Whole-brain efferent and afferent connectivity of mouse ventral tegmental area melanocortin-3 receptor neurons.

The mesolimbic dopamine (DA) system is involved in the regulation of multiple behaviors, including feeding, and evidence demonstrates that the melanocortin system can act on the mesolimbic DA system to control feeding and other behaviors. The melanocortin-3 receptor (MC3R) is an important component of the melanocortin system, but its overall role is poorly understood. Because MC3Rs are highly expressed in the ventral tegmental area (VTA) and are likely to be the key interaction point between the melanocortin and mesolimbic DA systems, we set out to identify both the efferent projection patterns of VTA MC3R neurons and the location of the neurons providing afferent input to them. VTA MC3R neurons were broadly connected to neurons across the brain but were strongly connected to a discrete set of brain regions involved in the regulation of feeding, reward, and aversion. Surprisingly, experiments using monosynaptic rabies virus showed that proopiomelanocortin (POMC) and agouti-related protein (AgRP) neurons in the arcuate nucleus made few direct synapses onto VTA MC3R neurons or any of the other major neuronal subtypes in the VTA, despite being extensively labeled by general retrograde tracers injected into the VTA. These results greatly contribute to our understanding of the anatomical interactions between the melanocortin and mesolimbic systems and provide a foundation for future studies of VTA MC3R neurons and the circuits containing them in the control of feeding and other behaviors.

Efferent projections of CGRP/Calca-expressing parabrachial neurons in mice.

The parabrachial nucleus (PB) is composed of glutamatergic neurons at the midbrain-hindbrain junction. These neurons form many subpopulations, one of which expresses Calca, which encodes the neuropeptide calcitonin gene-related peptide (CGRP). This Calca-expressing subpopulation has been implicated in a variety of homeostatic functions, but the overall distribution of Calca-expressing neurons in this region remains unclear. Also, while previous studies in rats and mice have identified output projections from CGRP-immunoreactive or Calca-expressing neurons, we lack a comprehensive understanding of their efferent projections. We began by identifying neurons with Calca mRNA and CGRP immunoreactivity in and around the PB, including populations in the locus coeruleus and motor trigeminal nucleus. Calca-expressing neurons in the PB prominently express the mu opioid receptor (Oprm1) and are distinct from neighboring neurons that express Foxp2 and Pdyn. Next, we used Cre-dependent anterograde tracing with synaptophysin-mCherry to map the efferent projections of these neurons. Calca-expressing PB neurons heavily target subregions of the amygdala, bed nucleus of the stria terminalis, basal forebrain, thalamic intralaminar and ventral posterior parvicellular nuclei, and hindbrain, in different patterns depending on the injection site location within the PB region. Retrograde axonal tracing revealed that the previously unreported hindbrain projections arise from a rostral-ventral subset of CGRP/Calca neurons. Finally, we show that these efferent projections of Calca-expressing neurons are distinct from those of neighboring PB neurons that express Pdyn. This information provides a detailed neuroanatomical framework for interpreting experimental work involving CGRP/Calca-expressing neurons and opioid action in the PB region.

Distribution of BDNF and trkB mRNA in the otic region of 3.5 and 4.5 day chick embryos as revealed with a combination of in situ hybridization and tract tracing.

We have used a recently developed technique which combines fluorescent tract tracing and in situ hybridization to study co-localization of neurotrophin mRNA and neurotrophin receptor mRNA expression simultaneously with the pattern of innervation in the developing chick ear. Efferent and afferent fibersfrom the VII/VIIIth cranial nerves were retrogradely and anterogradely filled with Dextran amines conjugated to Texas red and the brain stem was incubated for 2 hours in tissue culture medium. The tissue was subsequently fixed, sectioned frozen, mounted and subjected to in situ hybridization analysis using probes for brain-derived neurotrophic factor (BDNF) and its tyrosine kinase receptor, trkB. The results show that afferent and efferent fibers to the ear innervate areas of the developing otocyst which express BDNF mRNA. We also found that neurons in the stato-acoustic ganglion express high levels of trkB mRNA whereas the subset of facial motor neurons that is efferent to the ear only had no or very low levels of trkB mRNA. From our results we conclude that chicken otic efferent fibers preferentially project to areas with BDNF mRNA expression. The very low levels of trkB mRNA in these motor neurons compared to afferent neurons innervating the same region suggest that other factors, perhaps co-expressed with BDNF, may support efferents. A possible involvement of afferents in guiding efferents to specific areas of the ear is suggested.

