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Serotonin-immunoreactive varicosities in the cell body region and neural sheath of the snail, Helix pomatia, ganglia: An electron microscopic immunocytochemical study
0306-4522/91 $3.00 + 0.00 Pergamon Press plc IBRO
Neuroscience Vol. 42, No. 2, pp. 583-591, 1991 Printed in Great Britain
SEROTONIN-IMMUNOREACTIVE VARICOSITIES IN THE CELL BODY REGION AND NEURAL SHEATH OF THE SNAIL, HELIX POMATIA, GANGLIA: AN ELECTRON MICROSCOPIC IMMUNOCYTOCHEMICAL STUDY K. ELEKES Balaton Limnological Research Institute of the Hungarian Academy of Sciences, H-8237 Tihany, Hungary Abstract--The distribution and connections of serotonin-immunoreactive fibers in the cell body region and neural sheath of the central ganglia of the snail, Helix pomatia, have been examined. The cell body region of the ganglia is sf~pptied by an extremely dense network of varicose serotonin-immunoreactive fibers which surround neuronal perikarya in the ganglia. Immunoreactive processes also run to the neural sheath of both the ganglia and the peripheral nerve roots, forming a dense network. Electron microscopy revealed five different connections of serotonin-immunoreactive varicosities, according to their target: (i) non-specialized contacts with neuronal perikarya; (ii) non-specialized contacts with axon processes on the surface of the peripheral nerve roots; (iii) non-specialized neuromuscular connections with smooth muscle fibers in the neural sheath; (iv) varicosities engulfed by glial processes in both the cell body region and neural sheath; (v) varicosities embedded in the connective tissue elements of the sheath either partly or completely free of giial processes. In all cases of appositions no membrane specializations could be observed on either site of the contacts. These observations provide morphological evidence for non-synaptic regulatory actions of serotonincontaining neurons in Helix central nervous system: (i) modulation of the activity of neuronal perikarya; (ii) involvement in neuromuscular regulation; (iii) neurohormonal modulation of peripheral processes by release through the neural sheath.
Serotonin (5-HT) occurs in relatively high concentrations in the C N S o f the snail, Helix pomatia 17'37 and is considered to be a neurotransmitter substance. 37 The anatomical distribution of 5-HT-containing neurons in the C N S o f Helix pomatia has recently been described using the neurotoxic false transmitter substance, 5,6-dihydroxytryptamine, 39 and wholem o u n t immunocytochemistry) 6 In the course of the immunocytochemical analysis, a densely arranged network of 5-HT-immunoreactive (IR) fibers was observed in the neural sheath covering the ganglia and the peripheral nerves. Based on this observation, a neurohormonal role for 5-HT in control of peripheral processes was suggested.16 However, whole-mount immunolabeling analysis does not adequately provide insight into the precise arrangement of the 5-HT-containing elements. The exact relation of the 5 - H T - I R fibers to other cellular elements (neuronal cell bodies, glial and muscular components) of the sheath, as yet, have not been explored. Occurrence of 5-HT-IR fibers in the neural sheath and cell body layer have also been described in other gastropod species at the light microscopic level. 12"1s'3°To study the relationship of the 5 - H T - I R fibers to potential "targets" we applied a pre-embedding whole-mount technique combined Abbreviations: BSA, bovine serum albumin; DAB, 3,Ydiaminobenzidine; 5-HT, serotonin; 5-HT-IR, 5-HTimmunoreactive (-immunoreactivity); PAP, peroxidaseantiperoxidase; PB, phosphate buffer; PBS, phosphatebuffered saline. 583
with postosmification and araldite embedding37 This technique allows for the analysis o f the precise distribution of immunolabeled elements, and followed by re-embedding, the tissue can be further processed for electron microscopy. EXPERIMENTAL PROCEDURES
Twenty adult specimens of the snail, Helix pomatia, were used. The animals were freshly collected during Spring in the Tihany Peninsula and were kept under laboratory or natural conditions in the Institute's garden. The immunocytochemical method described by N/issel and Elekesz7was applied. The maximal intensity and overall labeling of the 5-HT-IR elements were obtained after three days of incubation in the primary antiserum. However, this resulted in a poor preservation ofultrastructure. In contrast, a one day incubation provided fairly good ultrastructure quality, but the labeling was less intense. The pattern of distribution of 5-HT-IR was the same in both types of experiments. The circumoesophageal ganglia were dissected and immediately immersed in 4% paraformaldehyde diluted in 0.1 M phosphate buffer (PB; pH 7.4) for 7 h at 4°C. For correlative light and electron microscopic investigations 0.25% glutaraldehyde was also added to the fixative. During the 24 h wash in PB the ganglia were either completely or partially desheathed (the outer layer of the sheath was removed). In some cases, the sheath was not removed at all from part of the CNS and peripheral nerve roots. For immtmocytoehemistry, the indirect peroxidase-antiperoxidase (PAP) method 4° was applied. Anti-5-HT antiserum (Incstar, Stillwater, U.S.A.) was diluted to 1:2000 and the incubation lasted either three days (light microscopy only) or 20 h (correlative light and electron microscopy) at 4°C. The secondary antibody (goat anti-rabbit IgG, Nordic Immune, Tilburg, The Netherlands) was diluted to 1:40 and the incubation
