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The patch clamp technique was used to study whole cell and single channel currents evoked by glycine in cerebellar granule cells in culture. Whole cell concentration response curve gave a Kd value for glycine of 73 microM and a Hill slope of 1.58. Glycine-activated currents reversed close to the predicted Cl- equilibrium potential. The responses to glycine were antagonized by strychnine and picrotoxin with an IC50 of 58 nM and 172 microM, respectively. Furthermore, glycine-evoked currents were potentiated by zinc in a dose-dependent way. In outside-out membrane patches, glycine opened channels with conductances of 32, 52, 84 and 96 pS. The most frequently occurring was the 52 pS channel. The single channel current/voltage relationship was linear in the potential range between -60 and 60 mV. The 52, 84 and 96 pS channels exhibited prolonged openings whereas the 32 pS was characterized by fast (< 10 ms) openings. Open and closed time histograms of the 52 pS channel could be fitted with the sum of two or three exponentials, respectively, whereas burst duration histograms could be fitted with the sum of two exponentials. Glycine current density change drastically during days in culture, the maximal expression being between day 4 and 7, suggesting that the expression of glycine receptor channels is developmentally regulated.

Activity-dependent enduring change in cellular communication is essential for specific connectivity during development of the nervous system and for adaptive responses of the mature nervous system. Here we report that glutamate activation of excitatory amino acid receptors induces the synthesis and release of proteoglycans (PGs) from fetal hippocampal-astrocytes in dissociated culture. PG synthesis and release are mediated via kainate and metabotropic receptor activation. Glutamate exposure did not regulate the release of a specific family of PG, but glutamate inhibited the synthesis of heparan sulfate (HS) PGs that appeared within the extracellular environment of the astrocyte. Particulate protein kinase C (PKC) activity was increased by glutamate and the PKC activator phorbol 10-myristate 13-acetate produced a dose-dependent increase in PG release. However, glutamate-induced PG release was not blocked by inhibition of PKC activity. These data suggest that PKC activation can lead to PG release, but is not necessary for it. Activity-dependent influences on a class of substrate-bound molecular species with growth-modulatory properties may be involved in spatial regulation of neuronal growth responses produced by excitatory amino acids.

N-Cadherin is a major cell adhesion molecule that is expressed in the developing nervous system where it has been implicated in neural migration and axon growth. Recently, a role for N-cadherin in oligodendrocyte differentiation has been identified [23]. Oligodendrocyte precursors adhere to N-cadherin and mature rapidly to produce myelin sheets. Since this implies that oligodendrocytes express N-cadherin, we examined the expression of N-cadherin by oligodendrocytes in culture. N-Cadherin was expressed by O-2A progenitors, immature oligodendrocytes and mature oligodendrocytes, but at a lower level than in type 1 astrocytes in the same cultures. On mature oligodendrocytes, the N-cadherin was concentrated on the major processes emerging from the soma. The ability of N-cadherin and merosin to promote oligodendrocyte precursor migration was also studied. Average migration rates were significantly higher on merosin (11.2 microns/h) than on N-cadherin (5.6 microns/h). These results suggest that N-cadherin is not likely to function predominantly as a substrate that stimulates migration of O-2A progenitors, but may be more important in initiating early oligodendrocyte-axon interactions that promote the process of myelination.

Developmental changes in glutamate receptor agonist-produced enhancement of 4-beta-[3H]phorbol-12,13-dibutyrate binding ([3H]-PDBu binding), indicative of an intracellular translocation of protein kinase C (PKC), were investigated in cerebellar granule cells. Our observations demonstrate that the magnitude of glutamate-, NMDA-, and kainate-produced enhancement of PKC translocation was dramatically decreased between 2 and 12 DIV, whereas there was only a minor reduction in the corresponding response caused by the non-NMDA receptor agonist, AMPA. The maximally enhanced stimulation of PKC translocation caused by glutamate and NMDA was significantly reduced already at 4 DIV, whereas a significant reduction of the kainate-induced enhancement of [3H]PDBu binding was not observed until 8 DIV. Glutamate- and NMDA-induced responses were effectively blocked by the specific NMDA receptor antagonists MK-801 (1 microM) and APV (100 microM) as well as by the addition of Mg2+ into assay media. In contrast, the non-NMDA receptor antagonist, CNQX (10 microM), effectively blocked the kainate-induced enhancement of [3H]PDBu binding, but had no effect on the NMDA- and glutamate-induced stimulation of PKC translocation. The metabotropic glutamate receptor agonist, ACPD (up to 250 microM), had no effect on the translocation of PKC. Taken together, our data support the working hypothesis that the rapidly occurring changes in the glutamate receptor agonist-produced translocation of PKC are most likely due to a differential maturation of glutamate ionotropic receptor subtypes and/or to development-dependent alterations in mechanisms responsible for the coupling between the glutamate receptor subtypes and the activation of PKC translocation in cerebellar granule neurons.

