Muscarinic cholinergic receptor binding in rat mast cells

Specific [³H]-QNB binding was present in isolated, purified, intact rat mast cells and in crude membrane preparations. The binding is saturable, time- and temperature-dependent. Cholinergic agents inhibit selectively the binding: atropine is the most effective of the antagonists while oxotremorine is the most potent of the muscarinic agonists. It is concluded that rat mast cells are provided with muscarinic cholinergic receptors.     

Distribution of neuronal cannabinoid receptor in the adult rat brain: a comparative receptor binding radioautography and in situ hybridization histochemistry.

The neuronal distribution of cannabinoid receptor in the adult rat brain is reported, combining receptor binding radioautography using the synthetic psychoactive cannabinoid ligand CP55,940 with in situ hybridization histochemistry using oligonucleotide probes complementary to rat cannabinoid receptor cDNA. In the cerebral cortex, especially in the frontal and cingulate cortex, dense binding was found in layers I and VI together with slight mRNA levels in a majority of both pyramidal and non-pyramidal-shaped neurons and of high mRNA levels in a moderate number of non-pyramidal-shaped neurons especially in layers II-III and V-VI. In the hippocampal dentate gyrus, very dense staining was found in the molecular layer together with high mRNA levels in a moderate number of hilar neurons close to the granular layer. In Ammon's horn, especially in the CA3 sector, very dense binding was found in the dendritic layers together with slight mRNA levels in the majority of the pyramidal cells and high mRNA levels in a moderate number of interneurons. In the basal ganglia, binding was very dense in the lateral putamen, substantia nigra pars reticulata, globus pallidus and entopeduncular nucleus, moderate in the medial putamen and caudate; and slight in the accumbens, together with slight to moderate mRNA levels in the striatal medium-sized neurons. Together with slight binding, slight to moderate mRNA levels were found in the majority of the neurons in the subthalamic nucleus. No binding and mRNA were found in the substantia nigra pars compacta and ventral tegmental area. Slight to moderate binding was found together with slight to moderate mRNA levels in the majority of neurons in the anterior olfactory nucleus; septum, especially medial septum and diagonal band of Broca; amygdala, especially basolateral amygdala; lateral habenula; ventromedial hypothalamic nucleus; lateral interpeduncular nucleus; central gray, dorsal cochlear nucleus; parabrachial nucleus; dorsal pontine tegmentum; pontine nuclei; commissural part of the nucleus tractus solitarius; inferior olive and dorsal horn of the spinal cord. In the cerebellum, very dense binding was found in the molecular layer together with slight mRNA levels in the majority of the granule cells and moderate mRNA levels in the basket and stellate cells. In conclusion, this study provides, for the first time, indirect assessment of the neurons containing cannabinoid receptor in the entire adult rat brain and will serve as a basis for future direct morphological confirmation using receptor immunohistochemistry and for functional studies.     

A comparison of D1 receptor binding and mRNA in rat brain using receptor autoradiographic and in situ hybridization techniques.

