Localization and ontogeny of the orphan receptor OR-1 in the rat brain

This study describes the expression of the OR-1 orphan receptor in embryonic, postnatal, and adult brain tissue studied byin situ hybridization. This newly characterized member of the nuclear receptor superfamily functions as a modulator of retinoic acid and thyroid hormone signalling by influencing gene activation by these hormones from a distinct promoter region. In the fetal brain OR-1 mRNA was observed from E13–E16 in the developing pons, tegmentum, pontine flexure, medulla, inferior and superior colliculi, cerebellum, hippocampus, thalamus, striatum, and cortical plate. At E18, OR-1 was expressed in the hippocampus, cerebellum, ventricular layer of the developing cortex and cortical plate, striatum, and olfactory bulb. In the E21 to early postnatal brain the highest expression of OR-1 mRNA was seen in the hippocampus, cerebellum, striatum, and olfactory bulb. The expression of OR-1 in the cerebellum increased during postnatal development and by d P21 OR-1 mRNA had reached the levels present in the adult in the cerebellar cortex. In the adult brain the highest expression of OR-1 mRNA was observed in the Ca1 area of the hippocampus and the cerebellar cortex. We conclude that OR-1 is widely expressed in the fetal brain, whereas in the postnatal and adult brains OR-1 mRNA is more discretely localized, and that the amount of OR-1 mRNA increases in the cerebellum during postnatal development. The results of this study suggest that, in the fetal brain, OR-1 has a spatially widespread role in modulating gene activation by retinoids and thyroid hormone, whereas in the adult brain this modulation occurs only in distinct neuronal populations.     

Expression of cadherin-8 mRNA in the developing mouse central nervous system

The expression of cadherin-8 was mapped by in situ hybridization in the embryonic and postnatal mouse central nervous system (CNS). From embryonic day 18 (E18) to postnatal day 6 (P6), cadherin-8 expression is restricted to a subset of developing brain nuclei and cortical areas in all major subdivisions of the CNS. The anlagen of some of the cadherin-8-positive structures also express this molecule at earlier developmental stages (E12.5–E16). The cadherin-8-positive neuroanatomical structures are parts of several functional systems in the brain. In the limbic system, cadherin-8-positive regions are found in the septal region, habenular nuclei, amygdala, interpeduncular nucleus, raphe nuclei, and hippocampus. Cerebral cortex shows expression in several limbic areas at P6. In the basal ganglia and related nuclei, cadherin-8 is expressed by parts of the striatum, globus pallidus, substantia nigra, entopeduncular nucleus, subthalamic nucleus, zona incerta, and pedunculopontine nuclei. A third group of cadherin-8-positive gray matter structures has functional connections with the cerebellum (superior colliculus, anterior pretectal nucleus, red nucleus, nucleus of posterior commissure, inferior olive, pontine, pontine reticular, and vestibular nuclei). The cerebellum itself shows parasagittal stripes of cadherin-8 expression in the Purkinje cell layer. In the hindbrain, cadherin-8 is expressed by several cranial nerve nuclei. Results from this study show that cadherin-8 expression in the embryonic and postnatal mouse brain is restricted to specific developing gray matter structures. These data support the idea that cadherins are a family of molecules whose expression provides a molecular code for the regionalization of the developing vertebrate brain. J. Comp. Neurol. 387:291–306, 1997. © 1997 Wiley-Liss, Inc.     

Expression of the orphan receptor TR4 during brain development of the rat

The orphan receptor TR4, member of the nuclear hormone receptor family, is related to the orphan receptors TR2, COUP-TFI and ARP-1, and was originally cloned from the adult rat brain. The latter two orphan receptors have been implicated in central nervous system (CNS) development. To investigate a possible role for TR4 in brain development, expression of TR4 was studied in rat embryos. At embryonic days 14.5 and 19.5, high expression of TR4 was found in the CNS, while low expression was detected throughout the embryo. In postnatal rats, TR4 was mainly expressed in the hippocampus and cerebellum, resembling the expression pattern found in adult brain. These data show that like COUP-TFI and ARP-1, expression of TR4 becomes restricted to distinct areas. In adult brain, TR4 is predominantly expressed in granule cells of both hippocampus and cerebellum. The data suggest a possible role for TR4 during proliferation and maturation of brain structures.     

