{"paper_id":"2529c65d-18fb-4f51-80cd-420ae7fa8553","body_text":"A hypothalamic circuit for circadian regulation of corticosterone secretion | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article A hypothalamic circuit for circadian regulation of corticosterone secretion Oscar D. Ramirez-Plascencia, Roberto De Luca, Natalia L. S. Machado, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4718850/v2 This work is licensed under a CC BY 4.0 License Status: Posted Version 2 posted You are reading this latest preprint version Show more versions Abstract The secretion of cortisol in humans and corticosterone (Cort) in rodents follows a daily rhythm which is important in readying the individual for daily activity. This rhythm is orchestrated by the suprachiasmatic nucleus (SCN), but how it ultimately regulates the circadian rhythm of activity of neurons in the paraventricular nucleus of the hypothalamus that produce corticotropin-releasing hormone (PVH CRH neurons) is not known. We hypothesized that the SCN may exert this influence by projections to the subparaventricular zone (SPZ), which in turn innervates neurons in the dorsomedial nucleus of the hypothalamus (DMH) that regulate PVH CRH neurons. First, we found that ablating SPZ Vgat neurons eliminates the circadian rhythm of Cort secretion, but that deleting Vgat from them does not, suggesting that they predominantly use some other transmitter. Next, we found that either ablating or acutely inhibiting the DMH glutamatergic (DMH Vglut2 ) neurons resulted in a 40-70% reduction in the daily peak of Cort. Deletion of the Vglut2 gene within the DMH produced a similar effect, highlighting the indispensable role of glutamatergic signaling. Chemogenetic stimulation of DMH Vglut2 neurons led to an increase of Cort levels, and optogenetic activation of their terminals in the PVH in hypothalamic slices directly activated PVH CRH neurons through glutamate action on AMPA receptors (the DMH Vglut2 → PVH CRH pathway). Similar to the disruption of DMH Vglut2 neurons, ablating, inhibiting, or disrupting GABA transmission by DMH GABAergic (DMH Vgat ) neurons diminished the circadian peak of Cort, particularly under constant darkness conditions. Chemogenetic stimulation of rostral DMH Vgat neurons increased Cort, although with a lower magnitude compared to DMH Vglut2 neuron stimulation, suggesting a role in disinhibiting PVH CRH neurons. Supporting this hypothesis, we found that rostral DMH Vgat neurons project directly to GABAergic neurons in the caudal ventral part of the PVH and adjacent peri-PVH area (cvPVH), which directly inhibit PVH CRH neurons, and that activating the rostral DMH Vgat terminals in the cvPVH in brain slices reduced GABAergic afferent input onto the PVH CRH neurons. Finally, ablation of cvPVH Vgat neurons resulted in increased Cort release at the onset of the active phase, affirming the pivotal role of the DMH Vgat → cvPVH Vgat → PVH CRH pathway in Cort secretion. In summary, our study delineates two parallel pathways transmitting temporal information to PVH CRH neurons, collectively orchestrating the daily surge in Cort in anticipation of the active phase. These findings are crucial to understand the neural circuits regulating Cort secretion, shedding light on the mechanisms governing this physiological process and the coordinated interplay between the SCN, SPZ, DMH, and PVH. corticosterone corticotropin-releasing hormone dorsomedial hypothalamus and paraventricular hypothalamus Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Full Text Additional Declarations The authors declare no competing interests. Supplementary Files FigS1SPZDTA.tif Extended figure 1. SPZ Vgat neuron ablation flattens the circadian rhythm of LMA and reduces the amplitude of the rhythm of Tb. (a) Representative micrograph of GABAergic neurons (native signal in green) from a Vgat-ires-Cre::L10-GFP control mouse ( left panel), and the density plots of the distribution injections of AAV-mCherry-DIO-DTA in the SPZ of the