Excitatory GABA Responses in Embryonic and Neonatal Cortical Slices Demonstrated by Gramicidin Perforated-Patch Recordings and Calcium Imaging

Abstract

Gramicidin perforated-patch-clamp recordings in brain slices were used to obtain an accurate assessment of the developmental change in the GABAA receptor reversal potential (EGABAA) in embryonic and early postnatal rat neocortical cells including neuroepithelial precursor cells, cortical plate neurons, and postnatal neocortical neurons. Our results demonstrate that there is a progressive negative shift in EGABAA with the most positive values found in the youngest cortical precursor cells. At the early stages of neocortical development, EGABAA is determined by the chloride (Cl-) gradient, and the internal chloride concentration ([Cl-]i) decreases with development. EGABAA is positive to the resting potential, indicating that GABA serves to depolarize developing neocortical cells. Consistent with this conclusion, GABAA receptor activation with muscimol was found-to increase the internal calcium concentration ([Ca2+]i) in both embryonic and early postnatal neocortical cells through the activation of voltage-gated calcium channels (VGCCs). Postnatal cells exhibit spontaneous postsynaptic synaptic currents, which are eliminated by bicuculline methiodide (BMI) but not glutamate receptor antagonists and reverse at the Cl- equilibrium potential. Likewise, brief spontaneous increases in [Ca2+]i, sensitive to BMI and TTX, are observed at the same ages, suggesting that endogenous synaptic GABAA receptor activation can depolarize cells and activate VGCCs. These results suggest that GABAA receptor-mediated depolarization may influence early neocortical developmental events, including neurogenesis and synaptogenesis, through the activation of Ca(2+)-dependent signal transduction pathways.

Imaging synaptic inhibition in transgenic mice expressing the chloride indicator, Clomeleon

“…Both the time course and the magnitude of the developmental decreases in [Cl − ] i reported by Clomeleon are consistent with previous estimates based on electrophysiological measurements (Owens et al, 1996) and the expression profiles of NKCC1 and KCC2 (Rivera et al, 1999;Stein et al, 2004;Yamada et al, 2004;Rivera et al, 2005). Some of these studies showed a rather large variability, both within a given preparation as well as among different cell types, consistent with our finding of a wide distribution of [Cl − ] i between individual neurons from young mice ( …”

“…A switch in GABAergic transmission, from depolarization to hyperpolarization, during neuronal development has been demonstrated in many studies (Ben-Ari et al, 1989;Cherubini et al, 1991;Owens et al, 1996) (Fig. 5b, top).…”

“…It was later established that the shift from depolarizing GABA responses in young animals to hyperpolarizing responses in adult animals correlated with changes of the equilibrium potential of Cl − towards more negative potentials (Cherubini et al, 1991;Owens et al, 1996), with the onset of the expression of Cl − -extruding transporters such as KCC2 (Rivera et al, 1999), and with a decrease in the expression of Cl − -accumulating transporters such as NKCC1 (Yamada et al, 2004). However, the presumed decrease in [Cl − ] i occurring during development has not yet been visualized in brain slices or in vivo.…”

A role for ion channels in glioma cell invasion

“…To the best of our knowledge, most immature cells that can migrate also accumulate intracellular Cl − and are, thus, well equipped to release KCl and to shrink, whereas there is no net force that drives Cl − out of the cell in most differentiated neurons. Because of the active accumulation of Cl − , immature neurons (Owens et al, 1996) and neuronal stem cells in the ventricular zone (LoTurco et al, 1995) depolarize in response to GABA, whereas differentiated neurons, which no longer move, either hyperpolarize or stabilize their membrane potential (Bormann et al, 1987). Although it is proposed that this depolarizing GABA response might be important physiologically in the context of synapse development (Ben Ari et al, 2004) we suggest that these cells might utilize GABA-gated Cl − channels to adjust their cell volume because they are still migrating through the brain.…”

A Sensitive Period of Mice Inhibitory System to Neonatal GABA Enhancement by Vigabatrin is Brain Region Dependent

“…However, under various conditions GABA facilitates neuronal depolarization, including human epileptogenesis (Cohen et al, 2002), after neuronal injury (van den Pol et al, 1996) and during normal early brain development (Ben-Ari et al, 1989;Khazipov et al, 2001;Owens et al, 1996). In the immature brain, activation of GABA A depolarizes the neuron membrane, and within the first and second postnatal weeks of life in mice E Cl À shifts toward a hyperpolarizing potential thereby leading to inhibitory GABA-mediated responses.…”