Abstracts

Rationale: The development of a balance between excitatory and inhibitory synapses is a critical step in the generation and maturation of functional circuits. Accumulating evidence suggests that neuronal activity is critical for achieving such a balance in the developing cortex. During corticogenesis, the principal inhibitory neurotransmitter GABA (Υ-aminobutyric acid) exerts an excitatory effect on newborn neurons due to the high intracellular chloride gradient made by the Na+-K+-2Cl- cotransporter (NKCC1). GABAergic signalling precedes glutamatergic signalling in the developing neocortex as newborn neurons express GABAA receptors and receive GABAergic inputs before forming glutamaterigic synapses with each other. While GABA-induced excitation may influence the sequential development of inhibitory and excitatory synaptic inputs, the mechanism that mediates this process is unknown. In this study, we attempt to elucidate the role of GABA signaling on circuit formation of newborn neurons and the mechanism by which inhibition and excitation is established in the cortex.Methods: To investigate the role of GABA in circuitry formation in the developing cortex, we used an RNA interference (RNAi) strategy to knockdown the expression of NKCC1, a Cl- importer responsible for GABA-induced depolarization in immature neurons. We performed in utero electroporation of plasmids co-expressing GFP and a previously characterized short hairpin RNA (shRNA) against Nkcc1 in 15-day-old embryonic mice. We used whole-cell recordings as well as confocal microscopy to study the physiological and anatomical evidence for synaptic integration of electroporated neurons during the first 3 weeks of postnatal life.Results: We demonstrate here that GABA-induced depolarization plays several important roles during synaptogenesis and early cortical circuit formation. Immature neurons expressing Nkcc1-shRNA show both temporal delay and defects in synaptic integration. Furthermore, these cells demonstrate anatomical evidence of altered circuit development by displaying fewer branches, smaller soma size, and decreased dendritic spine density. In addition, we show that depolarizing GABAergic transmission is required for the formation of glutamatergic synapses via GABAergic activation of NMDA-receptors.Conclusions: Here we show by knocking down NKCC1, that GABA-induced excitation regulates synapse formation and dendritic development of newborn cortical neurons in vivo. GABA-mediated depolarization also controls the formation of excitatory AMPA inputs via activation of NMDA channels. Our study identifies an essential role for GABA in the synaptic integration of newborn cortical neurons and suggests an activity-dependent mechanism for achieving the balance between excitation and inhibition in the developing cortex.