Comp. Biochem. Physiol. Vol. 78B, No. 2, pp. 453-460, 1984 0305-0491/84 $3.00 + 0.00 Printed in Great Britain © 1984 Pergamon Press Ltd

THE AMINO ACIDS OF THE LOCUST NERVOUS SYSTEM: THEIR CONCENTRATIONS A N D

RELEASE IN VITRO

A. JABBAR* and R. H. C. STRANG

Department of Biochemistry, University of Glasgow, Scotland, UK (Tel: 041-339-8855)

(Received 4 November 1983)

Abstraet--l. Amino acid concentrations were measured in the ganglia and haemolymph of the locust (Schistocerca americana gregaria) immediately after dissection and in the nervous tissue after incubation in various artificial media.

2. Alanine, aspartate, Gaba and glutamate were found to be present in greater concentration in the nervous tissue of the living insect than in the haemolymph, while the opposite was true of glycine, isoleucine, leucine, proline, phenylalanine, tyrosine and valine.

3. Efflux of the amino acids varied with the nature of the medium in such a way as to suggest that there was active uptake of aspartate, Gaba, glutamate and possibly alanine, while all the other amino acids except glycine were maintained in equilibrium due to processes of facilitated diffusion.

4. Electrical stimulation altered the proportions of the amino acids in the free pool, increasing the concentrations of glutamate and aspartate, while generally decreasing the efflux of the amino acids to the media.

INTRODUCTION

As the insect has no vascular system, biochemical precursors and nut r ien ts mus t reach all par ts of the ganglia of the nerve cord f rom the haemolymph sur rounding the tissue. This offers the potent ia l ad- vantage tha t isolated but otherwise intact por t ions of the ventra l nerve cord will behave in vitro in much the same way they do in vivo.

Previous publ ica t ions (Clement and Strang, 1978; St rang et al., 1979; S t rang and Clement, 1980) have explored various aspects of the behaviour of the locust ganglia in an a t t empt to see how closely the physiology and biochemistry in vitro mimic. the situ- a t ion in the living insect. The results reported here are concerned with ano the r aspect of the relat ionship of the nervous system to its enveloping haemolymph: the movemen t of amino acids into and out of the tissue. In par t icular it is the efflux of amino acids f rom the isolated nervous system and the effect of different media on tha t efflux, which have been examined. The work was carried out as a prel iminary to a s tudy of the effects of chemical and physical stress on the release of amino compounds from the locust nervous system.

As much as possible, the results are compared with those recorded for the same or closely comparab le insects, and also with analogous mammal i an tissues.

MATERIALS AND METHODS

Insects

Locusts were purchased from the Larujon Locust Suppli- ers, c/o Welsh Mountain Zoo, Colwyn Bay, North Wales. They were kept in cages at 30°C on a wheat bran diet.

*Currently at the Department of Biochemistry and Soil Science, University College of N. Wales, Bangor, N. Wales, UK.

Preparation of the nervous tissue for in vitro studies

Locusts were immobilised at -20°C for 15-20 min and ganglia were dissected out under a stereomicroscope while the insects were kept in crushed ice. After freeing them from adhering tracheal tissue, the ganglia were kept in ice-cold insect saline prior to incubation, or, alternatively, immedi- ately frozen in tubes kept on dry ice, prior to the mea- surement of endogeneous concentrations of the amino acids.

Composition of incubation medium

The insect saline used in these experiments was that of Hoyle (1953) except that MgCI 2 was omitted and NaHCO3 replaced by a phosphate buffering system (Clement, 1979). The saline contained approximately the same ionic concen- trations as present in the locust haemolymph. Glucose (10mM) was added as an exogenous energy source and sucrose (100 raM) was occasionally included to adjust the osmolarity to approximate to that of the haemolymph.