Immunolocalization of NFATc4 in the adult mouse brain

NFATc4 has recently been identified as playing an important role in variety of activity-dependent neuronal processes, including hippocampal plasticity, axonal growth, neuronal survival, and apoptosis. However, a systematic study examining the distribution of NFATc4 within the nervous system has yet to be conducted. With this in mind, we sought to determine the regional localization of NFATc4 within the adult mouse brain. Interestingly, NFATc4 was expressed broadly, but not uniformly, throughout various brain structures. The highest levels of NFATc4 expression were localized to the hippocampus, olfactory bulb, and various hypothalamic nuclei. Other brain regions that expressed NFATc4 included the cerebellum, striatum, globus pallidus, amygdala, neocortex, and brainstem nuclei. Given NFATc4's widespread expression, these results are consistent with the notion that NFATc4 may underlie activity-dependent neuronal plasticity throughout the adult brain.     

Expression of Na(+)-K(+)-Cl(-) cotransporter in rat brain during development and its localization in mature astrocytes

Na(+)-K(+)-Cl(-) cotransporter has been proposed to play an important role in the regulation of intracellular Cl(-) concentration in neurons during development. In this study, the expression pattern of the cotransporter in different regions of rat brain was examined at birth (P0), postnatal days 7 (P7), P14, P21, and adult by Western blotting analysis. In cortex, thalamus, cerebellum and striatum, the cotransporter expression level was low at P0 and significantly increased at P14 (P<0.05). The expression peaked at P21 and was maintained at the same level in adulthood. However, in hippocampus, a peak level of the cotransporter expression was detected in adult brain. The immunocytochemistry study of adult rat brain revealed that an intense staining of the Na(+)-K(+)-Cl(-) cotransporter protein was observed in dendritic processes of CA1-CA3 hippocampal pyramidal neurons. In contrast, abundant immuno-reactive signals of the cotransporter were found in somata of thalamic nucleus. Immunofluorescence double staining demonstrates that the Na(+)-K(+)-Cl(-) cotransporter was expressed in astrocytes within cortex, corpus callosum, hippocampus and cerebellum. In addition, co-localization of the cotransporter and glial fibrillary acidic protein (GFAP), or with aquaporin 4, was found in perivascular astrocytes of cortical cortex and white matter. The results indicate that a time-dependent expression of the Na(+)-K(+)-Cl(-) cotransporter protein occurs not only in cortex but also in hippocampus, striatum, thalamus and cerebellum. In addition, the cotransporter is expressed in astrocytes and perivascular astrocytes of adult rat brain.     

Developmental expression of voltage-gated potassium channel beta subunits.

Expression of potassium channel beta subunits (Kvbeta) was determined in the developing mouse CNS using an antiserum against an amino acid sequence present in the C-terminus of Kvbeta1, Kvbeta2, and Kvbeta3. Using the anti-Kvbeta antiserum, we determined that Kvbeta expression is restricted to the spinal cord and dorsal root ganglia in the embryonic CNS. At birth, Kvbeta expression is detected in brainstem and midbrain nuclei, but was not detected in the hippocampus, cerebellum or cerebral cortex. During the first postnatal week, Kvbeta expression is present in hippocampal and cortical pyramidal cells and in cerebellar Purkinje cells. Expression of Kvbeta subunits reaches adult levels by the third postnatal week in all of the brain regions examined. A rabbit antiserum directed against a unique peptide sequence in the N-terminus of the Kvbeta1 protein demonstrates that this subunit displays a novel expression pattern in the developing mouse brain. Kvbeta1 expression is high at birth in all brain regions examined and decreases with age. In contrast, Kvbeta2 expression is low at birth and increases with age to reach adult levels by the third postnatal week. These findings support the notion that the differential regulation of distinct potassium channel beta subunits, in the developing mouse nervous system, may confer the functional diversity required to mediate both neuronal survival and maturation.     

Expression of the calmodulin-dependent protein phosphatase, calcineurin, in rat brain: developmental patterns and the role of nigrostriatal innervation.

The distribution of neurons expressing the calmodulin-dependent protein phosphatase, calcineurin (CN) was characterized in developing and adult rat brain using a combination of immunocytochemical, immunoblot and in situ hybridization approaches. Immunoblot analysis revealed a strong increase postnatally in CN protein expression. Four differently-charged isoforms of CN were observed in adult brain with apparent regional differences in isoform expression. Immunocytochemistry showed highest levels of CN in hippocampus, striatum, substantia nigra, amygdala and septal nuclei with immunoreactivity first appearing in striatum and septal nuclei, followed by hippocampus, neocortex and limbic structures. In situ hybridization demonstrated that mRNA for the catalytic subunit of CN was seen as early as postnatal day (PND) 1 in striatum, cortex and hippocampus. Since immunoreactivity was not detectable until day 4, this suggests that mRNA expression may precede that of protein by several days in these regions. Lesioning of developing and adult nigrostriatal dopamine neurons either with 6-hydroxydopamine or by surgical hemitransection had little effect on expression of CN, suggesting that CN expression is not influenced transsynaptically by dopamine. Collectively, these findings demonstrate that CN protein and mRNA expression are subject to regional and temporal control during brain development suggesting that specific synaptic connections may influence CN gene expression. However, in striatum, dopaminergic innervation does not appear to affect CN levels.     

