Chronic fasudil treatment induces benzodiazepine-like tolerance via modulation of GABA-A receptor γ2 subunit expression and impairs contextual fear memory in mice

Background: Fasudil hydrochloride (FAS) is a selective Ras homologous (Rho) associated kinase (ROCK) inhibitor and vasodilator used to treat cerebral vasospasm. Owing to its neuroprotective effects in preclinical models of neurodegeneration, FAS is currently under clinical investigation for several neurological diseases. Although the precise mechanism of action of FAS remains unclear, some preclinical studies suggest that it may exert anxiolytic and anticonvulsant effects—pharmacological activities typically associated with positive allosteric modulators of the γ‑aminobutyric acid (GABA)-A ​ receptor, such as benzodiazepines. This study investigates whether FAS acts as a modulator of the GABA-A ​ receptor and examines the effects of its chronic administration. Methods: Protein comparative analyses and in silico docking assays were performed to assess the binding affinity of FAS for the GABA-A​ receptor. CD1 mice received daily oral administration of FAS for 12 weeks. One week after treatment cessation, behavioural tests and magnetic resonance (MR) analyses were conducted to evaluate brain function and metabolism. In addition, Rho guanosine triphosphatase activity, GABA-A receptor expression, and levels of postsynaptic density protein 95 (PSD‑95) were assessed. Results: The computational analysis predicted a strong binding affinity of FAS for the GABA-A​ receptor. In vivo assays revealed that FAS impaired contextual fear memory without affecting anxiety-like behaviour, motor coordination, or recognition memory. These behavioural alterations were accompanied by a reduction in the γ2 subunit of the GABA-A​ receptor in both the hippocampus and the prefrontal cortex (PFC). Within the PFC, PSD‑95 expression was decreased, whereas the activity of Ras-related C3 botulinum toxin substrate 1 was increased. MR analyses showed elevated glutamate levels in the PFC, together with a general increase in total choline and a mild impairment of white matter integrity. Conclusions: Our findings suggest that FAS influences GABA-A receptor expression and may act as an allosteric modulator. Chronic treatment in healthy mice appears to adversely affect the PFC, producing behavioural and molecular alterations resembling those observed following prolonged benzodiazepine exposure.