The distribution of cholinergic neurons and their co-localization with FMRFamide, in central and peripheral neurons of the spider Cupiennius salei.

The spider Cupiennius salei is a well-established model for investigating information processing in arthropod sensory systems. Immunohistochemistry has shown that several neurotransmitters exist in the C. salei nervous system, including GABA, glutamate, histamine, octopamine and FMRFamide, while electrophysiology has found functional roles for some of these transmitters. There is also evidence that acetylcholine (ACh) is present in some C. salei neurons but information about the distribution of cholinergic neurons in spider nervous systems is limited. Here, we identify C. salei genes that encode enzymes essential for cholinergic transmission: choline ACh transferase (ChAT) and vesicular ACh transporter (VAChT). We used in-situ hybridization with an mRNA probe for C. salei ChAT gene to locate somata of cholinergic neurons in the central nervous system and immunohistochemistry with antisera against ChAT and VAChT to locate these proteins in cholinergic neurons. All three markers labeled similar, mostly small neurons, plus a few mid-sized neurons, in most ganglia. In the subesophageal ganglia, labeled neurons are putative efferent, motor or interneurons but the largest motor and interneurons were unlabeled. Groups of anti-ChAT labeled small neurons also connect the optic neuropils in the spider protocerebrum. Differences in individual cell labeling intensities were common, suggesting a range of ACh expression levels. Double-labeling found a subpopulation of anti-VAChT-labeled central and mechanosensory neurons that were also immunoreactive to antiserum against FMRFamide-like peptides. Our findings suggest that ACh is an important neurotransmitter in the C. salei central and peripheral nervous systems.

Distribution of neuropeptides in the antennal lobes of male Spodoptera littoralis.

Olfaction is an important sensory modality that regulates a plethora of behavioural expressions in insects. Processing of olfactory information takes place in the primary olfactory centres of the brain, namely the antennal lobes (ALs). Neuropeptides have been shown to be present in the olfactory system of various insect species. In the present study, we analyse the distribution of tachykinin, FMRFamide-related peptides, allatotropin, allatostatin, myoinhibitory peptides and SIFamide in the AL of the male Egyptian cotton leafworm, Spodoptera littoralis. Immunocytochemical analyses revealed that most neuropeptides were expressed in different subpopulations of AL neurons. Their arborisation patterns within the AL suggest a significant role of neuropeptide signalling in the modulation of AL processing. In addition to local interneurons, our analysis also revealed a diversity of extrinsic peptidergic neurons that connected the antennal lobe with other brain centres. Their distributions suggest that extrinsic neurons perform various types of context-related modulation.

Temporal and histological evaluation of melatonin patterns in a 'basal' metazoan.

While recent advances suggest functional pleiotropy of melatonin in higher organisms, an understanding of the biological significance of this ancient molecule in early evolutionary groups is lacking. Here, endogenous melatonin production was identified for the first time in the sea anemone Actinia equina, a nonsymbiotic hexacorallian cnidarian. Day/night activity profiles of melatonin in this anemone indicated that melatonin levels oscillate with significant nocturnal peaks. However, dynamic changes in melatonin concentration did not persist under constant dark conditions and therefore were not circadian in nature. Thus, the oscillating pattern of melatonin in A. equina is presumed to be the result of alternative, simpler melatonin control mechanism that likely involves direct regulation by the daily photocycle. As nocturnal melatonin signals still potentially provide 'time-of-day' information and can illustrate the seasonally changing length of the biological night, we hypothesize that melatonin may be relevant to temporal coordination of timed processes also in anthozoans. Spatial patterns of melatonin distribution found in this study indicate abundant melatonin distribution in the endodermal filaments wrapped around gametes. This finding supports the possibility that one of the melatonin-responsive processes in this basal metazoan species may involve reproductive functions.

The neuromuscular system in freshwater furcocercaria from Belarus. II Diplostomidae, Strigeidae, and Cyathocotylidae.

