Adenosine A2A agonist CGS 21680 decreases the affinity of dopamine D2 receptors for dopamine in human striatum.

Adenosine A2A receptors (A2AR) and dopamine D2 receptors (D2R) are highly concentrated in the striatum, where they are co-localized and exert reciprocal antagonistic interactions. It has been suggested that the A2R/D2R interactions might provide a therapeutic approach for basal ganglia disorders, such as Parkinson's disease, and schizophrenia. In the present work evidence is presented for the existence of an A2AR/D2R interaction in human brain by using quantitative autoradi- ography. The areas analyzed were the dorsal caudate nucleus and putamen. Parallel studies were performed in rat striatal sections. The A2AR agonist CGS 21680 was found to significantly increase IC50 values of competitive inhibition curves of the D2R/D3R antagonist [125I]iodosulpiride vs dopamine both in rat striatal and human striatal brain sections.

CD34 immunoreactivity and interstitial cells of Cajal in the human and mouse gastrointestinal tract.

Immunoreactivity for the tyrosine kinase receptor Kit (Kit-ir) is an established marker for the interstitial cells of Cajal (ICC) of the gut. Recently, the presence of CD34 immunoreactivity (CD34-ir) has been reported in Kit-ir ICC around the myenteric plexus in human small intestine. Conversely, we observed that CD34-ir labeled Kit-negative fibroblast-like cells, closely adjacent to, but distinct from, the Kit-ir ICC. The existence of cells expressing both CD34-ir and Kit-ir remains controversial. CD34-ir and Kit-ir were studied by high-resolution confocal microscopy on cryostat sections of human and murine gut as well as murine whole-mounts, using specific antibodies raised to human and murine CD34, respectively. CD34-ir labeled numerous cells in all parts of the gut, in man and in mouse. CD34-ir was consistently observed in Kit-negative cells, distinct from the closely adjacent Kit-ir ICC. Thin processes of both cell types intermingled extensively, often at the limit of resolution for light microscopy. CD34-ir was also observed in Kit-negative mesenchymal cells in the submucosa, in capillaries and in mesothelial cells. CD34-ir is not a marker for Kit-ir ICC in the human and murine gut. No CD34-ir, Kit-ir-expressing cells were encountered. Conversely, CD34-ir cells, closely adjacent to, but distinct from, Kit-ir ICC were consistently identified. The intimate relationship between these cells may offer an alternative explanation for reports of CD34 and Kit co-localization. The ontogeny and function of CD34-ir cells in the gut, as well as the origin of gastrointestinal stromal tumors, remain unclear.

Caffeine-mediated induction of c-fos, zif-268 and arc expression through A1 receptors in the striatum: different interactions with the dopaminergic system.

Adenosine and the adenosine receptor antagonist, caffeine, modulate locomotor activity and striatal neuropeptide expression through interactions with the dopaminergic system by mechanisms which remain partially undetermined. We addressed this question by using quantitative immunocytochemistry and in situ hybridization, combined with retrograde tracing of striatal neurons, to characterize the mechanism(s) leading to the striatal increase in the immediate early genes (IEG), c-fos, zif-268 and arc, following a single injection of caffeine or the A1 antagonist, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX). Caffeine and DPCPX induced c-fos, zif-268 and arc expression, both at mRNA and protein levels, in large proportions of striatonigral and striatopallidal neurons. The involvement of dopamine systems was evaluated by manipulations of the dopaminergic transmission. Quinpirole, a D2 agonist, almost completely blocked the caffeine-induced IEG increase in both striatopallidal and striatonigral neurons. Conversely, the lesion of the nigrostriatal pathway and the D1 antagonist SCH23390 abolished the caffeine effects in striatonigral neurons but had no or slight effect, respectively, on its action in striatopallidal neurons. These observations demonstrate that caffeine- and DPCPX-mediated IEG inductions involved different mechanisms in striatonigral and striatopallidal neurons through blockade of A1 receptors. Immediate early gene inductions result from a stimulation of dopamine release in striatonigral neurons and from activation of glutamate release and probably also acetylcholine release in striatopallidal neurons. These results also support the idea that, besides A2A receptors, adenosine acting at the A1 receptor plays pivotal functions in the basal ganglia physiology and that blockade of these receptors by specific or nonspecific antagonists, DPCPX and caffeine, may influence a broad range of neuronal functions in the striatum.

