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PubMed2003

The proton/amino acid cotransporter PAT2 is expressed in neurons with a different subcellular localization than its paralog PAT1.

The new member of the mammalian amino acid/auxin permease family, PAT2, has been cloned recently and represents an electrogenic proton/amino acid symporter. PAT2 and its paralog, PAT1/LYAAT-1, are transporters for small amino acids such as glycine, alanine, and proline. Our immunodetection studies revealed that the PAT2 protein is expressed in spinal cord and brain. It is found in neuronal cell bodies in the anterior horn in spinal cord and in brain stem, cerebellum, hippocampus, hypothalamus, rhinencephalon, cerebral cortex, and olfactory bulb in the brain. PAT2 is expressed in neurons positive for the N-methyl-d-aspartate subtype glutamate receptor subunit NR1. PAT2 is not found in lysosomes, unlike its paralog PAT1, but is present in the endoplasmic reticulum and recycling endosomes in neurons. PAT2 has a high external proton affinity causing half-maximal transport activation already at a pH of 8.3, suggesting that its activity is most likely not altered by physiological pH changes. Transport of amino acids by PAT2 activity is dependent on membrane potential and can occur bidirectionally; membrane depolarization causes net glycine outward currents. Our data suggest that PAT2 contributes to neuronal transport and sequestration of amino acids such as glycine, alanine, and/or proline, whereby the transport direction is dependent on the sum of the driving forces such as substrate concentration, pH gradient, and membrane potential.

Amino Acid Transport Systems↗
PubMed2005

A novel phospholipase C, PLC(eta)2, is a neuron-specific isozyme.

Twelve phospholipase C (PLC) isozymes have been cloned so far, and they are divided into six classes, beta-, gamma-, delta-, epsilon-, zeta-, and eta-type, on the basis of structure and activation mechanisms. Here we report the identification of a novel PLC isozyme, PLC(eta)2. PLC(eta)2 is composed of conserved domains including pleckstrin homology, EF-hand, X and Y catalytic, and C2 domains and the isozyme-specific C-terminal region. PLC(eta)2 consists of 1164 amino acids with a molecular mass of 125 kDa. The PLC activity of PLC(eta)2 was more sensitive to calcium concentration than the PLC activity of the PLCdelta-type enzyme, which is thought to be the most calcium-sensitive PLC. Immunofluorescence analysis showed that PLC(eta)2 was localized predominantly to the plasma membrane at resting state via the pleckstrin homology domain. This observation was supported by Western blot analysis of cytosol and membrane fractions. In addition, expression of PLC(eta)2 was detected after birth and showed a restricted distribution in the brain; it was particularly abundant in the hippocampus, cerebral cortex, and olfactory bulb. The pattern was similar to that of the neuronal marker microtubule-associated protein 2 by Western blot. Furthermore, in situ hybridization showed positive signals for PLC(eta)2 in pyramidal cells of the hippocampus. Finally, we found that PLC(eta)2 was expressed abundantly in neuron-containing primary culture but not in astrocyte-enriched culture. These results indicate that PLC(eta)2 is a neuron-specific isozyme that may be important for the formation and/or maintenance of the neuronal network in the postnatal brain.

Amino Acid Sequence↗
PubMed1999

Some introductory notes on the organization of the forebrain in tetrapods.

As an introduction to the main theme of this conference an overview of the organization of the tetrapod forebrain is presented with emphasis on the telencephalic representation of sensory and motor functions. In all classes of tetrapods, olfactory, visual, octavolateral, somatosensory and gustatory information reaches the telencephalon. Major differences exist in the telencephalic targets of sensory information between amphibians and amniotes. In amphibians, three targets are found: the lateral pallium for olfactory input, the medial pallium for visual and multisensory input, and the lateral subpallium for visual, octavolateral and somatosensory information. The forebrains of reptiles and mammals are similar in that the dorsal surface of their cerebral hemisphere is formed by a pallium with three major segments: (a) an olfactory, lateral cortex; (b) a 'limbic' cortex that forms the dorsomedial wall of the hemisphere, and (c) an intermediate cortex that is composed entirely of isocortex in mammals, but in reptiles (and birds) consists of at least part of the dorsal cortex (in birds the Wulst) and a large intraventricular protrusion, i.e. the dorsal ventricular ridge. In birds, the entire lateral wall of the hemisphere is involved in this expansion. The intermediate pallial segment receives sensory projections from the thalamus and contains modality-specific sensory areas in reptiles, birds and mammals. The most important differences between the intermediate pallial segment of amniotes concern motor systems.

