Small Ganglion Research Articles

Morphologic evidence for three cell types in the human spiral ganglion

Although two types of spinal ganglion cells (large type I and smaller type II) have classically been described by anatomic studies in both animal and human spiral ganglion, there is physiologic and morphologic evidence for subtypes of the large type I ganglion cell. In addition, in the animal and human, a variety of morphologic differences based on cytoplasmic content, myelinization, immunostaining and morphometric analysis have suggested more than one variety of type I ganglion cell. Light and electron microscopic serial sections of the spiral ganglion in two human specimens in the basal, middle and upper middle turns were pooled for morphometric analysis of the cell area, nuclear area axon diameter. Analysis of variance, bivariate scatter plots and multivariate cluster analysis provided evidence for 3 types of ganglion cells in the human spiral ganglion: large, intermediate and small, varying from each other significantly on the basis of cell area. It was suggested, based on the morphologic findings and prevalence of the cell types, that the large and intermediate cells were subtypes of the classic type I spiral ganglion cell, whereas the small ganglion cell was consistent with the classically described type II ganglion cell.

Ret protein in the human fetal rectum

A major gene for Hirschsprung's disease (HD) recently has been mapped in chromosome 10q11.2 and identified to be the RET proto-oncogene. Mutations of the RET gene have occurred in HD patients, and abnormalities of expression and function of Ret protein (a receptor tyrosine kinase, which is the product of the RET gene) have been found in their intestines. In vitro studies of the biological effects of HD mutations suggest a loss of function effect, which may be negative-dominant. However, the developmental role of the Ret protein in the organogenesis of the enteric nervous system (ENS) and its role in the pathogenesis of HD remain unclear. The authors present a study of the expression of Ret protein in the human ENS during fetal development. Fresh rectal tissues were obtained from nine fetuses (gestational age range, 12 to 22 weeks). Ret protein expression was studied immunohistochemically, using antibodies against the car☐y-terminal 20 amino acids (anti-Ret C) and the extracellular domain (anti-Ret R5). The tyrosine kinase activity of the fetal ENS was investigated with antiphosphotyrosine mouse monoclonal antibody against the phosphorylated tyrosine residues. Anti-Ret C immunostaining was observed in ganglion cells at all ages, but intense activity was significantly higher among the cells of the younger fetuses. Intense anti-Ret R5 immunostaining was present in the enteric ganglion cells of the 12-week-old fetus. The tyrosine kinase activity of ganglion cells increases progressively with advancing gestational age. The results of this study support the hypothesis that the Ret protein receptor might play a crucial role in the cellular and molecular processes involved in the development and maturation of the ENS, abnormalities of which could result in HD. High Ret protein expression and low tyrosine kinase activity have been reported to occur in small ganglia of the HD hypoganglionic segment. In the present study, these markers were typical of the primitive and immature ENS during the early phase of hindgut development.

Distribution and origins of nitric oxide-producing nerve fibers in the dog tongue: correlated NADPH-diaphorase histochemistry and immunohistochemistry for calcitonin gene-related peptide using light and electron microscopy.

The distribution and origins of nitric oxide (NO)-producing nerves in the dog tongue with reference to calcitonin gene-related peptide (CGRP)-containing sensory fibers were investigated using NADPH-diaphorase (NADPH-d) histochemistry and immunohistochemistry for CGRP and NO synthase combined with retrograde axonal tracing and denervation experiments. The ultrastructural relationships between NADPH-d-positive and CGRP-immunoreactive neuronal elements were also examined electron microscopically. NADPH-d-positive and CGRP-immunoreactive varicose fibers were found within the taste buds and surrounding the epithelia of the fungiform papillae, and they disappeared completely after severance of the lingual nerve. Following injection of fast blue into the subepithelial layer of the anterior two thirds of the tongue, retrogradely labeled neurons possessing NO synthase and/or CGRP immunoreactivities were mainly detected in the trigeminal ganglion. Some of the retrogradely labeled trigeminal cells showed the coexistence of NADPH-d reactivity and CGRP immunoreactivity, but in the geniculate ganglion neither NADPH-d reactivity nor NO synthase immunoreactivity was found instead of retrogradely labeled CGRP-immunoreactive neurons. The lingual artery and its branches, including the arteriovenous anastomoses, showed dense distributions of NADPH-d-positive fibers, most of which were unaffected by the denervation experiments. There were many small ganglia in the tongue, and virtually all ganglionic neurons were NADPH-d reactive. CGRP-immuno-reactive varicose fibers were also found around the vascular walls and within the intralingual ganglia. Ultrastructural analysis revealed a close distribution of NADPH-d-positive and CGRP-immunoreactive varicose fibers within the arterial walls, and synaptic contacts between CGRP-immunoreactive terminals and NADPH-d-positive intralingual ganglionic neurons. These results indicated that the taste buds of epithelia of fungiform papillae in the anterior two thirds of the dog tongue receive NADPH-d-positive and CGRP-immunoreactive sensory fibers from the trigeminal ganglion, and that perivascular NADPH-d-positive fibers mainly originate from intrinsic ganglia in the tongue. The ultrastructural findings suggest an intrinsic peripheral nerve-reflex mechanism in the regulation of the lingual vascular function by NO-producing postganglionic parasympathetic neurons and CGRP-containing sensory fibers.