Incubation to follow the release of amino acids

Three pairs of thoracic ganglia were incubated at 37°C in 0.5 ml of fully oxygenated saline for 60 rain. In experiments to determine how release varied with time, samples of 50/11 were withdrawn at intervals up to 60 min of incubation. The volume of incubation medium was made up to 0.5 ml after each removal of the sample so as to maintain a constant volume. At the end of the experiment both the tissue (finally washed rapidly three times in fresh saline), and the incu- bation medium were analyzed quantitatively for the pres- ence of various amino acids. In calculating totals, cor- rections were made for the samples removed.

Extraction of amino compounds from nervous tissue

The procedure of Osborne (1973) was followed for the extraction of amino acids from the nervous tissue and haemolymph. Nervous tissue (2-10 mg) was homogenized in 20-200 #1 of extracting reagent in a thick walled glass tube in ice. The homogenization was achieved with the aid of a dental drill, fitted with a Teflon pestle.

Estimation of the amino acids

The quantitation of the amino compounds was achieved

453

454 A. JABBAR and R. H. C. STRANG

using the double isotope dansylation procedure of Jabbar and Strang (1979).

Electrical stimu&tion of thoracic gang/& in vitro

The thoracic ganglia (both meso- and meta-thoracic ganglia) were dissected out of the locust and after a thor- ough rinse in ice-cold insect saline, were placed in small plastic dish containing 50 #I of iso-osmotic saline. The dish was transferred to the Faraday cage. Stimulation was carried out at room temperature. A silver wire hook was placed under one of the connectives and adjusted to make a firm contact with the nerve cord. An indifferent electrode was placed in the saline to act as reference. The electrodes were connected to the stimulator. A continuous repeated stimulus of 1 msec duration with an amplitude of 5 V at 50 Hz was applied for 30 min (J. A. Miyan, personal com- munication). At the end of the experimental period the nerve cord was removed and washed three times in fresh saline, The amino acids in the nerve cord and in the bathing solution were estimated as given above. The radiochemicals used in these investigations were obtained from Amersham International Ltd., Amersham, England. The rest of the chemicals were of analytical grade and were purchased from BDH or Sigma Chemical Co., Poole, Dorset, England.

R E S U L T S

Concentration q/amino acids in the nervous tissue and in the haemolymph

In Table 1 are presented the concentrations in the freshly dissected ganglia and the ratios of the concen- trations in the :.issue to those in the haemolymph in the same insects, of a range of amino acids, both essential and non-essential (in a dietary sense). For ease of comparison the results of other authors for insect and mammalian tissues are also presented.

Considering first only the results of the present authors, the clearest and most statistically significant difference (P < 0.05) between the cerebral and tho- racic ganglia in terms of concentrations of the amino acids investigated was that the concentration of Gaba in the latter was about three times that in the former.

The ratios of the concentrations of the compounds in the thoracic ganglia to those in the haemolymph showed that about half of the amino acids under consideration are more concentrated in the tissue than in the surrounding plasma. This may argue either active uptake and retention, or a rate of production in the tissue which exceeds efflux. It is conspicuous that it is the non-essential amino acids which can be formed in the nervous system, that show the highest concentration ratios, while the essential amino acids, including such important precursors of neurotransmitters as phenylalanine and tyrosine, demonstrate no such positive accumulation.

When these results are compared to those for other closely comparable insects, locusts and cockroaches, there are the differences which seem inescapable in insect biochemistry, but the underlying similarities outweigh these. The major amino acids of the ner- vous system generally show remarkably similar con- centrations in both locust and cockroach. This is especially true of Gaba and, to a lesser extent, of aspartate, alanine and glutamate. An exception to the rule in the case of the last amino acid is the very high concentration found by Clarke and Donnellan (1982), but these authors point out that their method does not distinguish between glutamate and glu-

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Amino acids in the locust nervous system 455

tamine. Proline, a compound of great metabolic importance in many insects, is also usually present in high but rather variable concentrations in the ner- vous tissue. The figures in Table 1 range from 7-33/~mole/g. That the concentration in the tissue follows that in the haemolymph is shown by the appropriate ratio. Here the discrepancies between different authors largely disappear.

