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PubMed2002

Changes in immunoreactivity to calcium-binding proteins in the anterior olfactory nucleus of the rat after neonatal olfactory deprivation.

The effects of olfactory deprivation on the density of neuronal populations expressing the calcium-binding proteins calbindin D-28k, calretinin, and parvalbumin in the anterior olfactory nucleus of the rat were studied immunohistochemically in 60-day-old rats subjected to unilateral naris closure on the day of birth. The neuronal populations were characterized morphologically and topologically, and the density of each cell type was calculated in each subdivision of the anterior olfactory nucleus at seven rostrocaudal levels. Data were gathered into three groups: data from either the ipsilateral or contralateral anterior olfactory nucleus of experimental animals and data from control animals. Statistical analysis indicated that disruption of the normal afferent activity to one olfactory bulb affects the expression of the calcium-binding proteins investigated in the anterior olfactory nucleus, as revealed by variations in the density of certain neuronal populations. The observed effects were very heterogeneous and could not be related to any specific neuronal type, location, or to the expression of a given calcium-binding protein. Nevertheless, as a general rule the most affected neuronal populations were those expressing calbindin D-28k located in the rostral subdivisions. These subdivisions are the latest to develop in mammals and are those that receive the largest amount of inputs from the olfactory bulb.

Animals↗
PubMed2006

Mechanisms of neurodegeneration in neuronal ceroid-lipofuscinoses.

Neuronal ceroid-lipofuscinoses (NCL) are a group of neurodegenerative diseases and autosomal recessive lysosomal storage disorders. We examined the involvement of cell death, oxidative stress, and glutamate excitotoxicity using immunohistochemistry against Bcl-2, Bcl-x, oxidative products to proteins, lipids and DNA, calcium-binding proteins (calbindin-D28K, parvalbumin, calretinin), and glial glutamate transporters (excitatory amino acid transporters 1 and 2), in addition to terminal deoxynucleotidyl transferase-mediated dUTP-nick end labeling (TUNEL) in the brains from three cases of late infantile form of NCL (LINCL) and one case of juvenile form of NCL (JNCL) to investigate the neurodegenerative mechanisms. In the cerebral and cerebellar cortex, all of three LINCL cases demonstrated neurons with TUNEL-immunoreactive nuclei, whereas the JNCL case did not show TUNEL-immunoreactive nuclei. The coexistence of the nuclear TUNEL-immunoreactivity nuclei and cytoplasmic deposition of 4-hydroxy-2-nonenal-modified protein in the frontal cortex and hypoglossal nucleus may suggest a possible interrelationship between DNA fragmentation and lipid oxidation in LINCL. Additionally, glycoxidation of protein and oxidative stress to DNA seemed to be involved in the cerebellar and cerebral degeneration, respectively. Interneurons immunoreactive for calbindin-D28K and parvalbumin were severely reduced in the cerebral cortex, whereas those for calretinin were comparatively well preserved in LINCL, indicating the possibility of altered GABAergic system. The disturbance of expression of glial glutamate transporters seemed to be heterogeneous and mild. These findings suggest the possibility of new treatments for neurodegeneration in LINCL using antioxidative agents and/or GABAergic medications.

Adolescent↗
PubMed1996

Immunohistochemical characterization of subependymal giant cell astrocytomas.