[Autonomic angiotensinergic fibres in the human heart with an efferent sympathetic cophenotype]. / Le cophénotype sympathique efférent des fibres autonomes angiotensinergiques dans le cœur humain.

AIM: The autonomic innervation of the heart consists of sympathetic and parasympathetic nerve fibres, and fibres of the intrinsic ganglionated plexus with noradrenaline and acytylcholine as principal neurotransmitters. The fibres co-release neuropeptides to modulate intracardiac neurotransmission by specific presynaptic and postsynaptic receptors. The coexpression of angiotensin II in sympathetic fibres of the human heart and its role are not known so far. METHODS: Autopsy specimens of human hearts were studied (n=3; ventricles). Using immunocytological methods, cryostat sections were stained by a murine monoclonal antibody (4B3) directed against angiotensin II and co-stained by polyclonal antibodies against tyrosine hydroxylase, a catecholaminergic marker. Visualisation of the antibodies was by confocal light microscopy or laser scanning microscopy. RESULTS: Angiotensin II-positive autonomic fibres with and without a catecholaminergic cophenotype (hydroxylase-positive) were found in all parts of the human ventricles. In the epicardium, the fibres were grouped in larger bundles of up to 100 and more fibres. They followed the preformed anatomic septa and epicardial vessels towards the myocardium and endocardium where the bundles dissolved and the individual fibres spread between myocytes and within the endocardium. Generally, angiotensinergic fibres showed no synaptic enlargements or only a few if they were also catecholaminergic. The exclusively catechalominergic fibres were characterised by multiple beaded synapses. CONCLUSION: The autonomic innervation of the human heart contains angiotensinergic fibres with a sympathetic efferent phenotype and exclusively angiotensinergic fibers representing probably afferents. Angiotensinergic neurotransmission may modulate intracardiac sympathetic and parasympathetic activity and thereby influence cardiac and circulatory function.

Projection neurons of the mormyrid electrosensory lateral line lobe: morphology, immunohistochemistry, and synaptology.

This paper describes the morphological, immunohistochemical, and synaptic properties of projection neurons in the highly laminated medial and dorsolateral zones of the mormyrid electrosensory lateral line lobe (ELL). These structures are involved in active electrolocation, i.e., the detection and localization of objects in the nearby environment of the fish on the basis of changes in the reafferent electrosensory signal generated by the animal's own electric organ discharge. Electrosensory, corollary electromotor command-associated signals (corollary discharges), and a variety of other inputs are integrated within the ELL microcircuit. The organization of ELL projection neurons is analyzed at the light and electron microscopic levels based on Golgi impregnations, intracellular labeling, neuroanatomical tracer techniques, and gamma-aminobutyric acid (GABA), gamma-aminobutyric acid decarboxylase (GAD), and glutamate immunohistochemistry. Two main types of ELL projection neurons have been distinguished in mormyrids: large ganglionic (LG) and large fusiform (LF) cells. LG cells have a multipolar cell body (average diameter 13 microns) in the ganglionic layer, whereas LF cells have a fusiform cell body (on average, about 10 x 20 microns) in the granular layer. Apart from the location and shape of their soma, the morphological properties of these cell types are largely similar. They are glutamaterigic and project to the midbrain torus semicircularis, where their axon terminals make axodendritic synaptic contacts in the lateral nucleus. They have 6-12 apical dendrites in the molecular layer, with about 10,000 spines contacted by GABA-negative terminals and about 3,000 GABA-positive contacts on the smooth dendritic surface between the spines. Their somata and short, smooth basal dendrites, which arborize in the plexiform layer (LG cells) or in the granular layer (LF cells), are densely covered with GABA-positive, inhibitory terminals. Correlation with physiological data suggests that LG cells are I units, which are inhibited by stimulation of the center of their receptive fields, and LF cells are E units, excited by electric stimulation of the receptive field center. Comparison with the projection neurons of the ELL of gymnotiform fish, which constitute another group of active electrolocating teleosts, shows some striking differences, emphasizing the independent development of the ELL in both groups of teleosts.

Distribution of vasopressin and vasoactive intestinal polypeptide (VIP) fibers in the human hypothalamus with special emphasis on suprachiasmatic nucleus efferent projections.

