Physiological studies during formation and development of rat neuromuscular junctions in tissue culture

Developing neuromuscular junctions in tissue cultures of rat embryo spinal cord and muscle were studied with intracellular recording. Excitatory junctional potentials (ejps) were found during nerve or spinal cord stimulation, and also arising spontaneously. The time-course of the potentials tended to be shorter in cultures older than 9 days in vitro than in more recently innervated cultures. Evidence of multiple innervation was found in many cells. In order to test the hypothesis that transmitter was released in integral multiples of a quantal amount and according to a Poisson distribution, mean quantum content was calculated from the coefficient of variation of ejp amplitudes, the percentage of zero responses, and the ratio of mean amplitude to mean of the smallest class of amplitudes. The three independent measures were in fair or good mutual agreement, implying that the mechanism of transmitter release in newly developed junctions is the same as in the adult. In newly formed junctions, ejps were subthreshold for action potential generation, but afterwards mean quantum content increased and action potentials were generated by single ejps. In fibers developing both with and without innervation, the entire muscle cell surface was as sensitive to acetylcholine as the adult end plate region. Innervation was related to cross-striation: every cross-striated fiber tested was found to be innervated, and denervation in vitro led to loss of distinct cross-striations.     

Constant amplitude of postsynaptic responses for single presynaptic action potentials but not bursting input during growth of an identified neuromuscular junction in the lobster, Homarus americanus

As lobsters grow from early juveniles to adults their body size increases more than 20-fold, raising the question of how function is maintained during these ongoing changes in size. To address this question we studied the pyloric 1 (p1) muscle of the stomach of the lobster, Homarus americanus. The p1 muscle receives multiterminal innervation from one motor neuron, the lateral pyloric neuron of the stomatogastric ganglion. Staining with antibodies raised against synaptotagmin showed that as the muscle fibers increased in length, the spacing between the terminal innervation increased proportionally, so the number of synaptic contact regions/muscle fiber did not change. Muscle fibers were electrically coupled in both juveniles and adults. The amplitude of single intracellularly recorded excitatory junctional potentials evoked by motor nerve stimulation was the same in both juveniles and adults. Nonetheless, the peak depolarizations reached in response to ongoing pyloric rhythm activity or in response to high-frequency trains of stimuli similar to those produced during the pyloric rhythm were approximately twofold larger in juveniles than in adults. This suggests that homeostatic regulation of synaptic connections may operate at the level of the amplitude of the single synaptic potential rather than on the summed depolarization evoked during strong rhythmic activity.     

Electrophysiological investigation of the cat ciliary ganglion

Electrical responses of some nerves of the ciliary ganglion to stimulation of its other nerves were recorded, and intracellular recordings were also made from neurons of the ganglion (in situ). The overwhelming majority of preganglionic fibers terminate synaptically on neurons of the ganglion. Postganglionic fibers leave in the lateral and medial ciliary nerves, in which the velocity of conduction of excitation ranges from 1.9 to 9.0 m/sec. A few preganglionic fibers pass through the ciliary ganglion into the lateral ciliary nerve, giving off collaterals to neurons of the ganglion, so that stimulation of the lateral ciliary nerve evokes a response in the medial ciliary nerve (preganglionic axon reflex). The resting potential of neurons of the ciliary ganglion is 57±2.8 mV, and their action potential 68±3.6 mV. Single orthodromic stimulation usually evokes a single action potential in a neuron. The amplitude of the EPSP is increased during hyperpolarization of the postsynaptic membrane, confirming the chemical nature of synaptic transmission in the ganglion. The antidromic response consists of an IS-component and spike. The spike is followed by after-hyperpolarization, with a mean amplitude equal to 31% of the spike amplitude, and the time taken for it to fall to one–third of its initial amplitude is 75–135 msec.A. A. Bogomolets' Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 1, No. 1, pp. 101–108, July–August, 1969.     

Neurobiology of Polyorchis. I. Function of effector systems.

