Parabrachial neuron types categorically encode thermoregulation variables during heat defense - PubMed (original) (raw)

. 2020 Sep 2;6(36):eabb9414.

doi: 10.1126/sciadv.abb9414. Print 2020 Sep.

Xiaosa Du 3 4 5, Wen Zhang 4, Cuicui Gao 3 4 5, Hengchang Xie 3 4 5, Yan Xiao 6 7, Xiaoning Jia 3, Jiashu Liu 3, Jianhui Xu 8, Xin Fu 3 4 5, Hongqing Tu 3 4 5, Xiaoyu Fu 3 4 5, Xinyan Ni 3, Miao He 9, Jiajun Yang 6 7, Hong Wang 10, Haitao Yang 3, Xiao-Hong Xu 4, Wei L Shen 1

Affiliations

Parabrachial neuron types categorically encode thermoregulation variables during heat defense

Wen Z Yang et al. Sci Adv. 2020.

Abstract

Heat defense is crucial for survival and fitness. Transmission of thermosensory signals into hypothalamic thermoregulation centers represents a key layer of regulation in heat defense. Yet, how these signals are transmitted into the hypothalamus remains poorly understood. Here, we reveal that lateral parabrachial nucleus (LPB) glutamatergic prodynorphin and cholecystokinin neuron populations are progressively recruited to defend elevated body temperature. These two nonoverlapping neuron types form circuits with downstream preoptic hypothalamic neurons to inhibit the thermogenesis of brown adipose tissues (BATs) and activate tail vasodilation, respectively. Both circuits are activated by warmth and can limit fever development. The prodynorphin circuit is further required for regulating energy expenditure and body weight homeostasis. Thus, these findings establish that the genetic and functional specificity of heat defense neurons occurs as early as in the LPB and uncover categorical neuron types for encoding two heat defense variables, inhibition of BAT thermogenesis and activation of vasodilation.

Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).

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Figures

Fig. 1

Fig. 1. Heat defense variables and projection-specific analysis of the LPB→POA pathway.

(A to D) Changes of _T_core, _T_iBAT, and _T_tail under different ambient temperatures (_T_a) (n = 8 each). (E) Peak Δ_T_tail for different switches of ambient temperatures (_T_a) (n = 8 each). The target _T_a was indicated. Δ_T_tail, Δ_T_core, and Δ_T_iBAT are their current values subtracted by the value at t = 0. (F to H) Dynamics of Δ_T_core and Δ_T_iBAT over time under different _T_a switches (n = 8 each). (I) Scheme of optogenetic activation of glutamatergic (Vglut2) LPB terminals in the ventral medial POA (VMPO). (J) Expression of ChIEF from LPBVglut2 neural terminal in the VMPO. (K to M) Changes of _T_core (K), Δ_T_iBAT (L), and _T_tail (M) after photoactivation LPBVglut2 terminals in the VMPO (n = 4 each). Laser pattern: 473 nm, 6 mW, 20 Hz, 10 ms, 2-s on after 2-s off, 30 min. (N) Scheme of tissue-specific retro-TRAP, where translational ribosomes from VMPO-projected LPBVglut2 neurons were immunoprecipitated and associated mRNAs were used for sequencing. (O) Volcano plots (q value versus log2 fold change) for LPB mRNAs after retro-TRAP sequencing. Logarithmic ratios of mRNA enrichment fold (IP/Input, n = 3) plotted against the q value (where q ≤ 0.5 is considered significant) of the hierarchical linear model. Positive fold changes indicate an enrichment, and negative fold changes indicate a depletion in precipitated mRNAs. (P) Enrichment fold (IP/Input) of PB-expressed genes from Allen Institute. Scale bar, 200 μm. All data are shown as means ± SEM. All P values are calculated on the basis of repeated measures two-way analysis of variance (ANOVA) with Bonferroni’s corrections. *P ≤ 0.05, ***P ≤ 0.001, and ****P ≤ 0.0001. B, bregma; scp, superior cerebellar peduncle; MnPO, median preoptic nucleus; VMPO, ventromedial preoptic nucleus.

Fig. 2

Fig. 2. LPBPdyn/CCK neurons are candidate heat defense neurons.

