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Comparative quantitative aspects of putative neurotransmitters in the central nervous system of spiders (Arachnida: Araneida)
Camp. Biochem. Physiol. Vol. in Great Britain
78C, No.
2. pp. 357-362.
0306-4492184
1984
‘( 1984 Pergamon
Printed
$3.00 + 0.00 Press Ltd
COMPARATIVE QUANTITATIVE ASPECTS OF PUTATIVE NEUROTRANSMITTERS IN THE CENTRAL NERVOUS SYSTEM OF SPIDERS (ARACHNIDA: ARANEIDA) WILFRIED MEYER*, CHRISTA SCHLESINGER*, HANS MICHAEL POEHLINGJT and WOLFGANG RUGE$, *Institut fiir Zoologie, Tiedrztliche Hochschule Hannover, Bischofsholer Damm 15. D-3000 Hannover 1, FRG, tInstitut fiir Pflanzenkrankheiten und Pflanzenschutz, Universitlt Hannover, FRG and fzentrum
Innere
Medizin
und Dermatologie,
Medizinische
Hochschule
Hannover,
FRG
(Received 3 January 1984) Abstract-l. The amounts of eight putative neurotransmitters or modulators (acetylchohne, dopamine, noradrenaline, adrenaline, GABA, taurine, glutamic acid, glycine) were determined from the CNS of 12 species of five different spider families (Theraphosidae, Agelenidae, Araneidae, Lycosidae, Salticidae). 2. Comparatively high contents of acetylcholine and noradrenaline could be found in the CNS of hunting spiders, higher contents of GABA and taurine were visible in the web-building spider families, while extraordinarily high amounts of glutamic acid were confined to the Theraphosidae. 3. The results obtained are compared with findings from other arthropod groups and the role of putative transmitters or modulators in the spider CNS is discussed in relation to behavioural differences within the families investigated.
INTRODUCTION The results of numerous investigations in the vertebrate phyla clearly suggest that the full spectrum of putative neurotransmitters, mostly known from the vertebrate animals, is already present very early in evolution (Walker, 1982). This is especially true of the Arthropoda, where acetylcholine, catecholamines and several amino acids have been identified as transmitters in the Insecta, Crustacea and Xiphosura during the last 20 years (see e.g. Gerschenfeld, 1973; Walker and Kerkut, 1978; Evans, 1980; Evered et al., 1982; Walker, 1982). Corresponding information on the Arachnida, especially the Araneida, has been only recently available (see e.g. Meyer and Idel, 1977; Meyer and Pospiech, 1977; Meyer and Jehnen, 1980; Meyer et al., 1980). These studies have demonstrated that the neurotransmitter pattern here generally agrees with that broadly confirmed for the other arthropod groups mentioned above. The observations from the Araneida helped to clarify the specific structural and functional features of their central nervous system in relation to the most important modes of orientation using tactile and/or visual perception, In this way, it was possible, for example, to emphasize the physiological significance of protocerebral brain structures which are connected with associative functions and the transmission and utilization of visual stimuli, particularly in hunting spiders (Meyer and Idel, 1977; Meyer and Pospiech, 1977; Meyer, 1979; Meyer and Jehnen, 1980). The present study was designed to provide initial evidence concerning the comparative quantitative distribution of important putative neurotransmitters or neuromodulators in the central nervous system of the Araneida. This can be of specific interest regarding the different phylogenetic standards of the spider 357
families investigated, and their somewhat different ways of orientation, as well as with respect to the interaction of the various transmitters or modulators involved. MATERIALS AND
METHODS
Spider species of the following five different families were used: Theraphosidae-Aphonopeima eutylenum (Chamberlin) (8Op/2SS), Aciculuria sp. (6/l); Agelenidae-Tegenaricr derhami (Scopoli) (28/10), Tegenariu utrica (C. L. Koch) (10/6); Araneidae-Araneus sclopetarius (Clerck) (1713) Araneus diadematus (Clerck) (613) Araneus marmoreus (Clerck) (8/3), Zygiellu x-notata (Clerck) (I 5/6); Lycosidae-Pardosa amentata (Clerck) (45/10), Trochosa spinipalpis (Cambridge) (S/l); Salticidae--Murpissa muscosa (Clerck) (38/g), Sitticusfloricolu (C. L. Koch) (9/6). In order to avoid influences of diurnal variations, the whole central nervous system of adult animals was always removed at the same time (10.00 a.m.) in the cold from the cephalothorax, washed twice in buffered saline and immediately prepared according to the biochemical procedures noted below or stored in liquid nitrogen until further processing. (a) Aceiylcholine, ferase
acetylcholinesterase,
choline acery1tran.c-
Acetylcholine was extracted by acetoneeformic acid (15”; 1 N formic acid/85% acetone) after Toru and Aprison (1966) and assayed on a leech dorsal muscle in a microbath (Szerb, 1961; Dudar and Szerb, 1970). Acetylcholinesterase was determined photometrically according to Ellman et al. (1961) with a Zeiss PMQ 3 (substrate: acetylthiocholine iodide, Sigma). Activities of nonspecific cholinesterases were controlled by media containing butyrylthiocholine iodide (Sigma) as substrate and by addition of iso-OMPA (0. I mM) (Serva). Acetylcholinesterase activity was additionally inhibited by BW 284 c 51 (I mM) (Wellcome). Choline acetyltransferase was determined radiochemically
WILFKIEV MEYER rt al.
