AUTOMATED QUANTIFICATION OF RAPID SPINE SCALING FOLLOWING ACUTE DIAZEPAM‑INDUCED E/I IMBALANCE IN VIVO

Maintaining the fine balance between excitation and inhibition (E/I) is crucial for stable information processing and the prevention of pathological states like epilepsy. Diazepam, a GABA_A receptor modulator, directly shifts this balance by enhancing inhibitory transmission. While long-term adaptation to diazepam is well-documented, the rapid synaptic mechanisms that preserve network stability during acute perturbation remain poorly understood.
In this study, we investigated whether rapid alterations in excitatory synapses contribute to maintaining E/I balance following acute diazepam application. Using chronic two-photon microscopy in a transgenic mouse model, we tracked individual dendritic spines before and after pharmacological perturbation. To ensure an unbiased, large-scale analysis, we developed and validated an automated image-processing pipeline for longitudinal spine tracking, enabling robust quantification of over 6,600 spines across 9 mice.
Our results reveal that average dendritic spine size increased by 6% within one hour of diazepam administration, doubling the change observed in control conditions. This rapid enlargement occurred as a general scaling effect, as the relative size distribution of spines along the dendrites remained preserved. This rapid homeostatic adjustment at excitatory synapses could explain why inhibitory effects in vivo are significantly milder than those observed in vitro, providing a mechanistic basis for how the brain preserves network stability under acute challenge. Furthermore, our high-throughput pipeline provides a powerful platform for future research into circuit adaptation and resilience.