Evaluation of the total number of myenteric neurons in the developing chicken gut using cuprolinic blue histochemical staining and neurofilament immunocytochemistry

The aim of this study was to find an improved method with which to stain the entire population of myenteric neurons in the different segments of the developing chicken intestine. Histochemical staining with cuprolinic blue (quinolinic phthalocyanine) and immunostaining against neurofilament (NF) were performed on whole mounts prepared from intestinal segments of embryonic (day 19 of incubation) and hatched (1, 2, 4 and 7 days after hatching) chickens. Double labelling was performed to evaluate to what extent the two markers visualise the same nerve cell population. Cuprolinic blue stained neuronal somata highly selectively, whereas processes and glia cells were poorly labelled. The cuprolinic blue-positive neurons were uniform in shape. NF immunostaining revealed a morphologically highly variable neuron population. Double labelling with cuprolinic blue and NF resulted in an intensification of both stainings, allowing an accurate morphological classification of NF-stained myenteric neurons. Data obtained from the counting of cuprolinic blue-positive neurons were subjected to two-way ANOVA and the Tukey probe. The densities of ganglia and neurons were found to decrease, and the mean number of neurons per myenteric ganglion to increase, with different dynamics along the longitudinal axis of the gut during the examined time span. The variances in the number of NF-positive neurons were not homogeneous, and the data were therefore not suitable for ANOVA. Accordingly, only semiquantitative conclusions could be drawn.     

Synaptic interaction of serotonergic axons and corticotropin releasing factor (CRF) synthesizing neurons in the hypothalamic paraventricular nucleus of the rat. A light and electron microscopic immunocytochemical study

The morphological interrelationship between the central serotonergic and hypothalamic corticotropin-releasing factor (CRF) synthesizing systems was studied in the hypothalamic paraventricular nucleus (PVN) of colchicine pretreated male rats. The simultaneous immunocytochemical localization of the transmitter and peptide employed the peroxidase-antiperoxidase complex (PAP) technique using the silver-gold intensified (SGI) and non-intensified forms of the oxidized 3,3'-diaminobenzidine (DAB) chromogen. The paraventricular nucleus received a moderate serotonergic innervation as compared with other diencephalic structures. The distribution and arborization of serotonergic axons were more prominent in the parvocellular subnuclei than in the magnocellular units of the nucleus. Serotonin containing axons formed terminal bouton and en passant type synapses with dendrites and somata of parvocellular neurons. The immunocytochemical double labelling technique revealed the overlapping of serotonergic axons and CRF-immunoreactive neurons. Vibratome (40 micron) and semithin (1 micron) sections indicated that the interneuronal communication may take place on both dendrites and cell bodies of CRF-immunoreactive neurons. Ultrastructural analysis demonstrated that serotonin-containing terminals formed axo-dendritic and axo-somatic synapses with CRF-immunoreactive neurons. These findings indicate that the central serotonergic neuronal system can influence the function of the pituitary-adrenal endocrine axis via a direct action upon the hypophysiotrophic CRF synthesizing neurons.     

Retrograde neuronal tracing with cholera toxin B subunit: comparison of three different visualization methods

Summary In this report a comparison is made of three different visualization methods of rat cervical motoneurons retrogradely labelled with cholera toxin B subunit (CTb). CTb is a very sensitive retrograde neuro-anatomical tracer which can be detected either by immunochemical methods, or by the use of CTb conjugates such as CTb-HRP and CTb-FITC or CTb-TRITC, which can be visualized after histochemical detection and by fluorescence microscopy, respectively. The following results were obtained. (1) Immunochemical detection of CTb with peroxidase and DAB-Ni incubation provides the best labelling of the cell bodies and their processes, whereas immunochemical detection with FITC produces less effective labelling of the dendrites. (2) Histochemical visualization of CTb-HRP conjugate gives results similar to those of CTb immunochemistry but produces a much more granular appearance of the label, which may affect the identification of distal dendrites. In addition, direct electron-microscopic analysis of labelled structures can be achieved. (3) CTb-FITC and CTb-TRITC visualization permit double-labelling experiments but the labelled cells exhibit fluorescence only in their somata and proximal dendrites. (4) Factors other than labelling Intensity, e.g. double-labelling, preservation of the label, compatibility with other techniques and even economic reasons must be taken into consideration when a selection of visualization methods is to be made.     

