DOI: 10.5061/dryad.rn8pk0p7k
title: Dissociable control of unconditioned responses and associative fear learning by parabrachial CGRP neurons

description: 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 CGRPPBN 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 CGRPPBN 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.
description: Data were collected for behavioral measures, intersectional
 anterograde tracing, slice electrophysiology, and autonomic
 measures. 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.
 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.
 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.
 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.
description: Columns/rows of data for excluded animals have been highlighted
 in yellow.