358
Table I. Comparattve
weight aspects of brain, body and protein content (+_SEM)
Theraphosidae Brain wt (W) Body w! (ms) Brain wt Body wt (”,,) Brain protein Brain wt
10.530 (k2.280) I I.078 (k 1163.9) 0.09
Agelenidae
Araneidae
Lycosidae
0.767 (kO.157) 199.95 (245.383
0.620 (kO.157) 120.6X
0.920 (kO.190) 36.99 (k4.74)
0.38
0.51
2.53
5.20
5.170
5.025
4.145
3.995
according to Font-rum (1975) using [‘4C]acetyl-CoA in a final concentration of 0.4 mM. Ten ~1 of each homogenate and incubation medium were incubated for 30 min at 30’ C. The reaction was stopped by adding 2.5 ml ice-cooled phosphate buffer (10 mM, pH 7.4) followed by I ml acetonitrile containing 5 mg sodium tetraphenylborate (Sigma) and 3.5 ml scintillator (Quickszint, Zinsser). Radioactivity was counted using a Packard Prias PLD scintillator counter.
( i X.00)
ted against 1980).
the amounts
Salticidae 0.538
( + 0.024) 10.74 (k2.19)
of the amino acids (Poehling
et ~11..
(d) Protein determinations Protein determinations for (a) and (b) were carried out after Lowry et al. (1951). For the amino acid study protein content was determined after Neuhoff et al. (1979), i.e. the proteins were stained with Hoechst 2495 and evaluated by fluorometry, using bovine serum albumin as standard.
(b) Cutecholamines Dopamine, noradrenaline and adrenaline were determined radiochemically according to Passon and Peuler (1973) and Peuler and Johnson (1975) using the catecholamines radioenzymatic assay kit [3H] of Upjohn Diagnostics (USA). The tissue was homogenized in cold 0.4N perchloric acid containing 5 mM reduced glutathione and then centrifuged to produce a “protein-free” supernatant which was diluted 1:20 for further processing. The three catecholamines were simultaneously converted to their corresponding [‘Hlmethoxy-derivatives by the catalytic action of a partially purified preparation of COMT in the presence of [3H]SAM. Each labeled derivative was isolated by thin layer chromatography, converted by periodate oxidation to [‘H]vanillin and extracted. The radioactivity attributable to each catecholamine was determined by scintillation counting. (c) Amino
acids
For the determination of four amino acids (GABA, taurine, glutamic acid, glycine) the nervous tissue was transferred into 10 ~1 capillaries (Brand, Wertheim) or Pyrex glass tubes (+ 1.5 mm) sealed at one end. One mg tissue was homogenized in 20~1 0.05 M K,CO,. pH 9.0, using a dentist’s drill for capillaries and Teflon pestles for Pyrex tubes at 24,000 rpm, ice-cooled (Neuhoff, 1973). After centrifugation at 2000 rpm for 15 min at 4’C the supernatant was analysed for free amino acids. The amino acids were separated on 3 x 4cm micropolyamid sheets (A 1700 Schleicher and Schiill, Dassel FRG) as dansyl derivatives (1 -dimethylamino naphthalene-5-sulphonyl-chloride. Serva) as described by Meyer et al. (1980). The microchromatograms were evaluated by fluorescence scanning (Kronberg et al., 1978; Zimmer and Neuhoff. 1977). To obtain calibration curves for the quantitative analysis of GABA, taurine, glutamic acid and glycine, dilution series of these compounds were dansylated, separated as described above and the integrated fluorescence intensities were plot-
Table 2. Quantitattve Acetylcholine (nmol/mg protein) Acetylcholinesterase (nmol/mg protein mitt-‘) Choline acetyltransferase (nmol/mg protein nnn ‘)
RESULTS AND DISCUSSION