Subependymal giant cell astrocytoma (SEGA) is the most common neoplastic process involving the brain in patients with tuberous sclerosis complex (TSC). Morphologically, these tumors exhibit a wide range of cytoarchitecture with spindle and epithelioid cells resembling astrocytes, and also large, occasionally giant cells, some of which have a distinctly ganglion-like appearance. Unresolved questions regarding SEGAs center on: (a) their cytogenesis, i.e., whether they are derived from single or multiple precursors; and (b) their differentiating capacity along glial or neuronal lines. We sought to determine whether SEGAs represent truly mixed tumors or whether they consist of a single population of cells with a capacity for divergent differentiation. Twenty SEGAs were assessed for immunophenotypic features of either neuronal or glial differentiation or both. Only tumors from patients with a clinically confirmed diagnosis of TSC were included. Immunoreactivity for glial fibrillary acidic protein (GFAP) and/or S-100 protein was considered indicative of a glial phenotype, whereas the presence of neuronal differentiation was assessed by staining for cytoskeletal proteins [neurofilament epitopes, class III Beta-tubulin, microtubule-associated protein 2 (MAP2), synaptophysin], neurosecretory substances [serotonin, cholecystokinin, Beta-endorphin, substance P, somatostatin, metenkephalin, neuropeptide Y, vasoactive intestinal polypeptide (VIP), and for the 28-kDa neuron-associated calcium binding protein calbindin. Of the tumors examined, 18 exhibited both glial and neuronal epitopes, the staining pattern being variable. In 19 tumors, the constituent spindle, polygonal and giant or ganglion-like cells showed variable immunoreactivity for GFAP and S-100 proteins both within the cell body and processes. Neuron-associated cytoskeletal proteins were present in 18 cases. Class III Beta-tubulin immunoreactivity was demonstrated in 17 tumors, both within the bodies of all three cell types and to varying degrees within their processes. Neurofilament protein and calbindin staining was present in 8 tumors, with reactivity for the former being distributed in a phosphorylation-dependent manner. MAP2 was detected in a few cells of two tumors. Immunoreactivity for neuropeptides was observed in 17 lesions. Somatostatin and metenkephalin staining was noted in 10 tumors (50%) being present particularly within polygonal cells. Neuropeptide Y, serotonin and Beta-endorphin reactivity was found in 6 (30%), 5 (25%), and 4 tumors (20%), respectively; Beta-endorphin was lacking in giant cells, whereas neuropeptide Y and serotonin were seen within their cell bodies. Substance P and VIP were evident in only occasional polygonal cells of 2 (10%) and 1 tumor (5%), respectively. Stains for cholecystokinin were negative. The observation of immunoreactivity for both glial- and neuron-associated epitopes within tumor cells of the same morphology suggests that SEGAs represent proliferations of cell lineages with the capacity to undergo divergent glioneuronal as well as neuroendocrine differentiation to a greater extent than do other mixed glial-neuronal neoplasms.

Adolescent↗
PubMed2002

The distribution of purine P2X(2) receptors in the guinea-pig enteric nervous system.

The P2X(2) subtype of purine receptor was localised by immunohistochemistry to nerve cells of the myenteric ganglia of the stomach, small and large intestines of the guinea-pig, and nerve cells of submucosal ganglia in the intestine. Nerve cells with strong and with weak immunoreactivity could be distinguished. Immunoreactivity in both strongly and weakly immunoreactive neurons was absorbed with P2X(2) receptor peptide. In the myenteric plexus, strong immunoreactivity was in nitric oxide synthase (NOS)- and in calbindin-immunoreactive neurons. In all regions, over 90% of NOS-immunoreactive neurons were strongly P2X(2) receptor immunoreactive. The intensity of reaction varied in calbindin neurons; in the ileum, 90% were immunoreactive for the receptor, about one-third having a strong reaction. In the submucosal ganglia, all vasoactive intestinal peptide-immunoreactive neurons were P2X(2) receptor immunoreactive, but there was no receptor immunoreactivity of calretinin or neuropeptide Y neurons. Varicose nerve fibres with P2X(2) receptor immunoreactivity were found in the gastric myenteric ganglia. These fibres disappeared after vagus nerve section. It is concluded that the P2X(2) receptor is expressed by specific subtypes of enteric neurons, including inhibitory motor neurons, non-cholinergic secretomotor neurons and intrinsic primary afferent neurons, and that the receptor also occurs on the endings of vagal afferent fibres in the stomach.

Animals↗
PubMed2003

Protein kinase C isoforms in the enteric nervous system.