The human suprachiasmatic nucleus (SCN) is located in the basal part of the anterior hypothalamus and is considered as the biological clock that generates circadian rhythms and synchronizes the daily activity pattern with the environmental light-dark cycle. However, the mechanisms and pathways by which the SCN transmits its information to the other brain areas are unknown. Therefore, in the present study, we investigated the efferent projections of the SCN by the immunocytochemical staining of two major peptidergic SCN neurotransmitters: vasopressin (VP) and vasoactive intestinal polypeptide (VIP). It confirmed that these peptides are present in different subdivisions of the SCN. The results of this investigation show that VP and VIP fibers arising from the SCN were detected to branch extensively and hence seem to innervate the SCN itself and the central and medial part of the anteroventral hypothalamic area (AVH), the area below the paraventricular nucleus (sub-PVN), the ventral part of the paraventricular nucleus (PVN), and the dorsomedial nucleus of the hypothalamus (DMH). There appeared to be substantial congruity between the presumptive human SCN projections and those as observed by tracing in rat or hamster. Regarding the anatomical organization of the human SCN projections, the main projection areas appeared to be the AVH, the sub-PVN, the ventral part of the PVN, and the DMH. The observation that VIP and in particular VP fibers pass between the SCN and the PVN suggests that the human SCN and the PVN may have a direct anatomical connection. In addition, VP and VIP fibers were detected in several other hypothalamic areas that are not known to have clear direct connections to the SCN. The possible origin of these VP and VIP fibers is discussed.

Engrailed protein is expressed in interneurons but not motor neurons of the dorsal unpaired median group in the adult grasshopper.

We report that the homeodomain protein Engrailed (En) is differentially expressed by neuronal type. Expression was examined within identified midline neurons in T3, A1, and A2 neuromeres of the adult grasshopper by using immunohistochemistry. All save a few neurons in the adult dorsal unpaired median (DUM) group arise embryonically from a single precursor, the median neuroblast. DUM neurons are efferent neurons, local interneurons, or intersegmental interneurons, recognizable as such by their distinct morphologies and neurotransmitter phenotypes. We show that interneurons are En-positive, whereas efferents are En-negative. In the T3 DUM group, the 70 or so interneurons contained cytoplasmic immunoreactivity for gamma-aminobutyric acid (GABA) and glutamate decarboxylase. In double-labeling experiments, all GABA-immunoreactive neurons were also En-positive, and all En-positive neurons contained GABA immunoreactivity. In complementary experiments, the 20 or so efferents in the T3 DUM group, which are octopaminergic, were selectively labeled with a histological marker and then processed to reveal En immunoreactivity. No efferents in the group were En-positive. The abdominal DUM groups contain fewer neurons, but the same dichotomy of labeling was found. The En pattern is established during embryogenesis, with the type-specific pattern apparent by stage 90% of development, the earliest stage examined here. The differential expression of En in the embryo and its continued expression in the adult nervous system suggest a role in the development and maintenance of neuronal phenotype. Morphological differences between efferents and interneurons are discussed in light of a hypothesis that En mediates differential expression of cell adhesion or cell-affinity molecules.

Axonal transport of neurotrophins by visceral afferent and efferent neurons of the vagus nerve of the rat.

The receptor-mediated axonal transport of [125I]-labeled neurotrophins by afferent and efferent neurons of the vagus nerve was determined to predict the responsiveness of these neurons to neurotrophins in vivo. [125I]-labeled neurotrophins were administered to the proximal stump of the transected cervical vagus nerve of adult rats. Vagal afferent neurons retrogradely transported [125I]neurotrophin-3 (NT-3), [125I]nerve growth factor (NGF), and [125I]neurotrophin-4 (NT-4) to perikarya in the ipsilateral nodose ganglion, and transganglionically transported [125I]NT-3, [125I]NGF, and [125I]NT-4 to the central terminal field, the nucleus tractus solitarius (NTS). Vagal afferent neurons showed minimal accumulation of [125I]brain-derived neurotrophic factor (BDNF). In contrast, efferent (parasympathetic and motor) neurons located in the dorsal motor nucleus of the vagus and nucleus ambiguus retrogradely transported [125I]BDNF, [125I]NT-3, and [125I]NT-4, but not [125I]NGF. The receptor specificity of neurotrophin transport was examined by applying [125I]-labeled neurotrophins with an excess of unlabeled neurotrophins. The retrograde transport of [125I]NT-3 to the nodose ganglion was reduced by NT-3 and by NGF, and the transport of [125I]NGF was reduced only by NGF, whereas the transport of [125I]NT-4 was significantly reduced by each of the neurotrophins. The competition profiles for the transport of NT-3 and NGF are consistent with the presence of TrkA and TrkC and the absence of TrkB in the nodose ganglion, whereas the profile for NT-4 suggests a p75 receptor-mediated transport mechanism. The transport profiles of neurotrophins by efferent vagal neurons in the dorsal motor nucleus of the vagus and nucleus ambiguus are consistent with the presence of TrkB and TrkC, but not TrkA, in these nuclei. These observations describe the unique receptor-mediated axonal transport of neurotrophins in adult vagal afferent and efferent neurons and thus serve as a template to discern the role of specific neurotrophins in the functions of these visceral sensory and motor neurons in vivo.

