Regional and Cellular Localization of Presenilin-2 RNA in Rat and Human Brain

In situhybridization probes selective for presenilin-2 (PS-2) were used to determine the regional and cellular expression pattern of PS-2 mRNA in rat and human brain. In rat brain, the greatest expression of PS-2 mRNA is in the granule cell layers of the dentate gyrus and cerebellum. Molecular layers within these structures are virtually devoid of signal. Cortical expression of PS-2 message is restricted to neuronal layers, while the hybridization signal is weak or absent in molecular layers and white matter. Kidney, liver, and spleen display moderate levels of PS-2 message. A PS-2 sense strand probe produced no specific signals in any tissue. In human brain, the greatest hybridization signal for PS-2 is present in the granule cells of the cerebellum. Within hippocampus, the granule cell layer of dentate is strongly labeled, with CA3 pyramidal neurons also clearly visible. A laminar expression pattern is seen in the neuronal layers of human frontal and temporal cortex, with the deeper laminae having the strongest signals. These data are consistent with a primarily neuronal localization of PS-2 mRNA within the brains of both rat and human. Within the limitations of the analysis, it appears that virtually every neuron is labeled, and differences in the intensity of labeling are associated with both neuron size/density and brain region. The distribution of PS-2 RNA is not restricted to those regions having the greatest pathology in Alzheimer's disease. However, one unusual pathological feature of PS-2 mutations causing AD is the presence of cerebellar amyloid plaques in some cases. It is intriguing, in this context, that PS-2 RNA is enriched in the cerebellum, especially in human specimens.     

Expression of calmodulin mRNA in rat olfactory neuroepithelium.

A calmodulin (CaM) cDNA was isolated by differential hybridization screening of a lambda gt10 library prepared from rat olfactory mucosa. This cDNA fragment, containing most of the open reading frame of the rat CaMI gene, was subcloned and used to characterize steady-state expression of CaM mRNA in rat olfactory neuroepithelium and bulb. Within the bulb mitral cells are the primary neuronal population expressing CaM mRNA. The major CaM mRNA expressed in the olfactory mucosa is 1.7 kb with smaller contributions from mRNAs of 4.0 and 1.4 kb. CaM mRNA was primarily associated with the olfactory neurons and, despite the cellular complexity of the tissue and the known involvement of CaM in diverse cellular processes, was only minimally evident in sustentacular cells, gland cells or respiratory epithelium. Following bulbectomy CaM mRNA declines in the olfactory neuroepithelium as does olfactory marker protein (OMP) mRNA. In contrast to the latter, CaM mRNA makes a partial recovery by one month after surgery. These results, coupled with those from in situ hybridization, indicate that CaM mRNA is expressed in both mature and immature olfactory neurons. The program regulating CaM gene expression in olfactory neurons is distinct from those controlling expression of B50/GAP43 in immature, or OMP in mature, neurons respectively.     

Localization of mRNAs encoding alpha and beta subunits of soluble guanylyl cyclase in the brain of rainbow trout: comparison with the distribution of neuronal nitric oxide synthase

Detailed distribution of mRNAs encoding alpha and beta subunits of soluble guanylyl cyclase (sGC) was examined in the brain of rainbow trout by in situ hybridization. In addition, distribution of nitric oxide synthase (NOS) was mapped in adjacent parallel sections by neuronal NOS (nNOS) immunocytochemistry and NADPH-diaphorase (NADPHd) histochemistry. Following application of digoxigenin-labeled riboprobes for sGC alpha and beta subunit mRNAs, we found comparatively intense hybridization signals in the telencephalon, preoptic area, thalamus, hypothalamus, pretectum and tegmentum. Both nNOS immunocytochemistry and NADPHd histochemistry showed extensive distribution of nitroxergic neurons in various brain areas, although various degrees of dissociation of nNOS immunoreactivity (ir) and NADPHd staining were detected. In comparison with sGC subunit mRNAs, nNOS signals were more widely distributed in many neurons, including parvocellular neurons in the preoptic area, nucleus anterior tuberis in the hypothalamus, periventricular neurons in the optic tectum, most of the rhombencephalic neurons and pituitary cells. However, wide overlaps of sGC mRNA-containing neurons and nNOS-positive neurons were observed in the olfactory bulb, telencephalon, preoptic area, thalamus, hypothalamus, pretectum, optic tectum, tegmentum and cerebellum. The widespread overlapping in sGC subunit mRNAs and nNOS distribution suggests a role for sGC in various neuronal functions, such as processing of olfactory and visual signals and neuroendocrine function, possibly via NO/cGMP signaling in the brain of rainbow trout.     

