THE RESPIRATORY RATE OF THE SCIATIC NERVE OF THE FROG IN REST AND ACTIVITY

1. With the indicator method of Haas, the rates of carbon dioxide production have been measured in the case of the sciatic nerve, various parts of the brain, and the sartorius muscle of the frog. The rate of respiration of the sciatic nerve is from 10 to 30 per cent of that of the other tissues, varying somewhat with the individual. 2. Stimulation of the sciatic nerve with induction shocks sufficient to induce tetanus of the muscle does not increase the output of carbon dioxide from the sciatic nerve, even if continued as long as 30 minutes. Sartorius muscle used as a control showed a marked increase in carbon dioxide production upon relaxation after contraction resulting from such stimulation. 3. These facts indicate that the nerve impulse does not depend upon processes leading to the production of carbon dioxide.     

THE EXCRETION OF CARBON DIOXIDE BY FROG NERVE

1. Quiescent sciatic nerve of the frog discharges CO₂ at the average rate of 0.00876 mg. CO₂ per gram of nerve per minute. 2. Sciatic nerve steeped one minute in boiling water discharges CO₂ at first at a low rate and after an hour and a half not at all. 3. Degenerated sciatic nerve discharges CO₂ at a slightly higher rate than normal living nerve does. 4. Connective tissue from the frog discharges CO₂ at an average rate of 0.0097 mg. per gram of tissue per minute. 5. Assuming that a nerve is composed of from one-half to one-quarter connective tissue the CO₂ output from its strictly nervous components is estimated to be at a rate of 0.008 mg. CO₂ per gram of nerve per minute. 6. Stimulated sciatic nerve increases the rate of its CO₂ output over quiescent nerve by about 14 per cent. When this number is corrected for strictly nervous tissue the rate is about 16 per cent. 7. The increased rate of CO₂ production noted on stimulation in normal sciatic nerves was not observed when they were boiled, blocked, or degenerated. It was also not observed with stimulated strands of connective tissue.     

Electromagnetic stimulation blocks isometric twitch contraction in the frog sciatic nerve-gastrocnemius muscle preparation

The effect of electromagnetic stimulation (EMS) on nerve conduction and muscle twitch contraction was studied in isolated sciatic nerve-gastrocnemius muscle of the frog. Electrical stimulation (ES) of the sciatic nerve produced twitch contractions in the gastrocnemius muscle and these responses were reduced and eventually blocked by EMS, applied to the nerve simultaneously with ES, from a d.c. source at a certain frequency and duration of the induced current. The EMS-induced inhibition of the twitch contractions was reversible, and this depended on the induced current and its duration. The possibility that other factors may have contributed to the inhibition of twitch contractions, such as a rise in temperature, was also investigated. It was concluded that EMS inhibited indirectly-elicited twitch contractions produced by ES in the frog nerve-muscle preparation.     

THE PRODUCTION AND INHIBITION OF ACTION CURRENTS BY ALCOHOL

Suitable concentrations of ethyl alcohol (1 to 1.5 M) applied to a spot on a cell of Nitella lower the P.D. enough to cause action currents. The alcohol then suppresses action currents arriving from other parts of the cell and acts as a block. After the alcohol is removed the normal P.D. and irritability return. Similar experiments on the sciatic nerve and skin of the frog produced only a negative result.     

Effect of delta-aminolevulinic acid on frog nerve-muscle function

Effects of delta-aminolevulinic acid (ALA) on nerve-muscle function $\text{in vitro}$ have been examined using the frog sciatic gastrocnemius preparation. Levels of ALA that did not interfere with nerve conduction did inhibit the muscle's response to nerve stimulation for a period of 50 – 120 min. The amounts of ALA within muscle were declining at 2 hr after topical administration. It is suggested that some of the symptoms in acute attacks of intermittent porphyria may be attributable to effects from ALA.     

