Neurogenesis of cephalic sensory organs of <Emphasis Type="Italic">Aplysia californica</Emphasis>

The opisthobranch gastropod Aplysia californica serves as a model organism in experimental neurobiology because of its simple and well-known nervous system. However, its nervous periphery has been less intensely studied. We have reconstructed the ontogeny of the cephalic sensory organs (labial tentacles, rhinophores, and lip) of planktonic, metamorphic, and juvenile developmental stages. FMRFamide and serotonergic expression patterns have been examined by immunocytochemistry in conjunction with epifluorescence and confocal laser scanning microscopy. We have also applied scanning electron microscopy to analyze the ciliary distribution of these sensory epithelia. Labial tentacles and the lip develop during metamorphosis, whereas rhinophores appear significantly later, in stage 10 juveniles. Our study has revealed immunoreactivity against FMRFamides and serotonin in all major nerves. The common labial nerve develops first, followed by the labial tentacle base nerve, oral nerve, and rhinophoral nerve. We have also identified previously undescribed neuronal pathways and other FMRFamide-like-immunoreactive neuronal elements, such as peripheral ganglia and glomerulus-like structures, and two groups of conspicuous transient FMRFamide-like cell somata. We have further found two distinct populations of FMRFamide-positive cell somata located both subepidermally and in the inner regions of the cephalic sensory organs in juveniles. The latter population partly consists of sensory cells, suggesting an involvement of FMRFamide-like peptides in the modulation of peripheral sensory processes. This study is the first concerning the neurogenesis of cephalic sensory organs in A. californica and may serve as a basis for future studies of neuronal elements in gastropod molluscs. This work was supported by the German Science Foundation (DFG; Kl 1303/3-1 to A.K.K.), SYNTHESYS (DK-TAF-202 to T.W.), the German Academic Exchange Service (DAAD to T.W.), the Danish Natural Science Research Council (FNU; grants 21-04-0356 and 272-05-0174 to A.W.), and the Carlsberg Foundation (grant 2005-1-249 to A.W.).     

Serotonin-like immunoreactivity in the central and peripheral nervous systems of the interstitial acochlidean Asperspina sp. (Opisthobranchia)

Species of Acochlidea are common members of the marine interstitial environment and defined in part by their minuscule size and highly divergent morphology relative to other benthic opisthobranchs. Despite these differences, acochlideans such as species of Asperspina display many plesiomorphic characteristics, including an unfused condition of their neural ganglia. To gain insight into the distribution of specific neural subsets within acochlidean ganglia, a species of Asperspina was studied by using anti-serotonin immunohistochemistry and epifluorescence and confocal laser scanning microscopy. Results reveal similarities between Asperspina and larger opisthobranchs in the general distribution of serotonergic perikarya in the central nervous system. Specifically, the arrangement of perikarya into regional clusters within the cerebral and pedal ganglia and the absence of immunoreactive perikarya in the pleural ganglia are similar to the model species of Aplysia californica, Pleurobranchaea californica, and Tritonia diomedea. Moreover, serotonergic innervation of the rhinophores in all opisthobranchs, including Asperspina sp., originates from the cerebral ganglion instead of directly from the rhinophoral ganglion. Serotonergic innervation of the body wall, including the epithelium, muscles, and pedal sole, appears to arise exclusively from pedal and accessory ganglia. These observations indicate a general conservation of serotonin-like immunoreactivity in the central and peripheral nervous systems of acochlidean and other benthic opisthobranchs.     

Detection of FMRFamide-like immunoreactivities in the sea scallopPlacopecten magellanicus by immunohistochemistry and Western blot analysis

FMRFamide-like immunoreactivity was detected histochemically in the sea scallopPlacopecten magellanicus. Most immunoreactivity was concentrated in the cerebral, pedal, and parietovisceral ganglia, particularly in the cortical cell bodies and in their fibers which extend into the central neuropile. Whole-mount immunofluorescence studies were used to localize concentrations of immunoreactive cells on the dorsal and ventral surfaces of each ganglion. Immunoreactivity was also detected in nerves emanating from the ganglia. Strong immunoreactivity was localized in peripheral organs, including the gut and gills of juvenile and adult scallops. Weak immunoreactivity was detected in the gonads, heart, and adductor muscle of the adults. A broad FMRFamide-like immunoreactive band of 2.5–8.2 kDa was detected by Western blotting of acetone extracts of the parietovisceral ganglia. In the presence of protease inhibitors, two FMRFamide-like immunoreactive bands (7.2–8.2 kDa and >17 kDa) were obtained. Neither of these bands comigrated with the FMRFamide standard. It is concluded that peptides of the FMRFamide family are probably regulators of numerous central and peripheral functions inP. magellanicus.     

