Inhibitory and excitatory synaptic neuroadaptations in the diazepam tolerant brain

Benzodiazepine (BZ) drugs treat seizures, anxiety, insomnia, and alcohol withdrawal by potentiating γ2 subunit containing GABA type A receptors (GABA_ARs). BZ clinical use is hampered by tolerance and withdrawal symptoms including heightened seizure susceptibility, panic, and sleep disturbances. Here, we investigated inhibitory GABAergic and excitatory glutamatergic plasticity in mice tolerant to benzodiazepine sedation. Repeated diazepam (DZP) treatment diminished sedative effects and decreased DZP potentiation of GABA_AR synaptic currents without impacting overall synaptic inhibition. While DZP did not alter γ2-GABA_AR subunit composition, there was a redistribution of extrasynaptic GABA_ARs to synapses, resulting in higher levels of synaptic BZ-insensitive α4-containing GABA_ARs and a concomitant reduction in tonic inhibition. Conversely, excitatory glutamatergic synaptic transmission was increased, and NMDAR subunits were upregulated at synaptic and total protein levels. Quantitative proteomics further revealed cortex neuroadaptations of key pro-excitatory mediators and synaptic plasticity pathways highlighted by Ca²⁺/calmodulin-dependent protein kinase II (CAMKII), MAPK, and PKC signaling. Thus, reduced inhibitory GABAergic tone and elevated glutamatergic neurotransmission contribute to disrupted excitation/inhibition balance and reduced BZ therapeutic power with benzodiazepine tolerance.