Despite quite wide variations in the concentration ratios for different amino acids reported by different authors, the results are qualitatively the same in the insects examined, and there are many similarities to mammalian tissues. The amino acid showing the greatest concentration ratio is Gaba. This is because it is almost wholly confined to nervous system. Several authors, including the present, have been quite unable to find it in the haemolymph. Glutamate and aspartate have generally the next highest ratio, with alanine a long way behind. Considered across the spectrum of recorded results, proline averages the same concentration in the tissue as outside it. The essential amino acids isoleucine, leucine, phenyl- alanine, tyrosine and valine all show a lower concen- tration in the ganglia than in the haemolymph.

Despite the reasonable consistency shown in the two most investigated insects, locust and cockroach, the more fragmentary evidence from other species (e.g. the honeybee) suggests that at least in terms of the concentration of amino acids in the nervous

system, the results presented here do not represent the whole of the insect kingdom (Carta et al., 1961; Frontal±, 1964).

When the insects are compared to mammals, it is clear that the concentrations in the latters' nervous systems generally are lower, consistent with the much lower concentration of amino acids in mammalian plasma. Two amino acids stand out as exceptions to this rule: Gaba, which shows the same concentration as in the insects, and glutamate, whose concentration is higher than most of those recorded for the insects. In terms of the ratios, the picture in qualitative terms is very similar to that of the insects, except that glycine and to a lesser extent phenylalanine and tyrosine are more clearly concentrated in the mam- malian CNS. In quantitative terms, those concen- tration ratios greater than one are generally much higher in the mammal than in the insects, though this is by no means uniformly true, e.g. alanine.

Concentration and release of amino acids from the locust thoracic ganglia in vitro

Unstimulated tissue. The most obvious result of Table 2a is the steep rise in the concentration of alanine when the tissue is incubated in vitro. This amino acid doubles in concentration in the absence of glucose and trebles in its presence Both differences are significant at the level of P < 0.0005. The concen- tration of aspartate falls 36~ o (P <0.1) in simple

Table 2. (a) Total amino acid concentrations derived from thoracic ganglia after incubation in various media

Additions to simple saline KCN Glucose Glucose Glucose

+ + sucrose sucrose

Temp of incubation (°C) 0 0 37 37 37 37 37

Time of incubation (min) 0 60 60 60 60 60 60

Electrical stimulation . . . . +

Amino acid l 2 3 4 5 6 7

Alanine 11.60 ± 0.57 13.95 + 2.0 23.5 ± 2.02 24.87 ± 3.02 34.83 +- 0.96 32.96 ± 4.88 21.38 + 2.00 Aspartate 6.17 ± 0.48 6.34 ± 0.95 3.95 + 0.48 2.08 __. 0.16 6.54 ± 0.64 5.94 ± 1.23 10.96 + 2.07 Gaba 2.89 ± 0.25 2.09 __. 0.28 2.74 ± 0.17 3.53 ± 0.07 4.56 ± 0.97 3.06 ± 0.57 3.08 ± 0.26 Glutamate 7.08 ± 1.20 7.74 ± 0.44 6.77 ± 0.39 4.40 _ 0.48 7.51 ± 0.45 7.72 + 0.50 24.62 ± 3.80 Isoleucine 0.19 ± 0.03 0.13 ± 0.04 1.12 ± 0.02 0.80 ± 0.09 0.67 ± 0.14 0.94 ± 0.05 0.42 ± 0.08 Leucine 0.13±0.05 0.23+__0.06 0.88±0.17 0.93±0.13 0.75+0.21 1.10±0.15 0.21 ±0.05 Phenylalanine 0.55 ± 0.09 0.19 ± 0.05 0.59 ± 0.01 0.62 ± 0.03 0.67 ± 0.21 0.36 ± 0.03 0.18 ± 0.05 Proline 7.46±2.47 11.81±1.03 2.45±0.58 7.14±0.76 5.07 ± 0.68 4.32 ± 0.96 13.27±1.58 Tyrosine 0.97+0.40 0.49 ± 0.11 0.96 ± 0.13 0.58+_0.07 1.38 ± 0.12 1.36±0.22 0.28±0.03 Valine 0.89±0.25 0.76±0.17 1.31 ±0.12 1.05 ± 0.17 1.50±0.10 1.50 ± 0.25 0.66+_0.13 Total of

