Activity-Dependent Regulation of Na+,K+-ATPase α Isoform mRNA Expression In Vivo

To investigate the functional role of the different Na+, K(+)-ATPase alpha (catalytic) subunit isoforms in neuronal cells, we used quantitative in situ hybridization with riboprobes specific for alpha 1, alpha 2, and alpha 3 isoforms to measure the level of alpha isoform-specific expression in the neuroendocrine cells of the supraoptic (SON) and paraventricular (PVN) nuclei of rat hypothalamus. A prolonged increase in electrical activity of these cells, achieved by 5 days of salt treatment, increased the amount of alpha 1 isoform mRNA in the SON and PVN by 50%. Levels of alpha 1 mRNA in other brain regions and levels of alpha 2 and alpha 3 mRNAs were not affected by salt treatment. We conclude that the alpha 1 isoform Na+, K(+)-ATPase may be specifically adapted to pump out Na+, which enters the cells through voltage-gated channels during neuronal depolarization.     

C-fos expression in rat brain during heat stress

Rats, under urethane anesthesia, 0, 20, 40 or 80 min after the start of heat stress (42°C) were sacrificed for determination of c-fos expression in different brain regions. In situ hybridization and immunocytochemistry methods were used, respectively, for determination of c-fos mRNA and protein, respectively. In general, either colon temperature (T_CO), mean arterial pressure (MAP), local cerebral blood flow (CBF) or c-fos expression in different brain regions (including the preoptic area, supraoptic nuclei, paraventricular nuclei, thalamus, amygdala, nucleus tract solitarii, area postrema and ventrolateral medulla) increased at 20–40 min after the start of heat exposure. However, the heatstroke, which appears as profound decreases in both MAP and local CBF and increases in T_CO, was produced 80 min after heat stress. The c-fos expression was heavily induced in all these brain regions after the onset of heatstroke. The data suggest that c-fos expression in rat brain during heatstroke is associated with hyperthermia, arterial hypotension or cerebral ischemia.     

Na(+)-K(+)-ATPase gene expression in the avian eggshell gland: distinct regulation in different cell types

In lactating rats, ANG II receptor binding in the arcuate nucleus (ARH) and median eminence is decreased. To further evaluate brain angiotensinergic activity during lactation, we assessed angiotensinogen (AON) mRNA by in situ hybridization in forebrains of day 10 or 11 postpartum lactating and diestrous rats. AON mRNA was abundantly expressed in the ARH, preoptic, suprachiasmatic, supraoptic, paraventricular, and dorsomedial hypothalamic nuclei, and other regions, similar to that reported in male rat brains. AON mRNA levels were decreased 27% in the midcaudal ARH of lactating rats but did not differ between lactating or diestrous rats in any of the other brain areas examined. Immunofluorescence for AON and glial fibrillary acidic protein or tyrosine hydroxylase confirmed that the AON immunoreactivity in the ARH was limited to astrocytes. Confocal microscopy revealed close appositions of AON-positive astrocytes to dopaminergic neurons in the ARH. The decrease in AON mRNA in the midcaudal ARH during lactation coupled with decreased ARH ANG II receptor binding suggests that lactating rats are less subject to ANG II-mediated inhibition of prolactin secretion.     

Rapid, transient, and dose-dependent expression of hsp70 messenger RNA in the rat brain after morphine treatment