The results obtained on the comparative abundance of putative transmitters in the central nervous system of spiders are summarized in Tables 1-3 and Fig. I. The observed differences comparing the five spider families investigated are obviously related to the development of brain size and the somewhat contrasting habits of the animals. Spiders which rely mainly upon visual orientation and which can be characterized as wandering or hunting spiders (Lycosidae, Salticidae) clearly show definitely higher relative brain weights than the other families which predominantly use their tactile abilities for normal life (Table 1). This difference is based on the enormous increase of the optic ganglia in the protocerebrum, especially in the Salticidae, and a reduction in size of the optic masses and corpora pedunculata in the web-building spider families (e.g. Agelenidae, Araneidae) (Meyer and Idel, 1977; Meyer and Jehnen, 1980). In connection with these structural aspects, the relatively high amounts of acetylcholine in the brain of the hunting spider families (Tables 2, 3; Fig. 1) confirm the view that this substance can be considered the most important excitatory transmitter in sensory brain systems. This view correlates with histochemical observations in optic ganglia in these spider groups (Meyer and Idel, 1977; Meyer and Pospiech, 1977) and additionally with results obtained with histochemical and biochemical methods for the acetylcholine system in the central nervous system of the other great arthropod groups (see e.g. Florey, 1967; Mandelshtam, 1973; Callec, 1974;
aspects of the acetylcholine system in the CNS of spiders (k SEM) Theraphosidae
Agelenidae
Araneidae
Lycosidae
Salttcidae
0.599
I.004 (kO.032) 100.52 (k 2.959) 51.36 (i 1.978)
I.188 (kO.174) 247.87 (k65.815) 56.65 (k 13.256)
1.801 ( f 0.078) 278.04 (i22.51) 99.59 (i 15.088)
2.395 (iO.271) 107.81
(I 0.134) 203.10 (k 12.627) 20.43 (f0.589)
(*0.02) 51.99 (k 1.382)
Putative Table 3. Comparative
distribution
of putative
(&ol/mg
0.599 (kO.134) 17.2 (kO.25) 0.686 (kO.283) 20.01 (k2.55) 24.90 (k2.86) 42.90
protein)
Noradrenaline (pmol/mg protein) Adrenaline (pmol/mg protein) Dopamine (pmol/mg protein) GABA (nmol/mg protein) Taurine (nmol/mg protein) Glutamic acid (nmoljmg protein) Glycine (nmol/mg protein)
359
transmitters in the CNS of spiders in relation to brain protein content (+ SEM)
Theraphosidae Acetvlcholine
in CNS of spiders
transmitters
(k2.68)
100.83 (k42.25) 19.37 (+ 1.89)
Agelenidae
Araneidae
Lycosidae
Salticidae
1.004 ( f 0.032) 73.6 (i23.6) 1.85 (kO.403) 46.13 (k8.96) 101.86 (k 14.38) 194.33 (f 77.37)
1.188 (kO.174)
I.801 (kO.078)
2.395 (f0.271) 174.9 (k5.0) 3.487 (kO.454)
79.6
(kl4.1) 3.918 (kO.108) 82.38 (fl1.81) 98.95 (f7.36) 154.60 (f37.96)
40.67
35.19
24.50) 22.80 (kll.03)
(k 2.79)
(f
Hildenbrand et ul., 1974; Sugden and Newsholme, 1977). Catecholamines are widely distributed in the animal kingdom (Walker and Kerkut, 1978). Among the compounds concerned, dopamine is probably the most dominant catecholamine in invertebrates. It has been shown to exist in insects (e.g. Klemm, 1976; Evans, 1980), crustaceans (e.g. Elofsson et al., 1982) and also in arachnids (Araneida: see present study
45.64 (k9.61)
131.6
(+21.1) 4.33 (f0.167) 90.75 ( f 20.04) 89.98 (i 11.73) 86.13 (k 17.41) 72.0 (f16.38) 32.80 (i 14.04)
86.00
(k1.15) 27.33 (f0.762)
61.88 15.11) 35.61 (+ 12.76) 12.45 (i 8.93) (i
and Meyer and Jehnen, 1980; Atari: Megaw and Robertson, 1974; Binnington and Stone, 1977). In insects, crustaceans and xiphosurans the level of dopamine is higher than that of noradrenaline (Hiripi and S.-Rozsa, 1973; Elofsson et al., 1982; Roberts et al., 1983). The results of this study on the Araneida exhibit different aspects. The amounts of both substances are quite the same in Theraphosidae, Agelenidae and Araneidae, while the noradrenaline con-
6
ACh
0,lO
GABA
0.06
1 4
B
0.06 0.04 0.02
d
0i
TAU
4
2
0
-8
a-6
NA
d
t.-
4-
a-
o-
A 092 0.1 0 1
4
-2
-0
A
GLUT
1 1
-0
-6
dJIh
-4
-2
DA
-0
GLY
2
0 I
-4
D&L5 2
&II TAoAn
5
TAoAn
t 0
5
Fig. 1. Comparative distribution of putative neurotransmitters in the central nervous system of spiders. All data are given in nmol/mg wet wt, except for acetylcholine and adrenaline which are given in pmol/mg wet wt. Abbreviations: ACh-acetylcholine, NA-noradrenaline, A-adrenaline, DAAopamine, GABA--y-aminobutyric acid, TAU-taurine, GLUT-glutamic acid, GLY-glycine; T-Theraphosidae, AG-Agelenidae, AR-Araneidae, L-Lycosidae, S-Salticidae.
