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

Abstract

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.

Upregulation of Runt‐Related Transcription Factor‐2 Through CCAAT Enhancer Binding Protein‐β Signaling Pathway in Microglial BV‐2 Cells Exposed to ATP

“…Cultured cells were superficially washed with phosphate‐buffered saline (PBS) twice, followed by extraction of total RNA using ISOGEN according to the manufacturer's instruction (Fukui et al, ). Quantification of PCR products was conducted by real time‐based reverse transcription (RT)‐PCR using M × 3000P (Agilent Technologies, Santa Clara, California) with an iQ SYBR Green super mix (Bio‐Rad).…”

Promotion of Both Proliferation and Neuronal Differentiation in Pluripotent P19 Cells with Stable Overexpression of the Glutamine Transporter slc38a1

“…After blocking by 5% skimmed milk dissolved in 20 mM Tris-HCl buffer (pH 7.5) containing 137 mM NaCl and 0.05% Tween 20, the membrane was reacted with an antibody against MAP2 (1∶5000) or β-tubulin diluted with the buffer containing 1% skimmed milk to 1.0×10 4 -fold, followed by a reaction with an anti-mouse IgG antibody conjugated with peroxidase. Proteins reactive with those antibodies were detected with the aid of ECL™ detection reagents through exposure to X-ray films [40].…”

Selective Upregulation of Per1 mRNA Expression by ATP Through Activation of P2X7 Purinergic Receptors Expressed in Microglial Cells

“…Cultured cells were superficially washed with PBS twice, followed by extraction of total RNA using ISOGEN according to the manufacturer's instructions and subsequent synthesis of cDNA with 25 ng/ml oligo (dT)18 primer, 0.5 mM dNTP mix, and M-MLV Reverse Transcriptase (25). Reverse transcription polymerase chain reaction (RT-PCR) was conducted using upstream and downstream primers specific for each molecule ( Table 1).…”