Comparative immunohistochemistry of the cephalic sensory organs in Opisthobranchia (Mollusca,Gastropoda)

The structure and function of the central nervous systems of opisthobranch gastropods have been studied extensively. However, the organisation and function of the peripheral nervous system are poorly understood. The cephalic sensory organs (CSOs) are known to be chemosensory structures in the head region of opisthobranchs. In the present study, we used immunohistochemical methods and confocal laserscanning microscopy to comparatively examine the CSOs of different opisthobranchs, namely Acteon tornatilis, Aplysia punctata, Archidoris pseudoargus and Haminoea hydatis. We wanted to characterise sensory epithelia in order to infer the function of sensory structures and the organs they constitute. Immunoreactivity against the three antigens tyrosine hydroxylase, FMRFamide and serotonin was very similar in the CSOs of all investigated species. Tyrosine hydroxylase-like immunoreactivity was detected primarily in subepidermal sensory cell bodies, which were much more abundant in the anteriorly situated CSOs. This observation indicates that these cells and the respective organs may be involved in contact chemoreception and mechanoreception. The dominant features of FMRFamide-like immunoreactivity, especially in the posterior CSOs, were tightly knotted fibres which reveal glomerulus-like structures. This suggests an olfactory role for these organs. Serotonin-like immunoreactivity was detected in an extensive network of efferent fibres, but was not found within any peripheral cell bodies. Serotonin-like immunoreactivity was found in the same glomerulus-like structures as FMRFamide-like immunoreactivity, indicating a function of serotonin in the efferent control of olfactory inputs. Besides this functional implication, this study could also add some knowledge on the doubtful homology of the CSOs in opisthobranch gastropods.     

Observations of serotonin and FMRFamide-like immunoreactivity in palp sensory structures and the anterior nervous system of spionid polychaetes

Evidence suggests that ciliated sensory structures on the feeding palps of spionid polychaetes may function as chemoreceptors to modulate deposit-feeding activity. To investigate the probable sensory nature of these ciliated cells, we used immunohistochemistry, epi-fluorescence, and confocal laser scanning microscopy to label and image sensory cells, nerves, and their organization relative to the anterior central nervous system in several spionid polychaete species. Antibodies directed against acetylated alphatubulin were used to label the nervous system and detail the innervation of palp sensory cells in all species. In addition, the distribution of serotonin (5-HT) and FMRFamide-like immunoreactivity was compared in the spionid polychaetes Dipolydora quadrilobata and Pygospio elegans. The distribution of serotonin immunoreactivity was also examined in the palps of Polydora cornuta and Streblospio benedicti. Serotonin immunoreactivity was concentrated in cells underlying the food groove of the palps, in the palp nerves, and in the cerebral ganglion. FMRFamide-like immunoreactivity was associated with the cerebral ganglia, nuchal organs and palp nerves, and also with the perikarya of ciliated sensory cells on the palps.     

