current responses to all three substances increased in amplitude when the membrane potential was more negative than -60 mV and decreased in amplitude when the membrane potential was more positive. Responses were still present when the potential was held at -100 mV, indicating that the responses are not the result of a potassium conductance. Removal of external calcium from the perfusion medium abolished the outward currents. Our results indicate that the odorant-induced outward current is a calcium-dependent event that may be mediated by cyclic adenosine monophosphate.     

Ouabain blocks some rapid concentration-induced clamp acetylcholine responses onHelix neurons

1. The effects of ouabain, a potent inhibitor of Na(+)-K+ ATPase, were determined on the transmembrane responses of internally dialyzed Helix neurons to rapid acetylcholine (ACh) application using the "concentration clamp" technique. 2. Ouabain selectively depressed "A"-type responses to ACh, which are due to a selective increase in membrane permeability to chloride. In contrast, the "B"-type responses, due primarily to an increase in monovalent cation permeability, was unaffected. 3. The blockade of the Cl- responses was not associated with a change of the reversal potential of the response. Ouabain depressed the maximal response without shifting the dose-response curve. 4. Ouabain caused an increase in the time constant of decay of the ACh current, but the value in the presence of ouabain was not different from that of a lower concentration of ACh determined so as to give a response of the same peak amplitude. Therefore, the effect of ouabain is not on the process of receptor desensitization directly.     

Inactivation of calcium current in the somatic membrane of snail neurons

The decline of calcium inward currents evoked by a long-lasting membrane depolarization was studied on isolated snail neurons internally perfused with a K+-free solution. Two exponential components superimposed on a steady inward current could be distinguished, a slow decline with a time constant of several hundreds of milliseconds, observed at all the testing potentials used, and a fast one with a time constant of several dozens of milliseconds, which appeared at depolarizations to about -10 mV and above. When the calcium current was blocked by extracellular Cd2+ or verapamil, an outward current could be recorded at the same depolarizations. Subtraction of the latter current from the total current, recorded prior to the blockage, largely reduced the fast component of the decline of the total current. An increase in pHi from 7.3 to 8.1 led to the elimination of both the outward current and the fast component of the calcium current decline. The slow component remained practically unchanged, with its rate depending upon the current amplitude. It was slowed following intracellular administration of EDTA, and after equimolar substitution of Ba2+ for Ca2+. It is concluded that the fast component of the calcium inward current decline is mainly due to the superposition of the outward current produced by low selective channels. Only the slow component represents an actual decline of the inward current through calcium channels; it is due to ion accumulation at the inner surface of the cell membrane.     

Regional differences in cholinergic regulation of potassium current in feline esophageal circular smooth muscle

Potassium channels are important contributors to membrane excitability in smooth muscles. There are regional differences in resting membrane potential and K(+)-channel density along the length of the feline circular smooth muscle esophagus. The aim of this study was to assess responses of K(+)-channel currents to cholinergic (ACh) stimulation along the length of the feline circular smooth muscle esophageal body. Perforated patch-clamp technique assessed K(+)-channel responses to ACh stimulation in isolated smooth muscle cells from the circular muscle layer of the esophageal body at 2 (distal)- and 4-cm (proximal) sites above the lower esophageal sphincter. Western immunoblots assessed ion channel and receptor expression. ACh stimulation produced a transient increase in outward current followed by inhibition of spontaneous transient outward currents. These ACh-induced currents were abolished by blockers of large-conductance Ca(2+)-dependent K(+) channels (BK(Ca)). Distal cells demonstrated a greater peak current density in outward current than cells from the proximal region and a longer-lasting outward current increase. These responses were abolished by atropine and the specific M(3) receptor antagonist 4-DAMP but not the M(1) receptor antagonist pirenzipine or the M(2) receptor antagonist methoctramine. BK(Ca) expression along the smooth muscle esophagus was similar, but M(3) receptor expression was greater in the distal region. Therefore, ACh can differentially activate a potassium channel (BK(Ca)) current along the smooth muscle esophagus. This activation probably occurs through release of intracellular calcium via an M(3) pathway and has the potential to modulate the timing and amplitude of peristaltic contraction along the esophagus.     

