Electrophysiological investigation of the properties of acetylcholine receptors of frog sympathetic ganglionic neurons

Changes in membrane potential and conductance were studied in neurons of isolated sympathetic ganglia ofRana ridibunda during perfusion with cholinomimetics and cholinolytics. Activation of nicotinic (N) acetylcholine receptors by carbachol, suberyldicholine, and tetramethylammonium led to depolarization with an increase in conductance, whereas activation of muscarinic (M) acetylcholine receptors by perfusion with carbachol or 5-methylfurmethide, led to depolarization with a decrease or (less frequently) an increase in conductance. The M-cholinolytic atropine was shown to cause depolarization with an increase in conductance if perfusion with atropine was preceded by perfusion with carbachol.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 11, No. 5, pp. 475–482, September–October, 1979.     

Ionic mechanisms of the fast (nicotinic) phase of the acetylcholine response of identified Planorbarius corneus neurons

Responses to electrophoretic application of acetylcholine and suberyldicholine were investigated in identified neurons (LPed-2 and LPed-3) isolated from the left pedal ganglion ofPlanorbarius corneus. When microelectrodes filled with potassium chloride were used the reversal potentials of responses to acetylcholine and suberyldicholine were less negative than when microelectrodes filled with potassium sulfate were used; these reversal potentials were shifted toward depolarization if chloride ions in the medium were replaced by sulfate. These facts indicate that the responses in both LPed-2 and LPed-3 depend on chloride ions. Reversal potentials for acetylcholine and suberyldicholine in LPed-3 were virtually identical (–51 and –50 mV respectively), but in LPed-2 they differed significantly (–46 and –62 mV respectively). Replacement of sodium ions by Tris ions shifted the reversal potential for acetylcholine in LPed-2 toward hyperpolarization but did not change the reversal potential for suberyldicholine. Benzohexonium had the same action. The reversal potential for acetylcholine in medium with a reduced sodium concentration or in the presence of benzohexonium was the same as for suberyldicholine. It is concluded that on neuron LPed-2 acetylcholine activates both acetylcholine receptors which control conductance for chloride ions and acetylcholine receptors which change conductance for sodium ions, whereas suberyldicholine acts only on acetylcholine receptors responsible for the chloride conductance of the membrane.I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 12, No. 5, pp. 533–540, September–October, 1980.     

Visceral afferent pathways relaying synaptically in the caudal mesenteric sympathetic ganglion

The caudal mesenteric sympathetic ganglion of cats was isolated and perfused, and responses of the preganglionic trunks of the ganglion to electrical stimulation of the central end of the hypogastric nerve were studied. Stimulation of the nerve with single square pulses gives rise to early and late responses. Early responses appear after various latent periods and are the result of excitation of transit fibers of groups A, B, and C, whereas the appearance of late responses is associated with the synaptic transmission of excitation in the sympathetic ganglion from afferent sympathetic neurons at the first level (from the pelvic organs to the caudal ganglion) to afferent sympathetic neurons of the second level (from the caudal ganglion and above). Early responses are not blocked, but late responses are blocked by perfusion of the ganglion with azamethonium bromide and magnesium salts, and also by tetanization of the hypogastric nerve at 10–50 Hz. Other facts indicating the synaptic relaying of visceral sympatho-sympathetic afferent pathways in the ganglion are also described.Institute of Physiology, Academy of Sciences of the Belorussian SSR, Minsk. Translated from Neirofiziologiya, Vol. 2, No. 5, pp. 507–514, September–October, 1970.     

Synaptic effects of nicotinic and muscarinic agonists in sympathetic ganglia of the frog

Superfusion of the isolated sympathetic ganglia of the frog with nicotinic agonists (suberyldicholine, tetramethylamonium, and dimethylphenylpiperazinium), as well as acetylcholine in the presence of atropine led to a brief depolarization of the neurons and blockade of synaptic transmission. The muscarinic agonists methylfurmethide (MFM) and methyldilvasen, cis^–, L(+), as well as acetylcholine elicited a stable depolarization which is not accompanied by disturbance in transmission. Oxotremorine at a concentration of 1·10^–5 M did not lead to the depolarization of the post-synaptic membrane, but at a concentration of 1·10^–6 M decreased the quantal EPSP content twofold, which indicates that the presynaptic receptors belong to the M₂ subtype. Inhibition of acetylcholinesterase significantly intensified the postsynaptic effect of MFM: a shift of the concentration-effect curve took place toward the side of lower MFM concentrations. It was shown that the post-synaptic muscarinic receptors of the ganglionic neurons possess varied sensitivity to the enantiomers of methyldilvasen and, consequently, are stereospecific. The identified functional properties of the cholinoreceptors of the ganglionic neurons explain the set of changes in synaptic transmission under conditions of the prolonged presence of a mediator in the synaptic cleft.I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 20, No. 2, pp. 227–234, March–April, 1988.     