Overexpression of intercellular adhesion molecule-1 (ICAM-1) and MHC class II antigen on hypertrophic nerve trunks suggests an immunopathologic response in Hirschsprung's disease

The pathogenesis of Hirschsprung's disease (HD) is not fully understood. The authors studied expression of intercellular adhesion molecule-1 (ICAM-1) and major histocompatibility complex (MHC) class II antigen in the resected bowel specimes of 18 patients with HD who had no evidence of enterocolitis and in eight age- and site-matched controls, using indirect immunohistochemistry. There was strong expression of ICAM-1 and MHC class II antigen on hypertrophic nerve trunks, in both the submucous and myenteric plexuses of the aganglionic colon. The transition zone showed strong expression of ICAM-1 and MHC class II antigen on small ganglia in the myenteric and submucous plexuses. However, no staining of ganglia or nerve fibers was found in the submucous and myenteric plexuses of the colon from controls or in the ganglionic colon from patients with HD. The expression of both antigens on hypertrophic nerve trunks suggests the presence of an immunologic response in the pathogenesis of HD.

The cholinergic innervation of the adrenal gland and its relation to enkephalin and nitric oxide synthase.

Using a monoclonal antibody against rat brain choline acetyltransferase (ChAT) the cholinergic innervation of the adult rat adrenal gland was visualized. Almost all ChAT-positive fibres contained nitric oxide synthase (NOS), whereas enkephalin (ENK) was exclusively found in ChAT fibres among adrenaline chromaffin cells. The ChAT/NOS/ENK fibres disappeared after immunological sympathectomy, indicating a preganglionic origin. ChAT was not found in the superficial peptide- and NOS containing fibre plexus in the adrenal cortex or in small or large intra-adrenal ganglion neurones under control conditions. Even after colchicine treatment only one single ChAT-positive small ganglion neurone was found. It is possible, therefore that some small intra-adrenal ganglion neurones, which express NOS- and VIP-like immunoreactivities, are noncholinergic, nonadrenergic neurones.

Distribution of noradrenergic neurons in the female rat pelvic plexus and involvement in the genital tract innervation

The involvement of the pelvic plexus noradrenergic neurons in the innervation of the genital tract was studied in the female rat. Several small ganglia were observed in addition to the paracervical ganglion and immunocytochemistry for tyrosine hydroxylase was performed to examine the distribution and number of the noradrenergic neurons. 5069 ± 1525 nerve cell bodies were counted in the paracervical ganglion and 9.0 ± 0.8% of them were noradrenergic, displaying a clear somatotopic distribution in the ventro-medial part of the ganglion. Some accessory ganglia were located ventral to the main paracervical ganglion. 414 ± 149 nerve cell bodies were found in the accessory ganglia, of which 20.4 ± 3.1% were noradrenergic. Ganglia along the vesical branch of the hypogastric nerve, referred to as an hypogastric plexus, contained 233 ± 83 neurons among which 12.7 ± 7.2% were noradrenergic. Bilateral removal of the pelvic plexus produced degeneration of all the tyrosine hydroxylase-immunoreactive nerve fibres in the lower part of the uterus and in the cervix. In contrast, excision of the paracervical ganglia and the accessory ganglia caused no significant change in this innervation pattern. Combined retrograde tracing study and immunocytochemistry for tyrosine hydroxylase revealed a very small number of noradrenergic neurons also labelled with fluoro-Gold. Both findings suggest a limited involvement of the pelvic plexus noradrenergic neurons in the innervation of the lower genital tract.