FAST AXONAL TRANSPORT IN VITRO IN THE SCIATIC SYSTEM OF THE FROG

Abstract— An in vitro system from the frog has been used to study fast axonal protein transport. The preparation, which was incubated in a specially made chamber, consisted of the gastrocnemius muscle, the sciatic nerve, the dorsal ganglia and part of the spinal cord. The parts were separated from each other by silicone grease barriers, which made it possible to follow the migration of labelled proteins from the spinal cord and ganglia, along the sciatic nerve, towards the muscle. About 80 per cent of transported proteins in the sciatic nerve originated from the dorsal spinal ganglia and moved antidromically at a rate of 60–90 mm per day at 18°C. The rapidly transported proteins were 90 per cent particulate and mainly associated with structures sedimenting in the microsomal fraction.
The effects of cyclohexirnide showed that the synthesis of rapidly moving proteins and their transport were separate processes. A low concentration of colchicine inhibited the transport when it was present in the medium surrounding the ganglia, but had no effect even at a higher concentration, when it was added to the nerve compartment. The presence of vinblastine at a low concentration in either of the two compartments completely arrested the protein transport. Likewise N-ethylmaleimide or p-chloromercuribenzene sulphonic acid in the nerve medium effectively blocked the fast transport. Results from experiments performed to test the possibility of disto-proximal flow and of transfer of proteins from the muscle to the nerve are discussed.     

Effect of hyperglycaemia on muscle glycogen mobilization during muscle contractions in the rat

In the rat, muscle glycogen is mobilized during the first stage of exercise, despite normoglycaemia. The aim of the present study was to examine if this process could be prevented or reduced by hyperglycaemia. Three experiments were carried out: in the first, rats were forced to run on a treadmill; in the second the gastrocnemius muscle group was made to contract by stimulation of the sciatic nerve and in the third adrenaline was administered subcutaneously. Each group was divided into two subgroups: control and enriched with glucose (hyperglycaemic). It was shown that hyperglycaemia has no effect on running-induced glycogen mobilization in hind-limb muscles of different fibre composition but prevented it totally in diaphragm muscle. Hyperglycaemia also did not affect the glycogen mobilization induced by stimulation of the sciatic nerve. However, it delayed and reduced markedly the glycogenolytic effect of adrenaline. It is concluded that increased glycogenolysis in muscles at the beginning of exercise may be a consequence of a delay in the activation of glucose transporting mechanisms in muscle cells.     

THE EFFECT OF POTASSIUM ON THE ACID METABOLISM OF SURVIVING SKELETAL AND CARDIAC MUSCLES OF THE FROG

The potassium contraction of skeletal muscle and relaxation of cardiac muscle have been correlated with the carbon dioxide and total acid production of these tissues. 1. The immersion of surviving sartorius muscles of the frog in isotonic potassium chloride solution causes a marked increase in the rate of acid production. 2. It is probable that carbon dioxide is the principal acid involved in the above effect. 3. The immersion of surviving cardiac muscle of the frog in isotonic potassium chloride solution causes a pronounced depression in the rate of survival acid production. 4. Reasons are given for believing that these changes in metabolism may be independent of the stimulation and inhibition of contraction which potassium simultaneously produces in these tissues.     

Effects of a neurotoxin isolated from the sea anemone, Anemonia sulcata, at frog neuromuscular junction (author's transl)]

ATX II is a toxin extracted from tentacles of Anemonia sulcata. It was known that this protein displays neurotoxic effects on frog isolated neuromuscular preparation (Fig. 1, 2) and that muscular contractures observed with ATX II are blocked by d-tubocurarine (Fig. 3) or on a 40-days-denervated gastrocnemius (Fig. 4). Part of these experiments has already appeared. 1. These effects of ATX II depend on calcium concentration in the bathing medium, as is the case for transmitter release. The same results were observed when we substituted strontium to calcium. 2. On an intact sciatic sartorius preparation, ATX II does not act on the amplitude of the miniature endplate potentials (mepps, Fig. 6). The muscular action potential is not modified by this toxin. 3. ATX II increases the frequency of the mepps (Fig. 5). The evoked transmitter release (quantal content) after ATX II is also largely increased (Fig. 7). 4. In conclusion, it is suggested that ATX II acts indirectly on the muscle through an increase in acetylcholine release from the motor nerve terminals.     

