Developmental expression offlt3 mRNA in the mouse brain

The expression and localization offlt3 mRNA were investigated using Northern blotting andin situ hybridization during the developmental process of the mouse brain. By Northern blotting, the expression of theflt3 gene was not detected in embryonic, neonatal, or early postnatal brains, but was markedly increased with age. In adult mice,flt3 was abundantly expressed in the cerebellum, moderately in the pons, and faintly in the thalamus and the cerebral cortex. Byin situ hybridization, some neurons that expressedflt3 formed synaptic connections with each other. The present findings suggest that theflt3 gene expression is related to the cell type and the developmental process.     

Epsilon-sarcoglycan immunoreactivity and mRNA expression in mouse brain

Myoclonus dystonia (M-D) is a hereditary movement disorder caused by a maternally imprinted gene that is often associated with psychiatric symptoms. Most cases of M-D are believed to result from mutations of the epsilon-sarcoglycan protein. The neuroanatomical distribution of epsilon-sarcoglycan-like immunoreactivity in mouse was investigated by using an antiserum against the epsilon-sarcoglycan protein. The expression of epsilon-sarcoglycan mRNA was studied by a sensitive fluorescence in situ hybridization (FISH) method. Immunohistochemistry and FISH revealed a wide distribution of epsilon-sarcoglycan protein and mRNA throughout the mouse brain. High expression levels of epsilon-sarcoglycan mRNA and immunoreactivity were found in the mitral cell layer of the olfactory bulb, the Purkinje cell layer in cerebellum, and the monoaminergic neurons in the mouse midbrain. Immunohistochemistry revealed a similar distribution of epsilon-sarcoglycan protein. Double-labeling FISH showed colocalization of tyrosine hydroxylase and epsilon-sarcoglycan mRNAs within all the midbrain dopaminergic (DAergic) cell groups. By combining FISH with fluorescence immunohistochemistry, coexpression of epsilon-sarcoglycan mRNA and tryptophan hydroxylase immunoreactivity was found in the serotonergic (5-HTergic) neurons within the dorsal raphe nucleus. The distribution of epsilon-sarcoglycan in the mouse brain suggests that the symptom complex of M-D may be related to the effects of decreased epsilon-sarcoglycan activity on the development or function of monoaminergic neurons.     

Developmental expression and subcellular localization of glutaminyl cyclase in mouse brain

Glutaminyl cyclase (QC) converts N-terminal glutaminyl residues into pyroglutamate (pE), thereby stabilizing these peptides/proteins. Recently, we demonstrated that QC also plays a pathogenic role in Alzheimer's disease by generating the disease-associated pE-Abeta from N-terminally truncated Abeta peptides in vivo. This newly identified function makes QC an interesting pharmacological target for Alzheimer's disease therapy. However, the expression of QC in brain and peripheral organs, its cell type-specific and subcellular localization as well as developmental profiles in brain are not known. The present study was performed to address these issues in mice. In brain, QC mRNA expression was highest in hypothalamus, followed by hippocampus and cortex. In liver, QC mRNA concentration was almost as high as in brain while lower QC mRNA levels were detected in lung and heart and very low expression levels were found in kidney and spleen. In the developmental course, stable QC mRNA levels were detected in hypothalamus from postnatal day 5 to 370. On the contrary, in cortex and hippocampus QC mRNA levels were highest after birth and declined during ontogenesis by 20–25%. These results were corroborated by immunocytochemical analysis in mouse brain demonstrating a robust QC expression in a subpopulation of lateral and paraventricular hypothalamic neurons and the labeling of a significant number of small neurons in the hippocampal molecular layer, in the hilus of the dentate gyrus and in all layers of the neocortex. Hippocampal QC-immunoreactive neurons include subsets of parvalbumin-, calbindin-, calretinin-, cholecystokinin- and somatostatin-positive GABAergic interneurons. The density of QC labeled hippocampal neurons declined during postnatal development matching the decrease in QC mRNA expression levels. Subcellular double immunofluorescent analysis localized QC within the endoplasmatic reticulum, Golgi apparatus and secretory granules, consistent with a function of QC in protein maturation and/or modification. Our results are in compliance with a role of QC in hypothalamic hormone maturation and suggest additional, yet unidentified QC functions in brain regions relevant for learning and memory which are affected in Alzheimer's disease.     