The Brn-3 family of POU-domain factors: primary structure, binding specificity, and expression in subsets of retinal ganglion cells and somatosensory neurons

A search for POU domain sequences expressed in the human retina has led to the identification of three closely related genes: Brn-3a, Brn-3b, and Brn-3c. The structure and expression pattern of Brn-3b was reported earlier (Xiang et al., 1993); we report here the structures and expression patterns of Brn-3a and Brn-3c. Antibodies specific for each Brn-3 protein were generated and shown to label only ganglion cells in a variety of vertebrate retinas. A complex pattern of strongly and weakly immunolabeled ganglion cells was observed in mouse, cat, and monkey retinae. In mouse and cat retinae, Brn-3a and Brn-3b proteins are found in a large fraction of ganglion cells, whereas Brn-3c is present in fewer ganglion cells. In the cat retina, anti-Brn-3a immunoreactivity was strong in the small ganglion cells (gamma cells) and weak in the remaining ganglion cells (alpha and beta cells); anti-Brn-3b immunoreactivity was present in all ganglion cells; and anti-Brn3c immunoreactivity was confined to the small ganglion cells. Immunolabeling of macaque retinae following retrograde labeling from the lateral geniculate nucleus revealed strong anti-Brn-3a immunoreactivity in a minority of retrogradely labeled P-type ganglion cells, and weak Brn-3a immunoreactivity in all of the remaining P- and M-type ganglion cells. In the same retinae, strong anti-Brn-3b immunoreactivity was seen in nearly all P-type ganglion cells and weak immunoreactivity in nearly all M-type ganglion cells. Each of the Brn-3-specific antibodies also labeled subsets of neurons in the dorsal root and trigeminal ganglia, suggesting that primary somatosensory neurons and retinal ganglion cells share genetic regulatory hierarchies. In vitro selection of an optimal DNA binding site using the Brn-3b POU domain has revealed a consensus [(A/G)CTCATTAA(T/C)] that is recognized by each of the Brn-3 POU domains and is distinct from binding sites previously described for other POU domain proteins.

Nitric oxide synthase containing nerves in the atrioventricular node of the guinea pig heart

Immunoelectron microscopy was performed to localize immunoreactivity for nitric oxide synthase (NOS) in the guinea-pig atrioventricular node. Many small ganglia were found in the interatrial septum near the atrioventricular node. A small number of neurons in these ganglia were immunoreactive. NOS immunoreactive axons were observed in nerve bundles near or within the node. Very thick immunoreactive axons, 8–10 μm in diameter, were found between the conductive tissue and the ordinary cardiac muscles. Electron-microscopic examination confirmed that these axons were unmyelinated. Nerve bundles in this region contained both myelinated and unmyelinated axons; however, immunoreactivity was detected exclusively in unmyelinated axons. Fine NOS-immunoreactive nerve fibers with terminal varicosities were often seen in the atrioventricular node. Close contact between NOS-immunoreactive axon varicosity and specialized cardiac muscle cell was observed. Intranodal ganglia were observed among the specialized cardiac muscles. They were surrounded by numerous axons, some of them were immunoreactive. Direct axo-somatic synapses from NOS-immunoreactive terminals to the intranodal ganglion cells were observed. The present results indicate a possibility that nitric oxide plays a role in the neural control of the conductive tissue in the heart through direct neuromuscular contact.