P2X(2) receptor immunoreactivity in the dorsal vagal complex and area postrema of the rat.

Adenosine 5'-triphosphate (ATP) can function as a fast synaptic transmitter through its actions on ionotropic (P2X) and metabotropic (P2Y) receptors in neuronal tissue. The ionotropic receptors have been classified into seven subtypes (P2X(1)-P2X(7)) by molecular cloning. However, they are difficult to distinguish pharmacologically owing to an absence of specific agonists and antagonists. In this study we used neuroanatomical methods to determine the origin and neurochemical phenotype of the P2X(2) subtype of purinoceptor in the dorsal medulla of the rat. Using immunohistochemistry we observed dense networks of P2X(2) receptor immunoreactive labelled fibres and terminals in the dorsal vagal complex and area postrema, as well as labelled cell bodies in the dorsal vagal nucleus and the area postrema. The P2X(2) receptor was localized presynaptically in vagal afferent fibres and terminals in the nucleus tractus solitarius at the ultrastructural level by combining injections of an anterograde tracer (biotin dextran amine) into the nodose ganglion with pre-embedding immunogold visualization of P2X(2) immunoreactivity. Terminals immunoreactive for the P2X(2) receptor in the nucleus tractus solitarius were found to contain glutamate, but not GABA immunoreactivity by post-embedding immunogold-labelling techniques. In cell bodies in the area postrema, dual immunofluorescence also indicated that P2X(2) receptor immunoreactive cells are glutamatergic but not GABAergic. The P2X(2) receptor was localized to vagal preganglionic neurons in the dorsal vagal nucleus that were identified by prior intraperitoneal injections of the retrograde tracer FluoroGold. No cells immunoreactive for the P2X(2) receptor were observed in the nucleus tractus solitarius. The localization of P2X(2) receptor immunoreactivity presynaptically in vagal afferent terminals indicates that the receptor may be involved in modulating transmitter release from vagal afferent fibres. Furthermore, the presence of the P2X(2) receptor in vagal preganglionic cells and in glutamatergic cells of the area postrema implies that it may, respectively, play a role in regulation of vagal efferent cell activity and modulation of excitatory outputs from the area postrema to other brain regions.

Characterization of peptidergic efferents from the lateral parabrachial nucleus to identified neurons in the rat dorsal raphe nucleus.

The peptidergic content of the lateral parabrachial nucleus (LPB) efferents to the dorsal raphe nucleus (DRN) was studied by combining visualization of the anterogradely transported tracer Phaseolus vulgaris leucoagglutinin within fibers that were immunocytochemically stained for neurotensin (NT), calcitonin gene-related peptide (CGRP) or galanin (GAL). The identity of DRN target neurons was determined with simultaneous immunocytochemical labelling for serotonin, the major transmitter within the nucleus. Within the DRN, we estimated that about two-thirds of the anterogradely labelled fibers arising from the LPB also showed peptidergic immunoreactivity. NT was the most commonly observed neuropeptide in LPB neuronal efferents directed to the DRN, followed by CGRP and GAL. The peptidergic afferents in the DRN were oriented preferentially in the dorsoventral plane. Peptidergic fibers from the LPB possessed varicosities (diameters not exceeding 3 microns) and were apposed on serotoninergic neuronal somata. Some of the anterogradely labelled peptidergic fibers were not associated with cells showing immunoreactivity for serotonin. The present results suggest that NT-ergic, CGRP-ergic and GAL-ergic neurons within the LPB are in contact with serotoninergic and non-serotoninergic neurons within the DRN. Since the DRN is known to project to the LPB, it is likely that bi-directional interconnections between these nuclei exist. Such linkages may provide anatomical substrates for coordinated autonomic responses.

