THE PENETRATION OF ACETYLCHOLINE INTO THE CENTRAL NERVOUS TISSUES OF AN INSECT (PERIPLANETA AMERICANA L.)

Transcription

1 J. Exp. Biol. (1965), 43, With 4 text-figures Printed in Great Britain THE PENETRATION OF ACETYLCHOLINE INTO THE CENTRAL NERVOUS TISSUES OF AN INSECT (PERIPLANETA AMERICANA L.) BY J. E. TREHERNE* AND D. S. SMITH Department of Biology, University of Virginia, Charlottesville, Virginia, U.S.A. (Received 21 November 1964) INTRODUCTION The precise physiological role of acetylcholine in synaptic transmission in the insect central nervous system has been a subject of some speculation and controversy. One of the main difficulties involved in postulating the existence of a conventional cholinergic system in the insect central nervous system has been the relative insensitivity of intact ganglia to applied acetylcholine solutions (Roeder, 1948; Twarog & Roeder, 1956; Yamasaki & Narahashi, i960; Treherne, 1962ft). This apparent difficulty was at least partially resolved by the results of experiments which were interpreted as demonstrating that acetylcholine was effectively prevented from penetrating into the central nervous tissues by the presence of a peripheral diffusion barrier which was identified with the fibrous and cellular nerve sheath (Twarog & Roeder, 1956; O'Brien, 1957; O'Brien & Fisher, 1958). More recently it has been demonstrated that the exchanges of a variety of inorganic ions and molecules take place relatively rapidly between the haemolymph and the tissues of the central nervous system in Periplaneta (Treherne, 1961a, b, 1962a) and Carausius (Treherne, 1965). The present investigation was carried out in an attempt to throw some light on this paradoxical situation by direct measurement of the rate of penetration of acetylcholine into the central nervous tissues of Periplaneta americana. METHODS Adult male P. americana L. were used exclusively in this investigation. To irrigate the abdominal nerve cord with the experimental solution the integument was removed from the dorsal surface of a pinioned insect and the viscera were lifted from the body cavity. The body cavity was then filled with O-2-O-3 ml. of the radioactive acetylcholine solution. This solution was changed twice during the first minute and then once per minute during the remainder of the experiment. After an appropriate period of exposure the experimental solution was drained from the body cavity with filter paper and the whole abdominal nerve cord was carefully removed from the body. In each experiment the nerve cord was drawn across dry filter paper, to free it from any adhering fat body, before weighing to an accuracy of o-oi mg. on a Sartorius single-pan analytical balance. In some experiments single terminal abdominal Permanent address: A.R.C. Unit of Insect Physiology, Department of Zoology, University of Cambridge.

2 14 J- E. TREHERNE AND D. S. SMITH ganglia were de-sheathed, using finely ground watchmaker's forceps, and were then separated from the rest of the nerve cord for the assay of radioactivity. The radioactivity of the nerve sheath tissue was also measured in these experiments. The specific activity of the acetyl-i- M C-choline iodide used in these experiments was approximately 5-0 mc/mm, the concentration in the experimental solution being rox io~* M. In some experiments eserine was added to the solution at a concentration of io~* M. In these cases the nerve cords were treated with the eserine solution for 10 min. before the application of the radioactive solution containing this substance. The composition of the basic physiological solution was that given by Treherne (1962a) and was based on the solution devised by van Asperen & Esch (1956). The radioactive nerve cords were placed in o-i ml. of boiling distilled water for 1-5 min. before the addition of o-i ml. of 2-0% trichloracetic acid (Lewis & Smallman, 1956). The tissue was then subjected to ultra-sonication, the homogenate was centrifuged and the radioactivity was assayed using a Tricarb liquid scintillation counter. The liquid phosphor used in these experiments was the dioxane-methanol naphthaleneethylene glycol system devised by Bray (i960) which enabled the samples to be counted at 2-0 C. Tissues used for chromatography of radioactivity