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lasted 4 h at room temperature. Incubation overnight at 4°C was with PAP (Dakopatts, Copenhagen, Denmark) diluted to 1:100. All antisera were diluted in phosphate-buffered saline (PBS) containing 0.25% Triton X-100 and 0.25% bovine serum albumin (BSA). The histochemical reaction was performed with 0.06% 3,3'-diaminobenzidine(DAB) as chromogen and 0.01% H202 as substrate. After development, the ganglia were fixed with 1% OsO4 diluted with 0.1 M sodium cacodylate buffer (pH 7.2) for 1 h at 4°C, dehydrated in graded ethanol and propylene oxide and embedded in soft araldite (Durcupan ACM, Fluka, Switzerland). Following polymerization, 25 pm-thick sections were cut serially on a sliding microtome and then covered with Canada-balsam for light microscopy or with araldite for correlative light and electron microscopy. In this case, sections were re-embedded in araldite, cut with a diamond knife and then viewed with a TESLA BS500 electron microscope. Specificity of this antiserum has previously been proven both on Helix 16and on other gastropod CNS. 4,S,'s,3° RESULTS
Light microscopic immunocytochemistry The 3-day-long incubation with anti-5-HT antiserum resulted in intensive labeling of a dense network of varicose fibers in the entire cell body region of all ganglia. Also, a network of varicose fibers was found in the neural sheath of the ganglia and peripheral nerves (Fig. 1). The appearance of the 5-HT-IR fibers was similar but more precise than that described in whole-mount preparations. 16 The finer detail of the distribution of 5-HT-IR, especially in the cell body region, is revealed in the present study. The varicose fibers were observed to surround the neuronal cell bodies (Fig. 1c, d). The giant neurons were enveloped by these 5-HT-IR fibers (Fig. lb). The thick primary neurite was also engulfed by fine varicose immunoreactive axons. The 5-HT-IR fibers appeared frequently as branching processes in the multilayered glial sheath (trophospongium) covering the neuronal cell bodies. The varicose 5-HT-IR fibers entered the cell body region from the direction of the neuropile, showing that this innervation originated from the neuropile. The neural sheath around the ganglia and peripheral nerves contained a dense network of 5-HTIR fibers both in the innermost layer, perineurium, and in the thick, outer layer (Fig. le, f). In semithin sections, further detail of the distribution of 5-HT-IR fibers around the neuronal cell bodies was observed. The majority of the 5-HT-IR
fibers were located among the glial processes surrounding the cell bodies, rather than directly on the surface of the neurons (Fig. 2a, b). At a higher magnification the 5-HT-IR fibers among the glial processes, located closest to the surface of the neuronal cell bodies, gave rise to varicose endings embedded in the cytoplasm. These localizations were easy to find around large (80-100/~m in diameter) or giant (over 100/~m) neurons of the suboesophageal ganglion complex (Fig. 2b). Fine 5-HT-IR varicose fibers were traced branching off from thick immunoreactive axons of the neuropile area bordering the cell body layer. These fibers ran towards the neuronal cell bodies (Fig. 2c).
Electron microscopic immunocytochemistry The electron microscopic analysis of the ultrathin sections revealed five distinct locations for 5-HT-IR fibers and varicosities. (i) 5-HT-IR varicose fibers were observed surrounded by and embedded in the densely arranged multilayered system of glial processes (trophospongium) around the nerve cells. The majority of the nerve cell bodies were thickly covered by glial processes. A m o n g the fine glial processes both far and near to the cell body surface, 5-HT-IR varicosities containing synaptic vesicles were found (Fig. 3a). Membrane specializations were not observed between the 5-HTIR varicosities and the glial processes. (ii) 5-HT-IR varicosities intruded deeply into the cytoplasm of the nerve cells. The abundance of the 5-HT-IR varicosities with their "preterminal" axon segments appeared to penetrate the glial sheath and contact the nerve cell bodies (Fig. 3b-d). The varicosities did not form specialized synaptic contacts with the nerve cells. The nerve cell body membrane and the axolemma ran parallel without showing appositions of pre- or postsynaptic dense material, or a widening or narrowing of the intermembranous space (Fig. 3b, c). 5-HT-IR varicosities contacted different neuronal perikarya, which contained different populations of vesicles (Fig. 3b-d). (iii) Varicose 5-HT-IR fibers were observed on the surface of the peripheral nerves. In this case, the 5-HT-IR fibers and varicosities were located on axon processes of the outermost portion of the nerve trunks (Figs 4 and 5).
Fig. 1. Distribution of 5-HT-IR fibers in the cell body layer and neural sheath of Helix ganglia. Light micrographs of 25-/~m araldite sections. Scale bars = 100 #m (a); 50/~m (b-f). (a) Detail of the right parietal ganglion, showing a small part of the neuropile (np) and numerous varicose fibers (arrows) running to and around the neuronal cell bodies (P). (b) A giant neuron (P) is surrounded by several 5-HT-IR fibers (arrows). Note that both the levels of the axon hillock (AH) and the primary neurite (IS) are also densely packed by immunolabeled processes (arrowheads). (c), (d) Higher magnification detail from the right parietal (c) and right mesocerebrum (d) cell body region. Note the dense branching of 5-HT-IR processes (arrows) in the parietal ganglion. In the mesocerebrum, fine varicose fibers surround the cell bodies (P) almost individually. (e) 5-HT-IR varicose processes (arrowheads) in the neural sheath of the olfactory (on) and external peritentacular (epn) nerves. Arrow indicates labeled axons in the nerve. (f) Dense network of varicose 5-HT-IR fibers in the thick neural sheath on the right parietal ganglion.