D1, a subtype of the dopamine receptors, is widely distributed in the nervous system and has been shown to be positively coupled to adenylate cyclase. Using a combination of in vitro receptor autoradiographic and in situ hybridization techniques, the present study examines the co-distribution of D1 receptor binding sites and D1 receptor messenger RNA in adjacent rat brain sections. D1 receptor binding sites were labeled using the selective antagonist [3H]SCH23390 (4.6 nM) in the presence of 1 microM ketanserin, while the D1 receptor messenger RNA was visualized with a 35S-labeled riboprobe corresponding to a region between transmembrane domains III and VI of the rat D1 receptor (bp 383-843). Analysis of serial sections suggested a good agreement between D1 receptor binding and messenger RNA in several brain regions, including the paleocortex, caudate-putamen, nucleus accumbens, amygdala and suprachiasmatic nucleus. Marked discrepancies between D1 receptor binding and messenger RNA were observed in other brain regions including the entopeduncular and subthalamic nuclei, substantia nigra (pars reticulata), hippocampus and cerebellum. While technical considerations may contribute to these results, much of the discordance between the distributions is likely due to the differential localization D1 receptor messenger RNA in cell bodies and receptor binding sites on fibers and may provide insights into receptor synthesis, transport and membrane insertion. In the basal ganglia, for instance, D1 receptors are synthesized in the striatum and are either transported to efferent projections in areas such as the substantia nigra, or remain localized in striatal cells bodies. Ibotenic acid lesions in the striatum are consistent with these conclusions and demonstrate a coordinate loss of D1 receptor binding and messenger RNA in the caudate-putamen that is accompanied by a degeneration of fibers projecting to substantia nigra and a loss of D1 binding in the pars reticulata. Neurons in the dentate gyrus and in the granular layer of the cerebellum, on the other hand, synthesize D1 receptors and transport them entirely to either their dendritic or axonal fields, respectively, in the molecular layer. This analysis provides a better understanding of dopaminergic receptor systems in the CNS and their anatomical organization.     

A comparison of D1 receptor binding and mRNA in rat brain using receptor autoradiographic and in situ hybridization techniques.

D1, a subtype of the dopamine receptors, is widely distributed in the nervous system and has been shown to be positively coupled to adenylate cyclase. Using a combination of in vitro receptor autoradiographic and in situ hybridization techniques, the present study examines the co-distribution of D1 receptor binding sites and D1 receptor mRNA in adjacent rat brain sections. D1 receptor binding sites were labeled using the selective antagonist [3H](R)-(+)-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzaz epin- 7-ol (SCH23390) (4.6 nM), in the presence of 1 microM ketanserin, while the D1 receptor mRNA was visualized with a 35S-labeled riboprobe corresponding to a region between transmembrane domains III and VI of the rat D1 receptor (base pairs 383-843). Analysis of serial sections suggested a good agreement between D1 receptor binding and mRNA in several brain regions, including the paleocortex, caudate-putamen, nucleus accumbens, amygdala, and suprachiasmatic nucleus. Marked discrepancies between D1 receptor binding and mRNA were observed in other brain regions including the entopeduncular and subthalamic nuclei, substantia nigra (pars reticulata), hippocampus, and cerebellum. While technical considerations may contribute to these results, much of the discordance between the distributions is probably due to the differential localization of D1 receptor mRNA in cell bodies and receptor binding sites on fibers and may provide insights into receptor synthesis, transport, and membrane insertion. In the basal ganglia, for instance, D1 receptors are synthesized in the striatum and are either transported to efferent projections in areas such as the substantia nigra, or remain localized in striatal cells bodies. Ibotenic acid lesions in the striatum are consistent with these conclusions and demonstrate a coordinate loss of D1 receptor binding and mRNA in the caudate-putamen that is accompanied by a degeneration of fibers projecting to substantia nigra and a loss of D1 binding in the pars reticulata. Neurons in the dentate gyrus and in the granular layer of the cerebellum, on the other hand, synthesize D1 receptors and transport them entirely to either their dendritic or axonal fields, respectively, in the molecular layer. This analysis provides a better understanding of dopaminergic receptor systems in the CNS and their anatomical organization.     

Localization of parvalbumin mRNA in rat brain by in situ hybridization histochemistry

Summary Parvalbumin mRNA was localized in rat brain by in situ hybridization using a ³⁵S labelled rat parvalbumin cDNA and a synthetic oligodeoxyribonucleotide (corresponding to base sequences 140 to 183 of rat parvalbumin cDNA). Strongest hybridization signals were detected in the Purkinje cells of the cerebellum and in neurones of the reticular nucleus of the thalamus. Signal was also detected in the cerebral cortex, hippocampus, basal ganglia and brain stem in agreement with the distribution of parvalbumin immunoreactivity.     