Intracellular injection of dopamine enhances acetylcholine responses of neuron R2 in the Aplysia abdominal ganglion

1. At different levels of the holding potential on neuron R2 membrane in the Aplysia depilans abdominal ganglion, dopamine injected intracellularly increases the amplitude of both inward and outward currents recorded in response to the application of acetylcholine (ACh) to the ganglion surface. 2. The addition of dopamine to the external perfused solution produces generation of inward currents and a decrease in the cell response to the ACh. 3. The enhancing effect of injected dopamine on ACh responses is retained after inhibition of acetylcholinesterase (AChE) by a specific organophosphorous inhibitor, compound Gd-42. 4. The modulating effect of injected dopamine on ACh responses is discussed in terms of the existence of intracellular receptors of neurotransmitters in the differentiated cells.     

Effect of acetylcholine on ventrocaudal sensory neurons in the pleural ganglia ofAplysia

1. The effects of acetylcholine (ACh) on the soma of cultured ventrocaudal sensory neurons from the pleural ganglia of Aplysia kurodai were characterized. 2. Whole-cell recording was used for current and voltage clamping. ACh and other drugs were microapplied to the membranes of the cultured neurons. 3. Microapplication of ACh induced an outward current mediated by a conductance increase. No desensitization to repeated applications of ACh was detected. The threshold was 10(-7) M and the maximum response was at 10(-5) M. 4. The reversal potential in normal seawater is -80 mV, close to the K+ equilibrium potential. Increasing [K+]0 shifted the reversal potential by the amount predicted by the Nernst equation. Altering [Cl-]0 did not affect the reversal potential. Thus ACh opens a potassium channel in these sensory neurons and may act as a neurotransmitter on those neurons. 5. Atropine and d-tubocurarine partially blocked the ACh response. Hexamethonium had no obvious effect on this response. Tetraethylammonium reduced the response to 22% of control. Carbamylcholine and arecoline induced outward currents that were 71 and 12%, respectively, of the response to ACh. Nicotine and muscarine had almost no effect. 6. The ACh response was reduced by prior application of serotonin (5HT). The ACh response was also reduced by bath-applied 5HT, forskolin, and isobutylmethylxanthine. These data suggest that ACh activates an "S-like" channel in the ventrocaudal sensory neurons.     

Physiological and pharmacological properties of responses to GABA and ACh by abdominal motor neurons in Manduca sexta

1. GABA, ACh, and other agents were applied by pressure ejection to the neuropil of the third abdominal ganglion in the isolated nerve cord of Manduca sexta. Intersegmental muscle motor neurons with dendritic arborizations in the same hemiganglion were inhibited by GABA (Fig. 2) and excited by ACh (Fig. 5).
2. Picrotoxin was a potent antagonist of GABA (Fig. 4A). Bicuculline reduced GABA responses in some motor neurons (Fig. 4C), but had no effect on many other motor neurons. Curare reduced ACh responses (Fig. 6A). Bicuculline was an effective ACh antagonist in most motor neurons tested (Fig. 6B).
3. Motor neurons with dendrites across the ganglion from the ejection pipette exhibited different responses to GABA and ACh. Contralateral motor neurons often showed smaller, delayed hyperpolarizing GABA responses (Fig. 7). On two occasions, contralateral motor neurons had excitatory responses (Fig. 8). Contralateral motor neurons were hyperpolarized by ACh (Fig. 9). The inhibitory responses had only slightly longer latencies than ipsilateral excitatory ACh responses (Fig. 10A). The contralateral inhibitory ACh responses, but not the ipsilateral excitatory ACh responses, were eliminated by TTX (Fig. 10B).
4. A model, which includes inhibitory interneurons that cross the ganglionic midline to inhibit their contralateral homologs and motor neurons (Fig. 11), is proposed to account for contralateral responses to GABA and ACh and antagonistic patterns of activity of motor neurons during mechanosensory reflex responses.

