Primary sensory neurons containing command neuron peptide constitute a morphologically distinct class of sensory neurons in the terrestrial snail

In the central nervous system of the terrestrial snail Helix, the gene HCS2, which encodes several neuropeptides of the CNP (command neuron peptide) family, is mostly expressed in cells related to withdrawal behavior. In the present work, we demonstrate that a small percentage (0.1%) of the sensory cells, located in the sensory pad and in the surrounding epithelial region ("collar") of the anterior and posterior tentacles, is immunoreactive to antisera raised against the neuropeptides CNP2 and CNP4, encoded by the HCS2 gene. No CNP-like-immunoreactive neurons have been detected among the tentacular ganglionic interneurons. The CNP-like-immunoreactive fiber bundles enter the cerebral ganglia within the nerves of the tentacles (tentacular nerve and medial lip nerve) and innervate the metacerebral lobe, viz., the integrative brain region well-known as the target area for many cerebral ganglia nerves. The procerebral lobe, which is involved in the processing of olfactory information, is not CNP-immunoreactive. Our data suggest that the sensory cells, which contain the CNP neuropeptides, belong to a class of sensory neurons with a specific function, presumably involved in the withdrawal behavior of the snail.     

Comparative study of nerve elements and their relationships with Endocrine glands and muscle retractors in Ommatophores of snails and slugs

A comparative study of the total cytoarchitectonics and structural interrelations of receptor and nerve elements at the area of receptor cell bodies, in tentacular ganglia and their digital outgrowths as well as in the lateral wall of tentacular olfactory organs of several species of terrestrial snails and slugs from different families was carried out using Golgi and Colonnier silver nitrate impregnation technique and horseradish peroxidase labeling. The presence of a peculiar cluster of intraepithelial receptor cells has been established for the first time in sensory epithelium of ommatophores. A connection of endocrine cells of the optic gland with nerve and receptor elements of tentacular ganglia was shown. Peculiarities of innervation of muscle refractors and lateral wall of ommatophores were revealed. FMRFamide-positive nerve fibers were found to participate in innervation of muscle elements of ommatophores. Neurotensin-positive and occasional serotonin-positive nerve elements also were revealed in the tentacular organs. Electron microscopic study showed the presence of complicated synaptic complexes of either convergent or divergent type and of many symmetric synaptic structures in neuropil of tentacular ganglia. Many nervous fibers and synapses contain several types of granular and agranular vesicles, which indicates possible co-localization of several bioactive substances in the same nerve elements.Translated from Zhurnal Evolyutsionnoi Biokhimii i Fiziologii, Vol. 40, No. 6, 2004, pp. 556–568.     

The fine structure of the collar cells in the optic tentacles of Helix aspersa

Summary At the base of the optic tentacular ganglion there is a group of large monopolar cells containing numerous secretory inclusions. These are the collar cells. Secretory material can be seen accumulating in swollen portions of the granular endoplasmic reticulum. It is postulated that this material is transported to the Golgi bodies and thus the limiting membrane of the inclusions is derived from the Golgi membranes. The Golgi bodies appear to be polarized and small vesicles resembling secretory inclusions are associated with one face of these organelles. The secretory inclusions fuse together to form large membrane-bound secretory pools in the perikaryon. The collar-cell processes are packed with secretory inclusions. These processes traverse the digital extensions of the tentacular ganglion and pass into the epithelium covering the tip of the tentacle. The secretory inclusions do not resemble neurosecretory inclusions in other situations. The collar cell processes receive a nerve supply from single axons containing granular and agranular vesicles. The evidence that these cells may be modified neurons is only minimal.This work was supported by the Australian Research Grants Committee.     

