Extended amygdala-parabrachial circuits alter threat assessment and regulate feeding

Abstract

An animal's evolutionary success depends on the ability to seek and consume foods while avoiding environmental threats. However, how evolutionarily conserved threat detection circuits modulate feeding is unknown. In mammals, feeding and threat assessment are strongly influenced by the parabrachial nucleus (PBN), a structure that responds to threats and inhibits feeding. Here, we report that the PBN receives dense inputs from two discrete neuronal populations in the bed nucleus of the stria terminalis (BNST), an extended amygdala structure that encodes affective information. Using a series of complementary approaches, we identify opposing BNST-PBN circuits that modulate neuropeptide-expressing PBN neurons to control feeding and affective states. These previously unrecognized neural circuits thus serve as potential nodes of neural circuitry critical for the integration of threat information with the intrinsic drive to feed.

Fig. 1. Anatomical and molecular characterization of opposing BNST-PBN circuits.

(A) Schematic of viral injection and representative image depicting expression in BNST^vGAT soma and their terminals in the PBN. Scale bars, 100 μm. Blue, Nissl; green, eYFP; M, medial; L, lateral; V, ventral; D, dorsal. (B) Schematic of whole-cell patch clamp electrophysiology recordings of optically evoked inhibitory postsynaptic currents (IPSCs). (C) Photoactivation of BNST^vGAT terminals elicits IPSCs (five of eight cells responsive) in PBN neurons that are abolished by GABA type A (GABA_A) receptor antagonism (n = 5 cells, four mice). Ptx, picrotoxin; Bic, bicuculline. (D) Schematic of viral injection and representative image depicting expression in BNST^vGLUT2 soma and their terminals in the PBN. Scale bars, 100 μm. Blue, Nissl; green, eYFP. (E) Schematic of whole-cell patch clamp electrophysiology recordings of optically evoked excitatory postsynaptic currents (EPSCs). (F) Photoactivation of BNST^vGLUT2 terminals elicits EPSCs in PBN neurons (six of nine cells responsive) that are abolished by AMPA/N-methyl-d-aspartate (NMDA) receptor antagonism (n = 6 cells, five mice). (G) Cartoon of injection of translating ribosome affinity purification (TRAP) into PBN of vGLUT2-Cre or vGAT-Cre animals. Tagged mRNA was extracted from the BNST and sequenced. Inset: Representative image of TRAP–green fluorescent protein (GFP) expression in BNST. (H) Heatmap of transcripts enriched in either vGAT or vGLUT2 projections from BNST to PBN over input homogenate [preimmunoprecipitation (pre-IP)] (n = 3 vGLUT2-Cre samples; n = 2 vGAT-Cre samples). (I) Transcripts enriched in either vGAT or vGLUT2 projections from BNST to PBN, after normalization to respective input (pre-IP) homogenates. Positive log₂[fold change (FC)] values indicate transcript enrichment in BNST^vGAT-PBN neurons relative to BNST^vGLUT2-PBN neurons; negative log₂(FC) values indicate relative enrichment in BNST^vGLUT2-PBN neurons. *P < 0.05. Error bars indicate SEM. See also figs. S1 and S2.

Fig. 2. Feeding behavior engages an inhibitory BNST-PBN circuit.

(A) Schematic of in vivo fiber photometry and behavior. (B) Representative GCaMP6s expression in the BNST and PBN of a vGAT-Cre mouse. Scale bars, 100 μm (BNST) and 200 μm (PBN). (C) Representative responses of BNST^vGAT-PBN terminals during food consumption trials (high-sucrose chow; shaded areas represent periods of eating). (D and E) Average z-scored calcium response of BNST^vGAT-PBN terminals during consumption of normal chow under sated conditions (n = 57 bouts, seven mice) and averaged activity of 10-s preconsumption compared to postconsumption initiation over the testing period. (F and G) Average z-scored calcium response of BNST^vGAT-PBN terminals during consumption of sucrose (n = 93 bouts, seven mice) and averaged activity of 10-s preconsumption compared to postconsumption initiation over the testing period. (H and I) Average z-scored calcium response of BNST^vGAT-PBN terminals during consumption of high fat (n = 77 bouts, seven mice) and averaged activity of 10-s preconsumption compared to postconsumption initiation over the testing period. (J and K) Average z-scored calcium response of BNST^vGAT-PBN terminals during consumption of normal chow under anxiogenic novelty-suppressed feeding conditions (n = 121 bouts, seven mice) and averaged activity of 10-s preconsumption compared to postconsumption initiation over the testing period. ***P < 0.001, and ****P < 0.0001. Error bars indicate SEM. See also fig. S3.

Fig. 3. An excitatory BNST-PBN circuit is disengaged during feeding.