mean values 39.99 47.45 48.08 49.51 69.24 59.29 78.43

(b) ~ of total amino acids released to the medium

Alanine 3 1 + 4 72+1 63+1 3 6 + 4 5 7 + 7 ~ 28_+7 - - Aspartate 44 ± 4 4 ± 1 14 _+ 2 4 ± 4 1 __. 0.25 28 ± 10 Gaba 2 4 ± 5 2 9 ± 4 42__ I 1 5 ± 2 14-t-6 3 ± 1 Glutamate 38 ± 4 37 ± 9 53 ± 5 14 + 2 19 + 3 26 ± 7 Glycine 48-t-8 49_+ I 51 _+4 40_+3 47_+4 38_+ 11 Isoleucine 8 ± 9 68 _+ 1 66 ± 5 50 ± 6 59 ± 8 41 ± 9 Leucine 5 ± 10 52 ± 7 56_ 7 49 ± 11 48 ± 5 46 ± II Phenylalanine 29 _+ 17 62 ± 2 65 ± 1 38 ± 11 24 ± 6 36 ± 18 Proline 35 + 3 78 ± 2 87 _+ 1 74 ± 2 68 ± 6 39 ± 6 Tyrosine 25 ± 2 48 + 5 39 ± 7 46 + 7 45 + 5 27 ± 13 Valine 25 ± 7 49 ± 4 57 ± 2 39 ± 5 54 ± 5 18 ± 7

The total amino acid concentrations (a) are the amounts both in the tissue and released to the medium, derived from I g of tissue. All figures are the means (+ SD) of at least 4 separate experiments. KCN and glucose were added to the media at a concentration of 10 raM, sucrose at a concentration of 100 mM.

456 A. JABBAR and R. H. C. STRAXG

saline and by 67~, in the presence of KCN. Glu- tamate shows little change except to fall slightly (30%, P < 0.05) in the presence of KCN. In saline free of substrate or inhibitor proline falls in concentration by 67~o (P < 0.05) in the course of an hour's incubation at 37C. This fall is prevented equally by the presence of the inhibitor or glucose in the medium.

The addition of 100mM sucrose to make the medium iso-osmolar with insect haemolymph had no effect on the overall amounts of amino acids per unit weight of tissue.

As might be expected, incubation of the tissue at 0 C for 60min produced no change in the total amount of amino acids associated with the tissue.

Table 2b demonstrates that the efflux of amino acids to the medium varies with the conditions of incubation, often to a dramatic extent. The efflux at 0~C gives a basic level largely due to simple diffusion, uncomplicated by metabolism or active or facilitated diffusion mechanisms. Practically all the compounds measured diffused out of the tissue to about the same extent (between 25-45%), by the end of the period. The exceptions were isoleucine and leucine which showed a much lower efltux. These amino acids were in particularly low concentration in this experimental batch and in some of the four experiments whose results are averaged in the table, the amounts released to the medium were below the level of detectability. Individual experiments showed effluxes of between 17 201!;~, more in line with that of the other amino acids.