The neuromuscular system (NMS) in cercariae of Diplostomum pseudospathaceum, Cotylurus szidati, Australapatemon burti, Holostephanus volgensis, and Paracoenogonimus ovatus was studied with immunocytochemical methods and confocal scanning laser microscopy. The patterns of F-actin in the musculature, 5-HT immunoreactive (-IR), FMRF-amide-IR neuronal elements, and α-tubulin-IR in sensory receptors were investigated. The NMS in the five species studied were compared with each other and with three species of Schistosomatidae studied earlier (Bilharziella polonica, Trichobilharzia szidati, and Trichobilharzia franki). No major structural differences in the musculature, the 5-HT-IR or FMRF-IR neuronal elements were noticed between the cercariae. The minor variations observed in the musculature were related to the size and organization of the muscle fibers. The checked pattern formed by the transverse muscle fibers in the tail stems of D. pseudospathaceum, C. szidati, A. burti, H. volgensis, and P. ovatus was not observed in B. polonica, T. szidati, and T. franki. A trend in the differentiation of the longitudinal muscle fibers in the furca from evenly distributed fibers in H. volgensis and P. ovatus to many bundles in D. pseudospathaceum and two well-organized lateral bundles in C. szidati, A. burti, and Trichobilharzia spp. was observed. The transverse muscle fibers in the furca follow the same trend. The number of 5-HT-IR neurons in the cercarial bodies varied between 10 and 16. In cercariae of H. volgensis and P. ovatus, the central nervous system (CNS) was less centralized compared to the CNS in the other species studied, with only two 5-HT-IR marker neurons in each brain ganglion and the other neurons distributed evenly along the main cords. In the tails of H. volgensis and P. ovatus, many transverse 5-HT-IR comissures were found. In the tails of higher strigeidid cercariae, only a few crosslinks were observed. The number and distribution of sensory receptors on the bodies and tails of the cercarial species differed from each other. A trend in the differentiation of the sensory receptors in the tails was discerned. A process of grouping and decrease in number of ciliated receptors in the stem and in the furca from H. volgensis and P. ovatus to Schistosomatid cercariae took place.

The neuro-muscular system in fresh-water furcocercaria from Belarus. I Schistosomatidae.

The neuro-muscular system (NMS) in cercariae of the family Schistosomatidae from Belarus was studied with immunocytochemical methods and confocal scanning laser microscopy. The specimens of Bilharziella polonica were compared with Trichobilharzia szidati and Trichobilharzia franki. The patterns of F-actin in the musculature, 5-HT-immunoreactive (IR), FMRFamide-IR neuronal elements and α-tubulin-IR in sensory receptors and nerves were investigated. No indications of structural differences in the musculature, the 5-HT-IR, FMRF-IR neuronal elements and the general distribution of sensory receptors were noticed between cercariae of Trichobilharzia spp. The number of 5-HT-IR neurons in the cercarial bodies is 16. In cercaria B. polonica, the tail musculature is weaker than in Trichobilharzia spp. A detailed schematic picture of the NMS in the tail of Trichobilharzia spp. cercaria is given. The function of NMS elements in the tail is discussed.

The neuro-glandular brain of the Pyramicocephalus phocarum plerocercoid (Cestoda, Diphyllobothriidea): Immunocytochemical and ultrastructural study.

А novel type of a complex neuro-glandular brain structure including both nervous and glandular elements and associated with sensory ones is detected in Pyramicocephalus phocarum plerocercoid (Cestoda: Diphyllobothriidea), parasite of Gadus morua from the White Sea. The brain has two lateral lobes connected by a long cellular median commissure. The brain is tightly surrounded by glandular cells, which receive numerous synapses from the brain neurons. A complex of sensory organs associated with ducts and terminal pores of the frontal glands lies in the scolex tegument. Serotonin, FMRFamide- and GABA-like immunoreactive (IR) neurons are found in the brain, the main nerve cords, and the plexus of the plerocercoid. The innervation of the frontal gland ducts by FMRFamide-IR neurites is detected for the first time proving that they function under control of the nervous system and thus evidencing the eccrine nature of the secretion mechanism. Ultrastructural data show that light, dark and neurosecretory neurons are present in the brain lobes. The median commissure consists of loosely arranged thin parallel axons and several giant and small neurons. The commissure is stratified and penetrated by frontal glandular cells and their processes. Such neuro-glandular morpho-functional brain complex is suggested as a model for Diphyllobothriidae family. Five structural types of sensory organs are described in the scolex of P. phocarum; their colocalization with eccrine gland terminals is supposedly specific for Diphyllobothriidae family. Within the order Diphyllobothriidea, there are significant differences in the architecture of the plerocercoid brain at the family level. We suppose homology of giant commissural neurons among Diphyllobothriidea. Differences between diphyllobothriidean nervous system and that of other cestodes are discussed.

