Immunocytochemical analysis confirmed the validity of isolation procedures of neural progenitors capable of self-replication and differentiation from discrete fetal rat brain structures. A reverse transcription-polymerase chain reaction analysis revealed the expression of particular GABA A receptor (GA-BA A R), GABA B R-1 and GABA C R, but not GABA B R-2, subunits in neocortical cells before commitment. Sustained exposure to the GABA A R agonist muscimol at 100 lmol/L led to significant increases in the mitochondrial activity and the total areas of neocortical neurospheres formed during the cultivation for 12 days in a manner sensitive to a GABA A R antagonist, with lactate dehydrogenase release being unchanged. Moreover, prior sustained exposure to muscimol significantly facilitated the subsequent expression of an astroglial marker protein in cells differentiated by ciliary neurotrophic factor (CNTF) toward an astroglial lineage, with a concomitant decrease in the neuronal marker protein expression, in an antagonist-sensitive manner on Western blotting analysis. However, muscimol failed to significantly affect the expression of both marker proteins in cells differentiated in either the presence or absence of all-trans-retinoic acid. These results suggest that prior activation of GABA A R may preferentially facilitate the commitment by CNTF of neural progenitor cells toward an astroglial lineage after simulation of the self-replication activity in the developing rat brain.
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 evaluated the possible functional expression of metabotropic glutamate receptors (mGluRs) by neural progenitors from embryonic mouse neocortex. Constitutive expression was seen with group I, II, and III mGluRs in undifferentiated cells and neurospheres formed by clustered cells during culture with epidermal growth factor. The group III mGluR agonist, l‐2‐amino‐4‐phosphonobutyrate, drastically reduced proliferation activity at 1–100 μM without inducing cell death, with group I and group II mGluR agonists being ineffective, in these neurospheres. Both forskolin and a group III mGluR antagonist significantly increased the proliferation alone, but significantly prevented the suppression by l‐2‐amino‐4‐phosphonobutyrate. Activation of group III mGluR significantly decreased mRNA expression of the cell cycle regulator cyclinD1, in addition to inhibiting the transactivation mediated by cAMP of cyclinD1 gene in the pluripotent P19 progenitor cells. Prior activation of group III mGluR led to a significant decrease in the number of cells immunoreactive for a neuronal marker, with an increase in that for an astroglial marker irrespective of differentiation inducers. These results suggest that group III mGluR may be functionally expressed to suppress self‐renewal capacity through a mechanism related to cAMP formation with promotion of subsequent differentiation into astroglial lineage in neural progenitors.
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