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Discussion of Stimulation-Induced Release of Serotonin
Discussion of Stimulation-Induced Release of Serotonin P. ASCHER, J. GLOWINSKI, L. TAUC,AND J. TAXI Laboratoire de Neurophysiologie Cellulaire, Centre National de la Recherche Scienti$que. and Unit6 de Neuropharmacologie Biochimique, Colldge de France, and Labmatoire de Cytologie, FacultB des Sciences, Paris, France
As recalled by Chase and Kopin, there is some evidence suggesting that 5-HTmay serve as a neurotransmitter in the molluscan nervous system. We have also attempted to
demonstrate that exogenous 5-HT-3H can be accumulated by nervous structures of Helix and Aplysia, and can be liberated from these structures by electrical or chemical stimulation. Our experimental results agree quite well with those of the preceding authors; but autoradiographic pictures of incubated ganglia, in conjunction with biochemical data on ganglionic sheaths and other, apparently nonnervous, structures, seem to throw some doubt on the hypothesis that in mollusks both uptake and liberation of exogenous amines are features specific t o the synaptic structures or even to the nervous tissue in general.
Intact Ganglia ACCUMULATION AND RELEASE OF SEROTONlN-3H Accumulation of 5-HT-3H was observed with visceral, parietal, pleural, and cerebral ganglia of Aplysia and periesophageal ganglia of Helix. After 30 minutes of incubation, 3H concentrations up t o 15 times that of the incubation medium could be measured in the ganglia, 65% of which appeared to correspond to 5-HT-3H. This concentration did not increase very much after longer 'incubations. Transfer of incubated ganglia t o a perfusion chamber (2 ml/min) permitted the measurement of the spontaneous efflux of 3H, which appeared t o be rather slow (10%50% of the total initial content was liberated in the first hour after the beginning of the perfusion). If then an electrical stimulation (square pulses of 10 msec. duration, alternate polarities, lO/sec, 3-8 v/cm) was applied t o the ganglia, it evoked a striking incresse in the efflux of 3H (Fig. la). The rate of release thereafter progressively returned to values near or even lower than those preceding the stimulation. A similar release could be triggered by perfusion with a high K+ (100 mEq/liter) solution or with acetylcholine (10-3 gmlml), (Fig. lb).
PICTURES AUTORADIOGRAPHIC Aplysia and Helix ganglia were incubated for 47 minutes in 2 ml seawater containing 100 pcuries of S-HT-3H, washed with fresh seawater for 15 minutes and then'fixed in 6% glutaraldehyde buffered with cacodylate (Miller, 1962) for 1 hour and then 48 hours in Bouin's fluid. It can be seen in Fig. 2a that most of the radioactivity is localized in the connective sheath surrounding the ganglion. This sheath is a complex structure containing muscle fibers and axons (Schlote, 1957; Nisbet, 1961; Rosenbluth, 1963; Fernandez, 1966). Radioactivity, however, did not seem particularly concentrated around these elements. Inside the ganglion, some radioactivity could be detected in structures surrounding the neurons (Fig. 2b). But no radioactivity above the background noise could be detected 365
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FIG. 1. Release of 3H from intact visceral ganglia (a, b) ganglionic sheaths (c) or perihepatic connective tissue (d, e, f) of ApZysia induced by electrical stimulation (a, c, d), high K+ seawater (e) or acetylcholine (10-3 gm/ml (f).
either in the neuronal somata or in the central neuropil where the synapses have been localized on anatomical (Gerschenfeld, 1963) and physiological (Tauc, 1967) evidence. Thus, the radioactive 5-HT does not appear to be bound specifically by the nervous structures, but on the contrary seems to predominate in nonnervous tissues. This led us to investigate the possibility of uptake and release of 5-HT-3H in two “nonnervous” structures: ganglia deprived surgically of their neuron somata and synapses, connective tissue of the hepatopancreas.
“Nonnervous” Structures GANGLIONIC SHEATHS In snail infraesophageal ganglia it is relatively easy to open the connective sheath and to extract as a small globule the neuronal somata and the central neuropil. The “ganglionic sheath” which remains after this extraction contains some nerve fibers, among which those of the proximal parts of the pleuro-visceral connectives and branchial nerve, but can be presumed to contain no synapses. Endogenous 5-HT was found to be more concentrated in the central, neuronal portion (12 pg/gm) than in the ganglionic sheath ( 3 pglgm). However, for ganglia incubated with 5-HT-SH the findings were the
DISCUSSION
367
FIG.2. Autoradiographic photograph of s visceral ganglion of Aplysia incubated in 5-HT-3H. a. General view. b. Detail. The neuron somata and the neuropil appear devoid of radioactivity.
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P.ASCHER, J . QLOWINSKI, L. TAUC, A N D J . TAXI
opposite: the concentration of 3H/gm was 2 to 4 times higher in the ganglionic sheath, even when that sheath had been incised prior t o the incubation to permit a n easier access of the 3H amine t o the central core of the ganglion. Moreover, extraction of the neurons before the incubation did not impede the accumulation of 5-HT-3H in the ganglionic sheath, which radioactivity could be released either by electrical stimulation (Fig. lc) or chemical stirnulation.