360
WILFRIED MEYER et al
tent of the central nervous system exceeds that of dopamine in hunting spiders (Lycosidae, Salticidae; Table 3, Fig. 1). The latter group, as mentioned before, is equipped with well developed optical brain centres reacting positively for biogenic monoamines (Meyer and Jehnen, 1980). Thus, the results of this study suggest a difference in the potential importance of noradrenaline in different groups of arthropods. In contrast to the situation in insects, the levels of both amines appear to be closer in crustaceans and xiphosurans. In arachnids, as not only demonstrated by the present study in the Araneida, but also in the Atari by Megaw and Robertson (1974) and Stone et al. (1978) noradrenaline seems to play a major physiological role as either a transmitter or as a neurohormone. Amino acids constitute another distinctive class of neurotransmitters and/or neuromodulators in both the vertebrate as well as in the invertebrate central nervous system (de Feudis and Mandel, 1981; Walker, 1982; Fagg and Foster, 1983). In this substance group, GABA could be nominated as the major neurotransmitter and as demonstrated for vertebrates its function in insects was found to be exclusively inhibitory (Pitman, 1971; Roberts et ul., 1976). In the Araneida, as already assumed previously (Meyer et al., 1980) GABA, together with taurine, is the dominant amino acid in the most highly developed spider families. In terms of levels, it is interesting to note that the web-building spiders (Agelenidae, Araneidae) show the largest amounts of these compounds (Table 3, Fig. 1). Contrary to these findings, glutamic acid is, by far, the most important amino acid in the orthognath family investigated (Theraphosidae) (Fig. 1). Glycine, as in the mammalian central nervous system (Fagg and Foster, 1983; de Feudis and Mandel, 1981) obviously serves a more limited role as an inhibitory transmitter in spiders. Previous papers have indicated the modulatory functions of taurine in a variety of excitable tissues (Huxtable and Barbeau, 1976). In particular, Kuriyama et al. (1978) demonstrated that taurine significantly decreased the depolarization-induced release of labeled noradrenaline and acetylcholine from various rat tissues. On the basis of these effects of taurine, it has been suggested that this amino acid might have dual functions, both as a neurotransmitter and a neuromodulator (Kuriyama et al., 1978). Lately, it has been demonstrated that taurine also exerts modulatory effects on the release of other amino acid transmitters (GABA, glutamate) (Namima er al., 1983; Van Gelder, 1981). In spiders, as could be observed in this study, there is a clear quantitative relation between the amounts of taurine and those of noradrenaline, acetylcholine and glutamate (Fig. 1). Agelenidae and Araneidae, as families with mainly vibratory abilities, exhibit higher contents of taurine and GABA in their central nervous system and lower ones of acetylcholine and noradrenaline. In the spider families with more visual orientation this relation is inverse. These results and others obtained on the relative distribution of free amino acids in the spider brain (Meyer et al., 1980) indicate in this arthropod group both subgroup related, as well as, behaviourally based modulatory
effects of taurine. In particular, the latter influences have likewise been demonstrated in mammals (Rassin, 1981; Van Gelder, 1981). At the moment, evidence is clearly in favour of L-glutamate as a neurotransmitter (Walker, 1982). For vertebrates, particularly mammals, many have argued that this substance, together with aspartic acid, is one of the major excitatory transmitters in the central nervous system (Cotman et al., 1981). This may be substantiated for the arthropods by the distinctive relative abundance of glutamate and aspartate in spiders (Meyer et al., 1980). The substantial absolute amounts of glutamic acid occurring in the orthognath spider family Theraphosidae (Fig. l), in contrast to the low values measured for the labidognath families, seem astonishing, nevertheless. With regard to the possible link between putative acidic amino acid transmitters and specific behaviours (Cotman et al., 1981; de Feudis and Mandel, 1981) this difference may be associated with the basically different neurolocomotory patterns of the Theraphosidae compared to the other families investigated in this study (Wilson, 1967; Bowerman, 1977; Ward and Humphreys, 1981). The overall distribution of putative neurotransmitters and neuromodulators in the central nervous system of spiders shows clear parallels to other arthropod groups and other invertebrate and vertebrate animals. Several differences are visible where the absolute and relative amounts of these compounds in the Arthropoda as a whole, as well as the comparison of different spider families with each other are concerned. It seems possible that certain excitatory and inhibitory neurotransmitters, and neuromodulators, respectively, might act as substitutes for other ones within the same or a related functional range (acetylcholine-glutamic acid, web-building and hunting spiders vs Theraphosidae; GABA and taurinedatecholamines, in all families). However, it should be pointed out that the actual functions of these compounds, particularly in view of their interactions, is still widely speculative. Zoologically based i.e. further differentiated research investigations, based on phylogenetic aspects, would be very helpful in elucidating some of the problems discussed above (see e.g. Florey, 1967; Klemm, 1976; Evans, 1980; Walker, 1982). Acknowledgements-The skilful technical assistance of Miss Kristina Jeitner and Mrs Ilse Pfaffe is gratefully acknowledged. We thank Prof. Dr V. Neuhoff (MPI f. exp. Medizin, Forschungsstelle Neurochemie. Gettingen) for providing the scanning and evaluation facilities for the microchromatograms.
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78C, No.
2. pp. 357-362.