BET, a novel neuronal transmembrane protein with multiple EGF-like motifs

Using the signal sequence trap method, we have identified a cDNA clone coding for a type I transmembrane protein, BET, with 10 epidermal growth factor (EGF) motifs in the extracellular domain. In situ hybridization revealed that the bet mRNA is specifically expressed in the mitral/tufted cells in the olfactory bulb, Purkinje cells in the cerebellum, and pyramidal cells in the hippocampus. Using polyclonal antibodies, we have demonstrated that the BET protein is highly glycosylated and is localized in patches in the dendrites and in the somata of neurons. Since the predicted structure of BET shares many similarities with the Notch ligands, this novel protein may play crucial roles in establishing the neuronal networks in the olfactory system, cerebellum, and hippocampus. BET is the first transmembrane protein containing only multiple EGF-like repeats specifically expressed in the brain.     

Developmental changes in KCC1, KCC2 and NKCC1 mRNAs in the rat cerebellum

Cation chloride cotransporters are considered to play pivotal roles in controlling the intracellular and extracellular ionic environments of neurons, hence controlling neuronal function. To establish how these cotransporters are involved in cerebellum development, we investigated the expression of KCC1, KCC2 and NKCC1 mRNAs in the developing rat cerebellum using in situ hybridization histochemistry. In the external germinal layer, where premature cells exist, we found substantial KCC1 and NKCC1 mRNA expression on P7 and P14, while KCC2 mRNA was not detected. In contrast, KCC2 mRNA was already expressed in Purkinje cells on P1. We also observed KCC2 mRNA expression in postmigratory granule cells after P7. The expression of KCC1, KCC2, and NKCC1 mRNAs reached adult patterns by P21. In the adult cerebellum, KCC2 mRNA was expressed in most neurons, including Purkinje cells, granule cells, and stella/basket cells, while KCC1 and NKCC1 mRNAs were only detected in granule cells and glial cells. These findings suggest that in the rat cerebellum KCC2 mRNA expression is induced when neurons arrive their final destinations.     

In situ hybridization analysis of c-fos and c-jun expression in the rat brain following transient forebrain ischemia.

Early induction of the mRNAs encoding the c-Fos and c-Jun nuclear proteins was examined in rat brain by in situ hybridization at various timepoints following global forebrain ischemia by the method of four-vessel occlusion. All animals were subjected to 20 min of transient ischemia. This produced a pattern of proto-oncogene activation that was most intense in the granule cells of the dentate gyrus 30 min after ischemia, while the hilar cells in the dentate and the pyramidal cells of the CA3 region in the hippocampus showed a more delayed but robust expression of these immediate early genes at 1 h. The neurons of the CA1 region exhibited a more moderate hybridization signal at 1-2 h postischemia. Very little hybridization signal for either immediate early gene could be detected in animals perfused with fixative immediately following ischemia, suggesting that cellular energy levels may have to be restored to a certain level before efficient de novo mRNA synthesis can occur. In the cerebellum, a similar temporal pattern was observed: the granule cells exhibited a prompt but patchy expression of c-fos and c-jun that was followed by a delayed signal in the Purkinje cells. Without exception c-fos and c-jun appeared to be expressed in unison, although the time course of c-fos and c-jun mRNA accumulation and decay was different in various brain regions: invariably the cerebellum returned rapidly to its baseline with virtually no remaining signal at 3 h postischemia, while c-fos and c-jun activation in the hippocampus remained high at 3 h and returned to baseline by 6 h. Several other brain regions showed early production of c-fos and c-jun mRNAs, such as the medial habenula, piriform cortex, the amygdala, the centromedian, lateral posterior, paracentral, intermediodorsal and reuniens nuclei of the thalamus and the ventromedial and dorsal nuclei of the hypothalamus; in the brainstem, the trapezoid body and the noradrenergic neurons of the locus ceruleus as well as the adrenergic neurons in the ventrolateral medulla (C1 group) and nucleus tractus solitarius (C2 group) regions displayed slightly less intense hybridization signals. In addition, the ependyma of the lateral ventricles and the third ventricle showed a prompt albeit short-lived production of c-fos and c-jun mRNAs. Sham-operated animals as well as animals that had survived to one week postischemia showed either no or only trace levels of hybridization signal.(ABSTRACT TRUNCATED AT 400 WORDS)     

Localization of GDNF/neurturin receptor (c-ret, GFRalpha-1 and alpha-2) mRNAs in postnatal rat brain: differential regional and temporal expression in hippocampus, cortex and cerebellum

