Detlev W. Bronk

CHEMICAL EXCITATION OF NERVE

One of the noteworthy characteristics of neurones is their sensitivity to changes in the chemical environment. Even within the relatively protected interior of :he body, the properties of nerves are subject t o modification by variations in the composition of the body fluids. Indeed, the alterations of irritability and the trains of nerve impulses, which are the result of changes in the chemical environment, are among the most important factors involved in the regulation of the activity of the organism. This is one of t,he significant reasons for studying the chemical activation of nerve. A second reason derives from the current interest in the role of chemical agents in the mechanism of synaptic transmission. Furthermore, the investigation of the effects of various chemical agents is one of the most fruitful sources of information regarding the role of the several chemical components of the nerve structure and of the chemical processes involved in nervous action. This last consideration suggests that the most significant chemical agents for use in the study of the processes of activation are those which have an important part in the normal structure of nerve. Calcium is such an element. Potassium is another; it modifies the action of calcium, to which it is closely related in the regulation of nerve action, and i t has a marked influence on the electric potential difference across the interfaces a t which the nerve comcs in contact with its environment. Finally, the effects of acetylcholine on the initiation and conduction of the nerve impulse make an important and timely subject for investigation in such a study as this. It is with the effects of these agents that we shall be primarily concerned. There are others of significance for a general study of this probIem, but from these three we can derive many of the basic phenomena involved in chemical excitation. I

A continuous flow respirometer utilizing the oxygen cathode.

The method of construction and operating conditions are given for a respirometer suitable for determining steady‐state rates of oxygen uptake by peripheral nerve. The polarographic method is used to measure the change in oxygen concentration occurring when a solution of known oxygen concentration flows at a constant, predetermined rate over a tissue sample which removes oxygen from the stream. From a knowledge of the concentration change and volume‐flow rate the rate of oxygen uptake by the tissue is readily determined. Electrodes for stimulating the nerve sample and for recording the action potential are provided. By virtue of the flow system the respirometer maintains constant environmental conditions. The system is linear with respect to changes in the steady‐state rate of oxygen consumption, and the sensitivity is such that, for a 10‐mg sample of nerve, changes in the rate of oxygen uptake of 0.1 micromoles/g hr., (2 cu. mm/g hr.) are readily detected.

THE OXYGEN UPTAKE OF THE PERIPHERAL NERVE OF THE RAT

THE oxygen metabolism of the peripheral nerve of mammals has been studied much less extensively than has that of the nerves of frogs and invertebrates. In particular the relation between frequency of nerve impulses and magnitude of the increased oxygen consumption of active nerve is not known. The results described below include the determination of the rate of oxygen uptake of rat’s nerve at rest, evidence that the rate of oxygen uptake reaches a higher steady level when the nerve is conducting impulses, determination of these activity levels as a function of frequency, and a comparison of the effect of temperature on the rates of uptake of oxygen by nerve at rest and during activity.

Inhibition of Cardiac Accelerator Impulses by the Carotid Sinus.

It has been shown by Hering, 1 Heymans, 2 and others that stimulation of the afferent endings in the carotid sinus by an increase in the endosinusal pressure causes a slowing of the heart. Some have held that this is entirely due to a reflex stimulation of vagal fibers while others have presented evidence to show that there is a reflex inhibition of the cardiac accelerators as well. The second explanation involving a reciprocal mechanism has generally appeared to be the more probable. In the present investigation we have studied certain aspects of this reflex by observing the sympathetic impulses to the heart while varying the pressure in the carotid sinus. The experiments were performed on cats under nembutal anesthesia. The carotid sinus on each side was isolated and perfused by way of the common carotid and external carotid arteries. Inasmuch as the nerve supply remained intact an increase of pressure within the sinus caused an increase in the number of afferent impulses going to the medullary centers. 3 The effect of these impulses on the cardiac accelerator discharge was determined by recording with a vacuum tube amplifier and oscillograph the sympathetic impulses in one of the small nerves running from the stellate ganglion to the heart. The general nature of the cardiac sympathetic activity has been described 4 as a persistent tonic discharge of impulses grouped into large waves. We now find that as the pressure within the sinus is raised this discharge decreases until at about 125 to 150 mm. Hg. there is a complete inhibition of the sympathetic impulses. If the pressure within the sinus be maintained at such a level the period of complete inhibition lasts for some seconds, after which there is an escape with a return of the sympathetic discharge. The duration of this period of inhibition is a function of the endosinusal pressure.