Distribution of the nociceptin and nocistatin precursor transcript in the mouse central nervous system.

The distribution of prepronociceptin messenger RNA, the recently identified endogenous ligand of the ORL1 receptor (opioid receptor-like-1), has been studied in the adult mouse central nervous system using in situ hybridization. Prepronociceptin is a new peptide precursor that generates, upon maturation, at least three bioactive peptides: nociceptin, noc2 and the recently described nocistatin. Considering both the density of labeled neurons per region and their intensity of labeling, the distribution of prepronociceptin messenger RNA-containing neurons can be summarized as follows: the highest level of prepronociceptin messenger RNA expression was detected in the septohippocampal nucleus, bed nucleus of the stria terminalis, central amygdaloid nucleus, and in selective thalamic nuclei such as the parafascicular, reticular, ventral lateral geniculate and zona incerta. High to moderate levels of prepronociceptin messenger RNA expression were detected in the lateral, ventral and medial septum, and were evident in brainstem structures implicated in descending antinociceptive pathways (e.g., the gigantocellular nucleus, raphe magnus nucleus, periaqueductal gray matter), and also observed in association with auditory relay nuclei such as the inferior colliculi, lateral lemniscus nucleus, medioventral preolivary nucleus and lateral superior nucleus. A moderate level of prepronociceptin messenger RNA expression was observed in the medial preoptic nucleus, ventromedial preoptic nucleus, periventricular nucleus, pedonculopontine tegmental nucleus, solitary tract nucleus and spinal trigeminal nucleus. A weak level of prepronociceptin messenger RNA expression was present in some areas, such as the cerebral cortex, endopiriform cortex, hippocampal formation, medial amygdaloid nucleus, anterior hypothalamic area, medial mammillary hypothalamic nuclei, retrorubral field and substantia nigra pars compacta. No labeled cells could be found in the caudate-putamen, nucleus accumbens and ventral tegmental area. The present data confirm that nociceptin is expressed in a broad array of regions of the central nervous system. In good correlation with the presently known physiological actions of nociceptin, they include, amongst others, brain areas conveying/integrating pain and auditory sensory afferences.

CD34+ cells in human intestine are fibroblasts adjacent to, but distinct from, interstitial cells of Cajal.

Interstitial cells of Cajal (ICC) generate the pacemaker component of the gut and play important roles in the control of gut motility. The tyrosine kinase receptor Kit is an established marker for ICC. Recently, it has been reported that immunoreactivity for the sialomucin CD34 may be present on ICC in human intestine. Gastrointestinal stromal tumors express both Kit and CD34, suggesting that these tumors may derive from ICC. We characterized the distribution of CD34 immunoreactivity at the cellular level in the normal human gut, using double immunofluorescence immunohistochemistry and confocal microscopy. CD34 immunoreactivity identified previously unrecognized cells closely adjacent to, but distinct from, the Kit immunoreactive ICC. These CD34 immunoreactive cells expressed the fibroblast marker prolyl 4-hydroxylase-whereas ICC did not-and were also distinct from smooth muscle cells, glial cells, and macrophages. In the human gut, CD34 immunoreactivity is not expressed by ICC but by a population of fibroblasts, likely corresponding to the "fibroblast-like cells" described in previous ultrastructural studies. Our findings also challenge the hypothesis that stromal tumors originate from ICC.

Calcium binding protein calcyphosine in dog central astrocytes and ependymal cells and in peripheral neurons.