Effects of cholinergic compounds on spontaneous quantal transmitter release at the frog neuromuscular junction

The effects of nicotinic and muscarinic mimetics and lytics on spontaneous quantal transmitter secretion from the motor nerve endings were investigated during experiments on theRana temporaria sartorius muscle. Acetylcholine and carbachol reduced the frequency of miniature endplate potentials both in a normal ionic medium and in one with potassium ion concentration raised to 10 mM. Similar effects were produced by nicotinic agonists, namely nicotine, tetramethylammonium, and suberyldicholine, whereas muscarinic mimetics — methylfurmetide, oxotremorine, and F-2268 (L- and D-stereoisomers) — did not affect transmitter release. Neither d-tubocurarine, benzohexonium, nor atropine abolished the presynaptic effects of carbachol and acetylcholine. It is concluded that nicotinic cholinoreceptors are present at the frog motor nerve endings which modify spontaneous transmitter release and differ in their pharmacological properties from recognized N-cholinoreceptors of the motor and autonomic systems of the higher vertebrates.S. V. Kurashov Medical Institute, Ministry of Public Health of the RSFSR, Kazan'. Translated from Neirofiziologiya, Vol. 18, No. 5, pp. 586–593, September–October, 1986.     

Cholinoreceptor sensitivity of the mollusk neuron and frog skeletal muscle to acetylcholine and tetramethylammonium analogs

Studies have been made on the sensitivity of cholinoreceptors in identified isolated neuron from the pedal ganglion of the snail Planorbarius corneus and cholinoreceptors of m. rectus abdominis of the frog Rana temporaria to drugs which differ from acetylcholine by the structure either in cationic group, methylene chain, or ester group. Snail cholinoreceptors were found to be less sensitive to changes in the structure of cationic group and more sensitive to the increase in methylene chain from 3 to 4 groups, as compared to frog cholinoreceptors. The sensitivity of both preparations to changes in ester group, as well as to tetramethylammonium was found to be practically the same. Therefore, the sensitivity of neuronal cholinoreceptors in the snail to the effect of acetylcholine and tetramethylammonium analogues does not significantly differ from the sensitivity of cholinoreceptors in the abdominal muscle of the frog.     

Physiological properties of nicotinic acetylcholine receptors of sympathetic ganglionic neurons

The basic properties of nicotinic acetylcholine receptors of the neurons of a sympathetic ganglion responsible for the performance by these receptors of their main function — initiation of an electric current through the postsynaptic membrane — and determining the particular features of the acetylcholine receptors of these neurons by contrast with receptors of other objects, are described. Stoichiometric relations of the recognition center of the acetylcholine receptors with the transmitter, the relative strength of various agonists, and the method of action of -bungarotoxin on this center are indicated; the "life-time" and conductance of the ion channel are described. On the basis of "life-time" two groups of acetylcholine receptors are distinguished: synaptic (long-living) and extrasynaptic (short-living). Selective blockers of acetylcholine receptors of ganglionic neurons, namely bis-ammonium compounds, have two types of effect (competitive and channel-blocking), caused by the action of the blocker on two different regions of the receptor molecule, respectively. Since the channel-blocking action develops at lower concentrations than the competitive, and since it correlates closely with the ganglion-blocking effect, it is concluded that it is the first of these which determines the properties of selective blockers of acetylcholine receptors.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 16, No. 3, pp. 319–326, May–June, 1984.     

Action of local iontophoretic application of acetylcholine and serotonin to neurons of the rabbit superior cervical ganglion

The action of ionotophoretic application of acetylcholine and serotonin (5-hydroxytryptamine) on neurons of the isolated rabbit superior cervical ganglion was investigated by intracellular recording. The soma of neurons in the ganglion was shown to have no muscarinic receptors and to have only nicotinic receptors scattered irregularly over the whole surface of the neuron soma membrane. Acetylcholine has an excitatory action on presynaptic endings. In about half of the neurons of the ganglion the soma was shown to possess serotonin receptors.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 10, No. 5, pp. 519–524, September–October, 1978.     