Vgat-ires-cre mice ( n =8; right panels). (b) Daily LMA was reduced during the dark period in LD after SPZ Vgat ablation ( Repeated Measures [RM] Two-way ANOVA ; Šídák's multiple comparisons test. SPZ Vgat -GFP vs SPZ Vgat -DTA: *p<0.05, ***p<0.001), and (c) the periodogram showed reduced amplitude ( Two-way ANOVA ; Šídák's multiple comparisons test. SPZ Vgat -GFP vs SPZ Vgat -DTA: *p<0.05). (d) SPZ Vgat neuron ablation further reduced the circadian rhythm of LMA in DD (RM Two-way ANOVA ; Šídák's multiple comparisons test. SPZ Vgat -GFP vs SPZ Vgat -DTA: *p<0.05), (e) causing a dramatical reduction on the amplitude of the periodogram ( Two-way ANOVA ; Šídák's multiple comparisons test. SPZ Vgat -GFP vs SPZ Vgat -DTA: *p<0.05). (f) Bar graphs showing total LMA counts in the light and dark periods from the SPZ Vgat -GFP and SPZ Vgat -DTA mice. LMA was significantly reduced during the dark ( Two-way ANOVA ; Tukey's multiple comparisons: SPZ Vgat -GFP dark vs SPZ Vgat -DTA dark = ***p<0.001) or subjective dark periods ( Two-way ANOVA ; Tukey's multiple comparisons: SPZ Vgat -GFP subjective dark vs SPZ Vgat -DTA subjective dark = ***p<0.001). (g) The reduction of LMA during the dark or subjective dark period in SPZ Vgat -ablated mice reduced circadian index (CI, for method of calculation of CI, see Statistical Analysis in the Materials and Methods) by 51.1 ±3.5% in LD ( Unpaired t-test : t=6.381, df=14, ***p<0.001) and by 79.7 ±4.8% in DD ( Unpaired t-test : t=8.901, df=14, ***p<0.001), (h) while the cosinor amplitude was also reduced in LD ( Unpaired t-test : t=6.142, df=14, ***p<0.001) and DD ( Unpaired t-test : t=6.190, df=14, ***p<0.001) (i) Representative LMA actograms, showing LD and DD recordings from SPZ Vgat -GFP (blue) and SPZ Vgat -DTA (red) mice. (j-n) SPZ Vgat -ablated mice had a small increase in Tb during the light period and a small decrease during the dark period in both LD and DD, causing a significant change in the amplitude of the periodograms (LD: Two-way ANOVA ; Šídák's multiple comparisons test. SPZ Vgat -GFP vs SPZ Vgat -DTA: *p<0.05; DD: Two-way ANOVA ; Šídák's multiple comparisons test. SPZ Vgat -GFP vs SPZ Vgat -DTA: *p<0.05). However, (o) the CI was reduced by 50.4 ±5.6% in LD ( Unpaired t-test : t=6.551, df=14, ***p<0.001) and by 58.3 ±6.5% in DD ( Unpaired t-test : t=5.228, df=14, ***p<0.001), and (p) the cosinor amplitude of Tb was also reduced both in LD (Paired t-test: t=6.199, df=14, ***p<0.001) and DD ( Unpaired t-test : t=4.885, df=14, ***p<0.001). (q) Representative Tb actograms, red and blue lines as in panel i. LD, Light:Dark photoperiod; DD, Constant darkness. FigS2SPZFlFl.tif Extended figure 2. Vgat gene deletion from SPZ neurons only partially reduces LMA and Tb during the dark period. (a) Representative micrograph of Vgat mRNA expression (in red) from a control mouse ( left panel) and an iCre-EGFP injected mouse (in green; right panel). Notice the elimination of Vgat mRNA expression in the area where iCre-EGFP was expressed, whereas the Vgat expression in the SCN was preserved. The EGFP signal was enhanced with immunofluorescence for EGFP. Density plots of the distribution of injections of AAV-EGFP-iCre in the SPZ of the Vgat loxP/loxP mice ( n =7; right panels). (b) Neither daily total LMA nor (c) the LMA periodogram was affected by the Vgat deletion in the SPZ when animals were in LD, but (d) LMA was reduced during the subjective dark period in DD (RM Two-way ANOVA ; Šídák's multiple comparisons test. SPZ Vgat/flox -Control vs SPZ Vgat/flox -EGFP-iCre: *p<0.05), (e) with a reduction of the amplitude in the periodogram ( Two-way ANOVA ; Šídák's multiple comparisons test. SPZ Vgat/flox -Control vs SPZ Vgat/flox -EGFP-iCre: *p<0.05). (f) Total LMA counts in the light and dark periods from the SPZ Vgat/flox -Control and SPZ Vgat/flox -EGFP-iCre mice. Vgat ablation from SPZ neurons reduced the LMA during the subjective dark period under DD ( Two-way ANOVA ; Tukey's multiple comparisons: SPZ Vgat/flox -Control subjective dark vs