Calcyphosine is a calcium binding protein discovered in the dog thyroid in 1979. Calcyphosine mRNA and immunoreactivity were detected using Western and Northern blotting in the cerebral cortex, cerebral white matter and cerebellum. Using immunohistochemistry and in situ hybridization, both are present in ependymal cells, choroid plexus cells and several types of astrocytes of the subependymal cerebral layer, the cerebellar Bergmann layer, the retinal ganglion cell layer, the optic nerve and the posterior pituitary. Both are also present in neurons of nasal olfactory mucosa, enteric Auerbach and Meissner plexuses, orthosympathic and spinal cord ganglia as well as in endocrine cells of neural crest origin in the adrenal medulla. Calcyphosine immunoreactive astrocytes were also present mainly in hemispheric cerebral gray and white matter, hemispheric subcortical structures, brain stem and spinal cord. These results show that calcyphosine is a characteristic calcium binding protein of astrocytes and ependymal cells in the central nervous system and of neurons in the peripheral nervous system. This is of interest in view of the importance of calcium regulation in these cells, and since calcyphosine a calcium binding protein phosphorylated by cAMP dependent process, may be an intermediate between cAMP and inositol phosphate cascades.

Modulation of the voltage-gated sodium current in rat striatal neurons by DARPP-32, an inhibitor of protein phosphatase.

DARPP-32 is a cyclic adenosine monophosphate-regulated inhibitor of protein phosphatase 1, highly enriched in striatonigral neurons. Stimulation of dopamine D1 receptors increases phosphorylation of DARPP-32, whereas glutamate acting on N-methyl-D-aspartate receptors induces its dephosphorylation. Yet, to date, there is little direct evidence for the function of DARPP-32 in striatal neurons. Using a whole cell patch-clamp technique, we have studied the role of DARPP-32 in the regulation of voltage-gated sodium channels in rat striatal neurons maintained in primary culture. Injection of phospho-DARPP-32, but not of the unphosphorylated form, reduced the sodium current amplitude. This effect was similar to those induced by okadaic acid, with which there was no additivity and by tautomycin. Our results indicate that, in striatal neurons, sodium channels are under dynamic control by phosphorylation/dephosphorylation, and that phospho-DARPP-32 reduces sodium current by stabilizing a phosphorylated state of the channel or an associated regulatory protein. We propose that the DARPP-32-mediated modulation of sodium channels, via inhibition of phosphatase 1, contributes to the regulation of these channels by D1 receptors and other neurotransmitters which influence the state of phosphorylation of DARPP-32.

Effect of sensory deafferentation on the GABAergic circuitry of the adult cat visual system.

The effect of bilateral central retinal lesions on the GAD67 and GAD65 messenger RNA levels in the dorsal lateral geniculate nucleus, the perigeniculate nucleus and the visual cortex of the adult cat was investigated by in situ hybridization. Three days post-lesion, a decrease in the number of GAD67-expressing cells was apparent in the deafferented dorsal lateral geniculate nucleus. This decrease persisted until 7.5 months post-lesion and was more pronounced with longer survival times. The decrease in GAD67 mRNA was mirrored by a decrease in glutamate decarboxylase-immunoreactive cells. GAD65 messenger RNA expression levels were low in the dorsal lateral geniculate nucleus of both control and retinally-lesioned cats. In the perigeniculate nucleus the messenger RNA levels of both glutamate decarboxylase isoforms were clearly decreased over a restricted region. In the lesion-affected visual cortex, no changes at the messenger RNA level were observed for either GAD67 or GAD65 although changes in glutamate decarboxylase immunoreactivity have been previously described. Hence, in the dorsal lateral geniculate nucleus, the perigeniculate nuclcus and the visual cortex, different intracellular mechanisms seem to lead to decreased GABAergic inhibition in response to sensory deafferentation.