0306-4492184
1984
‘( 1984 Pergamon
Printed
$3.00 + 0.00 Press Ltd
COMPARATIVE QUANTITATIVE ASPECTS OF PUTATIVE NEUROTRANSMITTERS IN THE CENTRAL NERVOUS SYSTEM OF SPIDERS (ARACHNIDA: ARANEIDA) WILFRIED MEYER*, CHRISTA SCHLESINGER*, HANS MICHAEL POEHLINGJT and WOLFGANG RUGE$, *Institut fiir Zoologie, Tiedrztliche Hochschule Hannover, Bischofsholer Damm 15. D-3000 Hannover 1, FRG, tInstitut fiir Pflanzenkrankheiten und Pflanzenschutz, Universitlt Hannover, FRG and fzentrum
Innere
Medizin
und Dermatologie,
Medizinische
Hochschule
Hannover,
FRG
(Received 3 January 1984) Abstract-l. The amounts of eight putative neurotransmitters or modulators (acetylchohne, dopamine, noradrenaline, adrenaline, GABA, taurine, glutamic acid, glycine) were determined from the CNS of 12 species of five different spider families (Theraphosidae, Agelenidae, Araneidae, Lycosidae, Salticidae). 2. Comparatively high contents of acetylcholine and noradrenaline could be found in the CNS of hunting spiders, higher contents of GABA and taurine were visible in the web-building spider families, while extraordinarily high amounts of glutamic acid were confined to the Theraphosidae. 3. The results obtained are compared with findings from other arthropod groups and the role of putative transmitters or modulators in the spider CNS is discussed in relation to behavioural differences within the families investigated.
INTRODUCTION The results of numerous investigations in the vertebrate phyla clearly suggest that the full spectrum of putative neurotransmitters, mostly known from the vertebrate animals, is already present very early in evolution (Walker, 1982). This is especially true of the Arthropoda, where acetylcholine, catecholamines and several amino acids have been identified as transmitters in the Insecta, Crustacea and Xiphosura during the last 20 years (see e.g. Gerschenfeld, 1973; Walker and Kerkut, 1978; Evans, 1980; Evered et al., 1982; Walker, 1982). Corresponding information on the Arachnida, especially the Araneida, has been only recently available (see e.g. Meyer and Idel, 1977; Meyer and Pospiech, 1977; Meyer and Jehnen, 1980; Meyer et al., 1980). These studies have demonstrated that the neurotransmitter pattern here generally agrees with that broadly confirmed for the other arthropod groups mentioned above. The observations from the Araneida helped to clarify the specific structural and functional features of their central nervous system in relation to the most important modes of orientation using tactile and/or visual perception, In this way, it was possible, for example, to emphasize the physiological significance of protocerebral brain structures which are connected with associative functions and the transmission and utilization of visual stimuli, particularly in hunting spiders (Meyer and Idel, 1977; Meyer and Pospiech, 1977; Meyer, 1979; Meyer and Jehnen, 1980). The present study was designed to provide initial evidence concerning the comparative quantitative distribution of important putative neurotransmitters or neuromodulators in the central nervous system of the Araneida. This can be of specific interest regarding the different phylogenetic standards of the spider 357
families investigated, and their somewhat different ways of orientation, as well as with respect to the interaction of the various transmitters or modulators involved. MATERIALS AND
METHODS
Spider species of the following five different families were used: Theraphosidae-Aphonopeima eutylenum (Chamberlin) (8Op/2SS), Aciculuria sp. (6/l); Agelenidae-Tegenaricr derhami (Scopoli) (28/10), Tegenariu utrica (C. L. Koch) (10/6); Araneidae-Araneus sclopetarius (Clerck) (1713) Araneus diadematus (Clerck) (613) Araneus marmoreus (Clerck) (8/3), Zygiellu x-notata (Clerck) (I 5/6); Lycosidae-Pardosa amentata (Clerck) (45/10), Trochosa spinipalpis (Cambridge) (S/l); Salticidae--Murpissa muscosa (Clerck) (38/g), Sitticusfloricolu (C. L. Koch) (9/6). In order to avoid influences of diurnal variations, the whole central nervous system of adult animals was always removed at the same time (10.00 a.m.) in the cold from the cephalothorax, washed twice in buffered saline and immediately prepared according to the biochemical procedures noted below or stored in liquid nitrogen until further processing. (a) Aceiylcholine, ferase
acetylcholinesterase,
choline acery1tran.c-
Acetylcholine was extracted by acetoneeformic acid (15”; 1 N formic acid/85% acetone) after Toru and Aprison (1966) and assayed on a leech dorsal muscle in a microbath (Szerb, 1961; Dudar and Szerb, 1970). Acetylcholinesterase was determined photometrically according to Ellman et al. (1961) with a Zeiss PMQ 3 (substrate: acetylthiocholine iodide, Sigma). Activities of nonspecific cholinesterases were controlled by media containing butyrylthiocholine iodide (Sigma) as substrate and by addition of iso-OMPA (0. I mM) (Serva). Acetylcholinesterase activity was additionally inhibited by BW 284 c 51 (I mM) (Wellcome). Choline acetyltransferase was determined radiochemically
WILFKIEV MEYER rt al.