Neuromuscular system of the causative agent of dicrocoeliosis, Dicrocoelium lanceatum. II. Neuropeptide FMRFamide immunoreactivity in nervous system.

The parasitic flatworm, trematoda Dicrocoelium lanceatum or lancet fluke is the causative agent of a widespread parasite disease of grazing ruminants, dicrocoeliosis. The aim of this work is the study of the presence and localization of neuropeptide FMRFamide immunoreactive elements in the nervous system of D. lanceatum using immunocytochemical technique and confocal scanning laser microscopy. For the first time the data on the presence and distribution of the FMRFamide-immunopositive components in the central and peripheral departments of the nervous system of D. lanceatum has been obtained. FMRFamidergic neurons and neurites were identified in paired brain ganglia, in the brain commissure, longitudinal nerve cords and connective nerve commissures. The innervation of the oral and ventral suckers by peptidergic nerve structures was revealed. The distal part of the reproductive system is innervated by FMRFamide immunopositive neurites. The data obtained suggest that the neuropeptides of FMRFamide family can be involved in the regulation of functions of the attachment organs and the reproductive system in D. lanceatum. The study of neurotransmitters and their functions in flatworms expand our knowledge on the structure and function of the nervous system of trematodes of various taxonomic groups. The results obtained on the morphological organization of D. lanceatum nervous system support the exploitation of the FMRFamidergic components as an anthelmintic target.

The neuro-muscular system in cercaria with different patterns of locomotion.

The neuro-muscular system (NMS) of cercariae with different swimming patterns was studied with immunocytochemical methods and confocal scanning laser microscopy. Specimens of the continuously swimming Cercaria parvicaudata, Maritrema subdolum and Himasthla elongata were compared with specimens of the intermittently swimming Cryptocotyle lingua and the attached Podocotyle atomon. The patterns of F-actin in the musculature, 5-HT immunoreactive (-IR), FMRFamide-IR neuronal elements, α-tubulin-IR elements in the nervous and sensory systems and DAPI-stained nuclei were investigated. The general plan of the NMS was similar in all cercariae studied. No major structural differences in the patterns of muscle fibres were observed. However, in the tail of C. lingua, transverse muscle fibres connecting the bands of longitudinal muscles were found. No major structural differences in the 5-HT- or FMRFamide-IR nervous systems were observed. The number of 5-HT-IR neurones in the cercarial bodies varied between 12 and 14. The number and distribution of the α-tubulin-IR processes on the cercarial bodies and tails differed from each other. The relation between the number and structure of the α-tubulin-IR processes and the host finding strategy of the cercariae is discussed. A detailed schematic picture of the NMS in the tails of C. lingua and M. subdolum is presented.

FMRF-NH2 -related neuropeptides in Biomphalaria spp., intermediate hosts for schistosomiasis: Precursor organization and immunohistochemical localization.

