Modulation of cellular proliferation and differentiation through GABA_B receptors expressed by undifferentiated neural progenitor cells isolated from fetal mouse brain

Abstract

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.

Gradual Downregulation of Protein Expression of the Partner GABABR2 Subunit During Postnatal Brain Development in Mice Defective of GABABR1 Subunit

“…In contrast to the present in vivo study, a marked decrease is seen in the number of cells immunoreactive for MAP2 with a concomitant increase in GFAP-positive cells in cultured NPC isolated from embryonic GABA B R1-null mouse brains in our previous in vitro study (23). The present findings that Mash1 mRNA expression was similarly and predominantly decreased in cultured NPC from embryonic GABA B R1-null mouse brains in vitro and in whole brains of neonatal GABA B R1-null mice in vivo argue in favor of an idea that postnatal development could compensate both the suppressed neuronal differentiation and promoted astroglial differentiation mediated by impairment of the function of the activator type bHLH gene product Mash1 in murine brain NPC deficient of the GABA B R1 subunit.…”

“…GABA partially blocks the bFGF-induced increase in cell proliferation (19), but promotes cell proliferation in cultures of rat cerebellar progenitors (20). In our previous in vitro studies, activation of the ionotropic GABA A receptor (GABA A R) subtype leads to increased self-replication activity along with promotion of subsequent differentiation into astroglia in NPC isolated from embryonic rat (21) and mouse (22) brains, while activation of the metabotropic GABA B receptor (GABA B R) subtype results in the similarly efficient promotion of self-renewal activity together with the facilitation of subsequent differentiation into neurons in NPC isolated from neocortex of embryonic mice (23).…”

“…We usually used postnatal day 1 (P1), P7, and P56 Balb/c GABA B R1−null mice and their littermates (24). Wild-type and GABA B R-null mice were identified with reverse transcription polymerase chain reaction (RT-PCR) analysis using specific primers as described previously (23).…”

“…Sustained activation of GABA B R by different agonists, such as GABA and baclofen, promotes self-replication and subsequent neuronal differentiation in a manner sensitive to an antagonist in cultured NPC isolated from embryonic mouse brains, while a significant diminution is seen for proliferation and subsequent neuronal differ- entiation in cultured NPC isolated from GABA B R1-null mouse brains (23). To evaluate a possible mechanism underlying the suppressed neuronal differentiation, mRNA expression was at first determined with several proneural molecules endowed to modulate proliferation and/or differentiation of neural progenitors (29,30) in NPC cultured for 8 days under floating conditions.…”

“…Moreover, no significant changes were seen in mRNA and protein expression levels of both astroglial and microglial markers in brains between wild-type and GABA B R1-null mice when determined 1 to 7 days after birth (data not shown). Therefore, it is unlikely that deficiency of the GABA B R1 subunit would similarly lead to promoted astroglial differentiation along with suppressed neuronal differentiation of undifferentiated NPC during postnatal brain development in vivo as seen in those cultured in vitro (23).…”