were treated in boiling water for 1-5 min. before extraction in 60% ethanol. The chromatograms were made using Whatman No. 1 filter paper and a solvent system of ethyl acetatepyridine-water (50:30:20) (Malyoth & Stein, 1951). The radioactivity on the chromatograms was assayed by cutting the paper into i-o cm. strips and measuring the 14 C concentration in a toluene liquid phosphor. All experiments were carried out at a temperature of 20-0 C. (± o-i C). RESULTS Fig. 1 illustrates the accumulation of radioactivity in the tissues of the abdominal nerve cord following exposure to a io~* M solution of 14 C-labelled acetylcholine. These results show a very striking reduction in the rate of accumulation of radioactivity which was obtained in the presence of io~* eserine. In the presence of this cholinesterase inhibitor the uptake of 14 C originating as acetylcholine in the bathing medium showed only a slow increase after an initial influx, whereas in the untreated nerve cords the accumulation continued to rise sharply throughout the period of the experiment. The results summarized in Fig. 1 were obtained using unwashed preparations which were merely dried on filter paper after removal of the nerve cord from the radioactive solution. This procedure was adopted to avoid the possibility of losing any rapidly exchanging radioactivity from within the tissues during any washing in inactive solution. An estimate of the amount of radioactivity associated with the nerve sheath under these conditions was obtained by measuring the radioactivity of de-sheathed terminal abdominal ganglia and of the isolated sheath material. The results obtained with ganglia exposed to M C-labelled acetylcholine in the presence of eserine for 5-0 min. show that in these preparations approximately 90-3% of the radioactive material had penetrated into the underlying tissues of the ganglia (Table 1). Chromatograms of the tissue extracts of nerve cord exposed to "C-labelled acetylcholine in the presence of io" 4 M eserine, for the same 5*0 min. period as with the

3 Penetration of acetylcholine into central nervous tissues 15 experiments summarized in Table 1, showed that the greater part of the radioactivity within the tissues was present as acetylcholine (Fig. 3). The small peak of radioactivity obtained with the tissue extract in these experiments suggest that a limited amount of metabousm of the labelled acetylcholine might have occurred in the nerve cord tissues in the presence of io~* eserine r Time (min.) Fig. 1. The uptake of radioactivity by intact abdominal nerve cords when irrigated with a IO~*M solution of 14 C-acetylcholine (closed circles). The open circles illustrate the uptake observed with nerve cords exposed to the acetylcholine solution in the presence of IO~*M eserine. The vertical lines drawn through the points represent the extent of twice the standard error of the mean. Table 1. The distribution of radioactivity between the nerve sheath and the ganglion tissues in the terminal abdominal ganglion after exposure to io~* M u C-labelled acetylcholine in the presence of io~~* M eserine for 5-0 min. (radioactivity expressed as countsjmin.) rial S Activity in nerve sheath Activity in ganglion tissues Activity in ganglion tissues (%) 95-2") 9i-a 863 V J Data is presented in Fig. 2 showing the uptake of radioactivity per unit volume of tissue water in nerve cords treated with IO~*M eserine. In these experiments the accumulation of 14 C within the nerve cord tissues was compared with the uptake obtained during a i-osec. exposure to the radioactive medium. The mean value

4 16 J. E. TREHERNE AND D. S. SMITH obtained by this procedure is taken as an estimate of the surface contamination of the nerve cord and, as will be seen by comparison with the level of radioactivity in the tissues after 5 -o min., is in good agreement with the results obtained in the de-sheathing experiments summarized in Table 1. The level of radioactivity which would be expected in the event of the "C-acetylcholine approaching an equilibrium with the extracellular water of the nerve cord can be calculated from the measured value of 18-2% of the tissue water obtained for the s i 1 1 i Time (min.) Fig. 2. The uptake of radioactivity per unit volume of