Freshwater snails of the genus Biomphalaria serve as intermediate hosts for the digenetic trematode Schistosoma mansoni, the etiological agent for the most widespread form of intestinal schistosomiasis. As neuropeptide signaling in host snails can be altered by trematode infection, a neural transcriptomics approach was undertaken to identify peptide precursors in Biomphalaria glabrata, the major intermediate host for S. mansoni in the Western Hemisphere. Three transcripts that encode peptides belonging to the FMRF-NH2 -related peptide (FaRP) family were identified in B. glabrata. One transcript encoded a precursor polypeptide (Bgl-FaRP1; 292 amino acids) that included eight copies of the tetrapeptide FMRF-NH2 and single copies of FIRF-NH2 , FLRF-NH2 , and pQFYRI-NH2 . The second transcript encoded a precursor (Bgl-FaRP2; 347 amino acids) that comprised 14 copies of the heptapeptide GDPFLRF-NH2 and 1 copy of SKPYMRF-NH2 . The precursor encoded by the third transcript (Bgl-FaRP3; 287 amino acids) recapitulated Bgl-FaRP2 but lacked the full SKPYMRF-NH2 peptide. The three precursors shared a common signal peptide, suggesting a genomic organization described previously in gastropods. Immunohistochemical studies were performed on the nervous systems of B. glabrata and B. alexandrina, a major intermediate host for S. mansoni in Egypt. FMRF-NH2 -like immunoreactive (FMRF-NH2 -li) neurons were located in regions of the central nervous system associated with reproduction, feeding, and cardiorespiration. Antisera raised against non-FMRF-NH2 peptides present in the tetrapeptide and heptapeptide precursors labeled independent subsets of the FMRF-NH2 -li neurons. This study supports the participation of FMRF-NH2 -related neuropeptides in the regulation of vital physiological and behavioral systems that are altered by parasitism in Biomphalaria.

The New Data on the Serotonin and FMRFamide Localization in the Nervous System of Opisthorchis felineus Metacercaria.

BACKGROUND: Trematoda Opisthorchis felineus Rivolta, 1884 is the causative agent of dangerous parasite disease-opisthorchiasis, widespread in the Russian Federation. The details of the neuroanatomical localization of the serotoninergic and FMRFamidergic neurotransmitter elements as well as their functional roles remain not studied enough in both adult and larval forms of O. felineus. The studies in this area are important in term of the development of a new pharmacological strategy of the struggle with the causative agent of opisthorchiasis affecting the neuronal signal substances and the function of its nervous system. PURPOSE: The aim of this work was the immunocytochemical study of the neurotransmitters serotonin (5-HT, 5-Hydroxitryptamine) and neuropeptide FMRFamide localization in the nervous system of the opisthorchiasis causative agent-O. felineus metacercaria. To study the relationship between the detected neurotransmitters and the muscular elements of the parasite, the muscle staining was carried out simultaneously using fluorophore-conjugated phalloidin. METHODS: The localization of 5-HTergic and FMRFamidergic nerve structures was determined by immunocytochemical method. The staining samples were analyzed using a fluorescent and confocal laser scanning microscopies. RESULTS: The new data on the presence and distribution of the serotonin-immunopositive (IP)- and FMRFa-IP components in the central and peripheral departments of the nervous system of  O. felineus metacercaria has been obtained. Besides that a number of the new anatomical details of the nervous system organization and of the innervation of the organs and tissues in the investigated parasite have been revealed. CONCLUSION: The data obtained on the presence and localization of the 5-HTergic and peptidergic (FMRFamide) components in central and peripheral departments of the nervous system of O. felineus metacercaria elaborated and expanded the existing information about the nervous system as well as the innervations of the tissues and organs in the causative agent of opistchorchiasis.

Development of the larval muscle system in the mussel Mytilus trossulus (Mollusca, Bivalvia).

In the present study we examined muscle development throughout the complete larval cycle of the bivalve mollusc, Mytilus trossulus. An immunofluorescence technique and laser scanning confocal microscopy were used in order to study the organization of the muscle proteins (myosin, paramyosin, twitchin, and actin) and some neurotransmitters. The appearance of the muscle bundles lagged behind their nervous supply: the neuronal elements developed slightly earlier (by 2 h) than the muscle cells. The pioneer muscle cells forming a prototroch muscle ring were observed in a completed trochophore. We documented a well-organized muscle system that consisted of the muscle ring transforming into three pairs of velar striated retractors in the early veliger. The striations were positive for all muscle proteins tested. Distribution of FMRFamide and serotonin (5-HT) immunocytochemical staining relative to the muscle ring differed significantly: 5-HT-immunoreactive cells were situated in the center of the striated muscle ring, while Phe-Met-Arg-Phe-NH2 neuropeptide FMRFamid immunoreactive fibers were located in a distal part of this ring. Our data showed clearly that the muscle proteins and the neurotransmitters were co-expressed in a coordinated fashion in a continuum during the early stages of the mussel development. Our study provides the first strong evidence that mussel larval metamorphosis is accompanied by a massive reorganization of striated muscles, followed by the development of smooth muscles capable of catch-contraction.