Properties of the slow excitatory postsynaptic potential in mammalian sympathetic ganglion cells

Two types of slow excitatory postsynaptic potentials (EPSPs) with different properties were found in neurons of the rabbit superior cervical sympathetic ganglion. In our group of neurons slow EPSPs increased during artificial hyperpolarization and decreased during depolarization of the membrane. The input resistance of the cells fell or remained unchanged during the development of slow EPSPs. In the second group of cells slow EPSPs increased during depolarization and decreased during hyperpolarization. The reversal potential of these responses, determined by extrapolation, was –78.9±3.6 mV. Depolarization responses to activation of muscarinic cholinergic receptors by acetylcholine or carbachol developed in 53% of neurons with an increase in input resistance and had a reversal potential of –83.2±6.7 mV. It is suggested that in cells of the first group the ionic mechanism of the slow EPSPs is similar to that of the fast EPSPs, whereas in cells of the second group its main component is a decrease in the potassium conductance of the membrane.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 13, No. 4, pp. 371–379, July–August, 1981.     

Electrophysiological investigation of pathways in the middle cervical sympathetic ganglion of the turtle

Single unit responses in the middle cervical sympathetic ganglion ofEmys orbicularis to stimulation of other nerves and changes in these responses during the action of sympathetic blocking agents on the ganglion were investigated. The results showed that some fibers of the cervical sympathetic trunk of the turtle are interrupted in this ganglion. Postganglionic fibers pass out of the ganglion and enter the lateral branch and the sympathetic trunk. Other fibers pass through the ganglion without interruption and, together with postganglionic fibers, leave the ganglion in the cervical sympathetic trunk in a cranial direction. The velocity of conduction of excitation along the preganglionic fibers is between 4–3 and 2–1.5 m/sec and along the postganglionic fibers between 4–2.6 and 0.7–0.5 m/sec (fibers of types B₂ and C). Synaptic delay in the fast-conducting fibers averages 6.6 msec. Preganglionic fast-conducting fibers form synaptic contacts on neurons with type B₂ axons, while preganglionic slow-conducting fibers form contacts on neurons with type C axons. Terminals of two preganglionic fibers differing very slightly in their threshold of excitability, and probably constituting the same group, converge on some neurons.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukranian SSR, Kiev. Translated from Neirofiziologiya, Vol. 4, No. 1, pp. 83–89, January–February, 1972.     

Responses of rat spinal ganglion neurons mediated by activation of serotonin receptors of the third type

Application of serotonin (5-hydroxytryptamine; 5-HT) to rat dorsal root ganglion neurons under conditions in which potassium conductance was blocked by cesium ions elicited depolarizing responses followed by an increase in membrane conductance. The responses did not exhibit desensitization and were due to activation of 5-HT receptors of the third type (5-HT₃Rs), since they were insensitive to methysergide, the 5-HT₂R antagonist, but were inhibited by tropicetrone (ISC 205–930) and metoclopramide, the 5-HT₃R antagonists. The reversal potential of the 5-HT-induced depolarizing responses was –11.9 mV; their amplitude decreased following a decrease in extracellular Na⁺ concentration but remained constant after intracellular injection of GTP. The amplitude of the responses increased following elevation of intracellular cAMP concentration caused by theophylline or sodium fluoride whose potentiating effect was reduced by butamide, a protein kinase A inhibitor. Potentiation of the 5-HT-induced responses was also produced by increased intracellular Ca²⁺ concentration following either direct intracellular injections or a burst of action potentials. The potentiation could be prevented by trifluoroperazine, the calmodulin inhibitor. The 5-HT effects were also potentiated by methylfurmetide, an activator of muscarinic acetylcholine receptors. The effect of methylfurmetide was slightly decreased by trifluoroperazine and was markedly decreased by polymixin B, a protein kinase C inhibitor. The effects of 5-HT were also enhanced by ethanol.Neirofiziologiya/Neurophysiology, Vol. 25, pp. 258–263, July–August, 1993.     