Localization of m5 muscarinic receptor mRNA in rat brain examined by in situ hybridization histochemistry

The regional distribution of mRNA coding for the m5 muscarinic acetylcholine receptor subtype was investigated in tissue sections of rat brain by in situ hybridization histochemistry. The highest hybridization signal was observed in the hippocampus, but restricted to the ventral subiculum, pyramidal cells of the CA1 and, with lower intensity, of the CA2 subfields. Significant levels of hybridization were also seen in the substantia nigra pars compacta, ventral tegmental area, lateral habenula, ventromedial hypothalamic nucleus and mammillary bodies. An involvement of the m5 muscarinic receptors in the regulation of the dopaminergic nigrostriatal pathway is suggested.     

Comparison of neuronal inositol 1,4,5-trisphosphate 3-kinase and receptor mRNA distributions in the adult rat brain using in situ hybridization histochemistry.

As a result of its interaction with a specific receptor, inositol 1,4,5-trisphosphate mobilizes intracellular calcium. The metabolism of inositol 1,4,5-trisphosphate is rather complex: inositol 1,4,5-trisphosphate 3-kinase produces inositol 1,3,4,5-tetrakisphosphate, a putative second messenger. In order to elucidate inositol 1,3,4,5-tetrakisphosphate function, a comparative in situ hybridization study of the distributions of inositol 1,4,5-trisphosphate 3-kinase and receptor mRNAs was performed in the adult rat brain using oligonucleotides derived from their cDNA sequences. The neuronal distributions of the mRNA for the receptor were larger than for the kinase. Highest levels of both mRNAs were found in the cerebellar Purkinje cells, where they were enriched in their neuronal perikarya and to a lesser extent in their dendrites. In addition to the cerebellum, mRNAs were mainly detected in the hippocampal pyramidal cells of the CA1 sector of the Ammon's horn and in the granule cells of the dentate gyrus, and also in a majority of the neurons in the cortical layers II-III and V, especially in the frontal cortex and cingulate cortex; caudate-putamen, accumbens, olfactory tubercle and Calleja islets; claustrum; anterior olfactory nucleus; taenia tecta; piriform cortex; dorsolateral septum; bed nucleus stria terminalis; amygdala; hippocampal CA2-4 sectors and subiculum. The inositol 1,4,5-trisphosphate receptor mRNA but not kinase mRNA was found in a majority of the neurons in the thalamus, especially in the parafascicular nucleus; hypothalamus, especially the medial hypothalamus; substantia nigra pars compacta and ventral tegmental area; superior colliculus; lateral interpeduncular nucleus and central gray. Taking into account the limitation in sensitivity of the technique, both mRNAs were not detected in glial cells and in the olfactory bulb; basal nucleus of Meynert, diagonal band nuclei; medial septal nucleus; substantia innominata; globus pallidus; entopeduncular nucleus; substantia nigra pars reticulata; ventral pallidum; subthalamic nucleus; spinal cord and dorsal root ganglia. In conclusion, cerebellum and hippocampus appear to contain almost similar levels of kinase mRNA. This is in contrast to receptor mRNA levels which were at much higher levels in the cerebellum when compared with the hippocampus. For this reason, we have chosen hippocampal CA1 pyramidal cells and dentate gyrus granule cells for studying inositol 1,4,5-trisphosphate 3-kinase function.     

Phosphodiesterase type 4 isozymes expression in human brain examined by in situ hybridization histochemistry and[3H]rolipram binding autoradiography. Comparison with monkey and rat brain

We have examined the distribution of four different cyclic AMP-specific phosphodiesterase isozyme (PDE4A, PDE4B, PDE4C and PDE4D) mRNAs in the brain of different species by in situ hybridization histochemistry and by autoradiography with [³H]rolipram. We have compared the localization of each isozyme in human brain with that in rat and monkey brain. We have found that the four PDE4 isoforms display a differential expression pattern at both regional and cellular level in the three species. PDE4A, PDE4B and PDE4D are widely distributed in human brain, with the two latter appearing more abundant. In contrast, PDE4C in human brain, presents a more restricted distribution, limited to cortex, some thalamic nuclei and cerebellum. This is at variance with the distribution of PDE4C in rat brain, where it is found exclusively in olfactory bulb. In monkey brain, the highest expression for this isoform is found in the claustrum, and at lower levels in cortical areas and cerebellum. PDE4B presented a broad distribution, being expressed in both neuronal and non neuronal cell populations. In general, the distribution of binding sites visualized with [³H]rolipram correlated well with the expression of each PDE4 isozyme.     