Induction of Hsp70 in the brain has been reported after intake of drugs of abuse like amphetamine and lysergic acid diethylamide. In this investigation, gene expression of Hsp70 and other heat shock genes in the rat brain was studied in response to morphine. Twenty milligrams per kilogram morphine intraperitoneally resulted in a marked induction of Hsp70 messenger RNA (mRNA) expression in the frontal cortex with a maximum increase of 13.2-fold after 2 hours. A moderate increase of Hsp27 mRNA expression (6.7-fold) could be observed after 4 hours, whereas mRNA expression of Hsp90 and of the constitutive Hsc70 did not exceed a mean factor of 1.8-fold during the 24 hours interval. The increase in Hsp70 mRNA was dose dependent, showing a significant elevation after doses ranging from 10 to 50 mg/kg morphine. In situ hybridization revealed enhanced Hsp70 mRNA expression mainly in cortical areas, in the hippocampus, in the paraventricular and supraoptic nuclei of the hypothalamus, in the locus coeruleus, as well in the pineal body. The double in situ hybridization technique revealed increased Hsp70 mRNA expression mainly in VGLUT1-positive neurons and to a lesser extent in olig1-positive oligodendroglia. Immunohistochemistry revealed a marked increase of Hsp70 protein in neuronal cells and blood vessels after 12 hours. In contrast to animal experiments, morphine did not increase Hsp70 mRNA expression in vitro in micro-opioid receptor (MOR1)-expressing human embryonic kidney 293 cells, suggesting no direct MOR1-mediated cellular effect. To exclude a body temperature-related morphine effect on Hsp70 mRNA expression, the temperature was recorded. Five to 20 mg/kg resulted in hyperthermia (maximum 40.6 degrees), whereas a high dose (50 mg/kg) that produced the highest mRNA induction, showed a clear hypothermia (minimum 37.2 degrees C). These findings argue against the possibility that Hsp70 induction by morphine is caused by its effect on body temperature. It may be speculated that increased expression of Hsp70 after morphine application protects brain structures against potentially hazardous effects of opiates.     

Expression of dihydropyridine-sensitive brain calcium channels in the rat central nervous system.

We have localized dihydropyridine (DHP-sensitive calcium channels in rat brain by in situ hybridization and immunohistochemistry. The mRNA for the dihydropyridine-sensitive calcium channel alpha 1 subunit (DHPR-B) is prominently localized in neuronal cells in the olfactory bulb, dentate gyrus, hippocampus, arcuate nucleus, paraventricular nucleus, ventromedial nucleus, cerebral cortex, superior colliculus and the cerebellar Purkinje cell layer. Strong expression of DHPR-B mRNA was also found in the pituitary and pineal glands. DHP-sensitive calcium channel alpha 1 subunit distribution has also been examined immunohistochemically with polyclonal antibodies raised against synthetic peptides specific for the DHPR-B alpha 1 subunit protein. The results from immunohistochemistry were in good agreement with those from in situ hybridization. Thus, regional distribution and localization of DHPR-B mRNA and alpha 1 subunit protein in rat brain suggest that this type of DHP-sensitive brain calcium channel may play an important role in excitation-secretion coupling functions in the neuroendocrine system.     

Distribution of Preproatrial Natriuretic Peptide mRNA in Rat Brain Detected by In Situ Hybridization of DNA Oligonucleotides: Enrichment in Hypothalamic and Limbic Regions

The expression and distribution of mRNA encoding preproatrial natriuretic peptide (ppANP) in rat brain has been investigated by in situ hybridization of two 35S-labeled synthetic DNA oligonucleotides, based on a cDNA clone sequence that encodes rat ppANP. The highest relative concentrations of ppANP mRNA were detected in the medial preoptic hypothalamic nucleus ("anteroventral/third ventricle region") and the medial habenula. Moderate concentrations of ppANP mRNA were observed in the CA1 pyramidal cells of the hippocampus, the endopiriform nucleus, the arcuate nucleus, the zona incerta, and cells of the pontine tegmental and peduculopontine nuclei. Several of these regions, including the habenula and the hypothalamic areas, have previously been reported to contain atrial natriuretic peptide (ANP)-like immunoreactivity, but the expression of ppANP mRNA in CA1 pyramidal cells suggests the occurrence of differential translation of ppANP mRNA into protein product in different brain regions, or the existence of different immunological forms of the peptide. The abundance of ppANP mRNA in brain was relatively low in comparison with that previously reported for many other mRNA species encoding other brain neuropeptides. These results demonstrate that ANP gene expression occurs in discrete neuronal populations of the CNS and that studies of the regulation of this expression should now be possible using quantitative in situ hybridization.     

