Hypothalamic thermoregulatory neurons divergently modulate isoflurane anesthesia via temperature dependent and independent mechanisms.

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Learn more: PMC Disclaimer | PMC Copyright Notice iScience . 2026 Mar 30;29(5):115542. doi: 10.1016/j.isci.2026.115542 Search in PMC Search in PubMed View in NLM Catalog Add to search Hypothalamic thermoregulatory neurons divergently modulate isoflurane anesthesia via temperature dependent and independent mechanisms Shuang Cai Shuang Cai 1 Department of Anesthesiology, Shanghai Children’s Medical Centre, Shanghai Jiao Tong University, Shanghai 200127, China 2 Key Laboratory of Brain Science, Key Laboratory of Anesthesia and Organ Protection of Ministry of Education (In Cultivation), Zunyi Medical University, Zunyi, Guizhou 563006, China 3 Department of Anesthesiology, Affiliated Hospital of Zunyi Medical University, Zunyi, Guizhou 563000, China Find articles by Shuang Cai 1, 2, 3, 7 , Wen Z Yang Wen Z Yang 4 School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China Find articles by Wen Z Yang 4, 7 , Mao Xu Mao Xu 2 Key Laboratory of Brain Science, Key Laboratory of Anesthesia and Organ Protection of Ministry of Education (In Cultivation), Zunyi Medical University, Zunyi, Guizhou 563006, China 5 Zunyi Medical University, Zunyi, Guizhou 563006, China Find articles by Mao Xu 2, 5, 7 , Huimin Chen Huimin Chen 6 Department of Anesthesiology, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong 510515, China Find articles by Huimin Chen 6 , Liang Zhou Liang Zhou 2 Key Laboratory of Brain Science, Key Laboratory of Anesthesia and Organ Protection of Ministry of Education (In Cultivation), Zunyi Medical University, Zunyi, Guizhou 563006, China Find articles by Liang Zhou 2 , Tianyuan Luo Tianyuan Luo 2 Key Laboratory of Brain Science, Key Laboratory of Anesthesia and Organ Protection of Ministry of Education (In Cultivation), Zunyi Medical University, Zunyi, Guizhou 563006, China 3 Department of Anesthesiology, Affiliated Hospital of Zunyi Medical University, Zunyi, Guizhou 563000, China Find articles by Tianyuan Luo 2, 3 , Shouyang Yu Shouyang Yu 2 Key Laboratory of Brain Science, Key Laboratory of Anesthesia and Organ Protection of Ministry of Education (In Cultivation), Zunyi Medical University, Zunyi, Guizhou 563006, China Find articles by Shouyang Yu 2 , Kan Zhang Kan Zhang 1 Department of Anesthesiology, Shanghai Children’s Medical Centre, Shanghai Jiao Tong University, Shanghai 200127, China Find articles by Kan Zhang 1 , Haiying Wang Haiying Wang 3 Department of Anesthesiology, Affiliated Hospital of Zunyi Medical University, Zunyi, Guizhou 563000, China Find articles by Haiying Wang 3 , Tian Yu Tian Yu 2 Key Laboratory of Brain Science, Key Laboratory of Anesthesia and Organ Protection of Ministry of Education (In Cultivation), Zunyi Medical University, Zunyi, Guizhou 563006, China 3 Department of Anesthesiology, Affiliated Hospital of Zunyi Medical University, Zunyi, Guizhou 563000, China 5 Zunyi Medical University, Zunyi, Guizhou 563006, China Find articles by Tian Yu 2, 3, 5, ∗ , Jijian Zheng Jijian Zheng 1 Department of Anesthesiology, Shanghai Children’s Medical Centre, Shanghai Jiao Tong University, Shanghai 200127, China Find articles by Jijian Zheng 1, ∗∗ , Wei L Shen Wei L Shen 4 School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China 5 Zunyi Medical University, Zunyi, Guizhou 563006, China Find articles by Wei L Shen 4, 5, 8, ∗∗∗ Author information Article notes Copyright and License information 1 Department of Anesthesiology, Shanghai Children’s Medical Centre, Shanghai Jiao Tong University, Shanghai 200127, China 2 Key Laboratory of Brain Science, Key Laboratory of Anesthesia and Organ Protection of Ministry of Education (In Cultivation), Zunyi Medical University, Zunyi, Guizhou 563006, China 3 Department of Anesthesiology, Affiliated Hospital of Zunyi Medical University, Zunyi, Guizhou 563000, China 4 School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China 5 Zunyi Medical University, Zunyi, Guizhou 563006, China 6 Department of Anesthesiology, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong 510515, China ∗ Corresponding author zyyutian@126.com ∗∗ Corresponding author zhengjijian626@sina.com ∗∗∗ Corresponding author shenwei@shanghaitech.edu.cn 7 These authors contributed equally 8 Lead contact Received 2025 Oct 31; Revised 2026 Feb 1; Accepted 2026 Mar 26; Collection date 2026 May 15. © 2026 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). PMC Copyright notice PMCID: PMC13091562  PMID: 42004037 Summary Perioperative hypothermia is common clinically. The neural mechanisms underlying general anesthesia and the hypothermia it induces remain elusive. We found that lower core temperature (T core ) deepened anesthesia and delayed emergence, which showed a logarithmic relationship below 31.9°C. Chemogenetic activation of brain-derived neurotrophic factor in the lateral preoptic area (LPO BDNF ) neurons before isoflurane administration lowered T core and increased isoflurane induced burst-suppression ratio (BSR), δ power, and recovery time, while its optogenetic activation during isoflurane did not change T core and anesthesia depth. Conversely, chemogenetic activation of dorsomedial hypothalamus glutamatergic (DMH Vglut2 ) neurons increased T core , attenuated anesthesia depth and promoted recovery. Stage-specific optogenetic activation of DMH Vglut2 neurons and its downstream raphe pallidus (RPa) projection during isoflurane exposure induced cortical activation without T core alteration. Our study indicates that LPO BDNF neurons modulate isoflurane anesthesia primarily through temperature-dependent mechanism, while DMH Vglut2 neurons and the DMH Vglut2 to RPa pathway have a temperature-independent function in modulating isoflurane anesthesia. Subject areas: health sciences, human physiology, neuroscience Graphical abstract Open in a new tab Highlights • Anesthetic emergence delays logarithmically as T core falls below 31.9°C in mice • LPO BDNF neurons potentiates anesthetic depth via a temperature-dependent manner • The DMH Vglut2 →RPa pathway accelerates emergence by temperature-independent mechanism Health sciences; human physiology; neuroscience Introduction General anesthesia induces a state of unconsciousness often accompanied by suppression of multiple physiological functions, including thermoregulation, which frequently leads to hypothermia, respiratory inhibition, and circulatory inhibition. 1 , 2 Clinical studies report that approximately 70% of surgical patients develop intraoperative mild hypothermia with core temperature (T core ) less than 36.0°C, 3 , 4 which increases the risk of surgical complications, including coagulopathy, infection, shivering, and delayed recovery. 5 , 6 Recent studies reported that specific neural circuits that regulate sleep-wakefulness are involved in general anesthetics-induced unconsciousness and emergence, 7 , 8 , 9 and also share a significant overlap with thermoregulatory circuits. 10 , 11 , 12 These lead us to propose that the thermoregulatory neurons may also be involved in modulating the general anesthesia state, while the mechanisms remain elusive. T core is actively and tightly controlled by hypothalamic neurocircuits under physiological conditions. The hypothalamic preoptic area (POA) is a well-established center for thermoregulation. 13 , 14 , 15 Distinct regions and neuronal populations within the POA contribute to different aspects of thermoregulation. 16 Activation of γ-aminobutyric acid (GABAergic) neurons, mostly with BDNF expressing in the lateral preoptic area (LPO) induces torpor-like profound hypothermia without cold defense responses. 15 The dorsomedial hypothalamus glutamatergic (DMH) constitutes a principal efferent pathway of the LPO. 15 , 16 In contrast to the LPO, DMH is responsible for increasing T core in responding to cold. 17 , 18 The DMH contains both glutamatergic and GABAergic neurons. 19 , 20 Chemogenetic activation of dorsomedial hypothalamus glutamatergic (DMH Vglut2 ) neurons induces hyperthermia via their raphe pallidus (RPa) projections. 