Characterization of crustacean cardioactive peptide-like immunoreactivity in the horseshoe crab,Limulus polyphemus

The localization of crustacean cardioactive peptide-like immunoreactivity in the horseshoe crab Limulus polyphemus was investigated with enzyme-linked immunosorbent assay and fluorescence microscopy. Immunoreactivity was quantified in the opisthosomal nervous system (67.7 ± 11.4 ng/g), cardiac ganglion (45.0 ± 10.3 ng/g), prosomal nervous system (28. 5 ± 6. 6 ng/g), and midgut (24.6 ± 6.7 ng/g). In the brain, immunoreactive somata were observed in ganglion cells of the central body, in the medullary group and within the ventral medial group. Clusters of immunoreactive cells were found in each of the circumesophageal, pedal ganglia, and in the opisthosomal, abdominal ganglia. In the periphery, immunoreactive varicose fibers were observed in branches of the intestinal nerves, and near longitudinal and circular muscle fibers of the midgut. Immunoreactivity was observed in the cardiac ganglion and myocardium of the neurogenic heart. Synthetic crustacean cardioactive peptide had slight excitatory effects on the cardiac rhythm at doses up to 10^?6 M. This peptide had excitatory effects on the midgut at nanomolar doses. Ventral nerve cord extracts were partially purified with reverse phase high performance liquid chromatography. Two regions of immunoreactivity were detected, one of which coeluted with the authentic peptide. The distribution of crustacean cardioactive peptide immunoreactivity is compared with other transmitter systems in the Limulus nervous system, and myotropic actions of this peptide are discussed with respect to peptidergic modulation of intestinal motility. © 1995 Wiley-Liss, Inc.     

Serotoninlike immunoreactivity in the central and peripheral nervous system of the scale worm Harmothoe imbricata (Polychaeta)

The distribution of serotoninergic neurons in the nervous system of the scale worm Harmothoe imbricata was visualized in the anterior half of the body by the peroxidase-antiperoxidase (PAP) immunohistochemical method with a specific antiserotonin antibody. Immunoreactive neuronal somata were localized in discrete ganglion cell masses of the dorsally situated cerebral ganglion and in segmental ganglia of the ventral nerve cord. They also make up the majority of neurons present in the parapodial ganglia. Large and small varicose fibers stained in the neuropile of all the above-mentioned ganglia but also in interganglionic connectives and segmental nerves. On the basis of soma size and location and of fiber distribution, the reactive neurons were identified as primarily interneuronal with a few motoneurons and presumptive afferent neurons. The presence of a motor component was substantiated by observations of several reactive varicose fibers spread over longitudinal muscle layers of the trunk. In addition, neurites of the subepidermal nerve plexus and enterochromaffinlike cells of the gut epithelium reacted with the serotonin antibody. It is concluded that serotoninergic pathways are ubiquitous elements in the organization of the central and peripheral nervous system of this polychaete. The significance of these findings in relation to other annelid groups and to the physiological role of serotonin is discussed.     

Morphological and ultrastructural study of the ventral nerve cord in Branchiobdella pentodonta whitman (Annelida,oligochaeta)

The ventral nerve cord of Branchiobdella pentodonta consists of paired ganglia containing three different types of neurons, a central neuropil showing characteristic synapses and of an intersegmental apparatus made up of two large lateral connectives and a smaller ventral one. Some remarkable differences exist between the ventral nerve cord and that found in other oligochaetes; these include the presence of a large dorsal blood vessel, two dorsal muscular cells per metamere, and supportive glial cells ventral to the neuropil which have long transverse processes coming into contact with the segmental nerves.     

Neuroanatomy of the visual afferents in the horseshoe crab (Limulus polyphemus)