     

Effect of bradykinin on membrane properties of guinea pig bronchial parasympathetic ganglion neurons

The effect of bradykinin on membrane properties of parasympathetic ganglion neurons in isolated guinea pig bronchial tissue was studied using intracellular recording techniques. Bradykinin (1-100 nM) caused a reversible membrane potential depolarization of ganglion neurons that was not associated with a change in input resistance. The selective bradykinin B(2) receptor antagonist HOE-140 inhibited bradykinin-induced membrane depolarizations. Furthermore, the cyclooxygenase inhibitor indomethacin attenuated bradykinin-induced membrane depolarizations to a similar magnitude ( approximately 70%) as HOE-140. However, neurokinin-1 and -3 receptor antagonists did not have similar inhibitory effects. The ability of bradykinin to directly alter active properties of parasympathetic ganglion neurons was also examined. Bradykinin (100 nM) significantly reduced the duration of the afterhyperpolarization (AHP) that followed four consecutive action potentials. The inhibitory effect of bradykinin on the AHP response was reversed by HOE-140 but not by indomethacin. These results indicate that bradykinin can stimulate airway parasympathetic ganglion neurons independent of sensory nerve activation and provide an alternative mechanism for regulating airway parasympathetic tone.     

Cell-cell interaction during synaptogenesis

1. Neuromuscular synapse formation was studied using nerve and muscle cells dissociated from Xenopus embryos and kept in culture for 1 to 3 days. Within a few minutes of manipulated contact with isolated cholinergic neurons, miniature endplate potential-like depolarizations (MEPPs) due to spontaneous release of acetylcholine (ACh) from the neurons were detected in the muscle cells. 2. Addition of an antibody to a frog neural cell adhesion molecule (anti-NCAM) into the culture medium of nerve-muscle co-cultured for 1-3 days decreased the percentage of functional nerve-muscle contacts. 3. Acute exposure to anti-NCAM (1 hour) inhibited significantly muscle cell contact-triggered ACh release from initially identified cholinergic neurons. 4. Lysed muscle cells manipulated into contact with neurons induced ACh release, whereas lysed neurons did not, suggesting the presence of specific molecules on the muscle cell membrane capable of triggering ACh release from the cholinergic neuron. 5. Transient appearance of electrical coupling was detected between neuronal soma and muscle cell, suggesting the possibility of exchange of modulators for the formation and maintenance of neuromuscular synapses. 6. Neuromuscular synaptogenesis constitutes a complex process where at least two different types of direct cell-cell interaction seem to occur: a) cell surface molecule contact (and binding) for cell recognition and triggering of ACh release, and b) transient intercytoplasmic communication between the cells for possible passage of modulatory molecules.     

Phorbol esters that activate protein kinase C induce long-term changes of membrane excitability and postsynaptic currents in neocortical neurons

Intracellularly injected tumor promoter phorbol esters (PhEs) that activate protein kinase C (PKC) increased the excitability and altered the postsynaptic responses of neurons of the motor cortex of awake cats. PhEs increased the amplitude and duration of EPSPs and decreased the amplitude and durations of IPSPs. No consistent changes in resting membrane parameters that would account for these modifications were found. Corresponding changes in peak excitatory and inhibitory postsynaptic currents (EPSCs, IPSCs) were measured directly with the single electrode voltage clamp technique. The changes lasted for 50 min or longer. Quantitative analysis of EPSCs in response to ventrolateral thalamic stimulation and IPSCs in response to pyramidal tract stimulation made in a subgroup of fast PT cells suggested that PhE acted within the injected neuron rather than presynaptically to alter the synaptic currents. PhE also reduced a voltage-dependent, 3-aminopyridine sensitive fast outward current (IA) and an apamin and EGTA sensitive slow outward current (IK(Ca]. Control injections of a phorbol ester that did not activate PKC failed to induce changes in synaptic responses or resting membrane properties. These observations provide the first evidence that activation of PKC, in vivo, can induce long-lasting changes in synaptic responses of neocortical neurons by direct modification of postsynaptic ion channel conductivities.     

Changes in external Na induce a membrane current related to the Na-Ca exchange in cesium-loaded frog heart cells

The effects of transient alterations in Nao were investigated under voltage clamp conditions in frog heart cells previously loaded with Cs. Tetrodotoxin and Cs were used to inhibit Na and K currents. On applying a Na-poor solution (39.2 mM), an outward current was generated during both depolarizations and hyperpolarizations. The current amplitude described a U-shaped function of the membrane potential. On reapplying the standard solution after 15 min equilibration, an inward current was then induced that exhibited a bell-shaped function of the membrane potential. Current amplitude was sensitive to the external Ca concentration. Increasing pHi by 10 mM NH4Cl enhanced this current, while the internal acidification that occurred on switching back to the control solution greatly reduced it. Variations in the amplitude of this current during repetitive stimulations or long pauses are best explained by subsequent alterations in Nai and pHi; no evidence for a time dependence was found. This current was inhibited by La3+, Co2+, and D600, and was sensitive to adriamycin, quinidine, and disopyramide; lidocaine, another local anesthetic, and nifedipine had no effect. These observations extend previous work on intact heart cells and sarcolemmal vesicles. They suggest that the Na-Ca exchange may generate a current that is outward when Ca ions are moving into the cell.     