Some neurohistochemical properties of nerve elements in myenteric plexus of rabbit ileum: similarities and dissimilarities to the rodent pattern

Enteric neurons have distinct neurochemical codings in each species. The basal tone of the gastrointestinal tract of the rabbit is low and produces neurally evoked pendular movements. Therefore, it might have an innervation pattern different from that of other laboratory animals. We have characterised myenteric neuron populations in rabbit ileum with neurochemical markers that are known to be associated with distinct cell types and/or fibre systems in the myenteric plexus. The density of nerve cells estimated with the NADH-diaphorase technique was about 2500 cells/cm² and most, if not all, neurons contained microtubule-associated protein 2. NADPH-diaphorase-positive cells were numerous. One cell type was large and emitted long straight processes, whereas small cells bore thin filamentous dendrites. Neurons immunoreactive for 28-kDa calcium-binding protein were rare. Over 70% of them had very strongly labelled lamellar dendrites. Their axons were beaded and formed pericellular baskets around unstained somata. We found very few small tyrosine-hydroxylase-positive cells. The fibre network in the plexus was very strong; the axons formed many pericellular baskets. In double labelling studies, no co-localisation was revealed between the 28-kDa calcium-binding protein and NADPH-diaphorase. Some fibres containing 28-kDa calcium-binding protein formed only a few contacts on somata of NADPH-diaphorase-positive cells. None of the NADPH-diaphorase-labelled cells were found to be stained for tyrosine hydroxylase. Tyrosine-hydroxylase-positive fibres rarely made pericellular baskets on the surface of NADPH-diaphorase-positive somata. Strongly immunolabelled pericellular baskets were never observed around NADPH-diaphorase-positive cell somata. The results suggest that myenteric neurons in rabbit comprise distinct and characteristic neurochemical properties that are different from the rodent pattern. Therefore, the explanation of the motility pattern of rabbit intestine can be approached on a chemical neuroanatomical basis. Received: 6 August 1997/Accepted: 8 October 1997     

Misidentification of cardiac vagal pre-ganglionic neurons after injections of retrograde tracer into the pericardial space in the rat

We evaluated whether pericardial injections of the retrograde tracers cholera toxin subunit B (CTb) or Fast Blue (FB) reliably labelled cardiac vagal pre-ganglionic neurons. Injections of CTb into the pericardial space of the rat labelled neurons in both the external and compact formations of the nucleus ambiguus. Most labelled neurons were found in the compact formation of the nucleus ambiguus, and the majority of these, and only these, expressed immunoreactivity for calcitonin gene-related peptide. This distribution of labelled neurons and their immunohistochemical properties is characteristic of oesophageal motoneurons. Examination of the oesophagus following intra-pericardial CTb applications revealed strong labelling of motor end plates within the skeletal muscle of the thoracic but not the abdominal oesophagus. When a second retrograde tracer, FB, was injected into the abdominal oesophagus, labelled somata were found adjacent to CTb-labelled neurons in the compact formation of the nucleus ambiguus. No co-localisation of tracers was found, but identical proportions of calcitonin gene-related peptide (CGRP) immunoreactivity were observed in both groups of neurons. FB injected into the pericardial space labelled intra-cardiac neurons but not brainstem neurons. We conclude that intra-pericardial, and perhaps sub-epicardial, injections of some retrograde tracers are likely to label a subset of oesophageal, as well as cardiac, vagal motor neurons in the brainstem.This work was supported in part by grant No. G 00 M 0670 from the National Heart Foundation of Australia.     