SPZ Vgat/flox - EGFP-iCre subjective dark = **p=0.004), (g) resulting in a reduced circadian index (CI) by 39.7 ±4.8% in DD ( Unpaired t-test : t=4.674, df=10, ***p<0.001), (h) and a similar reduction the cosinor amplitude in DD ( Unpaired t-test : t=9.399, df=12, ***p<0.001) (i) Representative LMA actograms, showing LD and DD recordings from SPZ Vgat/flox -Control (blue) and SPZ Vgat/flox -EGFP-iCre (red). (j) The Vgat deletion from SPZ slightly reduced the Tb toward the end of the dark period in LD (RM Two-way ANOVA ; Šídák's multiple comparisons test. SPZ Vgat/flox -Control vs SPZ Vgat/flox -EGFP-iCre: *p<0.05), (k) reducing the amplitude in the periodogram ( Two-way ANOVA ; Šídák's multiple comparisons test. SPZ Vgat/flox -Control vs SPZ Vgat/flox - EGFP-iCre: *p<0.05). (l) Similar reduction was observed during the subjective dark period in DD in the SPZ Vgat/flox -EGFP-iCre mice (RM Two-way ANOVA ; Šídák's multiple comparisons test. SPZ Vgat/flox -Control vs SPZ Vgat/flox -EGFP-iCre: *p<0.05) (m) and in the periodogram ( Two-way ANOVA ; Šídák's multiple comparisons test. SPZ Vgat/flox -Control vs SPZ Vgat/flox -EGFP-iCre: *p<0.05). (n) The mean Tb was significantly reduced during the dark or subjective dark period in the SPZ Vgat/flox -EGFP-iCre mice either in LD ( Two-way ANOVA ; Tukey's multiple comparisons: SPZ Vgat/flox -Control vs SPZ Vgat/flox -EGFP-iCre dark = *p=0.021) or DD, ( Two-way ANOVA ; Tukey's multiple comparisons: SPZ Vgat/flox -Control subjective dark vs SPZ Vgat/flox -EGFP-iCre subjective dark = ***p<0.001). (o) The CI of Tb was reduced by 25.7 ±4.6% in LD ( Unpaired t-test: t=3.300, df=12, ***p=0.006) and by 38.6 ±7.3% in DD ( Unpaired t-test : t=4.689, df=12, ***p<0.001), (p) similar to the cosinor amplitude in LD ( Unpaired t-test : t=2.864, df=12, **p=0.014) and DD ( Unpaired t-test : t=5.318, df=12, ***p<0.001). (q) Representative Tb actograms, red and blue lines as in panel i. FigS3VglutDMHDTA.tif Extended figure 3. DMH Vglut2 neuron ablation reduces LMA during the subjective night and decreases Tb across the day. (a) Density plots of the distribution of injections of AAV-mCherry-DIO-DTA in the DMH of the Vglut2-ires-cre mice ( n =8). (b) No statistically significant change was detected in the Cort levels after the restraint stress protocol. (c) Daily LMA was reduced during the dark period in LD after DMH Vglut2 neuron ablation (RM Two-way ANOVA ; Šídák's multiple comparisons test. Pre-DTA vs DMH Vglut2 -DTA: *p<0.05), but (d) the periodogram showed no change. (e) DMH Vglut2 neuron ablation reduced LMA during the subjective dark in DD (RM Two-way ANOVA ; Šídák's multiple comparisons test. Pre-DTA vs DMH Vglut2 -DTA: *p<0.05), (f) reducing the amplitude of the periodogram ( Two-way ANOVA ; Šídák's multiple comparisons test. SPZ Vgat -GFP vs SPZ Vgat -DTA: *p<0.05). (g) Bar graphs showing total LMA counts in the light and dark periods before and after ablation of DMH Vglut2 neurons, during LD (left) and presumptive light and dark periods in DD (right). (h) The reduction of LMA during the dark period after DTA was sufficiently small that the circadian index (CI) was not significantly different. (i) However, the amplitude of the circadian rhythm of LMA as measured by cosinor analysis was reduced in DD after ablation ( Paired t-test : t=3.611, df=14, **p=0.002). (j) Representative LMA actograms, showing LD and DD recordings before (blue) and after (red) DTA. (k) Tb was reduced across the light phase and in the middle of the dark phase after DMH Vglut2 neuron ablation in LD ( Repeated Measures [RM] Two-way ANOVA ; Šídák's multiple comparisons test. Pre-DTA vs DMH Vglut2 -DTA: *p<0.05), while (l) the periodogram showed no change. (m) The reduction in the daily Tb was larger in both the presumptive light and dark periods in DD after DMH Vglut2 neuron ablation (RM Two-way ANOVA ; Šídák’s multiple comparisons test. Pre-DTA vs DMH Vglut2 -DTA: *p<0.05), (n) with no change in the periodogram. (o) The mean Tb was significantly reduced by DMH