Mass spectrometric analysis of FMRFamide-like immunoreactive neurons in the prothoracic and subesophageal ganglion of Periplaneta americana.

MALDI-TOF mass spectrometry combined with immunocytochemistry and retrograde labeling, was used to study the expression pattern and morphology of Pea-FMRFamide-related peptides in single neurons of the prothoracic ganglion and the subesophageal ganglion (SEG) of the American cockroach Periplaneta americana. In contrast to the postero-lateral cells (PLCs) of the meta- and mesothoracic ganglion, the prothoracic FMRFamide-related peptides expressing neurons not only extend in the posterior median nerve but also in an anterior median nerve, which is described herein. The peptidome of the prothoracic PLCs is identical with that of the meso- and metathoracic neurons, respectively. In this study, we identified a truncated form of Pea-FMRFa-24 which was found to be more abundant than the peptide originally designated as Pea-FMRF-24. FMRFamide-related peptides expressing postero-lateral cells were also detected in the labial neuromere of the SEG. Although their projection could not be solved, mass spectrometric analyses revealed the same peptide complement in these neurons as found in the thoracic postero-lateral cells. In all neurons which we studied no co-localized peptides of other peptide families were observed.

Distribution of FMRFamide-like immunoreactivity in the alimentary tract and hindgut ganglia of the barnacle Balanus amphitrite (Cirripedia, Crustacea).

In this study, the presence and distribution of FMRFamide-like immunoreactivity in the alimentary tract of barnacle Balanus amphitrite were investigated. A net of nerve fibers strongly immunoreactive to FMRFamide-like molecules was localized in the posterior midgut and hindgut. Positive varicose nerve terminals were also localized close to the circular muscle cells and, in the hindgut, close to the radial muscular fibers. Besides this nerve fibers network, one pair of contralateral ganglia was localized in the hindgut, each of them constituted by two strongly FMRFamide-labeled neurons and one nonlabeled neuron. Their immunoreactive axons directed toward the hindgut and posterior midgut suggest an involvement of FMRFamide-like substances in adult B. amphitrite gut motility. The hindgut associated ganglia of barnacles seem to correspond to the terminal abdominal ganglia of the other crustaceans. Since they are the only residual gut ganglia in the barnacle's reduced nervous system, we can hypothesize that gut motility needs a nervous system regulation partially independent of the central nervous system.

Development of embryonic and larval cells containing serotonin, catecholamines, and FMRFamide-related peptides in the gastropod mollusc Phestilla sibogae.

The present immunocytochemical study provides one of the first detailed descriptions of the development of cells containing a variety of neurotransmitters during much of the larval life of a nudibranch gastropod. Throughout much of early development, serotonergic cells were located only in the apical organ; as larvae approached metamorphosis, serotonergic cells were also detected in the cerebropleural and pedal ganglia. Cells exhibiting tyrosine hydroxylase immunoreactivity (indicative of catecholamine synthesis) were first located near the mouth but by late embryonic stages were also located in the apical organ and near the velum and eyes. By late larval stages, numerous catecholaminergic cells were found in the foot, with concentrations in the propodium. Finally, the first cells exhibiting FMRFamide immunoreactivity were detected posterior to the neuropil of the cerebropleural ganglia in the early embryo. Fibers that presumably originated from these cells subsequently invaded the cerebral and pedal ganglia and the apical organ. By early larval stages, a second pair of peptidergic neurons was located near the first pair, and additional peptidergic neurons were located in the apical organ and peripheral positions in the foot and medial and dorsal to the eyes. In addition to providing a unique phyletic perspective to our understanding of gastropod neural development, the present study also sets the stage for future studies into changes in the nervous system as this gastropod undergoes metamorphosis.