Octopamine-like immunoreactive neurones in locust genital abdominal ganglia

Using a well characterized anti-serum, the distribution of octopamine-like immunoreactive neurones is described in the locust seventh abdominal (A7) and terminal ganglia (TG), which are associated with genital organs. Apart from 4 paired ventral somata occasionally observed in the TG, all labelled cells could be identified as efferent dorsal- and ventral unpaired median (DUM/VUM) neurones by virtue of the characteristic large size and position of their somata, projections of their primary neurites in DUM-cell tracts, and bifurcating axons which arise from dorsal T-junctions and enter peripheral nerves. For the examined ganglia our data indicate that the whole population of efferent DUM and VUM-cells, defined here as progeny of the segment specific unpaired median neuroblast with peripheral axons, are octopaminergic, and that equal numbers of these cells occur in both sexes: 8 in A7 and 11 in TG. Sex-specific differences are probably restricted to the axonal projections of 5 octopamine-like immunoreactive DUM-somata in A7, and 5 in TG, which in females project into their segment specific sternal nerves, but in males into the genital nerve of the TG. Numerous intersegmentally projecting octopamine-like immunoreactive fibres traverse both ganglia. The majority probably stem from previously described octopamine-like immunoreactive neurones in the thoracic and suboesophageal ganglia.     

Fine structure of the larval rhinophores of the nudibranch,Rostanga pulchra,with emphasis on the sensory receptor cells

Summary The rhinophores of the veliger larva of Rostanga pulchra are located in the intravelar field near the base of the velar lobes. Each rhinophore is a cylindrical structure, tapering distally, and covered with a dense meshwork of microvilli. A conspicuous row of ciliary tufts runs along each side of the rhinophore and several stiffer tufts, composed of fewer cilia, are positioned around the tip or at the base. The rhinophoral epithelium consists of supporting cells, ciliated cells (giving rise to the ciliary rows), dendritic terminals (giving rise to the tufts around the apex), and sinuses containing occasional amebocytes. The lumen of the rhinophore is occupied by the rhinophoral ganglion and muscle cells that are oriented in two perpendicular planes. Cell bodies of the dendritic endings are located within the rhinophoral ganglion, which in turn joins into the optic and cerebral ganglia. Rhinophoral ganglionic neurons do not synapse with each other, but numerous neuromuscular synapses are found in the lumen of the rhinophore.Morphological evidence suggests that the dendritic endings are chemoreceptors and the ciliated cells are possibly mechanoreceptors but are not functional at this stage in development. The functional role of the rhinophores is discussed in relation to larval behavior at settlement and metamorphosis.     

Immunocytochemical demonstration of neuropeptides in the fish-gill parasite, Diclidophora merlangi (Monogenoidea)

Using the indirect immunofluorescence technique, immunoreactivity (IR) to three mammalian and one invertebrate regulatory peptide has been demonstrated in the nervous system of the monogenean gill parasite Diclidophora merlangi. IR to pancreatic polypeptide (PP), peptide tyrosine tyrosine (PYY) and FMRFamide was evident throughout central and peripheral nervous tissues, whereas vasoactive intestinal polypeptide (VIP)-IR was confined to a portion of the longitudinal ventral nerve cords. Staining patterns revealed the orthogonal arrangement of the nervous system consisting of paired cerebral ganglia, connecting post-pharyngeal commissure, three pairs of longitudinal nerve cords and associated neurones. PP-IR, PYY-IR and FMRFamide-IR were intense throughout the central nervous system of the worm. A small plexus of nerve fibres and somata in each peduncle was immunoreactive for FMRFamide and provided innervation to each of the eight posterior clamps. In the peripheral nervous system, PP-IR, PYY-IR and FMRFamide-IR occurred in an extensive nerve-net with fine, possibly sensory nerve endings in the tegument. PP-IR was also present in nerve fibres in the walls of the ootype, seminal vesicle and uterus. PYY- and FMRFamide-IRs, while evident in nerve fibres of the ootype wall, were also present in a distinct population of cells that encircles the ootype, and which are linked to it by fine cytoplasmic connectives. The majority of these somata were bipolar or multipolar. PYY-IR and FMRFamide-IR were also associated with nerve fibres and bipolar cells in the wall of the vitelline reservoir. Regulatory peptides would appear to play an integral role in neuronal functioning and egg development in D. merlangi.     