Aggressiveness, hypoalgesia and high blood pressure in mice lacking the adenosine A2a receptor.

Adenosine is released from metabolically active cells by facilitated diffusion, and is generated extracellularly by degradation of released ATP. It is a potent biological mediator that modulates the activity of numerous cell types, including various neuronal populations, platelets, neutrophils and mast cells, and smooth muscle cells in bronchi and vasculature. Most of these effects help to protect cells and tissues during stress conditions such as ischaemia. Adenosine mediates its effects through four receptor subtypes: the A1, A2a, A2b and A3 receptors. The A2a receptor (A2aR) is abundant in basal ganglia, vasculature and platelets, and stimulates adenylyl cyclase. It is a major target of caffeine, the most widely used psychoactive drug. Here we investigate the role of the A2a receptor by disrupting the gene in mice. We found that A2aR-knockout (A2aR-/-) mice were viable and bred normally. Their exploratory activity was reduced, whereas caffeine, which normally stimulates exploratory behaviour, became a depressant of exploratory activity. Knockout animals scored higher in anxiety tests, and male mice were much more aggressive towards intruders. The response of A2aR-/- mice to acute pain stimuli was slower. Blood pressure and heart rate were increased, as well as platelet aggregation. The specific A2a agonist CGS 21680 lost its biological activity in all systems tested.

The pathology of infantile hypertrophic pyloric stenosis after healing.

INTRODUCTION: Infantile hypertrophic pyloric stenosis (IHPS) is a common surgical affection of unknown etiology. The muscular hypertrophy is known to resolve within a few months after pyloromyotomy (PM). The pathology of IHPS has been studied extensively at the time of PM, but the fate of the pylorus after healing remains unknown. MATERIALS AND METHODS: We had the rare opportunity to study two pyloric biopsy specimens obtained 4 months and 2 years (respectively) after an uncomplicated PM for IHPS. They were compared with the initial specimen in one case, with 26 other specimens of IHPS, and with five normal controls. Immunohistochemistry using the avidin-biotin complex (ABC) system was performed for S-100 and nerve growth factor receptor, as markers for the enteric nervous system, and for the tyrosine kinase receptor c-kit, as a marker for the interstitial cells of Cajal (pacemaker cells). NADPH-diaphorase histochemistry was performed as a marker for the neuronal enzyme nitric oxide synthase, which produces the inhibitory neurotransmitter nitric oxide. RESULTS: In both cases of IHPS, after healing, the circular musculature was not hypertrophic. For all markers studied, the distribution appeared similar to that in the normal pylorus. In contrast, all specimens obtained at the time of PM displayed a severe reduction of the different markers in the hypertrophic musculature. DISCUSSION: The pathological features observed in the circular layer in IHPS appear to resolve within a few months after PM. This suggests that the involvement of the enteric nervous system in IHPS might be milder than generally assumed. The etiology remains obscure, but our occasional observations may provide new insight into the pathophysiology of IHPS, and are in agreement with the excellent longterm clinical outcome for IHPS.

Interstitial cells of Cajal in human colon and in Hirschsprung's disease.

BACKGROUND & AIMS: Subpopulations of interstitial cells of Cajal are regarded as the source of spontaneous slow waves of the gut musculature (pacemaker cells). Their ontogeny remains unclear, but a role of the tyrosine kinase receptor c-kit in their development has recently been recognized. This study examined the interstitial cells in the human colon and in Hirschsprung's disease (aganglionosis). METHODS: The distribution of the c-kit receptor was studied using specific antibodies in 5 normal patients, 10 patients with Hirschsprung's disease, and 3 patients with diversion loop enterostomies. c-kit immunohistochemistry was also combined with reduced nicotinamide adenine dinucleotide phosphate diaphorase histochemistry or with c-kit ligand (stem cell factor) immunohistochemistry. Transmission electron microscopy was performed in 1 patient with Hirschsprung's disease. RESULTS: c-kit immunoreactivity labeled a network of interstitial cells at the outer edge of the submucosa, in the muscular layers, and around the myenteric plexus. In aganglionic segments, interstitial cells were scarce and its network appeared disrupted. Interstitial cells of Cajal were identified in aganglionic regions by electron microscopy. Interstitial cells of Cajal are identifiable in newborns and exhibit similar distribution in diversion loops independent of contact with luminal nutrients. CONCLUSIONS: Our morphological data may explain the abnormal spontaneous electrical activity in aganglionic segments of Hirschsprung's disease and may give new insight into the ontogeny of interstitial cells.