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Fig. 2. 5-HT-IR processes in semithin sections taken from the cell body region. Scale bars = 20#m. (a) Detail of a giant neuron (P) surrounded by a thick layer of giial processes (trophospongium) (gl), in which 5-HT-IR fibers (arrowheads) are embedded. (b) Higher magnification of the surface of the giant neuron (other section plane). Immunoreactive varicosities (arrows) intrude into the perikaryon (P). Others, as well as longer intervaricose segments (long arrows), are embedded in the glial sheath (gi). (c) 5-HT-IR varicosities (arrowheads), originating from a thick fiber (arrow) in the neuropile bordering the cortex, contact neuronal perikarya (P). Double arrow indicates branching of a varicose fiber. (iv) 5-HT-IR varicosities were demonstrated contacting muscle fibers within the neural sheath (Fig. 6a, b). All muscle fibers found in the sheath showed a fine structure characteristic of smooth muscle fibers. Neither cross-striation nor dense Z-points were observed. At the sites of the neuromuscular contacts the muscle fibers always contained contractile elements. The neuromuscular contacts did not exhibit any membrane specializations (Fig. 6a). The contacting 5-HT-IR varicosities were at times accompanied by glial processes, which contained large electron-dense granules characteristic of the peripheral glial processes
in the molluscan nervous system. 2°'29 We have also found 5-HT-IR varicosities contacting muscle fiber(s), whose axolemma, not facing the muscle fiber, was completely free of glial process(es) (Fig. 6b). (v) 5-HT-IR varicosities were also observed in the neural sheath located relatively far (several micrometers) from muscle fibers and embedded in a mass of collagen fibers (Fig. 6c, d). Many of these varicosities were situated near the surface of the sheath. These 5-HT-IR varicosities were accompanied by glial processes; however, a part of the axon membrane was free of glial covering (Fig. 6d).
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Fig. 3. Electron microscopy of 5-HT-IR varicosities on the surface of neuronal perikarya. (a) Detail of the surface of the giant neuron also shown in Fig. 2b. 5-HT-IR elements (arrows) surrounded by glail processes (gl) and a varicosity (T) contacting the perikaryon (P) are seen. Scale bar = 1 #m. (b) 5-HT-IR varicosity (T) deeply embedded into the surface of the giant neuron (P). Neither the membrane of the varicosity nor of the perikaryon show any specialization. The varicosity contains small-sized agranular synaptic vesicles (v), many of them revealing immunoreactivity, m, mitochondrion. Scale bar = 0.5 #m. (c) 5-HT-IR varicosity (T) containing small agranular (arrow) and large granular (arrowhead) vesicles contacts a neuronal perikaryon 0a) without membrane specialization, gl, glial process. Scale bar = 0.5 #m. (d) 5-HT-IR varicosity (T) with small agranular (v) and large granular (arrowhead) vesicles deeply invades the surface of a neuronal perikaryon (P) containing large electron-dense granules (arrows). The varicosity contacts the perikaryon without membrane specialization, m, mitochondrion. Scale bar=0.5/~m.
The majority of all 5-HT-IR varicosities contained similar vesicle population: small-sized (50-60 nm) agranular clear vesicles comprised the majority of the population, and a few large granular vesicles (120150 nm) also occurred (Figs 3a-d and 5). The varicosities contacting the soma surface contained overa
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whelmingly agranular clear synaptic vesicles (Fig. 3a, b, d). 5-HT-IR varicosities were also occasionally found among the muscle fibers in the sheath which contained mostly large granular vesicles (Fig. 6c). All o f the vesicle types noted above revealed 5-HT-IR.
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Serotonin-immunoreactive terminals in Helix ganglia DISCUSSION The application of a highly specific antibody generated against 5-HT conjugated to BSA revealed the presence of a dense network of 5-HT-IR fibers both in the neural sheath and in the cell body region of the CNS of Helix pomatia. Recently, several immunocytochemical studies demonstrated the occurrence of 5-HT-containing fibers in the cortex and sheath of the nervous system of different gastropods (Cryptomphallus, 12 Helix, 16 Lymn~ea, TM and Aplysiaa°). The present report is the first to describe, in detail, the distribution of 5-HT-IR fibers in the sheath and cell body region of a gastropod mollusc. The study reveals their exact relation to the different cellular components in the CNS. The data on the ultrastructure of 5-HTIR nerve endings presented are also the first for the gastropod CNS. These findings provide fine structural support for a versatile role of 5-HT-containing neurons in regulatory processes in the snail nervous system: involvement in axosomatic and neuromuscular interactions, as well as in neurohormonal control of peripheral processes. The 5-HT-IR varicosities do not have specialized synaptic contacts either in the neural sheath or in the cell body layer. This indicates that 5-HT, which has generally been considered to be a neurotransmitter in the molluscan nervous system (e.g. Refs 13, 14, 31 and 37), can be involved in modulatory actions outside the primary region of neuronal communication, the neuropile. In this respect, the occurrence of 5-HT-IR varicosities contacting cell bodies appears to be important. This and the overall distribution of 5-HT-IR fibers around the neuronal cell bodies indicate a possible major involvement of 5-HT in affecting the activity of cell bodies. The modulatory action of 5-HT in gastropod feeding processes has been widely investigated. 19':3'24,43,'~ In Aplysia, the serotonergic metacerebral giant neuron was shown to terminate on the somata of buccal neurons.36 Non-specialized axosomatic contacts on the bimodal pacemaker bursting neuron of Helix have been described earlier, s'2: A similar situation can be proposed for the 5-HTIR varicose fibers along the surface of peripheral