tissue for intact nerve cords exposed to IO-*M 1 *C-tobelled acetylcholine in the presence of io~* M eserine. A. The level of radioactivity obtained after a i-o sec. exposure to the radioactive solution. The dotted lines represent the extent of twice the standard error of the mean. B. The level of radioactivity which would be expected in the event of the acetylcholine being at the same concentration in the extracellular fluid as in the outside medium. C. The level of radioactivity expected in the event of acetylcholine being distributed in the extracellular fluid according to a Donnan equilibrium with the outside medium. This level was calculated from the concentration ratio observed for inorganic cations in the nerve cord of PeripUtneta (Treherne, 1962 a). D. The level of radioactivity which would be achieved if the 14 C-labelled acetylcholine was at the same concentration in the total tissue water as in the outside medium. The volume of the extracellular water in this preparation was taken from the measured inulin space of 18-2% (Treherne 1962a). The vertical lines drawn through the points represent the extent of twice the standard error of the mean. inulin space with this insect (Treherne, 1962a). The results illustrated in Fig. 2 show that the radioactivity within the nervous tissues rapidly exceeded the level which would be expected in the event of the 14 C-labelled ions being at a concentration in the extracellular fluid similar to that in the outside medium (broken line B in Fig. 2). The

5 Penetration of acetylcholine into central nervous tissues 17 AcetyM-^C-chollne Distance of travel (cm.) Fig. 3. The distribution of radioactivity on chromatograms, developed with ethyl acetatepyridine-water (50:30:20), of 1 *C-labelled acetylcholine and of the tissue extract of nerve cords exposed to a solution of this substance in the presence of IO" 4 M eserine for 5-0 min I J_ I I I J Time (min.) Fig. 4. A semi-logarithmic plot of the data for the influx of "C-labelled acetylcholine plotted according to equation (1)., Exp. Biol. 43, 1

6 18 J. E. TREHERNE AND D. S. SMITH initial rapid influx of radioactivity does, however, rise to the level which would bl expected if the acetylcholine ions were distributed in the extracellular fluid according to a Donnan equilibrium with the outside medium (line C in Fig. 2). The extracellular level to be expected with this organic cation in Donnan equilibrium with the extracellular fluid represented in Fig. 3 was calculated from the concentration ratio of about i-8 obtained with monovalent inorganic cations in the nerve cord of this insect (Treherne, 1962 a). The results illustrated in Fig. 3 also indicate that the radioactivity originating as acetylcholine in the bathing medium had not entered into a rapid equilibrium with the bulk of the tissue water. The second slow rise in radioactivity within the nerve cord did not approach the level which would be expected in the event of the 14 C-labelled acetylcholine coming into a passive equilibrium with the tissue water in this preparation (line D in Fig. 2). The influx of ions and molecules from the outside medium into an effectively single-compartment system, such as a single cell (cf. Harris & Burn, 1949) or the blood of an aquatic animal (Treherne, 1954), can be represented by an equation of the form where k = transfer constant in the direction in-v out, t = time, and a, and a m are the radioactivities at time t and when complete exchange has taken place within the compartment. In such a single-compartment system the semi-logarithmic plot of (1 a t /a m ) with respect to time will result in a straight line. If in the present experiments the value for the radioactivity after i-o hr. is taken as an approximation for the asymptotic value (a w ) then the exchange of acetylcholine in eserinized nerve cords will be seen to occur as a two-stage process (Fig. 4), with an initial rapid phase eventually giving way to a slower phase. The uncertainty involved in the selection of the asymptote and the quality of the data make it impossible to assign any shape to the slow component, which in this case is merely represented by a broken line. (1) DISCUSSION The results described above clearly show that the penetration of acetylcholine