Vglut2 ablation for the light but not the dark period in LD, and for both in DD ( Two-way ANOVA ; Tukey's multiple comparisons. ** p<0.01, ***p<0.001). However, (p) the CI and (q) the cosinor amplitude of Tb was increased after ablation (by 46.65 ±12.8%) only during LD ( Paired t-test for CI : t=3.075, df=14, df=14, *p=0.008; for cosinor amplitude : t=2.415, df=14, *p=0.03). (r) Representative Tb actograms, red and blue lines as in panel j. LD, Light:Dark photoperiod; DD, Constant darkness. FigS4VglutDMHFl.tif Extended figure 4. Vglut2 gene deletion from DMH neurons reduces the peak of LMA and Tb during the transition from the dark to the light period. (a) Density plots of the injections of AAV-EGFP-iCre in the DMH of Vglut2 loxP/loxP mice ( n =7). (b) Daily distribution of LMA in LD ( RM Two-way ANOVA ; Šídák's multiple comparisons test. DMH Vglut2/flox -Control vs DMH Vglut2/flox -EGFP-iCre: *p<0.05). Vglut2 gene deletion from DMH neurons reduced LMA during the transition from the dark to the light period, and to a lesser extend in the early dark period, (c) reducing the amplitude of the periodogram peak at 24h ( Two-way ANOVA ; Šídák's multiple comparisons test. DMH Vglut2/flox -Control vs DMH Vglut2/flox -EGFP-iCre: *p<0.05). (d) In DD, the reduction in LMA during the transitions between the presumptive light and dark periods was similar to LD (RM Two-way ANOVA ; Šídák's multiple comparisons test. DMH Vglut2/flox -Control vs DMH Vglut2/flox -EGFP-iCre: *p<0.05), (e) with similar reduction in the amplitude of the periodogram ( Two-way ANOVA ; Šídák's multiple comparisons test. DMH Vglut2/flox -Control vs DMH Vglut2/flox -EGFP-iCre: *p<0.05). (f) However, for the entire light and dark periods in LD and presumptive light and dark periods in DD, the small changes in LMA did not reach statistical significance for total LMA counts, (g) CI or (h) cosinor amplitude. (i) Representative LMA actograms from the Control and DMH Vglut2 gene - deleted mice. (j) Tb was also reduced in LD during the transition from the dark to the light phase and to a lesser extent in the early dark phase in DMH Vglut2 gene-deleted mice (RM Two-way ANOVA ; Šídák's multiple comparisons test. DMH Vglut2/flox -Control vs DMH Vglut2/flox -EGFP-iCre: *p<0.05), (k) reducing the amplitude in the periodogram around the 24 period ( Two-way ANOVA ; Šídák's multiple comparisons test. DMH Vglut2/flox -Control vs DMH Vglut2/flox -EGFP-iCre: *p<0.05). (l) A similar pattern in Tb was seen during DD (RM Two-way ANOVA ; Šídák’s multiple comparisons test. DMH Vglut2/flox -Control vs DMH Vglut2/flox -EGFP-iCre: *p<0.05), (m) with similar reduction in the amplitude of the periodogram ( Two-way ANOVA ; Šídák's multiple comparisons test. DMH Vglut2/flox -Control vs DMH Vglut2/flox -EGFP-iCre: *p<0.05). (n) However, as for LMA, these changes were not large enough to produce a statistically significant difference in mean Tb, (o) CI or (p) cosinor amplitude of the circadian rhythm of Tb. (q) Tb actograms from the DMH Vglut2 gene-deleted mice included in this study. LD, Light:Dark photoperiod; DD, Constant darkness. FigS5VglutDMHhGlyR.tif Extended figure 5. Chemogenetic inhibition of the DMH Vglut2 neurons decreases the circadian index of LMA and reduces Tb. (a) Density plots of the distribution of injections of AAV-DIO-hGlyR-mCherry in the DMH of the Vglut2-ires-Cre mice ( n =5). (b-d) Chemo-inhibitions with IVM in LD induced higher LMA at the transition from the dark to the light phase and reduced the peak during the first hours of the dark phase (b: RM Two-way ANOVA ; Tukey's multiple comparisons test p<0.05. Line above the 24h graphs represent significative differences in Baseline vs IVM in green, and VEH vs IVM in blue; c: Two-way ANOVA ; Tukey's multiple comparisons test. light Baseline vs light IVM: **p=0.001, light VEH vs light IVM: *p=0.042, dark Baseline vs dark IVM: *p=0.03), reducing the CI (d: One-way ANOVA ; Tukey's multiple comparisons test. Baseline vs IVM: **p=0.001, VEH vs IVM: *p=0.016). (e-g) In