Study of the interstitial cells of Cajal in infantile hypertrophic pyloric stenosis.

BACKGROUND & AIMS: The interstitial cells of Cajal form a network in close association with the smooth muscle of the gut. They are regarded as pacemaker cells and might be involved in motility disorders. Their distribution was studied in a common disorder with a dysfunction of the pyloric sphincter called infantile hypertrophic pyloric stenosis. METHODS: Specimens from 27 infants with pyloric stenosis and 12 controls were processed for immunohistochemistry using a specific antiserum raised against c-kit, a tyrosine kinase receptor expressed by interstitial cells. RESULTS: In the normal pylorus, numerous interstitial cells were labeled throughout the tissue. In pyloric stenosis, c-kit immunoreactivity was absent in the major part of the tissue. Interstitial cells were observed only in the inner part of the musculature, near the submucosal edge, and in the antrum, at the proximal end of the biopsy specimens. CONCLUSIONS: The lack of interstitial cells in the pylorus possibly contributes to the motility disturbance of infantile pyloric stenosis.

NMDA receptor antagonist MK-801 down-regulates rat striatal proenkephalin and protachykinin mRNAs.

Using quantitative in situ hybridization, a significant decrease in expression of proenkephalin (27.1%) and of protachykinin mRNAs (20.0%) is observed in the rat caudate-putamen 14 days after daily intraperitoneal administration of N-methyl-D-aspartate receptor antagonist MK-801, 2 mg/kg.

Homolateral cerebrocortical changes in neuropeptide and receptor expression after minimal cortical infarction.

A cortical infarct of 2 mm diameter was obtained in the parietal cortex after a craniotomy, disruption of the dura mater and topical application of 3 M KCl. It has been shown previously that the presence of a small cortical infarct induces an increase in immediate early gene messenger RNA expression followed by an increase in neuropeptide and glutamic acid decarboxylase messenger RNA expression. Glutamate, acting at N-methyl-D-aspartate receptors, is held responsible for these changes, since they are blocked by pretreatment with dizocilpine. In the present study, we have analysed the consequences of the dramatic changes in messenger RNA expression on the level of immediate early gene products c-fos and zif 268, and on that of neuropeptides by using immunohistochemistry. After just 1 h, an increase in c-fos- and zif 268-like immunoreactivity is observed in the entire cortical hemisphere homolateral to the infarct, and is no longer detected after 6 h. An increase in cholecystokinin octapeptide-, substance P-, neuropeptide Y- and somatostatin-like immunoreactivity is observed in the entire cortical hemisphere homolateral to the infarct after three days, and is no longer detected after 30 days. To investigate if these dramatic increases in neuropeptide immunoreactivities may have functional consequences, we studied the level of cholecystokinin receptors by autoradiographic binding using [125I]cholecystokinin-8S and in situ hybridization for the detection of cholecystokinin-b receptor messenger RNA. A decrease in cholecystokinin binding sites and cholecystokinin-b receptor messenger RNA is observed in the entire cortical hemisphere homolateral to the infarct after three days, and is no longer detected after nine days. This study shows that a topical stimulation has diffuse effects, reaching regions far from the site of the lesion, and some of them are still strongly present after nine days. The increase in neuropeptide messenger RNAs is followed by an increase in the protein products of these genes, which may modify the neurotransmission. As a corollary to this, a decrease in cholecystokinin binding sites occurs. This may have further consequences on signal transduction pathways. This decrease in cholecystokinin binding sites is associated with a decrease in the cholecystokinin-b receptor messenger RNA, and this is the first example of a decrease in messenger RNA levels in this experimental model.