Recent studies have identified a multi-component receptor system for the neurotrophic factor, glial cell line-derived neurotrophic factor (GDNF) and its homolog, neurturin (NTN), comprising the signaling tyrosine kinase, Ret and multiple GPI-linked binding proteins, GDNF family receptor alpha-1 and alpha-2 (GFRalpha-1 and GFRalpha-2). In the present study the localization of c-ret and GFRalpha-1 and GFRalpha-2 mRNAs was assessed in the developing rat brain from postnatal day 4 to 70 by in situ hybridization histochemistry, using specific [35S]-labeled oligonucleotides. GFRalpha-1 and GFRalpha-2 mRNAs were differentially distributed throughout the brain at all ages studied, particularly in cerebral cortex, hippocampus, substantia nigra and regions of the thalamus and hypothalamus - both distributions overlapping but different to that of c-ret mRNA. C-ret mRNA was abundant in areas such as the lateral habenula, reticular thalamic nucleus, substantia nigra pars compacta, cranial motor nuclei, and the Purkinje cell layer of the cerebellum. GFRalpha-1 mRNA was abundant in dorsal endopiriform nucleus, medial habenula, reticular thalamic nucleus, pyramidal and granule cell layers of the hippocampus, substantia nigra pars compacta and in cranial motor nuclei. GFRalpha-2 mRNA was highly expressed in many regions including olfactory bulb, lateral olfactory tract nucleus, neocortical layers IV and VI, septum, zona incerta, and arcuate and interpeduncular nuclei. GFRalpha-2 mRNA was detected in the pyramidal cell layers (CA3) of hippocampus at P4 and P7, but was no longer detectable at P14 and beyond, including P70 (adult). GFRalpha-2 mRNA was also detected in Purkinje cells throughout the cerebellum in young postnatal rats, but was enriched in the posterior lobes at P28 and P70. These localization studies support evidence of GDNF/NTN as target-derived and autocrine/paracrine trophic factors in developing brain pathways and earlier suggestions of unique and complex signaling mechanisms for these factors via a family of receptors. Strong expression of GFRalpha-1 and GFRalpha-2 mRNAs in adult brain suggests possible non-trophic functions of GDNF/NTN, as described for other neurotrophins, such as brain-derived neurotrophic factor.     

Excitatory amino acid AMPA receptor mRNA localization in several regions of normal and neurological disease affected human brain. An in situ hybridization histochemistry study

In situ hybridization histochemistry was used to localize the mRNAs coding for four α-aminoisoxazole propionic acid-sensitive glutamate receptor subunits in human brain (age range 51–95 years, postmortem delay 4.5–10 h). High levels of the B receptor subunit mRNA were present in all the studied regions, followed by the A-subunit and the C-subunit. Only very low levels of the D-subunit mRNA were detected. In hippocampus, the mRNA coding for the B-subunits of the glutamate receptor was observed in granule cells of dentate gyrus and in the pyramidal cells of Ammon's horn. In cortex, the highest levels of glutamate receptor subunit mRNAs were found in layer I and layers III-IV of entorhinal and temporal cortex, although significant levels were also observed in the other cell layers. A differential distribution was seen in cerebellum where the A-subunit mRNA is expressed mainly by Purkinje cells, while the B-subunit mRNA is present in the internal granule cell layer. These results correlate well with previous data from autoradiographic studies on the localization of excitatory amino acid binding sites in human brain and pinpoint the cells where these receptors are synthesized. In situ hybridization in the hippocampus of patients affected by Alzheimer's disease (age range 77–82 years, postmortem delay 19–25.5 h) revealed a decrease on the content of the mRNAs coding for these excitatory amino acid receptors, while an increase was detected in surgically disected epileptic human hippocampi. These results corroborate and extend the previous data from in vitro autoradiography and suggest alteration of the excitatory amino acid disfunction during these neurodegenerative processes.     

Localization of mRNA for Ca/calmodulin-dependent protein kinase I in the brain of developing and mature rats

The gene expression of Ca²⁺/calmodulin-dependent protein kinase I (CaM kinase I) in the brain of developing and adult rats was examined by in situ hybridization histochemistry. During the development, CaM kinase I showed two chronological expression patterns; the persistent and relative high expression as observed in the olfactory bulb and cerebellar cortex, and the gradual decrease in the expression during the postnatal development as observed in most other brain regions. The gene expression was not detected in the germinal ventricular zone and cerebellar external granular layer. In the mature brain, CaM kinase I mRNA was expressed widely, though weakly in general, in almost all neurons, except for the olfactory bulb, cerebellum and hippocampus expressing at high intensity. These findings suggest that CaM kinase I may play a variety of neuronal Ca²⁺/calmodulin-mediated signaling processes in the developing and mature brains.     

Gene expression of Ca2+/calmodulin-dependent protein kinase of the cerebellar granule cell type or type IV in the mature and developing rat brain.