Ret-oncogene Expression Correlates with Neuronal Differentiation of Neuroblastic Tumors

An antibody to the ret proto-oncogene product (RET) was raised and applied to formalin-fixed, paraffin-embedded neuroblastic tumors (NBTs) to investigate its usefulness in diagnosis and evaluation of cell differentiation. In normal neural crest-derived tissues, most ganglion cells were moderately stained while large ganglion cells were weakly stained. In NBTs, the intensity of the staining in moderately differentiated neuroblasts and small ganglion cells was more prominent than in undifferentiated neuroblasts, while the cytoplasm of large ganglionic cells was weakly stained as in normal ganglion cells. The RET immunoreactivity was compared with that of nine neural and neuroendocrine markers. The results revealed a parallelism with the protein gene product 9.5 (PGP9.5), neuron specific enolase (NSE) and NF-150 kD (NF = M). These findings indicated that the RET immunoreactivity was correlated with ganglionic differentiation and maturation. Thus, RET was considered to be a new marker that would be implemented in diagnosis and estimation of neuronal differentiation of NBTs.

Immunohistochemical localization of RET protein in Hirschsprung's disease

A major gene causing Hirschsprung's disease was recently mapped in 10q11.2. Its physical localization was restricted to a 250-Kb interval containing the RET protooncogene (REarranged during Transfection). In 1994, point mutations affecting the RET proto-oncogene were identified in patients with Hirschsprung's disease. The authors present an immunohistochemical study on the expression and localization of the Ret protein (a receptor tyrosine kinase, which is the RET proto-oncogene product) in the intestinal plexuses of patients with Hirschsprung's disease. Ninety-two full-thickness intestinal wall pieces from 29 pediatric patients were studied (19 cases of classic Hirschsprung's disease, 5 of total colonic aganglionosis, and 5 controls). Ret protein immunohistochemical localization was obtained using c-Ret R5, anti-Ret K and anti-Ret C antibodies, respectively, against the extracellular domain, the tyrosine kinase domain, and the carboxy-terminal 20 amino acids of the Ret protein. A diffuse granular staining was present in the ganglia of normal colon, whereas the small ganglia of the hypoganglionic colon showed a reduced number of ganglion cells that were strongly stained with c-Ret R5 MoAb. A reduced synthesis of Ret protein was shown in the ganglionic and hypoganglionic segments of two cases of this series, the first with a complete deletion of the RET proto-oncogene and the second with a frameshift mutation and a stop codon in the extracellular domain. The activity of the receptor tyrosine kinases (RTKs) in intestinal ganglion cells was investigated using antiphosphotyrosine antibodies. A very low tyrosine kinase activity was shown in the small ganglia of the hypoganglionic segment. The results of this study suggest a primary role of this transmembrane receptor in the migration and differentiation of neural crest-derived neuroblasts that colonize the colon. The identification of the Ret protein ligand will better explain the role of this transmembrane receptor in the molecular and cellular mechanisms of Hirschsprung's disease.

A morphological study of the retinal ganglion cells of the Afghan pika ( Ochotona rufescens)

The distribution and morphology of the retinal ganglion cells was studied in a relative of the rabbit, the Afghan pika. The total number of retinal ganglion cells was approximately 170 000. The total number of optic nerve fibers was between 160 000 and 190 000, corresponding to the total number of retinal ganglion cells. Retinal ganglion cells were found to have a horizontal region of high-density. The maximum density was 5250 cells/mm 2. This region was located in the central retina below the optic disc. This area contained numerous closely packed small ganglion cells, while the peripheral retina (especially in the dorsal periphery) contained large ganglion cells more loosely dispersed. The retinal ganglion cells labeled by horseradish peroxidase (HRP) were morphologically classified into three types based on dendritic length and ramification pattern.

Extensive distribution of NADPH diaphorase activity in the nerve supply of the cat lower urinary tract

Nitric oxide is a neurotransmitter which causes smooth muscle relaxation and may contribute to this response in some regions of the lower urinary tract. In the present study the distributions of neurons and their axons which contain the synthetic enzyme for nitric oxide, nitric oxide synthase, were mapped by staining for NADPH diaphorase in sections of proximal urethra, bladder trigone and detrusor and whole-mounts of vesical ganglia from cats. Stained axons were present in the smooth muscle of all regions of bladder and proximal urethra, but were most common in the urethra and least prevalent in the detrusor. Stained axons were also present in the mucosa; most of these were associated with blood vessels, but some travelled close to the epithelium. Stained mucosal axons were much more numerous in the proximal urethra than in any bladder region. Darkly stained neuronal somata were found throughout the vesical ganglia, where they appeared to comprise the majority of neurons. Small ganglia containing stained neurons were also found in sections of various urinary tract regions, where they were located in the serosa, between muscle layers and, in the urethra, also in the mucosa. These studies have shown an extensive distribution of neurons and axons that stain for NADPH diaphorase (and are predicted to synthesize nitric oxide) throughout all tissues of the cat lower urinary tract. It is hypothesized that nitric oxide is an inhibitory transmitter in the cat bladder and proximal urethra and may also have a role as a sensory transmitter in the mucosa of these regions.