C kinases (PKCs) are a family of enzymes essential for the transduction of signals in a diverse range of cell types, including neurons. The different isoforms vary in their activation requirements. Therefore, cell-specific expression of different isoforms has implications for PKC-mediated control of organ function. This study has investigated the types and distributions of PKC isoforms in the small intestine of the guinea-pig, with particular emphasis on their localisation in myenteric neurons, using immunohistochemistry and western blotting techniques. Three PKC isoforms, gamma, eta and theta, were detected in the calbindin-immunoreactive subset of intrinsic primary afferent neurons, but not in other myenteric neurons. Both gamma and theta immunoreactivities were also located in interstitial cells of Cajal. In contrast to these isoforms, immunoreactivity for PKCs lambda and epsilon was present in all myenteric neurons of the ileum. PKCalpha immunoreactivity was detected primarily in the glial network, as shown through double labelling with antibodies to the glial filament protein, S100b. Myenteric neurons were also weakly immunoreactive for this isoform. PKCdelta immunoreactivity was very highly expressed in smooth muscle, but was largely absent from neurons. Immunoreactivity for RACK1, a binding protein for PKCbeta, was detected in both calbindin-immunoreactive neurons and in smooth muscle cells. This study indicates a selective distribution of PKC isoforms to specific cell types. Isoform-specific activity of these enzymes could provide a means through which targeted modulation of intestinal function is achieved.

Animals↗
PubMed2004

Development of nerves expressing P2X3 receptors in the myenteric plexus of rat stomach.

Development of neurones and fibres expressing P2X3 receptors in the myenteric plexus of rat stomach and coexistence of the P2X3 receptor with calbindin, calretinin and NOS during postnatal development, were investigated with immunostaining methods. Extrinsic nerves expressing P2X3 receptors appeared as early as E12 and were localised in the trunk and branches of the vagus nerve, which extended rapidly onto the whole rat stomach from E12 to E14. Intrinsic neurone cell bodies with P2X3-immunoreactivity in the myenteric ganglia were first demonstrated postnatally at P1, and at P14, when the number of neurones expressing the P2X3 receptor peaked at 45%. P2X3 receptor-immunoreactivity decreased subsequently, and at P60 only about 11% were P2X3-immunoreactive. Intraganglionic laminar nerve endings and intramuscular arrays were first demonstrated postnatally at P1 and P7, respectively. In the early postnatal days, there were many growth cone-like structures with strong P2X3 immunostaining associated with these endings and arrays. Double-immunostaining showed that 9-15% of P2X3-immunoreactive neurones in the gastric myenteric plexus expressed calbindin D-28 k only in the early postnatal days, while 14-21% of neurones from P1 to P60 increasingly expressed calretinin. About 20% of neurones with P2X3 immunoreactivity coexpressed NOS throughout perinatal development.

Animals↗
PubMed2006

Renal Ca2+ handling in sgk1 knockout mice.

Coexpression studies in Xenopus oocytes revealed the ability of the serum- and glucocorticoid-inducible kinase 1 (SGK1) to stimulate the renal epithelial Ca(2+) channel TRPV5. SGK1 increases the abundance of the channel protein in the plasma membrane, an effect requiring the participation of the Na(+)/H(+) exchanger regulating factor 2 (NHERF2). The present study was performed to explore the role of SGK1 in the regulation of renal Ca(2+) handling in vivo. To this end, TRPV5, calbindin D-28K abundance, and renal Ca(2+) excretion were analyzed in gene-targeted mice lacking functional SGK1 (sgk1( -/- )) and their age- and sex-matched littermates (sgk1( +/+ )). Immunohistochemistry revealed lower abundance of TRPV5 and calbindin D-28K protein in sgk1( -/- ) mice than in sgk1( +/+ ) mice, both fed with control diet. Feeding the mice a Ca(2+)-deficient diet marked ly increased TRPV5 protein abundance in both genotypes. Renal Ca(2+) excretion under control diet was significantly lower in sgk1 ( -/- ) than in sgk1( +/+ ) mice. The Ca(2+)-deficient diet decreased renal excretion of Ca(2+) to the same levels in both phenotypes. Furosemide increased fractional Ca(2+) excretion and dissipated the difference between phenotypes. We conclude that lack of SGK1 may lead to decrease in TRPV5 abundance in connecting tubules but does not abrogate TRPV5 regulation. The decrease in abundance of TRPV5 in connecting tubules of sgk1( -/- ) mice is presumably compensated for by enhanced Ca(2+) reabsorption in upstream nephron segments such as the loop of Henle, which may indirectly result from impaired SGK1-dependent Na(+) reabsorption in the aldosterone-sensitive distal part of the nephron, salt loss, and enhanced Na(+) (and Ca(2+)) reabsorption in those upstream nephron segments.