Small cardioactive peptide-like immunoreactivity and its colocalization with FMRFamide-like immunoreactivity in the central nervous system of the leech Hirudo medicinalis

Summary The distributions of small cardioactive peptide (SCP)- and FMRFamide-like immunoreactivities in the central nervous system of the medicinal leech Hirudo medicinalis were studied. A subset of neurons in the segmental ganglia and brains was immunoreactive to an antibody directed against SCP_B. Immunoreactive cell bodies were regionally distributed throughout the nerve cord, and occurred both as bilaterally paired and unpaired neurons. The majority of the unpaired cells displayed a tendency to alternate from side to side in adjacent ganglia. A small number of neurons were immunoreactive only in a minority of nerve cords investigated. Intracellular injections of Lucifer yellow dye and subsequent processing for immunocytochemistry revealed SCP-like immunoreactivity in heart modulatory neurons but not in heart motor neurons. FMRFamide-like immunoreactivity was also detected in cell bodies throughout the central nervous system. A subset of neurons contained both SCP- and FMRFamide-like immunoreactivities; others stained for only one or the other antigen. These data suggest that an antigen distinct from FMRFamide is responsible for at least part of the SCP-like immunoreactivity. This antigen likely bears some homology to the carboxyl terminal of SCP_A and SCP_B.     

Localization of substance P-like,leucine-enkephalin-like,methionine-enkephalin-like,and FMRFamide-like immunoreactivity in the eyestalk of the fiddler crab,Uca pugilator

Summary The occurrence and distribution of substance P (SP)-like, methionine-(Met)- and leucine-(Leu)-enkephalinlike, and FMRFamide-like immunoreactivities were determined in the neuroendocrine complex of the eyestalk of the fiddler crab, Uca pugilator, by immunocytochemistry. SP-like immunoreactivity was found in the optic peduncle, sinus gland, medulla externa, medulla interna, lamina ganglionaris, and retinular cells. Met-enkephalin-like and Leuenkephalin-like immunoreactivity was observed in most of the retinular cells, optic peduncle, sinus gland, medulla terminalis, and lamina ganglionaris. However, Met-enkephalin-like, but no Leu-enkephalin-like, immunoreactivity was seen in the medulla terminalis X-organ. FMRFamide-like immunoreactivity could be seen in all parts of the eyestalk except in the sinus gland, lamina ganglionaris, and retinular cells. FMRF-amide-like activity was especially strong in the three chiasmatic regions connecting the optic ganglia. The possibility that these four peptides may function as neuroregulators in the fiddler crab is discussed.This investigation was supported in part by Grant No. PCM-8300064 from the National Science Foundation to MF and Biomedical Research Support Grant No. 2 SO7RRO5373 SUB from the University of Kansas Medical Center to LLV     

Distribution of serotonin and FMRF-amide in the brain of Lymnaea stagnalis with respect to the visual system

Despite serotonin’s and FMRF-amide’s wide distribution in the nervous system of invertebrates and their importance as neurotransmitters,the exact roles they play in neuronal networks leaves many questions.We mapped the presence of serotonin and FMRF-amide-immunoreactivity in the central nervous system and eyes of the pond snail Lymnaea stagnalis and interpreted the results in connection with our earlier findings on the central projections of different peripheral nerves.Since the chemical nature of the intercellular connections in the retina of L.stagnalis is still largely unknown,we paid special attention to clarifying the role of serotonin and FMRF-amide in the visual system of this snail and compared our findings with those reported from other species.At least one serotonin-and one FMRF-amidergic fibre were labeled in each optic nerve,and since no cell bodies in the eye showed immunoreactivity to these neurotransmitters,we believe that efferent fibres with somata located in the central ganglia branch at the base of the eye and probably release 5HT and FMRF-amide as neuro-hormones.Double labelling revealed retrograde transport of neurobiotin through the optic nerve,allowing us to conclude that the central pathways and serotonin-and FMRF-amideimmunoreactive cells and fibres have different locations in the CNS in L.stagnalis.The chemical nature of the fibres,which connect the two eyes in L.stagnalis,is neither serotoninergic nor FMRF-amidergic.     

