The Kinetics of Sodium Transfer in the Central Nervous System of the Cockroach, Periplaneta Americana L.

Some previous investigations have shown that the exchanges of sodium and potassium ions between the haemolymph and the cockroach central nervous system occurred relatively rapidly (Treherne, 1961a) and appeared to be effected by a mechanism involving an active extrusion of sodium ions (Treherne, 1961b). More recently it has also been shown that the measured efflux of sodium ions was not significantly affected by the removal of substantial portions of the cellular and fibrous nerve sheath (Treherne, 1961 c). It was concluded from this that the rate-limiting factor measured in these experiments was not the transfer of ions across the perilemma but the extrusion of sodium from the underlying tissues of the central nervous system. Thus any rate-limiting movements of ions across the perilemma occurred too rapidly to be measured by the techniques used in the previous investigations. In the present experiments, therefore, an attempt has been made to measure the rapid component of ^MNa exchange by determining the rate of loss of radioactivity obtained on washing isolated nerve cords and single connectives and ganglia for relatively short periods in successive volumes of physiological solution.

The experiments described in this paper were carried out using the abdominal nerve cords of adult male Periplanata americana L. In these experiments the nerve cords were made radioactive by soaking them for varying periods in a solution containing ²⁴Na (0·1–0·5 mc./ml.). With short loading periods (20 sec.–5·0 min.) the isolated ligatured nerve cords were soaked in the oxygenated physiological solution ; for longer loading periods (5–20 min.) the nerve cords of decapitated individuals were perfused with the radioactive solution as described in a previous paper (Treherne, 1961c). The ligatures were tied with threads pulled from 15 denier nylon stockings. The composition of the radioactive solution used was that given by Treherne (1961a). On removal from the radioactive solution the ligatured nerve cords were carefully blotted and then washed for varying periods in successive 0·2 ml. amounts of inactive solution of the same composition. The amount of ²⁴Na remaining in the nerve cord at varying times was determined from the measured radioactivity of the washings. The radioactivity measurements were made with a Mullard MX 123 G.M. tube linked to a 100 c. Panax scaler.

Some preliminary measurements were made to estimate the extent of any ‘inulin space’ in the central nervous system. This was done by soaking ligatured isolated nerve cords for 1 hr. in a 3·0% solution of ¹⁴C-labelled inulin (3·0 mc./g.) made up in physiological solution. The nerve cords were then washed for 25 sec. and the ¹⁴C-inulin was extracted by soaking them for 24 hr. in the physiological solution. The washing time of 25 sec. used was found to be the minimum period necessary to remove 97 % of the radioactivity from the surface of a nerve cord exposed to ¹⁴C-inulin for 1 sec. These values are thus likely to be minimum estimates of the ‘inulin space’ of this organ for some radioactivity must have leaked from within the nerve cord during the washing procedure. In a limited number of cases the rate of loss of ¹⁴C-labelled inulin was determined by washing the ligatured isolated nerve cords in successive volumes of the physiological solution as for the ²⁴Na efflux experiments.

The results illustrated in Fig. 1 show the decline in radioactivity of some isolated abdominal nerve cords, previously soaked in the solution containing ²⁴Na, when maintained in an inactive solution of the same composition. In all cases semi-logarithmic plots of the results for varying loading times appeared to follow a complex course initially, eventually assuming an exponential form after a period of between 160–200 sec.

It was found possible to separate a fast component from the curves for the loss of ²⁴Na from the nerve cords by subtraction from the initial values lying above the line extrapolated to zero time. The separation of an efflux curve into fast and slow components with data plotted semi-logarithmically with respect to time is shown in Fig. 2. The fast component illustrated in Fig. 2 was complex initially, but assumed after a few seconds a simple exponential form with a half-time (t_0·5) of approximately 33·0 sec. The half-time for the slow component was, in this case, 260 sec.

The escape of ²⁴Na from the isolated abdominal nerve cords was also measured in the presence of 0·5 mM./l. 2:4-dinitrophenol. The poison was added to the physiological solution during the initial loading period with the ²⁴Na and was present at the same concentration in the inactive solution during the subsequent efflux experiments. Previous results (Treherne, 1961b) have shown that there was a slight delay period of a few minutes before the poison affected the rate of extrusion of sodium from the nerve cords. In the present experiments, therefore, the nerve cords which were loaded with ²⁴Na for only short periods (less than 5 min.) were pretreated with 0·5 mM./l. dinitrophenol to maintain a constant exposure to the poison of 5 min. before the efflux experiments were commenced. Fig. 3 shows the escape of ²⁴Na from a poisoned preparation loaded with ²⁴Na for 10 min. In this experiment the fast component was not abolished by the presence of the poison, in fact t_{0 6} in this case was 33-0 sec., which was the same as that for the normal preparation illustrated in Fig. 2. In this particular experiment the slow component for ²⁴Na efflux was, however, much reduced as compared with the normal preparation. The effects of 0·5 mM./l. 2:4-dinitrophenol on the escape of ²⁴Na from the isolated nerve cords are summarized in Table 1. The results clearly indicate that the presence of the poison affected the slow phase of sodium lo but not the initial fast component.

The total activity of the ²⁴Na in the slowly exchanging fraction was estimated by extrapolation of the slow component to zero time. Fig. 4 illustrates the estimated radioactivity of the slowly escaping fraction at varying times after exposure to the solution containing ²⁴Na. These data would appear to show that the poison had little effect on the rate of accumulation of the radioactive ions in the slowly exchanging fraction. The results are, however, too few to judge the equilibrium level of radioactivity as between the normal and poisoned preparations.