21 Beyond thermoregulation, the DMH promotes wakefulness via projecting to several key nuclei, including dorsal raphe nucleus (DR), ventral tegmental area (VTA), and locus coeruleus (LC). 22 , 23 Although the function of DMH GABA neurons in sevoflurane emergence has been implicated, 24 overall, how thermoregulatory DMH neurons contribute to anesthesia need deeper exploration. Thus, we hypothesized that brain-derived neurotrophic factor in the lateral preoptic area (LPO BDNF ) and DMH Vglut2 neurons play a substantial role of isoflurane-induced anesthesia. In this study, we find that low T core (less than 31.9°C) increased the depth of isoflurane anesthesia and delayed recovery. Chemogenetic activation of LPO BDNF neurons before isoflurane administration lowered T core and increased isoflurane induced electroencephalogram (EEG) burst-suppression ratio (BSR), δ power during anesthesia, and prolonged recovery time, while its optogenetic activation during isoflurane exposure did not change T core and anesthesia depth. Conversely, chemogenetic activation of DMH Vglut2 neurons increased T core , attenuated anesthesia depth during anesthesia and promoted recovery. Optogenetic activation of DMH Vglut2 neurons and its downstream RPa projection during anesthesia attenuated anesthesia depth without T core alteration, suggesting the existence of temperature-independent mechanism for direct modulation of anesthesia. Results Core temperature and anesthesia effect changes along with ambient temperature under isoflurane Under the various ambient temperatures (T a ) at 20°C, 25°C, 32°C, and 36°C ( Figures 1 A and 1B), T core was significantly changed under isoflurane exposure. Specifically, at 20°C, 0.8%, 1.1%, and 1.4% isoflurane reduced T core by 3.24°C ± 0.58°C, 3.54°C ± 0.69°C, and 4.51°C ± 0.93°C, respectively ( n = 7; Figures 1 C–1F). The same pattern changes were noted in other concentrations and ambient temperature ( Figures 1 C–1F). Quantitative analysis showed a linear relationship between T a and T core ( R 2 = 0.92; Figure 1 G). Figure 1. Open in a new tab T core negatively affects anesthesia depth in mice (A) Schematic diagram depicting the T a -controlling device for isoflurane-induced anesthesia. (B) Schematic of protocol for the isoflurane anesthesia procedure, with simultaneous T core monitoring and EEG recordings. (C–E) T core changes in response to different T a exposures (20°C/25°C/32°C/36°C) under 0.8% (C), 1.1% (D), and 1.4% (E) isoflurane, respectively. Gray shading indicates isoflurane anesthesia exposure (starting at t = 0, and ceasing at t = 32 min. LORR time is ∼2 min). (F) Changes in T core (measured at t = 32 min) under different isoflurane concentrations at different T a . (25°C T a : 0.8%, −1.36°C ± 0.21°C, 1.1%, −2.08°C ± 0.15°C, 1.4%, −2.22°C ± 0.23°C; 32°C T a : 0.8%, −0.27°C ± 0.19°C, 1.1%, −0.38°C ± 0.12°C, 1.4%, −1.11°C ± 0.36°C, 36°C T a : 0.8%, 1.70°C ± 0.20°C, 1.1%, 0.44°C ± 0.38°C, 1.4%, 0.84°C ± 0.18°C; 0.8%, 20°C vs. 25°C, p < 0.001, 32°C vs. 25°C, p < 0.001, 36°C vs. 25°C, p < 0.001; 1.1%, 20°C vs. 25°C, p < 0.001, 32°C vs. 25°C, p < 0.001, 36°C vs. 25°C, p < 0.001; 1.4%, 20°C vs. 25°C, p < 0.001, 32°C vs. 25°C, p < 0.001, 36°C vs. 25°C, p < 0.001; n = 7). (G) Linear regression model of T a and T core of mice at the time of isoflurane ceasing ( t = 32 min), Y = 0.66∗X + 14.79, R 2 = 0.92, p < 0.001. (H) LORR time plotted against T core at the start of isoflurane ( t = 0 min) for all tested mice as shown in (I). No linear relationship between LORR time and T core was detected ( R 2 = 0.01, p = 0.47). (I and J) Statistical analysis of RORR time across all groups. (25°C T a : 0.8%, 99.71 ± 25.45 s, 1.1%, 140.14 ± 16.36 s, 1.4%, 171.57 ± 27.08 s; 32°C T a : 0.8%, 39.43 ± 16.21 s, 1.1%, 46.00 ± 8.62 s, 1.4%, 45.71 ± 18.47 s; 0.8%, 20°C vs. 25°C, p < 0.001, 32°C vs. 25°C, p < 0.001, 36°C vs. 25°C, p < 0.001; 1.1%, 20°C vs. 25°C, p < 0.001, 32°C vs. 25°C, p < 0.001, 36°C vs. 25°C, p < 0.001; 1.4%, 20°C vs. 25°C, p < 0.001, 32°C vs. 25°C, p < 0.001, 36°C vs. 25°C, p < 0.001; n = 7). (J) Logarithmic regression model (one-phase decay) of RORR time as a function of T core at the time of isoflurane cessation ( t = 32 min) for all tested mice, Y = 20.3∗10 5 ∗eˆ(−0.27∗x) −69.06, R 2 = 0.93, p < 0.001. Inflection point (31.88°C) was calculated by nonlinear regression model: y = b 1 ∗( x < b 2 ) ∗ ( x − b 2 ) + b 3 ). (b 1 = −307.47, b 2 = 31.88, b 3 = 60.48). (K) Representative T core (top), raw EEG traces (middle), and EEG power spectra (bottom) at different T a under 1.4% isoflurane. (L) Raw EEG power density of mice in wake and 1.4% isoflurane at different T a (30 min). (M–O) EEG power percentage of different frequency bands during 0.8% (M; δ band, 20°C vs. 25°C, F (4, 130) = 153.02, p < 0.001, 32°C vs. 25°C, F (4, 130) = 4.96, p = 0.13; γ band, 20°C vs. 25°C, F (4, 130) = 6.92, p = 0.047; n = 7), 1.1% (N; δ band: 20°C vs. 25°C, F (4, 130) = 142.32, p < 0.001, 32°C vs. 25°C, F (4, 130) = 12.40, p = 0.030; n = 7), and 1.4% (O; δ band: 20°C vs. 25°C, F (4, 130) = 139.71, p < 0.001, 32°C vs. 25°C, F (4, 130) = 13.88, p = 0.0014; n = 7; α band, 20°C vs. 25°C, F (4, 130) = 8.30, p = 0.023; n = 7) isoflurane anesthesia procedures, respectively. LORR, the loss of righting reflex; RORR, the recovery of righting reflex; EEG, electroencephalogram. All data are presented as mean ± SEM, ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001, n.s., not significant (F) and (I) were analyzed by multiple t test, (G) and (H) were analyzed by linear regression analysis, (J) was analyzed by non-linear regression (one-phase decay) analysis, and (M–O) were analyzed by two-way RM ANOVA followed by Bonferroni’s multiple comparisons test. During anesthesia induction, T core remained stable (36°C–37°C) under various T a and showed no correlation with loss of righting reflex (LORR) time ( R 2 = 0.01, p = 0.47; Figure 1 H). However, recovery of righting reflex (RORR) time was inversely correlated with T core measured at the cessation of anesthesia ( t = 32 min; Figure 1 I). RORR time under 0.8%, 1.1%, and 1.4% isoflurane at 20°C T a were 348.57 ± 107.46 s, 568.57 ± 159.21 s, and 1074.29 ± 227.44 s, respectively, while at 36°C T a , they were only 9.43 ± 2.88 s, 10.57 ± 4.61 s, and 9.71 ± 3.64 s, respectively ( n = 7; Figure 1 I). Quantitative analysis showed a strong logarithmic relationship between RORR time and T core ( R 2 = 0.93, p < 0.001), and the turning point was identified at ∼31.88°C, beyond which further increase of T core had minimal effect on anesthesia emergence ( Figure 1 J). EEG spectral power distributions varied significantly with T a during isoflurane anesthesia ( Figures 1 K and 1L). Power spectral analysis further supported the negative impact of T core on anesthetic depth. Delta (δ) power (1–4 Hz) increased at lower T a (20°C) and reduced at higher T a (32°C). Under 0.8% isoflurane, δ power values were 56.66% ± 5.14% at 20°C T a , 33.16% ± 9.64% at 25°C T a , and 27.81% ± 6.46% at 32°C T a ( n = 7). The difference showed between 20°C and 25°C ( p < 0.001), while there was no difference between 32°C and 25°C ( p = 0.096; Figure 1 M). Under 1.1% and 1.4% isoflurane, δ power significantly varied with ambient temperature changes ( Figures 1 N and 1O). Chemogenetic activation of LPO BDNF neurons deepens anesthesia by decreasing T core Clozapine-N-oxide (CNO) administration (1 mg/kg, intraperitoneal [i.p.], Figure S1 A) to activate LPO BDNF prior to isoflurane exposure significantly reduced T core , and this hypothermic effect persisted throughout isoflurane exposure ( Figures 2 A–2D). While their activation did not affect LORR time ( Figure 2 E), it significantly prolonged anesthesia recovery time at 25°C T a (hM3Dq-CNO 446.00 ± 121.86 s vs. hM3Dq-saline 135.67 ± 15.15 s; n = 6, p < 0.001; Figure 2 F). Furthermore, chemogenetic activation significantly increased the burst suppression ratio (BSR) during anesthesia ( Figure 2 G). Power spectral analysis showed increased δ power (hM3Dq-CNO 55.34 ± 4.40% vs. hM3Dq-saline 44.59 ± 1.91%; p = 0.0091; Figure 2 H) during anesthetic maintenance, as well as elevated δ power (CNO 57.64% ± 4.16% vs. saline 43.64% ± 2.46%; p = 0.046; Figure 2 I) during recovery procedure at 25°C T a ( Figure S1 B). The same pattern changes were noted in the LPO BDNF activation mice at 20°C T a . Further T core reduction ( Figures 2 J and 2K) had no difference of LORR ( Figure 2 L) but prolonged RORR ( Figure 2 M), increased BSR ( Figure 2 N) and δ power ( Figure 2 O) during isoflurane exposure, and increased δ power during recovery phase ( Figure 2 P). At 32°C T a , activation of LPO BDNF , neurons also showed similar results to the changes in that of 25°C T a ( Figures 2 Q–2S and 2T–2W) but with no difference of RORR time ( n = 6, p > 0.05; Figure 2 T). Figure 2. Open in a new tab Chemogenetic activation of LPO BDNF neurons deepens isoflurane-induced anesthesia by decreasing T core (A) Protocol for chemogenetic experiments, CNO was delivered 30 min prior to isoflurane-induced anesthesia. T core and EEG were monitored during the anesthesia procedure. (B) Schematic representation of AAV-DIO-hM3Dq-mCherry (hM3Dq) injection into the LPO region of BDNF-ires-Cre mice (left). Representative image showing hM3Dq virus expressing in LPO region of mice (scale bars, 500 μm) (right). (C) T core changes at T a of 25°C under isoflurane in hM3Dq-saline and hM3Dq-CNO mice. T core was recorded every 2 min (measured by intraperitoneal implantation). CNO injected at −30 min, isoflurane started at t = 0 min, induced LORR of mice within 2 min, and maintained for 30 min, isoflurane ceased at t = 32 min. (D) The values of T core at different time under isoflurane in hM3Dq-saline and hM3Dq-CNO mice. T core at t = −30 min, the time of isoflurane starting ( t = 0 min), isoflurane cessation ( t = 32 min) and at the RORR moment (t RORR ). (E) Chemogenetic activation of LPO BDNF neurons at T a of 25°C did not influence LORR time. (F) Chemogenetic activation of LPO BDNF neurons at T a of 25°C prolonged the RORR time. (G) Burst suppression ratio (BSR) changes during isoflurane anesthesia procedure (30 min) in hM3Dq-saline and hM3Dq-CNO mice. BSR is plotted at every minute. (H) LPO BDNF neural activation during isoflurane procedure (0–30 min) at T a of 25°C displayed significant increase in δ power band (F (4, 50) = 26.59; p < 0.001; n = 6) analyzed from EEG recordings. (I) LPO BDNF neural activation in recovery procedure (from isoflurane ceasing to RORR) at T a of 25°C displayed increase in δ power band (F (4, 50) = 14.89; p = 0.046; n = 6) analyzed from EEG recordings. (J) T core changes at T a of 20°C under isoflurane in hM3Dq-saline and hM3Dq-CNO mice. (K) The values of T core at different time under isoflurane in hM3Dq-saline and hM3Dq-CNO mice. (L) Chemogenetic activation of LPO BDNF neurons at T a of 20°C did not influence the LORR time. (M) Chemogenetic activation of LPO BDNF neurons at T a of 20°C prolonged the RORR time. (N) Burst suppression ratio (BSR) changes during isoflurane anesthesia procedure (30 min) in hM3Dq-saline and hM3Dq-CNO mice. (O) LPO BDNF neural activation during isoflurane procedure (0–30 min) at T a of 20°C displayed significant increase in δ power band (F (4, 50) = 33.76; p = 0.0035; n = 6). (P) LPO BDNF neural activation in recovery procedure (from isoflurane ceasing to RORR) at T a of 20°C displayed decrease in β power band (F (4, 50) = 25.72; p < 0.001; n = 6). (Q) T core changes during isoflurane at T a of 32°C in hM3Dq-saline and hM3Dq-CNO mice. (R) The values of T core at different time under isoflurane in hM3Dq-saline and hM3Dq-CNO mice. (S and T) Chemogenetic activation of LPO BDNF neurons at T a of 32°C has no changes in LORR time (S) and RORR time (T). (U) BSR changes during isoflurane anesthesia procedure (0–30 min) at T a of 32°C in hM3Dq-saline and hM3Dq-CNO mice. (V) LPO BDNF neurons activation during isoflurane (0–30 min) at T a of 32°C displayed significant increase in δ power band (F (4, 50) = 11.38; p = 0.0021; n = 6). (W) LPO BDNF neural activation during recovery procedure (from isoflurane ceasing to RORR) at T a of 32°C displayed significant increase in δ power band and decrease in θ power band. (X) The linear regression model of mean BSR and mean T core of hM3Dq-saline and hM3Dq-CNO mice during isoflurane procedure (30 min). hM3Dq-saline, Y = −4.415∗X + 180.0, R 2 = 0.80, p < 0.001; hM3Dq-CNO, Y = −4.082∗X + 166.5, R 2 = 0.89, p < 0.001. Comparation within groups: slopes, p = 0.61; intercepts, p = 0.08. (Y) Logarithmic regression model (one-phase decay) of RORR time as a function of T core at the time of isoflurane ceasing ( t = 32 min) in hM3Dq-saline and hM3Dq-CNO groups. hM3Dq-saline, Y = 5.1∗10 5 ∗eˆ(−0.23∗x)−124.6, R 2 = 0.96, p < 0.001, hM3Dq-CNO, Y = 0.4∗10 5 ∗eˆ(−0.14∗x) −329.1, R 2 = 0.91, p < 0.001. k value within groups comparation showed no differences ( p = 0.41). All data are presented as mean ± SEM, ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001, ns, not significant, (D), (K), and (R) were analyzed by multiple t test, (E), (F), (L), (M), (S), and (T) were analyzed by paired t test, (G–I), (N–P), and (U–W) were analyzed by two-way RM ANOVA followed by Bonferroni’s multiple comparisons test, (X) was analyzed by linear regression analysis, and (Y) was analyzed by non-linear regression (one-phase decay) analysis. To determine whether the increased anesthetic depth resulted from reduced T core or a direct effect of LPO BDNF activation, we analyzed the temperature dependence of BSR and RORR time between CNO- and saline-treated groups ( Figures 2 X–2Y). BSR showed a linear correlation with mean T core in both groups ( Figure 2 X), without significant differences in the regression slopes ( p = 0.61) or intercepts ( p = 0.08). Also, there is no change in the logarithmic inverse relationship between T core and RORR time in the CNO and saline groups ( Figure 2 Y), suggesting that T core is the primary determinant of BSR and RORR. Chemogenetic activation of DMH Vglut2 neurons mitigates anesthesia by increasing T core and its intrinsic activity Inspired by neural modulation in the LPO, we focused on DMH Vglut2 neurons, which are major downstream targets of LPO BDNF neurons (∼53%, Figures S1 C–S1F). Indeed, optogenetic activation of the axonal terminals of LPO BDNF neurons in the DMH lowered T core and increased BSR ( Figures S1 G–S1O). Vglut2-ires-Cre mice were used to label glutamate neurons ( Figures S2 A–S2D). We employed chemogenetic activation of DMH Vglut2 neurons by injecting AAV-Ef1α-DIO-hM3Dq-mCherry into the DMH ( Figures 3 A and 3B). Viral expression was clearly seen in the DMH ( Figures 3 C and S3 A). CNO administration induced a ∼2°C increase in T core (from 36.88°C ± 0.18°C to 38.56°C ± 0.15°C; Figures 3 D and 3E), and this elevation persisted throughout anesthesia (0–32 min) and during RORR. At 25°C T a , T core was significantly higher in the DMH Vglut2 neural activation mice at anesthesia cessation (32.13°C ± 0.35°C vs. 30.88°C ± 0.23°C, p = 0.0096) and the RORR moment (30.75°C ± 0.43°C vs. 29.50°C ± 0.21°C, p = 0.021; n = 8; Figures 3 D and 3E), and RORR time was significantly shortened (hM3Dq-CNO 91.63 ± 9.55 s vs. hM3Dq-saline 127.50 ± 13.51 s; n = 8, p < 0.001; Figure 3 F). EEG analysis revealed significant alterations in BSR and power spectra ( Figure 3 G). During isoflurane exposure, BSR ( Figure 3 H) and δ power (38.69% ± 3.32% vs. 50.10% ± 3.40%, p = 0.031; Figure 3 I) showed significant reduction, and the decreased δ power also shown during recovery phase ( Figure 3 J). The same pattern changes were noted in the DMH Vglut2 neural activation mice at 20°C T a , such as decreased T core reduction ( Figures 3 K and 3L), shortened RORR ( Figure 3 M), decreased BSR ( Figures 3 N–3O) and δ power ( Figure 3 P) during isoflurane exposure, and decreased δ power in recovery phase ( Figure 3 Q). At 32°C T a , activation of DMH Vglut2 , neurons also showed similar results to the changes in that of 25°C T a ( Figures 3 R and 3S, 3U–3W), also no difference shown in RORR time ( n = 8, p > 0.05; Figure 3 T) and EEG power bands during recovery ( Figure 3 X). Figure 3. Open in a new tab Chemogenetic activation of DMH Vglut2 mitigates isoflurane-induced anesthesia largely by increasing T core (A) Protocol for chemogenetic experiment, CNO was intraperitoneally