Collar cells in planula and adult tentacle ectoderm of the solitary coral Balanophyllia regia (anthozoa eupsammiidae)

Summary The planula larva of the solitary coral Balanophyllia regia has an ectoderm of flagellate, diplosomal collar cells. The collar of these cells is composed of a ring of microvilli linked with mucus strands. Four types of flagellate gland cells, three types of nematocyst and spirocysts are present in the planula ectoderm. The function of these ectoderm cells is discussed. The mesogloeal muscular and packing tissues of the planula are briefly described. The tentacle of the adult coral, examined for comparison, has an ectoderm of flattened flagellate cells with a shallow collar. Collar cells similar to those of the planula are occasionally found on the tentacle and their function is not known. Independent sensory cells built on a modified collar cell plan with collar of thickened microvilli are common in the tentacle. These are quite separate from the three kinds of tentacular nematocyte. Distended glandular areas occur in the tentacle ectoderm. The flagellate tentacle gastrodermis, muscle and mesogloeal region are briefly described. The evolutionary significance of collar cell ectoderm in a planula is discussed and the occurrence of collar cells throughout the animal kingdom, reviewed.I am most grateful to Paul Tranter of the Plymouth Laboratory for providing material and to Gareth Morgan for assistance with electron microscopy.     

Why the ovotestis of Helix aspersa is innervated

Although Schmalz described the innervation of the ovotestis in pulmonate snails as early as 1914, no functions have been attributed to it. In H. aspersa, the intestinal nerve branches profusely within the ovotestis and terminates in the walls of the acini and in the sheath surrounding the early portion of the hermaphroditic duct. We found both sensory and motor functions for this innervation. Significantly, there is a tonic sensory discharge generated by the mechanical pressure of growing oocytes, and the level of tonic afferent activity is strongly correlated with the number of ripe oocytes; this is probably a permissive signal that gates ovulation. Tactile stimulation of the ovotestis causes a phasic sensory discharge and a pronounced cardio activation. Also, an efferent discharge is elicited in the ovotestis branch of the intestinal nerve. To study the motor consequences of efferent activity, the ovotestis branch was electrically stimulated. We found that such stimulation evokes peristaltic contractions of the initial portion of the hermaphroditic duct and increases beat frequencies of the cilia that line the interior of the duct. These effects could facilitate the transport of oocytes down the duct. Still other functions of afferent activity are implied by changes in the spontaneous activity of mesocerebral cells following nerve stimulation. Putative sensory neurons and putative motoneurons have been identified in the visceral and right parietal ganglia.     

Interaction of chemosensory, visual, and statocyst pathways in Hermissenda crassicornis

Neurons in the cerebropleural ganglia (CPG), photoreceptors in the eye, optic ganglion cells, and statocyst hair cells of the nudibranch mollusk Hermissenda crassicornis responded in specific ways, as recorded intracellularly, to stimulation of the chemosensory pathway originating at the tentacular chemoreceptors as well as to stimulation of the visual pathway originating at the photoreceptors. Synaptic inhibition of photoreceptors occurs via the chemosensory pathway. The possible significance of such intersensory interaction is discussed with reference to preliminary investigation of the animal's gustatory behavior and possible neural mechanisms of behavioral choice.     

Modulation of two oscillatory networks in the peripheral olfactory system by gamma-aminobutyric acid, glutamate, and acetylcholine in the terrestrial slug Limax marginatus

The digit-like extensions (the digits) of the tentacular ganglion of the terrestrial slug Limax marginatus are the cell body rich region in the primary olfactory system, and they contain primary olfactory neurons and projection neurons that send their axons to the olfactory center via the tentacular nerves. Two cell clusters (the cell masses) at the bases of the digits form the other cell body rich regions. Although the spontaneous slow oscillations and odor responses in the tentacular nerve have been studied, the origin of the oscillatory activity is unknown. In the present study, we examined the contribution of the neurons in the digits and cell masses to generation of the tentacular nerve oscillations by surgical removal from the whole tentacle preparations. Both structures contributed to the tentacular oscillations, and surgical isolation of the digits from the whole tentacle preparations still showed spontaneous oscillations. To analyze the dynamics of odor-processing circuits in the digits and tentacular ganglia, we studied the effects of gamma-aminobutyric acid, glutamate, and acetylcholine on the circuit dynamics of the oscillatory network(s) in the peripheral olfactory system. Bath or local puff application of gamma-aminobutyric acid to the cell masses decreased the tentacular nerve oscillations, whereas the bath or local puff application of glutamate and acetylcholine to the digits increased the digits' oscillations. Our results suggest the existence of two intrinsic oscillatory circuits that respond differentially to endogenous neurotransmitters in the primary olfactory system of slugs.     