The central nervous system of Limulus consists of a circumesophageal ring of fused ganglia and a paired ventral nerve cord. The anterior portion, the protocerebrum, receives sensory inputs including visual information. Three optic nerves, one each from the lateral eye, median ocellus, and ventral eye enter each side of the protocerebrum. The central connections of each optic nerve were determined by staining cut nerve trunks with cobalt chloride. The lateral optic nerve innervates the lamina, medulla, optic tract, ventral central body, and ocellar ganglion. The branching patterns of single axons, probably those of eccentric cells in the lateral eye retina, were observed. Single, large-diameter axons in the lateral optic nerve ramify at seven loci including sites in each of the structures innervated by the lateral optic nerve as a whole. The median optic nerve innervates the ocellar ganglion, central body, optic tract, and medulla. Three types of branching patterns were observed for single, large-diameter fibers in the median optic nerve. One type bypasses the ocellar ganglion and innervates the central body. A second type passes through the ocellar ganglion and optic tract without branching and innervates the posterior medulla. A third type innervates the ocellar ganglion, ventral central body, optic tract, and medulla. The ventral optic nerve is composed of large-diameter axons of ventral photoreceptors. Each axon enters the ganglion cell layer of the medulla and branches over a planar area less than 150 μm in diameter. We also observed that axons from mechanoreceptors on the anterior carapace innervate the posterior neuropil of the medulla, and that about 5% of the neurons in the medullar ganglion cell layer send axons to the ipsilateral circumesophageal connective.     

Mapping of serotonin-like immunoreactivity in the lobster nervous system

Serotonin exerts a wide range of physiological actions on many different lobster tissues. To begin the examination of the role of serotonin in lobsters at a cellular level, we have used immunohistochemical methods to search for presumptive serotonergic neurons, their central and peripheral projections, and their terminal fields of arborization. Whole mount preparations of the ventral nerve cord and various peripheral nerve structures have been used for these studies. With these tissues, more than 100 cell bodies have been found that show serotonin-like immunoreactivity. Although a few of the cell bodies are located peripherally (near the pericardial organs, a well known crustacean neurohemal organ), the vast majority are located in central ganglia. Every ganglion in the ventral nerve cord contains at least one immunoreactive cell body. The projections of many of the neurons have been traced, and we have constructed a map of the system of serotonin-immunoreactive cell bodies, fibers, and nerve endings. In addition, a dense plexus of nerve endings showing serotonin-like immunoreactivity surrounds each of the thoracic second roots in the vicinity of groups of peripheral neurosecretory neurons. These peripheral nerve plexuses originate from central neurons of the ventral nerve cord. In some cases we have been able to trace processes from particular central cell bodies directly to the peripheral nerve root plexuses; in other cases we have traced ganglionic neuropil regions to these peripheral endings.     

Development and distribution of serotonin in the central nervous system of Manduca sexta during embryogenesis. II. The ventral ganglia

The distribution of serotonin (5-HT) immunoreactive cells and their projections was mapped in the ganglia of the ventral nerve cord of the tobacco hornworm, Manduca sexta, during embryonic development, using an antiserum to 5-HT. Immunoreactive cells were first seen at 60% development. By 75% of embryonic development, a total of 94 immunoreactive cells were found in the ventral ganglia, including the suboesophageal ganglion. This number decreased to 80 neurons by 100% of development. About 50% of these cells were arranged in the abdominal ganglia and the rest were located in both the suboesophageal and thoracic ganglia. The suboesophageal ganglion exhibited immunoreactive segmental interneurons in areas corresponding to the mandibular, maxillary and labial neuromeres. Two pairs of immunoreactive interneurons were also observed to occur bilaterally in each of the thoracic and abdominal ganglia, with the exception of the prothoracic ganglion. This ganglion contained three pairs of bilaterally arranged immunoreactive neurons as early as 60% of embryonic development. Serotonin immunoreactivity was also found in a number of efferent neurons in the mandibular and labial neuromeres of the suboesophageal ganglion and in the prothoracic, mesothoracic and posterior abdominal ganglia. The occurrence of 5-HT in these efferent neurons suggests an involvement of serotonin in fore- and hindgut function via its effect on the visceral muscles. Immunoreactive lateral longitudinal fibers extended along the entire length of the ventral nerve cord together with dense segmental arborizations. The latter had regressed by the time the embryo was fully developed. This regression of the arborizations in the ganglia at the end of embryonic development indicates that a reorganization of 5-HT innervation occurs to support new larval functions. The time of appearance of 5-HT immunoreactive cells and fibers suggests that serotonin may play a role in the development of the ventral nerve cord.     