The electrical correlates of activity in the effector systems responsible for swimming, crumpling and postural changes have been recorded in the anthomedusan Polyorchis penicillatus. Motor spikes (pre-swim pulses), that initiate swimming contractions, appear without delay at distant sites on the inner nerve-ring in unstimulated preparations. Levels of Mg++ anaesthesia which block the neuromuscular junctions between PSP giant neurons and swimming muscle do not affect PSP activity. Swimming muscle potentials can be recorded from subumbrella and velar muscle sheets using extra- and intracellular electrodes. These action potentials have a distinct plateau and are propagated in a myoid fashion. Resting potentials average -70 mV with spikes overshooting zero by some 62 mV. The effects of repetitive stimulation are described. Extracellular recordings indicate that neuronal pathways may play a major role in mediating crumpling, unlike many other species where epithelial pathways are more important. Endodermal spikes recorded intracellularly from the radial and ring canals have amplitudes of some 92 mV arising from resting potentials that average -55 mV. Repetitive stimulation causes a decrease in amplitude and increase in duration of epithelial action potentials. Tentacle length is controlled by a pacemaker system located in both nerve rings. The frequency of spikes (PTPs) generated by this system determines the length and tonus of tentacles. The neuromuscular junctions between the motor neurons and tentacle muscle are Mg++ sensitive and show facilitating properties.     

THE INNERVATION AND PHYSIOLOGY OF THE EXTRINSIC BUCCAL RETRACTOR MUSCLES OF PHILINE APERTA (LINNAEUS)

Two buccal mass retractor muscles of Philine are innervatedby at least 4 excitatory motoneurons, whose cell bodies liein the buccal and the cerebral ganglia. The muscle fibres respondto action potentials generated in the motoneurons or their axonswith excitatory junction potentials (ejps), each of which isfollowed by a small twitch-like contraction. Both the electricaland mechanical responses facilitate and summate with repetitivestimulation. A large ventrally located cerebral neuron (VGC) inhibits tensiondevelopment in the muscle by reducing the amplitude of the excitatoryjunction potentials from and identified buccal motoneuron. Acetylcholinereversibly depolarises and causes tonic contraction of the muscles.This action is partially antagonised by hexamethonium, whichalso blocks the ejps from two axons in the buccal and one inthe pedal nerve 9. 5-Hydroxytryptamine potentiates the ejp fromthe identified buccal motoneuron and enhances the rate of relaxation.Histamine reduces the amplitude of the presumed cholinergicbuccal nerve ejps, but does not affect the hexamethonium sensitiveejp in the pedal nerve 9. In this respect its action resemblesthat of the ventral giant cell.     

Cerebral motoneurons mediating tentacle retraction in the land slug Ariolimax columbianus

(1) Tentacle retraction in the land slug Ariolimax columbianus can be elicited by stimulation of all nerves and connectives of the ipsi- and contralateral cerebral ganglia. (2) Six neurons in the left cerebral ganglion were classified as tentacle retraction motoneurons because their action potentials are followed one-for-one with constant delay by action potentials in the left tentacle retractor nerve and their depolarization causes retraction of the ipsilateral tentacle. The motoneurons can be identified by size, pattern of pigmentation, position, and physiological characteristics. (3) Each retractor motoneuron discharges at a rather constant rate and has more than one source of excitatory input, but no IPSPs were observed. No synaptic connections between the six retractor motoneurons were found. The nerve action potentials that correspond to each motoneurons are distinguishable by waveform and size rank. (4) Each motoneuron elicits visible contractions in a particular region of the ipsilateral retractor muscle, but the motor fields of some motoneurons overlap. Some motoneurons mediate relatively rapid contractions while others cause slower responses. (5) There is one-for-one correspondence between action potentials of the largest unit recorded extracellularly in the retractor nerve and exciatory junction potentials recorded from the retractor muscle. No evidence of a peripheral neural plexus was found in serial sections of the retractor muscle.     

Synaptic potentials of motoneurons of the masseter muscle

Postsynaptic potentials of 93 motoneurons of the masseter muscle evoked by stimulation of different branches of the trigeminal nerve were studied. Stimulation of the most excitable afferent fibers of the motor nerve of the masseter muscle evoked monosynaptic EPSPs with a latent period of 1.2–2.0 msec, changing into action potentials when the strength of stimulation was increased. A further increase in the strength of stimulation produced an antidromic action potential in the motoneurons with a latent period of 0.9 msec. In some motoneurons polysynaptic EPSPs and action potentials developed following stimulation of the motor nerve to the masseter muscle. The ascending phase of synaptic and antidromic action potentials was subdivided into IS and SD components, while the descending phase ended with definite depolarization and hyperpolarization after-potentials. Stimulation of cutaneous branches of the trigeminal nerve, and also of the motor nerve of the antagonist muscle (digastric) evoked IPSPs with a latent period of 2.7–3.5 msec in motoneurons of the masseter muscle. These results indicate the existence of functional connections between motoneurons of the masseter muscle and its proprioceptive afferent fibers, and also with proprioceptive afferent fibers of the antagonist muscle and cutaneous afferent fibers.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 1, No. 3, pp. 262–268, November–December, 1969.     