Three-dimensional spatial relationship of neuropeptides and receptors in the rat dorsal vagal complex.

Retrograde tracing, multi-label fluorescence immunohistochemistry, confocal microscopy and three-dimensional (3-D) reconstruction techniques were combined to examine the spatial relationship of immunoreactive nerve terminals containing either calcitonin gene-related polypeptide (CGRP) or substance P (SP) to identified gastric efferent neurons in the dorsal motor nucleus of the vagus (DMV) in the brainstem of the rat. The availability of an antibody to the receptor for SP (NK-1r) permitted observation of the association between peptide and receptor. Although both SP-IR and CGRP-IR nerve fibres came in close spatial proximity to identified gastric efferent neurons, few discrete contacts between these fibres and the neuronal membrane were observed. In addition, NK-1r-IR was localized to the somatic and dendritic membranes of a subpopulation of neurons within the DMV, with the majority of receptor labelling not in close spatial proximity to SP-IR nerve fibres. The methodology described in this study permitted the simultaneous observation of the spatial relationship between neuropeptide and an identified neuron (and the corresponding receptor in the case of SP) in 3-D, which is something that cannot be achieved using conventional microscopic techniques

Muscarinic receptor-mediated calcium signaling in spiral ganglion neurons of the mammalian cochlea.

Using indo-1 microspectrofluorometry, we examined the effects of cholinergic agonists on the concentration of intracellular Ca(2+) ions ([Ca(2+)](i)) in spiral ganglion neurons, isolated from rat cochleae at different stages of post-natal development (from P3 to P30). Extracellular application of acetylcholine (ACh) or carbamylcholine generated a rapid and transient increase in [Ca(2+)](i). The ACh concentration-response curve indicated an apparent dissociation constant (K(d)) of 8 microM and a Hill coefficient of 1.0. Removing extracellular free Ca(2+) did not suppress the ACh-induced Ca(2+) responses suggesting an intracellular Ca(2+)-release mechanism. When we compared the cholinergic response at different stages of postnatal development, there were no significant differences on the aspect of the Ca(2+) response and the percentage of responsive neurons, which ranged between 50 and 65% per cochlear preparation. The application of muscarine triggered reversible Ca(2+) responses similar to those observed with ACh, with an apparent K(d) of 10 microM and a Hill coefficient of 1.0. The cholinergic-induced Ca(2+)pirenzepine. Nicotine (10 to 100 microM) did not evoke Ca(2+) responses and the nicotinic antagonist curare (10 microM) did not block the ACh-evoked responses. The present study is the first direct demonstration of functional muscarinic receptors (mAChRs) in spiral ganglion neurons. These mAChRs activated by the cholinergic lateral efferent system may participate in the regulation of the electrical activity of the afferent auditory fibers contacting the inner hair cells.

Calcitonin gene-related peptide immunoreactivity in the rat efferent vestibular system during development.

The organization of the efferent fiber network during postnatal development was investigated by immunocytochemical detection of the calcitonin gene-related peptide (CGRP) in rat vestibular receptors from postnatal day 0 (PD 0) to adulthood. CGRP was detected at birth in a few efferent fibers below the sensory epithelia of cristae and maculae. Thereafter, the nerve fibers in the cristae progressively invaded the epithelia with an apex to base gradient from PD 2 to PD 4. There was also a rearrangement of the fibers during maturation of the efferent innervation, such that after reaching the surface of the epithelium, they turned back and moved towards the base of the sensory cells, producing numerous synaptic contacts. Analysis of surface preparations of utricules showed the irregular and asymmetric topographic organization of the efferent fiber network and the extensive, complex distribution of this innervation. The presence and broad distribution of CGRP in the epithelium at critical stages of development and synaptogenesis suggests that it is involved in the maturation of vestibular receptors.

Olivocochlear innervation of inner and outer hair cells during postnatal maturation: an immunocytochemical study.