Testosterone,gonadotropins and androgen receptor during spermatogenesis of Biomphalaria alexandrina snails (Pulmonata: Basommatophora)

Endocrine regulation of reproductive processes of the snail Biomphalaria alexandrina is poorly recognized. Thus, the aims of the study were: (1) to acquire histological images of the ovotestis; (2) to determine the hemolymph concentrations of testosterone (T) and gonadotropic hormones (luteinizing hormone: LH and follicle stimulating hormone: FSH), (3) to demonstrate androgen receptor (AR) immunolocalization in the ovotestis, and (4) to show LH and FSH protein expression in cerebral ganglia of small (diameter shell: 4–6 mm), medium (7–11 mm) and large (12–16 mm) B. alexandrina snails. These three groups represented different reproductive stages of the snail. The AR immunoexpression was found in the periphery and inside the acini of small (immature) snails as well as in spermatocytes, spermatids, Sertoli cells, the interstitial cells and the acinus lining epithelium of medium (mature) snails. Low AR immunoexpression was demonstrated in the interstitial cells of large (aged) snails. The neurons at the periphery of the cerebral ganglia and connective sheath of the ganglia showed a positive FSH and LH immunostaining. T concentration in the hemolymph was higher in medium snails than in small and large snails. In contrast, LH concentration was higher in medium snails than in small and large snails. These data suggests that gonadotropins and T play a role in the gonadal development in B. alexandrina.     

Cerebral parts of chemosensory systems of the snail: Structural bases of intersensory integration

Presented are data on distribution of afferent fibers from tentacular and labial nerves innervating chemosensory tentacular organs, lips, and mouth area in the cerebral ganglia of terrestrial pulmonary snails are in the article. By using Golgi impregnation and infusion of cobalt chloride and nickel, it has been shown that most terminal branches of afferent fibers of all chemosensory organs are located in several symmetric areas of both metacerebrums. A part of fibers both of tentacular and of labial nerves form peculiar tracts and terminate in the interior neuropil of procerebrums. In metacerebrums, various neurons providing connections with afferent fibers of chemosensory organs are revealed. Many of them also innervate interior neuropil and procerebrum cell bodies area. The data obtained allow considering procerebrums as higher integrative centers of chemosensory information.     

Modulation of two oscillatory networks in the peripheral olfactory system by γ‐aminobutyric acid,glutamate, and acetylcholine in the terrestrial slug Limax marginatus

The digit‐like extensions (the digits) of the tentacular ganglion of the terrestrial slug Limax marginatus are the cell body rich region in the primary olfactory system, and they contain primary olfactory neurons and projection neurons that send their axons to the olfactory center via the tentacular nerves. Two cell clusters (the cell masses) at the bases of the digits form the other cell body rich regions. Although the spontaneous slow oscillations and odor responses in the tentacular nerve have been studied, the origin of the oscillatory activity is unknown. In the present study, we examined the contribution of the neurons in the digits and cell masses to generation of the tentacular nerve oscillations by surgical removal from the whole tentacle preparations. Both structures contributed to the tentacular oscillations, and surgical isolation of the digits from the whole tentacle preparations still showed spontaneous oscillations. To analyze the dynamics of odor‐processing circuits in the digits and tentacular ganglia, we studied the effects of γ‐aminobutyric acid, glutamate, and acetylcholine on the circuit dynamics of the oscillatory network(s) in the peripheral olfactory system. Bath or local puff application of γ‐aminobutyric acid to the cell masses decreased the tentacular nerve oscillations, whereas the bath or local puff application of glutamate and acetylcholine to the digits increased the digits' oscillations. Our results suggest the existence of two intrinsic oscillatory circuits that respond differentially to endogenous neurotransmitters in the primary olfactory system of slugs. © 2004 Wiley Periodicals, Inc. J Neurobiol 59: 304–318, 2004     