Effect of acetycholine and cholinolytics on activity of pacinian corpuscles

The effect of acetylcholine and cholinolytics on excitability of the isolated mechanoreceptors of Pacinian corpuscles was studied by the external perfusion method. Addition of acetylcholine to the solution in a concentration of 10^–5–10^–3 g/ml increased the excitability of the receptors. The amplitude of the receptor potential fell in a solution with acetylcholine. Cholinolytics (D-tubocurarine, hexamethonium, and atropine) did not block receptor activity. It is concluded that acetylcholine does not participate in primary processes in Pacinian corpuscles.I. P. Pavlov Institute of Physiology, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 9, No. 2, pp. 185–190, March–April, 1977.     

Comparative research on synaptic transmission in the sympathetic ganglia of rabbits and frogs during acetylcholinesterase inhibition

Organophosphorus inhibitor of acetylcholinesterase (AChE) armin (1 x 10(-6) M) induced a variety of pre- and postsynaptic effects resulting from the AChE inhibition and subsequent accumulation of acetylcholine (ACh) in the synaptic cleft. The intensity of postsynaptic effects (level of neuron depolarization, degree of action potential depression) was shown to be different in the ganglia of frog and rabbit. This could be explained by differences in the total amount of ACh released in response to nerve stimulation as well as at rest. Both muscarinic and nicotinic cholinoreceptors were involved in the process of sustained depolarization of the neurons in the rabbit superior cervical ganglion after AChE inhibition. In frog ganglion neurons the nicotinic receptors did not participate in depolarization evidently due to their fast desensitization. The activation of presynaptic muscarinic receptors resulted in decrease of ACh released by nerve stimulation seems to weaken depolarization and blockade of synaptic transmission in sympathetic ganglia treated by AChE inhibitors.     

Electrophysiological investigation of the conduction of excitation in the pterygopalatine ganglion of the cat

When responses in some nerves of the pterygopalatine ganglion of the cat in situ to stimulation of its other nerves were recorded it was found that most fibers passing through the ganglion are continuous sympathetic postganglionic fibers (at least three groups). Most of the parasympathetic preganglionic fibers forming synapses on neurons of the ganglion constitute a group of fibers with the same threshold of excitation. Intracellular recording from single neurons of the pterygopalatine ganglion showed that stimulation of the Vidian nerve evokes orthodromic spike potentials in some neurons of the ganglion with a short latent period, and in others with a long latent period (2.5–6.0 and 10–44 msec, respectively). Evidently only fast-conducting fibers terminate synaptically on most neurons of the ganglion and only slow-conducting fibers on some of them. Recording from intact nerves of the pterygopalatine ganglion revealed no tonic activity in them. Microelectrode recording from single neurons of the ganglion showed that either the frequency of generation of spike potentials is relatively low (1–3/sec) or such potentials are absent altogether.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 8, No. 5, pp. 514–520, September–October, 1976.     

Conformation of acetylcholine at muscarinic nerve receptors: Crystal and molecular structure of 2-trimethylammoniummethyl-5-methyl furan iodide (5-methylfurmethide iodide)

2-Trimethylammoniummethyl-5-methyl furan (5-methylfurmethide) is a potent cholinergic agonist at muscarinic nerve receptors. The conformation of the molecule, as shown by crystal structure analysis, is restricted by steric hindrance. The only similar conformation of acetylcholine has τ(N⁺CCO) synclinal and τ(CCOC) antiplanar. This is the conformation found in solution and in crystals of the chloride, and it is believed to be the one relevant to interaction with muscarinic nerve receptors.     

Asymmetrical dendritic fields of retinal ganglion cells

By use of Golgi chrome—silver impregnation, studies were made of the dendritic branchings of feline and frog ganglion cells. It was shown that besides the known varieties of ganglion cells there were asymmetrical neurones whose dendrites lay all to one side. Essential differences distinguished these ganglion cells in the cat from those in the frog, differences depending upon the architectonics of the inner plexiform layer, which is broad and subdivided into layers in the frog, and narrow in the cat. We discuss the possible role of neurones with a unilateral arrangement of dendrites in relation to know electrophysiological data on retinal detectors and the receptive fields of ganglion cells.Brain Institute, Academy of Medical Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 3, No. 3, pp. 301–307, May–June, 1971.     

