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.     

Muscarinic M2 receptor mRNA expression and receptor binding in cholinergic and non-cholinergic cells in the rat brain: a correlative study using in situ hybridization histochemistry and receptor autoradiography.

The goal of the present study was to identify the cells containing mRNA coding for the m2 subtype of muscarinic cholinergic receptors in the rat brain. In situ hybridization histochemistry was used, with oligonucleotides as hybridization probes. The distribution of cholinergic cells was examined in consecutive sections with probes complementary to choline acetyltransferase mRNA. Furthermore, the microscopic distribution of muscarinic cholinergic binding sites was examined with a non-selective ligand ([3H]N-methylscopolamine) and with ligands proposed to be M1-selective ([3H]pirenzepine) or M2-selective ([3H]oxotremorine-M). The majority of choline acetyltransferase mRNA-rich (i.e. cholinergic) cell groups (medial septum-diagonal band complex, nucleus basalis, pedunculopontine and laterodorsal tegmental nuclei, nucleus parabigeminalis, several motor nuclei of the brainstem, motoneurons of the spinal cord), also contained m2 mRNA, strongly suggesting that at least a fraction of these receptors may be presynaptic autoreceptors. A few groups of cholinergic cells were an exception to this fact: the medial habenula and some cranial nerve nuclei (principal oculomotor, trochlear, abducens, dorsal motor nucleus of the vagus). Furthermore, m2 mRNA was not restricted to cholinergic cells but was also present in many other cells throughout the rat brain. The distribution of m2 mRNA was in good, although not complete, agreement with that of binding sites for the M2 preferential agonist [3H]oxotremorine-M, but not with [3H]pirenzepine binding sites. Regions where the presence of [3H]oxotremorine-M binding sites was not correlated with that of m2 mRNA are the caudate-putamen, nucleus accumbens, olfactory tubercle and islands of Calleja. The present results strongly suggest that the M2 receptor is expressed by a majority of cholinergic cells, where it probably plays a role as autoreceptor. However, many non-cholinergic neurons also express this receptor, which would be, presumably, postsynaptically located. Finally, comparison between the distribution of m2 mRNA and that of the proposed M2-selective ligand [3H]oxotremorine-M indicates that this ligand, in addition to M2 receptors, may also recognize in certain brain areas other muscarinic receptor populations, particularly M4.     

Autoradiographic localization of mas proto-oncogene mRNA in adult rat brain using in situ hybridization

The cellular localization and the distribution of the mas proto-oncogene/angiotensin receptor mRNA have been studied in the male rat brain using in situ hybridization with radiolabelled mas cRNA probes. Neuronal cell populations in the forebrain were selectively labelled. A strong specific labelling was demonstrated in the dentate gyrus, the CA3 and CA4 areas of the hippocampus, the olfactory tubercle (medical part), the piriform cortex and the olfactory bulb, while a weak to moderate labelling was present all over the neocortex and especially in the frontal lobe.     

Quantitative autoradiographic study on tyrosine hydroxylase mRNA with in situ hybridization and alpha 2 adrenergic receptor binding in the locus coeruleus of the spontaneously hypertensive rat.

alpha-2 Adrenergic (A2) receptors and tyrosine hydroxylase (TH) mRNA in the locus coeruleus (LC) were studied using [125I]iodoclonidine and [35S]TH oligonucleotide probe. Spontaneously hypertensive (SHR) rats contained less TH mRNA at their prehypertensive, but not at the well-established hypertensive stage, than age-matched Wistar-Kyoto rats. Furthermore, there is an up-regulation of A2 receptors in SHR rats which is parallel to their blood pressure elevation. The present data suggest that increased A2 receptors in conjunction with TH mRNA reduction in the LC are associated with initiation, but not maintenance of genetic hypertension.     

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.     

