The fine structure of the procerebrum of pulmonate molluscs, Helix and Limax

TISSUE 8- CELL 1970 2 (3) 399-411 Published by Oliver 5" Boyd, Edinburgh. Printed in Great Britain

I. Zs.-NAGY* and D. A. SAKHAROV-!-

THE FINE S T R U C T U R E OF THE P R O C E R E B R U M OF P U L M O N A T E MOLLUSCS, H E L I X A N D L I M A X ABSTRACT. Neuronal perikarya of the procerebrum of Helix and Limax are generally naked and lie side by side. The cell mass contains large numbers of axosomatic and axoaxonic synapses represented by boutons of t w o types; dense core vesicles ( 8 0 0 - 1 2 0 0 ~k in diameter) being characteristic of the first type and clusters of electron lucent vesicles ( 5 0 0 - 8 0 0 A) of the second. Endings of the t w o types occur also in the terminal mass of the neuropile while the internal mass contains peculiar axonal enlargements filled with fine twisted tubuli. Axons containing dense core vesicles seem to correspond to varicose monoaminergic fibres detected by a fluorescent histochemical method.

Introduction

THE procerebrum (PC) of pulmonate molluscs is a nervous centre of unique nature. Neurones in the PC are smaller than in the other ganglia of the circumoesophageal ring and their nuclei are notable for the abundance of chromatin (chromatic or globuli cells). Population of neurones seems to be homogeneous with respect to cell size while in other ganglia large and giant neurones occur together with small ones. it is also known that the PC attaches to the cerebral ganglion at a rather late stage of ontogenesis and that its morphogenesis differs from that of the rest of the brain. Light microscopic and comparative data are comprehensively treated by Bullock and Horridge (1965) and by Van Mol (1967). The PC has not been investigated physiologically. In former times however, such centres of invertebrate brains have been designated as 'Intelligenzsph/iren' (Haller,

1913; Hanstr6m, 1925), since Dujardin (1850) ascribed the function of intelligence to the corpora pedunculata of insects. This concept finds its modern development in the hypothesis of 'mnemons', memory units, implying small nerve cells---amacrines (Young, 1967). Apart from these far-fetched views, it should be admitted that the PC seems to have not only a peculiar structure but an unusual functional organization, too. With the intent of better understanding the organization of the PC, we resorted to electron microscopy. We faced also the need of histochemical localization of biogenic monoamines. Preliminary results have been published in a short communication (Zs.Nagy and Sakharov, 1969). Materials and Methods

The snail, Helix pornatia, and the slug, Limax cinerea-niger, were investigated electron microscopically while the histochemical demonstration of biogenic amines was * Biological Research Institute of the Hungarian carried out on H. pomatia, H. lucorum and Academy of Sciences, Tihany, Hungary. L. maximus. "~Institute of Developmental Biology, USSR The material for electron microscopy was Academy of Sciences, Moscow, USSR. fixed in 2% OsO4 buffered with s-collidine Received 21 November I969. (Bennett and Luft, 1959) for 30 rain. at 0°C Revised 16 March 1970. 399

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and subsequently for 10 rain. at room temperature. After dehydrating with ethanol and propylene oxide ganglia were embedded in Araldite (Durcupan ACM, Fluka). Sections were cut on an LKB Ultrotome 11I, stained with uranyl acetate and lead citrate (Reynolds, 1963). Micrographs were taken with a TESLA BS 413A electron microscope. Fluorescence microscopical demonstration of intraneuronal monoamines was carried out by an aqueous formaldehyde method. The procedure used (Sakharova and Sakharoy, 1968) was as follows: 1. Incubation of ganglia for 40 min. in ice-cold 1-4% formalin in snail Ringer; 2. cutting cryostat sections, mounting on slides, short drying in air stream

at about 0°C and then for 2-3 min. in 80"~C oven; 3. covering with paraffin oil and heating at 80'~C for 5-10 min.; 4. examination in fluorescence microscope.

General description and terminology i n the pulmonates studied, the PC consists of neural and glandular parts, the latter ('cerebral tube') occupying mainly an extraganglionic position (Van Mol, 1967). Nolte and Kuhlmann (1964) briefly described the ultrastructure of the epithelial cells of the 'cerebral tube'. Our description concerns only the neural part of the PC which is well developed in both Helix and Limax.