Animals↗
PubMed1999

Distribution of various neurochemicals within the zona incerta: an immunocytochemical and histochemical study.

To gain insight into the cellular organisation of the zona incerta, we have examined the chemoarchitectonic properties of this "uncertain zone". The brains of Sprague-Dawley rats and common cats were processed for immunocytochemistry or NADPH-diaphorase histochemistry using standard methods. For the immunocytochemistry, antibodies to y-aminobutyric acid (GABA), glutamic acid decarboxylase (GAD), parvalbumin, calbindin, tyrosine hydroxylase, somatostatin, serotonin and glutamate were used. Two general patterns of distribution in the zona incerta were seen. First, labelled cells were restricted largely to one of the cytoarchitectonically defined sectors of the zona incerta. For instance, GABA, GAD and parvalbumin-immunoreactive cells were found principally within the ventral sector, NADPH-diaphorase and glutamate-immunoreactive cells within the dorsal sector and tyrosine hydroxylase- and somatostatin-immunoreactive cells within the rostral sector. Second, labelled cells were scattered somewhat across all incertal sectors, with no clear region of concentration. This pattern included the calbindin- and serotonin-immunoreactive cell groups. These results indicate that the zona incerta is made up of many neurochemically distinct cell groups, some of which respect the well-defined cytoarchitectonic boundaries of the nucleus, whilst others do not. This rich neurochemical diversity in the zona incerta suggests that this nucleus may have differential effects on the different structures that it projects to.

Animals↗
PubMed2005

Changes in retinal neuronal populations in the DBA/2J mouse.

DBA/2J (D2) mice develop a form of progressive pigmentary glaucoma with increasing age. We have compared retinal cell populations of D2 mice with those in control C57BL/6J mice to provide information on retinal histopathology in the D2 mouse. The D2 mouse retina is characterized by a reduction in retinal thickness caused mainly by a thinning of the inner retinal layers. Immunocytochemical staining for specific inner retinal neuronal markers, viz., calbindin for horizontal cells; protein kinase C (PKC) and recoverin for bipolar cells, glycine, gamma-aminobutyric acid (GABA), choline acetyltransferase (ChAT), and nitric oxide synthase (NOS) for amacrine cells, and osteopontin (OPN) for ganglion cells, was performed to detect preferentially affected neurons in the D2 mouse retina. Calbindin, PKC, and recoverin immunoreactivities were not significantly altered. Amacrine cells immunoreactive for GABA, ChAT, and OPN were markedly decreased in number, whereas NOS-immunoreactive amacrine cells increased in number. However, no changes were observed in the population of glycine-immunoreactive amacrine cells. These findings indicate a significant loss of retinal ganglion and some amacrine cells, whereas glycinergic amacrine cells, horizontal, and bipolar cells are almost unaffected in the D2 mouse. The reduction in amacrine cells appears to be attributable to a loss of GABAergic and particularly cholinergic amacrine cells. The increase in nitrergic neurons with the consequent increase in NOS and NO may be important in the changes in the retinal organization that lead to glaucomain D2 mice. Thus, the D2 mouse retina represents a useful model for studying the pathogenesis of glaucoma and mechanisms of retinal neuronal death and for evaluating neuroprotection strategies.

Amacrine Cells↗
PubMed2005

Cocaine- and amphetamine-related transcript peptide and somatostatin in rat intracardiac ganglia.

The distribution of somatostatin and cocaine and amphetamine-regulated transcript (CART) was investigated in rat intracardiac ganglia. Somatostatin immunoreactivity was only present in nerve terminals, always colocalised with choline acetyltransferase immunoreactivity, surrounding approximately 10% of intracardiac neurons. Somatostatin-immunoreactive terminals particularly targeted intrinsic cardiac neurons that were immunoreactive for calbindin. Somatostatin was also present in sympathetic cholinergic neurons in the stellate ganglia, but could not be detected in neurons of the nucleus ambiguus and dorsal motor nucleus of the vagus in the brainstem. CART immunoreactivity was present in 46% of intracardiac neuronal somata, including those that expressed either NOS or calbindin immunoreactivity but was never present in terminals forming pericellular baskets around intracardiac neurons. CART immunoreactivity was absent from sympathetic cell bodies in the stellate ganglia, but was present in nerve terminals around sympathetic neurons. Based on the results of this study, additional chemical diversity was identified among elements of the rat cardiac nervous system that may define neural pathways of different function.