Pigment-dispersing hormone-like immunoreactive neurons in the central nervous system of the gastropods, Helix pomatia and Lymnaea stagnalis

By using an antiserum raised against a crustacean #-pigment-dispersing hormone (PDH), the distribution and chemical neuroanatomy of PDH-like immunoreactive neurons was investigated in the central nervous system of the gastropod snails, Helix pomatia and Lymnaea stagnalis. The number of immunoreactive cells in the Helix central nervous system was found to be large (700-900), whereas in Lymnaea, only a limited number (50-60) of neurons showed immunoreactivity. The immunostained neurons in Helix were characterized by rich arborizations in all central ganglia and revealed massive innervation of all peripheral nerves and the neural (connective tissue) sheath around the ganglia and peripheral nerve trunks. A small number of Helix nerve cell bodies in the viscero-parietal ganglion complex were also found to be innervated by PDH-like immunoreactive processes. Hence, a complex central and peripheral regulatory role, including neurohormonal actions, is suggested for a PDH-like substance in Helix, whereas the sites of action may be more limited in Lymnaea.     

Histochemical localization of FMRFamide, serotonin and catecholamines in embryonic Crepidula fornicata (Gastropoda, Prosobranchia)

 Recent reports indicate that neuronal elements develop in early larval stages of some Gastropoda from the Pulmonata and Opisthobranchia prior to the appearance of any ganglia of the future adult central nervous system (CNS). The present study describes similar early neuronal elements in Crepidula fornicata. A posterior FMRFamide-like immunoreactive (LIR) cell with anteriorly projected fibers was observed in the trochophore stage. Additional FMRFamide-LIR and serotonin-LIR cells and fibers were found in the apical organ in the trochophore and early veliger stages. FMRFamide-LIR and serotonin-LIR projections to the velum and foot were also detected at this time. As the veliger developed, peripheral FMRFamide-LIR and later catecholaminergic cells were located in the foot region. Also during this stage, catecholaminergic cells and processes were observed near the mouth. In addition, this study tentatively identified the first serotonin- and FMRFamide-LIR cells and fibers within the developing ganglia of the adult CNS, which appeared in close proximity to the earlier developing elements. These observations are consistent with the hypothesis that, in addition to its presumed role in the control of larval behaviors, the larval nervous system guides the development of the adult CNS. Larvae from the class Bivalvia and other invertebrate phyla also have neuronal elements marked by the presence of FMRFamide, serotonin, and catecholamines, and, therefore, this study may provide additional insights into phylogenetic relationships of the Gastropoda with other representatives of the Mollusca and different invertebrate phyla. Accepted: 10 February 1999     

GASTROPOD CHEMORECEPTION

(I). Gastropods use chemoreception for a wide variety of behaviours including feeding, homing, escape from predators and a variety of social and reproductive behaviours. Chemoreception is used to locate distant food sources, and to discriminate between potential foods. Responses to chemical food stimuli result from a combination of innate and experiential factors. Gastropods use chemical cues in mucus trails to home. They also home by direct olfactory orientation. Reproductive behaviour in a variety of gastropods appears to involve chemical cues. Evidence exists for pheromones controlling aggregation and mating. Numerous gastropods use chemical cues to avoid or escape from predators. (2). Amino acids appear as likely candidates for attractants and phagostimulants for gastropod feeding. Macromolecules are probably also involved. Amino acids have also been shown to stimulate reproductive behaviours in certain gastropods, thus suggesting a pheromonal function. However, the significance of this finding to the behaviour of the organisms in the field has yet to be evaluated. Saponins have been implicated as the active substances found in sea stars that elicit escape responses of marine gastropods. Choline esters may play a homologous role in gastropod—prey and gastropod-predator interactions. (3). Gastropods can apparently use a number of different methods to orient to olfactory cues. These include anemotaxis or rheotaxis, klinotaxis and tropotaxis. (4). The major chemosensory organs of gastropods have been identified. They include the anterior and posterior tentacles and lips of terrestrial pulmonates; the cephalic tentacles, the lips and buccal cavity lining, and possibly the osphradium of aquatic pulmonates; the cephalic and mantle tentacles, the anterior margin of the foot, the siphon tip, and the osphradium of prosobranchs; and the rhinophores, tentacles, oral veil and osphradium of opisthobranchs. (5). Many of the organs named above have been examined by both light and electron microscopy. The most common anatomical organization includes bipolar primary sensory cells with cell bodies located subepithelially, and a distal dendrite extending to the free surface. Often a peripheral ganglion is located deep to the sensory epithelium. It is unclear whether axons of the sensory cells project directly to the central ganglion or by way of interneurones located in the peripheral ganglia. (6). The dendritic specializations of the sensory cells vary considerably. Most bear cilia or a combination of cilia and microvilli. The functional significance of the variation in the types of sensory endings is unknown, although the chemosensory epithelia also respond to other sensory modalities, and it is often difficult to ascribe any one cell type to any one modality. Species-specific variations may also complicate the picture. (7). Prospects for and importance of future studies on gastropod chemoreception are discussed.     