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Fig. 1. Schematic drawing of cerebral ganglia of He~ix and Limax. M S C - mesocerebrum; MTC--metacerebrum; PC--procerebrum. This latter consists of cell mass (CM), terminal mass (TM) and internal mass (IM). cc--cerebral commissure; c-plc--cerebro-pleural connective; nt+no--tentacular and optic nerves. Black spots correspond to the regions of nerve cells, the white ones to the neuropile.

Fig. 2. Special inclusion of 'Randzellen' in Helix pomatia. × 27,500. Fig. 3. Granulated mitochondria (GM) of 'Randzellen' in H, pomatia, × 30,000. Fig. 4. Nerve cell situated in the superficial region of the cell mass of the

He~ix procerebrum. M--mitochondria; L---lipid droplets; MV--multivesicular bodies; ER--endoplasmic reticulum; FR--free ribosomes. × 27,500. Fig. 5. Compact region of the cell mass (H. pomatia). NC--nerve cells; NE--nerve ending forming axo-somatic synapse of the second type (arrows). C--cytosome-like body; SV--synaptic vesicles. × 27,500. TISSUE ~ CELL 1970 2 (3)

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in each cerebral ganglion, the PC is of foremost, anterio-lateral position. There are some anatomical differences in topography of PC between Helix and Limax (Fig. 1); these however are not of fundamental importance. The neural part of the PC consists of the cell mass occupying mainly a lateral position and of the fibre matter. The cell mass is represented by densely packed neurones (globuli cells) and rather scanty glial cells. Some authors believe that neuronal population of PC contains cells of two types with different forms and staining properties (Pelluet and Lane, 1961; Nolte and Kuhlmann, 1964). The processes of globuli cells join into bundles which leave the cell mass in groups and enter the fibre matter. In Helix, between the cell mass and the outer perineurium a layer of large cells is situated (called 'Randzellen' by Nolte and Kuhlmann, 1964) which seems to be absent in Limax. The fibre matter or neuropile has three distinctive regions. Two of t h e m ~ h e internal mass and terminal mass--are shown in Fig. 1. The third one, the external mass, is actually located in the anterior part of the metacerebrum and its fibres are reported to stem at least partially from the buccal ganglia (Bullock and Horridge, 1965, p. 13 l 5). It seems doubtful whether this region of the neuropile should be regarded as a part of the PC. The terminal mass is adjacent to the cell mass and is clearly demarcated from the internal mass rangeing medially as far as the mesocerebrum. The internal mass in Limax is more developed than in Helix. Results

Electron microscopic observations

Cell mass (Helix). Entering the cell mass from the side of the perineurium first of all

the 'Randzellen' are met. These cells have a lobulated nucleus with a very voluminous perinuclear space, expansions of which traverse the cytoplasm and occasionally can be traced up to the external membrane of the cell. The cytoplasm is densely packed with various structures and inclusions such as homogeneous lipid globules of moderate density, vacuoles of various diameter, multivesicular bodies and rosettes of glycogen. There are special inclusions of different diameter (Fig. 2) and of high density. They seem to be atypical nuclei, approaching dissolution. One cannot be sure, however, concerning their real nature. Another peculiarity of the 'Randzellen' is the presence of granulated mitochondria (Fig. 3). The matrix of these mitochondria contains variable numbers of granules of high density. The character of nerve cells underlying 'Randzellen' is different in various layers. The most peripheral neurones have an irregular form and are more rich in cytoplasm (Fig. 4) compared with those lying in deeper layers. Besides the axon these neurones have short processes; apparently this is why the neurones of PC have been referred to as 'astrocytes' (Haller, 1913; Nolte and Kuhlmann, 1964). The presence of numerous multivesicular bodies is typical of these cells (Fig. 4). In deeper layers of the cell mass, neurones have a smooth outline and the dense packing of the cells is fully represented. Here the neurones show a drop-like form with a single process extending toward the fibre matter. Neuronal perikarya either lie side by side or their external membranes are separated by glial processes which are usually very fine. Among the neurones, perikarya of glial cells can occasionally be seen. The distinction between nerve and glial

Fig. 6. Glio-neural connection in the cell mass (H. pomatia). NC--nerve cell; GC--glial cell; GM--glial mitochondria ; V--vesicles substituting the cell membranes; LE--lacunar expansion of the perinuclear space in glial cell. × 32,500. Fig. 7. Axon (AX) showing t w o enlargements (AE) atthe border between the cell mass and the terminal neuropile in Limax cinerea-niger. The enlargements contact with the soma (NS) and the axons as well (arrows), forming glomerular synapse. × 27,500. TISSUE ~ CELL 1970 2 (3)