Animals↗
PubMed1999

The cytoskeleton-associated neuronal calcium-binding protein caldendrin is expressed in a subset of amacrine, bipolar and ganglion cells of the rat retina.

Caldendrin is a novel calcium-binding protein confined to the somatodendritic compartment of neurons. Here we have studied the expression pattern of caldendrin in the rat retina. First we assessed the distribution of caldendrin transcripts in the adult and developing retina by in situ hybridization. In the adult retina, transcripts are expressed mainly in the inner half of the inner nuclear layer (INL) and to a lesser extent in the ganglion cell layer (GCL). During development labeling of the inner part of the cytoblast layer, where amacrine cells reside, is already present at postnatal day 1 (P1). The intensity of hybridization signal in this sublamina of the developing INL increases up to P8, whereas significant labeling in the GCL was first found at P14, coinciding with eye opening. Immunodetection with a polyclonal antibody revealed intensive staining of cells in the inner retina, which are presumably mainly amacrine and significantly fewer bipolar and ganglion cells. All parvalbumin-containing All amacrines were immunopositive for caldendrin. Colocalization with calbindin was found in cone bipolar cells, the majority of AII amacrines, and calbindin-positive cells in the GCL. In the GCL, caldendrin was also colocalized with calretinin-immunopositive cells. Most caldendrin-positive amacrine cells in the adult rat retina were glycinergic and only a few were GABAergic. In retinal flat mounts, it was confirmed that less than 10% of retrogradely labeled retinal ganglion cells (RGC) are caldendrin-positive. Caldendrin immunoreactivity does not colocalize with tyrosine hydroxylase, VIP, substance P and somatostatin immunoreactivity. In summary, caldendrin expression is regulated differentially in retinal cell types during development and is restricted to a subpopulation of amacrine, bipolar, and ganglion cells, suggesting specific functions in the developing and mature retina.

Animals↗
PubMed2006

Immobilization decreases duodenal calcium absorption through a 1,25-dihydroxyvitamin D-dependent pathway.

Immobilization induces significant and progressive bone loss, with an increase in urinary excretion and a decrease in intestinal absorption of calcium. These actions lead to negative calcium balance and the development of disuse osteoporosis. The aims of this study were to evaluate the molecular mechanisms of decreased intestinal calcium absorption and to determine the effect of dietary 1,25-dihydroxyvitamin D [1,25(OH)2D] and a high-calcium diet on bone loss due to immobilization. The immobilized rat model was developed in the Bollman cage III to induce systemic disuse osteoporosis in the animals. There was a significant decrease in lumbar bone mineral density (BMD) and intestinal calcium absorption in the immobilized group compared with the controls. Serum 25-hydroxyvitamin D concentration did not change, but 1,25(OH)2D concentration decreased significantly. The mRNA levels of renal 25-hydoxyvitamin D 24-hydroxylase (24OHase) increased, whereas those of renal 25-hydroxyvitamin D 1-alpha hydroxylase (1alpha-hydroxylase), duodenal transient receptor potential cation channel, subfamily V, member 6 (TRPV6), TRPV5, and calbindin-D9k were all decreased. A high-calcium diet did not prevent the reduction in lumbar BMD or affect the mRNA expression of proteins related to calcium transport. Dietary administration of 1,25(OH)2D increased the intestinal calcium absorption that had been downregulated by immobilization. TRPV6, TRPV5, and calbindin-D9k mRNA levels were also upregulated, resulting in prevention of the reduction in lumbar BMD. Therefore, it is concluded that dietary 1,25(OH)2D prevented decreases in intestinal calcium absorption and simultaneously prevented bone loss in immobilized rats. However, it remains unclear that calcium absorption and expression of calcium transport proteins are essential for the regulation of lumbar BMD.