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nerves where they are seen inside the sheath, contacting the axon processes of the nerve. At this site, 5-HT-containing elements may exert a regulating effect on the efferent and afferent information in the peripheral nerves. Axoaxonic synaptic contacts have been demonstrated previously in the intestinal nerve of Helix. 9 The 5-HT-IR varicosities contacting the smooth muscle fibers of the neural sheath suggest a serotonergic neuromuscular innervation. These varicosities never form specialized contacts with the muscle fibers, but axo- and sarcolemma segments do oppose each other. Neuromuscular contacts without membrane specializations have been described in the osphradial ganglion of the freshwater snail Planorbis corneus. 3 Smooth muscle elements have also been demonstrated in the ganglionic neural sheath of Helix aspersa ~ and Aplysia californica. 35 However, the exact functional significance of this phenomenon is not known. The unspecialized contacts of serotonergic innervation of the smooth muscle fibers in the sheath also suggest a modulatory role for this substance. Such a role for 5-HT has been suggested in the buccal and lip musculature of several gastropod species.26,33,42,43 5HT-containing varicosities have been found using autoradiography in the buccal musculature of Helix) e The occurrence of 5-HT-IR varicose fibers in other muscular tissue (intestine) of Helix has also been described at the light microscopic level.mSAnalysing the ultrastructure of the innervation of the Helix intestine, Benedeczky and Halasy 2 found that varicosities contacted the smooth muscle fibers without membrane specializations and intravesicular release occurred by exocytosis. Non-specialized neuromuscular contacts established by 5-HT-IR varicosities have been found in the alimentary tract 21 and genital chamber ~° musculature of the cricket. The electron microscopic observations on the occurrence of 5-HT-IR endings, partially or completely free of glial processes in the neural sheath, supports an earlier concept ~6that these elements may release 5-HT into the hemolymph. Thus, 5-HT may be participating in the neurohormonal control of different peripheral
Fig. 4. 5-HT-IR varicosities (T) contact (between arrowheads) axon processes on the surface of the left pallial nerve. GC, glia cell; gl, glial processes; A, internally located axon. Scale bar = 1 #m. Fig. 5. 5-HT-IR varicosity (arrow) embedded between surface axon processes (A) in the intestinal nerve. The varicosity contains small agranular and large granular vesicles. Arrowhead points to large granular vesicles in unlabeled axons, gl, glial processes. Scale bar = 0.5 #m. Fig. 6. 5-HT-IR varicosities in the neural sheath. (a) A small varicosity (T) surrounded by muscle fibers (M) contacts one of them (between arrowheads), gl, gliai process with electron-dense granules. Scale bar = 0.25 #m. (b) 5-HT-IR processes (T) between muscle fibers (M), contacting one of them (between arrowheads) and remaining from the others for a relatively large distance (c. 0.2#m). Note large omega-shaped invaginations along the axolemma (open arrowheads), possibly indicating sites of release. cf, collagen fibers; hc, hemocoel. Scale bar = 0.25 #m. (c) 5-HT-IR varicosity containing mainly large granular vesicles (arrows) is freely embedded in the mass of collagen fibers (cf) in the outer layer of the neural sheath. M, muscle fiber process; arrowhead, gliai process. Scale bar = 0.25 gin. (d) 5-HT-IR varicosity partly surrounded by gliai process faces freely along a segment of its axolemma the mass of collagen fibers (cf) of the neural sheath. Scale bar = 0.25 #m.
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organs or blood cells. The buccal and foot musculature nerves of the insect nervous system has been d e m o n strated. 1' Experiments designed to investigate whether located in the vicinity o f the C N S m i g h t be, for such a release-upake m e c h a n i s m exists in the neural example, p r i m a r y targets o f 5-HT released t h r o u g h sheath of the Helix ganglia are in progress in o u r the neural sheath. A n e u r o h o r m o n a l role for 5-HT in laboratory. invertebrates has been suggested as early as 1954 by Welsh. ~ Such a role o f 5-HT t h r o u g h o u t the release Acknowledgements--The skilful technical assistance of into the h e a r t following stimulation was suggested Ms V. Pintrr and Mr B. Bal~.zs is much appreciated. earlier in Helix. 3s The participation o f 5-HT in neuroThe author's thanks are also due to Dr G. B. Stefano for h o r m o n a l regulation o f peripheral functions has also correcting the English. This work was supported by an been suggested in a r t h r o p o d s . ~'6'7'25'28'41A release a n d OTKA grant (No. 1-600-2-89-1-667) from the Hungarian u p t a k e m e c h a n i s m for 5-HT existing in the peripheral Academy of Sciences. REFERENCES 1. Beltz B, and Kravitz E. A. (1983) Mapping of serotonin-like immunoreactivity in the lobster nervous system. J. Neurosci. 7, 533-546. 2. Benedeczky I. and Halasy K. (1988) Visualization of non-synaptic release sites in the myenteric plexus of the snail, Helix pomatia. Neuroscience 25, 163-170. 3. Benjamin P. R. and Peat A. (1968) Myoneural junction in the connective tissue sheath of a molluscan ganglion. Nature 219, 1371-1372. 4. Croll R. P. (1987) Distribution of monoamines in the central nervous system of the nudibranch gastropod, Hermissenda crassicornis. Brain Res. 405, 337-347. 5. Croll R. P. and Chiasson B. J. 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(Accepted 3 December 1990)
