Different mitochondrial nitric-oxide synthase (mt-NOS) isoforms have been described in rat and mouse tissues, such as liver, thymus, skeletal muscle, and more recently, heart and brain. The modulation of these variants by thyroid status, hypoxia, or gene deficiency opens a broad spectrum of mtNOS-dependent tissuespecific functions. In this study, a new NOS variant is described in rat brain with an M r of 144 kDa and mainly localized in the inner mitochondrial membrane. During rat brain maturation, the expression and activity of mt-NOS were maximal at the late embryonic stages and early postnatal days followed by a decreased expression in the adult stage (100 ؎ 9 versus 19 ؎ 2 pmol of [ 3 H]citrulline/min/mg of protein, respectively). This temporal pattern was opposite to that of the cytosolic 157-kDa nNOS protein. Mitochondrial redox changes followed the variations in mtNOS activity: mtNOSdependent production of hydrogen peroxide was maximal in newborns and decreased markedly in the adult stage, thus reflecting the production and utilization of mitochondrial matrix nitric oxide. Moreover, the activity of brain Mn-superoxide dismutase followed a developmental pattern similar to that of mtNOS. Cerebellar granular cells isolated from newborn rats and with high mtNOS activity exhibited maximal proliferation rates, which were decreased by modifying the levels of either hydrogen peroxide or nitric oxide. Altogether, these findings support the notion that a coordinated modulation of mtNOS and Mn-superoxide dismutase contributes to establish the rat brain redox status and participate in the normal physiology of brain development.

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In developing cerebellar interneurons, NMDA increases spontaneous GABA release by activating presynaptic NMDA receptors. We investigated the role of these receptors on differentiating basket/stellate cells in cerebellar cultures grown under conditions allowing functional synaptic transmission. Presynaptic GABAergic boutons were visualized either by GAD65 immunostaining or by using cells derived from GAD65-enhanced green fluorescent protein (eGFP) transgenic mice, in which cerebellar basket/stellate cells express eGFP. After the first week in culture, whole-cell recordings from granule cells reveal that acute application of NMDA increases miniature IPSC (mIPSC) frequency. Interestingly, after 2 weeks, the mIPSC frequency increases compared with the first week but is not modulated by NMDA. Furthermore, in cultures chronically treated with NMDA for 1 week, the size of the GABAergic boutons increases. This growth is paralleled by increased mIPSC frequency and the loss of NMDA sensitivity. Direct patch-clamp recording from these presynaptic terminals reveals single NMDA-activated channels, showing multiple conductance levels, and electronic propagation from the somatodendritic compartment. Our results demonstrate that NMDA receptors alter GABAergic synapses in developing cerebellar cultures by increasing the size of the terminal and spontaneous GABA release. These findings parallel changes in inhibitory synaptic efficacy seen in vivo in developing GABAergic interneurons of the molecular layer of the cerebellum.

During neuronal development, GABA A -mediated responses are depolarizing and induce an increase in the intracellular calcium concentration. Since calcium oscillations can modulate neurite outgrowth, we explored the capability of GABA to induce changes in cerebellar granule cell morphology. We find that treatment with GABA (1-1000 lM) induces an increase in the intracellular calcium concentration through the activation of GABA A receptors and voltage-gated calcium channels of the L-subtype. Perforated patch-clamp recordings reveal that this depolarizing response is due to a chloride reversal potential close to ) 35 mV. When cells are grown in depolarizing potassium chloride concentrations, a shift in reversal potential (Erev) for GABA is observed, and only 20% of the cells are depolarized by the neurotransmitter at day 5 in vitro.On the contrary, cells grown under resting conditions are depolarized after GABA application even at day 8. GABA increases the complexity of the dendritic arbors of cerebellar granule neurons via a calcium-dependent mechanism triggered by voltage-gated calcium channel activation. Specific blockers of calcium-calmodulin kinase II and mitogen-activated protein kinase kinase (KN93 and PD098059) implicate these kinases in the intracellular pathways involved in the neuritogenic effect of GABA. These data demonstrate that GABA exerts a stimulatory role on cerebellar granule cell neuritogenesis through calcium influx and activation of calcium-dependent kinases. Keywords: calcium influx, CaMKII, depolarization, erk1/2, GABA A receptors, neuritogenesis. GABA is the main inhibitory neurotransmitter in the adult central nervous system. During nervous system development, this neurotransmitter exerts depolarizing responses through the activation of GABA A receptors in many structures, including the hippocampus, hypothalamus, striatum and spinal cord (Ben-Ari et al. 1989;Fiszman et al. 1990Fiszman et al. , 1993Mandler et al. 1990;Obrietan and van den Pol 1995;Rohrbough and Spitzer 1996). A higher intracellular concentration of chloride yielding a depolarized chloride equilibrium potential explains these GABA-induced depolarizing responses during development in a number of cases (Reichling et al. 1994;Ben-Ari et al. 1997;Rivera et al. 1999;Ganguly et al. 2001

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