Delta Subunit-Containing Gamma-Aminobutyric Acid A Receptor Disinhibits Lateral Amygdala And Facilitates Fear Expression In Mice

Fear is an emotional response to an impending danger or threat situation.Reasonable fear can help organisms quickly respond to an environmental threat,which is a survival mechanism retained during species evolution.However,excessive fear can induce various mental illnesses,such as posttraumatic stress disorder(PTSD)and phobias.In the brain,the amygdala is a critical nucleus closely involved in regulating a series of emotional expressions such as fear and anxiety.Gamma-aminobutyric acidergic(GABAergic)inhibition is essential for regulating the activities of the amygdala.Many researchers have shown that GABAergic inhibition in the amygdala is involved in the regulation of fear emotion.The abnormal GABAergic inhibitory effect in the amygdala is often closely related to neuropsychiatric diseases,including anxiety disorders.Moreover,many existing anti-anxiety drugs target the GABAergic neurotransmissions within the amygdala and other brain regions.As the most important inhibitory system in the brain,GABA acts mainly by binding to the ionic A-type GABA receptor(GABA_AR)on the postsynaptic membrane and metabolic B-type GABA receptor(GABA_BR)on the extrasynaptic membrane.The ionic GABA_AR mediates not only transient and temporal inhibitory transmission but also persistent and tonic inhibition.Among all GABA_AR subtypes that mediate tonic suppression,?subunit-containing GABA_AR(GABA_A(?)R)is considered to be the only GABA_AR distributed on the extrasynaptic membrane.The GABA_A(?)R has a high sensitivity to GABA and low desensitization to prolonged GABA exposure.Thus,it has been indicated in mediating tonic inhibition in multiple brain regions such as the cerebellum and hippocampus.Recent studies have shown that in the intercalated cells(ITCs)of the amygdala and the lateral subdivision of the central amygdala,activation of GABA_A(?)R significantly affects neuronal excitability.However,whether the GABA_A(?)R is expressed in the lateral amygdala(LA)which is responsible for receiving and integrating various sensory information inputs?If so,what contribution do these receptors have to the inhibitory signal of the microcircuit in LA?And,how do these receptors regulate the expression of fear emotion?The neuronal mechanism by which these receptors act their function is unclear.To answer these questions,we occupied the transgenic mice expressing green fluorescent protein under the control of the glutamic acid decarboxylase 67promoter to specifically label GABAergic neurons(GAD67-GFP(?neo)mice)and crossed them with delta subunit knockout mice to construct the GFP^+/-Gabrd^+/+mice(WT group)and GFP^+/-Gabrd^-/-mice(KO group).The results of immunohistochemical staining showed that the positive signal of?subunit in WT mice was indiscrimination appeared on the surface of various types of interneurons,such as parvalbumin(PV),cholecystokinin(SSK)and somatostatin(SOM)interneurons.While for the KO mice,no positive signal was observed in both neuron types.Subsequently,we conducted outside-out patch electrophysiological recordings of PNs and INs in the LA of two genotypes of mice,and the results showed that the THIP which is the agonist of GABA_A(?)R could selectively increase the single-channel conductance of INs in WT mice.Subsequently,THIP and THIP+PTX were used to activate and inhibit GABA_A(?)R respectively.At the same time,the INs activity in the LA of WT and KO mice was recorded.It was found that THIP could significantly reduce the excitability of neurons in the WT group.This effect could be rescued by THIP+PTX.The above phenomenon was due to THIP and THIP+PTX sequentially decreasing and increasing neuron input resistance;for the KO group,there was no such effect.Furthermore,we used electrical stimulation to simulate the input of sensory information and found that knocking out the?subunit significantly enhanced the inhibitory neurotransmission on PNs in LA.This enhancement was due to the loss of GABA_A(?)R-mediated tonic inhibition in INs of LA,rather than affecting inhibitory synaptic transmission.The above-mentioned enhanced inhibitory effect,in turn,caused an impairment of LTP induction onto the LA PNs,and thus occluded the condition of fear memory of KO mice.It is well known that there are different stages of fear memory,include fear acquisition,fear retrieval,fear extinction,fear renewal and fear reinstatement.We test the fear conditioning of mice with two genotypes,and found that the KO mice froze significantly less often to CS than the WT mice did.However,both genotypes mice showed a similar response to the presentation of the unconditioned stimulus(US)and conditioned stimulus(CS)during habituation and CS-US pairing.Thus,the result indicating that the retrieval of long-term fear memory of KO mice was impaired.In summary,our work shows that GABA_A(?)R acts as a"brake"on the activity of LA,and the disinhibition mediated by it can effectively prevent the amygdala activity from being over-repressed and ensure the proper expression of emotions.