into the tissues of the central nervous system occurs relatively rapidly in this insect. The inward movement of this ion appears to take the form of a two-stage process, with an initial rapid influx eventually giving way to a slower accumulation. The rapid uptake of the radioactive acetylcholine cannot be attributed to any appreciable association with the nerve sheath, for the results of the experiments with the de-sheathed ganglia showed that the greater part of the radioactivity was situated in the underlying nervous tissues even in unwashed preparations. The rapid influx of 14 C-labelled acetylcholine in the presence of 10-1 M eserine also occurred in the absence of any appreciable hydrolysis of these ions entering the nerve cord. The present observations appear to be essentially similar to those which have been made on the exchanges of inorganic ions in the central nervous systems of Periplaneta (Treherne, 1961a, b, 1962a) and Carausius (Treherne, 1965). In both these insect species the exchanges taking place between the central nervous tissues and the haemolymph have been shown to occur as two-stage processes, the half-times for the rapid

7 Penetration of acetylcholine into central nervous tissues 19 Components being of the same order of magnitude as that measured for the influx of acetylcholine in the present investigation. In the previous studies the rapidly exchanging components were identified as the extracellular ion fractions of the central nervous tissues. Now it has already been shown that if the estimate of 18-2% for the extent of the extracellular water in the cockroach central nervous tissues (Treherne, 1962 a) is applied to the present data then the apparent concentration of the acetylcholine in the extra-cellular water exceeds that in the outside medium (Fig. 3). The concentration of the rapidly penetrating acetylcholine is, however, consistent with the extracellular level of this ion which would be exrjected if Donnan equilibrium is established, as has been demonstrated between the haemolymph and the extracellular fluid in the cockroach nerve cord (Treherne, 1962 a). The greatly increased uptake of radioactivity by normal preparations as compared with those treated with io~* M eserine must obviously have resulted from an intracellular accumulation of the labelled products of hydrolysis of the acetylcholine by the cholinesterases which have been shown to occur at high concentration in the central nervous tissues of this insect (cf. Colhoun, 1963). The sites of cholinesterase activity in the central nervous system of Rhodnius have been shown, in histochemical studies with the light microscope, to be effectively confined to the neuropile (Wigglesworth, 1958). An electron-microscope investigation on Periplaneta has also demonstrated a concentration in the neuropile, where the cholinesterase is associated with the axon membranes together with a relatively weak localization in the glial elements surrounding the nerve cell bodies (Smith & Treherne, 1965). There is a similar glial localization of eserine-sensitive esterase in the connectives in the latter species. It follows from the present experiments that the radioactive acetylcholine was able to penetrate rapidly to these regions of the central nervous system. These sites of cholinesterase activity border the narrow extracellular channels limited by the closely apposed glial surfaces and also those spaces found in the region of the axon surface which are generally ca A in width (Smith & Treherne, 1963). These restricted spaces appear to be confluent with the larger peripheral extracellular or 'glial lacunar spaces' (Wigglesworth, i960) and can be presumed to be the route by which the acetylcholine penetrates rapidly to the sites of cholinesterase activity. The chromatographic results showed that there was a possibility of only a limited metabolism of acetylcholine in the presence of io~* M eserine. Such a limited metabolism can perhaps be correlated with the demonstration of a relatively weak unspecific cholinesterase in the perineurium, the glial elements surrounding and within the neuropile and in the connectives of the central nervous system of this species (Smith & Treherne, 1965). Such a slow hydrolysis of the labelled