404 cells is easily made although these cells are approximately similar in size (10~). Unlike neurones, glial cells are multipolar, a few processes of about the same diameter leaving the cell body in various directions. The background of the glial cell nucleus is lighter while the osmiophilic sites are denser than in the nucleus of nerve cell (Fig. 6). Perinuclear space in the glial cell shows lacunar expansions which are absent in neurones (Fig. 6). Neuronal mitochondria both in the soma and in the axon are considerably smaller than glial ones. Finally, the glial cytoplasm occasionally shows bundles of fine fibrillar substance allowing the glial processes to be distinguished from the axons containing microtubules. Besides the above related structures, the neuronal perikaryon shows also endoplasmic reticulum of both agranular and granular types, free ribosomes, and sporadically compound globules reminiscent of the cytosomes (Fig. 5). These are about l-2t~ in diameter, usually have a limiting membrane and contain a fine fibrillar matrix as welt as lipid droplets with a rather dense surface layer. The homogeneous lipid inclusions (Fig. 4) are usually clustered at the axon hillock and have a high electron density. The perinuclear cytoplasm is as a rule very thin, thus indicating a high value of the nucleoplasmic ratio in the globuli cells. Occasionally, at the boundary between the nerve and glial cells, the external membranes are damaged and the intercellular cleft is replaced by a group of irregular vesicles (Fig. 6). At these sites, there is no morphologically manifested boundary between the two cells. Such pictures are usual for the PC, they can be traced in serial sections and occur only between nerve and glial cells, but they have never been observed between two neurones. It is characteristic that glial mitochondria are concentrated near these anomalous boundaries. The axons of nerve ceils of PC are fairly uniform as to their diameters (about 0'5~). Within the bundle, axons lie side by side, the gila[ processes isolating groups of axons rather than individual fibres. Outermost

ZS.-NAGY & SAKHAROV axons of a bundle pass sometimes close to the external membrane of the body of nerve cell, only the usual intercellular cleft being situated between the axon and the perikaryon. Within the bundle, there often occur one or a, few nerve fibres of greater diameter, containing dense-cored vesicles (DCV). After branching out of the stalk, these thick fibres run independently between nerve cells and form thereby axonal enlargements showing accumulations of DCVs. These enlargements come in contact with the somatic membranes of a few adjacent neurones. These axosomatic contacts have been referred to as synapses of the first type (Zs.-Nagy and Sakharov, 1969), since they are not the only form. Less often but regularly enough, there occur axonal enlargements containing agranular synaptic vesicles as well as one or a few mitochondria, forming axosomatic synapses of the second type (Fig. 5). The diameter of the agranular vesicles is about 500-800 ~ while that of the DCVs accounts about 800-1200 ~. Cell mass (Limax). Considering that the cell mass in Limax in general resembles that of Helix, we do not treat it in detail, only draw attention to the most interesting feature, i.e. the much more developed meshwork of synapses of the second type. Bouto•s containing agranular synaptic vesicles are most abundant in inner layers of the cell mass. These terminals occur, however, not only in the cell mass but also in the adjacent part of the fibre matter. The terminals of the second type distinctly differ from the thin part of the corresponding axon in their diameters and the ultrastructure of the axoplasm. Within the thin part, the fibre contains a bundle of microtubuli, scanty vesicles and lacunae of a variable character (Fig. 7). Such axons do not actually differ from the process emerging from a neuronal perikaryon. In the broad presynaptic part of the axon, the bundle of microtubuli breaks up, the outer zone of the enlargement contains one or a few clusters of electron-lucent vesicles accumulatedtoward the synaptic surface, while the centre is occupied by a group of mitochondria (Fig. 7). Glycogen-like granTISSUE 8" CELL 1970 2 (3)