358
Table I. Comparattve
weight aspects of brain, body and protein content (+_SEM)
Theraphosidae Brain wt (W) Body w! (ms) Brain wt Body wt (”,,) Brain protein Brain wt
10.530 (k2.280) I I.078 (k 1163.9) 0.09
Agelenidae
Araneidae
Lycosidae
0.767 (kO.157) 199.95 (245.383
0.620 (kO.157) 120.6X
0.920 (kO.190) 36.99 (k4.74)
0.38
0.51
2.53
5.20
5.170
5.025
4.145
3.995
according to Font-rum (1975) using [‘4C]acetyl-CoA in a final concentration of 0.4 mM. Ten ~1 of each homogenate and incubation medium were incubated for 30 min at 30’ C. The reaction was stopped by adding 2.5 ml ice-cooled phosphate buffer (10 mM, pH 7.4) followed by I ml acetonitrile containing 5 mg sodium tetraphenylborate (Sigma) and 3.5 ml scintillator (Quickszint, Zinsser). Radioactivity was counted using a Packard Prias PLD scintillator counter.
( i X.00)
ted against 1980).
the amounts
Salticidae 0.538
( + 0.024) 10.74 (k2.19)
of the amino acids (Poehling
et ~11..
(d) Protein determinations Protein determinations for (a) and (b) were carried out after Lowry et al. (1951). For the amino acid study protein content was determined after Neuhoff et al. (1979), i.e. the proteins were stained with Hoechst 2495 and evaluated by fluorometry, using bovine serum albumin as standard.
(b) Cutecholamines Dopamine, noradrenaline and adrenaline were determined radiochemically according to Passon and Peuler (1973) and Peuler and Johnson (1975) using the catecholamines radioenzymatic assay kit [3H] of Upjohn Diagnostics (USA). The tissue was homogenized in cold 0.4N perchloric acid containing 5 mM reduced glutathione and then centrifuged to produce a “protein-free” supernatant which was diluted 1:20 for further processing. The three catecholamines were simultaneously converted to their corresponding [‘Hlmethoxy-derivatives by the catalytic action of a partially purified preparation of COMT in the presence of [3H]SAM. Each labeled derivative was isolated by thin layer chromatography, converted by periodate oxidation to [‘H]vanillin and extracted. The radioactivity attributable to each catecholamine was determined by scintillation counting. (c) Amino
acids
For the determination of four amino acids (GABA, taurine, glutamic acid, glycine) the nervous tissue was transferred into 10 ~1 capillaries (Brand, Wertheim) or Pyrex glass tubes (+ 1.5 mm) sealed at one end. One mg tissue was homogenized in 20~1 0.05 M K,CO,. pH 9.0, using a dentist’s drill for capillaries and Teflon pestles for Pyrex tubes at 24,000 rpm, ice-cooled (Neuhoff, 1973). After centrifugation at 2000 rpm for 15 min at 4’C the supernatant was analysed for free amino acids. The amino acids were separated on 3 x 4cm micropolyamid sheets (A 1700 Schleicher and Schiill, Dassel FRG) as dansyl derivatives (1 -dimethylamino naphthalene-5-sulphonyl-chloride. Serva) as described by Meyer et al. (1980). The microchromatograms were evaluated by fluorescence scanning (Kronberg et al., 1978; Zimmer and Neuhoff. 1977). To obtain calibration curves for the quantitative analysis of GABA, taurine, glutamic acid and glycine, dilution series of these compounds were dansylated, separated as described above and the integrated fluorescence intensities were plot-
Table 2. Quantitattve Acetylcholine (nmol/mg protein) Acetylcholinesterase (nmol/mg protein mitt-‘) Choline acetyltransferase (nmol/mg protein nnn ‘)
RESULTS AND DISCUSSION