Characterization of TROY/TNFRSF19/TAJ-expressing cells in the adult mouse forebrain

A member of the tumor necrosis factor receptor superfamily (TNFRSF), TROY/TNFRSF19/TAJ, is highly expressed in the brain of adult mice. Northern blot analysis using mRNA taken from regions of the adult CNS showed the expression of TROY in all regions examined, including the olfactory bulb, cerebral cortex, striatum, and hippocampus. In situ hybridization and immunohistochemistry revealed that TROY mRNA and protein were strongly expressed in the rostral migratory stream (RMS) and subventricular zone (SVZ) of adult mice. In the adult SVZ, some glial fibrillary acidic protein (GFAP)-positive cells (type B cells) are thought to be multipotent neural stem cells. These type B cells divide slowly and generate epidermal growth factor receptor (EGFR)-positive transit-amplifying precursor cells (type C cells) in the presence of epidermal growth factor (EGF). Type C cells give rise to neuron-specific class III beta-tubulin (TuJ1)-positive neuroblasts (type A cells) that migrate to the olfactory bulb along the RMS. TROY-expressing cells were GFAP-positive, EGFR-positive, and TuJ1-negative in the adult SVZ. From these findings, TROY appears to be expressed in type B and type C cells, but not in type A cells, which was supported by immunoelectron microscopy. In addition, TROY was expressed in GFAP-positive astrocytes of the various regions, such as the cerebral cortex, striatum, and hippocampus. Thus, TROY was expressed in uncommitted precursor cells and astroglial lineage cells, suggesting that TROY plays some roles in the regulation of gliogenesis in the adult CNS.     

Developmental expression of Neuregulin-3 in the rat central nervous system

Neuregulin-3 (Nrg3) is a member of the Nrg family of growth factors identified as risk factors for schizophrenia. There are three Nrgs expressed in the nervous system (Nrg1-3) and of these Nrg1 has been the best characterized. To set the groundwork for elucidating neural roles for Nrg3, we studied its expression in the rat brain at both the RNA and protein levels. Using an antibody developed against Nrg3, we observed a developmental increase of Nrg3 protein expression from embryonic stages to adulthood and determined that it carries O-linked carbohydrates. In cortical neuronal cultures, transfected Neuro2a cells, and brain tissue sections Nrg3 protein was localized to the soma, neurites, and to the Golgi apparatus, where it is prominently expressed. Nrg3 was detected in excitatory, GABAergic and parvalbumin-expressing inhibitory neurons while expression in glia was limited. Nrg3 mRNA and protein were widely expressed during both embryonic and postnatal ages. At E17, Nrg3 was detected within the cortical plate and ventricular zone suggesting possible roles in cell proliferation or migration. At postnatal ages, Nrg3 was abundantly expressed throughout the cerebral cortex and hippocampus. Multiple thalamic nuclei expressed Nrg3, while detection in the striatum was limited. In the cerebellum, Nrg3 was found in both Purkinje cells and granule neurons. In the rodent brain, Nrg3 is the most abundantly expressed of the Nrgs and its patterns of expression differ both temporally and spatially from that of Nrg1 and Nrg2. These findings suggest that Nrg3 plays roles that are distinct from the other Nrg family members.     

Habrec1, a Novel Serine/Threonine Kinase TGF-β Type I-like Receptor, Has a Specific Cellular Expression Suggesting Function in the Developing Organism and Adult Brain

