Na ⁺ channel-mediated Ca ²⁺ entry leads to glutamate secretion in mouse neocortical preplate

Abstract

Before synaptogenesis, early excitability implicating voltagedependent and transmitter-activated channels is known to be crucial for neuronal development. We previously showed that preplate (PP) neurons of the mouse neocortex express functional Na ؉ channels as early as embryonic day 12. In this study, we investigated the role of these Na ؉ channels in signaling during early development. In the neocortex of embryonic-day-13 mice, activation of Na ؉ channels with veratridine induced a large Ca 2؉ response throughout the neocortex, even in cell populations that lack the Na ؉ channel. This Na ؉ -dependent Ca 2؉ activity requires external Ca 2؉ and is completely blocked by inhibitors of Na ؉ ͞Ca 2؉ exchangers. Moreover, veratridine-induced Ca 2؉ increase coincides with a burst of exocytosis in the PP. In parallel, we show that Na ؉ channel stimulation enhances glutamate secretion in the neocortical wall. Released glutamate triggers further Ca 2؉ response in PP and ventricular zone, as indicated by the decreased response to veratridine in the presence of ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor and NMDA-receptor inhibitors. Therefore, the combined activation of the Na ؉ channel and the Na ؉ ͞Ca 2؉ exchanger triggers Ca 2؉ signaling in the PP neurons, leading to glutamate secretion, which amplifies the signal and serves as an autocrine͞paracrine transmitter before functional synapses are formed in the neocortex. Membrane depolarization induced by glycine receptors activation could be one physiological activator of this Na ؉ channel-dependent pathway.Ca 2ϩ signaling ͉ Na ϩ ͞Ca 2ϩ exchanger ͉ exocytosis ͉ neurogenesis N eurons in the cerebral cortex are generated in a proliferative region known as the ventricular zone (VZ) (1). The first phase of neurogenesis occurs at embryonic days 11-13 (E11-E13) in mice and gives rise to a group of neurons that form the preplate (PP) above the VZ (2).During development, ligand-and voltage-gated ionic currents appear in a complex and progressive pattern, generating cell excitability. This early excitability has a widespread and important role in transmission of information and development of the nervous system. Combinations of ion channels usually generate Ca 2ϩ influx, which influences differentiation and regulates channel expression (for review, see ref.3). Neurotransmitters are reported to be present very early during neurogenesis (4). GABA and glutamate are both present in the neocortical wall and can regulate neuronal progenitor proliferation (4, 5) and neuronal migration (6). Taurine and glycine are also present and have been reported to be the most abundant neurotransmitters at E13 in mouse neocortex (7). Little is known about the channels and pathways that are involved in this early activity, and particularly little is known about how these neurotransmitters are secreted before the appearance of voltage-dependent Ca 2ϩ channels (8,9).Although cells are devoid of synaptic connection, spontaneous Ca 2ϩ activity is already present at E13 (J.-C.P. and M.A., u...

Glycine receptors control the generation of projection neurons in the developing cerebral cortex

“…In particular, GlyR a2 subunit transcripts were detected from E14 onwards in all cortical layers in rat embryos, 33 and membrane currents triggered by GlyRs were recorded on migrating projection neurons in E19 rat embryos. 34 These findings were further extended by experimental evidence supporting the existence of functional GlyRs in neurons located in the CP and IZ, 38,44,52,53 as well as in migrating cortical interneurons. 32 Our present study demonstrates that a2-containing GlyRs are functionally expressed in APs and BPs located in the developing cortical wall.…”

GABA and glutamate signaling: homeostatic control of adult forebrain neurogenesis

“…Free-floating sections were blocked (PBS + 0.3% Triton X100 + 2% BSA +/− 0.3% and incubated in the following primary antibodies for 2 hrs at RT or overnight at 4°C: goat anti-doublecortin (1:100, Santa Cruz, USA), guinea pig anti-glutamate-aspartate transporter (GLAST, 1:500, Chemicon, USA), mouse anti-glutamate (1:2000, Sigma, USA) (Platel et al, 2005), rabbit anti-GABA (1:500, Sigma, USA), rabbit anti-GAT1 (1:100, Chemicon, USA), and rabbit anti-VGAT (1:1000, Synaptic Systems, Germany). After several washes, slices were incubated with the appropriate secondary antibody (Alexa Fluor series at 1:1000, Invitrogen, USA; or Cyanine series at 1:500, Jackson Labs) for 1 hr at room temperature.…”

“…It is unlikely that neuroblasts, which are GABAergic (Bolteus and Bordey, 2004;Liu et al, 2005), release glutamate although immature cells may transiently release both glutamate and GABA (Gutierrez, 2005). We thus performed immunostaining for glutamate as previously described (Platel et al, 2005). Figure 4A shows that glutamate antibodies strongly labeled SVZ astrocytes while weakly labeled neuroblasts.…”

GABA and glutamate signaling: homeostatic control of adult forebrain neurogenesis

“…Free-floating sections were blocked (PBS + 0.3% Triton X100 + 2% BSA + /-0.3% Tween-20) and incubated in the following primary antibodies for 2 h at RT or overnight at 4°C: goat anti-doublecortin (1:100, Santa Cruz, USA), guinea pig anti-glutamate-aspartate transporter (GLAST, 1:500, Chemicon, USA), mouse anti-glutamate (1:2000, Sigma, USA) (Platel et al 2005), rabbit anti-GABA (1:500, Sigma, USA), rabbit anti-GAT1 (1:100, Chemicon, USA), and mouse anti-VGAT (1:1000, Synaptic Systems, Germany). After several washes, slices were incubated with the appropriate secondary antibody (Alexa Fluor series at 1:1000, Invitrogen, USA; or Cyanine series at 1:500, Jackson Labs) for 1 h at room temperature.…”