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

TLDR

Gramicidin perforated‑patch recordings in brain slices were used to measure developmental changes in the GABA_A receptor reversal potential in embryonic and early postnatal rat neocortical cells. E_GABA A progressively shifts negative during development, remaining positive to the resting potential in the youngest cells, so GABA depolarizes developing neocortical cells, elevates intracellular Ca via voltage‑gated calcium channels, and spontaneous GABAergic activity activates Ca‑dependent signaling that may influence neurogenesis and synaptogenesis.

Abstract

Gramicidin perforated-patch-clamp recordings in brain slices were used to obtain an accurate assessment of the developmental change in the GABA A receptor reversal potential ( E GABA A ) 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 E GABA A , with the most positive values found in the youngest cortical precursor cells. At the early stages of neocortical development, E GABA A is determined by the chloride (Cl − ) gradient, and the internal chloride concentration ([Cl − ] i ) decreases with development. E GABA A is positive to the resting potential, indicating that GABA serves to depolarize developing neocortical cells. Consistent with this conclusion, GABA A receptor activation with muscimol was found to increase the internal calcium concentration ([Ca 2+ ] 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 [Ca 2+ ] i , sensitive to BMI and TTX, are observed at the same ages, suggesting that endogenous synaptic GABA A receptor activation can depolarize cells and activate VGCCs. These results suggest that GABA A receptor-mediated depolarization may influence early neocortical developmental events, including neurogenesis and synaptogenesis, through the activation of Ca 2+ -dependent signal transduction pathways.