Light and electron microscopic localization of GABA-like immunoreactivity in myenteric plexus of chicken

Whole-mounts of 1-day-old chicken midgut were incubated with an antiserum against GABA-glutaraldehyde-BSA conjugate. The immunoreaction was visualized by using the peroxidase-antiperoxidase method, and processed for consecutive light and electronmicroscopic observation. GABA was selectively localized in some of the varicose and nonvaricose nerve fibres of the myenteric plexus. The varicose fibres formed dense networks within the myenteric ganglia, some of which--mainly in duodenum--also contained immunopositive nerve cell bodies. Some of the varicose fibres projected out from the myenteric plexus into the circular muscle layer. At the electronmicroscopic level, labelled axon terminals formed synaptic contact with unlabelled perikarya and vica versa. At the same time, no labelled terminals were found on immunostained cells. In a few cases, axon terminals with GABA positivity were situated close to the smooth muscle cells in the circular muscle layer, suggesting a prejunctional GABA effect on the neighbouring nerve terminals on the release of their transmitters.     

Light and electron microscopic localization of GABA-like immunoreactivity in myenteric plexus of chicken

Summary Whole-mounts of 1-day-old chicken midgut were incubated with an antiserum against GABA-glutaraldehyde-BSA conjugate. The immunoreaction was visualized by using the peroxidase-antiperoxidase method, and processed for consecutive light and electronmicroscopic observation. GABA was selectively localized in some of the varicose and nonvaricose nerve fibres of the myenteric plexus. The varicose fibres formed dense networks within the myenteric ganglia, some of which — mainly in duodenum — also contained immunopositive nerve cell bodies. Some of the varicose fibres projected out from the myenteric plexus into the circular muscle layer. At the electronmicroscopic level, labelled axon terminals formed synaptic contact with unlabelled perikarya and vica versa. At the same time, no labelled terminals were found on immunostained cells. In a few cases, axon terminals with GABA positivity were situated close to the smooth muscle cells in the circular muscle layer, suggesting a prejunctional GABA effect on the neighbouring nerve terminals on the release of their transmitters.     

Demonstration of GABA-like immunoreactivity in myenteric plexus of frog stomach

The GABAergic innervation of the frog stomach was studied by means of an indirect immunohistochemical method. Whole mount preparations were obtained from frog stomachs after the animals had been perfused with a mixture of picric acid, glutaraldehyde and glacial acetic acid. Samples were incubated with an antiserum specific for GABA coupled to BSA with glutaraldehyde. Anti-rabbit IgG-HRP was processed by the two step method (Eckert and Ude 1983). GABA-positive varicose fibers and also nerve cell bodies were revealed within the myenteric plexus. The density of GABA-immunoreactive neurons was not higher than 4-8 cell/cm2, which is approximately 1% of the total nerve cell number in the myenteric plexus.     

Demonstration of GABA-like immunoreactivity in myenteric plexus of frog stomach

Summary The GABAergic innervation of the frog stomach was studied by means of an indirect immunohistochemical method. Whole mount preparations were obtained from frog stomachs after the animals had been perfused with a mixture of picric acid, glutaraldehyde and glacial acetic acid. Samples were incubated with an antiserum specific for GABA coupled to BSA with glutaraldehyde. Anti-rabbit IgG-HRP was processed by the two step method (Eckert and Ude 1983).GABA-positive varicose fibers and also nerve cell bodies were revealed within the myenteric plexus. The density of GABA-immunoreactive neurons was not higher than 4–8 cell/cm², which is approximately 1% of the total nerve cell number in the myenteric plexus.     