DD, the daily increase in LMA at the beginning of the dark period was reduced (e: RM Two-way ANOVA ; Tukey's multiple comparisons test p<0.05. Lines above same as in B. f: Two-way ANOVA ; Tukey's multiple comparisons test. dark Baseline vs dark IVM: *p=0.036), reducing also the LMA CI (g: One-way ANOVA ; Tukey's multiple comparisons test. Baseline vs IVM: ***p<0.001, VEH vs IVM: *p=0.01). (h-m) Tb during the dark and presumptive dark period was reduced between 24-48 hr after IVM (h and k: RM Two-way ANOVA ; Tukey's multiple comparisons test p<0.05. Line above same as in B. I: Two-way ANOVA ; Tukey's multiple comparisons test. dark Baseline vs dark IVM: *p=0.01, dark VEH vs dark IVM: *p=0.024. l: Two-way ANOVA ; Tukey's multiple comparisons test. dark Baseline vs dark IVM: *p=0.01, dark VEH vs dark IVM: *p=0.014) resulting in a roughly 50% decrease in the CI of Tb during this same time period in LD and DD, although this only reached statistical significance in LD ( One-way ANOVA ; Tukey's multiple comparisons test. Baseline vs IVM: *p=0.015, VEH vs IVM: *p=0.024). FigS6CRHRabiesnew1k.tif Extended figure 6. Presumed monosynaptic inputs to the PVH CRH neurons based on conditional rabies virus tracing. (a) Schematic of the rabies infection of PVH CRH neurons to map their inputs. (b) Expression of both TVA (in red) and rabies EnvA (in green) marks doubly-transfected PVH CRH neurons (yellow) as “starter cells” to which neurons labeled only with green are presumed to project. (c) Representative micrographs of areas with retrogradely labeled neurons. (d) Total counts of the EnvA-rabies transfected neurons through the brain. The hypothalamus represents the most important source of inputs to the PVH CRH neurons. (e) Schematic of the EnvA-rabies experiment to map the monosynaptic input from the DMH Vgat neurons to PVH CRH neurons. (f) Mapping of the rabies- Vgat co-labeling distribution in the DMH at different rostro-caudal levels ( left panel), and representative images showing Vgat mRNA expression (in magenta, f’ and f’’) and rabies expression (in green; f’ and f’’’) within the DMH ( right panels). Neurons without Vgat mRNA (presumably glutamatergic) are shown by arrowheads and a doubly labeled cell indicated by the arrow is shown in a magnified inset at the lower right of each panel. The green signal from the Rabies infected cells was enhanced with immunofluorescence for EGFP. Reference scale bar: in c = 200µm, in b and f’-f’’’= 50µm, in f’-f’’’ insets = 10µm. 3V, third ventricle; AC, Anterior commissure; F, Fornix; OC, Optic Chiasm; SCP, Superior Cerebellar Peduncle; AHA, Anterior Hypothalamic Area; Arc, Arcuate Nucleus; BNST, Bed Nucleus of the Stria Terminalis; DMH, Dorsomedial Hypothalamus; LH, Lateral Hypothalamus; LPB, Lateral Parabrachial; LPO, Lateral Preoptic Area; MnPO, Median Preoptic Nucleus; MPA, Medial Preoptic Area; NTS, Nucleus of the Tractus Solitarius; PAG, Periaqueductal Gray Area; PE, Periventricular hypothalamic nucleus; POA, Preoptic Area; PVH, Paraventricular Hypothalamic nucleus ; RCh, Retrochiasmatic Nucleus; SCN, Suprachiasmatic Nucleus; SFO, Subfornical organ; SON, Supraoptic Nucleus; SPZ, Subparaventricular Zone; VLPO, Ventrolateral Preoptic Area; VMH, Ventromedial Hypothalamus; VMPO, Ventromedial Preoptic Area; VOLT, Vascular Organ of Lamina Terminalis; VP, Ventral Pallidum. Neuroanatomical regions and names were based on the Paxinos & Franklin Atlas 47 . FigS7VgatDMHDTAopt2.tif Extended figure 7. Ablation of DMH Vgat neurons dramatically reduces the total amount and circadian rhythm of LMA, but only reduces the daily level of Tb with little effect on its circadian rhythm. (a) Density plots of the distribution of injections of AAV-mCherry-DIO-DTA in the DMH of Vgat-ires-cre mice ( n =8). (b) Magnification of the representative micrograph showed in Fig 5b, showing few if any remaining Vgat-expressing neurons (green, native signal) within the area of the injection site (red, native signal). (c) The Cort levels were similar before and