Synaptic interaction of serotonergic axons and corticotropin releasing factor (CRF) synthesizing neurons in the hypothalamic paraventricular nucleus of the rat

Summary The morphological interrelationship between the central serotonergic and hypothalamic corticotropin-releasing factor (CRF) synthesizing systems was studied in the hypothalamic paraventricular nucleus (PVN) of colchicine pretreated male rats. The simultaneous immunocytochemical localization of the transmitter and peptide employed the peroxidase-antiperoxidase complex (PAP) technique using the silver-gold intensified (SGI) and non-intensified forms of the oxidized 3,3-diaminobenzidine (DAB) chromogen.The paraventricular nucleus received a moderate serotonergic innervation as compared with other diencephalic structures. The distribution and arborization of serotonergic axons were more prominent in the parvocellular subnuclei than in the magnocellular units of the nucleus. Serotonin containing axons formed terminal bouton and en passant type synapses with dendrites and somata of parvocellular neurons. The immunocytochemical double labelling technique revealed the overlapping of serotonergic axons and CRF-immunoreactive neurons. Vibratome (40 m) and semithin (1 m) sections indicated that the interneuronal communication may take place on both dendrites and cell bodies of CRF-immunoreactive neurons. Ultrastructural analysis demonstrated that serotonin-containing terminals formed axo-dendritic and axo-somatic synapses with CRF-immunoreactive neurons. These findings indicate that the central serotonergic neuronal system can influence the function of the pituitary-adrenal endocrine axis via a direct action upon the hypophysiotrophic CRF synthesizing neurons.Supported by NIH Grant NS19266     

Interrelations between monoaminergic afferents and corticotropin-releasing factor-immunoreactive neurons in the rat central amygdaloid nucleus: ultrastructural evidence for dopaminergic control of amygdaloid stress systems

Ample evidence implicates corticotropin-releasing factor (CRF)-producing neurons of the central amygdaloid nucleus (CeA) in vegetative, endocrine, and behavioral responses to stress and anxiety in laboratory rats. Monoaminergic systems are involved in modulating these responses. In the present paper, interrelations between CRF-immunoreactive (ir) neurons, and noradrenergic, serotonergic, and dopaminergic afferents were studied using single and double immunolabeling for light and electron microscopy in the rat CeA. Dopaminergic axons formed dense plexus in the CeA overlapping with the localization of CRF-ir neurons, and their terminals formed frequent associations with CRF-ir somata. Contacts of serotonergic axons on CRF-ir neurons were few, and contacts of noradrenergic axons were the exception. Ultrastructurally, symmetric synapses of dopaminergic terminals on CRF-ir somata and dendrites were found. More than 83% of CRF-ir somata were contacted in single ultrathin sections. About half of these possessed two or more contacts. Of non-ir somata, 37% were contacted by dopaminergic terminals, and only 13% of these had two or more contacts. Correlative in situ hybridization indicated that CeA CRF-ir neurons may express receptor subtype dopamine receptor subtype 2. In conclusion, dopaminergic afferents appear to specifically target CeA CRF neurons. They are thus in a position to exert significant influence on the rat amygdaloid CRF stress system.     

Anatomical relationship between neuropeptide-containing fibers and efferent vagal neurons projecting to the rat corpus.

Injections of the retrograde tracers into the posterior surface of the stomach at the greater curvature resulted in labelling of the right half of the dorsal motor nucleus of the vagus. Whereas injections into the anterior and posterior surfaces of the corpus resulted in bilateral labelling in the medulla. Immunocytochemical staining of the labelled sections using antisera to substance P was confined to a dense network of fibers within the dorsal motor nucleus of the vagus and the nucleus tractus solitarius with no cell bodies being detected. Calcitonin gene-related peptide-immunoreactivity was detected in nerve fibers in the nucleus tractus solitarius and cell bodies of the hypoglossal nucleus. Finally, neuropeptide Y-immunoreactivity was confined to nerve fibers within the vagal complex. Of the neurons labelled by the retrograde tracers injected into the corpus all were in close spatial contact with fibers containing substance P-immunoreactivity. A smaller number were associated with neuropeptide Y-containing fibers with a few adjacent to calcitonin gene-related peptide-immunoreactive fibers. These results indicate that substance P and neuropeptide Y may directly regulate efferent neurons controlling gastric motility and acid secretion. Calcitonin gene-related peptide, however, is unlikely to directly modulate the cell bodies of the neurons in the dorsal motor nucleus but may modulate the dendrites from these neurons in the nucleus tractus solitarius.     

