Circuit Breakers: α4βδ GABA-A Receptors Drive Adolescent Refinement of Neural Circuits in Prefrontal Cortex.

Abstract

Adolescence is a time when synaptic connections are sculpted to prepare for the cognitive challenges of adulthood, a process known as synaptic pruning. Although this process was first reported over 30 years ago, the initial trigger and functional reason for pruning remain unknown. This thesis provides a multifaceted investigation of α4βδ GABA-A receptor regulation of dendritic spine pruning within pyramidal neurons of the prelimbic prefrontal cortex across adolescent development in mice. The prelimbic cortex projects to the amygdala and drives anxiety states, making precise pruning of juvenile connections critical for proper maturation. Using high-resolution microscopic analysis of Golgi-stained samples, I report a dramatic developmental decrease in basilar dendritic spine density on layer 5 pyramidal neurons between early puberty (16.39 ± 1.55 spines/10μm) and young adulthood (6.10 ± 0.58 spines/10μm), reflecting a 63% reduction (p < 0.0001). This remodeling coincides with a transient 10-fold increase in α4 subunit expression precisely at puberty onset (p < 0.00001) within layer 5 pyramidal neuron dendrites, revealed by immunohistochemistry and amplified electrophysiological responses to a δ subunit-selective agonist (p = 0.00125). Convergent pharmacological, local knockdown (using viral shRNA knockdown and Cre-loxP deletion), and global knockout of α4βδ GABAARs prevented adolescent pruning, while augmenting α4βδ signaling via the selective agonist gaboxadol during early puberty significantly enhanced spine elimination (p<0.05). This demonstrates the causal role of this signaling pathway in mediating the extensive remodeling. The mechanism likely involves α4βδ receptor-mediated suppression of NMDA receptor activation of Kalirin-7 pathways which maintain the dendritic cytoskeleton. α4 knockout prevents the typical 50% decrease in Kalirin-7 levels at puberty (p<0.0001), suggesting dependence on α4βδ signaling. In contrast, increasing NMDAR expression prevents pruning. Critically, preventing pubertal pruning through localized α4βδ knockdown in the prelimbic cortex using AAV-Cre administration increases anxiety-like avoidance behaviors on the elevated plus maze test after an aversive stimulus by 60% in late adolescence (p<0.0001) and 40% in adulthood (p<0.05). This causally links excess prelimbic connectivity from disrupted juvenile synapse elimination to anxiety-related behavioral phenotypes. Furthermore, a similar developmental decrease in spine density occurs in layer 2/3 pyramidal neurons in female mice, aligned with a key role for α4-containing receptors as evidenced by a lack of pruning in α4 knockout mice. Taken together, these results suggest a role for the extrasynaptic α4βδ GABAARs in triggering synaptic pruning and further demonstrate one pathological outcome which can result from dysregulated pruning.