after the DMH Vgat ablation in the restraint stress protocol. (d-j) LMA was reduced during the dark phase after DMH Vgat neuron ablation in both LD ( RM Two-way ANOVA ; Šídák's multiple comparisons test. Pre-DTA vs DMH Vgat -DTA: *p<0.05) and DD ( RM Two-way ANOVA ; Šídák's multiple comparisons test. Pre-DTA vs DMH Vgat -DTA: *p<0.05), resulting in a reduced circadian index by 38.23 ±9.2% in LD (Paired t-test: t=3.252, df=14, ***p=0.005) and by 50.61 ±12.9% in DD ( Paired t-test : t=3.605, df=14, ***p=0.003) and similar reductions in cosinor amplitude in LD ( Paired t-test : t=5.138, df=14, ***p<0.001) and DD ( Paired t-test : t=5.503, df=14, ***p<0.001), with a reduction in peak amplitude but no change in tau in the periodogram (LD and DD: Two-way ANOVA ; Šídák's multiple comparisons test. SPZ Vgat -GFP vs SPZ Vgat -DTA: *p<0.05). (k) Representative LMA actograms before and after DMH Vgat neuron ablation (blue lines represents pre-ablation and red lines is post-ablation). (l) Tb was reduced by about 0.3 o C in LD, but (m) there was no change in the amplitude or period of the rhythm in periodogram analysis. (n) In DD, only the Tb during the presumptive dark period was reduced, but (o) this reduced the amplitude of the mean periodogram peak at 24h ( Two-way ANOVA ; Šídák's multiple comparisons test. SPZ Vgat -GFP vs SPZ Vgat -DTA: *p<0.05). (p) The reduction in mean Tb during the dark period is statistically significant in both LD and DD (LD: Two-way ANOVA ; Tukey's multiple comparisons. Dark Pre-DTA vs DMH Vgat -DTA: *p=0.032; DD: Dark Pre-DTA vs DMH Vgat -DTA: *p=0.012), but (q-r) there is no significant difference in the CI or cosinor amplitude of Tb. (s) Representative Tb actograms before and after DMH Vgat neuron ablation. FigS8VgatDMHFl.tif Extended figure 8. Vgat gene deletion in DMH neurons reduces the elevation of LMA and Tb during the middle of the dark and presumptive dark periods and the amplitude of their circadian rhythms. (a) Density plots of the injections of AAV-EGFP-iCre in the DMH of Vgat loxP/loxP mice ( n =7). (b-e) Vgat gene deletion in the DMH caused lower LMA during the middle of the dark phase in LD and presumptive dark phase in DD (LD: RM Two-way ANOVA ; Šídák's multiple comparisons test. DMH Vgat/flox -Control vs DMH Vgat/flox -EGFP-iCre: *p<0.05; DD: RM Two-way ANOVA ; Šídák's multiple comparisons test. DMH Vgat/flox -Control vs DMH Vgat/flox -EGFP-iCre: *p<0.05) with a reduction in the amplitude of in the mean periodogram peak at 24h (LD: Two-way ANOVA ; Šídák's multiple comparisons test. DMH Vgat/flox -Control vs DMH Vgat/flox -EGFP-iCre: *p<0.05; DD: Two-way ANOVA ; Šídák's multiple comparisons test. DMH Vgat/flox -Control vs DMH Vgat/flox -EGFP-iCre: *p<0.05). (f) Deletion of the Vgat gene in the DMH reduced movement during the dark or subjective dark period ( Two-way ANOVA ; Tukey's multiple comparisons test. LD DMH Vgat/flox -Control vs DMH Vgat/flox -EGFP-iCre: ***p<0.001; DD Dark DMH Vgat/flox -Control vs DMH Vgat/flox -EGFP-iCre: ***p<0.001). (g) The CI of LMA rhythm was reduced by 25.73 ±6.01% in LD ( Unpaired t-test t=4.275 df=12, **p=0.001) and 33.35 ± 12.63% in DD ( Unpaired t-test t=2.642 df=12, *p=0.021) and (h) the cosinor amplitude of LMA was similarly reduced in LD ( Unpaired t-test: t=5.194, df=12, ***p<0.001) and DD ( Unpaired t-test: t=5.232, df=12, ***p<0.001). (i) Representative LMA actograms from AAV-DIO-GFP and AAV-DIO-EGFP-iCre injected mice. (j-m) Tb is reduced during the middle of the dark phase in DMH Vgat gene-deleted mice in LD ( RM Two-way ANOVA ; Šídák's multiple comparisons test. DMH Vgat/flox -Control vs DMH Vgat/flox -EGFP-iCre: *p<0.05) and the presumptive dark phase in DD ( RM Two-way ANOVA ; Šídák's multiple comparisons test. DMH Vgat/flox -Control vs DMH Vgat/flox -EGFP-iCre: *p<0.05), with no change in the mean periodogram. (n) The reduction in mean Tb during the dark and presumptive dark phase in the DMH Vgat -gene deleted mice was statistically significant ( Two-way ANOVA ; Tukey’s multiple