(A) Schematic of in vivo fiber photometry and behavior. (B) Representative GCaMP6s expression in the BNST and PBN of a vGLUT2-Cre mouse. Scale bars, 100 μm (BNST) and 200 μm (PBN). (C) Representative responses of BNST^vGLUT2-PBN terminals during food consumption trials (high-sucrose chow; shaded areas represent periods of eating). (D and E) Average z-scored calcium response of BNST^vGLUT2-PBN terminals during consumption of normal chow under sated conditions (n = 133 bouts, six mice) and averaged activity of 10-s preconsumption compared to postconsumption initiation over the testing period. (F and G) Average z-scored calcium response of BNST^vGLUT2-PBN terminals during consumption of sucrose (n = 87 bouts, six mice) and averaged activity of 10-s preconsumption compared to postconsumption initiation over the testing period. (H and I) Average z-scored calcium response of BNST^vGLUT2-PBN terminals during consumption of high fat (n = 56 bouts, six mice) and averaged activity of 10-s preconsumption compared to postconsumption initiation over the testing period. (J and K) Average z-scored calcium response of BNST^vGLUT2-PBN terminals during consumption of normal chow under anxiogenic novelty-suppressed feeding conditions (n = 77 bouts, six mice) and averaged activity of 10-s preconsumption compared to postconsumption initiation over the testing period. **P < 0.01, ***P < 0.001, and ****P < 0.0001. Error bars indicate SEM. See also fig. S3.

Fig. 4. Distinct BNST-PBN circuits display opposing responses to aversive stimuli.

(A) Schematic of in vivo fiber photometry and behavior. (B) Average z-scored calcium transient responses of BNST^vGAT-PBN terminals to an aversive shock (n = 7 mice). (C) Mean z-scored calcium transient responses of BNST^vGAT-PBN terminals (n = 7 mice) 10 s before and after the shock initiation. (D) Representative heatmap of BNST^vGAT-PBN terminal calcium transient activity of a single mouse during aversive shock presentation. (E) Average z-scored calcium transient responses of BNST^vGLUT2-PBN terminals to an aversive shock (n = 6 mice). (F) Mean z-scored calcium transient responses of BNST^vGLUT2-PBN terminals (n = 6 mice) 10 s before and after the shock initiation. (G) Representative heatmap of BNST^vGLUT2-PBN terminal calcium transient activity of a single mouse during aversive shock presentation. (H) Average z-scored calcium transient responses of BNST^vGAT-PBN terminals to entry into the closed arm of an EZM (n = 6 mice). (I) Mean z-scored calcium transient responses of BNST^vGAT-PBN terminals (n = 6 mice) 5 s before and after entry into the closed arm of an EZM. (J) Mean z-scored calcium transient responses of BNST^vGAT-PBN terminals (n = 6 mice) 5 s before and after entry into the open arm of an EZM. (K) Average z-scored calcium transient responses of BNST^vGLUT2-PBN terminals to entry into the closed arm of an EZM (n = 5 mice). (L) Mean z-scored calcium transient responses of BNST^vGLUT2-PBN terminals (n = 5 mice) 5 s before and after entry into the closed arm of an EZM. (M) Mean z-scored calcium transient responses of BNST^vGLUT2-PBN terminals (n = 5 mice) 5 s before and after entry into the open arm of an EZM. *P < 0.05, and **P < 0.01. Error bars indicate SEM. See also fig. S3.

Fig. 5. Distinct BNST-PBN circuits drive opposing feeding and affective behaviors.

(A) Schematic of optogenetic approach to targeting BNST^vGAT-PBN and BNST^vGLUT2-PBN. (B) Schematic of food consumption assay. (C) Food deprivation increases food consumption in control animals. (D) BNST^vGAT-PBN activation increases consumption of normal chow under sated conditions, whereas BNST^vGLUT2-PBN activation decreases consumption of normal chow after food deprivation. (E) Representative heatmaps of time spent in the real-time place preference (RTPP) for Ctrl, BNST^vGAT-PBN:ChR2, and BNST^vGLUT2-PBN:ChR2 mice. (F) BNST^vGAT-PBN activation elicits an RTPP, while BNST^vGLUT2-PBN activation elicits a real-time place aversion, compared to control mice. (G) Schematic of positive (BNST^vGAT-PBN:ChR2) and negative (BNST^vGLUT2-PBN:ChR2) reinforcement tasks. (H) BNST^vGAT-PBN activation is positively reinforcing in an operant-self stimulation task. (I) BNST^vGLUT2-PBN activation is negatively reinforcing in an operant task to turn off photostimulation. (J) BNST^vGAT-PBN activation increases time spent in open arms of EZM. (K) BNST^vGLUT2-PBN activation decreases time spent in open arms of EZM. (L) Schematic of fear conditioning protocol. CS, conditioned stimulus; US, unconditioned stimulus. (M) BNST^vGAT-PBN activation suppresses cued-defensive responses (freezing) after conditioning. (N and O) Novelty-suppressed feeding. (N) Representative heatmaps depicting exploration of the open field. BNST^vGAT-PBN stimulation increases food consumed and BNST^vGLUT2-PBN stimulation decreases food consumed. (O) BNST^vGAT-PBN stimulation decreases latency to feed and BNST^vGLUT2-PBN stimulation increases latency to feed. (P) Conflict feeding in EZM. BNST^vGAT-PBN stimulation increases food consumption. (Q) Schematic of chemogenetic approach to inhibiting BNST^vGAT-PBN and BNST^vGLUT2-PBN. (R) Inhibition of BNST^vGLUT2-PBN increases feeding in sated mice. (S) Inhibition of BNST^vGAT-PBN decreases feeding in food-deprived mice. (T) Inhibition of BNST^vGAT-PBN decreases exploration of EZM. (U) Inhibition of BNST^vGAT-PBN decreases food consumption and inhibition of BNST^vGLUT2-PBN increases food consumption in novelty-suppressed feeding assay. (V) Inhibition of BNST^vGAT-PBN increases latency to feed in novelty-suppressed feeding assay. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Error bars indicate SEM. See also fig. S4 and table S1 for n.