3H-GABA uptake selectively labels identifiable neurons in the leech central nervous system

Segment ganglia of the leech ventral nerve cord synthesize the neurotransmitter γ-aminobutyric acid(GABA) when incubatyed in the presence of the prescursor glumate, suggesting that there may be GABA-ergic neurons in the leech nerve cord. GABA-accumulating neurons of the two taxonomically distant leech species, Haementeria ghilianii and Hirudo nedicinalis, have been labeled by takeing advantage of their high-affinity uptake system for the neurotransmitter. Autoradiography of sectioned segmental ganglia previously exposed to ³H-GABA reveals a reproducible pattern of about thirty ³labeled neurol cell bodies are bilaterally paired, although some apparently unpaired cell bodies also accumulate label. Neuronal processes were reproducibly labeled by GABA uptake and could be traced in the neuropil through commissures and fiber tracts into the segmental nerve roots and interganglionic connectives, respectively.     

Immunoreactivity in Limulus. II. Studies of serotoninlike immunoreactivity, endogenous serotonin, and serotonin synthesis in the brain and lateral eye

Serotoninlike immunoreactivity was examined by the fluorescein-isothiocyanate-labeled secondary antibody technique in the lateral eye and brain of Limulus. Endogenous serotonin was measured with high-performance liquid chromatography and electrochemical detection. The synthesis of [3H]serotonin from [3H]tryptophan was measured in the presence and absence of reserpine. Fibers with serotoninlike immunoreactivity were found in the proximal stalks of the corpora pedunculata, in the neuropil of the central body, in the neuropils of the visual centers (lamina, medulla, and ocellar ganglion), in the optic tract that connects the ocellar ganglion with the posterior medial medulla, and in the central neuropil of the brain. Immunoreactive somata were found in four groups in the brain. Up to 50 somata were scattered through each side of the dorsal medial group that lies centered on the dorsal surface within the curve of the central body. These neurons innervate the central body neuropil and send processes into the central neuropil. Three or four reactive somata formed the ventral pole of each medullar group. These may provide the innervation of the proximal stalk of the corpora pedunculata. Five to ten reactive neurons were observed anteriorly in the ventral posterior lateral group #2 on each side that send processes into the central neuropil. Ten to 15 reactive somata were found on either side of the midline in the dorsal anterior part of the ventral medial group that contribute processes to the central neuropil. The remainder of the brain was not immunoreactive. No immunoreactive fibers or somata were found in the lateral eye or in the lateral optic nerve. Serotoninlike and substance P-like immunoreactivities were not found to be colocalized anywhere in the brain. Significant amounts of endogenous serotonin were detected in the lamina and medulla whose neuropils are rich in immunoreactive fibers and in the central body and dorsal medial group that are also rich in immunoreactive somata and fibers. No endogenous serotonin was detected in either the lateral eye or the lateral optic nerve. The lamina, medulla, and central body and dorsal medial group also synthesized and stored [3H]serotonin from [3H]tryptophan. It is likely that serotonin is a neurotransmitter in the brain, but not in the lateral eye of the horseshoe crab. In particular, it appears that serotoninergic neurons may play a role in central visual processing.     

Mapping of octopamine-immunoreactive neurons in the central nervous system of the lobster

It has been suggested that serotonin and octopamine serve important roles in behavioral regulation in lobsters. In this paper the locations of octopamine-immunoreactive neurons were mapped in wholemount preparations of the ventral nerve cord of 4th stage lobster (Homarus americanus) larvae. Approximately 86 neurons were found, distributed as follows: brain, 12; circumesophageal ganglia, 2; subesophageal ganglion, 38; thoracic ganglia, 6 each; and 4th and 5th abdominal ganglia, 2 each. All the octopamine-immunoreactive neurons are paired and located along the midline. Of the 86 neurons, 28 were identified as neurosecretory, and 26 as intersegmental ascending thoracic, ascending abdominal, or descending interneurons. The neurosecretory system is arranged segmentally and located entirely within the thoracic and subesophageal neuromeres with extensive terminal fields of endings along 2nd thoracic and subesophageal nerve roots. This set of neurons shares the features of central and peripheral endings with 2 pairs of large serotonin-containing neurosecretory neurons found in the fifth thoracic and first abdominal ganglia. The intersegmental neurons include: (1) two cells in the brain and 2 pairs of cells in the 3rd and 4th neuromeres of the subesophageal ganglion, which project to the 6th abdominal ganglion; (2) a segmentally organized group of ascending internurons found in the subesophageal and in all thoracic ganglia; and (3) pairs of ascending interneurons found in the 4th and 5th ganglia in the abdominal nerve cord. By means of a biochemical assay, the cell bodies of octopamine-immunoreactive neurosecretory cells in the thoracic segment of the nerve cord were found to contain 40–100 fmol of octopamine, while control neurons had none. © 1993 Wiley-Liss, Inc.     