The D1 dopamine receptor in the rat brain: quantitative autoradiographic localization using an iodinated ligand

The distribution of dopamine D1 receptors in the rat, labeled with [125I]SCH 23982, was studied using a quantitative in-vitro light-microscopic autoradiographic method. The binding of [125I]SCH 23982 to slide-mounted tissue sections and membrane preparations of prefrontal cortex was saturable, specific and of high affinity. Scatchard analysis revealed a Kd of 1.15 +/- 0.47 nM and Bmax of 8.76 +/- 0.34 fmol/mg tissue in prefrontal cortex membranes and a Kd of 1.27 +/- 0.14 nM and Bmax of 67.6 +/- 3.75 fmol/mg tissue in slide-mounted tissue sections at the level of the striatum. [125I]SCH 23982 was found to predominantly label D1 receptors, but a small fraction of the binding was to serotonin receptors. D1 receptors were found throughout the forebrain and were concentrated in the substantia nigra pars reticulata, accumbens nucleus, caudate putamen, entopeduncular nucleus, olfactory tubercle and the major island of Calleja. [125I]SCH 23982 binding to serotonin receptors was concentrated in the cortices, dorsal raphe, central gray, anterior hypothalamic area and the molecular cell layer of the cerebellum. Knowledge of the distribution of D1 receptors may increase our understanding of the role of D1 receptors in central nervous system dopaminergic function. Furthermore, data on the potential sites of interaction of [125I]SCH 23982 with serotonin receptors may help to understand the complex physiology and pharmacology of the primarily D1 selective compound.     

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.     

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.     

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.     

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.     

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.     

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.     

Ⅰ型细胞间粘附分子在肝细胞癌中的表达及其意义

目的探讨肝癌细胞产生的Ⅰ型细胞间粘附分子１（ＩＣＡＭ－１）在肝细胞癌侵袭和转移过程中的作用。方法用原位分子杂交技术检测了３４例原发性肝癌及癌旁组织中ＩＣＡＭ－１ｍＲＮＡ的表达。结果２１例侵袭性生长的肝癌中１９例ＩＣＡＭ－１ｍＲＮＡ表达阳性,明显高于癌旁;１１例伴门静脉癌栓或肝内转移灶形成的肝癌,其癌组织均表达强阳性,癌栓及转移灶表达阳性;而１３例包膜完整的肝癌仅有４例表达阳性。结论肝癌细胞ＩＣＡＭ－１ｍＲＮＡ的表达与肝癌细胞的侵袭及转移相关。     

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 an endoplasmic reticulum calcium ATPase mRNA in rat brain by in situ hybridization

SERCA-2 is an endoplasmic reticulum Ca2+ ATPase present in brain [Gunteski-Hamblin A.-M. et al. (1988) J. biol. Chem. 263, 15032-15040]. We sought to map the distribution of this pump in the rat brain and investigate its relationship to Ca2+ uptake by brain endoplasmic reticulum. Using in situ hybridization and Northern blots with antisense oligonucleotide probes, we found that SERCA-2 is concentrated most densely in the cerebellum, especially in Purkinje cells, and in the hippocampus, with heavy labeling also in cortex, thalamus, pontine nuclei and the mitral cell layer of the olfactory bulb. 45Ca2+ uptake displayed a similar pattern with heaviest accumulation in cerebellum, hippocampus, cortex, thalamus and olfactory bulb. In corpus striatum and substantia nigra, relative 45Ca2+ accumulation was greater than SERCA-2 mRNA. Thus, SERCA-2 appears to be involved in Ca2+ uptake into endoplasmic reticulum in brain for release by inositol 1,4,5-trisphosphate and other agents.     

原位杂交法检测BP1基因在乳腺癌组织中表达

目的探讨BP1同源盒基因在乳腺癌中的表达及其与临床病理指标的关系。方法收集165例乳腺癌临床和病理资料,采用原位杂交法检测BP1的表达,同时用二步法进行ER、p53、PCNA、bcl2、cerbB2免疫组化染色。结果BP1基因表达率为67.88%,免疫组化:ER70.30%、p5349.09%、PCNA74.55%、bcl253.33%、cerbB275.52%。BP1与bcl2具有相关性(P<0.01),且均与ER相关(P<0.05),与p53呈负相关(P<0.05)。BP1与PCNA、cerbB2、患者年龄、组织学类型、淋巴结转移等无相关性。结论BP1可能通过某种非依赖p53基因调控机制,与bcl2、ER协同抑制肿瘤细胞的凋亡而参与乳腺癌的发生。