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.     

Regional localization of the mRNA coding for the neuropeptide cholecystokinin in the rat brain studied by in situ hybridization

The regional localization of mRNA coding for the neuropeptide cholecystokinin (CCK) has been studied in the rat brain by in situ hybridization using a 32P-labelled synthetic 32 mer oligonucleotide. Autoradiograms were quantified using computer-assisted microdensitometry. High levels of hybridization were observed in the neocortex, claustrum, endopiriform nucleus, cingular cortex, amygdala, olfactory bulb, hippocampus, ventral tegmental area, geniculate nucleus, several thalamic nuclei and substantia nigra compacta. Very weak signal was detected in the striatum, the cerebellum and the brainstem. The topographic distribution of CCK neurons observed overlaps in part with that previously described by immunohistochemical techniques. However, some discrepancies were also found, particularly in the thalamus. These results show that in situ hybridization with oligonucleotide probes together with a semiquantitative method described can be used to map the expression of the CCK mRNA in rat brain sections as well as its modification after pharmacological or physiological manipulations.     

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.     

In situ localization of S-phase-specific histone (H3) mRNA in Bowen's disease

PCNA and Ki-67 immunohistochemistry has been used to assess cell proliferation in place of tritiated thymidine or BrdU labeling of S-phase cells. Recently, it has been possible to reliably demonstrate histone H3 mRNA by in situ hybridization in formalin-fixed and paraffin-embedded tissue sections. We have compared this new proliferation marker with Ki-67 and PCNA with regard to distribution of positive cells and labeling indices (LI%) for 22 cases of Bowen's disease. In normal skin, Ki-67-IHC positive cells and histone mRNA positive cells were observed in the basal and suprabasal layers of the epidermis. In Bowen's disease, positive cells with each marker were more frequent in upper neoplastic epidermis than in suprabasal layers, and the average LI%s were markedly elevated with all markers, the scores decreasing in the following order: PCNA-IHC, Ki-67-IHC and H3mRNA-ISH. However, the results of double staining demonstrated that S-phase cells do not necessarily show exactly the same distributions as with PCNA and Ki-67-IHC labeling. H3mRNA-ISH showed three different degrees of reaction with significantly different LI%s, whereas PCNA and Ki-67 LI% did not vary essentially in the same areas. These results strongly suggest that Bowen's disease, which is well known as a low-grade neoplastic state with malignant potential, also demonstrates clear intratumoral heterogeneity of S-phase cells using the H3mRNA-ISH method.     

In situ hybridization demonstrates the stability of mRNA in post-mortem rat tissues.

In situ hybridization was used to detect messenger RNA (mRNA) in a variety of rat tissues which were fixed in formalin either immediately after death or after a 24 h period of storage at 5 degrees C. A synthetic polydeoxythymidine [poly d(T)] oligonucleotide probe was used to demonstrate polyadenylated [poly (A)] mRNA in the small intestine, pancreas, liver, cerebellum, and pituitary. Of these tissues, only the liver showed a small reproducible reduction in hybridization signal following delayed fixation. Synthetic oligonucleotide probes complementary to albumin and pro-opiomelanocortin (POMC) mRNAs were hybridized to liver and pituitary, respectively. There was no significant reduction in hybridization signal in post-mortem tissues. The results suggest that some mRNAs may be remarkably stable under certain post-mortem conditions and this should encourage the wider application of in situ hybridization techniques to post-mortem material.     

Transient expression of osteopontin mRNA and protein in amoeboid microglia in developing rat brain

To investigate a potential role of osteopontin (OPN) in developing rat brain, the expression of OPN mRNA and protein in the developing rat brain relative to the distribution of brain macrophages was investigated using in situ hybridization and immunohistochemistry, and the phagocytic capability of OPN-expressing cells was accessed using rhodamine isothiocyanate (RhIc) as a tracer. OPN-expressing cells appeared from embryonic day 16. During the first week of postnatal life, OPN-labeled cells increased markedly, and peaked around P7, then declined and had completely disappeared by the end of the second postnatal week. The spatiotemporal distribution pattern of OPN mRNA closely matched that of OPN protein. Their morphology and localization were compared with those of cells expressing the established microglial marker OX-42 in adjacent sections, and double-labeling studies demonstrated that OPN was localized to the amoeboid microglia which stain with the lectin GSI-B₄, another marker for microglia. Furthermore, OPN-labeled cells were confirmed to be active phagocytes emitting RhIc fluorescence indicating that the tracer into the brain tissues was engulfed by phagocytosis. Therefore, these results provide the first evidence that OPN is transiently expressed in active brain macrophages in the embryonic and early postnatal brain, and suggest that OPN may contribute to the migration and phagocytic function of brain macrophages in the developing brain.This work was supported by a Korea Research Foundation grant (KRF-2002-015-EP0106)     

The semi-quantitative distribution and cellular localization of angiotensinogen mRNA in the rat brain.