A topography and ultrastructural characterization ofin vivo 5,7-dihydroxytryptamine-labeled serotonin-containing neurons in the central nervous system ofAplysia californica

1. Several weeks after administration of 5,7-dihydroxytryptamine (5,7-DHT) to Aplysia, a dark pigmentation appears in serotonin-containing neurons, and this pigmentation allows visual identification of serotonergic neurons but does not appear to alter their physiology. 2. We have determined the distribution of labeled nerve cell bodies in the various ganglia of Aplysia and have characterized the pigment containing structures in both control and labeled neurons. 3. All neurons in this preparation, whether or not they utilize serotonin as a transmitter, contain pigment granules, and three types of pigment granules can be distinguished. After 5,7-DHT a new type of granule appears in serotonergic neurons, probably reflecting lysosomes that have accumulated serotonergic synaptic vesicles that contain the oxidized 5,7-DHT. 4. It remains unclear why this substance does not cause neurotoxicity in mollusks as it does in mammalian preparations.     

False transmitter, 5,6-dihydroxytryptamine as a tool in selecting serotonergic Helix neurons for studies with isolated,dialysed cells

1. Dialysed serotonergic neurons were identified, isolated from the ganglia of 5,6-dihydroxytryptamine (5,6-DHT) treated snail, Helix pomatia L. Twenty-four to 40 days after injection of 5,6-DHT into the animal, serotonergic neurons show a specific brown pigmentation, which stays there for several weeks. After protease digestion (0.5–1.0 mg/ml for 10–12 min) the labelled neurons can be easily separated. This method ensures the reliable identification of serotonergic neurons for intracellular dialysis.2. We showed that isolated serotonergic neurons maintain their membrane characteristics, and ion-currents can be registered under voltage clamp, just as from neurons of untreated animals. The threshold concentration of serotonin (10 ⁻⁷ M) and the survival time of pigment labelled dissociated cells were the same as for the control cells.3. Following 5-HT application, the voltage activated Ca-currents were either increased or decreased, depending on the neuron used.4. The different responses are probably caused by different receptors on the cell membrane or by the presence of different types of Ca-channels.5. The deactivation time constant of the Ca-current, calculated from the tail current, was also altered in the pigment labelled neuron following serotonin treatment.     

Central mechanisms of pain modulation.

Bulbospinal serotonergic neurons and two physiological classes of bulbospinal nonserotonergic cells interact to modulate pain transmission. Recent studies have begun to elaborate targets of descending pain modulation other than the well-studied flexion withdrawal pathways. Site-specific, naloxone-sensitive placebo analgesia, which is hard to reconcile with current models of descending pain modulation, presents an exciting challenge to the field.     

Compartmental culture of embryonic stem cell-derived neurons in microfluidic devices for use in axonal biology

Axonal pathology has been clearly implicated in neurodegenerative diseases making the compartmental culture of neurons a useful research tool. Primary neurons have already been cultured in compartmental microfluidic devices but their derivation from an animal is a time-consuming and difficult work and has a limit in their sources. Embryonic stem cell (ESC)-derived neurons (ESC_Ns) overcome this limit, since ESCs can be renewed without limit and can be differentiated into ESC_Ns by robust and reproducible protocols. In this research, ESC_Ns were derived from mouse ESCs in compartmental microfluidic devices, and their axons were isolated from the somal cell bodies. Once embryoid bodies (EBs) were localized in the microfluidic culture chamber, ESC_Ns spread out from the EBs and occupied the cell culture chamber. Their axons traversed the microchannels and finally were isolated from the somata, providing an arrangement comparable to dissociated primary neurons. This ESC_N compartmental microfluidic culture system not only offers a substitute for the primary neuron counterpart system but also makes it possible to make comparisons between the two systems.     