Neuronal localization of pancreatic polypeptide (PP) and vasoactive intestinal peptide (VIP) immunoreactivity in the earthworm (Lumbricus terrestris)

Nerve fibres and cell bodies displaying vasoactive intestinal polypeptide (VIP) or pancreatic polypeptide (PP) immunoreactivity were demonstrated in ganglia of the earthworm (Lumbricus terrestris). VIP cell bodies were found in the most anterior ganglion of the ventral nerve cord, the subpharyngeal ganglion. Immunoreactive nerves were seen running in the center of the cord until about the 10th segment. PP cell bodies were found in the cerebral ganglion where VIP was lacking, in the subpharyngeal ganglion and in more posteriorly located ganglia of the ventral nerve cord. PP nerve fibres could be followed below the 10th segment of the cord.     

Dopamine in the lobster Homarus gammarus. I. Comparative analysis of dopamine and tyrosine hydroxylase immunoreactivites in the nervous system of the juvenile

As a catecholamine, depamine belogs to a class of molecules that have multiple transmitter and hhormonal functions in vertebrate and invertebrate nervous systems. However, in the lobster, where many central beurons have been identified and the peripheral innervation pattern is well known, the distribution of dopamine-containing neurons has not been examined in detail. Therefore, immunocytochemical methods were used to identify neurons likey to contain dopamine and tyrosine hydroxylase in the central nervous system of the juvenile lobster Homarus gammarus.Approximately 100 neuronal somata stain for the catecholamine and/or its synthetic enzyme in the brain and ventral nerve cord. The systems of neurons labeled with dopamine and tyrosine hydroxylase natibodies have the following characteristic: (1) the two systems are nearly identical; (2) every segmental ganglion contains at least one pair of labeled neurons; (3)the positions and numbers of cell bodies labeled with each antiserum are similar in the various segmental ganglia; (4) six labeled neurons are anatomically identified; two interneurons from the brain project within the ventral cord to reach the last abdominal ganglion, two neurons from the commissural ganglia are presumably neurosecretory neurons, and two anterior unpaired medial abdominal neurons project to the hindgut muscles; and (5) nocell bodies are labeled in the stomatogastric ganglion, but fibers and terminals in the neuropil are stained. The remarkably small numbers of labeled neurons and the presence of very large labeled somata with far-reaching projections are distinctive features consistent with other modulatory aminergic systems in both vertebrates and invertebrates. © 1994 Wiley-Liss, Inc.     

Neuropeptides in insect sensory neurones: tachykinin-, FMRFamide- and allatotropin-related peptides in terminals of locust thoracic sensory afferents

Sensory afferents in the thoracic ganglia of the locust Locusta migratoria were labelled with antisera to different neuropeptides: locustatachykinins, FMRFamide and allatotropin. The locustatachykinin-immunoreactive (LTKIR) sensory fibres were derived from the legs and entered the ventral sensory neuropil of each of the thoracic ganglia via nerve 5. In the thoracic neuropil, the LTKIR sensory fibres formed a distinct plexus of terminations ventrally in the ipsilateral hemisphere. The peripheral cell bodies of the sensory neurones could not be revealed, but lesion experiments indicated that origin of the LTKIR fibres was the tarsus of each leg. Possibly the thin fibres are from tarsal chemoreceptors. Double labelling immunocytochemistry revealed that all the LTKIR sensory fibres contained colocalized FMRFamide immunoreactivity. A larger population of sensory fibres reacted with antiserum to moth (Manduca sexta) allatotropin. By means of double labelling immunocytochemistry, we could show that the LTKIR fibres constituted a subpopulation of the larger set of allatotropin-like immunoreactive fibres. Thus some sensory fibres may contain colocalized peptides related to locustatachykinins, FMRFamide-related peptide(s) and allatotropin-like peptide. A separate non-overlapping small set of sensory fibres in nerve 5 reacted with an antiserum to serotonin. Sensory fibres of the other nerves of the ventral nerve cord, including the abdominal ganglia, did not react with the peptide antisera. Since acetylcholine is the likely primary neurotransmitter of insect sensory fibres, it is possible that the peptides and serotonin are colocalized with this transmitter and serve modulatory functions in a subset of the leg afferents.     