PERIHEPATIC CONNECTIVE TISSUE The hepato-pancreas of Aplysia is surrounded by a thin layer of connective tissue in which until now no nerve endings have been described. Nevertheless, this tissue appears to contain relatively large amounts of 5-HT (2.3 pgglgm); in this tissue accumulation of 5-HT-3H is as rapid and as high as in ganglia, and release can be triggered by electrical stimulation (perfusion with high K+ seawater or perfusion with acetylcholine (Fig. 1, d, e, f).
Dopamine Experiments All the experiments described in the preceding sections have been repeated with dopamine-3H and have given the same results. I n particular, the autoradiographic picture is similar to that of Fig. 2. Previous work on mammalian tissues has shown that some of the biogenic amines are normally present in nonnervous structures, and furthermore that they can enter, when administered exogenously, in structures in which they, are not normally detectable (Hamberger et al., 1967). I n mollusks, this uptake by nonnervous structures is even more evident, and coexists with a failure of exogenously applied amines t o enter the neurons. This failure can be due to the complete absence of a n active uptake mechanism by the neurons, or to the fact that the uptake by peripheral structures impedes the accem of exogenous amines t o the neurons and the neuropil. This hypothesis is contradicted by the experiments in which incision of the ganglionic sheath did not increase the uptake by neurons. But even if one could obtain by some procedure an uptake of exogenous amines by neurons, the interpretation of stimulation experiments would still be complicated by the fact that electrical or chemical stimulation can liberate the amines from nonnervous structures. It remains to be determined whether uptake and liberation of amines from nonnervous structures constitute only a methodological artifact, or on the contrary, indicate a participation of the nonnervous structures in the metabolism of the amines. REFERENCES Fernandez, J. (1966). J . Comp. Neurol. 127, 157. Gerschenfeld, H. M. (1963). 2. ZetEforsch. Mikroskop. Anat. 60, 258. Hamberger, B., Norberg, K. A., and Olson, L. (1967). Actn Physiol. Scnnd. 69, 1. Miller, F. (1962). Intern. Congr. Electron Microscopy, 5th, Philadelphia, 1962 Q2. Nisbet, R. (1961). Proc. Roy. SOC.( L o n d o n ) B154, 267. Rosenbluth, J. (1963). 2. Zellforsch. Mikroskop. Anat. 60, 213. Schlote, F. W. (1957). 2. Zellforsch. Mikroskop. Anat. 45, 543. Tauc, L. (1967). In “Physiology of hlollusca” (K. M. Wilbur and C. M. Yonge, eds.), Vol. 11, p. 387. Academic Press, New York.
As recalled by Chase and Kopin, there is some evidence suggesting that 5-HTmay serve as a neurotransmitter in the molluscan nervous system. We have also attempted to
demonstrate that exogenous 5-HT-3H can be accumulated by nervous structures of Helix and Aplysia, and can be liberated from these structures by electrical or chemical stimulation. Our experimental results agree quite well with those of the preceding authors; but autoradiographic pictures of incubated ganglia, in conjunction with biochemical data on ganglionic sheaths and other, apparently nonnervous, structures, seem to throw some doubt on the hypothesis that in mollusks both uptake and liberation of exogenous amines are features specific t o the synaptic structures or even to the nervous tissue in general.
Intact Ganglia ACCUMULATION AND RELEASE OF SEROTONlN-3H Accumulation of 5-HT-3H was observed with visceral, parietal, pleural, and cerebral ganglia of Aplysia and periesophageal ganglia of Helix. After 30 minutes of incubation, 3H concentrations up t o 15 times that of the incubation medium could be measured in the ganglia, 65% of which appeared to correspond to 5-HT-3H. This concentration did not increase very much after longer 'incubations. Transfer of incubated ganglia t o a perfusion chamber (2 ml/min) permitted the measurement of the spontaneous efflux of 3H, which appeared t o be rather slow (10%50% of the total initial content was liberated in the first hour after the beginning of the perfusion). If then an electrical stimulation (square pulses of 10 msec. duration, alternate polarities, lO/sec, 3-8 v/cm) was applied t o the ganglia, it evoked a striking incresse in the efflux of 3H (Fig. la). The rate of release thereafter progressively returned to values near or even lower than those preceding the stimulation. A similar release could be triggered by perfusion with a high K+ (100 mEq/liter) solution or with acetylcholine (10-3 gmlml), (Fig. lb).