Immunohistochemical study on the distribution of serotonin-containing cell bodies in the brain stem of the dog

The distribution of serotonin-containing neurons in the brain of the dog was studied by use of PAP immunohistochemistry. The lower brain stem was endowed with extensively scattered serotonergic cell bodies, a large portion of which was located in the raphe nuclei. At the same time, prominent distribution of serotonergic neurons in lateral areas outside the raphe nuclei was also demonstrated. Our observations on the brain stem were, in principle, consistent with those on rats, cats and monkeys, with only minor differences.     

A rapid procedure for visualising the inner cell mass and trophectoderm nuclei of mouse blastocysts in situ using polynucleotide-specific fluorochromes

A rapid procedure has been devised to count the numbers of outer trophectoderm (TE) and inner cell mass (ICM) cells of mouse blastocysts by differentially labelling their nuclei in situ with polynucleotide-specific fluorochromes. The TE nuclei were labelled with propidium iodide (PI) by permeabilising the cells using selective antibody-mediated complement lysis (Solter and Knowles, '75). The blastocysts were then fixed in ethanol and the ICM nuclei labelled with bisbenzimide. These two fluorochromes have widely different fluorescent spectra. Thus, by using fluorescence microscopy with appropriate filter combinations, the PI-labelled TE nuclei appeared pink or red; the bisbenzimide-labelled ICM nuclei, blue or unlabelled. The total numbers of blastocyst nuclei and the numbers of ICM nuclei counted by differential labelling were similar to the numbers detected after spreading the nuclei of intact blastocysts or immunosurgically isolated ICMs by air-drying (Tarkowski '66). Differential labelling of TE and ICM nuclei in situ has two important advantages--that the numbers of both these cell types can be determined for individual blastocysts and that spatial relationships are partially preserved so that regional interactions can be studied.     

Synaptic communication between somatostatinergic axons and growth hormone-releasing factor (GRF) synthesizing neurons in the arcuate nucleus of the rat

Growth hormone (GH) production of the anterior pituitary gland is controlled by inhibiting and releasing hormones that are synthesized in the diencephalon. In order to elucidate the possible interrelationships between somatostatin and growth hormone-releasing factor (GRF) synthesizing neurons at the hypothalamic level, immunocytochemical double labelling studies were performed on sections containing the arcuate nucleus (ARC) of the rat. Somatostatin producing neurons were located in the dorsomedial part of the ARC, while somatostatin immunoreactive (IR) axons were found in the ventro-lateral part of the nucleus, an area containing GRF-synthesizing cells. The use of the dual antigen localization technique revealed the approach and juxtaposition of somatostatin containing axons to dendrites and cell bodies of GRF-synthesizing neurons. At the light microscopic level, several somatostatinergic axon varicosities were clustered around single GRF-synthesizing cells. Ultrastructural analysis of the ventro-lateral part of the ARC showed that (i), somatostatinergic axons established synaptic connections (ii), GRF-producing neurons received axons terminals on their somata and dendrites and (iii), somatostatin-IR axons formed asymmetric synaptic specializations with both dendrites and somata of GRF-synthesizing neurons. These morphological findings indicate that the hormone production and release of hypophysiotrophic GRF-IR neurons can be influenced by the central somatostatin system via direct synaptic mechanisms. The data support the concept, that the interaction of inhibiting and releasing hormones, which determines responses of the pituitary target cells, may take place also at the hypothalamic level.     