THE OXYGEN CONSUMPTION OF FROG NERVE DURING STIMULATION

1. The resting rate of oxygen consumption of the excised sciatic nerve of the frog is 1.23 c.mm. of oxygen per gm. of nerve per minute. 2. During stimulation with an induction coil with 100 make and 100 break shocks per second there is an excess oxygen consumption amounting on the average to 0.32 c.mm. of oxygen per gm. of nerve per minute of stimulation, or a 26 per cent increase over the resting rate. 3. The magnitude of the excess oxygen consumption in stimulation, in agreement with the all-or-none law, is not markedly influenced by considerable variations in the intensity of stimulation. 4. Increasing the frequency of stimulation from 100 to 200 shocks per second increases the extra oxygen used only 1.12–1.18 times. The same change in frequency of stimulation increases the negative variation 1.15 times and the heat production about 1.25 times (Hill). 5. This parallelism between the excess oxygen and the negative variation argues definitely for some causal connection between the excess oxygen and the nerve impulse itself. 6. Calculation shows that the oxygen tension inside these nerves was not zero.     

FUNCTIONAL ANALYSIS OF SWIM-BLADDER MUSCLES ENGAGED IN SOUND PRODUCTION OF THE TOADFISH

A functional analysis of the striated swim-bladder muscles engaged in the sound production of the toadfish has been performed by simultaneous recording of muscle action potentials, mechanical effects, and sound. Experiments with electrical nerve stimulation were made on excised bladder, while decerebrate preparations were used for studies of reflex activation of bladders in situ. The muscle twitch in response to a single maximal nerve volley was found to be very fast. The average contraction time was 5 msec. with a range from 3 to 8 msec., the relaxation being somewhat slower. The analysis of muscle action potentials with surface electrodes showed that the activity of the muscle fibers running transversely to the long axis of the muscle was well synchronized both during artificial and reflex activation. With inserted metal microelectrodes monophasic potentials of 0.4 msec. rise time and 1.2 to 1.5 msec. total duration were recorded. The interval between peak of action potential and onset of contraction was only 0.5 msec. Microphonic recordings of the characteristic sound effect accompanying each contraction showed a high amplitude diphasic deflection during the early part of the contraction. During relaxation a similar but smaller deflection of opposite phase could sometimes be distinguished above the noise level. The output from the microphone was interpreted as a higher order derivative function of the muscle displacement. This interpretation was supported by complementary experiments on muscle sound in mammalian muscle. The dependence of the sound effects on the rate of muscle contraction was demonstrated by changing the temperature of the preparation and, in addition, by a special series of experiments with repeated stimulation at short intervals. Results obtained by varying the pressure within the bladder provided further evidence for the view that the sound initiated in the muscle is reinforced by bladder resonance. Analysis of spontaneous grunts confirmed the finding of a predominant sound frequency of about 100 per second, which was also found in reflexly evoked grunts. During these, muscle action potentials of the same rate as the dominant sound frequency were recorded, the activity being synchronous in the muscles on both sides. Some factors possibly contributing to rapid contraction are discussed.     

Neuromuscular blocking in acutely tetanus intoxicated mice

The effects of tetanus toxin on neuromuscular transmission of mice in acute intoxication produced by intravenous injection of a large amount of the toxin were examined by (1) recording the phrenic nerve impulses, the electromyograms (EMGs) of the diaphragm and the electrocardiograms, and (2) the evoked EMGs of the gastrocnemius muscle in response to electrical stimulation of the sciatic nerve. The evoked EMGs of the gastrocnemius muscle were analyzed in terms of kinetic and tonic components by their different latencies. Just before death of animals, the EMGs of the diaphragm appeared with some delay relative to the corresponding phrenic discharges. Finally, the EMG of the diaphragm disappeared even in the presence of phrenic discharge, but cardiac electrical activities continued. The amplitudes of the evoked EMGs of the gastrocnemius muscle invariably became low before death, but the muscle action potential could be recorded by direct muscle stimulation for several minutes after death. The latencies of the evoked EMGs were constant until about the middle of the survival time when the latencies suddenly became prolonged. The longer latency was the same as that of the tonic action potentials. Thus, in acutely tetanus-intoxicated mice, neuromuscular transmission was blocked rapidly and the kinetic component of the muscle was blocked earlier than the tonic component.     

