Chronic heat exposure produces negative emotional and hyperarousal states but not depression-like behaviors.

(A) Experimental schematics. Mice (n=10 in each group) were divided into Control and Heat groups and conducted with chronic exposure to room temperature and heat conditions, respectively, followed by behavioral tests. (B, C) The heatmap of representative tracking trace examples in EPM test and the time spent in the open arms of EPM (Mann-Whitney unpaired two-tailed U test; U=0, ***p<0.001). (D, E) The heatmap of representative tracking trace examples in TCT and the interaction time with an unfamiliar male mouse relative to the inanimate object in TCT (Mann-Whitney unpaired two-tailed U test; U=13, ***p=0.0039). (F, G) The heatmap of representative tracking trace examples in FET and the time surrounding the unfamiliar female mouse compared to an unfamiliar male mouse in FET (Mann-Whitney unpaired two-tailed U test; U=10, **p=0.0015). (H, I) The 1st-time attack latency (Mann-Whitney unpaired two-tailed U test; U=21, *p=0.0288) and the attack durations (Mann-Whitney unpaired two-tailed U test; U=23, *p=0.0433) in the aggression test. (J, K) Visualized and representative acoustic startle response example (left panel) and corresponding labeled body parts’ skeletons (right panel) in ASR test from Control and Chronic Heat group.(L, M) The startle amplitude (Mann-Whitney unpaired two-tailed U test; U=6, ***p=0.0003) and the pre-pulse / pulse ratio (Mann-Whitney unpaired two-tailed U test; U=26, p=0.0753) in ASR test. (N-Q) The immobile time in the forced swim test (Mann-Whitney unpaired two-tailed U test; U=35, p=0.2799) and in the tail suspension test (Mann-Whitney unpaired two-tailed U test; U=26, p=0.0753; The percentage of sucrose intake in the sucrose preference test (Mann-Whitney unpaired two-tailed U test; U=49.5, p=0.4887); The time spent in the center of the open field test (Mann-Whitney unpaired two-tailed U test; U=35.5, p=0.2888). *p<0.05, **p<0.01, ***p<0.001, N.S.: not significant.

Involvement of the hypothalamic preoptic area to posterior paraventricular thalamus projections.

(A) The strategy of virus injection followed by heat exposure-induced c-Fos staining (n=3 mice). (B) The representative microphotograph showed the expression of rabies virus in the pPVT regions. (C) Rabies virus retro-labeled POA neurons. Upper: magnification: x4, scale bar: 400 μm; Lower: magnification: x10, scale bar: 40 μm; The top right corner of the lower picture: magnification: x60, scale bar: 5 μm. (D) The representative microphotograph showed that most of pPVT rabies virus retro-labeled POA neurons co-stained with heat exposure-induced c-Fos. Upper: magnification: x10, scale bar: 40 μm; Lower: magnification: x60, scale bar: 10 μm. (E) The strategy of virus injection (n=5 mice) and patch-clamp recording was performed on POA expressing ChR2-mCherry neurons and pPVT neurons using potassium and cesium, low chloride internal solutions, respectively. Representative traces showed that POA expressing ChR2-mCherry neurons exhibited robust firing in response to optical stimulation at different frequencies (n=15 neurons from 5 mice). (F, G) Representative traces showed that blue light stimulation evoked either excitatory postsynaptic current (EPSC) which could be blocked by cyanquixaline (CNQX, 10 μM) or inhibitory postsynaptic current (IPSC) which could be blocked by picrotoxin (PTX, 100 nM). (H) Pie chart showed the projection types recorded on pPVT neurons (n=63 neurons from 10 mice). (I) The representative recorded pPVT neuron was visualized by biocytin staining and was found being surrounded by POA expressing ChR2-mCherry terminals. Magnification: x60, scale bar: 20 μm. (J, K) The representative trace showed that the application of tetrodotoxin (TTX) eliminated the oPSC held at −70 mV while the addition of 4-amino-pyridine (4-AP, 1mM) recovered it and the quantification (n=24 neurons from 10 mice).

Activity changes of POA recipient pPVT neurons throughout chronic heat exposure.