Immunohistochemical evidence for different pathways immunoreactive to substance P and calcitonin gene-related peptide (CGRP) in the guinea-pig stellate ganglion

The colocalization of immunoreactivities to substance P and calcitonin gene-related peptide (CGRP) in nervous structures and their correlation with other peptidergic structures were studied in the stellate ganglion of the guinea pig by the application of double-labelling immunofluorescence. Three types of fibre were distinguished. (1) Substance P⁺/CGRP⁺ fibres, which sometimes displayed additional immunoreactivity for enkephalin, constituted a small fibre population of sensory origin, as deduced from retrograde labelling of substance P⁺/CGRP⁺ dorsal root ganglion cells. (2) Substance P⁺/CGRP^– fibres were more frequent; some formed baskets around non-catecholaminergic perikarya that were immunoreactive to vasoactive intestinal polypeptide (VIP). (3) CGRP⁺/substance P^– fibres were most frequent and were mainly distributed among tyrosine hydroxylase (TH)-immunoreactive cell bodies. The peptide content of fibre populations (2) and (3) did not correspond to that of sensory ganglion cells retrogradely labelled by tracer injection into the stellate ganglion. Therefore, these fibres are throught to arise from retrogradely labelled preganglionic sympathetic neurons of the spinal cord, in which transmitter levels may have been too low for immunohistochemical detection of substance P or CGRP. CGRP-immunoreactivity but no substance P-immunolabelling was observed in VIP-immunoreactive postganglionic neurons. Such cell bodies were TH-negative and were spared by substance P-immunolabelled fibre baskets. Retrograde tracing with Fast Blue indicated that the sweat glands in the glabrous skin of the forepaw were the targets of these neurons. The streptavidin-biotin-peroxidase method at the electron-microscope level demonstrated that immunoreactivity to substance P and CGRP was present in dense-cored vesicles of 50–130 nm diameter in varicosities of non-myelinated nerve fibres in the stellate ganglion. No statistically significant difference in size was observed between vesicles immunolabelled for substance P and CGRP. Immunoreactive varicosities formed axodendritic and axosomatic synaptic contacts, and unspecialized appositions to non-reactive neuronal dendrites, somata, and axon terminals. Many varicosities were partly exposed to the interstitial space. The findings provide evidence for different pathways utilizing substance P and/or CGRP in the guinea-pig stellate ganglion.     

An evaluation of retrograde tracing methods for the identification of chemically distinct cochlear efferent neurons.

We have compared retrograde labelling of rat olivocochlear neurons after unilateral cochlear injections of wheatgerm agglutinin conjugated horseradish peroxidase (WGA-HRP) and free HRP. After cochlear injection of WGA-HRP, labelling of nerve cell bodies in the brainstem can be explained not only as conventional retrograde labelling resulting from uptake by efferent nerve terminals synapsing on or near hair cells, but also as spurious labelling originating from tracer leakage, through the periotic duct and over the eighth nerve sheaths, into the cerebral-spinal fluid. Depending on the length of survival time, spurious labelling can involve small portions of the nucleus of the trapezoid body or the entire auditory brainstem and other non-auditory centers. On the contrary, moderate amounts of free HRP delivered to the cochlea do not lead to spurious labelling. With free HRP as the tracer of choice, we found that cochlear efferent cells were located not only in the ipsilateral LSO body and bilaterally within MVPO and RPO as already described by White and Warr, but also surrounding the ipsilateral LSO and in the ipsilateral LVPO. The allocation of these newly described olivocochlear neurons to the medial large cell or lateral small cell system is uncertain because they are located laterally in the brainstem and project ipsilaterally but are large spherical to fusiform or multipolar cells. A zinc salicylate-formol fixative and a metal intensified DAB reaction were found to be effective in visualizing retrogradely transported HRP in neurons and allowed immunocytochemical staining of the same sections with antisera to glutamic acid decarboxylase and choline acetyltransferase. This double label protocol can be used to produce a neurochemical map of the OC systems.     