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ules are sometimes very abundant in the central part of the enlargement. As in Helix, each axonal enlargement may contact with several nerve cells. The postsynaptic rnember of the contact is not always represented by a neuronal s o m a - - i t may be the proximal part of the axon as well. Naturally, these synapses occur not only in the cell mass, but also in the adjacent part of the fibre matter containing a bulk of proximal segments of axons of innermost nerve cells. The contacts of this type are sometimes so numerous that the somatic surface of a nerve cell is completely covered with boutons. Such pictures indicating a high degree of convergence of input fibres are unusual for gastropod nervous system, though c o m m o n in neurohistology of vertebrates. Invagination of a bouton into the soma and a local 'thickening' of synaptic membranes can also take place. Some presynaptic fibres cross at right angles the bundles of axons passing inward into the neuropile. Nerve cell perikarya, containing aggregations of agranular vesicles resembling those in the boutons of the second type, can be found now and again (Fig. 8). Lipid inclusions of the nerve cell cytoplasm in Limax differ morphologically from those found in Helix. Fibre matter (Helix). The neuropile is formed mainly by delicate axons of more or less uniform diameters (0"3-1"0t~). Glial processes are scanty. Multipolar perikarya of glial cells are comparatively numerous under the perineurium covering the medial pil ,rl, . . . . : neurium is formed by a ground substance impregnated with muscle and some other cell. Within the perineurium, nerve fibres containing D C V s can be seen. They are very likely involved in the innervation of the muscle cells of the brain sheath. The functional significance of these muscle cells is unclear. The character of the neuropile varies in different areas. There are extensive regions of thin parallel axons (Fig. 9) which apparently TISSUE 8- CELL 1970 2 (3)

405 should be regarded as connective tracts. Quite different are regions of crossing, interlacing and sometimes ramifying axons. Their axoplasm contains noticeably more mitochondria, vesicles of different types and glycogen than that of parallel axons. The region of crossing fibres might be a synaptic area (Fig. 10), however individual axoaxonic synapses cannot easily be recognized here on the usual morphological basis. Comparatively thick single axons containing D C V s occur in the fibre matter as well. Some of them pass just under the perineurium in a peculiar area rich in fibrous glia. Fibre matter (Limax). In general, the appearance of fibre matter resembles that of Helix. The area of terminal mass adjacent to the perineurium consists of a;{ons p o o r in inclusions of presynaptic types. These axons are not so strictly parallel as in the corresponding area of fibre matter in Helix. In other regions of the terminal mass the axons cross at various angles, often at right angles; large axona[ enlargements may contain aggregates of glycogen-like granules (Fig. 11), mitochondria and clusters of agranular synaptic vesicles (Fig. 12). Such enlargements are especially characteristic of the area adjacent to cell mass. An enlargement may make contacts with several postsynaptic fibres, forming in this way a synapse of glomerular type (Fig. 7). It should be noted that only a small number of similar structures was found in the Helix neuropile. We investigated also the internal mass of the Limax fibre matter. Fine and very fine axons also prevail here; unlike in the terminal mass, they havc irregular rather than round profiles thus leaving no expansions of interaxonal space. The ultrastructure of the axonal enlargements in the internal mass is extremely unusual: the axoplasm is filled with fine twisted tubuli (Fig. 13) whose diameter and density differ from those of microtubuli in the thin part of axons. It appears that the internal mass is the area of synaptic contacts whose nature and origin are different from the synapses of the terminal mass and of the cell mass.

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Fhlorescence microscopic observations Although the PC is the poorest of all parts of cerebral ganglion in monoaminergic elements, it regularly shows nerve fibres with fluorescence specific for biogenic amines (Fig. 14). In the terminal but not the internal mass of fibre matter, these fibres form a loose network of single fluorescing varicosities. The green colour of the fluorescence as well as reaction conditions used indicate the presence of a primary catecholamine. In Helix, a small amount of fluorescing axons produces a thin bundle along the inner boundary of the cell mass. Single fibres of the bundle enter the cell mass forming distinct bright varicosities among the neurones. A group of coarse fibres with green fluorescence passes over the dorsal part of the neuropile and continues into the tentacular nerve, constituting a bundle in its medial part. In Limax, the same green bundle can be seen in the medial part of nervus tentacularis. No boundary bundle was found in Limax, yet varicose fibres do occur in the cell mass, at least in its deepest layers. A small number of such fibres is dispersed all over the terminal mass of the slug. We have never observed perikarya with specific fluorescence of monoamines in the cell mass of the PC. However, monoaminergic neurones were found in the vicinity of the PC, both in medial and lateral regions of the anterior part of the metacerebrum. Some of these green fluorescing cells send their axons into the terminal mass of the neuropile of the PC. Coarse green fibres passing into the tentacular nerve seem to originate from fairly large fluorescing cells situated in the dorsal cellular rind of the visceral lobe of the metacerebrum.