A rise in temperature and the addition of com- pounds to the medium affected the efflux of almost all the amino acids. The greatly increased proportion of alanine lost to the medium (P < 0.005) at 37"C was reduced by the presence of the KCN and eliminated by the presence of glucose. In contrast, the higher temperature allowed the almost complete retention of aspartate in the tissue. The presence of KCN greatly increased the efllux of this amino acid to the medium (P <0.001). Gaba eftlux at 37 C was also greatly affected by the metabolic poison (P < 0.0005), while the presence of glucose allowed greater retention in the tissue (P < 0.0005 in the case of Gaba and <0.05 in the case of aspartate). The same pattern (and statistical significance) was shown for glutamate. Most of the other amino acids show a greatly in- creased etftux at the higher temperature in a way that is not much affected by the presence of the KCN or glucose. Glycine shows no change in efflux under any of the experimental conditions used. There was no evidence that increasing the osmolarity with sucrose had any effect in preventing the efflux of amino acids. Only aspartate was more completely retained in the tissue, but the difference was not statistically significant.

Stimulated tissue. The most obvious effect of stim- ulation in terms of the amounts of amino acids is the three-fold (P <0.0005) increase in glutamate. A smaller increase took place in the concentration of aspartate. The seeming rise in the concentration of proline is not statistically significant in relation to the initial concentration, but is certainly much higher than that of the unstimulated control at the end of the incubation period. In the case of the other amino acids there has been a fall in concentration ranging

from 40-50% although the range of values under- mines the statistical significance of these JElls.

When the eitlux is considered, in virtually every case the effect of stimulation has been to diminish the fraction of the amino acid lost from the tissue, and coupled with the lower total concentrations, this means that a smaller actual amount of the amino acid has left the tissue. As before, there are exceptions to this general[sat[on, and aspartate is the most obvious (P < 0.01) and, to a lesser extent, glutamate (P < 0.2).

Time course o[ release ~?] amino acid~. Figure 1 shows the time course of efflux into saline of the amino acids investigated. In almost all cases the greater part of the efflux had taken place within the first 10 rain from the start of the incubation and atker 20 min a new equilibrium of concentrations had been established between the tissue and the medium. Pro- line was exceptional in showing an almost linear efflux for 30 min. Gaba was unique among the amino acids studied, in that there seemed to be an initial efflux followed by a rapid reuptake into the ganglia.

DISCUSSION

The relative simplicity and low degree of special- [sat[on in the insect nerve cord would argue rather fewer differences in specialised metabolism between the cerebral and thoracic ganglia than is the case in analogous mammalian tissues. If the simple concen- trations of the amino acids under consideration are any guide, this seems to be true. The only significant difference between cerebral and thoracic ganglia was the three-fold greater concentration of Gaba in the latter. This echoes the findings of Clarke and Don- nellan (1982) for the same insect, although these authors found the difference to be rather less than two-fold. As Gaba is probably uniquely formed in the nervous tissue and mainly in the CNS and powerfully retained within it, this difference probably reflects a true neurophysiological difference between the ganglia.

As has been remarked in the previous section, there is reasonable agreement with other authors who have examined the amino acids in the nervous tissues o[ the locust and cockroach. How confident can we be that these concentrations represent the true in ~it'o situation? The present authors cooled the insects into immobility, and then worked as much as possible at 0"C. From their published methods, most of the other authors quoted in Table 1 took no precautions to prevent post mortem metabolic changes in the tissue prior to homogenisation. Only Clarke and Donnellan (1982) took the extreme precaution of rapidly tYeez- ing the whole insect in liquid nitrogen. A conspicuous difference between the results of these last two authors and the rest is the high concentration of glutamate/glutamine and aspartate/asparagine which Clarke and Donnellan found. Although the method used by these authors could not distinguish between the amino acids and their corresponding amides, the re- markably consistent findings of the present authors (unpublished) ['or the locust, and o1" Ray (1964) and Osborne and Neuhoff (1974) for the cockroach, suggest that glutamine constitutes 30 40!~, of the total of amino acid plus am[de in the nervous systems of the insects under consideration. This would mean

Amino acids in the locust nervous system 457

80

70

60

50

40

30

20

10

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have been introduced simply to help the clarity of the figure, and have no other significance.

that the concentration of glutamate in the thoracic ganglia of the locust used by Clarke and Donnellan would be approximately 25 #mole/g. It is perhaps significant that the present authors found equally high concentrations (glutamate 24 and aspartate 11 #mole/g) when the nerve cord was stimulated in vitro. It is possible therefore that the true concen- trations of glutamate and aspartate in the active insect are higher than most of those reported.