Expression of α and β parvalbumin is differentially regulated in the rat organ of corti during development

The expression of two calcium‐binding proteins of the parvalbumin (PV) family, the α isoform (αPV) and the β isoform known as oncomodulin (OM), was investigated in the rat cochlea during postnatal development and related to cholinergic efferent innervation. Using RT‐PCR analysis, we found that OM expression begins between postnatal day 2 (P2) and P4, and peaks as early as P10, while αPV mRNA begins expression before birth and remains highly expressed into the adult period. Both in situ hybridization and immunoreactivity confirm that OM is uniquely expressed by the outer hair cells (OHCs) in the rat cochlea and occurs after efferent innervation along the cochlear spiral between P2 and P4. In contrast to OM expression, αPV immunoreactivity is expressed in both inner hair cells (IHCs) and OHCs at birth. Following olivocochlear efferent innervation, OHCs demonstrate weak OM immunoreactivity beginning at P5 and diminished αPV immunoreactivity after P10. In organ cultures isolated prior to the efferent innervation of OHCs, OM immunoreactivity failed to develop in OHCs, but αPV immunoreactivity remained present in both IHCs and OHCs. In contrast, organ cultures isolated after efferent innervation of OHCs show OHCs with low levels of OM immunoreactivity and high levels of αPV immunoreactivity. This study suggests that OM and αPV are differentially regulated in OHCs during cochlear development. Our findings further raise the possibility that the expression of PV proteins in OHCs may be influenced by efferent innervation. © 2003 Wiley Periodicals, Inc. J Neurobiol 58: 479–492, 2004     

Structure of visual pathways in the nervous system of freshwater pulmonate molluscs

Central nervous system of freshwater pulmonate molluscs Lymnaea stagnalis and Planorbarius corneus was stained using retrograde transport of neurobiotin in the optic tract fibers. In both species, perikarya and fibers of the stained neurons are found in all ganglia except the buccal ones. Afferent fibers of the optic nerve form dense sensory neuropil located in relatively small volume of cerebral ganglia. Typical neuronal groups sending their processes into the optic nerves of ipsilateral and contralateral body halves are described. Among them, neurons of visceral and parietal ganglia innervating both eyes concurrently as well as sending projections into peripheral nerves are revealed. These neurons, supposedly, have a function to integrate sensory signals, which may be a basis for regulation of light sensitivity of retina and functioning of peripheral organs. Bilateral links of the molluscan eye with the pedal ganglia cells and statocysts are found, which is, likely, a structural basis of certain known behavioral patterns related to stimulation of visual inputs in the studied gastropod molluscs.     

Catecholaminergic Innervation of Central and Peripheral Auditory Circuitry Varies with Reproductive State in Female Midshipman Fish,Porichthys notatus

In seasonal breeding vertebrates, hormone regulation of catecholamines, which include dopamine and noradrenaline, may function, in part, to modulate behavioral responses to conspecific vocalizations. However, natural seasonal changes in catecholamine innervation of auditory nuclei is largely unexplored, especially in the peripheral auditory system, where encoding of social acoustic stimuli is initiated. The plainfin midshipman fish, Porichthys notatus, has proven to be an excellent model to explore mechanisms underlying seasonal peripheral auditory plasticity related to reproductive social behavior. Recently, we demonstrated robust catecholaminergic (CA) innervation throughout the auditory system in midshipman. Most notably, dopaminergic neurons in the diencephalon have widespread projections to auditory circuitry including direct innervation of the saccule, the main endorgan of hearing, and the cholinergic octavolateralis efferent nucleus (OE) which also projects to the inner ear. Here, we tested the hypothesis that gravid, reproductive summer females show differential CA innervation of the auditory system compared to non-reproductive winter females. We utilized quantitative immunofluorescence to measure tyrosine hydroxylase immunoreactive (TH-ir) fiber density throughout central auditory nuclei and the sensory epithelium of the saccule. Reproductive females exhibited greater density of TH-ir innervation in two forebrain areas including the auditory thalamus and greater density of TH-ir on somata and dendrites of the OE. In contrast, non-reproductive females had greater numbers of TH-ir terminals in the saccule and greater TH-ir fiber density in a region of the auditory hindbrain as well as greater numbers of TH-ir neurons in the preoptic area. These data provide evidence that catecholamines may function, in part, to seasonally modulate the sensitivity of the inner ear and, in turn, the appropriate behavioral response to reproductive acoustic signals.     