豚鼠肠系膜下神经节细胞电活动与结肠运动的关系

在豚鼠肠系膜下神经节(IMG)及其支配的结肠段联合标本上,对IMG细胞内电位与肠段纵肌或环肌舒缩活动进行了同步记录。实验结果表明:(1)肠段预置张力为零时,约50％IMG细胞有自发的快兴奋性突触后电位(EPSP)活动,切断结肠神经或以筒箭毒(50μmol/L)灌流IMG后消失;(2)筒箭毒或低钙高镁溶液阻断神经节传递时,环肌节律性收缩幅度增大,节律变慢,但对纵肌节律性收缩无明显影响,(3)串刺激节前神经,在IMG细胞引起一串快EPSP或动作电位并常跟随迟慢的EPSP,同时,纵肌在0.1-0.2s潜伏期后出现迅速的、时程基本与动作电位串一致的舒张波,后者在筒箭毒灌流IMG后消失,而环肌运动可见舒张、舒张波延长或收缩波增大。结果提示:IMG不仅中继经典的胆碱能传出功能,还参与以胆碱能传递为中介的肠-肠反射,该反射活动的传出效应主要在于抑制环肌收缩。     

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.     

Neural correlates of habituation of the proleg withdrawal reflex in larvae of the hawk moth, Manduca sexta

The larval proleg withdrawal reflex of the hawk moth, Manduca sexta, exhibits robust habituation. This reflex is evoked by deflecting one or more mechanosensory planta hairs on a proleg tip. We examined neural correlates of habituation in an isolated proleg preparation consisting of one proleg and its segmental ganglion. Repeated deflection of a single planta hair caused a significant decrease in the number of action potentials evoked in the proleg motor nerve (which carries the axons of proleg retractor motor neurons). Significant response decrement was seen for interstimulus intervals of 10 s, 60 s and 5 min. Response decrement failed to occur in the absence of repetitive stimulation, the decremented response recovered spontaneously following a rest, and electrical stimulation of a body wall nerve facilitated the decremented response (a neural correlate of dishabituation). Adaptation of sensory neuron responses occurred during repeated hair deflections. However, when adaptation was eliminated by direct electrical stimulation of sensory neurons, the response in the proleg motor nerve still decreased significantly. Muscle recordings indicated that the response of an identified proleg retractor motor neuron decreased significantly during habituation training. Thus, habituation of the proleg withdrawal reflex includes a central component that is apparent at the level of a single motor neuron. Accepted: 20 December 1996     

Conducting pathways of the superior cervical sympathetic ganglion of the cat

Individual nerves of the superior cervical sympathetic ganglion were stimulated in acute experiments on cats, and action potentials (AP) were recorded from other nerves of the ganglion in order to clarify whether or not there is transmission of excitation through the ganglion from one nerve to another and to establish whether this transmission is continuous or synaptic. The method of intracellular recording from neurons of the ganglion was also used. It is established that stimulation of the cervical sympathetic nerve evokes AP in all of the peripheral nerves of the ganglion, a circumstance that is the result of synaptic transmission of excitation. There is no transmission of excitation in the reverse direction or between any of the 12 peripheral nerves of the ganglion (including the four branches of the internal carotid nerve). Orthodromic excitation is recorded intracellularly from neurons of the ganglion during stimulation of the cervical sympathetic nerve, and antidromic excitation is recorded during stimulation of a peripheral nerve (the internal carotid nerve). It follows that the pathways through the ganglion which conduct excitation from the cervical sympathetic nerve into all of the remaining nerves of the ganglion are synaptic. Analysis of EPSP latent periods indicated that preganglionic fibers that differ sharply with respect to threshold and conduction rate (groups S₂ and S₄) converge on one and the same neurons of the ganglion.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 2, No. 2, pp. 216–224, March–April, 1970.     