Changes in the expression of several neurochemical markers associated with either axonal growth (GAP-43), synaptic vesicles (synaptophysin), or the cholinergic population of lateral olivocochlear (OC) efferents were investigated in the postnatal cochlea of hamsters. Growth-associated protein was expressed in the neonatal cochlea but not in the adult; immunoreactivity was found below inner hair cells (IHCs) from postnatal day (P) 2 through P14 and below outer hair cells (OHCs) from P5 to P14. In contrast, synaptophysin was expressed in both the neonate and adult cochlea; immunoreactivity was found below IHCs around P4 and below OHCs at P5. Both GAP-43 and synaptophysin immunoreactivities occurred first below IHCs in basal regions of the cochlea. Efferent fibers containing calcitonin gene-related peptide (CGRP) immunoreactivity were identified as early as P4 within the cochlear nerve but were not observed underneath IHCs until P7. Similar to GAP-43 and synaptophysin immunoreactivity, CGRP expression followed a basal to apical gradient; however, expression below OHCs appeared restricted to apical regions. These data raise the possibility that efferents expressing growth proteins and efferents expressing synaptic vesicle proteins co-exist during the first postnatal week. Furthermore, it is hypothesized that CGRP-containing lateral OC neurons form part of a later, secondary innervation to the cochlea.

Nervus terminalis projection to the retina in the 'four-eyed' fish, Anableps anableps.

The eye of the surface dwelling 'four-eyed' fish, Anableps possesses an aquatic and an aerial optical system. The aerial system is strongly hyperopic when the animal dives, i.e. during mating, and the dorsal pupil is submerged. We studied the retino-petal nervus terminals projection to the aerial and to the aquatic retina by Phe-Met-Arg-Phe-NH2 (FMRF) immunocytochemistry and found both to be equally innervated. This finding sheds doubt on the proposed functional significance of this projection for reproductive behaviour.

Enkephalin-like immunoreactivity in the chick brainstem: possible relation to the cochlear efferent system.

Mammalian lateral olivocochlear (LOC) neurons that are immunoreactive for choline acetyltransferase (ChAT) are also immunoreactive for enkephalin (Enk). To determine whether cochlear efferent neurons in birds might also contain Enk-like immunoreactivity (Enk-LI), we studied the auditory brainstem of the domestic chicken using antisera to ChAT, leucine-enkephalin (L-Enk) and methionine-enkephalin (M-Enk). Enk-LI terminals are found around, but not within, the superior olivary nucleus (SO) and the nucleus of the lateral lemniscus, pars intermedia (LLi). A moderate concentration of Enk-LI terminals is found ventromedial to the ventral facial nucleus (VIIv) where the ventrolateral group of ChAT-I cochlear efferent neurons is located. After colchicine injections into the lateral ventricle, a population of intensely stained Enk-LI perikarya was found in the nucleus of the lateral lemniscus, pars ventralis (LLv) with scattered cells in the LLi and the nucleus subceruleus ventralis (SCv). The distribution of Enk-LI and ChAT-I somata, however, never overlapped, even in adjacent sections. Thus, in the chick, Enk-LI perikarya are not distributed in areas where cochlear efferent neurons are found. Instead, a dense concentration of Enk-I terminals can be found in areas containing ChAT-I cochlear efferent neurons. The source of these enkephalinergic terminals may be a population of Enk-LI cells in the LLv.

Putative inhibitory collicular boutons contact large neurons and their dendrites in the dorsal cochlear nucleus of the rat.

Within the circuits of the acoustic nuclei, the inferior colliculus sends descending (collicular) terminals to control with a feedback mechanism, part of the activity of the dorsal cochlear nucleus (DCN). It is not known whether this descending projection is prevalently excitatory or inhibitory. Using the neuronal tracer Wheat Germ Agglutinin conjugated to Horse Radish Peroxidase (WGA-HRP) the connections between the inferior colliculus and the DCN of the rat have been investigated. By far most retrograde labelled large neurons were glycine and GABA negative (pyramidal and giant neurons) and rare medium-size cells were glycine positive. The ultrastructural immunocytochemical analysis for glycine and GABA shows that mainly large, excitatory, neurons innervate the inferior colliculus. Rare medium-size glycine-positive cells with intermediate characteristics between pyramidal and cartwheel cells, seem also to project to the colliculus. Few WGA-HRP labelled boutons contact the large cells or their dendrites, have symmetric pre- and post-synaptic thickenings, contain pleomorphic and/or flat vesicles, and are labelled for GABA or glycine. Since no GABA labelled cells in both the dorsal and ventral cochlear nucleus were retrograde labelled from the colliculus, the source of these intrinsic anterograde labelled boutons must be external to the cochlear nucleus. GABA positive neurons are both present in the inferior colliculus (injected with the tracer) and superior olivary complex (not injected with the tracer). This suggests that the double labelled boutons (WGA-HRP and GABA) are inhibitory GABA-ergic collicular terminals contacting the excitatory neurons of the DCN. Other few boutons or mossy fibers containing round vesicles and immunonegative for both glycine and GABA, were also seen contacting the large neurons and their dendrites in the DCN. As the round vesicles boutons may be derived from other retrograde cells of the cochlear nucleus (pyramidal and stellate cells) and those glycine positive from the glycinergic neurons in paraolivary nuclei, it is more likely that only the WGA-HRP and GABA labelled boutons are true collicular terminals.