comparisons test. LD Dark DMH Vgat/flox -Control vs DMH Vgat/flox -EGFP-iCre: **p=0.001; DD Dark DMH Vgat/flox -Control vs DMH Vgat/flox -EGFP-iCre: ***p<0.001). (o) As a result, the CI of Tb was also reduced in the DMH Vgat -gene deleted mice by 20.84 ±8.8% in LD ( Unpaired t-test t=2.342 df=12, *p=0.037) and by 26.6 ±8.9% under constant dark ( Unpaired t-test t=2.958 df=12, *p=0.012), and (p) the cosinor amplitude was lower in DMH Vgat gene-deleted mice in LD ( Unpaired t-test: t=2.482, df=12, *p= 0.028) and DD ( Unpaired t-test: t=3.902, df=12, **p=0.002). (q) Representative Tb actograms from AAV-DIO-GFP and AAV-DIO-EGFP-iCre injected mice. FigS9VgatDMHhGlyR.tif Extended figure 9. Chemogenetic inhibition of DMH Vgat neurons flattens the circadian rhythm of LMA and Tb. (a) Density plots of the AAV-DIO-hGlyR-mCherry injection sites in the DMH of Vgat-ires-Cre mice ( n =5). (b-g) There was a reduction in the amount of LMA during the dark and presumptive dark periods between 24-48 hr after IVM injection (b and e: RM Two-way ANOVA ; Tukey's multiple comparisons test p<0.05. Line above the 24h graphs represent significative differences in Baseline vs IVM in green, and VEH vs IVM in blue; c: Two-way ANOVA ; Tukey's multiple comparisons test. dark Baseline vs dark IVM: ***p<0.001, dark VEH vs dark IVM: **p=0.001. F: Two-way ANOVA ; Tukey's multiple comparisons test. dark VEH vs dark IVM: *p=0.04), resulting in a dramatic reduction in CI during the same time period (d: One-way ANOVA ; Tukey's multiple comparisons test. Baseline vs IVM: ***p<0.001, VEH vs IVM: **p=0.001. g: One-way ANOVA ; Tukey's multiple comparisons test. Baseline vs IVM: ***p<0.001, VEH vs IVM: **p=0.002). (h-m) Reductions in mean Tb after IVM administration were observed during the dark and subjective dark periods (h and k: RM Two-way ANOVA ; Tukey's multiple comparisons test p<0.05. i: Two-way ANOVA ; Tukey's multiple comparisons test. dark Baseline vs dark IVM: *p=0.041. l: Two-way ANOVA ; Tukey's multiple comparisons test. dark Baseline vs dark IVM: *p=0.038), driving a reduction in CI in both LD and DD (j: One-way ANOVA ; Tukey's multiple comparisons test. Baseline vs IVM: ***p<0.001, VEH vs IVM: **p=0.005. m: One-way ANOVA ; Tukey's multiple comparisons test. Baseline vs IVM: ***p<0.001, VEH vs IVM: **p=0.004). FigS10VgatPVHDTA.tif Extended figure 10. cvPVH Vgat neuron ablation increases the Cort response to restraint stress, but does not affect the circadian rhythm of LMA or Tb. (a) Density plots showing the distribution of injections of AAV-mCherry-DIO-DTA in the cvPVH of Vgat-ires-Cre mice ( n =5). (b) After 1 hour of movement restraint, mice with cvPVH Vgat ablation had higher levels of Cort (Unpaired t test: t=2.782, df=8, p= 0.047). (c) The 24h LMA counts and (d) mean periodogram under LD, as well as under DD (e, f) did not differ from control mice. (g-i) As a result, no differences were detected in the circadian rhythms of LMA after cvPVH Vgat neuron ablation (j) Representative LMA actograms from both controls and mice with ablation of cvPVH Vgat neurons. (k-n) There were no significant changes in mean Tb levels or their periodograms under either LD or DD. (o-q) Likewise, the circadian rhythm of Tb was undisturbed by the cvPVH Vgat ablation. (r) Representative Tb actograms from Vgat-Cre animals with injections of AAV-DIO-GFP and AAV-mCherrry-DIO-DTA the cvPVH. LD, Light:Dark photoperiod; DD, Constant darkness. FigS11DMHVGATcaudalPVHCRH.tif Extended Figure 11. In vitro optogenetic stimulation of the GABAergic input from the caudal DMH inhibits PVH CRH neurons. (a) A schematic of the experiment demonstrating connectivity between the caudal DMH (cDMH) Vgat neurons and ipsilateral PVH CRH neurons (cDMH Vgat → PVH CRH ; the DMH is shown on the opposite side of the brain to ease illustration). Vgat-ires-Cre::CRH-Venus mice were injected with AAV-DIO-ChR2-mCherry in the cDMH, and recordings were conducted in brain slices from Venus-labeled PVH CRH neurons while photostimulating the cDMH