In this study, we have attempted to evaluate the possible role of metabotropic GABA(B) receptors (GABA(B)R) expressed by neural progenitor cells prepared from neocortex of embryonic Std-ddY mice. Immunocytochemical analysis confirmed the validity of isolation procedures of neural progenitors, while round spheres were formed with clustered cells during culture with epidermal growth factor (EGF) for 10 days. A reverse transcription polymerase chain reaction analysis revealed constitutive expression of GABA(A)R, GABA(B)R, and GABA(C)R subtypes in undifferentiated progenitors and neurospheres formed within 10 days. Exposure to GABA led to concentration-dependent increases in the total area and proliferation activity of neurospheres at 10-300 microM, while the GABA(B)R agonist baclofen at 100 microM significantly increased the size of neurospheres expressing both GABA(B)R1 and GABA(B)R2 subunits in a manner sensitive to a GABA(B)R antagonist. By contrast, a significant decrease was seen in the total areas of neurospheres prepared from mice deficient of the GABA(B)R1 subunit. In neurospheres of GABA(B)R1-null mice, a significant increase was induced in the number of cells immunoreactive for a glial marker protein, with a concomitant decrease in that of a neuronal marker protein, upon spontaneous differentiation after the removal of EGF. These results suggest that GABA(B)R may be functionally expressed by neural progenitor cells to preferentially promote the commitment toward a neuronal lineage after the activation of cellular proliferation toward self-replication in the developing mouse brain.

We have previously shown significant potentiation of Ca(2+) influx mediated by N-methyl-D-aspartate receptors, along with decreased microtubules-associated protein-2 (MAP2) expression, in hippocampal neurons cultured under static magnetism without cell death. In this study, we investigated the effects of static magnetism on the functionality of neural progenitor cells endowed to proliferate for self-replication and differentiate into neuronal, astroglial, and oligodendroglial lineages. Neural progenitor cells were isolated from embryonic rat neocortex and hippocampus, followed by culture under static magnetism at 100 mT and subsequent determination of the number of cells immunoreactive for a marker protein of particular progeny lineages. Static magnetism not only significantly decreased proliferation of neural progenitor cells without affecting cell viability, but also promoted differentiation into cells immunoreactive for MAP2 with a concomitant decrease in that for an astroglial marker, irrespective of the presence of differentiation inducers. In neural progenitors cultured under static magnetism, a significant increase was seen in mRNA expression of several activator-type proneural genes, such as Mash1, Math1, and Math3, together with decreased mRNA expression of the repressor type Hes5. These results suggest that sustained static magnetism could suppress proliferation for self-renewal and facilitate differentiation into neurons through promoted expression of activator-type proneural genes by progenitor cells in fetal rat brain.

In this study, we evaluated the role of the GABA(A) receptor (GABA(A)R), expressed by undifferentiated neural progenitors isolated from fetal mouse neocortex, in the mechanisms relevant to self-replication and differentiation toward neuronal and astroglial lineages. Round spheres were formed with clusters of proliferating cells within 2-4 days during culture with epidermal growth factor (EGF), whereas the size of these clusters was drastically increased in proportion to increasing durations up to 10 days. Sustained exposure to the GABA(A)R agonist muscimol at 100 microM led to significant increases in the size of neurospheres cultured for 6-10 days, with increased proliferative activity and unchanged lactate dehydrogenase release in a manner sensitive to the GABA(A)R antagonist bicuculline. Muscimol also significantly increased the incorporation of 5-bromo-2'-deoxyuridine in neurospheres in a bicuculline-sensitive manner, whereas both high potassium and nifedipine significantly decreased the neurosphere area with increased numbers of apoptotic cells. Prior activation of GABA(A)R significantly promoted the subsequent expression of an astroglial marker protein in cells differentiated by ciliary neurotrophic factor (CNTF) toward an astroglial lineage after the removal of EGF, with a concomitant decrease in neuronal marker protein expression. In neurospheres with GABA(A)R activation, a significant and predominant increase was seen in mRNA expression of CNTF receptors. These results suggest that prior tonic activation of GABA(A)R may preferentially promote the differentiation by CNTF of neural progenitor cells toward an astroglial lineage through selective up-regulation of CNTF receptor expression in the developing mouse brain.

Abstract.We have previously shown the functional expression of GABA B receptors (GABA B R) composed of GABA B R1 and GABA B R2 subunits with ability to promote proliferation and neuronal differentiation in cultured neural progenitor cells (NPC) isolated from embryonic mouse brains. In this study, we evaluated postnatal changes in the expression profiles of different markers for progenitor, neuronal, astroglial, and microglial cells in brains of GABA B R1-null mice. Consistent with undifferentiated murine NPC cultured with epidermal growth factor, a significant and selective decrease was seen in mRNA expression of the proneural gene Mash1 in brains of GABA B R1-null mice at 1 day after birth. The expression of several NPC marker proteins was similarly decreased in brains of both wild-type and GABA B R1-null mice from 1 to 7 days after birth, while slight changes were induced in both mRNA and proteins for neuronal, astroglial, and microglial markers between wild-type and GABA B R1-null mouse brains within this developmental stage. In particular discrete brain structures of adult GABA B R1-null mice at 56 days after birth, a significant decrease was seen in neuronal marker protein levels along with a significant increase in both astroglial and microglial marker protein expression. Although no significant difference was found in mRNA expression of the partner GABA B R2 subunit between wild-type and GABA B R1-null mouse brains, GABA B R2 subunit protein levels were gradually declined during postnatal development within 56 days after birth in GABA B R1-null mouse brains. These results suggest that GABA B R2 protein levels are closely correlated with the partner subunit GABA B R1 protein levels in mouse brains during postnatal development in vivo.

This study was performed to investigate the expression profile of cytochrome P450 (CYP) isoforms and effects of polycyclic aromatic hydrocarbons (PAHs) and antiepileptic drugs on CYP1 expression in human astrocytoma MOG-G-CCM cells. Main methods: CYP1A1 and CYP1B1 expression were determined by quantitative real-time polymerase chain reaction, Western blotting, and immunocytochemistry. Key findings: MOG-G-CCM cells expressed various CYP isoforms. Among the CYP isoforms analyzed, CYP1B1 showed the highest expression level, followed by CYP1A1. Furthermore, CYP1B1 was localized in both the endoplasmic reticulum and mitochondria. 3-Methylcholanthrene (3-MC), benz[a]anthracene (B[a]A), benzo[a] pyrene (B[a]P), and valproic acid (VPA) increased the expression of CYP1B1 and CYP1A1. The potent aryl hydrocarbon receptor antagonist GNF351 significantly suppressed the 3-MC-and VPA-mediated upregulation of CYP1B1 and CYP1A1. In addition, VPA potentiated the induction of CYP1B1 and CYP1A1 by 3-MC, B[a]A, and B [a]P, although the augmentation of CYP1A1 was more remarkable than that of CYP1B1. In contrast, other antiepileptic drugs (carbamazepine, lamotrigine, levetiracetam, phenytoin) did not affect the 3-MC-mediated upregulation of CYP1B1 and CYP1A1. VPA is known to act as a histone deacetylase (HDAC) inhibitor. Therefore, the effects of trichostatin A, a representative HDAC inhibitor, on CYP1 induction by 3-MC were examined. Trichostatin A enhanced the 3-MC-mediated upregulation of CYP1A1 but not CYP1B1. Significance: These results partially indicated that VPA may augment the PAH-mediated induction of CYP1B1 and CYP1A1 through the activation of transcription by HDAC inhibition.