Animals↗
PubMed1999

PQBP-1, a novel polyglutamine tract-binding protein, inhibits transcription activation by Brn-2 and affects cell survival.

A novel gene, designated PQBP-1, which encodes a 265 residue protein that binds to the polyglutamine tract of the brain-specific transcription factor Brn-2, was identified. PQBP-1, which also interacts with the polyglutamine tract of triplet repeat disease gene products, binds with a higher affinity to an expanded polyglutamine tract. PQBP-1 has several functional domains, including hepta- and di-amino acid repeat sequences rich in polar residues essential for its interaction with the polyglutamine tract, a WWP/WW domain which binds to proline-rich motifs in other proteins, a putative nuclear localization signal sequence and a C2domain implicated in Ca2+-dependent phospholipid signaling. PQBP-1 is located in the nucleus and inhibits transcriptional activation by Brn-2. Overexpression of PQBP-1 in P19 embryonic carcinoma cells suppresses their growth rate and enhances their susceptibility to various stresses including serum deprivation, retinoic acid treatment and UV irradiation. Northern blot and in situ hybridization analyses revealed that PQBP-1 is a ubiquitous protein and is expressed primarily in neurons throughout the brain, with abundant levels in hippocampus, cerebellar cortex and olfactory bulb. These results suggest that PQBP-1 mediates important cellular functions under physiological and pathological conditions via its interaction with polyglutamine tracts.

Amino Acid Sequence↗
PubMed2003

Estrogen synthesis in fetal sheep brain: effect of maternal treatment with an aromatase inhibitor.

The aim of the present study was to determine whether the fetal lamb brain has the capacity to aromatize androgens to estrogens during the critical period for sexual differentiation. We also determined whether administration of the aromatase-inhibitor 1,4,6-androstatriene-3,17-dione (ATD) could cross the placenta and inhibit aromatase activity (AA) in fetal brain. Eight pregnant ewes were utilized. On Day 50 of pregnancy, four ewes were given ATD-filled Silastic implants, and the other four ewes received sham surgeries. The fetuses were surgically delivered 2 wk later (Day 64 of gestation). High levels of AA (0.8-1.4 pmol/h/mg protein) were present in the hypothalamus and amygdala. Lower levels (0.02-0.1 pmol/h/mg protein) were measured in brain stem regions, cortex, and olfactory bulbs. The Michaelis-Menten dissociation constant (K(m)) for aromatase in the fetal sheep brain was 3-4 nM. No significant sex differences in AA were observed in brain. Treatment with ATD produced significant inhibition of AA in most brain areas but did not significantly alter serum profiles of the major sex steroids in maternal and fetal serum. Concentrations of testosterone in serum from the umbilical artery and vein were significantly greater in male than in female fetuses. No other sex differences in serum steroids were observed. These data demonstrate that high levels of AA are found in the fetal sheep hypothalamus and amygdala during the critical period for sexual differentiation. They also demonstrate that AA can be inhibited in the fetal lamb brain by treating the mother with ATD, without harming fetal development.

Androgens↗
PubMed1994

Neuronal cell lineage in the vertebrate central nervous system.