(A) The overlapping between Pdyn-IRES-Cre and AAV-DIO-GFP and Pdyn staining in the LPB (n = 3 each). Arrows, examples of merged cells. (B) The overlapping between Pdyn-Cre and LSL-GFPL10 and glutamate staining in the LPB (n = 3 each). (C) The overlapping between Pdyn-Cre and LSL-GFPL10 and cold-induced cFos (10°C) in the LPB (n = 3 each). (D to F) The overlapping between Pdyn-Cre and LSL-GFPL10 and cFos induced by different warm ambient temperatures as indicated. Representative images are shown in [(D); n = 3 each). The relative cell numbers are shown in [(E) normalized to 30°C] and merge rates are shown in (F). (G to I) The overlapping between CCK-IRES-Cre and LSL-GFPL10 and cFos induced by different ambient temperatures as indicated. Representative images are shown in [(G); n ≥ 3 each]. The relative cell numbers are shown in [(H) normalized to 30°C] and merge rates are shown in (I). (J) The overlapping between CCK-Cre and LSL-GFPL10 and cold-induced cFos (10°C) in the LPB (n = 3 each). (K) The overlapping between CCK-Cre and LSL-GFPL10 and glutamate staining in the LPB (n = 3 each). (L) The overlapping between CCK-Cre and LSL-GFPL10 and DynA staining in the LPB (n = 3 each). Scale bars, 100 μm except in upper right boxes (50 μm). All data are shown as means ± SEM. All the P values are calculated based on ordinary one-way ANOVA with Tukey’s corrections. *P ≤ 0.05, **P ≤ 0.01, and ****P ≤ 0.0001. LPBC, LPB central part; LPBD, LPB dorsal part; LPBS, LPB superior part; LPBE, LPB external part.

Fig. 3

Fig. 3. LPBPdyn/CCK→POA circuitry is sensitive to warm temperatures.

(A) Scheme of Ca2+ fiber photometry. The floor temperature (T_floor) was controlled by a Peltier device. (B) Expression of DIO-GCaMP6s in LPBPdyn neurons. (C and D) Calcium dynamics from LPBPdyn soma after warming (C) or cooling (D) of the floor. Δ_F/_F_0 represents the change in GCaMP6s fluorescence from the mean level [t = (−120 to 0 s)] [as to (J), (K), (L), (N), (O), (T), and (U)]. The GFP was used as a control. (E) A representative trace of LPBPdyn soma to a 10-min warm stimuli. (F and G) A representative trace of LPBPdyn soma to different T_floor (F) and the correlation between peak Δ_F/F_0 and floor temperatures (G) (n = 5). (H) Scheme for calcium recording of LPBPdyn terminals in the VMPO. (I) Expression of DIO-GCaMP6s from LPBPdyn neural terminals in the VMPO and warm-induced cFos. (J and K) Calcium dynamics from LPBPdyn neural terminals in the VMPO after warming (J) or cooling (K), respectively. (L) Summary of responses from LPBPdyn neural terminals in the VMPO to indicated stimuli, including novel objects, chow food, HFD, mouse of the same sex, and temperatures (n = 4 each). (M) Scheme for soma recording of calcium signals in LPBCCK neurons. (N and O) Calcium dynamics from LPBCCK soma after warming (N) or cooling (O) of the floor, respectively. (P and Q) A representative trace of LPBCCK soma to different floor temperatures (P) and the correlation between peak Δ_F/_F_0 and floor temperatures (Q) (n = 6). (R) Scheme for calcium recording of LPBCCK neural terminals in the VMPO. (S) Expression of DIO-synapse-jGCaMP7b from LPBCCK terminals in the VMPO and warm-induced cFos. (T and U) Calcium dynamics from LPBCCK terminals in the VMPO after warming (T) or cooling (U) of the floor, respectively. Scale bars, 200 μm. All data are shown as means ± SEM.

Fig. 4

Fig. 4. LPBPdyn/CCK→POA circuits induce hypothermia.