Serotonin depletion by 5,7-dihydroxytryptamine alters deutocerebral development in the lobster,Homarus americanus

The olfactory and accessory lobes constitute prominent histological structures within the larval and mature lobster deutocerebrum, and both are associated with a dense innervation from paired serotonergic nerve cells, the dorsal giant neurons (DGNs). During development, the cell bodies of the DGNs are the first central somata to express serotonin (5-HT), and the onset of their 5-HT immunoreactivity coincides with the beginning of accessory lobe formation. In contrast, the olfactory lobe anlagen emerge much earlier and grow in the apparent absence of serotonin. The role of serotonergic input for the development of these brain structures was investigated in lobster embryos after serotonin had been depleted pharmacologically with the neurotoxin 5,7-dihydroxytryptamine. A ∼90% reduction of serotonin was confirmed in eggs using high-performance liquid chromatography with electrochemical detection. Morphometric analyses suggested that serotonin depletion dramatically slowed the growth of olfactory and accessory lobes, although glomeruli differentiated at the normal time in both areas. The toxin exhibited a high degree of specificity for serotonergic neurons and associated target regions, and serotonin depletion persisted for at least 2 months following treatment. The goal of future experiments is to determine which of the cell types that innervate the olfactory and accessory lobes are affected by toxin treatment, thereby resulting in the retarded growth of these areas. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 357–373, 1997     

Distribution of dopamine immunoreactive systems in brain stem and spinal cord of the chameleon

An immunohistochemical method, using glutaraldehyde fixation and a highly specific monoclonal antibody recently synthetized against dopamine (DA)-glutaraldehyde protein conjugate, permitted direct visualization of DA structures in the brainstem and spinal cord of a reptile (Chameleon). DA-immunoreactive cell bodies occurred in some contiguous areas of the midbrain tegmentum. The first one was located in the ventral tegmental area. Some somata intermingled with the oculomotor nucleus. The second group was the large round or oval DA-Immunostained neurons located in the substantia nigra. More caudally, a third group of round or fusiform DA-cell bodies was seen in an homologous area of so called mammalian A8 and were continuous with the substantia nigra group. In the medulla oblongata, the DA-containing cells were shown in the nucleus of solitary tract and in the dorsal lateral part of the dorsal motor nucleus of the vagus. The density of this DA-Immunoreactive neurons decreased more caudally. At the medullo-spinal level and upper cervical spinal cord, a few labelled cells were distinguished near the central canal. In the spinal cord DA-immunopositive cell bodies were observed in the vicinity of the central canal and formed a continuous column that extended throughout the rostral spinal cord. The apical processes of these neurons seemed to be in contact with the lumen of the central canal. This study constitute the first visualization of the immunoreactive DA-cell bodies at the medullo-spinal level which were already described, as TH immunoreactive in other species of reptiles.     

In vitro capsaicin-induced cytological changes and alteration in calcium distribution in giant serotonergic neurons of the snail Helix pomatia: a light- and electron-microscopic study

Morphological changes induced by capsaicin were studied in the serotonergic metacerebral giant neurons of the cerebral ganglia of Helix pomatia under in vitro conditions. Capsaicin at a concentration of 10^-4 M caused characteristic structural alterations in the giant serotonergic neurons but did not significantly influence serotonin immunoreactivity in the neurons. At the lightmicroscopic level, the most conspiciuous structural alterations were swelling of the cell bodies, which contained a swollen pale nucleus. Under the electron microscope, the nuclei,mitochondria and the cisternae of the endoplasmic reticulum were swollen in the capsaicin-affected metacerebral giant neurons. Electron-microscopic cytochemical techniques for calcium demonstration revealed electron-dense deposits in the swollen mitochondria and in the cisternae of the endoplasmic reticulum, suggesting an increased Ca²⁺ influx. The serotonergic metacerebral giant neurons could be labelled by cobalt (1 mM) in the presence of capsaicin (10^-4 M) suggesting that capsaicin opens the cation chanels of the capsaicin-sensitive neuronal membrane. The morphological and cytochemical alterations induced by capsaicin in the serotonergic metacerebral giant neurons of Helix pomatia closely resemble those induced in sensory neurons of mammalian dorsal root ganglion.This work was supported by OTKA grants No.: 2477, T016861, T017127 and ETT 587/93