Fundamental, yet unresolved, issues in developmental neurobiology concern the relative influence of genetic vs. epigenetic factors in determining cell phenotype and establishing positional relationships among clonally related cells in the developing and mature vertebrate central nervous system (CNS). Advances in our understanding of how cells acquire their identity awaited a means to introduce lineage tracers into dividing cells of the developing CNS where the progenitor cells, which are situated in a neuroepithelial layer adjacent to the ventricles, generally are small and inaccessible. The technique of retroviral-mediated gene transfer, whereby a heritable, easily detectable marker such as the gene for lacZ is integrated into the DNA of individual progenitor cells, has been used to analyze the lineage relationships of cells in the CNS and to derive the types of progenitor cells in the proliferative zones at different developmental stages. Collectively, these studies indicate that the CNS uses more than one strategy to achieve cell diversity. The analysis of the phenotypic composition of the cells within a clone indicates that there are predominantly separate progenitor cells for each of the main cell types comprising the cerebral cortex by the onset of cortical neurogenesis, although in other systems mostly multipotential progenitor cells persist throughout neurogenesis. Here I will compare and contrast the inferred role of cell lineage in the developing cerebral cortex and olfactory bulb, where postmitotic neurons migrate relatively long distances from their site of generation to their final destination, with that in other regions of the CNS in which the displacement of postmitotic neurons from their birthplace is significantly less.

Animals↗
PubMed1994

Time course of inhibition of brain nitric oxide synthase by 7-nitro indazole.

7-Nitro indazole (7-NI) inhibits rat striatal, cerebellar, hippocampal, cerebral cortex and olfactory bulb nitric oxide synthase (NOS) in vitro with IC50 values of 0.68 +/- 0.01 microM, 0.64 +/- 0.03 microM, 1.53 +/- 0.05 microM, 0.93 +/- 0.04 microM and 1.05 +/- 0.02 microM respectively (n = 6). Intraperitoneal (i.p.) or oral administration of 7-NI (30 mg kg-1) to rats inhibited NOS enzyme activity measured ex vivo in all five brain regions (n = 5-6). NOS inhibition (maximal effect, 0.5 h post-injection) was transient with complete recovery at either 4 h (oral administration) or 24 h (i.p. administration). Repeated i.p. injection of 7-NI (30 mg kg-1, every 4 h for 20 h) inhibited NOS enzyme activity at 24 h by 51-61% in all brain regions. The relatively transient NOS inhibitory effect of 7-NI following parenteral administration should be taken into account when using this drug to evaluate the central effects of nitric oxide.

Administration, Oral↗
PubMed2001

Immunological identification of the mammalian H3 histamine receptor in the mouse brain.

Affinity-purified antibodies raised against the peptide sequence H3 (349-358) receptor specifically recognized two protein species with Mr 62,000 and 93,000 in adult mouse forebrain membranes. Both immunoreactive species were suppressed greatly by preincubation of the antibody with the respective peptide. Immunohistochemical analysis using affinity-purified anti-H3 (349-358) antibodies yielded a high degree of coincidence with ligand-autoradiographical information, with high levels detected in the CA3 and dentate gyrus of the hippocampus, laminae V of the cerebral cortex, the olfactory tubercle, Purkinje cell layer of the cerebellum, substantia nigra, globus pallidus, thalamus and striatum. This study suggests further biochemical evidence for multiple H3 receptor subtypes and the widespread distribution of the H3 receptor in the mammalian brain.

Age Factors↗
PubMed2006

A novel high channel-count system for acute multisite neuronal recordings.

Multisite recording represents a suitable condition to study microphysiology and network interactions in the central nervous system and, therefore, to understand brain functions. Several different materials and array configurations have been proposed for the development of new probes utilized to record brain activity from experimental animal models. We describe new multisite silicon probes that broaden the currently available application base for neuroscientists. The array arrangement of the probes recording sites was extended to increase their spatial resolution. Probes were integrated with a newly developed electronic hardware and novel software for advanced real-time processing and analysis. The new system, based on 32- and 64-electrode silicon probes, proved very valuable to record field potentials and single unit activity from the olfactory-limbic cortex of the in vitro isolated guinea-pig brain preparation and to acutely record unit activity at multiple sites from the cerebellar cortex in vivo. The potential advantages of the new system in comparison to the currently available technology are discussed.

Action Potentials↗
PubMed1994

A human POU domain gene, mPOU, is expressed in developing brain and specific adult tissues.