The external mass is notable for the abundance of coarse green fibres. As we have already mentioned above, the external mass cannot be regarded as a part of the PC. The site of origin of the tentacular nerve in Helix is marked by intensive specific fluorescence, contrasting in this way to the rest of the PC. There is no corresponding area in the Limax PC. Discussion

The attention of many authors who studied the PC of stylommatophoran pulmonates was first of all drawn to the singularity of this nervous organ and to its distinction from the other ganglia of the central ring. The foremost position of PC in the brain and the fact that no similar organ had been found in lower gastropods led to the conclusion that the microscopical structure of the PC was a manifestation of the highest step of evolution of the central nervous system of gastropods (Haller, 1913; Hanstr6m, 1925). According to the latter author, the PC of Helix is a separated part of the tentacular ganglion, which shifted along the tentacular connective and came to adjoin the cerebral ganglion. It appears that there exists at least two types of ganglia in gastropods, showing different patterns of organization. Ganglia of the circumoesophagea[ ring, except the PC, and the buccal ganglia compose the first one, while the second includes the PC and accessory sensory ganglia of the head. The knowledge of the submicroscopic organization of nervous centers of gastropods has been based on numerous data on the first group of ganglia studied in pulmonates (Gerschenfeld, 1963; Amoroso et al., 1964),

Fig. 8. Perikaryon showing a cluster of agrunular vesicles (AV) in its cytoplasm. DCV dense core vesicles. Arrows point to the axo-somatic contact of the first type. N--nerve cell nuclei. Limax cinerea-niger, × 30,000. Fig. 9. Region of parallel axons (AX) in the terminal mass in He/ixpomatia. MT--microtubuli. × 27,500. Fig. 10. Region of crossing axons (AX) in the terminal mass in He/ixpomatia. L--lipid droplet ; G--glycogen-like granules, × 30,000. TISSUE 8- CELL 1970 2 (3)

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408 in a tectibranch, Aplysia (Rosenbluth, 1963; Coggeshall, 1967) and in some nudibranchs (Sakharov et al., 1965; Borovyagin and Sakharov, 1968; Nicaise et al., 1968; Schmeckel and Wechsler, 1968). In these centers, large, medium and small neurones covered by a glial trophospongium enclose the neuropile---a focus of all synaptic contacts. The absence of axosomatic synapses is a common feature in this kind of tissue. By contrast, in the PC the mass of small neurones devoid of satillite glia lies beside the neuropile which contains only a part of the synapses. Comparative ultrastructural data on neural organization of tentacular and other sensory ganglia would be of interest to verify how far such ganglia follow this line, It is not out of place to rnention here that in the corpora pedunculata of an insect there are also naked rather than sheathed small nerve cells (Landolt, 1965). Occurrence of axosomatic synapses has been reported in cephalopods on the basis of light microscopy (Young, 1939) and in pelecypods in electron micrographs (Zs.Nagy, 1964; /968b; Japha and Wachtel, 1969) while in gastropods the data are contradictory. The presence of pericellular fibres in the visceral ganglion of Aplysia was described by/~brfiham (1965) on the basis of silver impregnation; however electron microscopical investigations of the same ganglion could not confirm this finding (Rosenbluth, 1963; Coggeshall, 1967). Thus, typical axosomatic synapses in gastropod nervous system are found for the first time in the PC.

As to axoaxonic synapses, it should be mentioned that, in general, their morphological features are not conclusively distinct in the neuropile of gastropod ganglia; therefore such synapses appear to be less abundant than could be predicted from physiological data (Coggeshall, 1967; Borovyagin and Sakharov, 1968). On the contrary, the terminals of the second type in the Limax PC seem to represent a very distinct synaptic formation. It is one of the highly differentiated synapses of glomerular type described in neurohistology of invertebrates. Glomeruli of this kind have been found in the neuropile of corpora pedunculata in ants (Steiger, 1967) where the central presynaptic end knob aIso contains aggregations of glycogen granules. All the same, considerable difficulties remain in interpreting other axoaxonic contacts in the fibre matter. Only the general regions of synaptic interrelations can be identified with some confidence, but the attempts to detect individual synapses within these regions have no presently recognised uniform morphological basis. It should not be ruled out that the interaction between nerve fibres in such regions bears a generalized rather than one-to-one character. The origin of synapses found in the cell mass is not sufficiently clear. It seems most probable that fluorescing catecholamine varicosities correspond to synapses of the first type, since DCVs of molluscan nerve cells are known to represent the main storage sites of catecholamines (Zs.-Nagy,