Acceleratory nervous regulation of juvenile myogenic hearts in the isopod crustacean Ligia exotica

We examined regulation of the myogenic heart by two identified cardioacceleratory neurons (CA1, CA2) in early juveniles of the isopod Ligia exotica. Repetitive stimulation of either the CA1 or CA2 axon increased the frequency and plateau amplitude of the action potential and decreased the maximum hyperpolarization of the cardiac muscle. These effects were larger with increasing stimulus frequency. The rate of increase in the frequency caused by CA1 stimulation was significantly larger than that by CA2. No impulse activity of the cardiac ganglion was induced by acceleratory nerve stimulation. The frequency of the muscle activity was decreased by injection of a hyperpolarizing current into the muscle during stimulation of the acceleratory nerve. In a quiescent heart, acceleratory nerve stimulation caused an overall depolarization in the muscle membrane and the amplitude of the depolarization induced by CA1 stimulation was significantly larger than that by CA2. These results suggest that CA1 and CA2 neurons regulate the myogenic heart affecting directly the cardiac muscle; the CA1 neuron produces more potent effects than does the CA2 neuron.     

Morphologic and histochemical characteristics of frog skeletal muscle following application of colchicine to a motor nerve]

Experiments with application of colchicine to the muscle motor nerve carried out; this was done for the purpose of disturbance of rapid axoplasmic transport. A reduction of the areas of transverse sections of the muscle fibers, an increase in the number of fibers with a low succinic dehydrogenase (SDH) activity a greater homogeneity of the muscle fibers by the degree of optic density in staining for detection of the SDH activity was noted. Analogous changes were revealed under conditions of section of the motor nerve. However, denervation was accompanied by the block of conductivity and by degenerative changes in the nerve endings. As to the preparations treated with colchicine, transmission of excitation in the nerve and through the synapse was retained and was recorded by the end plate miniature potentials, end plate potentials and the action potentials of the muscle fibers. A conclusion was drawn that rapid axoplasmic transport brought substances maintaining differentiated state of the muscle fibers.     

Nerve,spinal cord and brain somatosensory evoked responses: a comparative study during electrical and magnetic peripheral nerve stimulation

Somatosensory evoked potentials (SEPs) and compound nerve action potentials (cNAPs) have been recorded in 15 subjects during electrical and magnetic nerve stimulation. Peripheral records were gathered at Erb's point and on nerve trunks at the elbow during median and ulnar nerve stimulation at the wrist. Erb responses to electrical stimulation were larger in amplitude and shorter in duration than the magnetic ones when ‘electrical’ and ‘magnetic’ compound muscle action potentials (cMAPs) of comparable amplitudes were elicited. SEPs were recorded respectively at Cv7 and on the somatosensory scalp areas contra- and ipsilateral to the stimulated side. SEPs showed a statistically significant difference in amplitude only for the brachial plexus response and for the ‘cortical’ N20-P25 complex; differences were not found between the magnetic and electrical central conduction times (CCTs) or for the peripheral nerve response latencies. Magnetic stimulation preferentially excited the motor and proprioceptive fibres when the nerve trunks were stimulated at motor threshold intensities.     

Modulation of the Aplysia gill withdrawal reflex by dopamine

The ability of dopamine to modulate gill contractions was tested in Aplysia. When dopamine was perfused through the gill vasculature, gill contractions caused by siphon stimulation (gill withdrawal reflex) and by depolarization of the gill motor neuron L7 were increased in amplitude, as compared with those evoked during seawater perfusion. Habituation of gill movements, brought about by repetitive stimulation of the siphon or of L7, was prevented by dopamine. Despite the absence of reflex habituation, the number of action potentials in central gill motor neurons, evoked by siphon stimulation, showed normal decrement. Dopamine's effects were blocked when the ctenidial nerve was cut or when L7 hyperpolarized. These data suggest that dopamine acts peripherally to increase the efficacy of L7's synaptic transmission onto gill muscle or elements of the gill neural plexus.     

Single neurone responses to tactile stimulation of the heart in the snail,Helix pomatia L.

Summary The electrical activity of the heart nerve and of single neurons in the suboesophageal ganglia were recorded during tactile stimulation of the heart. 15 neurons were identified which responded to heart stimulation by inhibiting or accelerating activity. Cells influenced by heart afferents are scattered in the visceral and in the right and left parietal ganglia.In most of the cases both decrease and increase of cell activity are caused by synaptic potentials, in some cases, however, the neuron is assumed to have a sensory character.The activity of three neurons influenced by heart stimulation was conducted into the heart nerve. These cells are central neurons of a heart-CNS-heart reflex.Some of the neurons located in the right parietal and visceral ganglia have no connection with the mechanoreceptors of the heart. Since their spikes propagate into the heart nerve, they probably take part in the extracardial regulation of heart activity.One of the neurons located in the visceral ganglion (cell V12) sends its axon into the heart nerve. The response of this neuron to heart stimulation was an increase in activity and an inhibition of the heart rate. This is an inhibitory neuron of the extracardial heart regulatory system.     