Distribution of biogenic amines in the slug,Limax maximus

Summary The distribution of monoamines inLimax maximus was studied by the histochemical fluorescent method of Falck and Hillarp. The number of 5-HT-containing and catecholamine-containing perikarya in the central nervous system is small compared with the non-fluorescent perikarya. However, all the ganglia except the proto-cerebral ganglia have some amine-containing neurons. There are relatively larger numbers of fluorescent cells in the cerebral, visceral, pedal and right parietal ganglia than in the other ganglia. A single, giant 5-HT-containing neuron was observed in each meta-cerebral ganglion.Monoamine neurons are localised in a number of peripheral tissues (heart, integument, tentacles, penis retractor muscle, sole of foot, kidney, alimentary canal, reproductive organs and tentacular, pharyngeal and cephalic retractor muscles). Neurons containing catecholamine are mostly associated with sensory structures such as the statocysts, the retina of the eye and the integument of the tentacles, whereas 5-HT-containing nerve fibres are mainly observed in muscle tissues.We wish to thank the Wellcome Trust for financial support.     

Immunocytochemical detection of substances related to methionine enkephalin in the tentacles and food of the snail (Helix aspersa)

Immunocytochemistry reveals the presence of methionine-enkephalin-like substance(s) in the collar cells of the two kinds of tentacles and in the foot of the snail Helix aspersa. The density of the immunoreactive material is higher in young animals than in adults. The greater part of the substance(s) is released at the surface of the epidermis and probably mixed with the mucus. A possible neuroendocrine and/or neuromodulatory function can be considered especially for the collar cells connected with the tentacular ganglia.     

The distribution of NADPH diaphorase activity and immunoreactivity to nitric oxide synthase in the nervous system of the pulmonate mollusc Helix aspersa

Enzyme histochemistry and immunocytochemistry were used to determine the distribution of neurons in the snail Helix aspersa which exhibited nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase activity and/or immunoreactivity to nitric oxide synthase (NOS). NADPH diaphorase-positive cells and fibres were distributed extensively throughout the central and peripheral nervous system. NADPH diaphorase-positive fibres were present in all neuropil regions of the central and peripheral ganglia, in the major interganglionic connectives and in peripheral nerve roots. NADPH diaphorase-positive cell bodies were found consistently in the eyes, the lips, the tentacular ganglia and the procerebral lobes of the cerebral ganglia; staining of cell bodies elsewhere in the nervous system was capricious. The distribution of NOS-like immunoreactivity differed markedly from that of NADPH diaphorase activity. Small clusters of cells which exhibited NOS-like immunoreactivity were present in the cerebral and pedal ganglia; fibres which exhibited NOS-like immunoreactivity were present in restricted regions of the neuropil of the central ganglia. The disjunct distributions of NADPH diaphorase activity and NOS-like immunoreactivity in the neurvous system of Helix suggest that the properties of neuronal NOS in molluscs may differ sigificantly from those described previously for vertebrate animals.     

Localization of a substance P-like material in the central and peripheral nervous system of the snail Helix aspersa

Summary A monoclonal antibody against substance P was used for immunocytochemical staining of the central ganglia of the snail Helix aspersa and several peripheral tissues including the gut, reproductive system, cardiovascular system, tentacle and other muscles.Within the central ganglia many neurones, and many fibres in the neuropile and the nerves entering the ganglia, were stained for the SP-like material. The largest numbers of reactive cell bodies were in the pleural ganglia and on the dorsal surfaces of the pedal ganglia. A group of cells was also found, surrounding the right pedal-cerebral connective, that did not fluoresce, but were enveloped by reactive processes terminating directly onto the neurone somata.Specific staining was observed in all peripheral tissues examined and always appeared to be concentrated in nerve terminals. Most particularly these occurred in the heart and aorta, the pharyngeal retractor muscle and the tentacle. Although mostly present in muscular tissues, some fluorescence was also observed in the nervous layer surrounding the retina. The tentacular ganglion also contained immunoreactive cell bodies.     