Neuroscience Vol. 42, No. 2, pp. 583-591, 1991 Printed in Great Britain
SEROTONIN-IMMUNOREACTIVE VARICOSITIES IN THE CELL BODY REGION AND NEURAL SHEATH OF THE SNAIL, HELIX POMATIA, GANGLIA: AN ELECTRON MICROSCOPIC IMMUNOCYTOCHEMICAL STUDY K. ELEKES Balaton Limnological Research Institute of the Hungarian Academy of Sciences, H-8237 Tihany, Hungary Abstract--The distribution and connections of serotonin-immunoreactive fibers in the cell body region and neural sheath of the central ganglia of the snail, Helix pomatia, have been examined. The cell body region of the ganglia is sf~pptied by an extremely dense network of varicose serotonin-immunoreactive fibers which surround neuronal perikarya in the ganglia. Immunoreactive processes also run to the neural sheath of both the ganglia and the peripheral nerve roots, forming a dense network. Electron microscopy revealed five different connections of serotonin-immunoreactive varicosities, according to their target: (i) non-specialized contacts with neuronal perikarya; (ii) non-specialized contacts with axon processes on the surface of the peripheral nerve roots; (iii) non-specialized neuromuscular connections with smooth muscle fibers in the neural sheath; (iv) varicosities engulfed by glial processes in both the cell body region and neural sheath; (v) varicosities embedded in the connective tissue elements of the sheath either partly or completely free of giial processes. In all cases of appositions no membrane specializations could be observed on either site of the contacts. These observations provide morphological evidence for non-synaptic regulatory actions of serotonincontaining neurons in Helix central nervous system: (i) modulation of the activity of neuronal perikarya; (ii) involvement in neuromuscular regulation; (iii) neurohormonal modulation of peripheral processes by release through the neural sheath.
Serotonin (5-HT) occurs in relatively high concentrations in the C N S o f the snail, Helix pomatia 17'37 and is considered to be a neurotransmitter substance. 37 The anatomical distribution of 5-HT-containing neurons in the C N S o f Helix pomatia has recently been described using the neurotoxic false transmitter substance, 5,6-dihydroxytryptamine, 39 and wholem o u n t immunocytochemistry) 6 In the course of the immunocytochemical analysis, a densely arranged network of 5-HT-immunoreactive (IR) fibers was observed in the neural sheath covering the ganglia and the peripheral nerves. Based on this observation, a neurohormonal role for 5-HT in control of peripheral processes was suggested.16 However, whole-mount immunolabeling analysis does not adequately provide insight into the precise arrangement of the 5-HT-containing elements. The exact relation of the 5 - H T - I R fibers to other cellular elements (neuronal cell bodies, glial and muscular components) of the sheath, as yet, have not been explored. Occurrence of 5-HT-IR fibers in the neural sheath and cell body layer have also been described in other gastropod species at the light microscopic level. 12"1s'3°To study the relationship of the 5 - H T - I R fibers to potential "targets" we applied a pre-embedding whole-mount technique combined Abbreviations: BSA, bovine serum albumin; DAB, 3,Ydiaminobenzidine; 5-HT, serotonin; 5-HT-IR, 5-HTimmunoreactive (-immunoreactivity); PAP, peroxidaseantiperoxidase; PB, phosphate buffer; PBS, phosphatebuffered saline. 583
with postosmification and araldite embedding37 This technique allows for the analysis o f the precise distribution of immunolabeled elements, and followed by re-embedding, the tissue can be further processed for electron microscopy. EXPERIMENTAL PROCEDURES
Twenty adult specimens of the snail, Helix pomatia, were used. The animals were freshly collected during Spring in the Tihany Peninsula and were kept under laboratory or natural conditions in the Institute's garden. The immunocytochemical method described by N/issel and Elekesz7was applied. The maximal intensity and overall labeling of the 5-HT-IR elements were obtained after three days of incubation in the primary antiserum. However, this resulted in a poor preservation ofultrastructure. In contrast, a one day incubation provided fairly good ultrastructure quality, but the labeling was less intense. The pattern of distribution of 5-HT-IR was the same in both types of experiments. The circumoesophageal ganglia were dissected and immediately immersed in 4% paraformaldehyde diluted in 0.1 M phosphate buffer (PB; pH 7.4) for 7 h at 4°C. For correlative light and electron microscopic investigations 0.25% glutaraldehyde was also added to the fixative. During the 24 h wash in PB the ganglia were either completely or partially desheathed (the outer layer of the sheath was removed). In some cases, the sheath was not removed at all from part of the CNS and peripheral nerve roots. For immtmocytoehemistry, the indirect peroxidase-antiperoxidase (PAP) method 4° was applied. Anti-5-HT antiserum (Incstar, Stillwater, U.S.A.) was diluted to 1:2000 and the incubation lasted either three days (light microscopy only) or 20 h (correlative light and electron microscopy) at 4°C. The secondary antibody (goat anti-rabbit IgG, Nordic Immune, Tilburg, The Netherlands) was diluted to 1:40 and the incubation