Effects of ghrelin on neuronal survival in cells derived from dorsal motor nucleus of the vagus.

BACKGROUND: The effects of intestinal inflammation on the central neurons projecting to the enteric nervous system are unknown. The dorsal motor nucleus of the vagus signals to the gastrointestinal system. Ghrelin is elevated in patients with inflammatory bowel disease and has been implicated as an inflammatory mediator. The purpose of this study was to investigate the effects of gastrointestinal inflammation on the dorsal motor nucleus of the vagus in rats, as well as the effects of proinflammatory cytokines and ghrelin on neurons from the dorsal motor nucleus of the vagus in vitro. METHODS: DiI was injected into the stomach wall of rats to retrogradely label neurons of the dorsal motor nucleus of the vagus. Intestinal inflammation was induced with indomethacin injection. Serial serum ghrelin measurements were performed. Tissue was examined under fluorescent microscopy. In vitro studies using primary culture of neurons from the dorsal motor nucleus of the vagus were performed. Reverse transcriptase-polymerase chain reaction for cytokine transcripts and immunohistochemistry for cytokine receptors were performed. Cell proliferation and apoptosis were measured by enzyme-linked immunosorbent assay. RESULTS: A significant decrease of DiI labeling was demonstrated in the dorsal motor nucleus of the vagus of animals injected with indomethacin. Serum levels of ghrelin were significantly elevated 2 days after induction of inflammation. In vitro, apoptosis and cell proliferation were measured after 24-hour exposure to experimental conditions. Ghrelin alone had no effect on apoptosis. Exposure to interleukin (IL)-1 beta or tumor necrosis factor (TNF)-alpha increased apoptosis. The addition of ghrelin to cytokine resulted in significant decreases in apoptosis compared to cytokine alone. Ghrelin significantly increased neuronal proliferation. Exposure to IL-1 beta, IL-6, or TNF-alpha significantly decreased proliferation. The addition of ghrelin to TNF-alpha or IL-6 significantly increased cellular proliferation compared to cytokine alone. CONCLUSIONS: Neurons from the dorsal motor nucleus of the vagus that project to the stomach are reduced in number after induction of colitis in rats. In vitro, proinflammatory cytokines increase apoptosis and decrease cell proliferation of neurons from the dorsal motor nucleus of the vagus. These effects are attenuated by ghrelin.

Acetylcholine receptors in spider peripheral mechanosensilla.

Peripherally located parts of spider mechanosensory neurons are modulated by several neurotransmitters released from apposed efferent fibers. Activities of acetylcholine (ACh) synthesizing enzyme choline acetyltransferase (ChAT) and ACh degrading enzyme acetylcholine esterase (AChE) were previously found in some efferent fibers. ChAT activity was also present in all the mechanosensory neurons, while AChE activity was only found in some. We show that spider mechanosensory neurons and probably some efferent neurons are immunoreactive to a monoclonal antibody against muscarinic ACh receptors (mAChRs). However, application of muscarinic agonists did not change the physiological responses or membrane potentials of neurons in the lyriform organ VS-3. Similarly, the sensitivities of the neurons of trichobothria (filiform hairs) remained unchanged after application of these agonists. Therefore, activation of mAChRs may only modulate the function of spider mechanosensory neurons indirectly, for example, by affecting the release of other transmitter(s). However, a subgroup of VS-3 neurons was inhibited by ACh, which also depolarized the membrane similar to these neurons' responses to GABA, suggesting that ACh activates anion channels in these neurons. Interestingly, all of the neurons responding to ACh were the rapidly adapting Type A neurons that were previously shown to express AChE activity.