Heterogeneous distribution of dopamine D2 receptor mRNA in the rat striatum: a quantitative analysis with in situ hybridization histochemistry.

Dopamine D2 receptor mRNAs have recently been cloned and their gross distribution in the central nervous system described. Quantitative in situ hybridization histochemistry with a cRNA probe complementary to the mRNAs encoding approximately 70% of the third intracellular loop of the rat D2 receptor was performed on sections of rat brain to determine whether differences previously observed in the density of ligand binding sites in subregions of the striatum were related to differences in mRNA levels. Film autoradiographic analysis demonstrated 30% more hybridization signal in the lateral compared to the medial caudate-putamen, a distribution parallel to that of binding of ligands specific for the D2 receptor. Inspection at the cellular level using emulsion autoradiography also indicated a differential distribution of the D2 receptor mRNA. Fewer positively labelled cells, as well as fewer silver grains per cell, were seen in the medial compared to the lateral half of the striatum. This suggests that the gradient seen in autoradiographic studies of the distribution of D2 receptors is related both to regional differences in D2 mRNA levels and to the density of cells expressing the receptor. In addition, the distribution of cells expressing D2 receptor mRNA in the extrastriosomal matrix was compared to that in striosomes identified by the presence of a high density of 3H-naloxone binding sites. Labelled cells were mainly found in the matrix (3H-naloxone binding-poor) but were also seen in striosomes (3H-naloxone binding-rich). The results suggest that differences in levels of D2 binding sites in subregions of the striatum are related to differences in the level of expression of this receptor in intrinsic striatal neurons, suggesting differential regulation of dopamine D2 receptor gene expression in topographically distinct striatal neurons.     

Localization of dopamine D2 receptor mRNA with non-radioactive in situ hybridization histochemistry.

A digoxigenin-labeled antisense 42-mer oligonucleotide was used for the localization of the dopamine D2 receptor mRNA in the rat brain. The digoxigenin label was identified with alkaline phosphatase conjugated sheep-anti-digoxigenin. In good analogy with the known terminal fields of the dopaminergic system, various nuclei throughout the brain were labeled. Positive in situ hybridization signals were also found in dopamine cell groups of the substantia nigra and ventral tegmental area and in regions where a dopaminergic innervation is controversial, like the cerebellar cortex and the hippocampus. The non-radioactive in situ hybridization procedure described, shows the localization of the dopamine D2 receptor mRNA with a very high contrast and an optimal histological resolution.     

Comparison of neuronal inositol 1,4,5-trisphosphate 3-kinase and receptor mRNA distributions in the human brain using in situ hybridization histochemistry.

The distribution of the messenger RNA coding for the recently cloned inositol 1,4,5-trisphosphate (InsP3) 3-kinase, the enzyme phosphorylating InsP3 to InsP4, was compared to the localizations of InsP3 receptor mRNA in the human brain using in situ hybridization histochemistry and oligonucleotide probes. InsP3 3-kinase and receptor mRNA levels were high in the cerebellar Purkinje cells. They were also observed, to a much lesser degree than in the cerebellum, in the hippocampal CA1 pyramidal cells and dentate gyrus granule cells, in the majority of the cortical neurons and in the striatal medium-sized neurons. Both mRNAs were not detected in the brainstem and in the glial cells.     

Localization of glycine receptor alpha 1 subunit mRNA-containing neurons in the rat brain: an analysis using in situ hybridization histochemistry.