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lasted 4 h at room temperature. Incubation overnight at 4°C was with PAP (Dakopatts, Copenhagen, Denmark) diluted to 1:100. All antisera were diluted in phosphate-buffered saline (PBS) containing 0.25% Triton X-100 and 0.25% bovine serum albumin (BSA). The histochemical reaction was performed with 0.06% 3,3'-diaminobenzidine(DAB) as chromogen and 0.01% H202 as substrate. After development, the ganglia were fixed with 1% OsO4 diluted with 0.1 M sodium cacodylate buffer (pH 7.2) for 1 h at 4°C, dehydrated in graded ethanol and propylene oxide and embedded in soft araldite (Durcupan ACM, Fluka, Switzerland). Following polymerization, 25 pm-thick sections were cut serially on a sliding microtome and then covered with Canada-balsam for light microscopy or with araldite for correlative light and electron microscopy. In this case, sections were re-embedded in araldite, cut with a diamond knife and then viewed with a TESLA BS500 electron microscope. Specificity of this antiserum has previously been proven both on Helix 16and on other gastropod CNS. 4,S,'s,3° RESULTS
Light microscopic immunocytochemistry The 3-day-long incubation with anti-5-HT antiserum resulted in intensive labeling of a dense network of varicose fibers in the entire cell body region of all ganglia. Also, a network of varicose fibers was found in the neural sheath of the ganglia and peripheral nerves (Fig. 1). The appearance of the 5-HT-IR fibers was similar but more precise than that described in whole-mount preparations. 16 The finer detail of the distribution of 5-HT-IR, especially in the cell body region, is revealed in the present study. The varicose fibers were observed to surround the neuronal cell bodies (Fig. 1c, d). The giant neurons were enveloped by these 5-HT-IR fibers (Fig. lb). The thick primary neurite was also engulfed by fine varicose immunoreactive axons. The 5-HT-IR fibers appeared frequently as branching processes in the multilayered glial sheath (trophospongium) covering the neuronal cell bodies. The varicose 5-HT-IR fibers entered the cell body region from the direction of the neuropile, showing that this innervation originated from the neuropile. The neural sheath around the ganglia and peripheral nerves contained a dense network of 5-HTIR fibers both in the innermost layer, perineurium, and in the thick, outer layer (Fig. le, f). In semithin sections, further detail of the distribution of 5-HT-IR fibers around the neuronal cell bodies was observed. The majority of the 5-HT-IR
fibers were located among the glial processes surrounding the cell bodies, rather than directly on the surface of the neurons (Fig. 2a, b). At a higher magnification the 5-HT-IR fibers among the glial processes, located closest to the surface of the neuronal cell bodies, gave rise to varicose endings embedded in the cytoplasm. These localizations were easy to find around large (80-100/~m in diameter) or giant (over 100/~m) neurons of the suboesophageal ganglion complex (Fig. 2b). Fine 5-HT-IR varicose fibers were traced branching off from thick immunoreactive axons of the neuropile area bordering the cell body layer. These fibers ran towards the neuronal cell bodies (Fig. 2c).
Electron microscopic immunocytochemistry The electron microscopic analysis of the ultrathin sections revealed five distinct locations for 5-HT-IR fibers and varicosities. (i) 5-HT-IR varicose fibers were observed surrounded by and embedded in the densely arranged multilayered system of glial processes (trophospongium) around the nerve cells. The majority of the nerve cell bodies were thickly covered by glial processes. A m o n g the fine glial processes both far and near to the cell body surface, 5-HT-IR varicosities containing synaptic vesicles were found (Fig. 3a). Membrane specializations were not observed between the 5-HTIR varicosities and the glial processes. (ii) 5-HT-IR varicosities intruded deeply into the cytoplasm of the nerve cells. The abundance of the 5-HT-IR varicosities with their "preterminal" axon segments appeared to penetrate the glial sheath and contact the nerve cell bodies (Fig. 3b-d). The varicosities did not form specialized synaptic contacts with the nerve cells. The nerve cell body membrane and the axolemma ran parallel without showing appositions of pre- or postsynaptic dense material, or a widening or narrowing of the intermembranous space (Fig. 3b, c). 5-HT-IR varicosities contacted different neuronal perikarya, which contained different populations of vesicles (Fig. 3b-d). (iii) Varicose 5-HT-IR fibers were observed on the surface of the peripheral nerves. In this case, the 5-HT-IR fibers and varicosities were located on axon processes of the outermost portion of the nerve trunks (Figs 4 and 5).
Fig. 1. Distribution of 5-HT-IR fibers in the cell body layer and neural sheath of Helix ganglia. Light micrographs of 25-/~m araldite sections. Scale bars = 100 #m (a); 50/~m (b-f). (a) Detail of the right parietal ganglion, showing a small part of the neuropile (np) and numerous varicose fibers (arrows) running to and around the neuronal cell bodies (P). (b) A giant neuron (P) is surrounded by several 5-HT-IR fibers (arrows). Note that both the levels of the axon hillock (AH) and the primary neurite (IS) are also densely packed by immunolabeled processes (arrowheads). (c), (d) Higher magnification detail from the right parietal (c) and right mesocerebrum (d) cell body region. Note the dense branching of 5-HT-IR processes (arrows) in the parietal ganglion. In the mesocerebrum, fine varicose fibers surround the cell bodies (P) almost individually. (e) 5-HT-IR varicose processes (arrowheads) in the neural sheath of the olfactory (on) and external peritentacular (epn) nerves. Arrow indicates labeled axons in the nerve. (f) Dense network of varicose 5-HT-IR fibers in the thick neural sheath on the right parietal ganglion.