Members of the TGF-β superfamily signal through a dual receptor system consisting of a type II receptor protein kinase that binds the ligand, after which this complex associates with a type I receptor to mediate intracellular signaling. In mammals, six type I and five type II receptors mediating responses to different TGF-β family members have been identified to date. Using primers from conserved regions of the protein kinase domain of the serine/threonine kinase receptors in a low-stringency polymerase chain reaction-based screening procedure, and deselecting known receptors with colony hybridization, we now report cloning a novel receptor member. The novel receptor was found in a cDNA library prepared from the habenular nucleus area and was designated Habrec1. Although only a partial sequence is available, it fits the criteria for a TGF-β type I serine/threonine kinase receptor.In situhybridization of Habrec1 reveals mRNA expression in several distinct areas of the developing central nervous system, including cortex cerebri, cerebellum, hippocampus, striatum, and thalamic nuclei. Expression is also seen in the anterior pituitary. In the periphery, strong expression prenatally includes brown fat, the gastrointestinal tract, liver, pancreas, thymus, and nasal cavity epithelium. In the adult brain Habrec1 mRNA is prominently found in cerebellum, cortex cerebri, and striatum, but at lower levels in several additional areas. We conclude that Habrec1 is a member of the TGF-β type I receptor family with expression patterns in the developing animal, suggesting specific functions in and outside the nervous system, and in the adult CNS, suggesting roles in both cortical and subcortical brain circuitry.     

Immunohistochemical localization of an inositol 1,4,5-trisphosphate receptor, P400, in neural tissue: studies in developing and adult mouse brain.

The immunohistochemical localization of P400/inositol 1,4,5-trisphosphate (InsP3) receptor protein was studied in developing and adult mouse brain by using monoclonal antibodies. The developmental expression pattern of P400/InsP3 receptor protein differed among different classes of neurons. It was first detected in the somata of immature Purkinje cells at embryonic day 17, in the ventrolateral region of the posterior vermis in the cerebellum. Axonal immunoreactivity within the cerebellar nuclei was first present at postnatal day 3. Neurons in the retrosplenial cortex, the anterior olfactory nucleus, and the CA1 region of the hippocampus expressed immunoreactivity earlier than other regions of the brain. In the adult brain, not only the Purkinje cell but also many other types of cells in many areas of the brain expressed P400/InsP3 receptor, though to a lesser extent. These included the neurons in the striatum, globus pallidus, nucleus accumbens septi, anterior olfactory nucleus, olfactory tubercle, precommissural hippocampus, hippocampus, substantia nigra, cerebral cortex, pons, and certain hypothalamic nuclei. Forebrain cortical regions that receive afferents from the olfactory bulb, such as the anterior olfactory nucleus, olfactory tubercle, prepiriform cortex, entorhinal cortex, and amygdala, exhibited distinct immunoreactivity, while olfactory bulb was almost devoid of staining. Immunoreactivity in the axonal pathways was also found in the limbic-hypothalamic pathways, strionigral projection, and part of the corpus callosum. Results of Western blot analysis and 3H-InsP3 binding assay were consistent with the qualitative regional differences of immunoreactivity demonstrated by immunohistochemical study. The location of InsP3 receptor in the brain correlates well with the InsP3 binding sites demonstrated by an autoradiographic study.     

Expression of DA11, a neuronal-injury-induced fatty acid binding protein,coincides with axon growth and neuronal differentiation during central nervous system development

DA11 is the first fatty acid binding protein (FABP) for which gene expression has been shown to be upregulated following neuronal injury in the adult peripheral nervous system. To understand better the potential regulatory role(s) of this unique FABP in axonal growth and neuronal differentiation, we undertook a temporal and spatial study of DA11 gene expression in the developing rat central nervous system (CNS). Transient upregulation of DA11 mRNA and protein levels in CNS tissues were quantified by Northern blot hybridization and Western immunoblot analyses at different developmental ages. Homogenates of embryonic and neonatal cerebral cortex, cerebellum, brainstem, and hippocampal tissues contained 100-fold more DA11 mRNA and protein than corresponding adult tissues. Significant increase in DA11 mRNA was observed as early as embryonic day (E) 14 in cerebral cortex and cerebellum and E19 in brain stem and hippocampus. Postnatal levels of DA11 remained elevated through postnatal day (P) 10 in cerebral cortex, P14 in brain stem and hippocampus, and P20 in cerebellum. Localization of DA11-like immunoreactivity to specific CNS tissues, cell types, and intracellular compartments at P9 revealed a spatial pattern of neuronal expression different than that reported for other FABPs. DA11 protein was detected in the nucleus, cytoplasm, axons, and dendrites of differentiating neurons in cerebral cortex, hippocampus, cerebellum, brain stem, spinal cord, and olfactory bulb. The strong association of DA11 gene expression with development throughout the CNS suggests that this unique FABP plays an important role in axonal growth and neuronal differentiation in many different neuronal populations. J. Neurosci. Res. 48:551–562, 1997. © 1997 Wiley-Liss Inc.     

