Endocytotic uptake of cationized ferritin tracer into glomus cells dissociated from the adult rat carotid body

Summary Glomus cells from carotid bodies of adult rats dissociated by means of collagenase or collagenase + trypsin were used to study by electron microscopy the endocytotic uptake of cationized ferritin (CF) tracer into subcellular compartments. The glomus cells were incubated with the tracer (1) in a basic salt medium (BM), or (2) in the BM into which calcium ionophore A23187 had been added, or (3) in a potassium-rich medium.Incubation of the cells in BM containing CF for 30 min resulted in attachment of the tracer to the cell membrane and uptake of a few solitary tracer particles into small vesicles and multivesicular bodies. No uptake into the cisternae of the Golgi apparatus was observed. Further incubation in BM containing CF for another 30 min resulted in increased uptake of the tracer into small vesicles and multivesicular bodies. A similar pattern of uptake was observed when the dissociated glomus cells were first preincubated in BM with CF for 30 min and then incubated for 1 min or 30 min in the BM solution containing both the ionophore and CF. Upon such incubation, CF particles were seen to penetrate into coated pits and sites of exocytosis at the cell surface. When the 30-min preincubation in BM was followed by incubation in a CF-containing potassium-rich medium for 15–30 min, uptake into vesicles, small lysosomes and occasionally also into profiles of the smooth endoplasmic reticulum was seen. Endocytotic mechanisms of the glomus cells are outlined.     

Sexual differentiation of rat liver carbonic anhydrase III

Using radioimmunoassay, the concentration of carbonic anhydrase III in the livers of adult male rats was found to be approx. 30-times greater than that observed in mature females. Castration of male rats led to a marked reduction in liver carbonic anhydrase III concentrations which could be partially restored to control levels by testosterone replacement. Administration of testosterone to ovariectomised female rats induced about a 5-fold increase in liver carbonic anhydrase III concentration. Immunoprecipitation analysis of the products of liver mRNA translation in vitro with antiserum specific for carbonic anhydrase III showed that hormonal control of the levels of carbonic anhydrase III in liver is mediated by changes in the amount of translatable carbonic anhydrase III mRNA. Marked changes in liver carbonic anhydrase III concentrations were also observed in developing and ageing male rats.     

Transplantation of the rat pineal organ to the brain: pinealocyte differentiation and innervation

Summary The pineal organ of neonatal rats was transplanted to the frontal part of the cerebral cortex or the cerebral interhemispheric fissure of an isogenic adult rat to determine whether pineal differentiation and pinealopetal innervation are affected by aberrant neuronal influences. Transplants were fixed for immunohistochemistry at 1, 2 and 6 months after transplantation. When treated with an anti-serotonin antibody, cells in transplants from both locations showed intense immunoreactivity and a morphology comparable to intact pinealocytes, indicating that the transplanted pinealocytes had differentiated normally. Tyrosine hydroxylase immunohistochemistry revealed that new catecholamine fibers of central nervous origin extended only into the periphery and not into the core of transplants grafted within the cortex. However, numerous catecholamine fibers were found in transplants placed in the interhemispheric fissure. These fibers were often accompanied by blood vessels, suggesting that they derived from sympathetic ganglia. Serotonin fibers, which are densely distributed in the cerebral cortex, were seldom found to enter transplants from both locations. These observations indicate that pineal cells express their characteristic properties even when transferred to a foreign milieu and that they do not receive novel innervation from the central nerves that normally do not innervate the intact pineal body; the transplant thereby retains the property of selective pinealopetal innervation.     