Distribution of GABA-like immunoreactivity in myenteric plexus of carp, frog and chicken

The distribution of GABA-like immunoreactivity was studied by means of indirect immunocytochemical methods in some lower vertebrate species (carp, frog, chicken). An immunoreactive network was revealed in the myenteric plexus of the alimentary canal of carp. GABA-positive nerve cells were attached closely to the fibres in the stomach. In other gut regions immunostained neurons were less frequent. Immunoreactive fibres often formed baskets on the surfaces of immunonegative neurons along the whole length of the alimentary canal. The number of immunopositive nerve fibres and pericellular baskets seemed to be lower in the mid- and hingut than in the foregut region. A similar distribution of GABA-immunoreactivity was revealed in the frog myenteric plexus. The ganglionated foregut region possessed a relatively dense GABAergic innervation. This part of the gut contained immunostained nerve cells and fibres, while the mid- and hindgut possessed only a scanty fibre system. Chicken exhibited an extensive immunoreactive plexus for GABA, although the GABA-stained perikarya were restricted mainly to the duodenum. Further regions of the small intestine were poor in immunoreactive cell bodies, which suggests a segmental origin and arrangement of GABAergic innervation within the plexus. In all three species studied, GABA-positive fibres run into the circular muscle layer. The varicosity suggests their influence on the movement of the smooth muscles through modifying the transmitter release of neighbouring terminals.     

Distribution of GABA-like immunoreactivity in myenteric plexus of carp,frog and chicken

Summary The distribution of GABA-like immunoreactivity was studied by means of indirect immunocytochemical methods in some lower vertebrate species (carp, frog, chicken). An immunoreactive network was revealed in the myenteric plexus of the alimentary canal of carp. GABA-positive nerve cells were attached closely to the fibres in the stomach. In other gut regions immunostained neurons were less frequent. Immunoreactive fibres often formed baskets on the surfaces of immunonegative neurons along the whole length of the alimentary canal. The number of immunopositive nerve fibres and pericellular baskets seemed to be lower in the mid- and hindgut than in the foregut region. A similar distribution of GABA-immunoreactivity was revealed in the frog myenteric plexus. The ganglionated foregut region possessed a relatively dense GABAergic innervation. This part of the gut contained immunostained nerve cells and fibres, while the mid- and hindgut possessed only a scanty fibre system. Chicken exhibited an extensive immunoreactive plexus for GABA, although the GABA-stained perikarya were restricted mainly to the duodenum. Further regions of the small intestine were poor in immunoreactive cell bodies, which suggests a segmental origin and arrangement of GABAergic innervation within the plexus. In all three species studied, GABA-positive fibres run into the circular muscle layer. The varicosity suggests their influence on the movement of the smooth muscles through modifying the transmitter release of neighbouring terminals.     

Neuronal and glial localization of NMDA receptors in the cerebral cortex

The crucial role of glutamate receptors of theN-methyl-d-aspartate (NMDA) type in many fundamental cortical functions has been firmly established, as has its involvement in several neuropsychiatric diseases, but until recently, very little was known of the anatomical localization of NMDA receptors in the cerebral cortex of mammals. The recent application of molecular biological techniques to the study of NMDA receptors has allowed the production of specific tools, the use of which has much increased our understanding of the localization of NMDA receptors in the cerebral cortex. In particular, immunocytochemical studies on the distribution of cortical NMDA receptors have:

1. Demonstrated the preferential localization of NMDA receptors in dendritic spines, in line with previous work;
2. Disclosed a thus far unknown fraction of presynaptic NMDA receptors on both excitatory and inhibitory axon terminals; and
3. Shown that cortical astrocytes express NMDA receptors.

These studies indicate that the effects of cortical NMDA receptor activation are not caused exclusively by the opening of NMDA channels on neuronal postsynaptic membranes, as previously assumed, and that the activation of presynaptic and glial NMDA receptors can contribute significantly to these effects.     