Two mechanisms of calcium oscillations in adipocytes

In non-excitable cells, several kinds of agonist-induced oscillations of cytosolic Ca²⁺ concentration ([Ca²⁺]_i) are known which differ in their form and generation mechanism. The oscillation source is, as a rule, the regulation of Ca²⁺ mobilization from intracellular stores through inositol 1,4,5-trisphosphate (IP₃) receptors (IP₃R) and in some cases through ryanodine receptors (RyR). In the present work, oscillations in single mature adipocytes of mice epididymal fat on the ninth day of cultivation are studied. Cells were stimulated by acetylcholine (ACh) or by fetal bovine serum (FBS). ACh at a concentration of 0.1–5 μM evoked a rise in [Ca²⁺]_i to a peak and subsequent oscillations whose peaks and troughs declined along with increasing amplitude while frequency decreased. In most cells oscillations lasted less than 5 min. The new constant or interspike level exceeded the initial one or was equal to it (at 1 μM ACh). The removal of ACh stopped oscillations immediately. An inhibitor of phospholipase C (U73122) or of IP₃R (Xestospongin C) did not affect the pattern of responses, which means that the generation of oscillations does not depend on IP₃. At the same time, suppression of responses by ryanodine, which blocks RyR, was observed. Besides, oscillatory responses were abolished by inhibitors of phosphatidylinositol 3-kinase, NO synthase, and cGMP-dependent protein kinase. FBS (1%) initiated oscillations characterized by return of [Ca²⁺]_i after each peak to the baseline level, occurring prior to stimulation, and by maintenance of roughly constant amplitude and frequency (of the order of 1 min⁻¹). Oscillations persisted longer (more than 15 min in 87% of cells) than with ACh. Repeated stimulation of cells by FBS revealed a strongly reduced sensitivity after 1 h of rest, whereas responses to ACh partially restored within 3 min. Investigation of the involvement of IP₃R and RyR in FBS-induced oscillations gave completely inverse results relative to ACh and demonstrated a leading role of IP₃R without a considerable contribution of RyR and of its activation pathways. With both stimuli, Ca²⁺ entry through the plasma membrane was necessary only as a support of oscillations. The results show that in adipocytes different agonists can engage distinct subsystems of Ca²⁺ signaling, each of them generating oscillations with a specific temporal pattern.     

Adaptation in the Visual Cortex: Influence of Membrane Trajectory and Neuronal Firing Pattern on Slow Afterpotentials

The input/output relationship in primary visual cortex neurons is influenced by the history of the preceding activity. To understand the impact that membrane potential trajectory and firing pattern has on the activation of slow conductances in cortical neurons we compared the afterpotentials that followed responses to different stimuli evoking similar numbers of action potentials. In particular, we compared afterpotentials following the intracellular injection of either square or sinusoidal currents lasting 20 seconds. Both stimuli were intracellular surrogates of different neuronal responses to prolonged visual stimulation. Recordings from 99 neurons in slices of visual cortex revealed that for stimuli evoking an equivalent number of spikes, sinusoidal current injection activated a slow afterhyperpolarization of significantly larger amplitude (8.5±3.3 mV) and duration (33±17 s) than that evoked by a square pulse (6.4±3.7 mV, 28±17 s; p<0.05). Spike frequency adaptation had a faster time course and was larger during plateau (square pulse) than during intermittent (sinusoidal) depolarizations. Similar results were obtained in 17 neurons intracellularly recorded from the visual cortex in vivo. The differences in the afterpotentials evoked with both protocols were abolished by removing calcium from the extracellular medium or by application of the L-type calcium channel blocker nifedipine, suggesting that the activation of a calcium-dependent current is at the base of this afterpotential difference. These findings suggest that not only the spikes, but the membrane potential values and firing patterns evoked by a particular stimulation protocol determine the responses to any subsequent incoming input in a time window that spans for tens of seconds to even minutes.     