POU domain genes constitute a family of transcription factors that exhibit distinct temporal and spatial patterns of expression. To investigate the possible functions that POU proteins may have in muscle development we have isolated four novel POU-domain-encoding sequences from human muscle tissue. One of these sequences, referred to as mPOU, encodes a new member of subclass VI of the POU family. In the embryo, mPOU is expressed exclusively in the developing brain, whereas in the adult its expression is restricted to brain, heart, skeletal muscle and lung. In the brain, the highest expression levels were found in specific cell layers of the cortex, the olfactory bulb, the hippocampus and the cerebellum. mPOU is shown to bind to DNA sequences containing the octamer motif and other POU factor target sites. The distinct expression pattern and divergent DNA-binding characteristics indicate that mPOU may regulate a distinct set of genes.

Amino Acid Sequence↗
PubMed2006

Targeted in utero delivery of a retroviral vector for gene transfer in the rodent brain.

In vivo application of viral vectors for gene transfer is a commonly used tool in anatomical and functional studies, as well as in development of neuroprotective and restorative strategies for therapy. Although the most common route of administration is via direct injection into the brain parenchyma in adult animals, a number of short-term studies have been performed in the developing central nervous system. Here we investigated the long-term transgene expression following in utero delivery of a retroviral vector encoding for the green fluorescent protein (GFP) marker gene at embryonic days 14.5-17.5 using an ultrasound-guided injection system. Intraparenchymal injections of the ganglionic eminence were compared with vector delivery to the intracerebroventricular space. Injections into the ganglionic eminences resulted in a predominantly unilateral transduction localized to the forebrain, giving rise to GFP-positive (GFP+) neurons and astrocytes in the striatum, olfactory bulb, cortex and hippocampus. When the vector was injected into the lateral ventricle, on the other hand, widespread expression of GFP was seen throughout the brain. The total number of GFP+ cells in the striatum was estimated to be between 20,000 and 50,000 cells using a computerized stereological quantification tool. Phenotypic characterization of these transduced cells using confocal microscopical analysis showed that 64% were NeuN+ neurons, 14% APC+ oligodendrocytes and 15% glial cells labelled with GFAP, S100beta and Iba1, when the vector injection was performed at E14.5. Delivery into later embryos resulted in a reduction in neuronal profiles with a reciprocal increase in glial cells.

Age Factors↗
PubMed1984

Regional calcium accumulation and cation shifts in rat brain by kainate.

Following local application of kainic acid, changes in the contents of Na+, K+, Ca2+, and Mg2+ of the striatum, cerebellum, and hippocampus of the rat were observed at various times after surgery. Within 1 h the levels of K+ decreased 20% whereas the levels of Na+ and Ca2+ increased at least 50% and 20%, respectively. These changes persisted for more than 8 weeks. Ca2+ levels rose further, to more than 10-fold during 8 weeks. The Mg2+ levels were slightly and only transiently decreased. Unilateral injections of kainate into the striatum affected the contents of these cations not only in this area, but also in the overlying cerebral cortex, the olfactory tubercle, and the ipsilateral substantia nigra. The Ca2+ increases were less when rats were kept on a diet deficient in Ca2+ and vitamin D. 45Ca2+, intravenously administered, accumulated significantly more in the kainate-lesioned striatum and substantia nigra than in the homotopic contralateral areas. Electron microscopic examination of the localization of Ca2+ with the oxalate-pyroantimonate technique showed the appearance of extracellularly located deposits and the accumulation of Ca2+ in (possibly degenerating) myelinated axons in kainate-lesioned striata. This study provides evidence that calcification of cerebral tissue is closely associated with neurodegenerative processes and shows that kainate may serve as a tool to elucidate the mechanism of brain calcification. The results are discussed in relation to idiopathic calcinosis (striopallidodentate calcinosis, Fahr's disease).

Animals↗
PubMed1989

Developmental regulation of microtubule-associated protein 2 expression in regions of mouse brain.