Response of a Sympathetic Ganglion to High Frequency Stimulation.

If a preganglionic mammalian nerve is stimulated at a low frequency, each volley of impulses initiates a fairly well synchronized discharge in the postganglionic nerve. 1 When the rate of stimulation is increased to 30 or 40 per second there is a progressive decrease in the height of the postganglionic spike potential. This apparent failure in the capacity of the ganglion to transmit impulses at a high frequency is a property of the synapse which must be taken into consideration in the formulation of any theory which attempts to explain the mechanism of synaptic transmission. All of our experiments were performed on cats in a room maintained at close to body temperature. The pregaglionic fibers in the third or fourth thoracic root were stimulated by means of a thyra-tron stimulator and the impulse discharge from the stellate ganglion was recorded in the inferior cardiac nerve. The effect which we are considering can be demonstrated by stimulating the preganglionic nerve at a frequency of about 60 per second. The successive postganglionic spike potentials rapidly decrease in height and after a variable number of stimuli they can no longer be detected at ordinary amplifications. There is no such rapid failure at these frequencies of the spike potential in either the pre- or postganglionic nerves when they are stimulated directly. This suggests that the ganglion blocks the transmission of impulses at high frequencies. Two observations argue against such a conclusion. In the first place we have found that stimulation of the preganglionic fibers at frequencies as high as 120 per second produces cardiac acceleration throughout long periods of stimulation. In the second place, the electrical record of postganglionic activity during such stimulation frequently shows a negative displacement of the base line which continues until the end of the stimulus. It would appear therefore that there is continued activity in the postganglionic fibers despite the absence of the usual spike potential.

A Sensitive Respirometer for the Measurement of Rapid Changes in Metabolism of Oxygen

A respirometer used to study the oxygen metabolism of peripheral nerve is described. The theory of its operation and its operating characteristics are given. The respirometer consists essentially of the nerve trunk itself, placed symmetrically across the tip of a polarized platinum microelectrode which is flush with the floor of the chamber. The sides of the nerve are exposed to moist gas of known oxygen content; the top surface of the nerve is covered. The current to the platinum electrode measures the concentration of oxygen in the nerve at the electrode tip. In the steady state the oxygen concentration near the electrode is determined by: (1) the configuration of the system, (2) the oxygen content of the gas phase, (3) the diffusion coefficient of oxygen in the tissue, and (4) the rate of oxygen consumption by the tissue. Starting from the steady state, any change in rate of oxygen uptake may be followed directly during the first 45 to 60 seconds of such a change; during this time the change is equal t...

Rhythmic Activity of Single Nerve Fibers Induced by Low Calcium

In the course of certain experiments on the chemical excitation of nerve we have observed the development of rhythmic volleys of impulses in the individual fibers which recur at regular intervals for long periods of time. Because of the possible relation of such a phenomenon to the mechanism of rhythmic activity of the nervous system in normal and pathological states we have studied this type of response in some detail. All of the experiments have been performed on the sciatic nerve of the frog. In order to facilitate the penetration of the solution used for stimulation the sheath of the excised nerve was removed for a length of about one cm. and here the fibers were carefully separated so as to form a large number of small bundles. The region thus treated with the solution was usually at one end, but it was equally satisfactory to do so anywhere along the course of the nerve. A single fiber was then isolated at the other end of the nerve and placed on non-polarizable electrodes leading to an amplifier and oscillograph. Partial removal of the ionized calcium in the nerve by sodium citrate or a reduction of its concentration by bathing the fiber bundles in calcium-free Ringers fluid initiates a continuous train of impulses from the treated end at the rate of about 1001 per sec. When the nerve fiber has been brought into this condition which causes a sustained discharge of impulses we find that the substitution of normal Ringers fluid for the citrate or the calcium-free solution ultimately transforms the discharge into a series of rhythmic volleys. The continuous discharge goes on for a time which is variable from preparation to preparation and then suddenly stops, followed after an interval of a few seconds by a burst of impulses.