Animals↗
PubMed1996

Amphetamine induces Fos-like immunoreactivity in the striatum of primates.

Injections of D-amphetamine (5 mg/kg) three hours before sacrifice were found to elicit robust Fos-like immunoreactivity in the striatum of cynomolgus monkeys and of the common marmoset. Labeled cells were most frequently observed in the medial portion of the caudate nucleus and were distributed in a patchy fashion. Comparison with adjacent sections stained for the calcium binding protein calbindin indicated that the patches of amphetamine induced labeling corresponded to the calbindin-poor striosomes. These results are consistent with those reported in rats and thus suggest similarities in the basic organization of striatal mechanisms underlying the response to amphetamine in rodents and primates.

Amphetamine↗
PubMed1995

Nuclear magnetic resonance assignments and secondary structure of bovine S100 beta protein.

S100 beta is a neurite extension factor and has been implicated in Alzheimer's disease and Down's syndrome. It belongs to a group of low molecular weight calcium-binding proteins containing the helix-loop-helix calcium binding motif. The structure of only one S100 protein, calbindin D9k, which has the lowest sequence similarity to the other members of the S100 group has been determined. We report the NMR assignments and secondary structure of calcium-free S100 beta. The secondary structure is similar to that of calbindin D9k, determined using NMR, except that there is clear evidence for an additional well ordered 5-residue alpha-helix in S100 beta.

Amino Acid Sequence↗
PubMed1994

Expression of horizontal cell phenotypes in monolayer cultures from immature rabbit retina.

Using the sandwich culture technique introduced by Brewer and Cotman we have studied the in vitro differentiation of A- and B-type horizontal cells which represent two well characterized cell types of the rabbit retina. Neurons from immature (postnatal day 3) rabbit retinae were dissociated and grown on inverted coverslips for up to 5 weeks in a chemically defined medium. On the basis of morphological criteria and the staining pattern for several immunocytochemical and autoradiographic horizontal cell markers we have examined to what extent expression of a distinct mature neuronal phenotype can take place under the artificial conditions of monolayer cultures. After 14 days in vitro neurons could be identified which had acquired elaborate morphological features closely resembling those of A- and B-type horizontal cells, respectively. Axonless A-like cells had 2-4 stout primary dendrites. In agreement with in situ observations these cells showed immunoreactivity for neurofilament proteins (68 kDa, 200 kDa), calbindin-28 kDa and less strongly for vimentin. B-like neurons reached varying states of development. Ideally, they had dendritic trees with 6-8 primary processes extending radially from the soma and a single axon-like process which branched extensively to form a profuse neuritic arbor strikingly similar to axon terminal systems of B-type cells in the intact retina. B-like cells also stained for vimentin, calbindin-28 kDa and unexpectedly also for neurofilament proteins. Interestingly, however, neurofilaments became redistributed during in vitro development eventually resulting in their restricted localization in the 'axon terminal system'. This apparently reflects a developmental process which has escaped detection in situ so far. Both cell types were intensely labelled with antibodies to gamma-aminobutyric acid (GABA), the presumed horizontal cell transmitter, but high affinity uptake of this transmitter was practically undetectable by [3H]-GABA autoradiography. This was in agreement with observations in intact retinae. These results support the notion that once a neuron has reached a certain developmental state further differentiation and maintenance of its particular morphological and functional properties are primarily governed by intrinsic factors, but do not exclude that extrinsic signals have important modulatory functions.

Animals↗
PubMed1995

Immunocytochemical localization of mu-opioid receptor in the rat caudate-putamen.

A guinea pig antibody against a C-terminal peptide of rat mu-opioid receptor (MOR) was produced to examine the distribution of MOR in the rat caudate-putamen (CP). The anti-peptide antibody recognized a protein of M(r) 69,000 in Triton X-100 extract of rat brain and in the membrane fraction of MOR-expressing culture cells. Intense MOR-like immunoreactivity (LI) was observed in island-like areas of the CP. Some MOR-LI was located on the cell bodies and dendrites of CP neurons. Double immunofluorescence study revealed that the intensely MOR-immunoreactive areas showed weak calbindin-LI, surrounded by intensely calbindin-positive regions. The results indicate that MOR-LI is enriched in the 'patches' of the neostriatal mosaic compartmentation.