Another sensitive indicator of the state of the nervous tissue is the concentration of alanine, which rises steeply in anaerobic conditions and even under the best conditions in vitro (Ray, 1964; Bradford et al., 1969; Strang, 1981). No significant differences in alanine concentration are apparent among the re- ports under discussion despite the fact that the au- thors [including the one reporting the lowest concen- tration of alanine (Ray, 1964)] took no particular precautions to prevent pos t m o r t e m changes. The question is complicated by the fact that most authors washed the tissue with saline before homogenising it. As shown in the Results section alanine diffuses readily out of the tissue, and this might be expected to have an effect on the apparent concentration. Nevertheless, the indications are that an average alanine concentration of between 7-8/~mol/g does represent the situation in the living insect. Proline is the other amino acid whose concentration in the nervous system varies widely between different re- ports. The consistency of the concentration ratios

with reference to the haemolymph, however, suggests that the nervous tissue responds passively to un- explained batch differences in the concentration of the compound in the haemolymph.

Most other amino acids studied show little net change in the course of incubation, and their averages are probably a good indication of concentrations in the living tissue. One which is particularly con- stant in all the nervous systems under discussion is Gaba. This probably reflects the fact that Gaba metabolism is so limited to the nervous system as to be unaffected by the metabolism of the rest of the organism.

Another aspect of amino acid occurrence which is remarkably similar in the two insect species and in mammals is the extent to which individual com- pounds have been concentrated in the nervous tissue. Alanine, aspartate, Gaba and glutamate all share this quality to some extent and have ratios well above unity. The rather low ratio for glutamate found by Clarke and Donnellan may be explained by the facts that (a) their method does not distinguish between glutamate and glutamine and (b) the ratio of glu- tamate to glutamine is greater than 1.0 in the nervous tissue, but lower than 1.0 in the haemolymph (present authors, unpublished; Schlesinger et al., 1977; Osborne and Neuhoff, 1974; Miller et al., 1973). Although the actual concentrations of amino acids in the mammalian nervous system are similar to those of the insect tissue, the much lower concentrations in

458 A. JABBAR and R. H. C. STRANG

the plasma of the former means that the ratio is much higher than is the case with the insects. This may suggest that the uptake processes in the mammal are more efficient than those in the insect. This is possibly exemplified by the finding that mammalian nervous tissue seems to possess two saturable uptake mech- anisms for glutamate with Km values of 1.5 x 10 ~ M and 3.6 × 10 ~M (Logan and Snyder, 1971), while cockroach nervous tissue has been reported as pos- sessing only one mechanism of intermediate affinity (Kin = 3.3 x 10 ~ M; Evans, 1978). It can of course be argued that the higher concentrations of amino acids characteristic of insect blood make high affinity up- take less necessary. One amino acid which is clearly different in the mammal and insect is glycine which is concentrated within the mammalian nervous tissue where it is thought to have a neurotransmitter action in the spinal column. The insect results provide no evidence for specific active uptake mechanisms such as are found in the mammal (Logan and Snyder, 1971), which would argue against glycine having any significant neural role. Tyrosine and phenylalanine are other amino acids for which there is evidence of specific active uptake in mammalian nervous tissue (Guroff et al., 1961), which is lacking in the insects under consideration.