Intraganglionic portal sinus located between small intensely fluorescent (SIF) cells and principal ganglionic neurons in the inferior mesenteric ganglion of the guinea pig

Summary The vascular system in the inferior mesenteric ganglion of the guinea pig was studied to clarify the transport pathway of transmitters released by the small intensely fluorescent (SIF) cells to the principal ganglionic neurons. Reconstruction of about 1500 1-m-thick serial sections of the ganglion demonstrated its portal system. SIF cells were tightly packed and formed two or three clusters under the capsule of the ganglion. Branches from the inferior mesenteric artery ran directly toward these clusters and broke up into a number of coiled and looped sinusoid capillaries among the SIF cells. They then drained into a large sinus surrounding the clusters in the ganglion. Capillaries were derived from this sinus and ramified among the principal ganglionic neurons. After supplying the neurons, these vessels drained into veins surrounding the ganglion. Therefore, as we observed two distinct groups of capillaries, we call this sinus the intraganglionic portal sinus. All the transmitters secreted from the SIF cells are collected into this intraganglionic portal sinus and are then conveyed through the capillaries to the principal ganglionic neurons.     

Effects of nicotine and Vitamin E on Carbonic anhydrase activity in some rat tissues In Vivo and In Vitro

Effects of nicotine, nicotine+vitamin E and nicotine+Hippophea rhamnoides L. extract (HRe-1) on muscle, heart, lungs, testicle, kidney, stomach, brain and liver carbonic anhydrase (CA; EC 4.2.1.1.) enzyme activities were investigated in vivo. Groups of rats were given nicotine (0.5?mg/kg/day, i.p.), nicotine+vitamin E (75?mg/kg/day, i.g.), nicotine+HRe-1 (250?mg/kg/day, i.g.) and a control group vehicle only. The results showed that nicotine inhibited the heart, lung, stomach and liver CA enzyme activities by ～80% (p?<?0.001), ～94% (p?<?0.001), ～47% (p?<?0.001) and ～81% (p?<?0.001) respectively, and activated muscle and kidney, but had no effects on the testicle and brain CA activities. Nicotine+vitamin E inhibited the heart and liver CA enzyme activities by ～50% (p?<?0.001), and ～50% (p?<?0.001), respectively, and nicotine+vitamin E activated the muscle CA activity. However, nicotine+vitamin E had no effect on lung, testicle, kidney, stomach and brain CA activities. Nicotine+HRe-1 inhibited the heart and stomach CA enzyme activities by ～51% (p?<?0.001), and ～32% (p?<?0.002), respectively, and activated the muscle and brain CA activities, but had no effects on the lung, testicle, kidney, and liver CA activities. In vitro CA inhibition results for similar experiments correlated well with the in vivo experimental results in lungs, testicles, kidney, stomach, brain and liver tissues.     

Nitric oxide synthase in the rat carotid body and carotid sinus

The participation of nitric oxide synthase (NOS) in the innervation of the rat carotid body and carotid sinus was investigated by means of NADPH-diaphorase histochemistry and NOS immunohistochemistry using antisera raised against purified neuronal NOS and a synthetic tridecapeptide. NOS was detected in 23% of neurons at the periphery of the carotid bodies. Some negative neurons were surrounded by NOS-positive terminals. NOS-containing varicose nerve fibres innervated the arterial vascular bed and, to a lesser extent, the islands of glomus cells. These fibres persisted after transection of the carotid sinus nerve and are probably derived from intrinsic neurons. Large NOS-positive axonal swellings in the wall of the carotid sinus were absent after transection of the sinus nerve, indicating their sensory origin. The results suggest a neuronal nitrergic control of blood flow, neuronal activity and chemoreception in the carotid body, and an intrinsic role of NO in the process of arterial baroreception.     

Coexistence of multiple peptides in small intensely fluorescent (SIF) cells of inferior mesenteric ganglion of the guinea pig

Summary Coexistence of peptides in the small intensely fluorescent cells was demonstrated by immunocytochemistry for met-enkephalin-Arg-Gly-Leu, vasoactive intestinal polypeptide, somatostatin, neuropeptide Y and dynorphin. In the extreme example, a single cell was immunoreactive to all 5 peptides examined. Four peptides coexisted in 8% and three peptides in 13% of SIF cells. In 10% of SIF cells no peptide immunoreactivity could be detected. The most prevalent peptide was met-enkephalin (in 46% of cells), then vasoactive intestinal polypeptide (45%), somatostatin (39%), neuropeptide Y (31%) and dynorphin (24%). Met-enkephalin and vasoactive intestinal polypeptide coexisted most commonly (25%).     