Animals↗
PubMed1991

N-methyl-D-aspartate evokes the release of somatostatin from striatal interneurons in primary culture.

Indirect immunocytochemistry of striatal neurons in primary culture, generated from the embryonic mouse brain, suggested that 2-4% of the neurons contained somatostatin-like immunoreactivity; the majority of these cells also contained neuropeptide Y immunoreactivity, characteristic of a subset of striatal interneurons. Although 10-15% of cultured striatal neurons showed moderate or intense immunoreactivity for calbindin-D28k, the majority of neurons with somatostatin-like immunoreactivity did not contain calbindin-D28k-like immunoreactivity; parvalbumin immunoreactivity was absent from the culture preparation. A highly sensitive radioimmunoassay was used to examine the actions of depolarizing agents and excitatory amino acids on the release of endogenous somatostatin-like immunoreactivity from striatal interneurons. During a 15 min incubation period, 47 +/- 10 fmol of somatostatin-like immunoreactivity were released from 14 days in vitro striatal neurons, cultured in 35 mm dishes. Depolarization with 56 mM KCl or 10 micrograms/ml veratrine resulted in an additional 105 +/- 9 and 56 +/- 5 fmol, respectively, of somatostatin-like immunoreactivity released; the release evoked by veratrine was blocked by 1 microM tetrodotoxin. In the presence of 100 microM N-methyl-D-aspartate, 112 +/- 21 fmol of somatostatin-like immunoreactivity (above basal) were released (+238%); the N-methyl-D-aspartate-evoked release was dose-dependent (EC50, 20 microM), attenuated in the absence of added Ca2+, potentiated in the absence of added Mg2+ and unaffected by the presence of 1 microM tetrodotoxin. The selective antagonists 2-amino-5-phosphonovalerate (100 microM) and MK-801 (1 microM) blocked the N-methyl-D-aspartate-evoked release of somatostatin-like immunoreactivity; KCl-evoked release was unaffected. Kainate was slightly more effective, yet five-fold less potent (EC50, 100 microM), than N-methyl-D-aspartate in evoking somatostatin-like immunoreactivity release; quisqualate was marginally effective. The results of this study suggest that N-methyl-D-aspartate and kainate receptors are present on striatal somatostatinergic interneurons in primary culture.

Animals↗
PubMed1993

Rectospinal neurons: cell bodies, pathways, immunocytochemistry and ultrastructure.

A novel class of enteric neurons projecting directly from the rectal wall to the spinal cord, "rectospinal neurons", was investigated in rats by combined retrograde neuronal tracing, immunocytochemistry and electron microscopy. Rectospinal neurons were almost confined to myenteric ganglia of the distal rectum below the pelvic diaphragm and were labeled preferentially by injections into spinal cord segments L6/S1. Injections into more rostral spinal cord segments resulted in hardly any labeled enteric neurons. Dorsal and ventral rhizotomy experiments indicated an almost exclusive projection of rectospinal neurons through dorsal roots L6/S1 to the respective spinal cord segments. Among various peptides immunostained, vasoactive intestinal polypeptide and calcitonin gene-related peptide were selectively found in rectospinal neurons, which were also shown to contain calbindin, neurofilament protein- and peripherin-immunoreactivity. Vasoactive intestinal polypeptide- and calbindin-immunostaining were frequently co-localized in the same perikarya, while calcitonin gene-related peptide-immunoreactive rectospinal neurons probably represented a separate population. Neonatal capsaicin treatment did not significantly reduce the number of rectospinal neurons. Electron microscopy revealed synaptic contacts on the surface of rectospinal neurons. Taken together, these results establish rectospinal neurons as an anatomically and neurochemically distinct class of enteric neurons. Synaptic contacts on rectospinal neurons suggest that these neurons may function as a direct link from the enteric to the central nervous system, thus indicating that connections between these two networks are reciprocal.

Afferent Pathways↗