The results obtained on the comparative abundance of putative transmitters in the central nervous system of spiders are summarized in Tables 1-3 and Fig. I. The observed differences comparing the five spider families investigated are obviously related to the development of brain size and the somewhat contrasting habits of the animals. Spiders which rely mainly upon visual orientation and which can be characterized as wandering or hunting spiders (Lycosidae, Salticidae) clearly show definitely higher relative brain weights than the other families which predominantly use their tactile abilities for normal life (Table 1). This difference is based on the enormous increase of the optic ganglia in the protocerebrum, especially in the Salticidae, and a reduction in size of the optic masses and corpora pedunculata in the web-building spider families (e.g. Agelenidae, Araneidae) (Meyer and Idel, 1977; Meyer and Jehnen, 1980). In connection with these structural aspects, the relatively high amounts of acetylcholine in the brain of the hunting spider families (Tables 2, 3; Fig. 1) confirm the view that this substance can be considered the most important excitatory transmitter in sensory brain systems. This view correlates with histochemical observations in optic ganglia in these spider groups (Meyer and Idel, 1977; Meyer and Pospiech, 1977) and additionally with results obtained with histochemical and biochemical methods for the acetylcholine system in the central nervous system of the other great arthropod groups (see e.g. Florey, 1967; Mandelshtam, 1973; Callec, 1974;
aspects of the acetylcholine system in the CNS of spiders (k SEM) Theraphosidae
Agelenidae
Araneidae
Lycosidae
Salttcidae
0.599
I.004 (kO.032) 100.52 (k 2.959) 51.36 (i 1.978)
I.188 (kO.174) 247.87 (k65.815) 56.65 (k 13.256)
1.801 ( f 0.078) 278.04 (i22.51) 99.59 (i 15.088)
2.395 (iO.271) 107.81
(I 0.134) 203.10 (k 12.627) 20.43 (f0.589)
(*0.02) 51.99 (k 1.382)
Putative Table 3. Comparative
distribution
of putative
(&ol/mg
0.599 (kO.134) 17.2 (kO.25) 0.686 (kO.283) 20.01 (k2.55) 24.90 (k2.86) 42.90
protein)
Noradrenaline (pmol/mg protein) Adrenaline (pmol/mg protein) Dopamine (pmol/mg protein) GABA (nmol/mg protein) Taurine (nmol/mg protein) Glutamic acid (nmoljmg protein) Glycine (nmol/mg protein)
359
transmitters in the CNS of spiders in relation to brain protein content (+ SEM)
Theraphosidae Acetvlcholine
in CNS of spiders
transmitters
(k2.68)
100.83 (k42.25) 19.37 (+ 1.89)
Agelenidae
Araneidae
Lycosidae
Salticidae
1.004 ( f 0.032) 73.6 (i23.6) 1.85 (kO.403) 46.13 (k8.96) 101.86 (k 14.38) 194.33 (f 77.37)
1.188 (kO.174)
I.801 (kO.078)
2.395 (f0.271) 174.9 (k5.0) 3.487 (kO.454)
79.6
(kl4.1) 3.918 (kO.108) 82.38 (fl1.81) 98.95 (f7.36) 154.60 (f37.96)
40.67
35.19
24.50) 22.80 (kll.03)
(k 2.79)
(f
Hildenbrand et ul., 1974; Sugden and Newsholme, 1977). Catecholamines are widely distributed in the animal kingdom (Walker and Kerkut, 1978). Among the compounds concerned, dopamine is probably the most dominant catecholamine in invertebrates. It has been shown to exist in insects (e.g. Klemm, 1976; Evans, 1980), crustaceans (e.g. Elofsson et al., 1982) and also in arachnids (Araneida: see present study
45.64 (k9.61)
131.6
(+21.1) 4.33 (f0.167) 90.75 ( f 20.04) 89.98 (i 11.73) 86.13 (k 17.41) 72.0 (f16.38) 32.80 (i 14.04)
86.00
(k1.15) 27.33 (f0.762)
61.88 15.11) 35.61 (+ 12.76) 12.45 (i 8.93) (i
and Meyer and Jehnen, 1980; Atari: Megaw and Robertson, 1974; Binnington and Stone, 1977). In insects, crustaceans and xiphosurans the level of dopamine is higher than that of noradrenaline (Hiripi and S.-Rozsa, 1973; Elofsson et al., 1982; Roberts et al., 1983). The results of this study on the Araneida exhibit different aspects. The amounts of both substances are quite the same in Theraphosidae, Agelenidae and Araneidae, while the noradrenaline con-
6
ACh
0,lO
GABA
0.06
1 4
B
0.06 0.04 0.02
d
0i
TAU
4
2
0
-8
a-6
NA
d
t.-
4-
a-
o-
A 092 0.1 0 1
4
-2
-0
A
GLUT
1 1
-0
-6
dJIh
-4
-2
DA
-0
GLY
2
0 I
-4
D&L5 2
&II TAoAn
5
TAoAn
t 0
5
Fig. 1. Comparative distribution of putative neurotransmitters in the central nervous system of spiders. All data are given in nmol/mg wet wt, except for acetylcholine and adrenaline which are given in pmol/mg wet wt. Abbreviations: ACh-acetylcholine, NA-noradrenaline, A-adrenaline, DAAopamine, GABA--y-aminobutyric acid, TAU-taurine, GLUT-glutamic acid, GLY-glycine; T-Theraphosidae, AG-Agelenidae, AR-Araneidae, L-Lycosidae, S-Salticidae.