Effects of intrauterine hypoxia-ischemia (HI) on receptor-stimulated phosphoinositide (PPI) hydrolysis were studied in rat cerebellar granule cell cultures prepared from an in utero HI model. On gestation day 17, HI conditions were achieved by complete clamping of the uterine vasculature for 30 min followed by removal of the clamps to permit reperfusion. Sham operation (SH, surgery without vasculature ligation) was performed as the control. Intrauterine HI did not affect the basal level of PPI hydrolysis (in the absence of stimulants) in cells prepared from either the SH or the HI group. PPI hydrolysis stimulated by quisqualate (QA) or trans-(1S,3R)-1-amino-1,3-cyclo-pentanedicarboxylic acid (trans-ACPD) was significantly reduced in cells prepared from the HI group, whereas intrauterine HI did not affect the PPI hydrolysis induced by ionotropic glutamate receptor agonists or by norepinephrine or serotonin. At a dose range of 100-300 microM, QA-stimulated PPI hydrolysis in cells prepared from the SH group increased by 3-to 4.5-fold, while this increase was only 2- to 2.5-fold in cells prepared from the HI group. Presence of L-NG-monomethyl-arginine (L-NMMA), a nitric oxide (NO) synthase inhibitor, did not increase QA-stimulated PPI hydrolysis in cells prepared from either the SH or the HI group, indicating that stimulation of NO formation is unlikely involved in the suppressive effects of intrauterine HI on QA-induced PPI hydrolysis. The QA-stimulated PPI hydrolysis in cells prepared from the HI group, but not from the SH group, was further inhibited by L-(+)-2-amino-3-phosphono-propionic acid (L-AP3). The overall results suggest that intrauterine HI has long-lasting suppressive effects on metabotropic glutamate receptor agonist-stimulated PPI hydrolysis and these effects might be associated with alterations in expression of metabotropic glutamate receptor subtypes.

In order to investigate genetic factors that interfere with hormone-mediated sex differentiation of dopaminergic neurons, we raised sex-specific primary cultures from embryonic day 13 diencephalon (D) or mesencephalon (M) of three different strains of mice, NMRI, CBA/J, and BALBc/J. Part of the cultures were maintained for 6 or 13 days in vitro (DIV) in medium containing 17 beta-estradiol or testosterone. The cultures were analyzed for sex differences in numbers of tyrosine hydroxylase-immunoreactive neurons, endogenous dopamine (DA) levels, and specific uptake of [3H]DA. Previous results obtained with cultures of embryonic Sprague-Dawley rats had shown that these parameters develop sex-specific characteristics in the absence of sex differences in hormone environment. Similar steroid-independent sex differences as they occur in the rat were found in M cultures of NMRI but not in CBA and BALBc mice. Long-term sex steroid treatment did not affect any of the above parameters in any strain. It is concluded that cell-autonomous realization of the genetic sex of dopaminergic neurons depends on the genetic background.

In this study we report the biochemical and initial molecular characterization of EAP-300, a developmentally regulated embryonal protein that has been shown previously to be expressed by radial glia in various regions of the CNS, including putative glial barriers. In the present study we have shown that the 300 kDa EAP-300 polypeptide is developmentally regulated in all tissues expressing the protein, which include various PNS and CNS tissues and muscle. In neural tissue the protein is readily detected during early embryogenesis, subsequently down-regulated at later stages, and is not detected in the adult. In contrast to neural tissue, small amounts of the protein are expressed in heart, consistent with earlier studies which showed that EAP-300 expression was maintained in the Purkinje cells of the heart conduction system. Metabolic labeling demonstrates that EAP-300 is a phosphoprotein, and is fatty acylated based on incorporation of [3H]palmitate. We also show that the normal developmental down-regulation of EAP-300 by glia does not occur in vitro, and these data therefore suggest that the signal(s) that regulates EAP-300 gene expression during development in vivo is absent in dissociated cell cultures. We have also initiated molecular studies of EAP-300 by screening embryonic brain cDNA expression libraries with a mixture of EAP-300 monoclonal antibodies. Sequence analysis of partial EAP-300 cDNAs indicate that the protein is related, if not identical, to IFAPa-400, a developmentally regulated intermediate filament-associated protein in chick that is proposed to participate in cell differentiation. These studies also indicate that EAP-300 mRNA is developmentally regulated and is expressed by glial cells in putative CNS barrier structures. Our studies also suggest that two pools of EAP-300 may exist in cells, implying that unlike IFAPa-400 the EAP-300 protein may not always be associated with intermediate filaments. Interestingly, our studies demonstrate that EAP-300 contains a novel repeat amino acid domain comprised of multiple leucine-zipper motifs, which may contribute to its function during glial differentiation.