Localization of GABA-like immunoreactivity in the central nervous system of Aplysia californica.

Gamma-aminobutyric acid (GABA) is present in the central nervous system of Aplysia californica (Gastropoda, Opisthobranchia) where its role as a neurotransmitter is supported by pharmacological, biochemical, and anatomical investigations. In this study, the distribution of GABA-immunoreactive (GABAi) neurons and fiber systems in Aplysia was examined by using wholemount immunohistochemistry and nerve backfill methods. GABAi neurons were located in the buccal, cerebral, and pedal ganglia. Major commissural fiber systems were present in each of these ganglia, whereas more limited fiber systems were observed in the ganglionic connectives. Some of the interganglionic fibers were found to originate from two unpaired GABAi neurons, one in the buccal ganglion and one in the right pedal ganglion, each of which exhibited bilateral projections. No GABAi fibers were found in the nerves that innervate peripheral sensory, motor, or visceral organs. Although GABAi cells were not observed in the pleural or abdominal ganglia, these ganglia did receive limited projections of GABAi fibers originating from neurons in the pedal ganglia. The distribution of GABAi neurons suggests that this transmitter system may be primarily involved in coordinating certain bilateral central pattern generator (CPG) systems related to feeding and locomotion. In addition, the presence of specific interganglionic GABAi projections also suggests a role in the regulation or coordination of circuits that produce components of complex behaviors.     

Distribution and partial characterization of FMRFamide-like peptides in the stomatogastric nervous systems of the rock crab, Cancer borealis, and the spiny lobster, Panulirus interruptus

The distribution of FMRFamide-like peptides was studied in the complete stomatogastric nervous system [the paired commissural ganglia, single oesophageal ganglion, and the single stomatogastric ganglion (STG)] of two decapod crustacean species, the spiny lobster Panulirus interruptus and the rock crab Cancer borealis, by using immunocytochemical techniques. Antiserum 231 from the O'Donohue laboratory and antiserum 671C (described here) gave essentially the same staining patterns. In the commissural ganglia of both species there were ten to 20 stained neurons and dense neuropilar staining. The oesophageal ganglion of the crab had four stained neurons. Lucifer Yellow backfills followed by immunostaining showed that the two larger stained neurons of the oesophageal ganglion sent processes into the inferior ventricular nerve. The two smaller neurons sent processes into the inferior oesophageal nerves. The oesophageal ganglion of the lobster had two stained neurons that sent processes into the inferior ventricular nerve as well. None of the somata of the STG stained in either species, but in both species stained fibers were seen in the stomatogastric nerve that entered the STGs and ramified profusely throughout the neuropil. In some preparations of the crab, a stained fiber was visible in the dorsal ventricular nerve. The amounts of the FMRFamide-like peptides found in all regions of the nervous system of P. interruptus and C. borealis were determined by radioimmune assay (RIA). Column chromatography and high-performance liquid chromatography suggest that, in both species, much if not all of the RIA-assayable material is accountable for by peptides that are larger and more hydrophobic than FMRFamide.     

Serotonin immunoreactivity in the central nervous system of the marine molluscs Pleurobranchaea californica and Tritonia diomedea

The central nervous systems of the marine molluscs Pleurobranchaea californica (Opisthobranchia: Notaspidea) and Tritonia diomedea (Opisthobranchia: Nudibranchia) were examined for serotonin-immunoreactive (5-HT-IR) neurons and processes. Bilaterally paired clusters of 5-HT-IR neuron somata were distributed similarly in ganglia of the two species. In the cerebropleural ganglion complex, these were the metacerebral giant neurons (both species), a dorsal anterior cluster (Pleurobranchaea only), a dorsal medial cluster including identified neurons of the escape swimming network (both species), and a dorsal lateral cluster in the cerebropleural ganglion (Pleurobranchaea only). A ventral anterior cluster (both species) adjoined the metacerebral giant somata at the anterior ganglion edge. Pedal ganglia had the greatest number of 5-HT-IR somata, the majority located near the roots of the pedal commissure in both species. Most 5-HT-IR neurons were on the dorsal surface of the pedal ganglia in Pleurobranchaea and were ventral in Tritonia. Neither the buccal ganglion of both species nor the visceral ganglion of Pleurobranchaea had 5-HT-IR somata. A few asymmetrical 5-HT-IR somata were found in cerebropleural and pedal ganglia in both species, always on the left side. The clustering of 5-HT-IR neurons, their diverse axon pathways, and the known physiologic properties of their identified members are consistent with a loosely organized arousal system of serotonergic neurons whose components can be generally or differentially active in expression of diverse behaviors. J. Comp. Neurol. 395:466–480, 1998. © 1998 Wiley-Liss, Inc.     