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Fig. 2. 5-HT-IR processes in semithin sections taken from the cell body region. Scale bars = 20#m. (a) Detail of a giant neuron (P) surrounded by a thick layer of giial processes (trophospongium) (gl), in which 5-HT-IR fibers (arrowheads) are embedded. (b) Higher magnification of the surface of the giant neuron (other section plane). Immunoreactive varicosities (arrows) intrude into the perikaryon (P). Others, as well as longer intervaricose segments (long arrows), are embedded in the glial sheath (gi). (c) 5-HT-IR varicosities (arrowheads), originating from a thick fiber (arrow) in the neuropile bordering the cortex, contact neuronal perikarya (P). Double arrow indicates branching of a varicose fiber. (iv) 5-HT-IR varicosities were demonstrated contacting muscle fibers within the neural sheath (Fig. 6a, b). All muscle fibers found in the sheath showed a fine structure characteristic of smooth muscle fibers. Neither cross-striation nor dense Z-points were observed. At the sites of the neuromuscular contacts the muscle fibers always contained contractile elements. The neuromuscular contacts did not exhibit any membrane specializations (Fig. 6a). The contacting 5-HT-IR varicosities were at times accompanied by glial processes, which contained large electron-dense granules characteristic of the peripheral glial processes
in the molluscan nervous system. 2°'29 We have also found 5-HT-IR varicosities contacting muscle fiber(s), whose axolemma, not facing the muscle fiber, was completely free of glial process(es) (Fig. 6b). (v) 5-HT-IR varicosities were also observed in the neural sheath located relatively far (several micrometers) from muscle fibers and embedded in a mass of collagen fibers (Fig. 6c, d). Many of these varicosities were situated near the surface of the sheath. These 5-HT-IR varicosities were accompanied by glial processes; however, a part of the axon membrane was free of glial covering (Fig. 6d).
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Fig. 3. Electron microscopy of 5-HT-IR varicosities on the surface of neuronal perikarya. (a) Detail of the surface of the giant neuron also shown in Fig. 2b. 5-HT-IR elements (arrows) surrounded by glail processes (gl) and a varicosity (T) contacting the perikaryon (P) are seen. Scale bar = 1 #m. (b) 5-HT-IR varicosity (T) deeply embedded into the surface of the giant neuron (P). Neither the membrane of the varicosity nor of the perikaryon show any specialization. The varicosity contains small-sized agranular synaptic vesicles (v), many of them revealing immunoreactivity, m, mitochondrion. Scale bar = 0.5 #m. (c) 5-HT-IR varicosity (T) containing small agranular (arrow) and large granular (arrowhead) vesicles contacts a neuronal perikaryon 0a) without membrane specialization, gl, glial process. Scale bar = 0.5 #m. (d) 5-HT-IR varicosity (T) with small agranular (v) and large granular (arrowhead) vesicles deeply invades the surface of a neuronal perikaryon (P) containing large electron-dense granules (arrows). The varicosity contacts the perikaryon without membrane specialization, m, mitochondrion. Scale bar=0.5/~m.
The majority of all 5-HT-IR varicosities contained similar vesicle population: small-sized (50-60 nm) agranular clear vesicles comprised the majority of the population, and a few large granular vesicles (120150 nm) also occurred (Figs 3a-d and 5). The varicosities contacting the soma surface contained overa
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whelmingly agranular clear synaptic vesicles (Fig. 3a, b, d). 5-HT-IR varicosities were also occasionally found among the muscle fibers in the sheath which contained mostly large granular vesicles (Fig. 6c). All o f the vesicle types noted above revealed 5-HT-IR.
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Serotonin-immunoreactive terminals in Helix ganglia DISCUSSION The application of a highly specific antibody generated against 5-HT conjugated to BSA revealed the presence of a dense network of 5-HT-IR fibers both in the neural sheath and in the cell body region of the CNS of Helix pomatia. Recently, several immunocytochemical studies demonstrated the occurrence of 5-HT-containing fibers in the cortex and sheath of the nervous system of different gastropods (Cryptomphallus, 12 Helix, 16 Lymn~ea, TM and Aplysiaa°). The present report is the first to describe, in detail, the distribution of 5-HT-IR fibers in the sheath and cell body region of a gastropod mollusc. The study reveals their exact relation to the different cellular components in the CNS. The data on the ultrastructure of 5-HTIR nerve endings presented are also the first for the gastropod CNS. These findings provide fine structural support for a versatile role of 5-HT-containing neurons in regulatory processes in the snail nervous system: involvement in axosomatic and neuromuscular interactions, as well as in neurohormonal control of peripheral processes. The 5-HT-IR varicosities do not have specialized synaptic contacts either in the neural sheath or in the cell body layer. This indicates that 5-HT, which has generally been considered to be a neurotransmitter in the molluscan nervous system (e.g. Refs 13, 14, 31 and 37), can be involved in modulatory actions outside the primary region of neuronal communication, the neuropile. In this respect, the occurrence of 5-HT-IR varicosities contacting cell bodies appears to be important. This and the overall distribution of 5-HT-IR fibers around the neuronal cell bodies indicate a possible major involvement of 5-HT in affecting the activity of cell bodies. The modulatory action of 5-HT in gastropod feeding processes has been widely investigated. 19':3'24,43,'~ In Aplysia, the serotonergic metacerebral giant neuron was shown to terminate on the somata of buccal neurons.36 Non-specialized axosomatic contacts on the bimodal pacemaker bursting neuron of Helix have been described earlier, s'2: A similar situation can be proposed for the 5-HTIR varicose fibers along the surface of peripheral