Peptidomic analysis of the larval Drosophila melanogaster central nervous system by two-dimensional capillary liquid chromatography quadrupole time-of-flight mass spectrometry.

Peptides are the largest class of signalling molecules found in animals. Nevertheless, in most proteomic studies peptides are overlooked since they literally fall through the mazes of the net. In analogy with proteomics technology, where all proteins expressed in a cell or tissue are analyzed, the peptidomic approach aims at the simultaneous visualization and identification of the whole peptidome of a cell or tissue, i.e. all expressed peptides with their post-translational modifications. In this paper we describe the analysis of the larval fruit fly central nervous system using two-dimensional capillary liquid chromatography/quadrupole time-of-flight tandem mass spectrometry (LC/Q-TOF-MS/MS. Using the central nervous systems of only 50 larval Drosophila as starting material, we identified 38 peptides in a single analysis, 20 of which were not detected in a previous study that reported on the one-dimensional capillary LC/MS/MS analysis of the same tissue. Among the 38 sequenced peptides, some originate from precursors, such as the tachykinin and the IFamide precursor that were entirely missed in the first study. This clearly demonstrates that the two-dimensional capillary LC approach enhances the coverage of the peptidomic analysis.

The distribution of cells containing FMRFamide- and 5-HT-related molecules in the embryonic development of Viviparus ater (Mollusca, Gastropoda).

The timing and spatial distribution of cells containing FMRFamide- and 5-HT-related molecules in the embryonic development of the mollusc Viviparus ater are examined using immunohistochemistry. FMRFamide-like molecules emerge in the early stage E8 (8% of embryonic development) before the 5-HT immunoreactivity, and they are not only found during nervous system ontogeny. As the parts of the digestive tract differentiated, the pattern of the diffuse gut endocrine cells, present in adults, start to be established (E20-E30), and both open and closed cell types are immunoreactive to anti-FMRFamide antibody. From their appearance (E20), cells with a 5-HT-like phenotype are distributed in the central nervous ganglia and progressively assembled during embryonic development. The early occurrence of both these molecules in V. ater embryos reinforces the growing view that neurotransmitters play a regulatory role in embryogenic processes. In particular, the very early presence of FMRFamide-related factors suggests an involvement of these molecules in the regulation of basic, not only neuronal, cell behaviours, while 5-HT seems to be a more specific neural development signal.

Ontogenetic organization of the FMRFamide immunoreactivity in the nervus terminalis of the lungfish, Neoceratodus forsteri.

The development of the nervus terminalis system in the lungfish, Neoceratodus forsteri, was investigated by using FMRFamide as a marker. FMRFamide immunoreactivity appears first within the brain, in the dorsal hypothalamus at a stage around hatching. At a slightly later stage, immunoreactivity appears in the olfactory mucosa. These immunoreactive cells move outside the olfactory organ to form the ganglion of the nervus terminalis. Immunoreactive processes emerge from the ganglion of the nervus terminalis in two directions, one which joins the olfactory nerve to travel to the brain and the other which courses below the brain to enter at the level of the preoptic nucleus. Neither the ganglion of the nervus terminalis nor the two branches of the nervus terminalis form after surgical removal of the olfactory placode at a stage before the development of FMRFamide immunoreactivity external to the brain. Because this study has confirmed that the nervus terminalis in lungfish comprises both an anterior and a posterior branch, it forms the basis for discussion of homology between these branches and the nervus terminalis of other anamniote vertebrates.

Organization and neural connections of the anterior optic tubercle in the brain of the locust, Schistocerca gregaria.