Roles of carbonic anhydrase 8 in neuronal cells and zebrafish

Background

Carbonic anhydrase 8 (CA8) is an isozyme of α-carbonic anhydrases (CAs). Previous studies showed that CA8 can be detected in human adult brain, with more intense expression in the cerebellum. Single mutations in CA8 were reported to cause novel syndromes like ataxia, mild mental retardation or the predisposition to quadrupedal gait.

Methods

In the present study, we examine the functions of CA8 in neuronal cell lines, mouse cerebellar granule neurons and zebrafish.

Results and conclusions

We demonstrated that overexpression of CA8 in neuronal cells significantly decreased cell death under staurosporine treatment. Moreover, CA8 overexpression significantly increased cell migration and invasion ability in neuronal cells and in mouse cerebellar granule neurons, implicating that CA8 may be involved in neuron motility and oncogenesis. By using zebrafish as an animal model, motor reflection of 3 dpf zebrafish embryos was significantly affected after the down-regulation of CA8 through ca8 morpholino.

Conclusions

We concluded that CA8 overexpression desensitizes neuronal cells to STS induced apoptotic stress and increases cell migration and invasion ability in neuronal cells. In addition, down-regulated CA8 decreases neuron mobility in neuronal cells and leads to abnormal calcium release in cerebellar granule neurons. Knockdown of the ca8 gene results in an abnormal movement pattern in zebrafish.

General significance

Our findings provide evidence to support that the impaired protective function of CA8 contributes to human neuropathology, and to suggest that zebrafish can be used as an animal model to study the biological functions of human CA8 in vivo.     

Colocalization of neuropeptides and NADPH-diaphorase in the intra-adrenal neuronal cell bodies and fibres of the rat

Colocalization of vasoactive intestinal peptide, neuropeptide Y, calcitonin gene-related peptide, substance P, and tyrosine hydroxylase, respectively, with NADPH-diaphorase staining in rat adrenal gland was investigated using the double labelling technique. All vasoactive intestinal peptide- and some neuropeptide Y-immunoreactive intrinsic neuronal cell bodies seen in the gland were double stained with NADPH-diaphorase. Double labelling also occurred in some nerve fibres immunoreactive to vasoactive intestinal peptide and neuropeptide Y in the medulla and cortex. No colocalization of calcitonin gene-related peptide, substance P or tyrosine hydroxylase immunoreactivity with NADPH-diaphorase staining was observed. However, nerve fibres with varicosities immunoreactive for all the neuropeptides examined were closely associated with some of the NADPH-diaphorase-stained neuronal cell bodies. Thus, in rat adrenal gland, nitric oxide is synthesized in all ganglion cells containing vasoactive intestinal peptide and in some containing neuropeptide Y, but not in those containing calcitonin gene-related peptide, substance P or tyrosine hydroxylase.     

Mitochondrial carbonic anhydrase in the nervous system: expression in neuronal and glial cells

Carbonic anhydrase (CA) V is a mitochondrial enzyme that has been reported in several tissues of the gastrointestinal tract. In liver, it participates in ureagenesis and gluconeogenesis by providing bicarbonate ions for two other mitochondrial enzymes: carbamyl phosphate synthetase I and pyruvate carboxylase. This study presents evidence of immunohistochemical localization of CA V in the rodent nervous tissue. Polyclonal rabbit antisera against a polypeptide of 17 C-terminal amino acids of rat CA V and against purified recombinant mouse isozyme were used in western blotting and immunoperoxidase stainings. Immunohistochemistry showed that CA V is expressed in astrocytes and neurons but not in oligodendrocytes, which are rich in CA II, or capillary endothelial cells, which express CA IV on their plasma face. The specificity of the immunohistochemical results was confirmed by western blotting, which identified a major 30-kDa polypeptide band of CA V in mouse cerebral cortex, hippocampus, cerebellum, spinal cord, and sciatic nerve. The expression of CA V in astrocytes and neurons suggests that this isozyme has a cell-specific, physiological role in the nervous system. In astrocytes, CA V may play an important role in gluconeogenesis by providing bicarbonate ions for the pyruvate carboxylase. The neuronal CA V could be involved in the regulation of the intramitochondrial calcium level, thus contributing to the stability of the intracellular calcium concentration. CA V may also participate in bicarbonate ion-induced GABA responses by regulating the bicarbonate homeostasis in neurons, and its inhibition could be the basis of some neurotropic effects of carbonic anhydrase inhibitors.     

