Auxiliary α2δ1 and α2δ3 subunits of calcium channels drive excitatory and inhibitory neuronal network development.

VGCCs are multisubunit complexes that play a crucial role in neuronal signaling. Auxiliary alpha 2 delta subunits of VGCCs modulate trafficking and biophysical properties of the pore-forming alpha 1 subunit and trigger excitatory synaptogenesis. Alterations in the expression level of alpha 2 delta subunits were implicated in several syndromes and diseases, including chronic neuropathic pain, autism, and epilepsy. However, the contribution of distinct alpha 2 delta subunits to excitatory/inhibitory imbalance and aberrant network connectivity characteristic for these pathologic conditions remains unclear. Here, we show that alpha 2 delta 1 overexpression enhances spontaneous neuronal network activity in developing and mature cultures of hippocampal neurons. In contrast, overexpression, but not downregulation, of alpha 2 delta 3 enhances neuronal firing in immature cultures, whereas later in development it suppresses neuronal activity. We found that alpha 2 delta 1 overexpression increases excitatory synaptic density and selectively enhances presynaptic glutamate release, which is impaired on alpha 2 delta 1 knockdown. Overexpression of alpha 2 delta 3 increases the excitatory synaptic density as well but also facilitates spontaneous GABA release and triggers an increase in the density of inhibitory synapses, which is accompanied by enhanced axonal outgrowth in immature interneurons. Together, our findings demonstrate that alpha 2 delta 1 and alpha 2 delta 3 subunits play distinct but complementary roles in driving formation of structural and functional network connectivity during early development. An alteration in alpha 2 delta surface expression during critical developmental windows can therefore play a causal role and have a profound impact on the excitatory-to-inhibitory balance and network connectivity.