Localization of a substance P-like material in the central and peripheral nervous system of the snail Helix aspersa

A monoclonal antibody against substance P was used for immunocytochemical staining of the central ganglia of the snail Helix aspersa and several peripheral tissues including the gut, reproductive system, cardiovascular system, tentacle and other muscles. Within the central ganglia many neurons, and many fibres in the neuropile and the nerves entering the ganglia, were stained for the SP-like material. The largest numbers of reactive cell bodies were in the pleural ganglia and on the dorsal surfaces of the pedal ganglia. A group of cells was also found, surrounding the right pedal-cerebral connective, that did not fluoresce, but were enveloped by reactive processes terminating directly onto the neurone somata. Specific staining was observed in all peripheral tissues examined and always appeared to be concentrated in nerve terminals. Most particularly these occurred in the heart and aorta, the pharyngeal retractor muscle and the tentacle. Although mostly present in muscular tissues, some fluorescence was also observed in the nervous layer surrounding the retina. The tentacular ganglion also contained immunoreactive cell bodies.     

Fine Structure of Tentacles,Arms and Associated Coelomic Structures of Cephalodiscus gracilis (Pterobranchia,Hemichordata)

The tentacles of the pterobranch Cephalodiscus, a hemisessile ciliary feeder, originate from the lateral aspects of the arms and are covered by an innervated epithelium, the majority of its cells bearing microvilli. Each side of a tentacle has two rows of ciliated cells and additional glandular cells. The coelomic spaces in the tentacles are lined by cross-striated myoepithelial cells, allowing rapid movements of the tentacles. One, possibly two, blood vessels accompany the coelomic canal. On their outer sides the arms are covered by a simple ciliated epithelium with intra-epithelial nerve fibres; the inner side is covered by vacuolar cells. On both sides different types of exocrine cells occur. The collar canals of the mesocoel are of complicated structure. Ventrally their epithelium is pseudostratified and ciliated; dorsally it is lower and forms a fold with specialized cross-striated myoepithelial cells of the coelomic lining. Arms, tentacles, associated coelomic spaces and the collar canal of the mesocoel are considered to be functionally interrelated. It is assumed that rapid regulation of the pore width is possible and even necessary when the tentacular apparatus is retracted, which presumably leads to an increase of hydrostatic pressure in the coelom.     

Cellular structure and ultrastructure of the black coral Antipathes aperta: 1. Organization of the tentacular epidermis and nervous system

The tentacular epidermis of the black coral Antipathes aperta is organized into three distinct regions, containing at least nine different types of cells. The outermost region is dominated by spirocytes along with two types of nematocytes, organized into discrete wart-like batteries. The two nematocyte types both contain microbasic b-mastigophore nematocysts. The outer boundary of the wart is marked by the presence of both spumous and vesicular mucus cells. The ciliation of the wart is contributed principally by the spirocytes. Warts are enveloped and separated from one another by an unusual neurosensory cell complex that extends from the tentacular surface to the mesogleal connective tissue foundation. Funnel-like, flagellated cells composing the complex connect with ganglion cells composing the dominant portion of the nerve net system. Branches of this complex also penetrate the central portion of the wart, making direct contact with the cnidae. The tentacular mid-region is composed of nematocytes and spirocytes in various stages of maturation, along with epitheliomuscular cell (EMC) somata. The EMC's narrow apically extend toward the tentacle surface, forming contacts with the cnidae. The basal end of the EMC expands to form the larger portion of the tentacular musculature. The inner region of tentacular epidermis is marked by a neuromuscular complex sheathed by extensions of mesoglea. The ganglion cells occur as a plexus deep within the tentacle and form polarized junctions with the EMC's, but neuromuscular synapses are not well enough defined for documentation. Polarized synapses lacking well-defined membrane thickenings characterize the interneuronal junctions. Granular cells lining the mesogleal surface appear to be responsible for mesogleal fibrillogenesis.     