Ganglionic distribution of inputs and outputs of C-PR, a neuron involved in the generation of a food-induced arousal state inAplysia

Cerebral neuron C-PR is thought to play an important role in the appetitive phase of feeding behavior ofAplysia. Here, we describe the organization of input and output pathways of C-PR. Intracellular dye fills of C-PR revealed extensive arborization of processes within the cerebral and the pedal ganglia. Numerous varicosities of varying sizes may provide points of synaptic inputs and outputs.Blocking polysynaptic transmission in the cerebral ganglion eliminated the sensory inputs to C-PR from stimuli applied to the rhinophores or tentacles, indicating that this input is probably mediated by cerebral interneurons. Identified cerebral mechanoafferent sensory neurons polysynaptically excite C-PR. Stimulation of the eyes and rhinophores with light depresses C-PR spike activity, and this effect also appears to be mediated by cerebral interneurons.C-PR has bilateral synaptic actions on numerous pedal ganglion neurons, and also has effects on cerebral neurons, including the MCC, Bn cells, CBIs and the contralateral C-PR. Although the somata of these cerebral neurons are physically close to C-PR, experiments using high divalent cation-containing solutions and cutting of various connectives indicated that the effects of C-PR on other cerebral ganglion neurons (specifically Bn cells and the MCC) are mediated by interneurons that project back to the cerebral ganglion via the pedal and pleural connectives. The indirect pathways of C-PR to other cerebral neurons may help to ensure that consummatory motor programs are not activated until the appropriate appetitive motor programs, mediated by the pedal ganglia, have begun to be expressed.     

Peptide immunocytochemistry of neurons projecting to the retrocerebral complex in the blow fly, <Emphasis Type="Italic">Protophormia terraenovae</Emphasis>

Antisera against a variety of vertebrate and invertebrate neuropeptides were used to characterize neurons with somata in the pars intercerebralis (PI), pars lateralis (PL), and subesophageal ganglion (SEG), designated as PI neurons, PL neurons, and SEG neurons, respectively, all of which project to the retrocerebral complex in the blow fly, Protophormia terraenovae. Immunocytochemistry combined with backfills through the cardiac-recurrent nerve revealed that at least two pairs of PI and SEG neurons for each were FMRFamide-immunoreactive. Immunoreactivity against [Arg7]-corazonin, beta-pigment-dispersing hormone (beta-PDH), cholecystokinin8, or FMRFamide was observed in PL neurons. Immunoreactive colocalization of [Arg7]-corazonin with beta-PDH, [Arg7]-corazonin with cholecystokinin8, or beta-PDH with FMRFamide was found in two to three somata in the PL of a hemisphere. Based on their anatomical and immunocytochemical characteristics, PI neurons were classified into two types, PL neurons into six types, and SEG neurons into two types. Fibers in the retrocerebral complex showed [Arg7]-corazonin, beta-PDH, cholecystokinin8, and FMRFamide immunoreactivity. Cholecystokinin8 immunoreactivity was also detected in intrinsic cells of the corpus cardiacum. The corpus allatum was densely innervated by FMRFamide-immunoreactive varicose fibers. These results suggest that PI, PL, and SEG neurons release [Arg7]-corazonin, beta-PDH, cholecystokinin8, or FMRFamide-like peptides from the corpus cardiacum or corpus allatum into the hemolymph, and that some PL neurons may simultaneously release several neuropeptides.