The a3 isoform of the 100-kDa V-ATPase subunit is highly but differentially expressed in large (>or=10 nuclei) and small (<or= nuclei) osteoclasts

Osteoclasts dissolve bone through acidification of an extracellular compartment by means of a multimeric vacuolar type H+-ATPase (V-ATPase). In mammals, there are four isoforms of the 100-kDa V-ATPase "a" subunit. Mutations in the a3 isoform result in deficient bone resorption and osteopetrosis, suggesting that a3 has a unique function in osteoclasts. It is thus surprising that several studies show a basal level of a3 expression in most tissues. To address this issue, we have compared a3 expression in bone with expression in other tissues. RNA blots revealed that the a3 isoform was expressed highest in bone and confirmed its expression (in decreasing order) in liver, kidney, brain, lung, spleen, and muscle. In situ hybridization on bone tissue sections revealed that the a3 isoform was highly expressed in multinucleated osteoclasts but not in mononuclear stromal cells, whereas the a1 isoform was expressed in both cell types at about the same level. We also found that a3 expression was greater in osteoclasts with 10 or more nuclei as compared with osteoclasts with five or fewer nuclei. We hypothesize that these differences in a3 expression may be associated with previously demonstrated differences between large and small osteoclasts with reference to their resorptive activity.     

Novel Alternative Splicing Predicts a Truncated Isoform of the NMDA Receptor Subunit 1 (NR1) in Embryonic Rat Brain

The expression of mesencephalic brain derived neurotrophic factor (BDNF) has been shown to be regulated by dopaminergic neuronal functioning and glutamate receptors (GluRs). In turn, BDNF participates in the regulation of mesencephalic GluRs’ expression. In the present study we analyzed, using semi-quantitative RT-PCR, the effect of BDNF as well as of the GluRs agonists NMDA and trans-(±)-1-Amino-(1S,3R)-cyclopentane dicarboxylic acid (t-ACPD), on the expression levels of the NMDA GluR subunit 1 (NR1) mRNA, using rat cultured mesencephalic neurons. In the course of this study, a novel rat mRNA splice variant of NR1 was identified. This new NR1 mRNA isoform is characterized by the insertion of an 82 base pair intron containing an inframe stop codon, thus predicting the expression of a putative truncated protein of 465 amino acids. The RT-PCR and in situ hybridization reveals that the novel NR1 mRNA is expressed in various brain regions of the rat embryo, whereas no expression was detected in the adult rat brain. The modulation of the novel NR1 mRNA isoform by both BDNF and the metabotropic GluR agonist t-ACPD, suggests that the resulting putative NR1 truncated protein may be relevant in the regulatory network of glutamatergic neurotransmission in the developing central nervous system.     

The Monoamine Neurons of the Rat Brain Preferentially Express a Splice Variant of α1B Subunit of the N-Type Calcium Channel

The N-type voltage-dependent calcium channels play a significant role in neurotransmitter release. The alpha1B subunit of the N-type calcium channel functions as the primary subunit that forms the pore and contains the structural motifs that mediate the pharmacological and gating properties of the channel. We report on an isoform of the alpha1B subunit that is preferentially expressed by the monoaminergic neurons of the rat brain. This isoform contains a 21-amino acid cassette in the synprint site present in the cytoplasmic loop between domains IIS6 and IIIS1. RT-PCR of micropunched tissue was used to show preferential expression of this isoform in regions of the brain containing monoaminergic neurons and to a lesser extent in the cerebellum. Double-label in situ hybridization was used to show expression of this isoform mRNA in dopaminergic neurons of the ventral mesencephalon. The expression of two distinct N-type calcium channels containing these alpha1B subunit isoforms by the monoaminergic neurons may provide for synapse-specific regulation of neurotransmitter release.     

Calcyclin (S100A6) binding protein (CacyBP) is highly expressed in brain neurons.

The expression of a novel calcyclin (S100A6) binding protein (CacyBP) in different rat tissues was determined by Western and Northern blotting. Polyclonal antibodies against recombinant CacyBP purified from E. coli exhibited the highest reaction in the brain and weaker reaction in liver, spleen, and stomach. CacyBP immunoreactivity was also detected in lung and kidney. Densitometric analysis showed that the concentration of CacyBP in the soluble fractions of total brain and cerebellum is approximately 0.17 and 0. 34 ng/microg protein, respectively. Northern blotting with a specific cDNA probe confirmed the high level of CacyBP expression in the rat brain and lower levels in other tissues examined. Immunohistochemistry and in situ hybridization of rat brain sections revealed strong expression of CacyBP in neurons of the cerebellum, hippocampus, and cortex. The in situ hybridization detected CacyBP in hippocampus as early as P7 (postnatal day 7) and a peak of expression at P21, and the expression signal was preserved until adulthood. In the entorhinal cortex, the peak of expression was observed at P7, whereas in the cerebellum it was seen at P21. The results presented here show that CacyBP is predominantly a neuronal protein. (J Histochem Cytochem 48:1195-1202)     