Fig. 6. Dynorphin- and CGRP-expressing PBN neurons receive both excitatory and inhibitory BNST input.

(A) Fluorescent in situ hybridization of pDyn and CGRP in the PBN. Scale bar, 100 μm. (B) pDyn and CGRP neurons are genetically and anatomically distinct populations in the PBN (n = 452 cells). Plots represent the percentage of all labeled cells. (C) Schematic of virus injection depicting CAG-ChR2 in the BNST and DIO-mCherry in the PBN of pDyn-Cre mice. (D) Photoactivation of BNST terminals evoked monosynaptic IPSCs (top trace, +10 mV) and EPSCs (bottom trace, −70 mV) in PBN-Dyn neurons that were restored with bath application of 4-aminopyridine (4-AP) and tetrodotoxin (TTX) (purple trace; n = 13 cells, seven mice). (E) Photoactivation of BNST terminals evoked polysynaptic IPSCs (top trace, +10 mV) and EPSCs (bottom trace, −70 mV) in PBN-Dyn neurons that were blocked with bath application of TTX and could not restored with 4-AP (n = 9 cells, seven mice). (F) Schematic of virus injection depicting CAG-ChR2 in the BNST and DIO-mCherry in the PBN of Calca-Cre mice. (G) Photoactivation of BNST terminals evoked monosynaptic IPSCs (top trace, +10 mV) and EPSCs (bottom trace, −70 mV) in PBN-Calca neurons that were restored with bath application of 4-AP and TTX (purple trace; n = 12 cells, six mice). (H) Photoactivation of BNST terminals evoked polysynaptic IPSCs (top trace, +10 mV) and EPSCs (bottom trac, −70 mV) in PBN-Calca neurons that were blocked with bath application of TTX and could not restored with 4-AP (n = 3 cells, six mice). (I) Pie charts depicting the proportion of PBN-Dyn neurons receiving monosynaptic, polysynaptic, or no input from the BNST, as well as the proportion of the evoked monosynaptic currents that are inhibitory or excitatory. (J) Pie charts depicting the proportion of PBN-Calca neurons receiving monosynaptic, polysynaptic, or no input from the BNST, as well as the proportion of the evoked monosynaptic currents that are inhibitory or excitatory.

Fig. 7. Dynorphin-expressing PBN neurons mediate feeding and affective behavior.

(A) Schematic of in vivo fiber photometry. (B) Average z-scored calcium response of PBN^pDyn terminals during consumption of normal chow under sated conditions (n = 68 bouts, six mice) and averaged activity of 10-s preconsumption versus postconsumption initiation. (C) Average z-scored calcium response of PBN^pDyn terminals during consumption of normal chow under anxiogenic conditions (n = 39 bouts, six mice) and averaged activity of 10-s preconsumption versus postconsumption initiation. (D) Average z-scored calcium response of PBN^pDyn terminals to an aversive shock (n = 25 bouts, six mice) and averaged activity of 5 s before shock initiation compared to 5 s after (top) and 5 to 10 s after (bottom) shock initiation. (E) Average z-scored calcium response of PBN^pDyn terminals to entry into the closed arm of an EZM and averaged activity of 5 s before and after entry into the closed arm (top; n = 75 bouts, six mice) and open arm (bottom; n = 75 bouts, six mice). (F) Schematic of optogenetic approach to targeting PBN^pDyn neurons. (G) ChR2-eYFP expression in PBN^pDyn neurons. Scale bar, 200 μm. (H) Representative heatmaps of time spent in RTPP for Ctrl and PBN^pDyn:ChR2 mice. (I and J) PBN^pDyn activation elicits a real-time place aversion in a (I) frequency-dependent manner, including (J) 20 Hz (ChR2, n = 8; Ctrl, n = 6). (K) PBN^pDyn activation decreases food consumption after food deprivation (ChR2, n = 8; Ctrl, n = 6). (L) Schematic of chemogenetic approach to targeting PBN^pDyn neurons. (M) hM4Di-mCherry expression in PBN^pDyn neurons. Scale bar, 100 μm. (N to Q) Chemogenetic inhibition of PBN^pDyn neuron (N) increases food consumption and (O) decreases latency to eat while (P) increasing food consumed in a novelty-suppressed feeding task and (Q) increasing time spent in the open arms of EZM (DREADD, n = 7 to 8; Ctrl, n = 6 to 7). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Error bars indicate SEM. See also fig. S5.