Central nervous sensitization and dishabituation of reflex action in an insect by the neuromodulator octopamine

Habituation of excitatory synaptic inputs onto identified motor neurons of the locust metathoracic ganglion, driven electrically and by natural stimuli, was examined using intracellular recording. Rapid progressive reduction in amplitude of EPSPs from a variety of inputs onto fast-type motor neurons occurred. The habituated EPSPs were quickly dishabituated by iontophoretic release of octopamine from a microelectrode into the neuropilar region of presumed synaptic action. The zone within which release was effective for a given neuron was narrowly-defined. With larger amounts of octopamine applied at a sensitive site the EPSP became larger than normal, and in many instances action potentials were initiated by the sensitized response. Very small EPSPs onto a motor neuron, which were associated with proprioceptive feedback, and which were originally too small to be detected above the noise, were potentiated to a level of several mV by the iontophoresed octopamine. A DUM neuron (presumed to be octopaminergic) was found, whose direct stimulation was followed by a strong dishabituating and sensitizing action leading to spikes, of inputs to an identified flexor tibiae motor neuron. The action and its time course were closely similar to those evoked by octopamine iontophoresed into the neuropil in the region of synaptic inputs to the motor neuron. It is concluded that DUM (octopaminergic) neurons exert large potentiating actions on central neuronal excitatory synaptic transmission in locusts.     

Analysis of tonic activity in sympathetic ganglionic neurons in rabbits

Tonic activity of neurons of the rabbit superior cervical ganglion was studied by intracellular recording. This activity was compared with responses of these same neurons to single stimulation of preganglionic fibers. Neurons of the ganglion were found to have two synaptic inputs — principal and accessory. The principal input consists of one preganglionic fiber, the discharge in which evokes an action potential in the neuron with a high safety factor. The accessory input consists of two or more converging fibers, and the neuron discharges only as a result of summation of EPSPs evoked by activation of these fibers.     

Arsenazo III and antipyrylazo III calcium transients in single skeletal muscle fibers

The metallochrome calcium indicators arsenazo III and antipyrylazo III have been introduced individually into cut single frog skeletal muscle fibers from which calcium transients have been elicited either by action potential stimulation or by voltage-clamp pulses of up to 50 ms in duration. Calcium transients recorded with both dyes at selected wavelengths have similar characteristics when elicited by action potentials. Longer voltage-clamp pulse stimulation reveals differences in the late phases of the optical signals obtained with the two dyes. The effects of different tension blocking methods on Ca transients were compared experimentally. Internal application of EGTA at concentrations up to 3 mM was demonstrated to be efficient in blocking movement artifacts without affecting Ca transients. Higher EGTA concentrations affect the Ca signals' characteristics. Differential effects of internally applied EGTA on tension development as opposed to calcium transients suggest that diffusion with binding from Ca++ release sites to filament overlap sites may be significant. The spectral characteristics of the absorbance transients recorded with arsenazo III suggest that in situ recorded signals cannot be easily interpreted in terms of Ca concentration changes. A more exhaustic knowledge of the dye chemistry and/or in situ complications in the use of the dye will be necessary.     

Modulation of a feeding neural circuit by microinjection of K+ channel expression genes into a single identified neuron in Aplysia kurodai

In Aplysia buccal ganglion expression genes for voltage-dependent K(+) channels (AKv1.1a) were injected into one of four electrically coupled multi-action (MA) neurons that directly inhibit jaw-closing (JC) motor neurons and may cooperatively generate their firing pattern during the feeding response. Following the DNA injection, the firing threshold increased and the spike frequency at the same current decreased in the current-induced excitation of the MA neuron; indicating a decrease in excitability of the MA neuron. This procedure also reduced the firing activity of MA neurons during the feeding-like rhythmic responses induced by the electrical nerve stimulation. Moreover, the firing pattern in JC motor neurons was remarkably changed, suggesting the effective contribution of a single MA neuron or electrically coupled MA neurons to the generation of the firing pattern in the JC motor neurons. This method appears useful for exploring the functional roles of specific neurons in complex neural circuits.