(A) Experimental schematics. Mice (n=5) were stereotaxically injected with Cre-dependent GCaMP into the pPVT, followed by the implantation of optical fiber and chronic calcium recording. (B) Representative microphotographs showed the virus expression in the POA and pPVT regions, scale bar: all 40 μm. (C) From the top to the bottom: the representative calcium events (on the left panel) and the calcium events from different mice (on the right panel) on different days. Each vertical stripe in the heatmap represents one calcium event for each mouse. Statistical analysis of the frequency and amplitude of POA recipient pPVT neurons’ calcium events compared for (D) Pre and During heat exposure on day 1 (Paired, parametric, two tailed t-test; Frequency (t=6.278, df=4, P=0.0033); Amplitude (t=4.344, df=4, *p=0.0122)); (E) Pre heat on day 1 and After heat on day 2 (Paired, parametric, two tailed t-test; Frequency (t=0.2787, df=4, p=0.7943); Amplitude (t=1.726, df=4, p=0.1595)); (F) Pre heat on day 1 and During heat exposure on day 21 (Paired, parametric, two tailed t-test; Frequency (t=6.124, df=4, **p=0.0036); Amplitude (t=4.704, df=4, **p=0.0093)); (G) Pre heat on day 1 and after Chronic heat on day 22 (Paired, parametric, two tailed t-test; Frequency (t=6.216, df=4, **p=0.0034); Amplitude (t=1.36, df=4, p=0.2454)); (H) During heat exposure on day 1 and day 21 (Paired, parametric, two tailed t-test; Frequency (t=1.242, df=4, p=0.2821); Amplitude (t=0.9424, df=4, p=0.3993)). * p<0.05, ** p<0.01, N.S.: not significant.

POA recipient pPVT neurons are sufficient and necessary for chronic heat exposure-induced negative emotional and hyperarousal states in mice.

(A) Experimental schematics. Mice (n=10 in each group) were stereotaxically injected with either AAV1-hSyn-EGFP or AAV9-CaMKII-ChR2-mCherry into the POA, followed by the implantation of optical fiber into pPVT, chronic optogenetic activation, and behavioral tests. (B, C) The heatmap of representative tracking trace examples in EPM test and the time spent in the open arms (Mann-Whitney unpaired two-tailed U test; U=2, ***p<0.001). (D, E) The heatmap of representative tracking trace examples in TCT and the interaction time with an unfamiliar male mouse relative to the inanimate object (Mann-Whitney unpaired two-tailed U test, U=8; ***p=0.0007). (F, G) The heatmap of representative tracking trace examples in FET and the time surrounding the unfamiliar female mouse compared to an unfamiliar male mouse (Mann-Whitney unpaired two-tailed U test; U=13, **p=0.0039). (H, I) The 1st-time attack latency (Mann-Whitney unpaired two-tailed U test; U=8, p=0.007) and the attack durations (Mann-Whitney unpaired two-tailed U test; U=19, *p=0.0185) in the aggression test. (J, K) Visualized acoustic startle response examples (upper panel) and corresponding labeled body parts’ skeletons (lower panel) from hSyn-EGFP group and ChR2-mCherry group. (L, M) The startle amplitude (Mann-Whitney unpaired two-tailed U test; U=17, *p=0.0115) and the pre-pulse / pulse ratio (Mann-Whitney unpaired two-tailed U test; U=48, p=0.9118) in ASR test. (N) Experimental schematics. Mice (n≥6 in each group) were stereotaxically injected with AAV1-hSyn-Cre-EGFP in the POA and either AAV9-Ef1a-Dio-eNpHR3.0-EGFP or AAV8-hSyn-Dio-EGFP into the pPVT, followed by the implantation of optical fiber and behavioral tests. (O) Representative microphotographs showed the expression of AAV1-hSyn-Cre-EGFP and AAV9-hSyn-Dio-eNpHR3.0 within the POA and pPVT, respectively. Magnification: x4, scale bar: 100 μm (upper panel) and 250 μm (lower panel). (P, Q) The time (One-way repeated measures ANOVA with Tukey post-hoc test; F(2, 27)=83.03, ***p<0.001; Control vs. Chronic heat, ***p<0.001; Chronic heat vs. Chronic heat + inhibition, ***p<0.001) and the entry numbers (One-way repeated measures ANOVA with Tukey post-hoc test; F(2, 20.92)=27.96, ***p<0.001; Control vs. Chronic heat, ***p<0.001; Chronic heat vs. Chronic heat + inhibition, ***p<0.001) into the open arms of EPM. (R) The time spent with an unfamiliar male mouse compared to an inanimate object in TCT (One-way repeated measures ANOVA with Tukey post-hoc test; F(2, 27)=5.381, *p=0.0108; Control vs. Chronic heat, *p=0.0292; Chronic heat vs. Chronic heat + Inhibition, *p=0.0175). (S) The ratio of time with an unfamiliar female mouse compared to an unfamiliar male mouse in FET (One-way repeated measures ANOVA with Tukey post-hoc test; F(2, 27)=10.94, ***p=0.0003; Control vs. Chronic Heat, ***p=0.0002; Chronic Heat vs. Chronic Heat + Inhibition, *p=0.0335). (T, U) The 1st attack latency (One-way repeated measures ANOVA with Tukey post-hoc test; F(2, 17)=7.426, *p=0.0048; Control vs. Chronic Heat, *p=0.0157; Chronic Heat vs. Chronic Heat + Inhibition, *p=0.0063) and the attack durations (One-way repeated measures ANOVA with Tukey post-hoc test; F(2, 17)=13.38, ***p=0.0003; Control vs. Chronic Heat, ***p=0.0003; Chronic Heat vs. Chronic Heat + Inhibition, **p=0.0051) in the aggression test. (V, W) The startle amplitude (One-way repeated measures ANOVA with Tukey post-hoc test; F(2, 21)=21.03, ****p<0.0001; Control vs. Chronic Heat, ***p<0.001; Chronic Heat vs. Chronic Heat + Inhibition, ***p<0.001) and the pre-pulse / pulse ratio (One-way repeated measures ANOVA with Tukey post-hoc test; F(2, 21)=3.802, *p=0.039; Control vs. Chronic Heat, p=0.2616; Chronic Heat vs. Chronic Heat + Inhibition, p=0.2246) in ASR test. *p<0.05, **p<0.01, ***p<0.001, N.S.: not significant.