The localization and ontogenic changes of expression of the mRNA for Ca2+/calmodulin-dependent protein kinase of the cerebellar granule cell type or type IV (CaM kinase Gr or IV) in the rat brain were examined by in situ hybridization histochemistry. At the young adult stage, intense expression signals for this kinase mRNA were detected in the cerebellar granule cells, the hippocampal pyramidal cells, the dentate granule cells, and the piriform cortex. Moderate levels of the mRNA were expressed in the thalamic nuclei and the cerebral cortex. No distinct expression signals were detected in the Purkinje cells and most brainstem nuclei except for the pontine nuclei, locus ceruleus and inferior olive which showed weak expression. During development, two chronological patterns of changes in the gene expression for this kinase were discerned. The first was a high and persistent expression from the developing stages till the adult stage, which was observed in the cerebellar granule cells, the hippocampal pyramidal cells and the dentate granule cells. The other was a transiently high expression during limited developmental periods, which was observed in the Purkinje cells, neurons in the inferior olive, various brain stem nuclei, and the subventricular neuronal cells. These findings suggest that this Ca2+/calmodulin-dependent protein kinase is involved differentially in multiple Ca2+ signaling pathways in different developing and mature neurons.     

Expression of mRNA species encoding heat shock protein 90 (hsp90) in control and hyperthermic rabbit brain

Northern blot and in situ hybridization were employed to investigate regional and cell type differences in the expression of hsp90 mRNA species in control and hyperthermic rabbit brain. Riboprobes specific to hsp90 α and β mRNA species were utilized in time-course Northern blot studies on cerebral hemispheres and the cerebellum. Following hyperthermia, levels of hsp90 α and β mRNA were elevated in both brain regions; however, the magnitude of induction was more robust in the cerebellum than in cerebral hemispheres. The pattern of expression of hsp90 genes in rabbit brain was analyzed by in situ hybridization. These studies revealed that hsp90 genes are preferentially expressed in neuronal cell populations in the unstressed mammalian brain. The distribution of hsp90 α and β mRNA was similar, though the signal for the latter was stronger. Following hyperthermia, changes were not detected in the pattern of hsp90 β mRNA expression in the hippocampus. In the cerebellum, a rapid induction of hsp90 β mRNA was apparent in the neuron-enriched granule cell layer, followed by a delayed accumulation in Purkinje neurons. Unlike hsp70, induction of hsp90 was not detected in glial cells of hyperthermic rabbit brain. The localization of hsp90 to neurons suggests that this heat shock protein plays an important role in neuronal function. © 1996 Wiley-Liss, Inc.     

Differential distribution of cAMP-specific phosphodiesterase 7A mRNA in rat brain and peripheral organs

We investigated the regional distribution and cellular localization of mRNA coding for the cAMP-specific phosphodiesterase 7A (PDE7A) in rat brain and several peripheral organs by in situ hybridization histochemistry. The regional expression of two splice variants, PDE7A1 and PDE7A2, was examined by RT-PCR using RNA extracted from several brain regions. PDE7A mRNA was found to be widely distributed in rat brain in both neuronal and nonneuronal cell populations. The highest levels of hybridization were observed in the olfactory bulb, olfactory tubercle, hippocampus, cerebellum, medial habenula nucleus, pineal gland, area postrema, and choroid plexus. Positive hybridization signals were also detected in other areas, such as raphe nuclei, temporal and entorhinal cortex, pontine nuclei, and some cranial nerve motor nuclei. Both mRNA splice forms were differentially distributed in several areas of the brain with the striatum expressing only PDE7A1 and the olfactory bulb and spinal cord expressing PDE7A2 exclusively. In peripheral organs the highest levels of PDE7A hybridization were seen in kidney medulla, although testis, liver, adrenal glands, thymus, and spleen also presented high hybridization signal. These results are consistent with PDE7A being involved in the regulation of cAMP signaling in many brain functions. The consistent colocalization with PDE4 mRNAs suggests that PDE7A could have an effect on memory, depression, and emesis. The results offer clear anatomical and functional systems in which to investigate future specific PDE7 inhibitors.     

Localization of mRNA for protein phosphatase 2C in the brain of adult rats.

Type 2C protein phosphatase (PP2C) is one of four major serine-threonine specific phosphoprotein phosphatases which modulate various intracellular activities. By in situ hybridization analysis of the adult rat, expression signals of mRNA for PP2C were observed most highly in the granule cells and Purkinje cells of the cerebellum, the pyramidal cells of the hippocampus and granule cells of the dentate gyrus, and plexus choroideus of the lateral ventricle, whereas moderate levels of its expression were observed in the medial habenula, piriform cortex and the pineal body. Several discrete nuclei of the brainstem including pars compacta of the substantia nigra, the pontine nuclei, and the locus ceruleus expressed the mRNA moderately. Weak expression of PP2C mRNA was observed in mitral and internal granule cells of the olfactory bulb, spinal cord gray matter, the cerebral neocortex, thalamic and hypothalamic nuclei. Only faint expression was detected in the caudate putamen. These patterns of expression are different from that of calcineurin/PP2B reported by other immunohistochemical studies and it is suggested that various neuronal proteins are differentially dephosphorylated by the different types of PP.