Any discussion of the metabolism of the amino acids must be confined simply to net changes in concentration in vitro and so be rather superficial. The clearest effect of incubation, even under aerobic conditions, is the rapid accumulation of alanine. It is well known that in many intact invertebrates, includ- ing insects, alanine is the chief initial product of anaerobiosis (Stokes and Awapara, 1968; Felbeck and Grieshaber, 1980; Price, 1963). That this is also true of the isolated insect nervous system was shown by Ray (1964) for the cockroach nerve cord. The rate of accumulation found by Ray was much less than that reported here for the locust, although the pro- portions under different conditions are much the same. The fact that glucose in the medium greatly increases the accumulation of a[anine suggests that the amino acid is derived from pyruvate by trans- amination. (This is confirmed by the authors" un- published observation that the increase in alanine is prevented by the presence of amino oxyacetic acid, an inhibitor of transaminase enzymes.) Pyruvate has been shown in increase in vitro (Clement and Strang, 1978). If alanine accumulation does represent a re- sponse to anaerobiosis, then even constant bubbling of O2 through the medium does not compensate for the disruption of the tracheal system.

The decline in the concentration of proline in the absence of exogenous substrates prevented by the presence of KCN is consistent with a role for the amino acid as an aerobic substrate for the nervous tissue (Strang, 1981). The presence of glucose pre- venting this decline suggests inhibition of proline metabolism by the sugar or some of its immediate metabolites. Ray (1964) found the same utilization of proline in aerobic conditions, but did not find that it was prevented by glucose. This result, however was rather contradicted by a later report (Ray, 1965) that proline utilization only took place when glycolysis was blocked by iodoacetate. The evidence at the moment favours glucose oxidation taking precedence

over proline oxidation in the nervous systems of cockroach and locust.

Turning to the efflux of amino acids from the tissue into the artificial media under different conditions, a variety of effects is apparent. The degree of release which is more or less the same at 0 C shows extreme individual differences when the temperature is raised. The efflux of alanine for instance, has a clearly temperature-dependent component which is slightly (97,,,) but significantly reduced (P < 0.0005), in the presence of KCN. This suggests a small energy- requiring component in the efftux. The presence of glucose seems to reverse the efflux, but this is compli- cated by the fact of the much greater overall pro- duction of alanine in the presence of the mono- saccharide. This means that although the percentage of the total amino acid released to the medium is diminished, the actual amount/g of tissue released is almost the same as in the absence of glucose. Thus the apparently diminished efflux may be due to a rapid rate of production and a limiting rate of efflux, rather than active retention or uptake. The drawback to this argument is that the best in ~'it,o measurements always indicate a higher concentration of alanine in the tissue than in the haemolymph and there is no indication that there is a high rate of alanine pro- duction in the living insect which might account for this. On balance, it seems likely that in the insect nervous system, as in that of the mammal (Neame and Smith, 1965), the equilibrium for alanine is maintained by both facilitated efflux and influx, with possibly some active component in this process. Many of the other amino acids studied show the same temperature dependence on release which is relatively unaffected by KCN or glucose. This includes iso- leucine, leucine, proline, lyrosine and valine. This suggests that these amino acids are maintained in equilibrium by means of facilitated diffusion, with no active component to the process. Glycine seems to respond only to simple concentration gradients and diffusion processes.

In contrast to those amino acids which show increased efttux at high temperature, glutamate, as- partate and Gaba are more firmly retained within the tissue under these conditions. This degree of retention is diminished by KCN and increased by glucose suggesting that the retentive process (which probably represents a reuptake) is a strongly active process.

These results are consistent with previous reports on the uptake of amino acids into these insect tissues. Osborne and Neuhoff (1974) discovered that radio- labelled glutamate and Gaba were quite rapidly taken up into the cockroach nervous system in t'itro. Proline uptake was much more slow. This uptake was affected by metabolic inhibitors. The specific satur- able uptake of glutamate into cockroach nerve cord already mentioned (Evans, 19751 was also dimin- ished by KCN and iodoacetate, but by no means abolished.