Electrical activity of the sympathetic ganglia during the depressor reflex

Tonic activity of neurons of the superior cervical sympathetic ganglion was recorded by the "sucrose gap" method and in the 4th and 5th lumbar sympathetic ganglia with the aid of focal nonpolarizing electrodes in acute experiments on anesthetized cats and rabbits. The preganglionic fibers of the ganglia were left intact. Stimulation of the depressor nerve not only sharply inhibited the tonic activity of the ganglia but also led to the appearance of electropositive potentials of 0.7 ± 0.2 mV in the superior cervical ganglion and 20–250 µV in the lumbar ganglia. The amplitude of this potential was unchanged by atropine (1 · 10^–6M). A similar effect occured without stimulation of the depressor nerve, after division of the preganglionic fibers or blocking of their conduction; it is attributed to the cessation of preganglionic tonic impulses which induce not only spikes, but also many EPSPs in neurons of the ganglion. Their frequency in the lumbar ganglia was 4/sec. Summation of these EPSPs leads to constant electronegativity of the ganglion surface relative to the postganglionic fibers, and its disappearance is recorded as a positive potential. Stimulation of the depressor nerve thus does not induce IPSPs in the ganglion; consequently, the inhibition of synaptic activity observed under these circumstances is located in the CNS and not in the ganglion.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 6, No. 5, pp. 519–524, September–October, 1974.     

The effect of adrenaline on potentials in the isolated gills of the flounder (Platichthys flesus L.)

Summary A technique is described for simultaneously measuring perfusion pressure and transepithelial potentials in isolated perfused gills.In this preparation the potentials measured in external media of Ringer or sea water were both reduced by 4 mV to 5 mV in the presence of 10^–6 M adrenaline. This effect was reversed by 10^–5 M phentolamine in both situations; 10^–5 M propanolol was without effect (Figs. 2, 3). Both potentials were reduced by oxygen lack (Figs. 1, 6).The preparations showed inconsistent vasomotor responses to adrenaline but 10^–5 M acetylcholine consistently produced an increase in branchial vascular resistance (Figs. 7, 8).It is suggested that adrenaline directly inhibits the branchial ion extrusion pump via a mechanism involving -adrenergic receptors. It is further suggested that the biogenic potential produced by the activity of the branchial ion pump makes a significant contribution to the total potential in sea water. The significance of these findings in relation to other work is discussed.     

Substance P and acetylcholine are co-localized in the pathway mediating mucociliary activity in Rana pipiens

Mucociliary activity is an important clearance mechanism in the respiratory system of air breathing vertebrates. Substance P (SP) and acetylcholine play a key role in the stimulation of the mucociliary transport in the frog palate. In this study, retrograde neuronal tracing was combined with immunocytochemistry for SP and choline acetyl transferase (ChAT) in the trigeminal ganglion and for neurokinin-1 receptor (NK1R) in the palate of Rana pipiens. The cells of origin of the palatine nerve were identified in the trigeminal ganglion using the retrograde tracer Fluorogold (FG). Optimal labeling of FG cells in the trigeminal ganglion was obtained at 96 h of exposure. Immunoflorescent shows that SP and acetylcholine are co-localized in 92% of the cells labeled with FG in the trigeminal ganglion. NK1 receptors were found in the membrane of epithelial and goblet cells of the palate. Ultrastructural study of the palate showed axonal-like endings with vesicles in connection with epithelial and goblet cells. These results further support the concerted action of both neurotransmitters in the regulation of mucociliary activity in the frog palate.     

Ion channel block by acetylcholine, carbachol and suberyldicholine at the frog neuromuscular junction

Three nicotinic agonists, suberyldicholine, acetylcholine and carbachol, have been investigated by single channel recording at the endplates of adult frog muscle fibres. All three agonists can block the channels that they open. Suberyldicholine is the most potent blocker; it has an equilibrium constant for binding to the open channel of about 6 microM and blockages last for about 5 ms on average, at -105 mV. A plot of the mean number of blockages per unit open time against concentration ('blockage frequency plot') suggests that suberyldicholine does not produce long-lived blocked states such as might occur, for example, if it could be trapped within a shut channel. The characteristics of the 'blockage frequency plot' are analysed in Appendix 2. Block by acetylcholine and carbachol has much lower affinity (the equilibrium constants being a few millimolar for both), and blockages are much briefer, so that blockage appears to produce noisy single channel currents of reduced amplitude. A method based on the spectral density of the excess 'open' channel noise has been used to investigate the rate of blocking and unblocking. The basis of this method is discussed in Appendix 1. It is estimated that the mean duration of a blockage is about 18 microseconds for acetylcholine and 9 microseconds for carbachol.