injected 30 min prior to isoflurane-induced anesthesia. T core and EEG were monitored during the anesthesia procedure. (B) Schematic representation of AAV-DIO-hM3D-EYFP or AAV-DIO-EYFP injection into the DMH region of Vglut2-ires-Cre mice. (C) Representative images showing the expression of hM3Dq receptors (green) in the DMH of Vglut2-ires-Cre mice. Scale bars, 100 μm. (D) T core changes at T a of 25°C under isoflurane in hM3Dq-saline and hM3Dq-CNO mice. T core was recorded every 2 min (measured by intraperitoneal implantation). CNO injected at −30 min, isoflurane started at t = 0 min, induced LORR of mice within 2 min, and maintained for 30 min, isoflurane ceased at t = 32 min. (E) The values of T core at different time under isoflurane in hM3Dq-saline and hM3Dq-CNO mice. T core at t = −30 min, the time of isoflurane starting ( t = 0 min), isoflurane cessation ( t = 32 min) and at the RORR moment (t RORR ). (F) Chemogenetic activation of DMH Vglut2 neurons at T a of 25°C shortened the RORR time. (G) Representative raw EEG traces (top) and EEG power spectra (bottom) at T a of 25°C under isoflurane in hM3Dq-saline and hM3Dq-CNO mice. (H) BSR changes during isoflurane anesthesia procedure (30 min) at T a of 25°C in hM3Dq-saline and hM3Dq-CNO mice. BSR is plotted at every minute. (I) DMH Vglut2 neural activation during isoflurane (0–30 min) displayed significant decrease in δ power band (F (4, 70) = 15.92; p = 0.031; n = 8). (J) DMH Vglut2 neural activation during recovery procedure (from isoflurane ceasing to RORR of mice) displayed significant decrease in δ power band (F (4, 70) = 19.26; p = 0.031; n = 8). (K) T core changes at T a of 20°C under isoflurane in hM3Dq-saline and hM3Dq-CNO mice. (L) The values of T core at different time under isoflurane in hM3Dq-saline and hM3Dq-CNO mice. (M) Chemogenetic activation of DMH Vglut2 neurons shortened the RORR time. (N) Representative raw EEG traces (top) and EEG power spectra (bottom) at T a of 20°C under isoflurane in hM3Dq-saline and hM3Dq-CNO mice. (O) BSR changes during isoflurane anesthesia procedure at T a of 20°C in hM3Dq-saline and hM3Dq-CNO mice. (P) DMH Vglut2 neural activation during isoflurane anesthesia procedure (0–30 min) displayed significant decrease in δ power band (F (4, 70) = 19.89; p = 0.027; n = 8) and increase in θ power band (F (4, 70) = 4.18; p = 0.036; n = 8). (Q) DMH Vglut2 neural activation during recovery procedure (from isoflurane ceasing to RORR of mice) displayed significant decrease in δ power band (F (4, 70) = 15.59; p = 0.040; n = 8). (R) T core changes at T a of 32°C under isoflurane in hM3Dq-saline and hM3Dq-CNO mice. (S) The values of T core at different time under isoflurane in hM3Dq-saline and hM3Dq-CNO mice. (T) Chemogenetic activation of DMH Vglut2 neurons has no influence on RORR time. (U) Representative raw EEG traces (top) and EEG power spectra (bottom) at T a of 32°C under isoflurane in hM3Dq-saline and hM3Dq-CNO mice. (V) BSR changes during isoflurane anesthesia procedure at T a of 32°C in hM3Dq-saline and hM3Dq-CNO mice. (W) DMH Vglut2 neural activation during isoflurane (0–30 min) displayed significant decrease in δ power band (F (4, 70) = 19.71; p = 0.014; n = 8) and increase in α power band (F (4, 70) = 2.55; p = 0.019; n = 8). (X) DMH Vglut2 neural activation during recovery procedure (from isoflurane ceasing to RORR of mice) displayed no significant differences. (Y) The linear regression model of mean BSR and mean T core of hM3Dq-saline and hM3Dq-CNO mice during isoflurane procedure (30 min). hM3Dq-saline, Y = −3.999∗X + 167.0, R 2 = 0.51, p < 0.001; hM3Dq-CNO, Y = −3.271∗X + 137.7, R 2 = 0.6, p < 0.001. Comparation within groups: slopes, p = 0.47; intercepts, p = 0.023. (Z) Logarithmic regression model (one-phase decay) of RORR time as a function of T core at the time of isoflurane ceasing ( t = 32 min) in hM3Dq-saline and hM3Dq-CNO mice. hM3Dq-saline, Y = 1.1∗10 5 ∗eˆ(−0.19∗x) +118.2, R 2 = 0.90, p < 0.001, hM3Dq-CNO, Y = 6.4∗10 5 ∗eˆ(−0.27∗x) +63.91, R 2 = 0.90, p < 0.001. k value within groups comparation showed no differences ( p = 0.47). All data are presented as mean ± SEM, ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001, ns, not significant, (E), (L), and (S) were analyzed by multiple t test, (F), (M), and (T) were analyzed by paired t test, (H–J), (O–Q), and (V–X) were analyzed by two-way RM ANOVA followed by Bonferroni’s multiple comparisons test, (Y) was analyzed by linear regression analysis, and (Z) was analyzed by non-linear regression (one-phase decay) analysis. BSR in both the groups exhibited a linear relationship with mean T core during anesthesia, showing as comparable slopes of the fitted lines ( p = 0.47), while the intercepts of these lines differed significantly ( p = 0.024; Figure 3 Y). The temperature dependence of RORR time across the CNO and saline groups followed a logarithmic relationship with T core , and no significant differences were found between the fitted curves ( p = 0.47; Figure 3 Z). These data suggest that temperature is still the dominant modulatory factor for anesthesia depth after DMH Vglut2 activation, but its intrinsic neuronal activation also influences the isoflurane anesthesia. Chemogenetic inhibition of DMH Vglut2 neurons decreases T core and deepens anesthesia Conversely, we also performed chemogenetic inhibition to further define the role of DMH Vglut2 neurons in anesthesia regulation ( Figure S3 B). AAV-Ef1α-DIO-hM4Di-EYFP (hM4Di) or AAV-Ef1α-DIO-EYFP (EYFP) was injected into the DMH of Vglut2-ires-Cre mice ( Figure S3 C). After 3 weeks of viral expression, c-Fos staining confirmed robust neuronal inhibition in hM4Di-expressing mice following CNO administration (hM4Di-CNO: 17.33% ± 2.11% vs. hM4Di-saline: 4.00% ± 1.39%; n = 6; p < 0.001; Figure S3 C). DMH Vglut2 neural inhibition induced hypothermia with a reduction of T core by ∼2°C (37.50°C ± 0.20°C to 35.81 ± 0.16°C; Figures S3 D and S3E). Under 1.4% isoflurane, this hypothermia was exacerbated, with significantly lower T core at anesthesia cessation (29.56°C ± 0.15°C vs. 30.75°C ± 0.41°C; p = 0.017) and at RORR (28.81°C ± 0.27°C vs. 30.00°C ± 0.41°C; p = 0.029; n = 8; Figure S3 E). Consistent with deeper anesthesia, hM4Di-CNO mice exhibited prolonged RORR time (142.25 ± 15.93 s vs. 120.25 ± 14.90 s; n = 8; p = 0.013; Figure S3 F). EEG analysis ( Figure S2 G) demonstrated an elevated BSR ( Figure S3 H) and increased δ power during maintenance phase ( Figure S3 I). This increased δ power also occurred during recovery ( Figure S3 J). LPO BNDF and DMH Vglut2 neuronal activity changes under isoflurane To determine the potential role of LPO and DMH neurons in anesthesia procedure, we examined c-Fos expression during isoflurane exposure, and anesthesia awake state separately. The LPO exhibited significantly lower c-Fos activation during both the awake and anesthesia stages ( Figures 4 A and 4B), while the DMH showed substantially greater c-Fos activation during the awake stage compared to the anesthesia stage ( Figures 4 C and 4D). These data are consistent with a possible direct and temperature-independent function of DMH in emergence. Figure 4. Open in a new tab LPO BDNF and DMH Vglut2 neuronal activity changes under isoflurane (A) Representative images showing the c-Fos expression (red) under neural state (left), anesthesia (middle) and awake (right) in LPO region. Scale bars, 200 μm. (B) Quantification of c-Fos expression in LPO in different anesthetic stages as indicated. (C and D) Representative images (C) and quantification (D) showing the c-Fos expression (red) under neural state (left), anesthesia (middle) and awake (right) in DMH region. Scale bars, 100 μm. (E) Schematic diagram of calcium signal recordings via fiber photometry. (F) Representative images showing the expression of AAV2/9-hSyn-DIO-GCaMP6s-WPRE virus in the LPO of BDNF-ires-Cre mice. Scale bars, 250 μm. (G) Representative raw Ca 2+ signal trace of LPO BDNF neurons in 0.8% isoflurane at 25°C (up), 1.4% isoflurane at 25°C (middle), and 32°C (bottom). (H and I) Z scored calcium ΔF/F traces of LPO BDNF neurons (H) and quantification of the peak calcium Z scores (I) at LORR