Age-related changes of the nitric oxide system in the rat brain

This work examines the age-related changes of the NO pathway in the central nervous system (CNS), analyzing nitric oxide synthase (NOS) isoform expression, the level of nitrotyrosine-modified proteins, and the NOS activity in the cerebral cortex, decorticated brain (basal ganglia, thalamus, hypothalamus, tegtum and tegmentum) and cerebellum of young, adult and aged rats. Our data demonstrate that the different NOS isoforms are not uniformly expressed across the CNS. In this sense, the nNOS and eNOS isoenzymes are expressed mainly in the cerebellum and decorticated brain, respectively, while the iNOS isoenzyme shows the highest level in cerebellum. Concerning age, in the cerebral cortex nNOS significantly increased its expression only in adult animals; meanwhile, in the cerebellum the eNOS expression decreased whereas iNOS increased in adult and aged rats. No age-related changes in any isoform were found in decorticated brain. NOS activity, determined by nitrate plus nitrite quantification, registered the highest levels in the cerebellum, where the significant increase detected with aging was probably related to iNOS activity. The number of nitrotyrosine-modified immunoreactive bands differed among regions; thus, the highest number was detected in the decorticated brain while the cerebellum showed the least number of bands. Finally, bulk protein nitration increased in cerebral cortex only in adult animal. No changes were found in the decorticated brain, and the decrease detected in the cerebellum of aged animals was not significant. According to these results, the NO pathway is differently modified with age in the three CNS regions analyzed.     

Insulin-like growth factor-II/mannose-6-phosphate receptor: widespread distribution in neurons of the central nervous system including those expressing cholinergic phenotype

The insulin-like growth factor-II/mannose-6-phosphate (IGF-II/M6P) receptor is single transmembrane glycoprotein that plays a critical role in the trafficking of lysosomal enzymes and the internalization of circulating IGF-II. At present, there is little information regarding the cellular distribution of the IGF-II/M6P receptor within the adult rat brain. With the use of immunoblotting and immunocytochemical methods, we found that the IGF-II/M6P receptor is widely but selectively expressed in all major brain areas, including the olfactory bulb, striatum, cortex, hippocampus, thalamus, hypothalamus, cerebellum, brainstem, and spinal cord. Intense IGF-II/M6P receptor immunoreactivity was apparent on neuronal cell bodies within the striatum, deeper layers (layers IV and V) of the cortex, pyramidal and granule cell layers of the hippocampal formation, selected thalamic nuclei, Purkinje cells of the cerebellum, pontine nucleus and motoneurons of the brainstem as well as in the spinal cord. Moderate neuronal labeling was evident in the olfactory bulb, basal forebrain areas, hypothalamus, superior colliculus, midbrain areas, granule cells of the cerebellum and in the intermediate regions of the spinal gray matter. We also observed dense neuropil labeling in many regions, suggesting that this receptor is localized in dendrites and/or axon terminals. Double-labeling studies further indicated that a subset of IGF-II/M6P receptor colocalizes with cholinergic cell bodies and fibers in the septum, striatum, diagonal band complex, nucleus basalis, cortex, hippocampus, and motoneurons of the brainstem and spinal cord. The observed widespread distribution and colocalization of IGF-II/M6P receptor in the adult rat brain provide an anatomic basis to suggest a multifunctional role for the receptor in a wide-spectrum of central nervous system neurons, including those expressing a cholinergic phenotype.     

Gas6, a ligand for the receptor protein-tyrosine kinase Tyro-3, is widely expressed in the central nervous system

Gas6 (growth arrest specific gene-6) is a ligand for members of the Axl subfamily of receptor protein-tyrosine kinases. One of these receptors, Tyro-3, is widely expressed in the central nervous system. We have used biochemical and histological techniques, including in situ hybridization, to determine the expression patterns of Gas6 mRNA and protein during development. Gas6 is widely expressed in the rat central nervous system (CNS) beginning at late embryonic stages and its levels remain high in the adult. Gas6 is detected as a single 85 kDa protein, which is encoded by a single 2.5 kb mRNA species. At embryonic day 14 it is detected in the heart, blood vessels, testes, choroid plexus, and in the ventral spinal cord. In the adult, Gas6 is expressed in the cerebral cortex, (predominantly in layer V), the piriform cortex, and the hippocampus (areas CA1, CA3 and the dentate gyrus). It is also expressed in thalamic and hypothalamic structures, the midbrain, and in a subset of motor and trigeminal nuclei. In the cerebellum, it is expressed in Purkinje neurons and deep cerebellar nuclei. Protein S, a protein related to Gas6, is only detected at low levels in the CNS. The spatial and temporal profiles of Gas6 expression suggest that it could potentially serve as the physiologically relevant ligand for Tyro-3 in the postnatal rat nervous system.