acetylcholine could contribute to the slow accumulation of radioactivity observed with the eserinized preparations. The demonstration of a rapid influx of acetylcholine into the intact nerve cord of Periplaneta does not accord with some earlier hypotheses which have been suggested to explain the relative ineffectiveness of this compound on synaptic transmission in insects. In Periplaneta synaptic transmission in the intact terminal abdominal ganglion has been shown to be unaffected by the presence of io~ 2 M acetylcholine (Roeder, 1948), whereas in de-sheathed ganglia a rapid and reversible conduction block developed at concentrations of between 3-0 and 5-0 x IO~ 3 M acetylcholine (Twarog & Roeder,

8 20 J. E. TREHERNE AND D. S. SMITH 1956; Yamasaki & Narahashi, i960). These results were interpreted as demonstrating that the peripheral nerve sheath functioned as a diffusion barrier restricting the entry of acetylcholine into the underlying tissues. This conclusion was also apparently supported by the experiments of O'Brien (1957) and O'Brien & Fisher (1958) demonstrating a correlation between the toxicity and the degree of ionization of a series of neuropharmacological compounds. It was postulated that because of its ionized condition acetylcholine was unable to penetrate the tissues of the intact nerve cord. There were, however, some exceptions to the above generalization. Boccacci, Natalizi & Bettini (i960), for example, showed that iodoacetic acid entered the tissues of the intact nerve cord without difficulty. Furthermore, the exchanges of a variety of inorganic ions were found to take place relatively rapidly between the haemolymph and the central nervous tissues in Periplaneta (Treherne, 1961a, b, 1962 a) and Carausius (Treherne, 1965). Finally, there are also the observations that injection of acetylcholine beneath the nerve sheath did not cause the development of a rapid conduction block in ganglia of Locusta (Harlow, 1958) and Periplaneta (Treherne, 19626). The results of the present experiments appear to add conclusive proof to these earlier observations that the nerve sheath does not, in fact, function as a significant diffusion barrier restricting the entry of acetylchoene into the tissues of the central nervous sy9tem of this insect. It appears likely that the increased sensitivity of the de-sheathed ganglia to acetylcholine resulted from some of the drastic secondary changes which have been shown to result from the de-sheathing procedure (Treherne, 1962 a, b). Finally, it is. relevant to consider the possibilities which remain to explain the relative insensitivity of the intact insect nervous system to applied acetylcholine in the absence of an appreciable diffusion barrier to this substance. The possibility exists that there may be some local protection of the regions of synaptic activity, although it should be mentioned that there appears to be no morphological basis for such a mechanism. Electronmicrographs of cockroach central nervous tissues show that the most likely regions of synaptic activity (i.e. those containing axoplasmic vesicles which are associated with membrane-bound regions of cholinesterase (Smith & Treherne, 1965)), are in fact confluent with the general extracellular system (Smith & Treherne, 1963). Alternatively, as Yamasaki & Narahashi (i960) suggested, the insitivity of the central nervous system to applied acetylcholine may result from a combination of high cholinesterase activity of these tissues (cf. Chadwick, 1963; Colhoun, 1963) with a low susceptibility of the post-synaptic membrane to the transmitter substance. Such a situation could permit the existence of a cholinergic synaptic mechanism in the absence of a peripheral diffusion barrier. SUMMARY C-labelled acetylcholine was found to penetrate rapidly into the tissues of the intact abdominal nerve cord. Uptake in the presence of io~* M eserine occurred as a two-stage process, the initial rapid influx being identified as the penetration into the extracellular system of the nerve cord. 2. There was a more rapid accumulation of radioactivity in normal preparations as compared with those treated with io~* M eserine, presumably as a result of intracellular uptake of the products of hydrolysis of the acetylcholine.

