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.     

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 tau mRNA in developing rat brain

A microtubule-associated protein, tau, promotes microtubule assembly, forms characteristic short cross-bridges (less than 20 nm) between microtubules, and switches isoforms from juvenile to adult at the end of the first postnatal week in the rat brain. The developmental expression of tau was studied in rat central nervous system, mainly the cerebrum and cerebellum, by in situ hybridization. Tau mRNAs were localized in a wide variety of neural cells. The expression of tau mRNAs in the spinal cord appeared to precede that in the brain, and the expression in the brainstem appeared to precede that in the cerebral cortex and cerebellum. On neural cells throughout the cortical plate of the cerebral cortex, tau mRNAs were expressed in large amounts during the first postnatal week, but by the third postnatal week the expression had become reduced. In the cerebellum, tau mRNAs were enriched in granule cells. The expression in the internal granular layer peaked during the second and third postnatal weeks, and the relatively high level of expression persisted to young adulthood. Thin section transmission electron microscopic study revealed that the proportion of neighboring microtubules in parallel fiber axons of cerebellar granule cells with the distance less than 20 nm was as low as 10% at the end of the first postnatal week, but this proportion increased to as high as 35% at the end of the second postnatal week. Northern blot analysis showed that tau mRNAs were congruent to 6 kb as was reported previously, and those detected in the first postnatal week were three- to five-fold more abundant and approximately 0.2 kb smaller than those detected in the second or third postnatal weeks. The data suggest that (a) tau mRNAs are abundantly expressed in a wide variety of neurons in the central nervous system at the stage of neurite formation, and (b) tau mRNAs are expressed in more basal levels at later stages, but may be important in the formation and maintenance of characteristic microtubule bundles typically found in parallel fiber axons and in other axons.     

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.     

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.     

Circovirus-infected geese studied by in situ hybridization.

It has now been established that circovirus infection is common in farmed geese, but little is known about the clinicopathological significance of such infections. Ten clinically diseased geese suspected of being infected by circovirus were studied by in situ hybridization using a goose circovirus DNA probe. Circovirus DNA was demonstrated in the bursa of Fabricius (BF), spleen, thymus, bone marrow, liver, kidney, lung and heart, indicating that infection can be multisystemic. In some birds, virus DNA was present in very large quantities, most notably in the BF, liver and small intestine. With the exception of BF and thymus, there were no histological findings that would have suggested the presence of such quantities of circovirus DNA. In view of the very large quantities of virus DNA labelling present in some tissues, and by analogy to porcine circovirus type 2 infection and psittacine beak and feather virus infections, which are known to cause severe disease, and which have similar virus distribution to that found in our geese, it seems probable that the circovirus was important in the disease manifestations shown by the infected geese.     

Developmental changes in tropoelastin gene expression in the rat lung studied by in situ hybridization.

Gene expression for tropoelastin, the proprotein for elastin, was examined in the rat lung from 17 days of gestation (pseudoglandular stage) to adulthood by in situ hybridization using a rat-specific 35S-radiolabeled riboprobe. The tropoelastin message was present in vascular and airway smooth muscle, endothelial, septal interstitial, alveolar wall, and mesothelial cells but not in epithelial cells. With alveolar septal formation, the message in the interstitium increased progressively from 17 days of gestation, reaching a peak at 7 to 11 days postnatal. The signal in the arterial walls, in contrast, peaked between 19 days of gestation to 1 day postnatal and thereafter declined first from the outer media. The signal in general declined significantly by 21 days postnatal, and elastogenesis was virtually absent in the adult. These results support the idea that tropoelastin gene expression in the interstitium is closely associated with the centripetal progression of alveolarization, and the early postnatal decrease of tropoelastin expression in blood vessels corresponds with the sudden postnatal changes in the pulmonary hemodynamics. Furthermore, in the rat fetus and neonate, endothelial cells expressed the gene for tropoelastin and hence probably play a significant role in the formation of internal elastic lamina in vivo.     

Repeated electroconvulsive shocks cause transient changes in rat hippocampal somatostatin and neuropeptide Y immunoreactivity and mRNA in situ hybridization signals

Increased levels of somatostatin (SS) and neuropeptide Y (NPY) have been demonstrated in the hippocampal formation after kindling. The increase might be specifically associated with kindling, or be an effect of repeated seizures per se. In order to separate these two components we studied the effects of repeated electroconvulsive shocks (ECS) on hippocampal SS-like and NPY-like immunoreactivity and SS mRNA and NPY mRNA in situ hybridization. ECS elicit seizures without having a demonstrable kindling effect. Rats were subjected to 10, 20, or 36 ECS (50 mA, 0.5 s), given as one shock per day, 5 days per week. One, 2 and 30 days after the last ECS, the rats were killed, together with sham-treated control rats, and processed for immunocytochemistry and non-radioactive in situ hybridization. There was a bilateral increase in SS-like and NPY-like immunoreactivity 1 and 2 days after the last ECS in the outer part of the dentate molecular layer. This is the terminal field of the hilar SS-containing and NPY-containing neurons, which displayed both increased immunoreactivity and hybridization signal of the cell bodies. There was also a bilateral de novo expression of NPY-like immunoreactivity in the mossy fiber system, but this was not accompanied by the appearance of a detectable NPY hybridization signal over the parent dentate granule cell bodies. The increase in SS-like immunoreactivity and hybridization signal was most pronounced in the rats that had received the largest number of ECS. This was not observed for the NPY-like immunoreactivity and hybridization signal, where the increase appeared similar after 10, 20 and 36 ECS. One month after the last ECS, both the SS-like and NPY-like immunoreactivity and the in situ hybridization signals had decreased towards normal levels. Since increased SS and NPY levels are also induced by repeated ECS, these changes are accordingly not specific to kindling-induced seizures. In a second experiment, the perforant path to the fascia dentata was transected 1 month prior to the ECS treatment. Removal of such major afferent input did not abolish the ECS-induced increase in hippocampal SS-like and NPY-like immunoreactivity, suggesting that the neuropeptide changes were not caused by afferent stimulation via the perfant path fibers, but rather may be an effect of direct electrical activation of the relevant cells.     

