A peptidergic amygdala microcircuit modulates sexually dimorphic contextual fear

Abstract

Trauma can cause dysfunctional fear regulation leading some to develop disorders like post-traumatic stress disorder (PTSD). The amygdala regulates fear, and, PACAP and PAC1 receptors are linked to PTSD symptom severity at genetic/epigenetic levels, with a strong link in females with PTSD. We discovered a PACAPergic projection from the basomedial amygdala (BMA) to the medial intercalated cells (mICCs). In vivo optogenetic stimulation of this pathway increased cfos expression in mICCs, decreased fear retention and increased fear extinction. Selective deletion of PAC1 receptors from the mICCs in females reduced fear acquisition, but enhanced fear generalization and reduced fear extinction in males. Optogenetic stimulation of the BMA-mICCs PACAPergic pathway produced excitatory postsynaptic currents (EPSCs) in mICCs neurons, which was enhanced by PAC1 receptor antagonist, PACAP 6-38. Our findings show that mICCs modulate contextual fear in a dynamic and sexdependent manner via the microcircuit containing the BMA and mICCs, dependent on behavioral state. Results 1.PACAP-expressing neurons in the BMA innervate the medial ICCs We examined EGFP expression in the Adcyap1-EGFP mice, which restricts EGFP expression to PACAP expressing neurons [12]. Although distributed broadly in the amygdala, EGFP + cells were enriched in the lateral and basomedial nucleus (BMA) subregion with fibers in the mICCs (Fig. 1A and Fig. S1A, B). We evaluated the local afferents of BMA and found a majority of PACAPergic neurons in the BMA innervate the dorsal and ventral mICCs (Fig.1A). By comparison, we found little to no innervation of the CN by EGFP + neurons (Fig 1A. and Fig. S1B). The pattern of expression corresponds with PACAP mRNA expression, which is high in the BMA and with some expression in the mICCS (Allen Brain Atlas Mouse Brain In Situ hybridization) (Fig. S1C). To further confirm the existence of monosynaptic PACAPergic projections from BMA to mICC we used an intersectional approach as described in Fenno et. al. 2014 [13]. For this, we injected a retrogradely trafficked Cre-dependent HSV virus (hEF1α-LS1L-mCherry-IRES-flpo) into the mICCs (dorsal portion) and a Flp-dependent AAV5-EF1a-fDIO-ChR2-eYFP-WPRE into the BMA of Adcyap1-2A-Cre. Adcyap1-2A-Cre mice express Cre specifically in PACAP-containing neurons. With the intersectional approach, the HSV virus, which is Credependent and expresses FLP, retrogradely transports allowing expression specifically in PACAPergic neurons in the BMA. The AAV viruses in turn are FLP-dependent, so therefore expresses only in neurons that have FLP. This allows labeling specific projections from the BMA to mICCs. Thus, using two different approaches, we were able to confirm that PACAPergic (Adcyap1-EGFP) neurons in the BMA innervate mICCs (Fig. 1B and Fic. 1C). 2.In vivo optogenetic stimulation of BMA PACAPergic input to the mICCs decreases fear retention and increases fear extinction