Distribution and coexistence of urotensin I and urotensin II peptides in the cerebral ganglia of Aplysia californica.

Urotensin I (UI) and urotensin II (UII) were demonstrated in the cerebral ganglia of Aplysia californica by applying immunocytochemical and radioimmunoassay procedures. Sequential analysis of adjacent sections of the cerebral ganglia of Aplysia demonstrated that the UI-immunoreactive (UI-IR) neurons of the F cluster of the cerebral ganglia also contained UII immunoreactivity (UII-IR). Both UI-IR and UII-IR were also observed in a cuff-like arrangement of fibers surrounding the proximal portion of the supralabial nerve, as well as in a few fibers in the anterior tentacular nerves. The UI-IR perikarya of the cerebral ganglia appeared to project to the entire CNS of Aplysia, but the UII-IR fibers appeared only in the neuropile and commissure of the cerebral ganglia. The UI-IR staining was abolished by previous immunoabsorption of the UI antiserum with sucker (Catastomus commersoni) UI, but not with ovine corticotropin-releasing factor (CRF), rat/human CRF, or goby (Gillichthys mirabilis) UII. Immunostaining with UII antiserum was quenched by goby UII, but not by sucker UII-A, UII-B, UII-A(6-12), or carp (Cyprinus carpio) UII-alpha and UII-gamma. The UII staining was not abolished by UI or somatostatin. The F cluster was not stained when a somatostatin antiserum was applied. Radioimmunoassay of dilutions of cerebral ganglia extract, using UII antiserum, revealed a parallel displacement curve to synthetic goby UII.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential localization of vasopressin- and oxytocin-immunoreactive cells and conventional neurosecretory cells in the ganglia of the earthworm Pheretima hilgendorfi

Cells containing arginine vasopressin (AVP)- and oxytocin (OXT)-like substances were immunohistochemically visualized in the cerebral, subesophageal, and ventral nerve cord ganglia of the earthworm Pheretima hilgendorfi. Whether these anti-AVP– and anti-OXT–reactive cells are identical with classical aldehyde fuchsin (AF)-positive neurosecretory cells was tested in serial sections. In all ganglia, groups of scattered neuronal cell bodies and axons strongly reactive to AVP and OXT antisera were observed, but AF-positive cells consisting of type a (dark blue) and type b (purple) cells were predominantly present in the cerebral and subesophageal ganglia. In the cerebral and subesophageal ganglia anti-AVP– and anti-OXT–reactive cells were generally larger than AF-positive cells. Some AF-positive cells were reactive either to anti-AVP or anti-OXT serum, but some failed to react to either serum. Anti-AVP– and anti-OXT–reactive cells were not immunoreactive to OXT and AVP antisera, respectively. Electron microscopic observations showed that the granules of type a cells were larger and less electron dense than those of type b cells and anti-AVP–reactive cells. The present cytological observations clearly showed that AVP- and OXT-like substances were widely present in the ganglionic cells of the earthworm     

A cytologic analysis of the bag cell control of egg laying in Aplysia

A fine structural analysis of the ovotestis in Aplysia was undertaken in order to analyze the site of action of the bag cell hormone. Five stages of oocyte development are described. Of particular interest is the fact that the yolk seems to be synthesized primarily by the granular endoplasmic reticulum. In addition, small muscle cells whose long, thin processes surround the follicle of the ovotestis have been pointed out. This paper suggests that bag cell extract has a direct action on these small muscle cells causing them to contract and thus expel oocytes from the ovotestis. The evidence for this suggestion is that (1) these muscle cells are the most obvious effector cells in the ovotestis, (2) there are no signs of neural innervation of these muscles, (3) the time course for the liberation of the oocytes is so short that any other method of oocyte release is unlikely, (4) there is no cytologic evidence for any other expulsion process except muscular contraction, and (5) the ripe oocytes are attached to other cells of the wall of the ovotestis only by very small, simple junctions, thus making them the most likely cells to be expelled by muscular contraction.