moment, isoflurane started at t = 0 s, the baseline is −50 to 0 s, and the dashed line represent the LORR moment of 0.8% isoflurane at 25°C (purple), 1.4% isoflurane at 25°C (black), and 32°C (pink). (J) LORR time of mice under different conditions. (K–L) Z scored calcium ΔF/F traces of LPO BDNF neurons (K) and quantification of the peak calcium Z scores (L) at RORR moment after isoflurane ceasing ( t = 0 s) under different conditions, and the dashed line represent the RORR moment of 0.8% isoflurane at 25°C (purple), 1.4% isoflurane at 25°C (black), and 32°C (pink). (M) RORR time of mice under different conditions. (N) RORR time and the peak time of Ca 2+ signal after isoflurane ceasing at 25°C and 32°C T a . (O) Representative images showing the expression of AAV2/9-hSyn-DIO-GCaMP6s-WPRE virus in the DMH of Vglut2-ires-Cre mice. Scale bars, 100 μm. (P) Representative raw Ca 2+ signal trace of DMH Vglut2 neurons in 0.8% isoflurane at 25°C (up), 1.4% isoflurane at 25°C (middle), and 32°C (bottom). (Q and R) Z scored calcium ΔF/F traces of DMH Vglut2 neurons (Q) and quantification of the peak calcium Z scores (R) at LORR moment, isoflurane started at t = 0 s, the baseline is −50 to 0 s, and the dashed line represent the LORR moment of 0.8% isoflurane at 25°C (purple), 1.4% isoflurane at 25°C (black), and 32°C (pink). (S) LORR time of mice under different conditions. (T and U) Z scored calcium ΔF/F traces of DMH Vglut2 neurons (T) and quantification of the peak calcium Z scores (U) at RORR moment after isoflurane ceasing ( t = 0 s) under different conditions, and the dashed line represent the RORR moment of 0.8% isoflurane at 25°C (purple), 1.4% isoflurane at 25°C (black), and 32°C (pink). (V) RORR time of mice under different conditions. (W) RORR time and the peak time of Ca 2+ signal after isoflurane ceasing at 25°C and 32°C T a . All data are presented as mean ± SEM, ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001, n.s., not significant, (B), and (D) were analyzed by unpaired t test, and (I), (J), (L–M), (R), (S), and (U–W) were analyzed by one-way multiple comparisons test. Because c-Fos labeling provides limited temporal resolution of neural activity, we next directly recorded Ca 2+ signal from LPO BDNF and DMH Vglut2 neurons separately via fiber photometry ( Figure 4 E). Genetically encoded calcium indicators (GCaMPs) was selectively expressed in the LPO BDNF ( Figure 4 F) and DMH Vglut2 neurons ( Figure 4 O). Isoflurane started at t = 0 and lasted for 10 min. The calcium signal of LPO BDNF neurons revealed clear transient activation in the first 20 s of isoflurane exposure, and followed by a gradual decline ( Figures 4 G–4J). In the recovery process, the Ca 2+ signals began to increase when isoflurane ceased ( Figures 4 K–4M), and peaked approximately at the moment of RORR ( Figure 4 N). These results suggest that LPO BDNF neurons are not the primary direct target of isoflurane for inducing hypothermia and unconsciousness; rather, they modulate anesthetic depth secondarily through their influence on T core . DMH Vglut2 neurons Ca 2+ signal displayed similar suppression ( Figures 4 P–4S) to that of LPO BDNF neurons under the anesthesia stage. In contrast to LPO, in the awake stage, DMH Vglut2 neurons displayed strong activity prior to the RORR time point ( Figures 4 T–4W), suggesting these neurons play an initiative role in driving emergence. This further implies that isoflurane-induced hypothermia and unconsciousness may be partially mediated by inhibiting this pro-arousal neural population. Stage-specific optogenetic activation of DMH Vglut2 neurons reveals its effect on anesthesia independent of temperature We photoactivated LPO BDNF and DMH Vglut2 neurons 15 min after LORR during isoflurane exposure for 15 min ( Figure 5 A). Optogenetic activation of LPO BDNF neurons did not alter T core during anesthesia ( Figures 5 B and 5C). EEG δ power and BSR also showed no change by photoactivation during anesthesia ( Figures 5 G–5I), suggesting undetected role in anesthetic depth. Another, similar changes showed when experiment was conducted at 20°C T a ( Figures S4 A–S4F), but no changes in T core and RORR at 32°C T a ( Figures S4 G–S4L). Notably, photoactivation of LPO BDNF neurons produced a post-stimulation effect, leading to reduced T core during RORR at 25°C T a (ChR2 28.25°C ± 0.21°C vs. EYFP 29.92°C ± 0.20°C, p < 0.001; Figures 5 D and 5E), and prolonged RORR (ChR2 150.43 ± 11.86 s vs. EYFP 113.14 ± 16.05 s, p < 0.001, Figure 5 F). The δ power was increased during recovery (ChR2 53.80% ± 1.16% vs. EYFP 46.71% ± 2.05%, p = 0.026; n = 7, Figure 5 J). We propose that the post-stimulation effects likely arise from accumulated neuromodulators or a primed circuit state whose influence is unmasked after isoflurane withdrawal. Future studies using real-time neurotransmitter sensing or delayed antagonist application could directly test this priming model versus a slow neurohumoral release. Figure 5. Open in a new tab Stage-specific optogenetic activation of DMH Vglut2 neurons reveals its effect on anesthesia independent of temperature (A) Protocol for the optogenetic stimulation experiment. Blue shading indicates the optical stimulation phase, starting from 17 min after isoflurane induction and ending with isoflurane cessation at t = 32 min. (B) Schematic representation of AAV-DIO-ChR2-EYFP injection and optical fiber implantation into the LPO region of BDNF-ires-Cre mice. (C) Representative images showing the expression of ChR2-EYFP (green) in the LPO of BDNF-ires-Cre mice. Scale bars, 500 μm (right). (D) T core changes of ChR2 and EYFP mice under isoflurane. T core was recorded every 2 min. Isoflurane administration started at t = 0 min, maintained for 30 min and ceased at t = 32 min. Photo-stimulation started at t = 17 min and ceased at t = 32 min. (E) The values of T core at the RORR moment under isoflurane in ChR2 and EYFP mice. (F) Optogenetic activation of LPO BDNF neurons prolonged the RORR time. (G) Representative raw EEG traces (top) and EEG power spectra (bottom) at 25°C of T a under isoflurane in ChR2 and EYFP mice. (H) BSR changes of ChR2 and EYFP mice during isoflurane anesthesia procedure (30 min). (I) LPO BDNF neural activation during isoflurane (0–30 min) displayed no change. (J) LPO BDNF neural activation during recovery procedure displayed significant increase in δ power band (F (4, 60) = 21.22; p = 0.011; n = 7). (K) Schematic representation of AAV-DIO-ChR2-EYFP or AAV-DIO-EYFP injection and optical fiber implantation into the DMH region of Vglut2-ires-Cre mice. (L) Representative images showing the expression of ChR2-EYFP (green) in the DMH of Vglut2-ires-Cre mice. Scale bars, 100 μm (right). (M) T core changes of ChR2 and EYFP groups under isoflurane. (N) The values of T core at the RORR moment under isoflurane in ChR2 and EYFP mice. (O) Optogenetic activation of DMH Vglut2 neurons shortened the RORR time of mice. (P) Representative raw EEG traces (top) and EEG power spectra (bottom) at 25°C of T a under isoflurane in ChR2 and EYFP mice. (Q) BSR changes of ChR2 and EYFP mice during isoflurane anesthesia procedure (30 min). (R) DMH Vglut2 neural activation during isoflurane (0–30 min) displayed significant decrease in δ power band (F (4, 50) = 18.05; p = 0.035; n = 6). (S) DMH Vglut2 neural activation during recovery procedure (from isoflurane ceasing to RORR of mice) displayed significant decrease in δ power band (F (4, 50) = 9.36; p = 0.032; n = 6) and increase in γ power band (F (4, 50) = 6.59; p = 0.047; n = 6). All data are presented as mean ± SEM, ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001, ns, not significant, (E), (F), (N), and (O) were analyzed by unpaired t test, and (H–J) and (Q–S) were analyzed by two-way RM ANOVA followed by Bonferroni’s multiple comparisons test. Conversely, DMH Vglut2 neural activation ( Figures 5 K and 5L) significantly increased T core during RORR at 25°C T a (−5.52°C ± 0.25°C vs. −6.97°C ± 0.53°C, n = 6; Figures 5 M and 5N), which accelerated emergence from anesthesia (69.67 ± 5.41 s vs. 107.83 ± 4.83 s, n = 6, p < 0.001; Figures 5 O–5S). Interestingly, EEG analysis revealed immediate BSR