Morphology of horseradish peroxidase (HRP)-injected glial cells in the myenteric plexus of the guinea-pig

Glial cells of the myenteric plexus from guinea pig small intestine were intracellulary filled with horseradish peroxidase (HRP), and histochemically stained. Camera lucida-like drawings of twenty cells were morphologically and morphometrically analyzed. The cells have very small ellipsoid, somata (85±0.7 m equivalent diameter, i.e., about 330 m³ volume), and send up to 20 thin and short processes (less than 26 to about 110 m in length). The morphology of the cells appears to depend on their location within the plexus. Glial cells located within the ganglia are similar to CNS protoplasmic astrocytes; they are star-shaped, and their very short processes are irregularly, branched. In contrast, glial cells within the interganglionic fiber tracts resemble CNS fibrous astrocytes. They extend longer processes that are parallel to the fiber tracts, and show less tendency to branch. We propose that the morphology of enteric glia is determined by the structure of the microenvironment. Both cell types form several flat endfeet at a basal lamina either surrounding blood vessels or at the ganglionic border. Furthermore, the occurrence of holes in the glial cell processes suggests that particular neuronal cell processes may be enwrapped in a specific manner. Fractal analysis of camera lucida-like drawings of the cells showed that the cells have a highly complex surface structure, comparable to that of protoplasmic astrocytes in the brain. These tiny cells may possess a membrane surface area of 2000 m², almost 90% of which are contributed by the cell processes. This geometry may enable an intense exchange of metabolites and ions between neurons, glial cells, and the capillaries and/or environment of enteric ganglia.     

Phosphatase activity in the myenteric plexus

Intense and very intense reactions were obtained for acid phosphatase, calcium activated ATP-ase (pH 9.4), magnesium activated ATP-ase (pH 7.2) and glucose-6-phosphatase in the cytoplasms of the myenteric plexus nerve cells of the small intestine of Macacus rhesus and rabbit. Nucleotidase activity was moderate or slight and unspecific alkaline phosphatase activity absent. Both ATP-ases presented an intense activity in the myenteric plexus nerve cells of human fetuses 30, 33, and 34 weeks old; 5-nucleotidase activity, slight in the 30-week-old fetuses became more intense in the 33- and 34-week-old fetuses. The satellite neuroglial cells, nerve fibers and blood capillaries presented negative alkaline phosphatase reactions and intense or very intense activities of the other phosphatases.     

Neuronal distribution and subcellular localization of HCNP-like immunoreactivity in rat small intestine

A novel peptide, hippocampal cholinergic neurostimulating peptide (HCNP), originally purified from young rat hippocampus, affects the development of specific cholinergic neurons of the central nervous system in vitro. In this study, HCNP-like-immunoreactive nerve processes and nerve cell bodies were identified by electron microscopic immunocytochemistry in the rat small intestine. Labeled nerve processes were numerous in the circular muscle layer and around the submucosal blood vessels. In the submucosal and myenteric plexuses, some HCNP-like-immunopositive nerve cell bodies and nerve fibers were present. The reaction product was deposited on the membranes of various subcellular organelles, including the rough endoplasmic reticulum, Golgi saccules, ovoid electron-lucent synaptic vesicles in axon terminals associated with submucosal and myenteric plexuses, and the outer membranes of a few mitochondria. The synaptic vesicles of HCNP-like-positive terminals were 60–85 nm in diameter. The present data provide direct immunocytochemical evidence that HCNP-like-positive nerve cell bodies and nerve fibers are present in the submucosal and myenteric plexuses of the rat small intestine. An immunohistochemical light microscopic study using mirror-image sections revealed that in both the submucosal and myenteric ganglia, almost all choline acetyltransferase (ChAT)-immunoreactive neurons were also immunoreactive for HCNP. These observations suggest (i) that HCNP proper and/or HCNP precursor protein is a membrane-associated protein with a widespread subcellular distribution, (ii) that HCNP precursor protein may be biosynthesized within neurons localized in the rat enteric nervous system, and (iii) that HCNP proper and/or HCNP precursor protein are probably stored in axon terminals.     

Distribution of neurons with high-affinity uptake sites for GABA in the myenteric plexus of the guinea-pig,rat and chicken

The occurrence of neurons possessing high-affinity uptake sites for GABA was studied in the myenteric plexus of the guinea-pig ileum, caecum, and proximal and distal colon, the rat proximal colon, and the chicken gizzard with the use of 3H-GABA and autoradiography. Experiments were carried out on plexuses that had been freshly isolated from the gut wall or on isolated plexuses that had been maintained as explant cultures for 7 to 14 days. Scattered neurons selectively labelled with 3H-GABA were found in the myenteric plexuses from all the areas examined. The results suggest that GABAergic neurons are widely distributed in the enteric nervous system.     