Actions of a histaminergic/peptidergic projection neuron on rhythmic motor patterns in the stomatogastric nervous system of the crab Cancer borealis

Histamine is a neurotransmitter with actions throughout the nervous system of vertebrates and invertebrates. Nevertheless, the actions of only a few identified histamine-containing neurons have been characterized. Here, we present the actions of a histaminergic projection neuron on the rhythmically active pyloric and gastric mill circuits within the stomatogastric ganglion (STG) of the crab Cancer borealis. An antiserum generated against histamine labeled profiles throughout the C. borealis stomatogastric nervous system. Labeling occurred in several somata and neuropil within the paired commissural ganglia as well as in neuropil within the STG and at the junction of the superior oesophageal and stomatogastric nerves. The source of all histamine-like immunolabeling in the STG neuropil was one pair of neuronal somata, the previously identified inferior ventricular (IV) neurons, located in the supraoesophageal ganglion. These neurons also exhibited FLRFamide-like immunoreactivity. Activation of the IV neurons in the crab inhibited some pyloric and gastric mill neurons and, with inputs from the commissural ganglia eliminated, terminated both rhythms. Focal application of histamine had comparable effects. The actions of both applied histamine and IV neuron stimulation were blocked, reversibly, by the histamine type-2 receptor antagonist cimetidine. With the commissural ganglia connected to the STG, IV neuron stimulation elicited a longer-latency activation of commissural projection neurons which in turn modified the pyloric rhythm and activated the gastric mill rhythm. These results support the hypothesis that the histaminergic/peptidergic IV neurons are projection neurons with direct and indirect actions on the STG circuits of the crab C. borealis.     

NADPH-diaphorase localization in the CNS and peripheral tissues of the predatory sea-slug Pleurobranchaea californica

The distribution of putative nitric oxide synthase (NOS)-containing cells in the opisthobranch mollusc Pleurobranchaea californica was studied histochemically via NADPH-diaphorase (NADPH-d) reduction of Nitro Blue Tetrazolium (NTB). Whole mounts and cryostat sections were prepared from the central nervous system and peripheral organs, including the buccal muscles, esophagus, salivary glands, foot, mantle, and gills. NADPH-d-positive neurons were localized predominantly to the buccal and pedal ganglia as well as to distinct areas of the cerebropleural and visceral ganglia. A variety of identified neurons were positive for NADPH-diaphorase in various central ganglia, including the metacerebral cells of the cerebropleural ganglion, putative locomotor neurons of the pedal ganglia, and buccal motoneurons. Specific staining was observed only in somata of central neurons, whereas neuropil areas remained unstained. However, NADPH-d-reactive axons were dense in buccal ganglion nerves, whereas peripheral nerves and connectives of other ganglia had few or no NADPH-d positive terminals. In the periphery, NADPH-d activity was detected only in a few neurons of the rhinophore and tentacle ganglia. NADPH-d staining was marked in the salivary glands and gills, but there was no or very little staining in the esophagus, buccal mass, and foot. Histochemical stain production required the presence of both β-NADPH and NBT; α-NADPH could not substitute for β-NADPH. The inhibitor of NOS, 2,6-dichlorophenol-indophenol, at 10⁻³ M, totally abolished NADPH-d-positive staining. The apparent high activity of central NADPH-d contrasts with much lower activity in the ganglia of the related gastropod Tritonia. These data suggest a role for nitric oxide as a signal molecule in the central nervous system of Pleurobranchaea. © 1996 Wiley-Liss, Inc.     