suppression following photo-stimulation ( Figures 5 P and 5Q) and reduced δ power without altering T core during isoflurane exposure (36.95% ± 3.66% vs. 48.54% ± 3.38%, n = 6, p = 0.035; Figure 5 R). Comparable effects on T core and EEG parameters were observed at 20°C T a ( Figures S5 A–S5F), while only BSR suppression showed in DMH Vglut2 neural activation mice at 32°C T a ( Figures S5 G–S5L). Critically, under thermoneutral conditions (T a = 36°C), where T core was maintained, normothermia (T core in between 36°C and 37°C) throughout the procedure, optogenetic activation of DMH Vglut2 neurons still elicited immediate BSR suppression without any change in T core ( Figures S5 M–S5R). Collectively, these results demonstrate that DMH Vglut2 neurons exert a robust, direct wake-promoting effect during isoflurane anesthesia that is temperature-independent. The DMH Vglut2 to RPa pathway is essential for the temperature-independent effect on anesthesia regulation Building on the demonstrated temperature-independent effects of DMH Vglut2 neurons on anesthesia regulation, we sought to characterize the specific neural pathways mediating these effects. Given the established anatomical connection between DMH Vglut2 neurons and RPa under physiological conditions, 25 we selectively manipulated this pathway by optogenetics ( Figures 6 A, 6B, and S6 A–S6C). The pathway activation during isoflurane exposure neither affected T core during anesthesia maintenance nor induced significant changes at RORR ( Figures 6 C and 6D). However, photo-stimulation significantly accelerated isoflurane recovery (ChR2 110.5 ± 11.39 s vs. EYFP 133.88 ± 16.75 s, n = 8, p < 0.001; Figure 6 E) and transiently suppressed BSR ( Figure 6 F), while δ power remained unchanged ( Figures 6 G and 6H). The same pattern changes were noted in the pathway activation mice at 32°C T a ( Figures S6 F–S6K). In the inhibition experiments, optogenetic suppression of DMH Vglut2 to RPa pathway only reduced T core during the early stage of anesthesia ( Figure 6 I), with no differences observed at anesthesia cessation or RORR ( Figure 6 J). Further, inhibition significantly prolonged emergence time (eNpHR: 143.50 ± 7.74 s vs. EYFP 129.17 ± 13.62 s, n = 6, p = 0.0067; Figure 6 K). Figure 6. Open in a new tab Optogenetic manipulation of the DMH vglut2 to RPa pathway reveals its temperature-independent effect on anesthesia (A) Schematic representation of AAV-DIO-ChR2-EYFP (ChR2) or AAV-DIO-EYFP (EYFP) injection into the DMH, and optical fiber implantation into the RPa of Vglut2-ires-Cre mice. (B) Protocol for the pathway optogenetic stimulation experiment. Blue shading indicates the optical stimulation phase, starting from 17 min after isoflurane induction and ending with isoflurane cessation at t = 32 min. (C) T core changes of ChR2 and EYFP groups under isoflurane. (D) The values of T core at different time under isoflurane in ChR2 and EYFP mice. T core at the time of isoflurane cessation ( t = 32 min), and at the RORR moment (t RORR ). (E) Optogenetic activations of the DMH Vglut2 to RPa pathway shortened the RORR time. (F) BSR changes of ChR2 and EYFP mice during isoflurane anesthesia procedure. (G and H) Optogenetic activations of the DMH Vglut2 to RPa pathway displayed no significant changes in EEG power bands during isoflurane exposure (G), and recovery procedure (H). (I) T core changes of eNpHR and EYFP mice under isoflurane. Photo-inhibition started at t = −20 min. (J) The values of T core at different time under isoflurane in eNpHR and EYFP mice. (K) Optogenetic inhibition of the DMH Vglut2 to RPa pathway prolonged the RORR time. (L) Schematic representation of AAV-DIO-ChR2-EYFP virus and optical fiber implantation into the DMH region, and AAV-DIO-hM4Di-EYFP virus injection into RPa region of Vglut2-ires-Cre mice. (M) T core changes of RPa-hM4Di mice (saline vs. CNO) with concurrent DMH Vglut2 activation under isoflurane. CNO injected at −20 min, isoflurane started at t = 0 min, induced LORR of mice within 2 min, and maintained for 30 min, isoflurane ceased at t = 32 min. (N) BSR changes of ChR2-saline and ChR2-CNO mice during isoflurane anesthesia procedure. (O) RPa Vglut2 neural inhibition increased δ power band (F (4, 60) = 17.8; p = 0.0024; n = 7) during isoflurane anesthesia procedure (0–30 min) in DMH Vglut2 neural activation mice. All data are presented as mean ± SEM, ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001, ns, not significant, (D) and (J) were analyzed by multiple t test, (E) and (K) were analyzed by unpaired t test, and (F–H) and (N–O) were analyzed by two-way RM ANOVA followed by Bonferroni’s multiple comparisons test. To validate the functional connection of the DMH Vglut2 to RPa projection, we performed additional experiments combining optogenetic activation of DMH Vglut2 and chemogenetic inhibition of the RPa ( Figure 6 L). In awake mice, this projection contributed to thermoregulation, as evidenced by a hyperthermia effect that was partially suppressed upon RPa inhibition ( Figures S6 D and S6E). Under isoflurane anesthesia, chemogenetic inhibition of RPa neurons (CNO injected at −20 min) showed an initial reduction of T core ( Figure 6 M), and the mitigated anesthetic depth by DMH Vglut2 activation was also partially attenuated by RPa inhibition ( Figures 6 N and 6O). Collectively, these results demonstrate that the DMH Vglut2 to RPa pathway modulates anesthetic emergence independently of thermoregulatory mechanisms. And the arousal-promoting effect of DMH Vglut2 neurons is partly through Vglut2 neurons in RPa region. Discussion Our study showed a linear correlation between T a and T core during anesthesia, and an exponential inverse correlation between T core and anesthetic emergence, with a critical T core threshold of ∼31.9°C. We speculate that a similar critical temperature threshold likely exists in humans, significantly influencing the timing of emergence from anesthesia. Elucidating such a threshold would hold important implications for clinical anesthesia management. Importantly, thermoregulatory neurons of LPO BDNF and DMH Vglut2 were closely associated with anesthetic modulation. Specifically, while LPO BDNF neurons mediated anesthetic effects inclusively through thermoregulation, DMH Vglut2 neurons exerted modulation via both temperature-dependent and temperature-independent mechanism. This work establishes a different paradigm for understanding how anesthesia is modulated by thermoregulatory neurons through defined neural circuit mechanisms, which are in some ways analogous to the sleep-wake circuits implicated in general anesthesia, 8 , 9 thereby offering another insights into the mechanisms underlying anesthetic states. The POA is a complex brain region composed of heterogeneous neuronal populations that regulate diverse functions and behaviors, including body temperature homeostasis, 26 , 27 sleep-wake cycles, 28 , 29 and anesthetics-induced states. 30 , 31 BDNF protein is most expressed in the hippocampus and hypothalamus, 32 and its expressing neurons are known to regulate feeding and energy expenditure. 33 , 34 Recently, more studies have proved its thermoregulation function in the hypothalamus. 35 BDNF neurons in MnPO were progressively activated during warm exposure, and optogenetic activation of those neurons resulted in hypothermia. 36 Our data demonstrated that pre-anesthetic activation of LPO BDNF neurons significantly decreased T core and the hypothermic effect persisted throughout isoflurane exposure. However, the activation during isoflurane exposure did not induce body temperature change. This might be due to the impairment of behavioral regulation (e.g., shivering and vasoconstriction) induced by isoflurane. 2 Previous studies reported that POA neurons (ventrolateral and median preoptic nucleus) modulated sleep-wake transitions, but do not influence general anesthesia-induced unconsciousness (mice with T core maintained at 37°C–38°C). 