Fig. 11. Region of crossing axons (AX) in the terminal mass in Limax cinerea niger. Note the aggregations of glycogen-like granules (G) in the axonal enlargements. × 30,000. Fig. 12. Axonal enlargement (AE) of the second type in theterminal mass in Limax. SV--synaptic vesicles; A X - - a x o n ; Arrows point to the axo-axonic synapse. × 44,000. Fig. 13. The internal mass of Limax procerebrum. AX--axons; TT--twisted tubu]i in axonal enlargements, x 32,500. Fig. 14. Fluorescence of fine varicose fibres in procerebrum of Helix/ucorum. Aqueous formaldehyde technique for the demonstration of biogenic amines. CM--cell mass, TM--terminal mass of the neuropile. × 600. TISSUE 8- CELL 1970 2 (3)

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410 1968a). The endings of the first type appear to originate from nerve cells containing a primary catecholamine and situated in the anterior part of the metacerebrum. On the contrary, nerve terminals of the second type may originate from cells situated in the PC. Bullock and Horridge (1965, Fig. 23.18, bottom right) refer to a figure taken from Veratti (1900) showing the G olgi-impregnated cells of the Limax PC. These cells give rise to processes branching among the neuronal perikarya. It seems likely that such a cell may be the presynaptic neurone forming endings of the second type. The explanation of the anomalous boundary between nerve and glial cells (Fig. 6) seems to be questionable. First of all, the possibility should be kept in mind that fragile membranes can break up into vesicles during the fixation (Gray, 1966). However, the fact that it never occurs between two nerve cells might suggest some specific significance of the phenomenon in the glioneural relationship. Even in the case of being a fixation artifact, it indicates some kind of lability of cell membranes which perhaps might represent a certain specific functional state.

Summary Submicroscopic morphology of the Helix and L i m a x procerebrum differs in some respects from that of previously studied ordinary nerve centres of gastropods. Neuronal perikarya of procerebrum are mainly naked and lie side by side while those of other ganglia are known to be separated by capsules of satellite glia. The neuropile of the procerebrum is not the only region of synaptic contacts: axosomatic and axoaxonic synapses are also to be found in the cell mass, represented by boutons of two types. Dense cored vesicles are characteristic of the first, and clusters of electron lucent vesicles of the second type. Synapses of the second type are much more developed in Limax than in Helix. The presynaptic enlargements may contact with several postsynaptic elements, forming glomerulus-like synapses. A scanty network of varicose monoaminergic fibres can be found both in the neuropile and in the cell mass of procerebrum by a fluorescent histochemical method; they seem to correspond to the presynaptic endings of the first type. Glial cells, 'Randzellen' and regional morphological features of the neuropile and the cell mass are also described.

References JkBRAHAM, A. 1965. Die Struktur der Synapsen im Ganglion viscerale yon Aplysia cal~fornica. Z. mikroskop.-anat. Forsch., 73, 45-59. AMOROSO, E. C., BAXTER, M. 1., CHIQUO1NE,A. D. and NISBET, R. H. 1964. The fine structure of neurons and other elements in the nervous system of the giant African land snail Archachatina marginata. Proc. Roy. Soc. (B), 160, 167-180. BENNETT, H. S. and LUFT, J. H. 1959, S-Collidine as a basis for buffering fixatives. J. Biophys. Biochem. Cytol., 6, 113-114. BOROVVAGIN,V. L. and SAKHAROV,D. A. 1968. The fine structure of giant neurons of Tritonia. Nauka, Moscow (in Russian). BtJL•OCg:, T. H. and HORRIDGE, G. A. 1965. Structure and function in the nervous s'ystems qf invertebrates, vol. 2. Freeman, San Francisco and London. COC,GrS•ALL, R. E. 1967. A light and electron microscope study of the abdominal ganglion of Aplysia californica. J. Neurophysiol., 30, 1263-1287. DUJARDIN, F. 1850. M6moire sur le syst6me nerveux des insectes. Ann. Sci. nat. (Zool.), (3), 14, 195205. GERSCUENFELD, H. M. 1963. Observations on the ultrastructure of synapses in some pulmonate molluscs. Z. Zellforseh., 60, 258-275. TISSUE 8" CELL 1970 2 (3)

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TISSUE 8- CELL 1970 2 (3)