A hypothalamic circuit that controls body temperature

Abstract

The homeostatic control of body temperature is essential for survival in mammals and is known to be regulated in part by temperature-sensitive neurons in the hypothalamus. However, the specific neural pathways and corresponding neural populations have not been fully elucidated. To identify these pathways, we used cFos staining to identify neurons that are activated by a thermal challenge and found induced expression in subsets of neurons within the ventral part of the lateral preoptic nucleus (vLPO) and the dorsal part of the dorsomedial hypothalamus (DMD). Activation of GABAergic neurons in the vLPO using optogenetics reduced body temperature, along with a decrease in physical activity. Optogenetic inhibition of these neurons resulted in fever-level hyperthermia. These GABAergic neurons project from the vLPO to the DMD and optogenetic stimulation of the nerve terminals in the DMD also reduced body temperature and activity. Electrophysiological recording revealed that the vLPO GABAergic neurons suppressed neural activity in DMD neurons, and fiber photometry of calcium transients revealed that DMD neurons were activated by cold. Accordingly, activation of DMD neurons using designer receptors exclusively activated by designer drugs (DREADDs) or optogenetics increased body temperature with a strong increase in energy expenditure and activity. Finally, optogenetic inhibition of DMD neurons triggered hypothermia, similar to stimulation of the GABAergic neurons in the vLPO. Thus, vLPO GABAergic neurons suppressed the thermogenic effect of DMD neurons. In aggregate, our data identify vLPO→DMD neural pathways that reduce core temperature in response to a thermal challenge, and we show that outputs from the DMD can induce activity-induced thermogenesis.

Keywords: dorsomedial hypothalamus; energy expenditure; fiber photometry; preoptic area; thermoregulation.

Fig. 1.

Requirement of preoptic GABAergic neurons in reducing T_core. (A) Heat-induced (38 °C, 2 h) cFos colocalized with the GABAergic marker GAD67 in the vLPO (no. of cFos⁺ and GAD67⁺/no. of cFos⁺ = 36.3 ± 2.4%, n = 3). The dashed white lines indicate boundaries between subregions. (B) Scheme of optogenetic modulation and viral expression of ChR2 (excitatory) or hGtACR1 (inhibitory) in vLPO^Vgat neurons. The dashed yellow lines indicate the positions of optical inserts. (C) Slice recordings of neurons expressing ChR2 (Upper) or hGtACR1 (Lower). Blue light (blue, 6 mW, 40 Hz) faithfully elicited photocurrents in ChR2-expressing neurons in the vLPO. A blue light pulse (6 mW) completely silenced hGtACR1-expressing neurons in the vLPO. (D and E) T_core (D) and activity (E) changes after optogenetic stimulation in mice expressing ChR2 (Upper, n = 4) or hGtACR1 (Lower, n = 3) in vLPO^Vgat neurons. Stimulation protocol for ChR2: unilateral light pulses for 2 s (473 nm, 10 mW, 20 Hz, 40% on) followed by a 2-s break, with the sequence repeating for 30 min. For hGtACR1: bilateral light on for 30 s (473 nm, 6 mW) followed by a 90-s break, with the sequence repeating for 30 min. Bar graph of activity changes (average of 10-min interval) are shown in the right. Baselines (b.s.) represents the average counts between t = –30 and –20 min. (20–30 min) represents the average of counts between t = 20 and 30 min. (Scale bars: A, 100 μm; B; 200 μm.) All data are plotted as mean ± SEM. The P values compared with control group (eYFP) are calculated based on statistical tests listed in SI Appendix, Table S1. *P ≤ 0.05; **P ≤ 0.01. aca, anterior commissure, anterior part; B, bregma; dLPO and vLPO, dorsal and ventral part of lateral preoptic nucleus respectively; MPO, medial preoptic nucleus; 3V, third ventricle; VLPO, ventrolateral preoptic nucleus.

Fig. 2.