Gonadotropin releasing hormone (GnRH) neurons project to growth hormone and somatolactin cells in the steelhead trout

Analysis of gene expression using gonadotropin-releasing hormone (GnRH) antisense oligonucleotide confirmed by immunocytochemical localization the occurrence of GnRH neurons along the nervus terminalis in the steelhead trout (Oncorhynchus mykiss). Double-label immunocytochemistry revealed the distribution of mammalian (m), salmon (s) and chicken II (cII)-type GnRHs and various pituitary hormones. Both sGnRH and mGnRH appeared to be colocalized in the same cells of the nervus terminalis. Chicken GnRH II-immunoreactivity was found only in fibers and terminals. In the younger fish [73 and 186 days after fertilization (DAF)] GnRH neurons were seen rostral to the olfactory bulb. A novel GnRH ganglion, along the nervus terminalis, was found at the cribiform bone (gCB). A few non-immunoreactive rounded cells were seen among the GnRH neurons. A second smaller ganglion was seen at the most rostrally located part of the ventromedial olfactory bulb (gROB). In the older fish (850 DAF) GnRH neurons were also observed in the basal forebrain. A small group of neurons (2-3 cells), at the caudoventromedial border of the olfactory bulb, formed the ganglion terminale. Occasionally isolated GnRH-immunoreactive cells were seen at the base of the olfactory epithelium, along the ventromedial margins of the olfactory nerve. GnRH-immunoreactive and GnRH mRNA expressing neurons were absent from midbrain regions at the ages observed. GnRH-immunoreactive fibers were present only in older fish. The pattern of distribution of fibers that were immunoreactive to all three forms of GnRH was identical. Fibers were seen along the medial side of the olfactory nerve, throughout the brain and in the pituitary, associated with growth hormone and somatolactin cells.(ABSTRACT TRUNCATED AT 250 WORDS)

A qualitative and quantitative analysis of the response of the retinal ganglion cell soma after stretch injury to the adult guinea-pig optic nerve

The development of a model for focal axonal injury in the optic nerve of the adult guinea-pig has allowed a qualitative and quantitative analysis of the response of the retinal ganglion cell soma to this type of injury. Large and medium sized retinal ganglion cells show classic 'central chromatolysis' in about 30% of ganglion cells between three and seven days after injury, a high proportion of which undergo degeneration between seven and 14 days. Small ganglion cells and small neurons do not demonstrate any morphological response to stretch injury of the optic nerve. However, a small number of larger ganglion cells demonstrate enlargement of the cell soma and nucleolus together with reconstitution of the rough endoplasmic reticulum between seven and 14 days after stretch injury. We suggest that these cells are either recovering from or regenerating after a non-disruptive lesion to their axons. We suggest that some of these morphological changes parallel documented regenerative responses in peripheral/extrinsic neurons after injury to their axons. We conclude that the time course of the 'axon reaction' after stretch injury to axons is longer than that obtained after crush or transection. We provide good morphological evidence that the level of injury after application of non-disruptive mechanical strain to axons is less severe than in the former two models of axonal injury and that a proportion of damaged neurons do not die but rather demonstrate either/or recovery or a regenerative response.

Vagal innervation of the rat pylorus: an anterograde tracing study using carbocyanine dyes and laser scanning confocal microscopy.