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.     

RAPID AXONAL TRANSPORT IN VITRO IN THE SCIATIC SYSTEM OF THE FROG OF FUCOSE-, GLUCOSAMINE- AND SULPHATE-CONTAINING MATERIAL

—An in vitro system from the frog has been used to study fast axonal transport of glycoproteins. The migration of [³H]fucose-, [³H]glucosamine- and [³⁵S]sulphate-labelled material was followed from the dorsal ganglia, along the sciatic nerve towards the gastrocnemius muscle. The distribution in different subcellular fractions, effect of cycloheximide and transport kinetics did not differ very much between fucose- and glucosamine-incorporation into the nerve. Cycloheximide blocked the synthesis of TCA-insoluble radioactivity, which was transported at a rate of 60–90 mm per day at 18°C, more effectively than the synthesis of stationary proteins in the ganglia. About 10 per cent of the TCA-insoluble and transported radioactivity was extracted by chloroform-methanol (2:1, v/v) and might be glycolipids and the rest glycoproteins. Results suggest that TCA-soluble activity, which was recovered in the nerve, originated in part from labelled macromolecules consumed along the axons. The rapidly transported TCA-insoluble radioactivity was 85 per cent particulate and mainly associated with structures sedimenting in the microsomal fraction. [³⁵S]Sulphate-labelled TCA-insoluble material was resistant towards chloroform-methanol (2:1, v/v) extraction and rapidly transported from the ganglia into the nerve. The synthesis was inhibited by cycloheximide. The material, probably proteoglycans, represented a quantitatively minor part of transported glycoproteins.     

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological techniques, in the understanding of the underlying pathophysiology¹. Here we describe a method to perform electrophysiological studies on mouse sciatic nerves in vivo.The animals are anesthetized with isoflurane in order to ensure analgesia for the tested mice and undisturbed working environment during the measurements that take about 30 min/animal. A constant body temperature of 37 °C is maintained by a heating plate and continuously measured by a rectal thermo probe². Additionally, an electrocardiogram (ECG) is routinely recorded during the measurements in order to continuously monitor the physiological state of the investigated animals.Electrophysiological recordings are performed on the sciatic nerve, the largest nerve of the peripheral nervous system (PNS), supplying the mouse hind limb with both motoric and sensory fiber tracts. In our protocol, sciatic nerves remain in situ and therefore do not have to be extracted or exposed, allowing measurements without any adverse nerve irritations along with actual recordings. Using appropriate needle electrodes³ we perform both proximal and distal nerve stimulations, registering the transmitted potentials with sensing electrodes at gastrocnemius muscles. After data processing, reliable and highly consistent values for the nerve conduction velocity (NCV) and the compound motor action potential (CMAP), the key parameters for quantification of gross peripheral nerve functioning, can be achieved.     

Rapid vasodilation in response to a brief tetanic muscle contraction.

To test the hypothesis that vasodilation occurs because of the release of a vasoactive substance after a brief muscle contraction and to determine whether acetylcholine spillover from the motor nerve is involved in contraction-induced hyperemia, tetanic muscle contractions were produced by sciatic nerve stimulation in anesthetized dogs (n = 16), instrumented with flow probes on both external iliac arteries. A 1-s stimulation of the sciatic nerve at 1. 5, 3, and 10 times motor threshold increased blood flow above baseline (P < 0.01) for 20, 25, and 30 s, respectively. Blood flow was significantly greater 1 s after the contraction ended for 3 and 10 x motor threshold (P < 0.01) and did not peak until 6-7 s after the contraction. The elevations in blood flow to a 1-s stimulation of the sciatic nerve and a 30-s train of stimulations were abolished by neuromuscular blockade (vecuronium). The delayed peak blood flow response and the prolonged hyperemia suggest that a vasoactive substance is rapidly released from the contracting skeletal muscle and can affect blood flow with removal of the mechanical constraint imposed by the contraction. In addition, acetylcholine spillover from the motor nerve is not responsible for the increase in blood flow in response to muscle contraction.     