(A to F) Light-evoked EPSCs recorded from VMPO neurons innervated by LPBPdyn (A to C) and LPBCCK (D to F) neurons. Light patterns: blue, 7 mW, 10 ms. EPSCs were blocked by GluR antagonists, AP5, and CNQX (B and E). EPSCs were blocked after TTX and were recovered by TTX and 4-AP treatment (C and F). (G) Scheme for optogenetic stimulation of LPBPdyn&ChIEF neural terminals. (H) The LPBPdyn&ChIEF neural terminals in the VMPO. (I) Changes of _T_core after photoactivation of LPBPdyn neural terminals in the VMPO (n = 5 each). (J) Quantification of Δ_T_core after photoactivation of LPBPdyn terminals in the VMPO (n = 5 each), the vLPO (n = 4 each), and the DMH (mRuby, n = 4; ChIEF, n = 3). Laser pattern: 473 nm, 6 mW, Hz as indicated, 30 min. (K) Scheme for optogenetic stimulation of LPBCCK&ChIEF terminals. (L) The LPBCCK&ChIEF neural terminals in the VMPO. (M) Changes of _T_core after photoactivation of LPBCCK terminals in the VMPO. (N) Quantification of Δ_T_core after photoactivation of LPBCCK terminals in the VMPO (tdTomato, n = 5; ChIEF, n = 11) and the DMH (n = 4 each). Laser pattern: 473 nm, 3 mW, 30 min. Scale bars, 500 μm. All data are shown as means ± SEM. The P values are calculated on the basis of repeated measures two-way ANOVA with Bonferroni’s corrections (I and M) and ordinary two-way ANOVA with Dunnett’s corrections (J and N). *P ≤ 0.05, **P ≤ 0.01, and ****P ≤ 0.0001; ns, not significant. DMH, dorsomedial hypothalamus; vLPO, ventral part of lateral preoptic nucleus.

Fig. 5

Fig. 5. LPBPdyn→POA circuit regulates iBAT thermogenesis and muscle shivering.

(A) Scheme for optogenetic stimulation of LPBPdyn&ChIEF terminals in the VMPO (terminal photostimulation). (B and C) Dynamics of Δ_T_core, Δ_T_iBAT, and Δ_T_tail after terminal photostimulation (B) (n = 5, 5, and 7, respectively) and controls (C) (n = 4 each). Δ_T_tail, Δ_T_core, and Δ_T_iBAT are their current values subtracted by the value at t = 0. (D) Shivering EMG of the nuchal muscle after terminal photostimulation (ChIEF, n = 5; GFP, n = 6; _T_skin = 10°C.). (E) Changes of physical activity after terminal photostimulation (n = 7 each). (F) Scheme for simultaneous recording of _T_core and _T_iBAT by pluggable T-type thermocouples. (G to I) Representative traces of _T_core and _T_iBAT after terminal photostimulation (G) and mean change rates (first 5 min) of _T_core and _T_iBAT during the phase of body cooling (H) and rewarming (I) (n = 4 each). (J) _T_core changes induced by terminal photostimulation in sham group and iBAT sympathetic nerves denervated group (n = 4 each). (K) Scheme to block POA glutamatergic neurons while photoactivation of LPBPdyn neuron terminals in the VMPO. (L and M) POA glutamatergic blocking abolished the photoactivation-induced effect in _T_core reduction (L) (ChIEF replotted from Fig. 4I.) and T_iBAT reduction (M) (ChIEF replotted from (B), POA blocking, n = 4. Δ_T(t = i) = T(t = i) − T(t = 0), Laser patterns: 473 nm, 6 mW, 20 Hz, 2-s on after 2-s off, time as indicated. All data are shown as means ± SEM. The P values are calculated on the basis of repeated measures two-way ANOVA with Bonferroni’s corrections (D, J, L, and M), ordinary two-way ANOVA with Bonferroni’s corrections (E), and paired t tests (H and I). *P ≤ 0.05, **P ≤ 0.01, and ****P ≤ 0.0001.

Fig. 6

Fig. 6. LPBCCK→POA circuit regulates vasodilation and muscle shivering.