In an attempt to identify the distribution and structure of vagal fibers and terminals in the gastroduodenal junction, vagal efferents were labeled in vivo by multiple injections of the fluorescent carbocyanine dye DiA into the dorsal motor nucleus (dmnX), and vagal afferents were anterogradely labeled by injections of DiI into the nodose ganglia of the same or separate rats. Thick frontal cryostat sections were analysed either with conventional or laser scanning confocal microscopy, using appropriate filter combinations and/or different wavelength laser excitation to distinguish the fluorescent tracers. Vagal efferent terminal-like structures were present in small ganglia within the circular sphincter muscle, which, in the absence of a well-developed, true myenteric plexus at this level, represent the myenteric ganglia. Furthermore, vagal efferent terminals were also present in submucosal ganglia, but were absent from mucosa, Brunner's glands and circular muscle fibers. Vagal afferent fibers and terminal-like structures were more abundant than efferents. The most prominent afferent terminals were profusely branching, large net-like aggregates of varicose fibers running within the connective tissue matrix predominantly parallel to the circular sphincter muscle bundles. Profusely arborizing, highly varicose endings were also present in large myenteric ganglia of the antrum and duodenum, in the modified intramuscular ganglia, and in submucosal ganglia. Additionally, afferent fibers and terminals were present throughout the mucosal lining of the gastroduodenal junction. The branching patterns of some vagal afferents suggested that individual axons produced multiple collaterals in different compartments. NADPH-diaphorase positive, possibly nitroxergic neurons were present in myenteric ganglia of the immediately adjacent antrum and duodenum, and fine varicose fibers entered the sphincter muscle from both sides, delineating the potential vagal inhibitory postganglionic innervation. These morphological results support the view of a rich and differentiated extrinsic neural control of this important gut region as suggested by functional studies.

Nitric oxide synthase containing nerves in the atrioventricular node of the guinea pig heart

Immunoelectron microscopy was performed to localize immunoreactivity for nitric oxide synthase (NOS) in the guinea-pig atrioventricular node. Many small ganglia were found in the interatrial septum near the atrioventricular node. A small number of neurons in these ganglia were immunoreactive. NOS immunoreactive axons were observed in nerve bundles near or within the node. Very thick immunoreactive axons, 8-10 microns in diameter, were found between the conductive tissue and the ordinary cardiac muscles. Electron-microscopic examination confirmed that these axons were unmyelinated. Nerve bundles in this region contained both myelinated and unmyelinated axons; however, immunoreactivity was detected exclusively in unmyelinated axons. Fine NOS-immunoreactive nerve fibers with terminal varicosities were often seen in the atrioventricular node. Close contact between NOS-immunoreactive axon varicosity and specialized cardiac muscle cell was observed. Intranodal ganglia were observed among the specialized cardiac muscles. They were surrounded by numerous axons, some of them were immunoreactive. Direct axo-somatic synapses from NOS-immunoreactive terminals to the intranodal ganglion cells were observed. The present results indicate a possibility that nitric oxide plays a role in the neural control of the conductive tissue in the heart through direct neuromuscular contact.

Distribution of cytoskeletal proteins (neurofilaments, peripherin and MAP-tau) in the cochlea of the human fetus.

We report here an immunohistochemical study of the distribution of intermediate filaments (neurofilament, peripherin) and a microtubule-associated protein, tau, in the human fetal cochlea at 27 weeks of gestation. Neurofilament immunoreactivity (160 and 200 KDa) was localized in afferent and efferent fibers of the cochlear innervation and restricted to a few small spiral ganglion neurons. Peripherin immunoreactivity was specifically distributed in some small ganglion neurons and in their central and peripheral extensions, particularly in fibers reaching the lower part of the outer hair cells. Double immuno-labelling studies with these neurofilaments and peripherin antibodies show that only small neuron cell bodies were stained. Morpholometrical data indicate that immunostained neurons could be related to the Type II neuron population in the spiral ganglion. Tau protein was localized in intraganglionic spiral bundle fibers and in fibers that reach the lower part of hair cells. These observations suggest that neurofilament and peripherin antibodies stain a particular population of human spiral ganglion neurons with Type II characteristics. Moreover, the specificity of peripherin labelling in Type II cells and their processes suggest that peripherin could be used as a probe for the developmental study of this system in the human cochlea. On the other hand, tau antibody appeared as a marker for efferent fibers during development and could give information on the ontogenesis of efferent innervation.