Trophoblastic invasion and modification of uterine veins during placental development in macaques

Summary Immunoreactivity to the rate limiting enzyme of catecholamine synthesis, tyrosine hydroxylase, has been described in the inferior sensory (= nodose) ganglion of the vagal nerve in the rat. The aim of the present study was to characterize further this neuronal population. The neurons do not represent displaced autonomic efferent neurons, since they do not receive synaptic input, as indicated by the absence of synaptophysin-immunoreactive terminals. In addition to the immunoreactivity to tyrosine hydroxylase, a tyrosine hydroxylase cRNA probe hybridizes with nodose ganglion neurons as demonstrated by in situ hybridization and Northern blotting. Many but not all of the tyrosine hydroxylase-immunoreactive neurons are also immunoreactive to the dopamine synthesizing enzyme, aromatic-l-amino-acid-decarboxylase, but lack the noradrenaline-synthesizing enzyme, dopamine--hydroxylase, thus favoring synthesis of dopamine. Neuropeptide Y, which is often colocalized with catecholamines, is also present in a subset of nodose ganglion neurons, as indicated by immunohistochemistry, in situ hybridization and Northern blotting. However, double-labeling immunofluorescence has revealed that these two antigens are localized in different cell populations. Retrograde neuronal tracing utilizing fluorescent dyes (Fast blue, Fluoro-gold) combined with tyrosine hydroxylase immunohistochemistry has demonstrated that the esophagus and stomach are peripheral targets of tyrosine-hydroxylase-containing vagal visceroafferent neurons.     

Ontogeny of the carotid body and glomus cells distributed in the wall of the common carotid artery and its branches in the chicken

Summary Developmental patterns of immunoreactivity for serotonin and neuropeptide Y were investigated immunohistochemically in the carotid body and glomus cells in the wall of the common carotid artery and around its branches of chickens at various developmental ages. The development of peptidergic nerve fibers was also studied. Serotonin immunoreactivity began to appear in the glomus cells of the carotid body and around arteries at 10 days of incubation and became very intense from 12 days onwards. Neuropeptide Y immunoreactivity also appeared in these cells at 10 days, became intense at 14 days, and was sustained until 20 days. After hatching, neuropeptide Y immunoreactivity in the carotid body rapidly decreased with age and almost cisappeared at posnatal day 10. However, it persisted for life in the glomus cells distributed in the wall of the common carotid artery. Substance P- and calcitonin gene-related peptide (CGRP)-immunoreactive fibers first penetrated into the carotid body parenchyma at 12 days of incubation. These peptidergic nerve fibers in the carotid body and glomus cell groups in and around arteries gradually increased with age, and approached the adult state at 18 days of incubation. Only a few galanin-and vasoactive intestinal peptide (VIP)-immunoreactive fibers were observed in the late embryonic carotid bodies. They rapidly developed after hatching and reached adult numbers at postnatal day 10. During late embryonic and neonatal development, considerable numbers of met-enkephalin-immunoreactive fibers were detected in the connective tissue encircling the carotid body.     