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.     

Preprogastrin-releasing peptide messenger ribonucleic acid: neuroanatomical localization in rat brain by in situ hybridization with synthetic oligodeoxynucleotide probes.

Gastrin-releasing peptide (GRP) is a 27 amino acid peptide that is present in both the central and peripheral nervous systems and that shares immunological and functional properties with the amphibian peptide, bombesin. GRP has multiple putative biological functions including effects on feeding behaviour and carbohydrate metabolism, body temperature, and effects on hormone release, but little is known about the regulation of GRP gene expression in the brain. This study examined the distribution of neurones expressing preproGRP mRNA in rat brain by in situ hybridization of [35S]-labelled DNA oligonucleotides. PreproGRP mRNA was detected in several regions of brain, with highest concentrations in the parvocellular paraventricular and suprachiasmatic nuclei of the hypothalamus, the lateral and basolateral nuclei of the amygdala, the amygdaloid-hippocampal area and the ventral part of the granule cell layer of the dentate gyrus. Moderate levels were seen in layers II and III of the cingulate and retrosplenial cortex, the medial and mediobasal nuclei of the amygdala, the anteroventral thalamic nucleus; medial geniculate nucleus and the parabrachial nucleus. These findings are largely consistent with the cellular localization of GRP-like immunoreactivity in rat brain and recent studies of preproGRP mRNA localization using cRNA probes. The distribution of preproGRP mRNA observed further suggests the involvement of GRP in the central regulation of several functions including regulation of hypothalamic/pituitary hormone release.     

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.     

Neurotransmitter and neuromodulator systems of the rat inferior colliculus and auditory brainstem studied by in situ hybridization

This study was concerned with the distribution of a variety of putative neuromodulator and neurotransmitter systems in auditory regions of the rat brainstem using in situ hybridization histochemistry. Serial brain sections were screened for the presence of mRNAs for (i) precursors of the neuroactive substances cholecystokinin, somatostatin, proenkephalin and substance P (preprotachykinin), (ii) glutamic acid decarboxylase, the key synthesizing enzyme for GABA, or (iii) subunits l, 2 and 3 of the GABA_A receptor. Detectable message for all of these probes was found in at least one auditory brainstem area. There were clear differences in the distribution of the various mRNAs in subregions of the inferior colliculus, superior olivary complex, lateral lemniscus and cochlear nucleus. Cells expressing mRNA for glutamic acid decarboxylase were most prominent in the inferior colliculus, but were also present in all lower auditory brainstem nuclei, except the medial superior olivary nucleus and medial nucleus of trapezoid body. The mRNA for GABA_A1 receptor subunits was detectable in all auditory regions investigated, although at different levels of expression. GABA_A2 and 3 mRNA signals were seen in inferior colliculus, lateral lemniscus and in almost all superior olivary complex regions, but in fewer cells and at lower levels than the GABA_A1 subtype. Moderate to high levels of preprocholecystokinin mRNA expression were seen in all subregions of the inferior colliculus. In other auditory brainstem areas, preprocholecystokinin mRNA levels were either low or absent. With regard to mRNAs for the neuroactive peptides somatostatin, preprotachykinin and preproenkephalin, all were expressed in the inferior colliculus but there were differences in their cellular distribution. For example, there were almost no preprotachykinin mRNA expressing cells in the central nucleus of inferior colliculus and levels of somatostatin mRNA were especially high in the dorsal cortex and in layer 3 of the external cortex of inferior colliculus. There were also differences in the pattern of expression of these mRNAs in the various brainstem auditory nuclei; there was no preprotachykinin mRNA in any part of the superior olivary complex, only somatostatin mRNA was found in the ventral cochlear nucleus, and expression of preproenkephalin mRNA was pronounced in the ventral nucleus of the trapezoid body and the rostral periolivary zone. The data are considered in light of the connectivity and functional organization of the auditory brainstem.     

Developmental changes in tropoelastin mRNA levels in rat lung: evaluation by in situ hybridization

Alveolarization of the immature lung is thought to be influenced by the presence of elastic fibers that could provide structural support for developing septa. Although morphometric studies have established that alveolar septal development occurs from days 4 to 13 in the neonatal rat, the precise time period over which elastin synthesis occurs has proved difficult to determine. We have evaluated the usefulness of in situ hybridization techniques to follow tropoelastin message expression in parenchymal tissue, small vessels, and bronchioles in the developing rat lung from days 4 through 18. This method proved to be sufficiently sensitive to detect differences in rates of tropoelastin message expression from days 4 through 18 (P less than 0.0001). Peak tropoelastin message expression was observed in the small vessels on day 4 and in parenchymal tissue on days 9 through 11. Because the time course of tropoelastin message expression in small vessels differs from that in parenchymal tissue, the use of lung extracts to analyze rates of tropoelastin synthesis in the developing lung may be in error.     

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.