Lymphocyte-mediated regulation of beta-endorphin in the myenteric plexus

Lymphocytes are antinociceptive and can modulate visceral pain perception in mice. Previously, we have shown that adoptive transfer of CD4+ T cells to severe combined immune-deficient (SCID) mice normalized immunodeficiency-related visceral hyperalgesia. Pain attenuation was associated with an increase in beta-endorphin release by T cells and an upregulation of beta-endorphin in the enteric nervous system. In this study, we investigated the relationship between T cells and opioid expression in the myenteric plexus. We examined opioid peptide and receptor expression in the myenteric plexus in the presence and absence of mucosal T cells. We found a positive association between T cells and beta-endorphin expression; this was accompanied by a downregulation of the micro-opioid receptor (MOR). In vitro, T helper (Th) type 1 and type 2 cytokine stimulation of CD4+ T cells or isolation of T cells from in vivo Th-polarized mice did not increase T cell release of beta-endorphin or the induction of beta-endorphin expression in the myenteric plexus. However, exogenous beta-endorphin did upregulate beta-endorphin expression, and both cycloheximide and naloxone methiodide inhibited peptide upregulation. Therefore, our results suggest that nonpolarized CD4+ T cells release beta-endorphin, which, through an interaction with MOR, stimulates an upregulation of beta-endorphin expression in the myenteric plexus. Thus, we propose that the mechanism underlying lymphocyte modulation of visceral pain involves T cell modulation of opioid expression in the enteric nervous system.     

Neuronal and glial cytoskeletons

Long-awaited evidence for in vivo functions of the major neuronal microtubule associated proteins indicates that they are directly involved in neurite extension. Companion evidence reveals an intrinsic role for glial intermediate filaments in glial cell extension along neurites and for neurofilaments in establishing axonal caliber. New fluorescence and photoactivation experiments require a re-thinking of models of slow axonal transport and of the part the cytoskeleton plays in axonal guidance.     

Basic fibroblast growth factor, neurofilament, and glial fibrillary acidic protein immunoreactivities in the myenteric plexus of the rat esophagus and colon

The enteric nervous system consists of a number of interconnected networks of neuronal cell bodies and fibers as well as satellite cells, the enteric glia. Basic fibroblast growth factor (bFGF) is a mitogen for a variety of mesodermal and neuroectodermal-derived cells and its presence has been described in many tissues. The present work employs immunohistochemistry to analyze neurons and glial cells in the esophageal and colic enteric plexus of the Wistar rat for neurofilament (NF) and glial fibrillary acidic proteins (GFAP) immunoreactivity as well as bFGF immunoreactivity in these cells. Rats were processed for immunohistochemistry; the distal esophagus and colon were opened and their myenteric plexuses were processed as whole-mount preparations. The membranes were immunostained for visualization of NF, GFAP, and bFGF. NF immunoreactivity was seen in neuronal cell bodies of esophageal and colic enteric ganglia. GFAP-immunoreactive enteric glial cells and processes were present in the esophageal and colic enteric plexuses surrounding neuronal cell bodies and axons. A dense net of GFAP-immunoreactive processes was seen in the ganglia and connecting strands of the myenteric plexus. bFGF immunoreactivity was observed in the cytoplasm of the majority of the neurons in the enteric ganglia of esophagus and colon. The two-color immunoperoxidase and immunofluorescence methods revealed bFGF immunoreactivity also in the nucleus of GFAP-positive enteric glial cells. The results suggest that immunohistochemical localization of NF and GFAP may be an important tool in the study of the plasticity in the enteric nervous system. The presence of bFGF in neurons and glia of the myenteric plexus of the esophagus and the colon indicates that this neurotrophic factor may exert autocrine and paracrine actions in the enteric nervous system.