The relative levels of microtubule-associated protein 2(MAP2) were determined during postnatal development of the mouse in six different discrete brain regions: cerebellum, cortex, hippocampus, olfactory bulb, brainstem, and hypothalamus. Brain homogenates were electrophoresed on sodium dodecyl sulfate-containing gels and analyzed by immunoblotting with MAP2-specific antibodies. The levels of MAP2 in each region were determined using radiolabeled secondary antibodies and densitometric quantification of the autoradiograms over a range that was determined to have a linear response. The results indicated that in all regions and at all ages there was only one high-molecular-weight polypeptide of MAP2, which did not change in electrophoretic mobility after dephosphorylation. In most regions, the levels of MAP2 increased during the first 2 postnatal weeks. However, there were differences in the time course and relative levels of MAP2 between regions. In addition, all regions of the brain expressed the low-molecular-weight form of MAP2 (MAP2c) that was present at birth as a heterogeneous group of polypeptides with an apparent molecular weight of 70K. Most of the heterogeneity of MAP2c, however, was eliminated after dephosphorylation. The levels of MAP2c decreased dramatically after 2 weeks postnatally, except for the olfactory bulb, where the levels of MAP2c remained relatively high even in adults.

Aging↗
PubMed1990

Ventricular cerebrospinal fluid monoamine transmitter and metabolite concentrations reflect human brain neurochemistry in autopsy cases.

Concentrations of dopamine (DA), its metabolites 3-methoxytyramine and homovanillic acid (HVA), noradrenaline (NA), its metabolites normetanephrine (NM) and 3-methoxy-4-hydroxyphenylglycol (MHPG), 5-hydroxytryptamine (5-HT, serotonin), and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) were measured in 14 brain regions and in CSF from the third ventricle of 27 human autopsy cases. In addition, in six cases, lumbar CSF was obtained. Monoamine concentrations were determined by reversed-phase liquid chromatography with electrochemical detection. Ventricular/lumbar CSF ratios indicated persistence of rostrocaudal gradients for HVA and 5-HIAA post mortem. Ventricular CSF concentrations of DA and HVA correlated positively with striatal DA and HVA. CSF NA correlated positively with NA in hypothalamus, and CSF MHPG with levels of MHPG in hypothalamus, temporal cortex, and pons, whereas CSF NM concentration showed positive correlations with NM in striatum, pons, cingulate cortex, and olfactory tubercle. CSF 5-HT concentrations correlated positively with 5-HT in caudate nucleus, whereas the concentration of CSF 5-HIAA correlated to 5-HIAA levels in thalamus, hypothalamus, and the cortical areas. These data suggest a specific topographic origin for monoamine neurotransmitters and their metabolites in human ventricular CSF and support the contention that CSF measurements are useful indices of central monoaminergic activity in man.

Adult↗
PubMed1991

Developmental changes in distribution of acidic fibroblast growth factor in rat brain evaluated by a sensitive two-site enzyme immunoassay.

We developed a sensitive two-site enzyme immunoassay (EIA) system for acidic fibroblast growth factor (aFGF), using a polyclonal antibody raised in rats. This assay is based on the sandwiching of the antigen between anti-aFGF antibody immunoglobulin G (IgG) coated on plates and biotinylated anti-aFGF antibody IgG; the detection of biotinylated IgG was performed by enzyme reaction of streptavidin-conjugated beta-D-galactosidase (beta-D-galactoside hydrolase; EC 3.2.1.23). Our system was specific for aFGF, because basic fibroblast growth factor, which shares a 55% homology of amino acid sequence with aFGF, hardly cross-reacted at all. The sensitivity of this system (0.2 ng/ml) enabled us to quantify endogenous immunoreactive aFGF in the CNS. Using this two-site EIA system, we examined the levels of aFGF in various regions of rat brain and their developmental changes. At the early stage of neonatal development, i.e., 2 days after birth, all brain regions registered low aFGF levels (less than 10 ng/g tissue). However, at the young adult stage (21- to 49-day-old animals), an extremely high level of aFGF (75-90 ng/g tissue) was found in the ponsmedulla; relatively high levels (30-40 ng/g tissue) were found in the diencephalon and mesencephalon; and comparatively low aFGF levels (5-15 ng/g tissue) were found in various other brain regions such as the frontal cortex, piriform cortex, hippocampus, olfactory bulb, cerebellum, and striatum. This marked change in the regional distribution of aFGF in the rat brain during postnatal development from 2 to 21 days after birth suggests that this factor plays a significant role in the brain during this period.

Aging↗