Acute and Consolidation Transcriptional Programs in the Central Amygdala Following Parabrachial Pain-circuit Activation

Abstract

The central nucleus of the amygdala (CeA) receives nociceptive input from parabrachial nucleus(PBN) neurons expressing the CGRP precursor encoded by the Calca gene, and plasticity at this synapse has been implicated in chronic pain development. Which CeA populations respond to parabrachial input, and how their transcriptional programs evolve from acute activation to consolidation, remains unclear. Here, we used single-cell RNA sequencing to characterize CeA GABAergic neuron responses after chemogenetic activation of CalcaPBN neurons at acute (D0.01, 30 minutes) and consolidation (D3, 3 days) timepoints. Analysis of 19,544 neurons resolved 21 clusters including 13 core CeA populations. Acute activation preferentially engaged C9, a population co-expressing Prkcd and Calcrl, which showed significant upregulation of immediate early genes including Arc, Egr1, and Egr4. Differential expression analysis revealed striking temporal asymmetry: D0.01 showed 1,872 differentially expressed genes (DEGs) with balanced directionality, while D3 showed 4,787 DEGs with a 25-fold bias toward downregulation. Suppressed genes included ionotropic receptors (Gria4, Gabra1), postsynaptic scaffolds (Camk2b, Shank3, Homer2), and the transcription factor Foxo3. Temporal overlap was minimal: just 15 genes sustained significance in C8 (Calcrl neurons) across both timepoints. Gene ontology analysis showed 87% of enriched terms were D3-specific, including synaptic plasticity regulation and negative regulation of long-term potentiation. These findings demonstrate that acute activation and early consolidation engage distinct transcriptional programs, with consolidation characterized by widespread suppression consistent with homeostatic refinement rather than simple potentiation. This molecular taxonomy identifies specific CeA populations and gene programs as potential intervention targets in the transition from acute to nociplastic pain.