The localization of glycine receptors in the rat brain was examined by means of in situ hybridization histochemistry using an oligonucleotide probe to the sequence of the alpha 1 subunit. Strongly- or moderately-labeled neurons were found in the cranial nuclei, sensory nuclei such as the spinal trigeminal nucleus, principal trigeminal nucleus, gracile and cuneate nuclei, dorsal and ventral cochlear nuclei, superior olivary nucleus, medial and lateral trapezoid nuclei, lateral lemniscus and vestibular nuclei, red nucleus, parabrachial area, cerebellar nuclei, dorsal tegmental nucleus, reticular formation and parafascicular nucleus. This study thus demonstrated the localization of neurons which are regulated by glycine via strychnine-sensitive glycine receptors in the rat brain.     

Muscarinic acetylcholine receptor subtype mRNA expression and ligand binding in the aged rat forebrain

Previous studies indicate that a 20-30% decline in muscarinic acetylcholine receptor binding occurs in localized areas of rat brain during aging. In this study, reduced [3H]-quinuclidinyl benzilate binding was observed in striata from 24-25-month-old rats relative to 5-6-month-old animals using homogenate binding assays. To determine if the decline in receptor concentration occurs as a result of decreased receptor synthesis, the expression of the m1, m3, and m4 muscarinic receptor mRNAs as well as [3H]-QNB binding were determined in adjacent sections of young and old male rats using in situ hybridization and in vitro receptor autoradiography respectively. A significant decline in collective muscarinic receptor binding as assessed by [3H]-QNB was observed in the caudate putamen, olfactory tubercle, nucleus accumbens, and several frontal and parietal cortical areas. The only difference observed in muscarinic mRNA expression for any of the three subtypes examined was a decline in m1 hybridization in the olfactory tubercle. The results of this study demonstrate that the regional brain areas displaying age-related decreases in receptor binding do not correlate with those areas showing a decrease in muscarinic receptor expression. Apparently, the decline in muscarinic acetylcholine receptor density with age does not result from a decline in receptor gene expression.     

The distribution and cellular localization of the serotonin 1C receptor mRNA in the rodent brain examined by in situ hybridization histochemistry. Comparison with receptor binding distribution

The regional distribution and cellular localization of mRNA coding for the serotonin 1C receptor were investigated in tissue sections of mouse and rat brain by in situ hybridization histochemistry. Several 32P-labelled riboprobes derived from mouse genomic clones were used. The serotonin 1C receptor binding sites were visualized autoradiographically and quantified using [3H]mesulergine as ligand, in the presence of spiperone to block serotonin 1C receptors. Strong hybridization signal was observed in the choroid plexus of all brain ventricles. High levels of hybridization were also seen in the anterior olfactory nucleus, pyriform cortex, amygdala, some thalamic nuclei, especially the lateral habenula, the CA3 area of the hippocampal formation, the cingulate cortex, some components of the basal ganglia and associated areas, particularly the nucleus subthalamicus and the substantia nigra. The midbrain and brainstem showed moderate levels of hybridization. The distribution of the serotonin 1C receptor mRNA corresponded well to that of the serotonin 1C receptors. The highest levels of serotonin 1C receptor binding were observed in the choroid plexus. In addition, significant levels of the serotonin 1C receptor binding were seen in the anterior olfactory nucleus, pyriform cortex, nucleus accumbens, ventral aspects of the striatum, paratenial and paracentral thalamic nuclei, amygdaloid body and substantia nigra pars reticulata. The cingulate and retrosplenial cortices as well as the caudal aspects of the hippocampus (CA3) were also labelled. Binding in brainstem and medulla was low and homogeneously distributed. No significant binding was seen in the habenular and subthalamic nuclei. Similar findings were obtained in rat brain. These results demonstrate that, in addition to their enrichment in the choroid plexus, the serotonin 1C receptor mRNA and binding sites are heterogeneously distributed in the rodent brain and thus could be involved in the regulation of many different brain functions. The combination of in situ hybridization histochemistry with receptor autoradiography opens the possibility of examining the regulation of the serotonin 1C receptor synthesis after pharmacological or physiological alterations.     