Chronically activated POA recipient pPVT neurons exhibited exaggerated response to stressful situations.

(A) Experimental schematics. Mice (n=5) were stereotaxically injected with AAV1-hSyn-Cre-EGFP in the POA and AAV9-hSyn-Flex-jGCaMP8F-WPRE in the pPVT, followed by the implantation of optical fiber and calcium recording. (B) The number of entries into the open arms of EPM (Paired, parametric, two tailed t-test; t=6.901, df=4, **p=0.0023). (C) The number of instances where the mice paused at the center area followed by back to closed arms of EPM (Paired, parametric, two tailed t-test; t=9, df=4, ***p=0.0008). (D) Pie chart showed the changes of the percentage of pause and then running back to closed arms. (E) The changes of running speed in EPM (Mann-Whitney unpaired two-tailed U test; U=139, **p=0.0028). (F-H) Heatmaps showed the calcium activities of POA recipient pPVT neurons when mice performed the pause-and-run-back-to-closed-arms behavior for pre (n=13 trials from 5 mice) and post conditions (n=47 trials from 5 mice), Z-Score calcium signals, and statistical comparison (Mann-Whitney unpaired two-tailed U test; U=194, *p=0.0455). (I) The time spent in open arms of EPM (Paired, parametric, two tailed t-test; t=3.649, df=4, *p=0.0218). (J) The total number of the behavioral event: back from open to closed arms (Paired, parametric, two tailed t-test; t=6.901, df=4, **p=0.0023). (K) Pie chart showed the changes of number of running episodes from open to closed arms for pre and post conditions. (L) The changes of running speed in EPM (Mann-Whitney unpaired two-tailed U test; U=23, ***p=0.0005). (M-O) Heatmaps showed the calcium activities of POA recipient pPVT neurons when mice performed fast running from open to closed arms for pre (n=14 trials from 5 mice) and post conditions (n=13 trials from 5 mice), Z-Score calcium signals, and statistical comparison (Mann-Whitney unpaired two-tailed U test; U=42, *p=0.0168). (P) The total distances of mice travelled in the chamber previously subjected to chronic heat (Paired, parametric, two tailed t-test; t=6.876, df=4, **p=0.0023). (Q) The changes of motion speed for pre and post conditions from one representative mouse. (R) The number of instances with motion speed exceeding 400 mm/s (Paired, parametric, two tailed t-test; t=5.100, df=4, ***p=0.007). (S) The number of fast running episodes for pre and post conditions. (T-V) Heatmaps showed the calcium activities of POA recipient pPVT neurons when the mice performed fast running in the previous chronic heat-exposed chamber (n=90 trials from 5 mice) compared to pre-heat condition (n=38 trials from 5 mice), Z-Score calcium signals, and statistical comparison (Mann-Whitney unpaired two-tailed U test; U=1218, *p=0.01). *p<0.05, **p<0.01, ***p<0.001.