Developmental changes in expression of small GTPase RhoG mRNA in the rat brain

We have recently reported that RhoG, a member of Rho family small GTPases, is involved in neurite outgrowth in cultured neuronal cells. Here, we report the expression of RhoG mRNA in the developing rat brain by in situ hybridization analysis. At embryonic day 16, RhoG expression was observed throughout the ventricular zone, but was down-regulated in the region at birth. On the other hand, RhoG expression at postnatal day 20 was highly enriched in white matter tracts, including the corpus callosum, the anterior commissure, and the cerebellar white matter, and double-labeling experiments demonstrated that major RhoG-expressing cells in white matter tracts were oligodendrocytes. These results suggest distinct pre- and postnatal roles of RhoG in the development of the central nervous system.     

Developmental expression of orphan g protein-coupled receptor 50 in the mouse brain

Mental disorders have a complex etiology resulting from interactions between multiple genetic risk factors and stressful life events. Orphan G protein-coupled receptor 50 (GPR50) has been identified as a genetic risk factor for bipolar disorder and major depression in women, and there is additional genetic and functional evidence linking GPR50 to neurite outgrowth, lipid metabolism, and adaptive thermogenesis and torpor. However, in the absence of a ligand, a specific function has not been identified. Adult GPR50 expression has previously been reported in brain regions controlling the HPA axis, but its developmental expression is unknown. In this study, we performed extensive expression analysis of GPR50 and three protein interactors using rt-PCR and immunohistochemistry in the developing and adult mouse brain. Gpr50 is expressed at embryonic day 13 (E13), peaks at E18, and is predominantly expressed by neurons. Additionally we identified novel regions of Gpr50 expression, including brain stem nuclei involved in neurotransmitter signaling: the locus coeruleus, substantia nigra, and raphe nuclei, as well as nuclei involved in metabolic homeostasis. Gpr50 colocalizes with yeast-two-hybrid interactors Nogo-A, Abca2, and Cdh8 in the hypothalamus, amygdala, cortex, and selected brain stem nuclei at E18 and in the adult. With this study, we identify a link between GPR50 and neurotransmitter signaling and strengthen a likely role in stress response and energy homeostasis.     

Adenosinergic modulation of caffeine-induced c-fos mRNA expression in mouse brain

The adenosine receptor antagonist, caffeine, transiently induced proto-oncogene c-fos mRNA in mouse brain in a dose-dependent fashion. In situ hybridization revealed that caffeine-induced c-fos expression was high in caudate-putamen and olfactory tubercle at both subconvulsive and convulsive doses. The pattern of c-fos mRNA distribution following caffeine administration differs from that reported after seizures induced by electroconvulsive shock (ECS) or other chemical convulsants, and closely parallels the distribution of adenosine A₂ receptors. Furthermore, the potent adenosine A₂ receptor agonist,5′-N-ethylcarboxamide adenosine (NECA) blocked caffeine-induced c-fos expression whereas the adenosine A₁ receptor ligand, N⁶-cyclohexyladenosine (CHA), had no effect. This study suggests that the caffeine-induced expression of c-fos mRNA may be mediated by the adenosine A₂ receptor in mouse brain.     

Age-dependent differences in flurothyl-induced c-fos and c-jun mRNA expression in the mouse brain

Numerous studies have examined the brain regional distribution of the immediate early gene (IEG), c-fos, following seizures induced by a variety of chemical or electrical provocations in the rat. Very little is known concerning the regional and temporal distribution of IEG expression following seizures in mice, and even less regarding the effects of development. In the present study, seizures of varying severities were induced in immature (postnatal day 17-18) and mature male (postnatal day 55-60) C3H mice with flurothyl, a volatile convulsant. In the immature mouse, neither c-fos nor c-jun mRNA were statistically elevated following any type of acute seizure activity. In the mature mouse, seizures of different severity resulted in differential effects on regional c-fos and c-jun mRNA expression. We conclude that the c-fos and c-jun are not reliable indicators of seizure activity in immature mice, whereas they remain indirect markers of neuronal activity in mature mice.     