The escape of ²⁴Na from isolated ligatured fragments of the central nervous system was studied in some experiments. The loss of radio-sodium from the terminal abdominal ganglion and from the connective between the fourth and fifth abdominal ganglia was found to occur as a two-stage process as for the whole abdominal nerve cord. The results for the escape of ²⁴Na from a single isolated connective are illustrated in Fig. 5. In this case, as for the whole nerve cord, the fast component appeared to be initially complex eventually becoming a simple exponential function. The halftimes for ²⁴Na loss were in this experiment: 41·0 sec. for the fast phase and 338·0 sec. for the slow.

The effect of removal of a substantial portion of the perilemma on the rate of loss of sodium was studied in this investigation, by comparison of sodium loss as between an intact isolated terminal abdominal ganglion and one in which the dorsal portion of the cellular and connective tissue sheath was removed with sharpened watchmaker’s forceps. Fig. 6 illustrates the fast components for the escape of ²⁴Na from normal and partially desheathed ganglia. The effects of removal on ²⁴Na loss were not obvious, certainly the final exponential phase was not significantly affected by the desheathing. Any effect of this procedure must, therefore, be sought in the initial complex phase which is at the moment very difficult to analyse, especially as the initial portion will also be affected by varying amounts of surface radioactivity.

Preliminary experiments were carried out in an attempt to discover the extent of any extracellular space in the central nervous system of this insect. This was done by the conventional method of measuring the inulin space. ¹⁴C-labelled polysaccharide was used. Table 2 shows the inulin space as a percentage of nerve-cord water in washed nerve cords previously soaked for 1 hr. in the radioactive solution.

The movements of the ¹⁴C-labelled inulin molecules within spaces demonstrated in the previous experiments was studied by determining the rate of loss of radioactivity from the abdominal nerve cord. Fig. 7 shows the measured radioactivity associated with the nerve cord plotted against time when the preparation was washed in inactive physiological solution. These data when plotted logarithmically showed an initial curved portion which eventually gave way to an exponential decline, with a half-time of 214’0 sec.

The results outlined above showed that the escape of ²⁴Na from the isolated abdominal nerve cord occurred as a two-stage process–an initial rapid phase, with a half-time of about 28·5 sec., eventually giving way to a slow component, with a halftime of approximately 277·0 sec. The experiments with dinitrophenol clearly indicated that only the slow phase of sodium efflux was reduced by the presence of a metabolic inhibitor, the initial rapid phase being unaffected. The slow phase of sodium loss demonstrated by the present technique thus corresponds both qualitatively and quantitatively with the outward movement of sodium ions from the central nervous system measured in previous investigations (Treherne, 1961b; 1961c). In the earlier studies in which the radioactive nerve cords were suspended in flowing saline above a GM tube, the technique was not capable of measuring the extremely rapid initial escape of ²⁴Na demonstrated in the present investigation. In these earlier studies then the fast phase of sodium efflux was not recognized.

The demonstration of a rapid exchange of sodium ions between an extracellular space in the central nervous system and the external solution helps to clarify the results obtained in a previous investigation (Treherne, 1961c). In this earlier study it was found that the rate of sodium extrusion from the isolated terminal abdominal ganglion was not significantly affected by the removal of substantial portions of the cellular and fibrous nerve sheath. It has now been shown that the efflux of ²⁴Na measured in the previous experiments corresponded to the slow phase of sodium loss obtained in the present experiments and, in fact, represents an extrusion from the cellular components of the central nervous system. Thus as the relatively slow extrusion takes place into an extracellular space from which ions escape very rapidly it is not surprising that in the previous study removal of the perilemma did not affect the measured loss of sodium ions.

The exchange of sodium ions taking place between the extracellular space and the various cellular components of the cockroach central nervous system appeared to be essentially similar to those taking place in cephalopod giant axons (cf. Hodgkin, 1958). As in the squid axon the accumulation of radioactive sodium within the various cells of the central nervous system of the cockroach appeared to be a passive process, unaffected by the presence of a metabolic inhibitor. The efflux of sodium from the cellular components of the central nervous system was, however, reduced in the presence of metabolic inhibitors as in the cephalopod giant axon. In the absence of external potassium ions (Treherne, 1961b, 1961c) the extrusion of sodium ions from the cellular components was also reduced indicating that, as in the squid axon, the efflux is effected by some sort of linked sodium pump.

The demonstration of the rapid exchanges of sodium ions between the external solution and an extracellular space in the central nervous system of this insect is perhaps rather unexpected in view of the observations of Hoyle (1953) and of Twarog & Roeder (1956) that the blocking time for insect nerve is dramatically reduced in solutions of high potassium concentration when the perilemma is removed. Twarog & Roeder, for example, demonstrated that in the intact cockroach nerve cord the blocking time in a solution containing 140 mM./l. potassium was between 22–30 min., as compared to 60–90 sec. in the desheathed preparation. The results have been generally interpreted as indicating that the perilemma functions as a diffusion barrier of some kind. The present experiments have demonstrated, however, an efflux from an extracellular space through the nerve sheath which appears to be a purely passive process and which takes place relatively rapidly (t_0·5 = 28·5 sec.). It is hoped that some further researches on the cockroach central nervous system may help to throw some light on this apparently paradoxical situation.