Parabrachial CGRP neurons receive diverse threat-related signals and contribute to multiple phases of adaptive threat responses in mice, with their inactivation attenuating both unconditioned behavioral responses to somatic pain and fear-memory formation. Because CGRP^PBN neurons respond broadly to multi-modal threats, it remains unknown how these distinct adaptive processes are individually engaged. We show that while three partially separable subsets of CGRP^PBN neurons broadly collateralize to their respective downstream partners, individual projections accomplish distinct functions: hypothalamic and extended amygdalar projections elicit assorted unconditioned threat responses including autonomic arousal, anxiety, and freezing behavior, while thalamic and basal forebrain projections generate freezing behavior and, unexpectedly, contribute to associative fear learning. Moreover, the unconditioned responses generated by individual projections are complementary, with simultaneous activation of multiple sites driving profound freezing behavior and bradycardia that are not elicited by any individual projection. This semi-parallel, scalable connectivity schema likely contributes to flexible control of threat responses in unpredictable environments.

Data were collected for behavioral measures, intersectional anterograde tracing, slice electrophysiology, and autonomic measures.

1. Data for behavioral measures were collected using video and have been fully processed: freezing behavior was measured based on absence of locomotion and is given as a semi-continuous measure across session epochs. Anxiety measures were obtained by tracked video data comparing time spent in various locations within an elevated plus maze. Excel sheets summarizing the various measures across individual experimental and control animals have been provided.
2. Intersectional anterograde tracing data were collected from sectioned brain tissue from mice injected with retrogradely transported Flp in distinct target regions, and INTRSECT viruses restricting expression to Cre-expressing neurons either expressing or not expressing Flp injected into the PBN. All regions of interest were sectioned such that each imaged section was 180um apart and were mounted and imaged in caudal to rostral order. Each section that contained an area of interest was imaged and analyzed for each experimental animal. Data were processed by outlining each downstream target region with an ROI in imageJ with an off-target ROI from the same section for background-subtraction. Mean pixel intensity was measured within the ROI, then this value had the background intensity subtracted before being multiplied by ROI size to generate a value for total pixel intensity for that ROI. Both the mean pixel intensity, backgroun intensity, ROI size, and total pixel intensity are included for each section of each target-region.
3. Slice electrophysiology data consists of the mean EPSC frequencies and amplitudes obtained from current clamp recordings while delivering light pulses at terminals expressing excitatory or inhibitory opsins and recording from putative post-synaptic neurons.
4. Autonomic data was collected using pulse oximeters with collar sensors or in a plethysmography chamber for some respiratory measures in awake, freely moving animals. Reported data is the average heart rate, blood oxygenation, respiratory rate, and pulse distension during baseline and stimulation periods. Vasodilation measures are the result of analyzing infrared camera images of mice where ROIs were drawn to extract temperature readings 1/3 below base of tail and measuring temperature difference before and during 30-Hz photostimulation. Original infrared image files are also provided.

Columns/rows of data for excluded animals have been highlighted in yellow.