Tissue-specific expression of insulin receptor isoforms in obesity/type 2 diabetes mouse models

The two insulin receptor (IR) isoforms IR-A and IR-B are responsible for the pleiotropic actions of insulin and insulin-like growth factors. Consequently, changes in IR isoform expression and in the bioavailability of their ligands will impact on IR-mediated functions. Although alteration of IR isoform expression has been linked to insulin resistance, knowledge of IR isoform expression and mechanisms underlying tissue/cell-type-specific changes in metabolic disease are lacking. Using mouse models of obesity/diabetes and measuring the mRNA of the IR isoforms and mRNA/protein levels of total IR, we provide a data set of IR isoform expression pattern that documents changes in a tissue-dependent manner. Combining tissue fractionation and a new in situ mRNA hybridization technology to visualize the IR isoforms at cellular resolution, we explored the mechanism underlying the change in IR isoform expression in perigonadal adipose tissue, which is mainly caused by tissue remodelling, rather than by a shift in IR alternative splicing in a particular cell type, e.g. adipocytes.     

Autoradiographic Visualization of the Receptor Subclasses for Vasoactive Intestinal Polypeptide (VIP) in Rat Brain

Vertongen, P., S. N. Schiffmann, P. Gourlet and P. Robberecht. Autoradiographic visualization of the receptor subclasses for Vasoactive Intestinal Polypeptide (VIP) in rat brain. Peptides 18(10) 1547–1554, 1997.—Vasoactive Intestinal Polypeptide (VIP) exerts its biological effects through interaction with two high affinity receptors named the VIP₁- and the VIP₂ receptors. Their messenger RNAs have been mapped in rat brain by in situ hybridization. A cyclic peptide (RO 25-1553) and a secretin analogue ([R¹⁶]chicken secretin) were identified as selective agonist peptides for the VIP₂- and VIP₁ receptors, respectively. The iodinated peptides retained the high affinity and selectivity of the unlabelled peptides and were used for the mapping of each receptor subclass in rat brain. VIP₁ receptors were present in the cerebral cortex, the piriform cortex, the claustrum, the caudate-putamen, the dentate gyrus, the lateral amygdaloïd nucleus, the anteroventral thalamic nucleus, the rhomboïd nucleus, the supraoptic nucleus and the choroïd plexus. VIP₂ receptors were present in the cerebral cortex, the claustrum, the caudate-putamen, the nucleus accumbens, the lateral septal nuclei, the bed nucleus of the stria terminalis, the basolateral amygdaloïd nucleus, the Ammon’s horn, the thalamic nuclei except some centromedial nuclei, the medial habenula, the suprachiasmatic nucleus, the periventricular nucleus, the mammilary nucleus, the superior colliculus and the choroïd plexus.     

An In Vivo Kinetic Model with L-[35S]Methionine for the Determination of Local Cerebral Rates for Methionine Incorporation into Protein in the Rat

A method has been developed for the simultaneous in vivo measurement of local rates for methionine incorporation into cerebral protein in the rat. It is based on the use of L-[35S]methionine as a tracer for reflecting the bidirectional exchange of methionine between plasma and brain and its incorporation into cerebral protein, using a dynamic three-compartment model. An operational equation based on this model has been derived in terms of determinable variables. The method has been applied to the normal freely moving rat and to the rat under chloral hydrate anesthesia. In the freely moving rat, the values of methionine incorporation into cerebral protein in the gray matter vary widely from structure to structure (50-300 nmol/100 g/min), with the highest values in structures related to neurosecretory functions, e.g., supraoptic and paraventricular nuclei. The values for white matter are more uniform (24-28 nmol/100 g/min) at levels approximately six- to seven-fold lower than for gray matter. Chloral hydrate anesthesia depresses the rate of methionine incorporation in all the structures examined. Anesthesia did not reduce the heterogeneity normally present within gray matter.     

