Gephyrin Palmitoylation in Basolateral Amygdala Mediates the Anxiolytic Action of Benzodiazepine

Background: Benzodiazepines (BZDs) have been used to treat anxiety disorders for more than five decades as the allosteric modulator of the gamma-aminobutyric acid A receptor (GABA_AR). Little is known about other mechanisms of BZDs. Here, we describe how the rapid stabilization of postsynaptic GABA_AR is essential and sufficient for the anxiolytic effect of BZDs via a palmitoylation-dependent mechanism.

Methods: Palmitoylated proteins in the basolateral amygdala (BLA) of rats with different anxious states were assessed by a biotin exchange protocol. Both pharmacological and genetic approaches were used to investigate the role of palmitoylation in anxiety behavior. Electrophysiological recording, reverse transcription polymerase chain reaction, Western blotting, and coimmunoprecipitation were used to investigate the mechanisms.

Results: Highly anxious rats were accompanied by the deficiency of gephyrin palmitoylation and decreased the synaptic function of GABA_AR in the BLA. We then identified that the dysfunction of DHHC12, a palmitoyl acyltransferase that specifically palmitoylates gephyrin, contributed to the high-anxious state. Furthermore, diazepam, as an anxiolytic drug targeting GABA_ARs, was found to increase gephyrin palmitoylation in the BLA via a GABA_AR-dependent manner to activate DHHC12. The anxiolytic effect of diazepam was nearly abolished by the DHHC12 knockdown. Specifically, similar to the effect of BZD, the overexpression of DHHC12 in the BLA exerted a significant anxiolytic action, which was prevented by flumazenil.

Conclusions: Our results support the view that the strength of inhibitory synapse was controlled by gephyrin palmitoylation in vivo and proposes a previously unknown palmitoylation-centered mode of BZD's action.