GABA‐induced neurite outgrowth of cerebellar granule cells is mediated by GABA_A receptor activation, calcium influx and CaMKII and erk1/2 pathways

Abstract

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

Differential Outgrowth of Axons and their Branches Is Regulated by Localized Calcium Transients

“…It seems likely that differential activation of calcium frequency-dependent effectors such as CaMKII and calcineurin would occur in each axonal process. Positive effectors such as CaMKI and CaMKII, which have been shown to promote neurite outgrowth (Borodinsky et al, 2003;Wayman et al, 2004Wayman et al, , 2006 are preferentially activated in axonal processes with higher frequencies of Ca 2ϩ transients (Tang and Kalil, 2005). CaMKII␤ has been shown to bind directly to actin filaments and increase filopodial dynamics (Fink et al, 2003), consistent with results indicating that direct elevation of Ca 2ϩ activity by photolysis of caged calcium can rapidly evoke protrusion of new filopodia through polymerization of actin filaments (Lau et al, 1999).…”

GABA Regulates Dendritic Growth by Stabilizing Lamellipodia in Newly Generated Interneurons of the Olfactory Bulb

“…It remains that, in some experiments, we used higher doses of GABA. In accordance with previous reports (Bird and Owen, 1998;Borodinsky et al, 2003), we had to take into consideration the possibility of a rapid GABA degradation and used relatively high concentrations of GABA (100 M) for chronic treatment experiments (Ͼ5 h). Similarly, in slice cultures, high doses were required for inducing lamellipodia on a timescale similar to that of dissociated cultures.…”

Single-Cell Characterization of Retrograde Signaling by Brain-Derived Neurotrophic Factor

“…This capping of excitability has been proposed to be critical for growth-promoting excitation without causing excitotoxicity (Ben-Ari, 2002). It has been shown that this depolarizing GABAergic input can be critical for normal neuronal development (Borodinsky et al, 2003;Ge et al, 2006), and a possible mediator of this effect could be regulated release of BDNF, the expression of which has been shown to be enhanced by GABA application (Berninger et al, 1995). Although it has been reported that depolarization-induced release of BDNF is essentially eliminated by exposure to TTX (Blochl and Thoenen, 1995;Katz, 2000, 2002;Kojima et al, 2001;Gartner and Staiger, 2002) and would therefore not be expected to occur in response to moderate sustained depolarization, it is possible that the methodology used was not sensitive enough to detect it.…”