Immunocytochemical localization of a growth-associated protein (GAP-43) in rat adrenal gland

We have localized at light and electron-microscopic level the growth-associated protein GAP-43 in adrenal gland using single and double labelling immunocytochemistry. Clusters of GAP-43-immunofluorescent chromaffin cells and many immunofluorescent fibres were observed in the medulla. GAP-43-immunoreactive fibres also formed a plexus under the capsule, crossed the cortex and ramified in the zona reticulata. Double labelled sections showed the coexpression of GAP-43 with a subpopulation of tyrosine hydroxylase-and of dopamine--hydroxylase-immunoreactive chromaffin cells. Dual colour immunofluorescence for GAP-43 and calcitonin gene-related peptide (CGRP) revealed that some of the GAP-43-immunoreactive fibres also express CGRP. Pre-embedding electron microscopy showed GAP-43 immunoreactivity associated with the plasma membranes and cytoplasm of noradrenaline-producing chromaffin cells, and with processes of nonmyelin-forming Schwann cells. Immunoreactive unmyelinated axons and terminals were also observed. The immunostained terminals made symmetrical synaptic contacts with chromaffin cells. Immunoreactive unmyelinated fibres and small terminals were present in the cortex. Our results show that GAP-43 is expressed in noradrenergic chromaffin cells and in various types of nerve fibres that innervate the adrenal. Likely origins for these fibres include preganglionic sympathetic fibres which innervate chromaffin cells, postganglionic sympathetic fibres in the cortex, and CGRP containing sensory fibres.     

Immunohistochemical study on fetal raphe samples transplanted into the leptomeningeal tissue of 5,6-dihydroxytryptamine-treated adult rats

Summary Pieces of fetal midbrain raphe containing serotonergic and dopaminergic neurons were transplanted into the leptomeningeal tissue (see Fig. 3) of adult host rats that had previously been denervated by treatment with 5,6-dihydroxytryptamine. One, 2 and 5 months after transplantation, the rate of neuronal survival in the grafted tissue and the extent of axonal outgrowth into the host brain were studied by use of serotonin and tyrosine hydroxylase (TH) immunohistochemistry. The survival rate of the grafts in the 1-month group was approximately 70%. Neurons containing either serotonin or catecholamine were demonstrated by means of immunocytochemical procedures in the grafts. Two and 5 months after transplantation, serotonin-immunoreactive nerve fibers were densely distributed throughout the graft tissue, while TH-immunoreactive fiber elements were restricted to an area near the somata of TH-positive neurons. Numerous serotonin-immunoreactive fibers derived from the transplant were found in the leptomeningeal tissue surrounding the graft, on the wall of neighboring blood vessels, and also in the adjacent parenchyma of the host brain. Outgrowing TH-immunoreactive nerve fibers were not observed in the host brain, although such elements occurred in the leptomeningeal tissue and the wall of the larger blood vessels. These results suggest that the serotonergic and catecholaminergic (dopaminergic) neurons located in transplants of the raphe nuclei show different patterns when reinnervating the host tissue.     

Immunohistochemical characterisation of sympathetic and parasympathetic pelvic neurons projecting to the distal colon in the male rat

The pelvic ganglia are mixed ganglia containing both sympathetic and parasympathetic neurons that receive spinal input via the hypogastric (lumbar cord) and pelvic nerves (sacral cord), respectively. A recent study has utilised immunohistochemistry against synaptophysin (a protein associated with small vesicles) to visualise the preganglionic terminals in these ganglia. By selectively cutting the hypogastric or pelvic nerves and allowing subsequent terminal degeneration, the populations of parasympathetic and sympathetic preganglionic terminals, respectively, can be visualised. The present study has used this method in conjunction with retrograde labelling of pelvic neurons from the distal colon and double label immunofluorescence against tyrosine hydroxylase and vasoactive intestinal polypeptide (VIP) to identify and characterise the sympathetic and parasympathetic neurons projecting to the distal colon from the major pelvic ganglia of the male rat. Approximately equal numbers of distal colonic-projecting pelvic neurons are sympathetic and parasympathetic. Almost all noradrenergic neurons are sympathetic. Of the VIP neurons that project to the distal colon approximately one third are sympathetic, one third parasympathetic and the remaining third are possibly innervated by both the lumbar and sacral cord. Extrapolation from our results also suggests that the majority of non-noradrenergic neuropeptide Y neurons (which are known to comprise the remainder of the neurons) are parasympathetic. These studies have demonstrated that the pelvic ganglia are a major source of sympathetic innervation to the distal bowel and have further shown that the distal colon is another target for the non-noradrenergic sympathetic neurons of the pelvic ganglia.     