The pharmacological characteristics of two types of cholinoreceptors in the membrane of dialyzed neurons

1. The ramped voltage clamp technique was developed as a rapid means of studying the effects of certain nicotinic and muscarinic agents on ionic involvement and conductance changes during acetylcholine (ACh) responses of Helix pomatia neurons. 2. Atropine was found to be a potent cholinolytic on A-type neurons, ACh responses of which are blocked by ouabain and mediated by Na+ and Cl- permeabilities, while d-tubocurarine blocked B-type ACh responses which are insensitive to ouabain and mediated by Na+ and K+ permeabilities. 3. Nicotinic agent butyrylcholine was found to be a potent cholinomimetric on B-type cells. 4. The results suggest that ACh receptors on A-type cells are more "muscarinic" while those on B-type cells are more "nicotinic". 5. It was also suggested that both muscarinic and nicotinic ACh receptors may coexist in the Helix neuronal membrane and the possibility of ACh interacting with one of them is determined by the level of phosphorylation of the membrane proteins.     

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.     

Response to 5-hydroxytryptamine on neurons of guinea pig celiac and inferior mesenteric ganglia in primary culture.

The electrophysiological effects of serotonin, a putative neurotransmitter in prevertebral sympathetic ganglia, were evaluated in cultured celiac and inferior mesenteric ganglia (IMG) neurons. Intracellular microelectrode recordings were performed in neurons that were maintained in culture an average of 26 days. Seventy-eight of 85 neurons responded when serotonin (10 microM) was applied by pressure ejection from a micropipette to the surface of the isolated cells. The majority of the neurons (n = 48) generated fast depolarizations, although slow depolarizations (n = 17), bipolar responses (n = 5), hyperpolarizations (n = 7), and a biphasic response (n = 1), were also seen. Hyperpolarizing responses were evoked in celiac neurons only. All responses were inhibited by the 5-HT3 antagonist MDL 72,222 (5 microM). Fast responses were not inhibited by tetrodotoxin (n = 3). These results demonstrate that serotonin evokes a variety of membrane potential changes in cultured prevertebral sympathetic neurons by activating 5-HT3 receptors.     

Acetylcholine receptors in spider peripheral mechanosensilla

Peripherally located parts of spider mechanosensory neurons are modulated by several neurotransmitters released from apposed efferent fibers. Activities of acetylcholine (ACh) synthesizing enzyme choline acetyltransferase (ChAT) and ACh degrading enzyme acetylcholine esterase (AChE) were previously found in some efferent fibers. ChAT activity was also present in all the mechanosensory neurons, while AChE activity was only found in some. We show that spider mechanosensory neurons and probably some efferent neurons are immunoreactive to a monoclonal antibody against muscarinic ACh receptors (mAChRs). However, application of muscarinic agonists did not change the physiological responses or membrane potentials of neurons in the lyriform organ VS-3. Similarly, the sensitivities of the neurons of trichobothria (filiform hairs) remained unchanged after application of these agonists. Therefore, activation of mAChRs may only modulate the function of spider mechanosensory neurons indirectly, for example, by affecting the release of other transmitter(s). However, a subgroup of VS-3 neurons was inhibited by ACh, which also depolarized the membrane similar to these neurons’ responses to GABA, suggesting that ACh activates anion channels in these neurons. Interestingly, all of the neurons responding to ACh were the rapidly adapting Type A neurons that were previously shown to express AChE activity.     

The Effect of Glycerol Treatment of Voltage-Clamped Snake Muscle Fibers

The effect of glycerol treatment on the membrane currents and tension development was studied in voltage clamped snake muscle fibers. In muscle fibers which were exposed for 1 h to a normal saline containing 400 mM glycerol and then returned to a normal medium, graded depolarizations did not accompany contractile responses. However, when the fiber was depolarized to a certain level, an increment of outward current appeared which partially inactivated with time. The threshold for delayed rectification in glycerol-treated fibers was almost the same as that of intact fibers in spite of the absence of contractile tension. The results suggest that the delayed rectification may be attributed at least in part to the surface membrane and that the contractile activation probably does not depend simply on the inactivating outward currents through the delayed rectification channel.