37 This is consistent with our results that LPO BDNF neuronal optogenetic activation and chemogenetic activation at 32°C induced no change of anesthetic effect. However, the activation of LPO BDNF neuron at room temperature conditions (20°C–25°C) increased anesthetic depth shown in our study, and this further supports the notion that LPO BDNF neurons modulate anesthetic effect in a temperature-dependent way. While our experimental approach (e.g., acute optogenetic modulation) effectively captures immediate pharmacodynamic changes (like BSR suppression), it is not designed to dissect the intertwined roles of pharmacokinetics (e.g., isoflurane metabolism) and pharmacodynamics over longer periods such as recovery. A key future goal is to disentangle these effects, potentially through direct drug monitoring or closed-loop experimental paradigms. Optogenetic activation of the axonal terminals of LPO BDNF neurons in the DMH lowered T core and increased BSR during isoflurane, while whether LPO BDNF neurons functioning through inhibiting DMH neurons needs further studies. Activation of GABAergic or Vglut2-expressing neurons in the DMH contributed to hyperthermia. 15 , 36 , 38 Our data showed that chemogenetic activation before isoflurane administration increased T core during anesthesia procedure. We also found that optogenetic Vglut2 neurons during isoflurane suppressed BSR immediately and decreased δ power bands without altering T core , indicating its intrinsic consciousness regulation. It should be noted that although the stimulation parameters evoked robust responses, they may not capture the precise endogenous dynamics of these neuronal populations. Previous study reported that optogenetic activation of GABAergic neurons in DMH facilitate sevoflurane emergence. 24 Although the DMH is a major downstream target of LPO, it also integrates inputs from other sleep-wake related nuclei, such as the ventromedial prefrontal cortex (vmPFC) and suprachiasmatic nucleus (SCN). 22 , 39 This broader connectivity may explain its ability to influence anesthetic states independently of thermoregulation. Previous studies demonstrated that DMH Vglut2 neurons project to the RPa, driving T core elevation via brown adipose tissue (BAT) thermogenesis, shivering, and tail vasomotion. 16 , 38 , 40 In line with these findings, we showed that optogenetic activation of the DMH Vglut2 to RPa pathway increased T core during the awake state, but without altering T core during isoflurane exposure. It remains to be determined whether isoflurane causes its effect through disruption of this neural pathway or through suppression of peripheral thermoregulatory functions. Intriguingly, under isoflurane anesthesia, this pathway modulates anesthetic depth, indicating a temperature-independent mechanism of isoflurane induced anesthesia. Although we demonstrated that the temperature-independent arousal-promoting effect of DMH Vglut2 neurons is partly mediated by Vglut2 neurons in the RPa, further investigation employing neuronal staining and cell-type-specific functional assays will be required to fully elucidate the diverse downstream effectors of this pathway. We also speculate that this temperature-independent effect may affect by elevated heart rate and blood pressure, as previous study showing that DMH-RPa pathway activation under anesthesia still raises both parameters. 41 Given that the DMH Vglut2 neurons project to multiple downstream targets, including VTA and DR region, 22 future studies are required to determine whether these complementary pathways also contribute to anesthetic state control, potentially forming a coordinated multi-synaptic network for arousal. Our study may explain the high occurrence of hypothermia clinically, as the thermoregulatory system is profoundly affected by general anesthetics. Recent advances in targeted neuromodulation, including POA-mediated hypothermia induction in non-human primates 42 and non-invasive T core modulation via ultrasound 43 highlight the translational potential of neural circuit-based temperature management. These approaches may inform the development of individualized anesthetic strategies that minimize the adverse effects of hypothermia while leveraging its potential advantages in some special medical conditions (e.g., cardiac surgery, organ transplantation, and neonatal encephalopathy 44 , 45 , 46 , 47 ). Collectively, our findings provide critical insights into the neural mechanisms of isoflurane-induced hypothermia and general anesthesia. Limitations of the study This study did not examine the peripheral vascular changes, which is regarded as another important factor that influences body temperature during anesthesia. Incorporating observations of the peripheral circulatory system, measuring BAT, monitoring T core in brain or with higher sensitivity would provide more comprehensive understanding in further studies. 48 The enhanced anesthetic effect observed under 20°C T a may inevitably include neuroendocrine and metabolic alterations induced by cold stress. 49 , 50 To avoid additional differences in gender, 51 we prioritized male cohorts in experiments. Resource availability Lead contact Further information and requests for resources should be directed to and will be fulfilled by the lead contact, shenwei@shanghaitech.edu.cn (Wei L. Shen). Materials availability This study did not generate new unique reagents. Data and code availability • All data reported in this paper will be shared by the lead contact upon request. • This paper does not report original code. • Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request. Acknowledgments The authors thank Prof. Daqing Ma, Imperial College London, for critical comments during manuscript preparation. This work was supported by grants from National Natural Science Foundation of China (no. 82430042 to T.Y.; nos. 32425028, 92357304, and 32330042 to W.L.S; nos. 82060653, and 82460707 to T.L.; no. 82160682 to S.Y.; and no. 32471076 to W.Z.Y.), Scientific and Technological Innovation 2030 under grant 2021ZD0204300. Noncommunicable Chronic Diseases-National Science and Technology Major Project (2024ZD0530300), the project (no. 2023-107 to W.Z.Y.) of Key Laboratory of Anesthesia and Organ Protection of Ministry of Education (In Cultivation), Zunyi Medical University. We thank the Shanghai Municipal Government and ShanghaiTech and Shanghai Frontiers Science Center for Bomacromolecules and Precision Medicine University for financial support. Author contributions Study concept and design, W.L.S., T.Y., J.Z., S.C., and H.W.; acquisition of data, S.C., M.X., S.Y., H.C., and L.Z.; data and project management, W.Z.Y., T.L., and J.Z.; data cleaning and analysis, W.L.S., S.C., W.Z.Y., M.X., and K.Z.; drafting of the manuscript, S.C., W.L.S., W.Z.Y., J.Z., and T.Y. All authors participated in the interpretation and revision of critically important contents of the review. All authors approved the version of the manuscript submitted. Declaration of interests The authors declare that they have no conflicts of interest. STAR★Methods Key resources table REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit anti- c -Fos Synaptic systems Cat # 226003; RRID: AB_2231974 Rabbit anti-Glutamate Sigma Cat #G6642; RRID: AB_259946 Goat anti-rabbit IgG Alexa Fluor 594 Invitrogen Cat # 111-585-144; RRID: AB_2307325 Bacterial and virus strains rAAV-Ef1α-DIO-hM3Dq-EYFP/mCherry Brain Case Cat BC-0145/0146 rAAV-Ef1α-DIO-hM4Di-EYFP Brain Case Cat BC-0154 rAAV-Ef1α-DIO-EYFP Brain Case Cat BC-0302 AAV-Ef1α-DIO-hChR2 (H134R)-EYFP Brain Case Cat BC-0107 AAV-Ef1α-DIO-eNpHR-EYFP Brain Case Cat BC-0125 AAV-hSyn-DIO-GCaMP6s Brain Case Cat BA-0238 rAAV-EF1α-DIO-WGA-Flpo Brain Case Cat BC-1400 rAAV-EF1α-FDIO-EGFP Brain Case Cat BC-0474 Chemicals, peptides, and recombinant proteins Isoflurane RWD Life science Cat R510-22-10 Clozapine-N-Oxide (CNO) Sigma-Aldrich Cat#C0832 Software and algorithms Thermochron iButton Dallas Semiconductor DS1922L Wdsen Wdsen Electronic Technology http://wdsen.com/iButtonData.html Spike2 Cambridge Electronic Design https://ced.co.uk/upgrades/spike2demo MATLAB MathWorks https://www.mathworks.com/products/matlab GraphPad Prism 9.0 GraphPad Software https://www.graphpad.com/ Open in a new tab Experimental model and study participant details Animals All experimental procedures were approved by the Animal Care and Use Committees