Critical role of the vLPO^Vgat→DMD connection in reducing T_core. (A) Scheme for anterograde tracing using ChR2 and retrograde tracing using CTb. (B) vLPO^{Vgat & ChR2} terminals in the DMD. (C) vLPO neurons were retrogradely labeled by CTb injected in the DMD, which colocalized with heat-induced cFos. White arrows indicate the colocalization of cFos and CTb. (Scale bars: 100 μm.) (D) Scheme for terminal optogenetic stimulation in the DMD after ChR2 injection into vLPO^Vgat neurons. Both injection and stimulation were bilateral. (E and F) Bilateral terminal optogenetic stimulation in the DMD-reduced T_core (E) and activity (F) (n = 5). Illumination protocol: light pulses for 2 s (473 nm, 10 mW, 20 Hz, 40%) followed by a 2-s break, with the sequence repeating for 1 h. Bar graph of activity changes (average of 20-min interval) are shown in F. Baselines (b.s.) represents the average of counts in between t = –30 and –10 min. (40–60 min) represents the average of counts between t = 40 and 60 min. (G) Induction of inhibitory postsynaptic currents (IPSCs) in DMD neurons by light stimulation (blue, 6 mW, 2 ms) of ChR2-expressing terminals projected from vLPO^Vgat neurons. IPSCs were blocked by bicuculline (bic.), and partially recovered after wash. Thick lines indicate the mean, whereas the shaded areas indicate SD. (H) Single-cell RT-PCR analysis of recorded cells. All data are plotted as mean ± SEM (except in G); P values compared with control group (532 nm) are calculated based on statistical tests listed in SI Appendix, Table S1. *P ≤ 0.05; ***P ≤ 0.001. DMD and DMV, dorsal and ventral part of dorsomedial hypothalamic nucleus respectively; VMH, ventromedial hypothalamic nucleus.

Fig. 3.

DMD neural dynamics in response to thermal stimuli. (A) Scheme of fiber photometry setup. (B) The expression of GCaMP6f in the DMD driven by Vglut2-IRES-Cre or Vgat-IRES-Cre. The dashed yellow lines indicate the positions of optical inserts. The dashed white lines indicate the boundary of the DMH. (Scale bars: 200 μm.) (C) Controlled floor temperature (T_floor) with a Peltier controller. (Left) Cooling traces (25–13 °C). (Right) Heating traces (25–38 °C). (D) Cooling, but not warming-activated DMD^Vglut2 neurons. Thick lines indicate the mean, whereas the shaded areas indicate SEM. ΔF/F_o represents change in GCaMP6f fluorescence from the mean level before the floor temperature change. b.s. represents baseline, which is the averaged ΔF/F_o between t = –20 and –120 s. (0–10) and (110–120 s) represent the averaged ΔF/F_o between t = (0–10 s) and t = (110–120 s), respectively (n = 3). (E) Cold, but not warmth, activated DMD^Vgat neurons (n = 4). All data are plotted as mean ± SEM. The P values, compared with baselines, are calculated based on statistical tests listed in SI Appendix, Table S1. *P ≤ 0.05; ***P ≤ 0.001; ns, not significant. DMD and DMV, dorsal and ventral part of dorsomedial hypothalamic nucleus, respectively; 3V, third ventricle.

Fig. 4.

Activation of DMD neurons increases body temperature. (A) Schematic and representative images of viral expression of hM3D in DMD Vglut2-IRES-Cre⁺ neurons. (B–D) Activation of DMD^Vgut2 neurons (n = 5) by CNO injection (i.p. at t = 0, 1.5 mg/kg body weight) resulted in significant increases in T_core (B), EE (C), and activity (D). (E) Schematic and representative images of viral expression of hM3D in DMD Vgat-IRES-Cre⁺ neurons. (F–H) Activation of DMD^Vgat neurons (n = 8) by CNO injection (i.p. at t = 0, 1.5 mg/kg body weight) resulted in significant increases in T_core (F), EE (G), and activity (H). (Scale bars: 100 μm.) All data are plotted as mean ± SEM. The P values, compared with the saline group, are calculated based on statistical tests listed in SI Appendix, Table S1. **P ≤ 0.01; ***P ≤ 0.001.

Fig. 5.

Optogenetic inhibition of DMD neurons induces hypothermia. (A and B) Bilateral inhibition of DMD^Vgut2 neurons via hGtACR1 resulted in significant decreases in T_core (A) and activity (B) (n = 4). ΔT_core represents the T_core changes from the mean level before light delivery (t = –30 to –10 min). The baseline (b.s.) (average of t = –30 to –10 min) and t = 60 min are in the bar graph. The average of activity in 30-min intervals between t = –40 and –10 min (baseline, b.s.) and between t = 30 and 60 min are shown in the bar graph. Stimulation protocol: light on for 30 s (473 nm, 10 mW) followed by a 90-s break, with the sequence repeating for 1 h. (C and D) Bilateral inhibition of DMD^Vgat neurons (n = 4) via hGtACR1 resulted in significant decreases in T_core (C) and activity (D). Stimulation protocol is the same as in A. (E) Model for heat-induced suppression of thermogenesis. Sold line represents the connection verified in the current study. Dash lines represents proposed connections based our data and other reports. (+), activation. (–), inhibition. All data are plotted as mean ± SEM. The P values, compared with baseline (b.s.), are calculated based on statistical tests listed in SI Appendix, Table S1. *P ≤ 0.05; **P ≤ 0.01. DMD, dorsal part of dorsomedial hypothalamic nucleus; POA, preoptic area; rMR, rostral medullary region; vLPO, ventral part of lateral preoptic nucleus.