Subtle sex differences in vasopressin mRNA expression in the embryonic mouse brain

Arginine vasopressin (AVP) is a neuropeptide which acts centrally to modulate numerous social behaviors. One receptor subtype through which these effects occur is the AVP 1a receptor (AVPR1A). The modulatory effects of Avp via the AVPR1A varies by species as well as sex, since both AVP and the AVPR1A tend to be expressed more prominently in males. Beyond these neuromodulatory effects there are also indications that the AVP system may play a role in early development to, in part, organize sex‐specific neural circuitry that is important to sexually dimorphic social behaviors in adulthood. However, to date, AVP's role in early development is poorly understood, particularly with respect to its differential effect on males and females. In order to determine the timing and distribution of the AVP system in early brain development, we examined the brains of male and female C57BL/6J mice between embryonic day (E) 12.5 and postnatal day (P) 2 and quantified Avp and Avpr1a mRNA using qPCR and AVPR1A protein using receptor autoradiography. The mRNA for Avp was measurable in males and females starting at E14.5, with males producing more than females, while Avpr1a mRNA was found as early as E12.5, with no difference in expression between sexes. AVPR1A binding was observed in both sexes starting at E16.5, and while there were no observed sex differences, binding density and the number of neuroanatomical areas did increase over time. These data are significant as they provide the first whole‐brain characterization of the vasopressin system in the embryonic mouse. Further, these findings are consistent with data from other species, that have documented a sex difference in the vasopressin system during early brain formation.     

Developmental and cell type-specific expression of thyroid hormone transporters in the mouse brain and in primary brain cells

Cellular thyroid hormone uptake and efflux are mediated by transmembrane transport proteins. One of these, monocarboxylate transporter 8 (MCT8) is mutated in Allan-Herndon-Dudley syndrome, a severe mental retardation associated with abnormal thyroid hormone constellations. Since mice deficient in Mct8 exhibit a milder neurological phenotype than patients, we hypothesized that alternative thyroid hormone transporters may compensate in murine brain cells for the lack of Mct8. Using qPCR, Western Blot, and immunocytochemistry, we investigated the expression of three different thyroid hormone transporters, i.e., Mct8 and L-type amino acid transporters Lat1 and Lat2, in mouse brain. All three thyroid hormone transporters are expressed from corticogenesis and peak around birth. Primary cultures of neurons and astrocytes express Mct8, Lat1, and Lat2. Microglia specifically expresses Mct10 and Slco4a1 in addition to high levels of Lat2 mRNA and protein. As in vivo, a brain microvascular endothelial cell line expressed Mct8 and Lat1. 158N, an oligodendroglial cell line expressed Mct8 protein, consistent with delayed myelination in MCT8-deficient patients. Functional T(3)- and T(4)-transport assays into primary astrocytes showed K(M) values of 4.2 and 3.7 μM for T(3) and T(4). Pharmacological inhibition of L-type amino acid transporters by BCH and genetic inactivation of Lat2 reduced astrocytic T(3) uptake to the same extent. BSP, a broad spectrum inhibitor, including Mct8, reduced T(3) uptake further suggesting the cooperative activity of several T(3) transporters in astrocytes.     

Kainic acid upregulates uncoupling protein-2 mRNA expression in the mouse brain

Intraperitoneal injection of kainic acid (KA) in C57BL/6J and 129T2SvEmsJ mice led to a transient induction of uncoupling protein-2 (Ucp2) mRNA expression in several brain regions, which included the CA1 subfield of the hippocampus, the dorsal endopiriform nucleus and the piriform cortex in both strains. In all those regions, levels of Ucp2 mRNA expression, as determined by in situ hybridization, peaked at 24 h and returned to basal levels within 72 h post-injection. The increase in mRNA expression was mainly observed in neurons, with microglial cells displaying only scattered expression of the gene. The neuronal induction of Ucp2 in response to KA was stronger in 129T2SvEmsJ mice than in C57BL/6J, which suggests a role for Ucp2 in excitotoxic challenges and neuroprotection.     

Developmental expression of somatostatin in mouse brain. I. Immunocytochemical studies

The postnatal development of the distribution of somatostatin immunoreactive (SOMLI) neurons and fibers in the forebrain of the Balb/C mouse and their relationship to cholinergic afferents have been examined. SOMLI was first discernable in the hypothalamus on postnatal day (PND) 3 and increased gradually to reach adult levels by PND 30. In the limbic system, SOMLI is detectable at birth. In all other structures of the forebrain, SOMLI could be observed by PND 3 but the distribution, density and morphology of the immunoreactive neurons evolved over the following 2-3 weeks. In general, SOMLI cells and fibers increased for 1-3 weeks after their initial appearance and subsequently declined to achieve adult levels. The distribution pattern of SOMLI elements in adult mouse brain was similar to previous reports in rat with a few notable differences in thalamus, olfactory structures and, to a lesser degree, cortex and hippocampus. The temporal pattern of SOMLI expression in extrahypothalamus forebrain regions, during development, suggests a role of this peptide in differentiation and synapse formation. Such an hypothesis receives further support from neonatal lesions of the basal forebrain which resulted in transient cortical cholinergic deafferentation, a delay of cortical differentiation and a transient increase in the number of SOMLI cells in cortex.     