Cavernous oxygen tension and smooth muscle fibers: relation and function.

PURPOSE: We studied the effect of intracavernous oxygen tension on the alteration of cavernous smooth muscle fibers in potent and impotent men. MATERIALS AND METHODS: Intracavernous oxygen tension (mm. Hg) was measured during flaccidity and 10 minutes after intracavernous prostaglandin E1 injection in psychogenic control patients, and those with venous leakage and arterial lesions. Cavernous biopsies were performed and the percent of smooth muscle fibers was analyzed objectively using immunohistochemical actin anti-actin staining. Simultaneously brachial oxygen tension (mm. Hg) was measured and the cavernous brachial oxygen tension index was then determined. RESULTS: At flaccidity no significant difference was noted in oxygen tension values among the 3 groups of patients. After prostaglandin E1 injection cavernous oxygen tension and the cavernous brachial oxygen tension index in the control group were significantly different (p < 0.01) from those of the venogenic and arteriogenic groups (p < 0.01). The mean percent of cavernous smooth muscle fibers in the control group was significantly different from those of the venous leakage and arterial lesion groups (p < 0.01). There was a good correlation between the percent of cavernous muscle fibers and the value of oxygen tension before (p < 0.05) and after prostaglandin E1 injection (p < 0.01). A similar correlation was noted between cavernous muscle fibers and cavernous brachial oxygen tension index in the different groups of patients (p < 0.01). CONCLUSIONS: Reduction of the intracavernous smooth muscle fibers in impotent patients could be explained by low intracavernous oxygen tension.

Prostate capsule: computerized morphometric analysis of its components.

OBJECTIVES: There is little objective information on what is designated as being the prostate capsule. We have quantified objectively with computerized image analysis the percentage of smooth muscle fibers (SMFs) surrounding the surgical specimens of radical prostatectomies. METHODS: Ten radical prostatectomy specimens were used. Six different parts from prostate lobe were analyzed. Thirty different fields were examined from each tissue section. For tissue sections, different stains were used: hematoxylin and eosin, Masson's trichrome, and immunohistochemical staining with antiactin for SMFs. RESULTS: The mean thickness of the prostate capsule was 0.5 to 2 mm. The mean percentages of SMFs in the different regions of the prostate capsule was 30.6%. No difference was noted between the percentage of SMFs in the prostatic capsule compared with that in the prostate parenchyma itself (30.7%). CONCLUSIONS: This study provides objective evidence to demonstrate that the prostate does not have a true capsule. This capsule represents a fibromuscular band that probably should be considered as the extension of the internal parenchyma.

Decrease of zif-268 and c-fos and increase of c-jun mRNA in the cat areas 17, 18 and 19 following complete visual deafferentation.

We used in situ hybridization to investigate the effect of complete visual deafferentation on immediate early gene expression in adult cat visual cortex. Deafferentation was obtained by unilateral section of the optic tract and sections of both the corpus callosum and anterior commissure. In this model, one hemisphere served as control for the other within the same animal. A decrease in zinc finger protein (zif)-268 and c-fos mRNA was observed in the superficial and deep layers of areas 17 and 18, and all layers of area 19 in the deafferented hemisphere. This decrease, present 3 days after surgery, was maximal after 30 days. An increase of c-jun mRNA was observed in the deep layers of areas 17, 18 and 19 in the deafferented hemisphere 3, 10 and 30 days after surgery. These results suggest that visual input activates zif-268 and c-fos expression and tonically depresses c-jun expression in the primary visual complex yielding similar levels of c-jun and c-fos expression in normal conditions.