360
WILFRIED MEYER et al
tent of the central nervous system exceeds that of dopamine in hunting spiders (Lycosidae, Salticidae; Table 3, Fig. 1). The latter group, as mentioned before, is equipped with well developed optical brain centres reacting positively for biogenic monoamines (Meyer and Jehnen, 1980). Thus, the results of this study suggest a difference in the potential importance of noradrenaline in different groups of arthropods. In contrast to the situation in insects, the levels of both amines appear to be closer in crustaceans and xiphosurans. In arachnids, as not only demonstrated by the present study in the Araneida, but also in the Atari by Megaw and Robertson (1974) and Stone et al. (1978) noradrenaline seems to play a major physiological role as either a transmitter or as a neurohormone. Amino acids constitute another distinctive class of neurotransmitters and/or neuromodulators in both the vertebrate as well as in the invertebrate central nervous system (de Feudis and Mandel, 1981; Walker, 1982; Fagg and Foster, 1983). In this substance group, GABA could be nominated as the major neurotransmitter and as demonstrated for vertebrates its function in insects was found to be exclusively inhibitory (Pitman, 1971; Roberts et ul., 1976). In the Araneida, as already assumed previously (Meyer et al., 1980) GABA, together with taurine, is the dominant amino acid in the most highly developed spider families. In terms of levels, it is interesting to note that the web-building spiders (Agelenidae, Araneidae) show the largest amounts of these compounds (Table 3, Fig. 1). Contrary to these findings, glutamic acid is, by far, the most important amino acid in the orthognath family investigated (Theraphosidae) (Fig. 1). Glycine, as in the mammalian central nervous system (Fagg and Foster, 1983; de Feudis and Mandel, 1981) obviously serves a more limited role as an inhibitory transmitter in spiders. Previous papers have indicated the modulatory functions of taurine in a variety of excitable tissues (Huxtable and Barbeau, 1976). In particular, Kuriyama et al. (1978) demonstrated that taurine significantly decreased the depolarization-induced release of labeled noradrenaline and acetylcholine from various rat tissues. On the basis of these effects of taurine, it has been suggested that this amino acid might have dual functions, both as a neurotransmitter and a neuromodulator (Kuriyama et al., 1978). Lately, it has been demonstrated that taurine also exerts modulatory effects on the release of other amino acid transmitters (GABA, glutamate) (Namima er al., 1983; Van Gelder, 1981). In spiders, as could be observed in this study, there is a clear quantitative relation between the amounts of taurine and those of noradrenaline, acetylcholine and glutamate (Fig. 1). Agelenidae and Araneidae, as families with mainly vibratory abilities, exhibit higher contents of taurine and GABA in their central nervous system and lower ones of acetylcholine and noradrenaline. In the spider families with more visual orientation this relation is inverse. These results and others obtained on the relative distribution of free amino acids in the spider brain (Meyer et al., 1980) indicate in this arthropod group both subgroup related, as well as, behaviourally based modulatory
effects of taurine. In particular, the latter influences have likewise been demonstrated in mammals (Rassin, 1981; Van Gelder, 1981). At the moment, evidence is clearly in favour of L-glutamate as a neurotransmitter (Walker, 1982). For vertebrates, particularly mammals, many have argued that this substance, together with aspartic acid, is one of the major excitatory transmitters in the central nervous system (Cotman et al., 1981). This may be substantiated for the arthropods by the distinctive relative abundance of glutamate and aspartate in spiders (Meyer et al., 1980). The substantial absolute amounts of glutamic acid occurring in the orthognath spider family Theraphosidae (Fig. l), in contrast to the low values measured for the labidognath families, seem astonishing, nevertheless. With regard to the possible link between putative acidic amino acid transmitters and specific behaviours (Cotman et al., 1981; de Feudis and Mandel, 1981) this difference may be associated with the basically different neurolocomotory patterns of the Theraphosidae compared to the other families investigated in this study (Wilson, 1967; Bowerman, 1977; Ward and Humphreys, 1981). The overall distribution of putative neurotransmitters and neuromodulators in the central nervous system of spiders shows clear parallels to other arthropod groups and other invertebrate and vertebrate animals. Several differences are visible where the absolute and relative amounts of these compounds in the Arthropoda as a whole, as well as the comparison of different spider families with each other are concerned. It seems possible that certain excitatory and inhibitory neurotransmitters, and neuromodulators, respectively, might act as substitutes for other ones within the same or a related functional range (acetylcholine-glutamic acid, web-building and hunting spiders vs Theraphosidae; GABA and taurinedatecholamines, in all families). However, it should be pointed out that the actual functions of these compounds, particularly in view of their interactions, is still widely speculative. Zoologically based i.e. further differentiated research investigations, based on phylogenetic aspects, would be very helpful in elucidating some of the problems discussed above (see e.g. Florey, 1967; Klemm, 1976; Evans, 1980; Walker, 1982). Acknowledgements-The skilful technical assistance of Miss Kristina Jeitner and Mrs Ilse Pfaffe is gratefully acknowledged. We thank Prof. Dr V. Neuhoff (MPI f. exp. Medizin, Forschungsstelle Neurochemie. Gettingen) for providing the scanning and evaluation facilities for the microchromatograms.
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Zimmer H. G. and NeuhotT V. (1977) Quantitative Auswertung von zweidimensionalen Mikro-DfinnschichtChromatogrammen. In Informatik Fachherichte, Vol. 8: Digitale Bildverarbeitung (Edited by Nagel H. H.). pp. 12-20. Springer Verlag, Berlin.