Developmental expression of glycine receptor subunits in rat cerebellum

The distribution of the glycine receptor subunits alpha1-3 and beta in the developing rat cerebellum was studied from postnatal day 1 to adulthood by means of quantitative RT-PCR and immunohistochemistry. qRT-PCR of postnatal cerebella indicated the presence of mRNA for each subunit, with a relative expression of alpha2>alpha3>alpha1>beta. The immunohistochemistry indicated a strong alpha2 signal in the Purkinje cells, internal and external granular layers. The alpha1-3 subunits had weak signals in the Purkinje cells and molecular layer. The alpha1 subunit was expressed at a low level and was also found in the white matter. The function of these receptors in neuronal and glial plasma membranes in early postnatal development remains to be determined.     

Differential expression of BDNF mRNA splice variants in mouse brain and immune cells

For the neurotrophin BDNF several splice variants were recently described. We analyzed the expression of mBDNF mRNA splice variants in cells of the immune system in comparison to the central nervous system (CNS). Whereas all splice variants are expressed in the CNS, only mBDNF 3 mRNA could be detected in primary and secondary lymphoid organs as well as in purified T cells and macrophages. After activation, only mBDNF 3 mRNA expression was upregulated in T cells without the additional appearance of other mBDNF splice variants. Therefore, mBDNF mRNA is differentially regulated in the CNS and the immune system opening the possibility of specific manipulation of mBDNF expression in immune cells without globally affecting mBDNF in the CNS.     

Effects of D-cycloserine and midazolam on the expression of the GABA-A alpha-2 receptor subunits in brain structures of fear conditioned rats

The role of the GABA-A alpha-2 receptor subunit in the basolateral amygdala (BLA), dentate gyrus of the hippocampus (DG) and prefrontal cortex (M2 area) during a fear session (performed one week after the conditioned fear test), was studied. We employed a model of high (HR) and low anxiety (LR) rats divided according to their conditioned freezing response. Pretreatment of rats with d-cycloserine immediately before the fear session attenuated fear response in HR and LR rats and increased the density of alpha-2 subunits in the BLA, M2 area and DG of HR animals. The less potent behavioural influence of midazolam (in HR group only) was linked to the increased expression of alpha-2 subunit in M2 area and DG. These results support a role of the GABA-A receptor alpha-2 subunit in processing of emotional cortico-hippocampal input to the BLA.     

Developmental expression of somatostatin in mouse brain. II. In situ hybridization

The distribution and the levels of expression of preprosomatostatin (PPSOM) mRNA were examined during pre- and postnatal development of the mouse brain using the in situ hybridization technique. The signal obtained by in situ hybridization of embryonic tissues at day 14 and day 17 of gestation was highest over the neurons of the pyriform cortex, amygdala, and entopeduncular nucleus. The signal was very low over cells of the neocortex and the developing hippocampal formation. The density of grains overlying the neurons of the amygdala and pyriform cortex continued to be high during early postnatal life, but decreased as the animals became adults. A progressive increase of PPSOM mRNA expression was observed in postnatal animals in the stratum oriens and dentate gyrus of the hippocampal formation. In the cerebral cortex and striatum, the number of these neurons became maximal between postnatal weeks 1 and 3. In the diencephalon, the highest densities of grains were found over neurons in the nucleus reticularis thalami and zona incerta at postnatal day 21; these levels declined slightly thereafter. The cells of the periventricular nucleus of the hypothalamus had high densities of grains as early as postnatal week 1 and continued to have high densities of grains in adult animals. These patterns of hybridization density parallelled the distribution of SOM-like immunoreactivity in the mouse brain. When PPSOM mRNA expression was examined in the cerebral cortices of mice that received lesions of the nucleus basalis of Meynert as neonates, a transient increase in the number of cells expressing PPSOM mRNA was observed in the frontoparietal cortex ipsilateral to the lesion at postnatal day 10, but not at postnatal day 30. Importantly, the density of grains over the individual cells was not altered in lesioned animals at these two ages.