Demonstration of rat preprotachykinin A mRNA in the rat trigeminal ganglion by in situ hybridization histochemistry

The localization of gamma-preprotachykinin A mRNAs in the rat trigeminal ganglion was demonstrated by in situ hybridization histochemistry using the 32P and 35S labelled gamma-preprotachykinin A complementary DNA. In situ hybridization using 32P allowed shorter exposure times, whereas higher resolution of the hybridization signal on both film and emulsion autoradiograms was obtained using 35S. Preprotachykinin A mRNA detected by the gamma-preprotachykinin A probe was localized in about 15 per cent of the trigeminal ganglion cells, most of which were small or medium sized. Immunohistochemical studies using anti-substance P antibodies demonstrated that 15-20 per cent of total trigeminal ganglion cells were positive. These cells were small or medium sized. The result of immunohistochemistry coincided well with that of in situ hybridization histochemistry. The present study showed that the cellular localization of preprotachykinin A mRNA could be analysed by in situ hybridization histochemistry.     

Localization of preprogalanin mRNA in rat brain: in situ hybridization study with a synthetic oligonucleotide probe

Galanin is a peptide containing 29 amino acid residues that is present in both the central and peripheral nervous systems. Galanin has multiple putative biological functions including regulation of hormone release, stimulation of feeding behaviour, and effects on blood pressure. This study examined the distribution of neurones expressing preprogalanin mRNA in the rat brain by in situ hybridization of a specific 35S-labelled oligonucleotide. Preprogalanin mRNA was detected in several regions of brain, with high concentrations in the paraventricular, periventricular, supraoptic, dorsomedial and arcuate nuclei of the hypothalamus; the locus coeruleus and dorsal raphe nucleus in the pons; and the nucleus tractus solitarii and ventrolateral reticular nucleus in the medulla. These findings are consistent with studies of the cellular localization of galanin-like immunoreactivity in rat brain, and further suggest the involvement of galanin in the regulation of several functions ranging from water balance to blood pressure control.     

The distribution of histidine decarboxylase mRNA in the rat brain: an in situ hybridization study using synthetic oligonucleotide probes

L-Histidine decarboxylase catalyzes the formation of histamine from the amino acid L-histidine. We have studied the distribution of neurons expressing mRNA for histidine decarboxylase in adult rat brain using in situ hybridization with synthetic oligonucleotide probes. The expression of mRNA for histidine decarboxylase was detected in the hypothalamic tuberomammillary nucleus that has been shown to contain histidine decarboxylase-like and histamine-like immunoreactivity, but not in any other brain area. This method may prove useful in studying the physiological role of central histaminergic neurons.     

Localization of M(2) muscarinic acetylcholine receptor protein in cholinergic and non-cholinergic terminals in rat hippocampus

The muscarinic receptor family (M(1)-M(4)) mediates cholinergic modulation of hippocampal transmission. Pharmacological and physiological studies have indicated that a presynaptic receptor on cholinergic terminals plays a key role in regulating ACh release, although the molecular identity of this subtype is uncertain. In this study, the localization of the M(2) receptor is described in detail for the pyramidal cell layer in the CAl region of the hippocampus. Electron microscopic analysis of M(2) immunoreactivity in this area revealed mainly presynaptic expression of this subtype. Double-labeling experiments using antibodies to M(2) and to the vesicular acetylcholine transporter, a novel, specific marker of cholinergic terminals, were used to investigate the nature of these presynaptic receptors. These studies have revealed that M(2) is located in cholinergic and non-cholinergic terminals. This is the first direct anatomical evidence that suggests that M(2) may indeed function as a cholinergic autoreceptor in the hippocampus. The distribution of the M(2) receptor in non-cholinergic terminals also suggests functional roles for M(2) as a presynaptic heteroreceptor.