Increased pre- and post-synaptic excitability of pPVT neurons but saturated circuitry neuroplasticity capacity following chronic heat exposure.

(A) Experimental schematics. In vitro brain slice recording was performed on mice from three groups (n=16 neurons from 3 mice in the Room temperature group, n=20 neurons from 4 mice in the Acute heat group, and n=20 neurons from 4 mice in the Chronic heat group). (B) Representative traces of miniature postsynaptic currents from three groups. Duration: 12 s. Scale bar: 10 pA. (C, D) The changes of mIPSCs’ amplitude (One-way repeated measures ANOVA with Tukey post-hoc test; F(2, 54)=8.226, ***p=0.0008, Room temperature vs. Acute heat, ***p=0.0005; Room temperature vs. Chronic heat, *p=0.0268) and frequency (One-way repeated measures ANOVA with Tukey post-hoc test; F(2, 53)=1.346, p=0.269, Room temperature vs. Acute heat, p=0.4327; Room temperature vs. Chronic heat, p=0.9787) of pPVT neurons. (E, F) The changes of mEPSCs’ amplitude (One-way repeated measures ANOVA with Tukey post-hoc test; F(2, 52.98)=0.1995, p=0.8198, Room temperature vs. Acute heat, p=0.8433; Room temperature vs. Chronic heat, p=0.8433) and frequency (One-way repeated measures ANOVA with Tukey post-hoc test; F(2, 50.1)=2.981, p=0.0598, Room temperature vs. Acute heat, p=0.2931; Room temperature vs. Chronic heat, *p=0.0473) of pPVT neurons. (G) The representative traces of action potential of pPVT neurons upon 100 pA current injection. (H) The changes of excitability of pPVT neurons when different currents were injected to the patched pPVT neurons (n=11 neurons from 3 mice in each groups; Mann-Whitney unpaired two-tailed U test; 30 pA: U=22.5, **p=0.0091; 40 pA: U=31, *p=0.048; 50 pA: U=29.5, *p=0.0412; 60 pA: U=25, *p=0.0167; 70 pA: U=29.5, *p=0.0396). (I) The changes of rheobase of action potential (Mann-Whitney unpaired two-tailed U test; U=28.5, *p=0.0345). (J) Experimental schematics. Mice (n=5 mice in each group) stereotaxically injected with AAV9-CaMKII-ChR2-mCherry into the POA were then divided into the Room temperature and Chronic heat groups. Sagittal slices of mice were prepared for LTP induction and recording. (K) The representative traces showed pPVT neurons from mice exposed to room temperature and chronic heat exhibited different amplitude of oEPSCs after blue light-mediated high frequency stimulation. (L) The LTP induction and recording of POA to pPVT pathway from slices of mice exposed to room temperature and chronic heat conditions after blue light stimulation at 30 Hz (n=14 neurons from control group and 18 neurons from chronic heat group). (M) Statistical comparison of the amplitude of oEPSCs at different time points (Two-way repeated measures ANOVA with Sidak post-hoc test; Interaction: F(3, 120)=0.3486, p=0.7902; Optical stimulation main effect: F(3, 120)=76.2, ***p<0.001; Time points effect: F(3, 120)=0.8215, p=0.4844; 1st min: Room temperature vs. Chronic heat: ***p<0.001; 10th min: Room temperature vs. Chronic heat: p=0.0001; 20th min: Room temperature vs. Chronic heat: ***p<0.001; 30th min: Room temperature vs. Chronic heat: **p=0.0024). *p<0.05, **p<0.01, ***p<0.001, N.S.: not significant.

A working model of neural circuit mechanisms underlying POA recipient pPVT neurons-mediated chronic heat exposure-induced negative emotional and hyperarousal states.

Different from acute heat exposure, chronic heat exposure-induced enhancement in excitatory inputs to pPVT and saturated neuroplasticity contributed to the increased membrane excitability underlie the heightened activities of POA recipient pPVT neurons, rendering mice become more susceptible to stressful situations manifested as negative emotions and hyperarousal states.