9 Penetration of acetylcholine into central nervous tissues The level of radioactivity in the rapidly exchanging fraction was consistent with the hypothesis that the acetylcholine ions were distributed in the extracellular fluid according to a Donnan equilibrium with the haemolymph in eserinized preparations. 4. These results are discussed in relation to the possible physiological role of acetylcholine in synaptic transmission in this insect. REFERENCES VAN ASPEREN, K. & ESCH, I. VAN (1956). The chemical composition of the haemolymph in Periplaneta americana. Arch, nierl. Zool. II, BOCCACCI, M., NATALIZI, G. & BETTINI, S. (i960). Research on the mode of action of halogen containing alkylating agents on insects. J. Intt. Phyiiol. 4, BRAY, G. A. (i960). A simple efficient liquid scintillator for counting aqueous solutions in a liquid scintillation counter. Anal. Biochem. 1, CHADWICK, L. E. (1963). Actions on insects and other invertebrates. In Handbuch der Experimentellen Pharmakologie Erganzungswerk (ed. G. B. Koelle), 15, Berlin: Springer-Verlag. COLHOITN, E. H. (1963). The physiological significance of acetylcholine in insects and observations upon other pharmacologically active substances. In Advances in Inject Physiology (eds. J. W. L. Beament, J. E. Treherne & V. B. Wigglesworth), 1, London: Academic Press. HARLOW, P. A. (1958). The action of drugs on the nervous system of the locust (Locusta ndgratoria). Ann. Appl. Biol. 46, HARRIS, E. J. & BURN, G. P. (1949). The transfer of sodium and ions between muscles and the surrounding medium. Trans. Faraday Soc. 45, LEWIS, S. E. & SMALLMAN, B. N. (1956). The estimation of acetylcholine in insects. J. Physiol. 134, MALYOTH, G. & STEIN, H. W. (1951). Beitrag zur Papierchromatographie der Cholinester und der Zucker. Biochem. Z. 333, O'BRIEN, R. D. (1957). Esterasea in the semi-intact cockroach. Ann. Ent. Soc. Amer. 50, O'BRIEN, R. D. & FISHER, R. W. (1958). The relation between ionization and toxicity to insects for some neuropharmacological compounds. J. Econ. Ent. 51, ROEDER, K. D. (1948). The effect of anticholinesterases and related substances on nervous activity in the cockroach. Johns Hopkins Hosp. Bull. 83, SMITH, D. S. & TREHERNE, J. E. (1963). Functional aspects of the organization of the insect nervous system. In Advances in Insect Physiology, (eds. J. W. L. Beament, J. E. Treherne & V. B. Wigglesworth) 1, London: Academic Press. SMITH, D. S. & TREHERNE, J. E. (1965). An electron microscopic study of acetylcholinesterase localization in the central nervous system of an insect (Periplaneta americana L.). (In preparation.) TREHERNE, J. E. (1954). The exchange of labelled sodium in the larva of Aedes aegypti L. J. Exp. Biol. 3i» TREHERNE, J. E. (1961 a). Sodium and potassium fluxes in the abdominal nerve cord of the cockroach, Periplaneta americana L. J. Exp. Biol. 38, TREHERNE, J. E. (19616). The kinetics of sodium transfer in the central nervous system of the cockroach, Periplaneta americana L. J. Exp. Biol. 38, TREHERNE, J. E. (1962a). The distribution and exchange of some ions and molecules in the central nervous system of Periplaneta americana L. J. Exp. Biol. 39, TREHERNE, J. E. (19626). Some effects of the ionic composition of the extracellular fluid on the electrical activity of the cockroach abdominal nerve cord. J. Exp. Biol. 39, TREHERNE, J. E. (1965). The distribution and exchange of inorganic ions in the central nervous system of the stick insect. Carausius morosus. J. Exp. Biol. (In the Press.) TWAROO, B. M. & ROEDER, K. D. (1956). Properties of the connective tissue sheath of the cockroach abdominal nerve cord. Biol. Bull., Woods Hole, in, WIGGLESWORTH, V. B. (1958). The distribution of esterase in the nervous system and other tissues of the insect Rhodnius prolixus (Hemiptera). Quart. J. Micr. Sci. 99, WIGGLESWORTH, V. B. (1960). The nutrition of the central nervous system in the cockroach, Periplaneta americana L. The role of the perineurium and glial cells in the mobilization of reserves. J. Exp. Biol. 37, YAMASAXI, T. & NARAHASHI, T. (i960). Synaptic transmission in the last abdominal ganglion of the cockroach. J. Inst. Physiol. 4, 1-13.