Sciatic nerve regeneration by transplantation of menstrual blood-derived stem cells

This is the first study demonstrating the efficacy of menstrual blood-derived stem cell (MenSC) transplantation via a neural guidance conduit, for peripheral nerve regeneration. The synthesized poly (?-caprolactone)/Gelatin conduit, filled with collagen type I and seeded with 3?×?10⁴ MenSCs, was implanted into a rat’s 10 mm sciatic nerve defect. The results of hot plate latency, sciatic functional index and weight-loss percentage of wet gastrocnemius muscle demonstrated that the MenSC transplantation had comparable nerve regeneration outcome to autograft, as the gold standard of nerve bridging. The transplantation of MenSCs via a synthetic conduit could ameliorate the functional recovery of sciatic nerve-injured rats which make them a potential candidate for cell therapy of peripheral nervous system disorders.     

A new method for excitation-contraction uncoupling in frog skeletal muscle

The mechanical activity of frog sartorius muscle fibers can be uncoupled from the electrical activity of their surface membranes by immersing the preparation in Ringer solution containing either 1.5 or 2.0 M of formamide for 15--20 min. This uncoupling is not reversed when the muscle is transferred to normal frog Ringer solution. Formamide does not affect the electrical activity of the sciatic nerve branch, and both endplate potentials and miniature endplate potentials may be recorded from the uncoupled muscles. Prolonged exposure to formamide, beyond the time needed to paralyze, causes neuromuscular block.     

Multielectrode spiral cuff for ordered and reversed activation of nerve fibres

In this paper we present the modelling, design, and experimental testing of a nerve cuff multielectrode system for selective activation of fibres in superficial peripheral nerve trunk regions which is capable of activating fibres in physiological order. The multielectrode system consists of 45 platinum electrodes embedded within a self-curling spiral silicone sheet organized in fifteen longitudinal groups consisting of three electrodes spaced equidistally around the circumference of the cuff. Electrodes in the centre band acted as stimulating cathodes while the two electrodes of the same group in the two outer bands were connected together and corresponded to the position of a particular cathode, serving as anodes to block the nascent action potentials by membrane hyperpolarization. The interpolar distance was 6 mm on both sides, resulting in a total cuff length of about 20 mm. The cuff was constructed with a diameter to fit the size of the dog sciatic nerve. Preliminary animal testing of the nerve cuff was performed on the sciatic nerve of a Bigley female dog. In the 45-electrode stimulation system, biphasic cathodic first pulses with quasitrapezoidal-shaped cathodic and square anodic parts were delivered through the particular group of tripolar electrodes to effect both selective stimulation of motor axons within the gastrocnemius muscle fascicle, and differential block by membrane hyperpolarization. The test was repeated using rectangular cathodic first biphasic current pulses delivered monopolarly on the central electrode of the same group while connected anodes were replaced by a common anode situated elsewhere in the surrounding tissue. In both experiments an isometric torque in the ankle joint elicited by the gastrocnemius muscle was measured and compared. It was shown that tripolar activation with quasitrapezoidal stimulation pulses elicited an isometric torque with a peak value of 0.83 Nm in 65 ms after onset of delivering stimulating pulses in comparison to the monopolar activation with rectangular biphasic pulses where the peak of the same value was observed in 45 ms after onset. Thus, the multipolar cuff stimulating monopolarly provided an effective means of activating motor axons selectively within the gastrocnemius muscle fascicle, while more physiological recruitment of the muscle fibres was evident when stimulating tripolarly.     

ELECTRON MICROSCOPE AND X-RAY DIFFRACTION STUDIES OF THE EFFECTS OF DEHYDRATION ON THE STRUCTURE OF NERVE MYELIN : II. Optic Nerve

The dehydration of rat optic nerve has been studied by allowing specimens to become partially or fully dried before fixation and preparation for electron microscopy. A correlation is established between electron micrographs of the myelin sheath and corresponding small-angle x-ray diffraction patterns. The modifications of the optic nerve myelin layers during drying were very similar to those described in more detail for the myelin of frog sciatic nerve. The most striking difference was that the system of fine layers characteristic of the fully dried myelin was much more extensive in the case of the optic nerve, and the layer thickness was significantly greater than the corresponding layer in the frog sciatic nerve preparation. The significance of these correlations is discussed.