Hindbrain PBN to CeC and ovBNST HiFi circuits modulate medial and lateral preoptic hypothalamus for defensive locomotion: the discovery of perisomatic PACAP‐glutamatergic Calyx‐of‐Held‐like synapse in the rodent pallidum nuclei
Authors: Zhang Limei , Hernández Vito S. , Eiden Lee E. 
Published: April 2020

Defensive behaviors, including fight‐flight and freezing, are critical for animal survival. There has been significant progress in elucidating the brain regions that control decision making and locomotion, which have implicated an extended network of interconnected brain regions, local circuit computations, and cell types that interact to generate emotion‐driven behaviors. However, the roles of neuropeptides in those circuits for functional connectivity are largely unclear. We describe here a direct PACAP‐glutamatergic projection from the hindbrain parabrachial complex (PBN) to the some nuclei of pallidum, i. e. central amygdala and the bed nucleus of stria terminalis (BNST), selectively targeting GABAergic somatostatin expressing neurons. This projection establishes perisomatic glutamatergic “Calyx of Held” like synapses, in addition to axon‐dendritic synapses onto the targeting CeC and ovBNST neurons – a “high‐fidelity” (HiFi) arrangement for insuring a high response‐to‐stimulus ratio. The targeted neurons were found to project to the lateral and medial preoptical hypothalamus where the locomotor initiator and controller centers are located in rodent. To assess the behavioral implication of this pathway, we devised an experiment assessing the effect of predator odor (cat urine) exposure on defensive behavior of wild‐type compared to PACAP knock‐out C57Bl6/N mice. Mice were placed in a glass‐lidded box containing a sample of cat urine within a contained whose lid could be opened remotely. Video recording was made during a 10 min period of urine odor exposure, after which animals were euthanized by cervical dislocation and brains rapidly removed and processed for double in situ hybridization histochemistry using theRNAscope 2.5 HD Duplex Assay. The neuronal activation in the olfactory bulb (OB), piriform cortex (Pir), medial and central amygdala (MeA and CeA), bed nucleus of stria terminalis (BNST), medial and lateral preoptic hypothalamus, parabrachial nucleus (PBN) and hippocampus were assessed for fos expression within PACAP, PAC1, VGluT2 and VGAT‐positive cells. Cat urine exposure triggered purposeful movement such as object exploration/retreat and freezing behaviors in both wild‐type and knock‐out mice, with the PACAP‐deficient (knock‐out) group showing significantly less of both behaviors than the wild‐type group (p < 0.05 and p < 0.01 respectively). Fos mRNA expression was significantly less in the PACAP‐deficient compared to the wild‐type group in PBN, CeC, BNST, MPO and LPO (p < 0.001 in all cases), with similar expression in both groups in the dorsal and ventral DG and in LS. These data suggest that PACAP‐Pac1 projections to CeC are required for the expression of defensive responses elicited by predator threats and offer an initial step towards identifying the PACAP‐PAC1 signaling in sensory‐emotional circuits with locomotor consequences.Support or Funding InformationGrants CONACYT‐CB‐238744, and IN216918 to L.Z. and L.E.E. acknowledges the support of the NIMH Intramural Research Program through MH002386.