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nerves where they are seen inside the sheath, contacting the axon processes of the nerve. At this site, 5-HT-containing elements may exert a regulating effect on the efferent and afferent information in the peripheral nerves. Axoaxonic synaptic contacts have been demonstrated previously in the intestinal nerve of Helix. 9 The 5-HT-IR varicosities contacting the smooth muscle fibers of the neural sheath suggest a serotonergic neuromuscular innervation. These varicosities never form specialized contacts with the muscle fibers, but axo- and sarcolemma segments do oppose each other. Neuromuscular contacts without membrane specializations have been described in the osphradial ganglion of the freshwater snail Planorbis corneus. 3 Smooth muscle elements have also been demonstrated in the ganglionic neural sheath of Helix aspersa ~ and Aplysia californica. 35 However, the exact functional significance of this phenomenon is not known. The unspecialized contacts of serotonergic innervation of the smooth muscle fibers in the sheath also suggest a modulatory role for this substance. Such a role for 5-HT has been suggested in the buccal and lip musculature of several gastropod species.26,33,42,43 5HT-containing varicosities have been found using autoradiography in the buccal musculature of Helix) e The occurrence of 5-HT-IR varicose fibers in other muscular tissue (intestine) of Helix has also been described at the light microscopic level.mSAnalysing the ultrastructure of the innervation of the Helix intestine, Benedeczky and Halasy 2 found that varicosities contacted the smooth muscle fibers without membrane specializations and intravesicular release occurred by exocytosis. Non-specialized neuromuscular contacts established by 5-HT-IR varicosities have been found in the alimentary tract 21 and genital chamber ~° musculature of the cricket. The electron microscopic observations on the occurrence of 5-HT-IR endings, partially or completely free of glial processes in the neural sheath, supports an earlier concept ~6that these elements may release 5-HT into the hemolymph. Thus, 5-HT may be participating in the neurohormonal control of different peripheral
Fig. 4. 5-HT-IR varicosities (T) contact (between arrowheads) axon processes on the surface of the left pallial nerve. GC, glia cell; gl, glial processes; A, internally located axon. Scale bar = 1 #m. Fig. 5. 5-HT-IR varicosity (arrow) embedded between surface axon processes (A) in the intestinal nerve. The varicosity contains small agranular and large granular vesicles. Arrowhead points to large granular vesicles in unlabeled axons, gl, glial processes. Scale bar = 0.5 #m. Fig. 6. 5-HT-IR varicosities in the neural sheath. (a) A small varicosity (T) surrounded by muscle fibers (M) contacts one of them (between arrowheads), gl, gliai process with electron-dense granules. Scale bar = 0.25 #m. (b) 5-HT-IR processes (T) between muscle fibers (M), contacting one of them (between arrowheads) and remaining from the others for a relatively large distance (c. 0.2#m). Note large omega-shaped invaginations along the axolemma (open arrowheads), possibly indicating sites of release. cf, collagen fibers; hc, hemocoel. Scale bar = 0.25 #m. (c) 5-HT-IR varicosity containing mainly large granular vesicles (arrows) is freely embedded in the mass of collagen fibers (cf) in the outer layer of the neural sheath. M, muscle fiber process; arrowhead, gliai process. Scale bar = 0.25 gin. (d) 5-HT-IR varicosity partly surrounded by gliai process faces freely along a segment of its axolemma the mass of collagen fibers (cf) of the neural sheath. Scale bar = 0.25 #m.
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organs or blood cells. The buccal and foot musculature nerves of the insect nervous system has been d e m o n strated. 1' Experiments designed to investigate whether located in the vicinity o f the C N S m i g h t be, for such a release-upake m e c h a n i s m exists in the neural example, p r i m a r y targets o f 5-HT released t h r o u g h sheath of the Helix ganglia are in progress in o u r the neural sheath. A n e u r o h o r m o n a l role for 5-HT in laboratory. invertebrates has been suggested as early as 1954 by Welsh. ~ Such a role o f 5-HT t h r o u g h o u t the release Acknowledgements--The skilful technical assistance of into the h e a r t following stimulation was suggested Ms V. Pintrr and Mr B. Bal~.zs is much appreciated. earlier in Helix. 3s The participation o f 5-HT in neuroThe author's thanks are also due to Dr G. B. Stefano for h o r m o n a l regulation o f peripheral functions has also correcting the English. This work was supported by an been suggested in a r t h r o p o d s . ~'6'7'25'28'41A release a n d OTKA grant (No. 1-600-2-89-1-667) from the Hungarian u p t a k e m e c h a n i s m for 5-HT existing in the peripheral Academy of Sciences. REFERENCES 1. Beltz B, and Kravitz E. A. (1983) Mapping of serotonin-like immunoreactivity in the lobster nervous system. J. Neurosci. 7, 533-546. 2. Benedeczky I. and Halasy K. (1988) Visualization of non-synaptic release sites in the myenteric plexus of the snail, Helix pomatia. Neuroscience 25, 163-170. 3. Benjamin P. R. and Peat A. (1968) Myoneural junction in the connective tissue sheath of a molluscan ganglion. Nature 219, 1371-1372. 4. Croll R. P. (1987) Distribution of monoamines in the central nervous system of the nudibranch gastropod, Hermissenda crassicornis. Brain Res. 405, 337-347. 5. Croll R. P. and Chiasson B. J. 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(Accepted 3 December 1990)