PICTURES AUTORADIOGRAPHIC Aplysia and Helix ganglia were incubated for 47 minutes in 2 ml seawater containing 100 pcuries of S-HT-3H, washed with fresh seawater for 15 minutes and then'fixed in 6% glutaraldehyde buffered with cacodylate (Miller, 1962) for 1 hour and then 48 hours in Bouin's fluid. It can be seen in Fig. 2a that most of the radioactivity is localized in the connective sheath surrounding the ganglion. This sheath is a complex structure containing muscle fibers and axons (Schlote, 1957; Nisbet, 1961; Rosenbluth, 1963; Fernandez, 1966). Radioactivity, however, did not seem particularly concentrated around these elements. Inside the ganglion, some radioactivity could be detected in structures surrounding the neurons (Fig. 2b). But no radioactivity above the background noise could be detected 365
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FIG. 1. Release of 3H from intact visceral ganglia (a, b) ganglionic sheaths (c) or perihepatic connective tissue (d, e, f) of ApZysia induced by electrical stimulation (a, c, d), high K+ seawater (e) or acetylcholine (10-3 gm/ml (f).
either in the neuronal somata or in the central neuropil where the synapses have been localized on anatomical (Gerschenfeld, 1963) and physiological (Tauc, 1967) evidence. Thus, the radioactive 5-HT does not appear to be bound specifically by the nervous structures, but on the contrary seems to predominate in nonnervous tissues. This led us to investigate the possibility of uptake and release of 5-HT-3H in two “nonnervous” structures: ganglia deprived surgically of their neuron somata and synapses, connective tissue of the hepatopancreas.
“Nonnervous” Structures GANGLIONIC SHEATHS In snail infraesophageal ganglia it is relatively easy to open the connective sheath and to extract as a small globule the neuronal somata and the central neuropil. The “ganglionic sheath” which remains after this extraction contains some nerve fibers, among which those of the proximal parts of the pleuro-visceral connectives and branchial nerve, but can be presumed to contain no synapses. Endogenous 5-HT was found to be more concentrated in the central, neuronal portion (12 pg/gm) than in the ganglionic sheath ( 3 pglgm). However, for ganglia incubated with 5-HT-SH the findings were the
DISCUSSION
367
FIG.2. Autoradiographic photograph of s visceral ganglion of Aplysia incubated in 5-HT-3H. a. General view. b. Detail. The neuron somata and the neuropil appear devoid of radioactivity.
368
P.ASCHER, J . QLOWINSKI, L. TAUC, A N D J . TAXI
opposite: the concentration of 3H/gm was 2 to 4 times higher in the ganglionic sheath, even when that sheath had been incised prior t o the incubation to permit a n easier access of the 3H amine t o the central core of the ganglion. Moreover, extraction of the neurons before the incubation did not impede the accumulation of 5-HT-3H in the ganglionic sheath, which radioactivity could be released either by electrical stimulation (Fig. lc) or chemical stirnulation.
PERIHEPATIC CONNECTIVE TISSUE The hepato-pancreas of Aplysia is surrounded by a thin layer of connective tissue in which until now no nerve endings have been described. Nevertheless, this tissue appears to contain relatively large amounts of 5-HT (2.3 pgglgm); in this tissue accumulation of 5-HT-3H is as rapid and as high as in ganglia, and release can be triggered by electrical stimulation (perfusion with high K+ seawater or perfusion with acetylcholine (Fig. 1, d, e, f).
Dopamine Experiments All the experiments described in the preceding sections have been repeated with dopamine-3H and have given the same results. I n particular, the autoradiographic picture is similar to that of Fig. 2. Previous work on mammalian tissues has shown that some of the biogenic amines are normally present in nonnervous structures, and furthermore that they can enter, when administered exogenously, in structures in which they, are not normally detectable (Hamberger et al., 1967). I n mollusks, this uptake by nonnervous structures is even more evident, and coexists with a failure of exogenously applied amines t o enter the neurons. This failure can be due to the complete absence of a n active uptake mechanism by the neurons, or to the fact that the uptake by peripheral structures impedes the accem of exogenous amines t o the neurons and the neuropil. This hypothesis is contradicted by the experiments in which incision of the ganglionic sheath did not increase the uptake by neurons. But even if one could obtain by some procedure an uptake of exogenous amines by neurons, the interpretation of stimulation experiments would still be complicated by the fact that electrical or chemical stimulation can liberate the amines from nonnervous structures. It remains to be determined whether uptake and liberation of amines from nonnervous structures constitute only a methodological artifact, or on the contrary, indicate a participation of the nonnervous structures in the metabolism of the amines. REFERENCES Fernandez, J. (1966). J . Comp. Neurol. 127, 157. Gerschenfeld, H. M. (1963). 2. ZetEforsch. Mikroskop. Anat. 60, 258. Hamberger, B., Norberg, K. A., and Olson, L. (1967). Actn Physiol. Scnnd. 69, 1. Miller, F. (1962). Intern. Congr. Electron Microscopy, 5th, Philadelphia, 1962 Q2. Nisbet, R. (1961). Proc. Roy. SOC.( L o n d o n ) B154, 267. Rosenbluth, J. (1963). 2. Zellforsch. Mikroskop. Anat. 60, 213. Schlote, F. W. (1957). 2. Zellforsch. Mikroskop. Anat. 45, 543. Tauc, L. (1967). In “Physiology of hlollusca” (K. M. Wilbur and C. M. Yonge, eds.), Vol. 11, p. 387. Academic Press, New York.