(A) Scheme for optogenetic stimulation of LPBCCK&ChIEF terminals in the VMPO. (B and C) Dynamics of Δ_T_core, Δ_T_iBAT, and Δ_T_tail after photoactivation of LPBCCK terminals in the VMPO (B) (n = 11, 5, and 6, respectively) and controls are shown in (C) (n = 4 each). Δ_T_core, Δ_T_iBAT, and Δ_T_tail are the values of _T_core, _T_iBAT, and _T_tail subtracted by the value at t = 0, respectively. (D) _T_core changes induced by photoactivation of LPBCCK terminals in the VMPO in the sham group and iBAT sympathetic nerves denervated group (n = 8 each). (E) Shivering EMG of the nuchal muscle after photoactivation of LPBCCK terminals in the VMPO (ChIEF, n = 4; tdTomato, n = 6). (F) Changes of physical activity after photoactivation of LPBCCK terminals in the VMPO (ChIEF, n = 8; tdTomato, n = 5). Laser patterns: 473 nm, 6 mW [3 mW in (D)], 20 Hz, 2-s on after 2-s off, time as indicated. (−30 to 0 min), (0 to 30 min), and (30 to 60 min) in (F) represents the averaged physical activity between t = (−30 to 0 min), t = (0 to 30 min), and t = (30 to 60 min), respectively. All data are shown as means ± SEM. The P values are calculated on the basis of repeated measures two-way ANOVA with Bonferroni’s corrections (E) and ordinary two-way ANOVA with Bonferroni’s corrections (right panels in F). *P ≤ 0.05, **P ≤ 0.01.

Fig. 7

Fig. 7. LPBPdyn→POA circuit is required for heat defense.

(A) Scheme for blocking POA-projected LPBPdyn neurons using neurotoxin TeNT. Retrograde AAVs carrying Cre-dependent FlpO were injected in the VMPO, which drives expression of FlpO-dependent TeNT in the LPB. (B and C) Changes of _T_core after warm (B) and cold (C) exposures, respectively (n = 6 each), where mCherry is the control. (D) Changes of EE after warm exposure (n = 6 each). (0 to 360 min) represents the averaged EE between t = (0 to 360 min). (E and F) Changes of _T_iBAT (E) and their quantification (F) under different ambient temperatures (_T_a) recorded by an infrared camera (TeNT, n = 8; mCherry, n = 7). The coat hair on top of the iBAT was shaved. (0 to 60 min), (90 to 270 min), and (300 to 450 min) in (F) represent the averaged _T_iBAT between t = (0 to 60 min), t = (90 to 270 min), and t = (300 to 450 min), respectively. (G) Changes of _T_tail under different _T_a recorded by an infrared camera (TeNT, n = 8; mCherry, n = 7). All data are shown as means ± SEM. The P values are calculated on the basis of repeated measures two-way ANOVA with Bonferroni’s corrections (B, C, and G), ordinary two-way ANOVA with Bonferroni’s corrections (F), and unpaired t tests (D). *P ≤ 0.05, **P ≤ 0.01, and ****P ≤ 0.0001.

Fig. 8

Fig. 8. LPBCCK→POA circuit is required for heat defense and fever limiting.

(A) Scheme for blocking POA-projected LPBCCK neurons using TeNT. (B and C) Changes of _T_core after warm (B) and cold (C) exposures, respectively (TeNT, n = 7; mCherry, n = 8). (D) Fever responses after injection of IL-1β (n = 7 each group; 10 to 12 weeks after viral injection). AUC, area under the curve. (E and F) Changes of _T_iBAT (E) and their quantification (F) under different _T_a (n = 7 each). (0 to 60 min), (90 to 270 min), and (330 to 480 min) in (F) represent the averaged _T_iBAT between t = (0 to 60 min), t = (90 to 270 min), and t = (330 to 480 min), respectively. (G and H) Changes of _T_tail under different _T_a (n = 7 each). (I) Proposed model for LPBPdyn/CCK circuits in regulating iBAT thermogenesis, vasodilation, muscle shivering, and body weight. All data are shown as means ± SEM. The P values are calculated on the basis of repeated measures two-way ANOVA with Bonferroni’s corrections (B, C, G, and H), ordinary two-way ANOVA with Bonferroni’s corrections (F), and unpaired t tests (D). *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001.

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