刺激大鼠穹窿下器官诱发饮水和脑内c—fos表达的胆碱能机制

实验以饮水行为脑内c-fos表达为指标,,观察刺激大鼠穹窿下器官（SFO）的效应,结果显示,刺激SFO能诱发明显的饮水行为,与此同时,前脑8个部位（终板血管器官,正中视前核,室旁核,视上核,下丘脑外侧区,穹窿周核背侧区,丘脑联合核和无名质）和后脑3个部位（最后区,孤束核和壁旁外侧核）的Fos蛋白表达明显增强,免疫组化双重染色结果显示,刺激SFO能诱导视上核和室旁核中部分神经元呈Fos蛋白和加压素共同表达。脑室注射阿托品能部分阻断刺激SFO诱发的饮水行为,脑内上述各部位所诱导的Fos蛋白表达也明显减弱,以上结果提示,M胆碱能机制参与 刺激SFO诱发的饮水行为和脑内Fos蛋白的表达。     

Cortical neurons express PSI,a novel isoform of phosphacan/RPTPbeta

Phosphacan is a chondroitin sulfate proteoglycan representing the secreted extracellular part of a transmembrane receptor protein tyrosine phosphatase (RPTP-). These isoforms have been implicated in cell-extracellular matrix signaling events associated with myelination, axon growth, and cell migration in the developing central nervous system and may play critical roles in the context of brain pathologies. Recently, we have reported the identification of a new isoform of phosphacan, the phosphacan short isoform (PSI), the expression of which peaks in the second postnatal week. PSI interacts with the neuronal receptors L1 and F3/contactin and can promote neurite growth of cortical neurons. In this study, we have assessed, by in situ hybridization, the expression profile of PSI in the rat brain at postnatal day 7. PSI is largely expressed in the gray matter of the developing cerebral cortex in which it colocalizes with phosphacan, whereas the expression of RPTPbeta receptor forms is restricted to the ventricular area in which PSI has not been observed. Neurons from all layers of the cortex express PSI. In the cerebellum, on the other hand, no expression of PSI has been detected, although the other phosphacan/RPTP-beta isoforms show strong PSI expression here. Overall, our study suggests that PSI is expressed during the postnatal period in differentiated neurons of the cortex but is absent from structures in which proliferation and migration occur. The significance of these observations is discussed in the context of previous models of phosphacan/RPTP-beta functions.The authors thank the German Research Council (DFG) for grant support (SFB 509 and SPP 1048 to A.F.) and for a graduate training grant to Alice Klausmeyer (GK 736).     

Localization and osmotic regulation of vesicular glutamate transporter-2 in magnocellular neurons of the rat hypothalamus

In this report we present immunocytochemical and in situ hybridization evidence that magnocellular vasopressin and oxytocin neurons in the hypothalamic supraoptic and paraventricular nuclei express type-2 vesicular glutamate transporter, a marker for their glutamatergic neuronal phenotype. To address the issue of whether an increase in magnocellular neuron activity coincides with the altered synthesis of the endogenous glutamate marker, we have introduced a new dual-label in situ hybridization method which combines fluorescent and autoradiographic signal detection components for vasopressin and vesicular glutamate transporter-2 mRNAs, respectively. Application of this technique provided evidence that 2% sodium chloride in the drinking water for 7 days produced a robust and significant increase of vesicular glutamate transporter-2 mRNA in vasopressin neurons of the supraoptic nucleus. The immunocytochemical labeling of pituitary sections, followed by the densitometric analysis of vesicular glutamate transporter-2 immunoreactivity in the posterior pituitary, revealed a concomitant increase in vesicular glutamate transporter-2 protein levels at the major termination site of the magnocellular axons. These data demonstrate that magnocellular oxytocin as well as vasopressin cells contain the glutamatergic marker vesicular glutamate transporter-2, similarly to most of the parvicellular neurosecretory neurons examined so far. The robust increase in vesicular glutamate transporter-2 mRNA and immunoreactivity after salt loading suggests that the cellular levels of vesicular glutamate transporter-2 in vasopressin neurons are regulated by alterations in water–electrolyte balance. In addition to the known synaptic actions of excitatory amino acids in magnocellular nuclei, the new observations suggest novel mechanisms whereby glutamate of endogenous sources can regulate magnocellular neuronal functions.