In addition to being the major inhibitory neurotransmitter, gamma-aminobutyric acid (GABA) is thought to play a morphogenetic role in embryonic development. During the last decade, considerable progress has been made in elucidating the molecular mechanisms involved in GABA synthesis and biological action. The present review is an attempt to summarise recent results on the ontogeny of the different components of embryonic GABA signalling with an emphasis on the synthesis of GABA by different molecular forms of glutamic acid decarboxylase (GAD).

The schedule of NMDA receptor subunit expression and the appearance of functional NMDA-gated ion channels were investigated during the retinoic acid (RA) induced neuronal differentiation of NE-4C, a p53-deficient mouse neuroectodermal progenitor cell line. NR2A, NR2B, and NR2D subunit transcripts were present in both nondifferentiated and neuronally differentiated cultures, while NR2C subunits were expressed only transiently, during the early period of neural differentiation. Several splice variants of NR1 were detected in noninduced progenitors and in RA-induced cells, except the N1 exon containing transcripts that appeared after the fourth day of induction, when neuronal processes were already formed. NR1 and NR2A subunit proteins were detected both in nondifferentiated progenitor cells and in neurons, while the mature form of NR2B subunit protein appeared only at the time of neuronal process elongation. Despite the early presence of NR1 and NR2A subunits, NMDA-evoked responses could be detected in NE-4C neurons only after the sixth day of induction, coinciding in time with the expression of the mature NR2B subunit. The formation of functional NMDA receptors also coincided with the appearance of synapsin I and synaptophysin. The lag period between the production of the subunits and the onset of channel function suggests that subunits capable of channel formation cannot form functional NMDA receptors until a certain stage of neuronal commitment. Thus, the in vitro neurogenesis by NE-4C cells provides a suitable tool to investigate some inherent regulatory processes involved in the initial maturation of NMDA receptor complexes.

NE-7C2 neuroectodermal cells derived from forebrain vesicles of p53-de®cient mouse embryos (E9) produce neurons and astrocytes in vitro if induced by all-trans retinoic acid. The reproducible morphological stages of neurogenesis were correlated with the expression of various NMDA receptor subunits. RT-PCR studies revealed that GluR11 and GluR14 subunit mRNAs were transcribed by both non-induced and neuronally differentiated cells. GluR13 subunit mRNAs were not synthesized by NE-7C2 cells and increased numbers of messages from the GluR12 gene were detected only after neural network formation. The presence of the GluRz1 protein was detected throughout neural induction, whereas retinoic acid-induced neuron formation elevated the amount of exon 21 (C1)-and exon 22 (C2)-containing GluRz1 mRNAs and resulted in the appearance of exon 5 (N1)-containing transcripts. NMDA-elicited Ca 21 -signals were detected only in cells displaying neuronal morphology, but preceding the appearance of synapsin-I immunoreactivity. Our ®ndings demonstrated that, in spite of the presence of subunits necessary for channel formation, functional channels were formed by NE-7C2 cells no sooner than the time of neurite maturation. The data show that the cell line provides a suitable model to analyse the mechanisms involved in NMDA receptor gene expression before the appearance of synaptic communication. Glutamic acid, one of the most abundant small molecular neurotransmitters, has multiple roles during the early stages of neural differentiation, well before synapses become functionally active (Mattson and Hauser 1991;Meier et al. 1991). In the early phases of neural tissue formation, neural progenitors and young neurons express various glutamate receptors and display low vulnerability to glutamate toxicity (LoTurco et al. 1991(LoTurco et al. , 1995Scherer and Gallo 1998).The physiological effects of glutamate are mediated by metabotropic and ionotropic glutamate receptors (Nakanishi 1992; Seeburg 1993). Ionotropic glutamate receptor channels have been classi®ed into three structurally and pharmacologically distinct subtypes: the a-amino-3-hydroxy-5-methylisoxazole-4-propionate, kainate and NMDA receptors.Cloning the genes encoding NMDA receptor subunits has provided molecular evidence for the existence of heteromeric receptors (Monyer et al. 1992;Nakanishi 1992). Functional NMDA receptors contain the GluRz1-type subunit (GluRz1 in mouse; NR1 in rat) and any of the four GluR1-type subunits (GluR11±4 in mouse, NR2A±D in rat; Moriyoshi et al. 1991;Meguro et al. 1992). The presence of the GluRz1 subunit is necessary for the assembly of functional channels (Schoepfer et al. 1994). Several properties of NMDA receptors, however, appear to be modulated by particular GluR1 subunits (Monyer et al. 1992. Abbreviations used: ATRA, all-trans retinoic acid; d,l-AP5, dl-2-amino-5-phosphonopentanoic acid; FCS, fetal calf serum; FITC, uorescein isothiocyanate; GAPDH, glycerol-aldehyde-3-phosphate dehydrogenase; GFAP, glial ®brillary acidic protein; GluR1...

The expression of different forms of glutamate decarboxylases and GABA was investigated in the course of retinoic acid-induced neuronal differentiation of NE-7C2 cell-line established from brain vesicles of 9-day-old mouse embryos lacking functional p53 gene. Non-induced NE-7C2 cells expressed embryonic GAD mRNAs with a low level of embryonic GAD25 protein and did not contain detectable amounts of GABA. Addition of 10 )6 M retinoic acid induced the expression of N-tubulin and a significant increase in the level of embryonic GAD messages and GAD25 protein in early stage differentiating neurones. The enzymatically active embryonic GAD44 was detected at later stages of induction in neurone-like cells and showed a maximum of expression at the time of neurite elongation and network formation. With the advance of neuronal maturation, the expression of embryonic forms declined while the adult GAD65 and GAD67 transcripts became dominant. GABA-containing neurones were first demonstrated on the sixth day of induction coinciding with the peak of GAD44 expression and the beginning of GAD65 expression. The sequential induction of different GAD forms and the stage-dependent GABA synthesis in NE-7C2 cells is highly reminiscent of the temporal pattern found in vivo and suggests that these processes might be involved in the differentiation of neuronal progenitors.

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