Subpopulations of corticotropin-releasing factor containing neurons and internal circuits in the chicken central extended amygdala

In mammals, the central extended amygdala is critical for regulation of the stress response. This regulation is extremely complex, involving multiple subpopulations of GABAergic neurons and complex networks of internal and external connections. Two neuron subpopulations expressing corticotropin-releasing factor (CRF), located in the central amygdala and the lateral bed nucleus of the stria terminalis (BSTL), play key roles in the long-term components of fear learning and in sustained fear responses akin to anxiety. Very little is known on the regulation of stress by the amygdala in non-mammals, hindering efforts for trying to improve animal welfare. In birds, one of the major problems relates to the high evolutionary divergence of the telencephalon, where the amygdala is located. In the present study, we aimed to investigate the presence of CRF neurons of the central extended amygdala in chicken and the local connections within this region. We found two major subpopulations of CRF cells in BSTL and the medial capsular central amygdala of chicken. Based on multiple labeling of CRF mRNA with different developmental transcription factors, all CRF neurons seem to originate within the telencephalon since they express Foxg1, and there are two subtypes with different embryonic origins that express Islet1 or Pax6. In addition, we demonstrated direct projections from Pax6 cells of the capsular central amygdala to BSTL and the oval central amygdala. We also found projections from Islet1 cells of the oval central amygdala to BSTL, which may constitute an indirect pathway for regulation of BSTL output cells. Part of these projections may be mediated by CRF cells, in agreement with expression of CRF receptors in both Ceov and BSTL. Our results show a complex organization of the central extended amygdala in chicken, and open new venues for studying how different cells and circuits regulate stress in these animals.