Fine structural study of interstitial cells associated with the deep muscular plexus of the rat small intestine,with special reference to the intestinal pacemaker cells

Two types of interstitial cells have been demonstrated in close association in the deep muscular plexus of rat small intestine, by electron microscopy. Cells of the first type are characterized by a fibroblastic ultrastructure, i.e. a well-developed granular endoplasmic reticulum, Golgi apparatus and absence of the basal lamina. They form a few small gap junctions with the circular muscle cells and show close contact with axon terminals containing many synaptic vesicles. They may play a role in conducting electrical signals in the muscle tissue. Cells of the second type are characterized by many large gap junctions that interconnect with each other and with the circular muscle cells. Their cytoplasm is rich in cell organells, including mitochondria, granular endoplasmic reticulum and Golgi apparatus. They show some resemblance to the smooth muscle cells and have an incomplete basal lamina, caveolae and subsurface cisterns. However, they do not contain an organized contractile apparatus, although many intermediate filaments are present in their processes. They also show close contacts with axon terminals containing synaptic vesicles. These gap-junction-rich cells may be regular components of the intestinal tract and may be involved in the pacemaking activity of intestinal movement.     

Studies on colocalization of neuropeptide Y,vasoactive intestinal polypeptide,catecholamine-synthesizing enzymes and acetylcholinesterase in the larynx of the rat

Summary In the present immunohistochemical study, the distribution of nerve fibers containing neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) in the larynx was examined and compared with that of fibers containing tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (BDH), and with that of acetylcholinesterase (AChE)-positive nerve fibers, in intact and vagotomized rats and in rats subjected to removal of the superior cervical ganglion (SCG). Fibers showing TH/DBH-like immunoreactivity (LI) were only found in the walls of arteries and arterioles, whereas AChE-positive nerve fibers were located close to the acini and ducts of the glands, in blood vessel walls, in the perichondrium and in the lamina propria. NPY-LI and VIP-LI coexisted in local AChE-positive ganglionic cells and in a subpopulation of the AChE-positive fibers, NPY-LI also being present in some periarterial fibers showing TH/DBH-LI. Unilateral removal of the SCG eliminated the TH/DBH-innervation in the upper but not the lower parts of the larynx ipsilaterally, whereas the NPY-innervation of the arteries in the upper parts only partly disappeared and the NPY-innervation of the other structures remained unchanged. The distribution of VIP-innervation was unchanged after vagotomy and removal of the SCG. The results suggest that VIP is present in the postganglionic parasympathetic innervation, whereas NPY is present in both the postganglionic parasympathetic and sympathetic innervation of the rat larynx.     

Localization of acetylcholinesterase in dissociated cell cultures of the carotid body of the rat

Summary The localization of acetylcholinesterase (AChE) was investigated at the cellular and subcellular levels in dissociated cell cultures of the carotid body of the neonatal rat, prepared by the methods of Fishman and Schaffner (1984). In the presence of iso-OMPA, which blocks non-specific cholinesterase, staining was confined almost exclusively to glomus-cell clusters and occasional isolated cells. These clusters grow as discrete islands scattered throughout the culture and display typical catecholamine (CA) fluorescence as in vivo. AChE staining was abolished or reduced by the cholinesterase inhibitors eserine (30–100 M), or (the poorly lipid soluble) echothiophate (8 (M). Processing of the same culture sequentially for the demonstration of both AChE and CA revealed that glomus-cell clusters and individual glomus cells were consistently positive for both. In electron micrographs AChE reaction product was associated intracellularly with the nuclear envelope and cytoplasm of glomus cells (identified by their characteristic dense cored granules), as well as extracellularly with the boundaries of contiguous glomus cells. Significantly, reaction product occurred in some glomus cell profiles that had both dense-cored and clear (cholinergic-like) vesicles. These findings are discussed in the context of a possible dual (adrenergic/cholinergic) function status of glomus cells in the rat's carotid body.