The action of strychnine on sensory end organs in muscle and skin of the frog

STRYCHNINE has such a profound effect upon reflex activity that it is of some interest to enquire into its influence upon the discharge of impulses from the peripheral sense organs. There are, of course, many reported observations as to the action of this drug upon organs of special sense. They are, however, conflicting, many of them being subjective, and the recent work of Adrian and Matthews(i) on the impulse discharge from the eye of the Conger eel makes it likely that here the effect of strychnine is due to its action on the synaptic connections of the retina rather than on the excitability of the light receptors. On the other hand, Lapicque(2) has shown that strychnine shortens the chronaxie of motor nerve fibres and Danilewsky and Perich anjanz(3) that it increases the irritability of motor nerve fibres: it is unlikely therefore that it should be entirely without effect on the sensory end organ. The methods recently developed for recording the discharge of;impulses from sense organs make it a fairly simple matter to study. the action; of diugs upon them. In the present work the discharge of proprioceptive impulses from a muscle under tension has been recorded before and after strychnine, and the impulses from strychninised frog skin have been studied by way of comparison. The preparation employed was the excised muscleftexor superficialis digitorum with the connecting nerve tibialis superficialis of mediumsized frogs (R. temporaria). The muscle was left attached to the tibia and thereby mounted in one compartment of a double moist chamber. This compartment was then partly filled with Ringer fluid or Ringerstrychnine fluid so that the muscle was bathed with the desired solution. A thread from the tendon of the muscle was connected to a loading lever on which was hung a weight that was lowered at a constant rate by an oil dash-pot. The nerve was so arranged as not to come in contact with the fluid bathing the muscle and was suspended between two nonpolarisable electrodes in the second compartment of the moist chamber.

Afferent Impulses in the Carotid Sinus and Aortic Nerves.

Afferent impulses in the carotid sinus nerve of the rabbit have been recorded by means of a vacuum tube amplifier and either a string galvanometer or Matthews oscillograph and the arterial pulse curve registered simultaneously with a Wiggers manometer. The experiments show that there is a burst of impulses accompanying each heart cycle followed by comparative inactivity. This discharge is coincident with the rapid rise in arterial pressure revealed by the carotid pulse curve. The duration of the nerve activity extends usually to about the incisura although frequently there are scattered impulses continuing throughout diastole. In some cases there is a second smaller outburst which coincides with the rise in pressure following the incisura. At high blood pressures the discharge becomes continuous, an effect which is likewise produced as a result of asphyxia. The general character of the discharge in the carotid sinus nerve agrees closely with that found in the aortic (cardiac depressor) nerve. The impulses in this nerve have been recorded by Adrian 1 and several other workers and simultaneous records of the impulses and pulse curves have recently been reported by the author. 2 Here, too, there is a large outburst of impulses synchronous with the rapid rise in pressure the major part of the discharge usually continuing to the incisura. The activity of the sensory nerve endings in both the aorta and the carotid sinus appears, on the basis of these experiments, to be a function of both the absolute level of pressure and the rate of change of pressure.

Impulses in Cardiac Sympathetic Nerves.

A better understanding of the mechanism of the reflex regulation of heart rate and blood pressure is to be gained from a study of the afferent nerve impulses from the circulatory system and of the efferent impulses to the heart and blood vessels. Previous communications have described the nature of the nervous discharge from the arch of the aorta and from the carotid sinus, 1 , 2 and its relation to blood pressure. On the efferent side, sympathetic impulses concerned in maintaining the tone of the blood vessels have been recorded. 3 The present report is concerned with the activity of the sympathetic fibers to the heart. One of the small nerve twigs running to the heart from the stellate or inferior cervical ganglion in a cat under urethane anesthesia was freed from the surrounding tissue and cut close to the heart, all other cardiac sympathetic fibers remaining intact. The nerve was then slung onto electrodes and the action potentials, after amplification, were recorded by means of an oscillograph. Figure 1A shows a typical discharge. It will be observed that the impulses tend to come in bursts but in general we have not been able to identify the frequency of these volleys with the heart rate or respiratory rhythm, although it has been shown 3 that in the case of sympathetic nerves carrying fibers to the blood vessels, there is usually a grouping that is synchronous with either the respiratory or cardiac rhythm. The records further show that under the conditions of these experiments there is normally a ”tonic” sympathetic discharge to the heart. The relation of the discharge in the cardiac sympathetic fibers to the heart rate is shown by a comparison of Figures 1A and B. In A, the frequency of the heart beat was 132 per min.