Distribution of NADPH diaphorase in intramural plexuses of cat and opossum esophagus

Nonadrenergic noncholinergic (NANC) nerves, releasing nitric oxide (NO), regulate peristalsis in esophageal smooth muscle. The NADPH diaphorase reaction identifies nitric oxide synthase (NOS). NO-synthesizing (NANC) nerves should be more abundant in esophageal smooth muscle than in striated muscle. We used NADPH diaphorase histochemistry in whole-mounts of the esophagus of cat (predominantly striated muscle) and opossum (predominantly smooth muscle) to compare striated and smooth muscle regions. In the opossum myenteric plexus, 90% of nerve cells were stained, with two populations, dark-staining and light-staining cells. Dark cells constituted 35–51% of stained cells with no difference between striated muscle and smooth muscle regions. In the cat myenteric plexus, 50% of cells were unstained in the striated muscle segment, but only 10% in the smooth muscle segment. Stained cells constituted two populations, dark cells and light cells, dark cells making up 48–60% of stained cells in both striated and smooth muscle segments, with no difference between segments. Both dark cells and light cells lay in clusters in opossum, but not in cat. Dark cells showed no selectivity for large or small ganglia of myenteric plexus in either region in opossum. Many more NADPH diaphorase positive nerve fibers lay in the circular smooth muscle layer than in the other layers. They were not found in striated muscle. Interstitial cells of Cajal in the circular layer of smooth muscle were also positive. In the submucosal plexus of opossum, all cells were stained, 10% being dark cells in both striated and smooth muscle segments. The cat has almost no nerve cells in the submucosal plexus. The similarity of the myenteric plexus in striated muscle and smooth muscle regions of the esophagus suggests that NADPH diaphorase positive nerves have other functions besides regulation of smooth muscle. Also, a positive reaction may not dictate that NO is the sole transmitter released.

Visualization of Human Choroidal Ganglion Cells with the Supravital Fluorescent Dye 4-(4-Diethylamino-styryD-N-methylpyridium Iodide

The distribution and morphology of ganglion cell bodies and nerve fibers in supravital human choroidea is studied by staining with the fluorescent dye 4-(4-diethylaminostyryl)-N-methylpyridium iodide (4-Di-2-ASP). We have used the choroids from human donor eyes donated for cornea transplantation. The isolated tissues were rinsed in ringer solution and processed within up to 24 h after death with 3 microM of 4-Di-2-ASP in Ringer for 5-60 min at different temperatures (4-37 degrees C). After incubation, choroid whole flat mounts were observed in a fluorescence microscope. A delicate network of nerve fibers could be distinguished as soon as 5 min after immersion in 4-Di-2-ASP. In addition, labelled ganglion cell perikarya could be seen after longer incubation periods. They occurred individually or grouped in small ganglia of up to 8 cells and were linked together by various processes, which could also be traced to blood vessels. The fluorescent dye 4-Di-2-ASP provides a quick and simple method to visualize the innervation of the human choroid, including local ganglion cells. This nontoxic fluorescent technique will permit further manipulation and electrophysiological investigation of choroidal innervation.

Cutaneous and subcutaneous sensory receptors of the hagfish Myxine glutinosa with special respect to the trigeminal system

The integument of the hagfish Myxine glutinosa is described with respect to the topography and the fine structural organization of the dermal and hypodermal nerve fiber plexus. Both nerve fiber plexuses contain small ganglion cells with axodendritic and axosomatic synapscs. The six barbels of the head (4 nasal and 2 oral barbels) are supplied with about 5600 afferent trigeminal nerve fibers via the right and left ophthalmic nerve. With respect to the topography of the sensory nerve terminals in the barbels different types of receptors are termed the external cuff receptor, internal cuff receptor, and perichondrial receptor. Free nerve terminals occur within the epidermal layer, especially at the tip region of the barbels and in the glassy membrane of the dermis. The hypodermal edge receptor organ extends from the ventral nasal barbel to the oral barbel. A mechanoreceptive function of the different receptor types is discussed. The innervation pattern of the barbel is similar to the innervation of the mammalian sinus hair. In this context, the barbel is a highly differentiated receptor organ able to explore the nearest surroundings with high stereognostic perception. The ganglion cells of the skin seem to represent a part of the peripheral autonomic nervous system, which is involved in the control of secretion mechanisms.