GABA-immunoreactive neurons in the nematode Ascaris

gamma-Aminobutyric acid (GABA) immunoreactive neurons in the cephalic, somatic, and caudal regions of the Ascaris nervous system were visualized with serial section and whole-mount GABA immunocytochemistry. In the ventral and dorsal nerve cords, GABA-like immunoreactivity (GLIR) is localized to the neurites and cell bodies of identified inhibitory motor neurons and to two fibers, one in each cord, that arise from neurons in the nerve ring. GLIR is absent from identified excitatory motor neurons and from ventral cord interneurons. In neurons containing GLIR, immunoreactivity was present throughout the cell, which argues against an exclusive localization of GABA at conventional synapses. In whole mounts, ten GABA-immunoreactive neurons were present in the cephalic region. These include four nerve ring-associated cells (the RME-like cells), two bilaterally symmetrical pairs of lateral ganglia neurons (the amphid-GABA and deirid-GABA cells) and one bilaterally symmetrical pair of ventral ganglion cells (the VG-GABA cells). In sections, the RME-like cells and the VG-GABA cells were consistently stained through the cephalic region. However, anti-GABA staining of the lateral ganglia cells in sections was light, thus suggesting that they contain less GLIR than the other more intensely stained GABA-immunoreactive neurons. In the caudal region, a single GABA-immunoreactive neuron was present in the dorsal rectal ganglion. Our data suggest that these ten cephalic neurons, and a single dorsal rectal ganglion neuron, use GABA as a neurotransmitter.     

Embryonic differentiation of serotonin-containing neurons in the enteric nervous system of the locust (Locusta migratoria)

The enteric nervous system (ENS) of the locust consists of four ganglia (frontal and hypocerebral ganglion, and the paired ingluvial ganglia) located on the foregut, and nerve plexus innervating fore- and midgut. One of the major neurotransmitters of the ENS, serotonin, is known to play a vital role in gut motility and feeding. We followed the anatomy of the serotonergic system throughout embryonic development. Serotonergic neurons are generated in the anterior neurogenic zones of the foregut and migrate rostrally along the developing recurrent nerve to contribute to the frontal ganglion. They grow descending neurites, which arborize in all enteric ganglia and both nerve plexus. On the midgut, the neurites closely follow the leading migrating midgut neurons. The onset of serotonin synthesis occurs around halfway through development-the time of the beginning of midgut closure. Cells developing to serotonergic phenotype express the serotonin uptake transporter (SERT) significantly earlier, beginning at 40% of development. The neurons begin SERT expression during migration along the recurrent nerve, indicating that they are committed to a serotonergic phenotype before reaching their final destination. After completion of the layout of the enteric ganglia (at 60%) a maturational phase follows, during which serotonin-immunoreactive cell bodies increase in size and the fine arborizations in the nerve plexus develop varicosities, putative sites of serotonin release (at 80%). This study provides the initial step for future investigation of potential morphoregulatory functions of serotonin during ENS development.     

Radioimmunoassay determination of tachykinin-related peptide in different portions of the central nervous system and intestine of the cockroach Leucophaea maderae

A radioimmunoassay was developed for insect tachykinin-related peptides with the use of an antiserum raised to the locust neuropeptide locustatachykinin I (LomTK I). Determination of tachykinin-related peptide was performed in different tissues of the cockroach Leucophaea maderae. The largest amounts of LomTK-like immunoreactivity (LomTK-LI) reside in the brain and in the midgut. Relatively large amounts were also found in the suboesophageal ganglion and throughout the ganglia of the ventral nerve cord, whereas smaller amounts of LomTK-LI were detected in the corpora cardiaca, foregut and hindgut. Extracts of unfused abdominal ganglia and midguts, respectively, were analysed by a combination of reversed phase high performance liquid chromatography, and radioimmunoassay for LomTK-LI. The extracts of abdominal ganglia and midguts both contain LomTK-LI material which separates in at least two major components. This LomTK-LI material had retention times corresponding approximately to those of synthetic LomTK I and II. Since the cellular source of LomTK-LI material in the foregut and hindgut was not known from earlier studies, we investigated these tissues by immunocytochemistry. We found that the LomTK-LI material associated with the foregut was in arborizing fibres in the oesophageal and gastric nerves and in the ingluvial ganglion. In the hindgut the muscle layer was innervated by immunoreactive fibres derived from cell bodies in the terminal ganglion. The amount of LomTK-LI material in other portions of the nervous system correlates well with previous immunocytochemical data. We conclude that L. maderae have two or more isoforms of tachykinin-related peptides in the nervous system and intestine and that these are present in various amounts in different parts of the central nervous system and intestine. The relative large amounts of LomTK-LI material in the suboesophageal ganglion, oesophageal nerve and associated ganglia and intestine indicate important roles of tachykinin-related peptides in feeding and digestion.