The anterior optic tubercle is a small neuropil in the insect brain and a major target of visual interneurons from the optic lobe. The functional role of the tubercle is poorly understood, but recent evidence from locusts points to a possible involvement in polarization vision. The present study examines the organization of the anterior optic tubercle in the locust Schistocerca gregaria and its connections with other brain areas. The tubercle of the locust consists of an upper and a lower subunit. Both units are connected in parallel with the medulla and lobula of the optic lobe, with the contralateral tubercle, and with the lateral accessory lobe in the median protocerebrum. Wide-field transmedullary neurons provide input from the medulla. Neurons with processes in the dorsal rim of the medulla, a relay station in the polarization vision pathway, project exclusively to the lower unit of the tubercle. Visual input from the lobula to the upper and lower unit originates from topographically distinct strata. The most prominent output target of the tubercle is the lateral accessory lobe in the median protocerebrum. Neurons from the upper unit project widely in the lateral accessory lobe, whereas neurons from the lower unit have focused projections confined to the median olive and to the lateral triangle. The two subunits of the anterior optic tubercle are, therefore, processing stages in two parallel visual pathways from the optic lobe to the median protocerebrum. Pathways via the lower unit of the tubercle appear to be involved in polarization vision.

Distribution of FMRFamide-like immunoreactivity in the amphibian brain: comparative analysis.

FMRFamide is a small neuropeptide present in particular neurons of the basal forebrain and midbrain of the vertebrate groups studied, especially fishes and mammals. In order to assess interspecies variation, the distribution of FMRFamide-like immunoreactivity was studied in the brains of 13 species of amphibian. Although FMRFamide-immunoreactive (IR) terminals occurred throughout much of the brain, IR cell groups were noted in circumscribed regions of the CNS. In the eight anuran species studied, two major populations of labeled perikarya were observed: one in the septopreoptic area and another one in the caudal portion of the diencephalon. The rostrocaudal extent of both and the number of labeled somata in each neuronal group displayed species-specific differences. In urodeles and gymnophiones, labeled perikarya were located in the diencephalon, but there were remarkable species differences in the number of such cells. It is discussed whether sex or season of collection may account for some of the differences observed. The distribution of FMRFamide-IR perikarya, fibers, and pathways in the brain of anurans, urodeles, and gymnophiones was compared. The existence of FMRFamide perikarya in the anterior preoptic neuropil and medial septum appeared to be a feature common to all anurans; labeled neurons in the dorsal thalamus, however, may be present only in the (viviparous) gymnophione Typhlonectes compressicauda. Cerebrospinal fluid contacting FMRFamide neuronal cell bodies and fibers were observed in each of the three taxonomic orders. The data are compared with those previously obtained for other groups of vertebrates.

Centrifugal visual system of Crocodylus niloticus: a hodological, histochemical, and immunocytochemical study.

The retinopetal neurons of Crocodylus niloticus were visualized by retrograde transport of rhodamine beta-isothiocyanate or Fast Blue administered by intraocular injection. Approximately 6,000 in number, these neurons are distributed in seven regions extending from the mesencephalic tegmentum to the rostral rhombencephalon, approximately 70% being located contralaterally to the injected eye. None of the centrifugal neurons projects to both retinae. The retinopetal neurons are located in rostrocaudal sequence in seven regions: the formatio reticularis lateralis mesencephali, the substantia nigra, the griseum centralis tectalis, the nucleus subcoeruleus dorsalis, the nucleus isthmi parvocellularis, the locus coeruleus, and the commissura nervi trochlearis. The greatest number of cells (approximately 93%) is found in the nucleus subcoeruleus dorsalis. The majority are multipolar or bipolar in shape and resemble the ectopic centrifugal visual neurons of birds, although a small number of monopolar neurons resembling those of the avian isthmo-optic nucleus may also be observed. A few retinopetal neurons in the griseum centralis tectalis were tyrosine hydroxylase (TH) immunoreactive. Moreover, in the nuclei subcoeruleus dorsalis and isthmi parvocellularis, both ipsilaterally and contralaterally, approximately one retinopetal neuron in three (35%) was immunoreactive to nitric oxide synthase (NOS), and a slightly higher proportion (38%) of retinopetal neurons were immunoreactive for choline acetyltransferase (ChAT). Some of them contained colocalized ChAT and NOS/reduced nicotinamide adenine dinucleotide phosphate-diaphorase. Fibers immunoreactive to TH, serotonin (5-HT), neuropeptide Y (NPY), or Phe-Met-Arg-Phe-amide (FMRF-amide) were frequently observed to make intimate contact with rhodamine-labeled retinopetal neurons. These findings are discussed in relation to previous results obtained in other reptilian species and in birds.