of Zunyi Medical University, Guizhou, China ((2021) 2–581) and conducted in accordance with the ARRIVE guideline. Wild type adult C57BL/6 J mice (purchased from the Changsha Tianqin Biotechnology Co., Ltd, license number: SCXK2012-0005; Changsha, China), Vglut2-ires-Cre mice (JAX stock no. 016963), and BDNF-ires-Cre mice 52 were used. All experiments were performed on male adult mice (8–16 weeks old). All animals were housed at an ambient temperature of 22 ± 2°C with a relative humidity of 60 ± 10% and a 12-h light/12-h dark cycle (light on at 8:00 p.m.). They freely access food and water. Method details Stereotaxic brain surgeries and viral injection Mice were anesthetized with pentobarbital (40 mg/kg, intraperitoneal [i.p.]) and then placed on a stereotaxic apparatus (RWD Life Science, Shenzhen, China). Lidocaine (1%) was subcutaneously injected before exposing the surface of the skull. The coordinates of viral injection sites include the LPO (AP, +0.5 mm; ML, ±0.82 mm; DV, −5.2 mm) and DMH (AP, −1.75 mm; ML, ±0.3 mm; DV, −5.1 mm). AAV virus was delivered (speed: 25 nL/min) through a glass micropipette (1-mm glass stock, tapering to a 10–20 microns tip) using a micro-syringe pump (Legato R130, KD Scientific, United States). The pipette was kept in the site for 10 min to allow the virus to diffuse before slowly withdrawn. In chemogenetic experiments, virus was bilaterally and respectively injected 120 nL into the LPO of BDNF-ires-Cre mice , and DMH of Vglut2-ires-Cre mice. In optogenetics experiments, virus was unilaterally injected into LPO and DMH respectively, and the optical fibers (200 μm O.D., 0.37 numerical aperture, Newton Inc.) were positioned 0.2 mm above the injection site. The electroencephalographic electrodes were placed on the skull (AP: +1.0 mm, ML: ±1.5 mm; AP: −3.5 mm, ML: ±1 mm), and attached to the skull with a skull-penetrating screw and dental acrylic. In calcium signal recording experiments, virus was unilaterally and respectively injected into LPO and DMH, and optical fibers were positioned 0.2 mm above the injection site. During all the surgical procedures, a heating pad with a rectal temperature probe was used to keep the body temperature of mice around at 37°C. After surgery, a subcutaneous injection of carprofen (5 mg/kg) was administered for postoperative analgesia. Mice were then transferred to their housing cages for 3–4 weeks before next experiments. Core body temperature recording Abdominal temperature probe (Thermochron iButton DS1922L, Dallas Semiconductor, USA) was implanted into the mice under isoflurane anesthesia two days before experiments. After surgery, a subcutaneous injection of carprofen (5 mg/kg) was administered for postoperative analgesia. The data were recorded at every 2 min throughout experiments and analyzed offline ( Wdsen Electronic Technology Co., Ltd , Shanghai). EEG recording and analysis Electroencephalography signals were captured during all the experiment with a neuronal recording system (Apollo, Bio-Signal Technologies, USA). These data were then digitized and analyzed using the Spike2 software package (Cambridge Electronic Design, Cambridge, United Kingdom). Delta (δ), theta (θ), alpha (α), beta (β), gamma (γ), and total spectral powers were calculated using the frequency bands of 1–4, 5–8, 9–12, 13–25, 26–60, and 1–60 Hz, respectively. Relative power within the frequency range of each band was divided by the total power in 1–60 Hz for data analysis. The burst suppression ratio (BSR) was the duration of the suppression wave divided by the total recording time, which computed with the MATLAB (MathWorks R2023a). Ambient temperature controlling A plastic water bag equipped with a thermostatic water was placed at the bottom of the anesthesia chamber to manipulate ambient temperature at 20°C, 25°C, 32°C, and 36°C during experiments. Isoflurane exposure Mice were placed in the anesthesia chamber (RWD Company, Shenzhen, China) and anesthetized with 1.4% isoflurane in oxygen enriched air at a rate of 1.5 L/min. We flipped the cage 90° every 15 s, the time of loss of righting reflex (LORR) and recovery of righting reflex (RORR) was determined when isoflurane exposure started and exposure stopped during ambient temperature at 20, 25, 32 or 36°C, respectively. All experiments were performed between 9:00 a.m. and 6:00 p.m. In the chemogenetics groups, CNO (1 mg/mL, 1 mg/kg, i.p.) or saline (0.9%, equal volume, i.p.) were injected randomly 30 min before and then, EEG, LORR and RORR were recorded during and after isoflurane administration. In the optogenetics groups, the photo-stimulation (473 nm, 8 mW, 25 Hz, 10 ms duration, 1s off-cycle) (Thinker Tech, China) was conducted for 15 min, EEG, LORR and RORR were recorded during isoflurane administration. Calcium fiber photometry recordings AAV2/9-hSyn-DIO-GCaMP6s virus was injected and optical fibers (200 μm O.D., 0.37 numerical aperture, Inper Inc., China) was implemented in the LPO in the BDNF-ires-Cre mice and DMH of Vglut2-ires-Cre mice, respectively. The fluorescence signals were collected by a multichannel fiber photometry system (Inper Inc., China), equipped with a 488-nm and 410-nm lasers to collect signals and noise at a sampling rate of 50Hz, respectively, two weeks after injection, under 0.8% or 1.4% isoflurane exposure at 20, 25 or 32°C of ambient temperature, respectively, each for 10 min. The values of fluorescence change (ΔF/F) were derived by calculating (F(t) − F)/F, where F is the baseline fluorescence signal averaged in a 50 s time window prior to the starting and ceasing of isoflurane, ΔF/F and Z score (Z(t) = (F(t) - μ)/σ), Z(t) = Z score at time t, F(t) = Raw fluorescence value at time t, μ = Mean baseline fluorescence, σ = Standard deviation of baseline fluorescence) represents changes in fluorescence. Immunohistochemistry Some cohort mice after above procedure or the hM3Dq and hM4Di mice injected with CNO for 2 h were killed with terminated pentobarbital anesthesia and perfused with the phosphate-buffered saline (PBS) followed by 4% paraformaldehyde (PFA). Their brains were removed and post-fixed in PFA overnight at 4°C and put in 30% sucrose in PBS at 4°C until they sank. The brains were coronally sectioned into 30-μm slices with a cryostat (Leica CM1950). The sections were first incubated in blocking solution (PBS containing 2.5% normal goat serum, 1.5% bovine serum albumin, and 0.1% Triton X-100) for 2 h at room temperature. They were then incubated with the primary antibody (Rabbit anti- c -Fos, Synaptic systems, # 226003, 1:1000; Rabbit anti-Glutamate, Sigma, #G6642, 1:200) in a blocking solution overnight at 4°C and then with the related secondary antibody (Alexa Fluor 594 conjugated goat anti-rabbit IgG, Invitrogen, # 111-585-144, 1:1000) at room temperature for 2 h after washing with PBS. After washed with PBS, the sections were mounted on glass slides and cover-slipped with a mounting media (Gold antifade reagent with DAPI, Life Technologies, USA). All images were captured with the virtual microscopy system (Olympus BX63). Quantification and statistical analysis All data were presented as mean ± SEM. Student’s t test were applied for statistical comparisons between the two groups, Multiple t -test and two-way ANOVA followed by Bonferroni’s multiple comparisons test were used in multiple groups comparison where appropriate. Linear regression analysis was used to analysis the relation of T core and T a , T core and LORR time, the BSR and T core . Non-linear regression (One phase decay) analysis was used to analysis the relation of T core and RORR time. The statistical details are shown in results and figure legends. A significance threshold was set at p less than 0.05. All statistical analysis was carried out with the GraphPad Prism software (version 9.0, GraphPad Software Inc., San Diego, CA, USA). Published: March 30, 2026 Footnotes Supplemental information can be found online at https://doi.org/10.1016/j.isci.2026.115542 . Contributor Information Tian Yu, Email: zyyutian@126.com. Jijian Zheng, Email: zhengjijian626@sina.com. Wei L. Shen, Email: shenwei@shanghaitech.edu.cn. 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