Although there are clear differences in the extent of etllux of different amino acids, no amino acid leaves the tissue to such an extent that equality of concen- tration within and without the tissue is achieved. The elflux curves for all the amino acids show that an equilibrium has been achieved alter about 311 rain in the artificial medium. The maxim um amount of tissue

Amino acids in the locust nervous system 459

used in one experiment was 15 mg, containing about 12 #1 tissue fluid. Thus the volume inside the tissue is only about 3~o of that outside (500#1). It would therefore require the elttux of more than 97~ of the amino acid to achieve equal concentration in tissue and medium. Thus even in the case of the least retained amino acid (proline), there are processes which maintain a favourable gradient with reference to the nervous tissue.

Addition of sucrose to ensure that the osmolarity of the saline is the same as that of haemolymph had no effect either on the production or effiux of any of the amino acids examined. Despite suggestions that controlled production and diffusion of amino acids constitutes an important mechanism of osmolar regu- lation in insects (Berridge, 1970, Gilles and Schoffeniels, 1964), there is no evidence to support this with reference to the locust nervous system in vitro.

Elech'ical stimulation had a number of well defined effects when compared to the unstimulated control. The rise in concentration of glutamate and aspartate has already been commented on. A similar rise in the concentration of glutamate was found by Giacobini et al. (1966) in the stimulated crayfish nerve in vitro. It is interesting that in the present case the rise in the concentration of glutamate over the control (+16.9#mole/g) is almost equal and opposite to the fall in alanine ( -13 .5 #mole/g). Glutamate and alanine are linked by alanine aminotransferase which is present in the thoracic ganglia of locust and cockroach in high activity (Sugden and Newsholme, 1975; Jabbar, 1982) which almost certainly ensures that the precursors and products are constantly at equilibrium. A rise in glutamate and a fall in alanine must reflect and amplify an increase in the concen- tration of ~t-oxoglutarate and a fall in that of pyru- vate. This again would agree with the findings of Giacobini et al. (1966) with stimulated crayfish nerve.

An inexplicable result of stimulation is that the concentration of proline in stimulated tissue seems to rise, or at least not to fall. This is difficult to reconcile with the role of proline as an energy substrate for the tissue. Even if glucose is the preferred substrate and carbohydrate metabolism somehow inhibits the utili- zation of proline, this does not account for the difference between the stimulated and unstimulated tissue.

In general, however, stimulation has caused a net decline in the concentration of most individual amino acids and also in the proportions released. The only exceptions to this last point are aspartate and glu- tamate. This provides a contrast with mammalian tissue. Stimulation of mammalian tissue in situ and in various preparations including slices, synaptosomes and whole cervical ganglia, causes the release of a wide range of amino acids, and the effect is not limited to possible neurotransmitters (Katz et al., 1969; Hardy et al., 1980; McBride and Klingman, 1972).

The decline in concentration of many amino acids almost certainly merely represents a redistribution of amino groups, catalysed by various amino transfer- ases, as the total sum of average concentrations of amino acids is greater after stimulation than before. This increase in the free amino acid pool is similar to

that found by Gilles and Schoffeniels (1964) for the stimulated central nerve cord of the lobster. As quoted previously for the specific case of alanine, unpublished observations by the authors confirm that amino oxyacetic acid, had a general effect in pre- venting changes in the composition of the amino acid pool in the course of incubation. The results also suggest rather little utilisation of amino acids as a source of energy, despite the evidence in favour of proline.

The lower release of most amino acids to the medium in stimulated tissue suggests that the intact blood/brain barrier is operating in favour of the tissue under these circumstances. The stimulation experiments were carried out partly to re-examine the reports that stimulation of the isolated insect nervous system caused the release of neurotoxic compounds containing amino groups (Sternburg et al., 1959; Hawkins and Sternburg, 1964; Tashiro et al., 1972, 1975). It is difficult to know whether or not the present results contradict the previous ones. Only Tashiro identified released compounds as amino acids: leucine, isoleucine and tyrosine. These amino acids are certainly released from the locust nervous system, but stimulation did not increase this effect. Other authors simply concentrated on unknown toxic compounds and did not consider the release of identifiable amino acids.

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