The present study describes the regional distribution and cellular localization of angiotensinogen-mRNA in the rat brain as investigated by means of in situ hybridization also in combination with immunocytochemistry for glial fibrillary acidic protein. The angiotensinogen gene expression seemed to be restricted to astroglia and showed marked regional differences. In some areas angiotensinogen-mRNA was present in almost all astrocytes with a strong signal (e.g. hypoglossal nucleus), whereas in other areas the angiotensinogen gene was expressed only in a certain population of glial cells. Some areas like the lateral septum were devoid of any detectable angiotensinogen-mRNA. A semi-quantitative atlas of the regional distribution of brain angiotensinogen-mRNA was obtained by using computer-assisted microdensitometry and revealed considerable rostro-caudal fluctuations of the angiotensinogen-mRNA content of certain regions (e.g. the subfornical organ). Furthermore, a semi-quantitative analysis on the cellular level of angiotensinogen gene expression was performed showing a correlation of the angiotensinogen gene expression to the glia content of the regions examined. It was also demonstrated that the angiotensinogen gene expression had its highest levels in several distinct areas of the brain (e.g. the preoptic region and the hypothalamus), whereas other areas showed only low to moderate levels (e.g. the thalamus). The expression of the angiotensinogen gene in the rat brain was not only restricted to areas involved in cardiovascular and neuroendocrine control, but was also present in functionally different regions. Our data thus indicate that, based on the regional distribution of angiotensinogen-mRNA, angiotensin peptides may have other functions besides participation in cardiovascular and neuroendocrine control.     

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.     

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.     

Nogo-A在大鼠脑组织中的亚细胞定位

目的Nogo-A在中枢神经系统的髓鞘和神经元中广泛表达,并且有多种亚分布形式。在以往研究的基础上,将通过检测Nogo-A/B(N-18)在大鼠脑组织中的亚细胞定位,进一步获得Nogo-A在细胞核中表达的有力证据。方法通过差速离心法和NP-40处理得到大鼠皮层和小脑样本的纯化细胞核亚组分,利用Westernblot来检测各亚细胞成分中Nogo-A的表达。通过免疫荧光双标染色,共聚焦显微镜观察Nogo-A在大鼠皮层和小脑组织中的亚细胞定位。结果WesternBlot在纯化细胞核中可检测到清晰的Nogo-A条带。免疫荧光染色显示胞核内有免疫阳性物质表达,并且和Hoechst复染的胞核着色有广泛重合。结论Nogo-A在大鼠皮层和小脑细胞核中有明确表达,并且很有可能以全长形式入核。     

Dysregulation of human brain microtubule-associated tau mRNA maturation in myotonic dystrophy type 1

Intraneuronal aggregates of hyperphosphorylated tau proteins, referred to as pathological tau, are found in brain areas of demented patients affected by numerous different neurodegenerative disorders. We previously described a particular biochemical profile of pathological tau proteins in myotonic dystrophy type 1 (DM1). This multisystemic disorder is characterized by an unstable CTG repeat expansion in the 3'-untranslated region of the DM protein kinase gene. In the human central nervous system, tau proteins consist of six isoforms that differ by the presence or absence of the alternatively spliced exons 2, 3 and 10. Here we show that the pattern of tau isoforms aggregated in DM1 brain lesions is characteristic. It consists mainly of the aggregation of the shortest human tau isoform. A disruption in normal tau isoform expression consisting of a reduced expression of tau isoforms containing the exon 2 was observed at both the mRNA and protein levels. Large expanded CTG repeats were detected and showed marked somatic heterogeneity between DM1 cases and in cortical brains regions analysed. Our data suggest a relationship between the CTG repeat expansion and the alteration of tau expression showing that DM1 is a peculiar tauopathy.     

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.     

Ultrastructural localization of enkephalin-like immunoreactivity in developing rat cerebellum

Methionine enkephalin, an endogenous opioid peptide, participates in the regulation of growth in the developing brain. In the present study, enkephalin-like immunoreactivity was localized in the cerebellum of developing and adult rats by immunoelectron microscopy. In 10-day-old animals, enkephalin-like immunoreactivity was found in the somata of proliferating, migrating and differentiating neural cells, and was associated with the plasma membrane, microtubules, filaments, mitochondria, endoplasmic reticulum and nuclear envelope. Both neurons and glia in the cerebellum of the preweaning rat displayed a similar profile of immunoreactivity. Reaction product was also detected in the dendrites and dendritic spines of Purkinje cells where it was concentrated in postsynaptic densities. The majority of internal granule neurons in 10-day-old animals were not immunoreactive, nor were axons, glial processes and postsynaptic elements (with the exception of mossy fiber terminals). At weaning (Day 21), enkephalin-like immunoreactivity was confined primarily to the somata of Purkinje, basket and stellate neurons, and to Purkinje cell dendrites and synaptic spines. Adult rats (day 75) exhibited no enkephalin-like immunoreactivity. These results establish that enkephalin or an enkephalin-like substance can be detected during the ontogeny of both neurons and glia in the cerebellar cortex, and appears to be associated with certain structural elements.