Basic Fibroblast Growth Factor Increases the Number of Excitatory Neurons Containing Glutamate in the Cerebral Cortex Flora M. Vaccarino,' Michael L. Schwartz, 2 Dennis Hartigan,' and James F. Leckman 1 1 Child Study Center and 2 Section of Neurobiology, Yale University, New Haven, Connecticut 06510 Stem cells isolated from the ventricular zone of embryonic day 12.5 rat telencephalon progressively proliferate and differentiate in vitro into three major classes of amino acid-containing neurons, glutamate, aspartate, and GABA. We quantitatively examined the effect of basic fibroblast growth factor (bFGF) and nerve growth factor (NGF) on amino acid-containing neurons. bFGF caused a threefold increase in gluta- mate-containing neurons, while the number of GABA- and aspartate- containing neurons was not significantly changed. In contrast NGF did not alter the number of amino acid-containing neurons. The ratio of glutamate- to GABA-containing neurons in untreated or NGF-treated cultures was 0.6:1. In the bFGF-treated cultures, this ratio was 1.4:1, which closely approximates the ratio in the cerebral cortex in vivo. Treatment with antisense oligonucleotides targeted to bFGF mRNA provoked a 50% decrease in the number of glutamate-containing neu- rons but had no significant effect on the GABA-containing neurons. Thus, diffusible factors such as bFGF may play an important role in determining the relative proportion of excitatory versus inhibitory neu- rons in the cerebral cortex by selectively regulating the proliferation of stem cells committed to different neurotransmitter phenotypes. Pyramidal and nonpyramidal cells represent the two main classes of neurons of the cerebral cortex and are distin- guished by their use of excitatory versus inhibitory neuro- transmitters. Pyramidal neurons, which are the projection neurons, utilize the excitatory neurotransmitters glutamate or aspartate, while most local circuit neurons (nonpyramidal neurons) use the inhibitory transmitter GABA (Jones and Pe- ters, 1984; Rakic and Singer, 1988). The balance of excitatory and inhibitory neuronal phenotypes is important for the proper functioning of the cortical circuitry. For example, the activation of either GABA or glutamate receptors produces antagonistic effects on the induction of long-term potentia- tion at cortical synapses (Artola and Singer, 1987) and on in- tracellular transduction systems, such as protein kinase C (Vaccarino et al., 1991). Glutamate- and aspartate-containing neurons represent approximately 35-40% of all neurons of the cerebral cortex of rats, cats, and monkeys (Conti et al., 1987, 1989). Despite considerable cytological, connectional, and biochemical differences between cortical areas, the ratio of glutamatergic to GABAergic neurons is remarkably uniform (1.2:1) (Jones and Peters, 1984; Conti et al., 1987; Hendry et al., 1987; Meinecke and Peters, 1987). These data suggest that the generation of a proper ratio of excitatory and inhibitory classes of cortical neurons is tightly controlled and may be independently regulated within the cerebral cortex, as well as in other regions. The results of a number of recent studies indicate that many features of the pyramidal versus nonpyramidal neuronal phenotype may be determined prior to the arrival of neurons in the developing cortical plate. For example, neurons may be committed to a specific pattern of axonal projections be- fore they reach their final position in the cortex (Caviness, 1982; Jensen and Killackey, 1984; Schwartz et al., 1991). Sim- ilarly, studies in which early-generated cortical neurons are transplanted into older host brains indicate that the commit- ment to a particular laminar fate is made during the final mitosis of the progenitor cells within the ventricular zone (McConnell and Kaznowski, 1991). Finally, retrovirus studies of cell lineage suggest that mitotically active stem cells of the cortical ventricular zone are committed to give rise to neu- rons of either a pyramidal or a nonpyramidal phenotype (Par- navelas et al., 1991; Luskin et al., 1993) or to cells containing either GABA or glutamate (Mione et al., 1994). Currently, little is known about the microenvironmental factors that regulate the commitment to a glutamate or a GABA phenotype or that act selectively to control the prolif- eration and differentiation of precursors within these lineages to achieve the observed ratio of these cell classes within the mature cortex. In the PNS, as well as for glial cells of die forebrain, growth factors and other cytokines play an impor- tant role in the determination of cell fate (Iilien and Raff, 1990; McKinnon et al., 1990; Nawa et al., 1991;Jessell and Melton, 1992). Multiple subtypes of the fibroblast growth fac- tor receptor (FGF-R) are expressed in the ventricular germi- native zone of the embryonic CNS but not in die mande layer (Heuer et al., 1990; Orr-Urtreger et al., 1991; Wanaka et al., 1991; Peters et al., 1992). Later in development, FGF-R is ex- pressed in selected populations of mature neurons (Heuer et al., 1990;Wanaka et al., 1991;Peters et al., 1992).These studies suggest that growth factors of the FGF family, in addition to being trophic factors for differentiated neurons (Morrison et al., 1986;Hatten et al., 1988;Walicke, 1988;Grothe et al., 1989) may be important for initial events in neurogenesis. Indeed, studies of primary cultures isolated from forebrain have dem- onstrated a role for basic FGF (bFGF) in the proliferation of neuronal progenitors (Gensburger et al., 1987; Cattaneo and McKay, 1990; Murphy et al., 1990; Ray et al., 1993). Given the specific localization of FGF-R in the forebrain ventricular zone during neurogenesis, we decided to investi- gate whether bFGF might influence die determination of ex- citatory and inhibitory neuronal fates in the cerebral cortex. To explore diis question, we developed an in vitro primary culture model that mimics the temporal pattern of neurogen- esis seen in situ. In this cell culture system, bFGF, but not NGF, appears to play a role in the proliferation and/or differ- entiation of glutamate-containing neuron precursors. Materials and Methods Primary Culture Primary cell cultures were made from the telencephalon of embry- onic rats taken on day 12.5 postconception (E12.5) using a modifi- cation of previously published procedures (Cattaneo and McKay, 1990; Murphy et al., 1990). The day on which the vaginal plug was found was considered E0.5. Embryos were dissected in sterile Hank's balanced salt solution maintained at 4°C. The telencephalic neuroepi- thelium of the ventricular zone was dissected away from mesodermal tissues and then dispersed into a single-cell suspension by gentle trituration with fire-polished Pasteur pipettes. The cell suspension was then centrifuged at 300 x g (4°Q for 5 min and cells were resuspended in scrum-free medium (SFM). The SFM was a modified Cerebral Conn Jan/Feb 1995;1.64-78; 1047-3211/95/M.OO