Vgat input. (b) An example of ChR2-mCherry expression in the cDMH ( top left, native signal) and density plots of the AAV-DIO-ChR2-mCherry injection sites ( n = 4 mice; right and bottom ). (c) Opto-evoked inhibitory post-synaptic currents (oIPSCs) recorded in the PVH CRH neurons. (d) Percentages of PVH CRH neurons responding (Connected) and not responding (Not Connected) to photostimulation of the cDMH Vgat input ( n = 23 PVH CRH recorded neurons from 4 mice). (e) Amplitude ( left ; filled markers, cells responding to photostimulation, n =22, open markers, cells not responding to photostimulation, n =1 neurons, from 4 mice; mean and ± SEM of responding neurons) and latency ( right ) of oIPSCs in PVH CRH neurons in response to photostimulation of the cDMH Vgat input (mean and ± SEM; n =22 from 4 mice). (f) Raster plot of IPSCs in a representative PVH CRH neurons with photostimulation of the cDMH Vgat → PVH CRH input (bin duration: 50ms). (g) IPSC probability in response to photostimulation of the cDMH Vgat → PVH CRH input (black, n = 23). Reference scale bar: in (b) = 250 µm. f, fornix; 3V, third ventricle. Cite Share Download PDF Status: Posted Version 2 posted You are reading this latest preprint version Show more versions Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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SPZ\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eVgat\\u003c/em\\u003e\\u003c/sup\\u003e\\u003cem\\u003e neuron ablation flattens the circadian rhythm of LMA and reduces the amplitude of the rhythm of Tb. \\u003c/em\\u003e(\\u003cstrong\\u003ea\\u003c/strong\\u003e) Representative micrograph of GABAergic neurons (native signal in green) from a \\u003cem\\u003eVgat-ires-Cre::L10-GFP\\u003c/em\\u003e control mouse (\\u003cem\\u003eleft\\u003c/em\\u003e panel), and the density plots of the distribution injections of AAV-mCherry-DIO-DTA in the SPZ of the \\u003cem\\u003eVgat-ires-cre\\u003c/em\\u003e mice (\\u003cem\\u003en\\u003c/em\\u003e=8; \\u003cem\\u003eright\\u003c/em\\u003e panels). (\\u003cstrong\\u003eb\\u003c/strong\\u003e) Daily LMA was reduced during the dark period in LD after SPZ\\u003csup\\u003e\\u003cem\\u003eVgat\\u003c/em\\u003e\\u003c/sup\\u003e\\u003csup\\u003e \\u003c/sup\\u003eablation (\\u003cem\\u003eRepeated Measures [RM]\\u003c/em\\u003e \\u003cem\\u003eTwo-way ANOVA\\u003c/em\\u003e; \\u003cem\\u003eŠídák's\\u003c/em\\u003e multiple comparisons test. SPZ\\u003csup\\u003eVgat\\u003c/sup\\u003e-GFP vs SPZ\\u003csup\\u003eVgat\\u003c/sup\\u003e-DTA: *p\\u0026lt;0.05, ***p\\u0026lt;0.001), and (\\u003cstrong\\u003ec\\u003c/strong\\u003e) the periodogram showed reduced amplitude (\\u003cem\\u003eTwo-way ANOVA\\u003c/em\\u003e; \\u003cem\\u003eŠídák's\\u003c/em\\u003e multiple comparisons test. SPZ\\u003csup\\u003eVgat\\u003c/sup\\u003e-GFP vs SPZ\\u003csup\\u003eVgat\\u003c/sup\\u003e-DTA: *p\\u0026lt;0.05). (\\u003cstrong\\u003ed\\u003c/strong\\u003e) SPZ\\u003csup\\u003eVgat\\u003c/sup\\u003e neuron ablation further reduced the circadian rhythm of LMA in DD (RM \\u003cem\\u003eTwo-way ANOVA\\u003c/em\\u003e; \\u003cem\\u003eŠídák's\\u003c/em\\u003e multiple comparisons test. SPZ\\u003csup\\u003eVgat\\u003c/sup\\u003e-GFP vs SPZ\\u003csup\\u003eVgat\\u003c/sup\\u003e-DTA: *p\\u0026lt;0.05), (\\u003cstrong\\u003ee\\u003c/strong\\u003e) causing a dramatical reduction on the amplitude of the periodogram (\\u003cem\\u003eTwo-way ANOVA\\u003c/em\\u003e; \\u003cem\\u003eŠídák's\\u003c/em\\u003e multiple comparisons test. SPZ\\u003csup\\u003eVgat\\u003c/sup\\u003e-GFP vs SPZ\\u003csup\\u003eVgat\\u003c/sup\\u003e-DTA: *p\\u0026lt;0.05). (\\u003cstrong\\u003ef\\u003c/strong\\u003e) Bar graphs showing total LMA counts in the light and dark periods from the SPZ\\u003csup\\u003eVgat\\u003c/sup\\u003e-GFP and SPZ\\u003csup\\u003eVgat\\u003c/sup\\u003e-DTA mice. LMA was significantly reduced during the dark (\\u003cem\\u003eTwo-way ANOVA\\u003c/em\\u003e; \\u003cem\\u003eTukey's\\u003c/em\\u003e multiple comparisons: SPZ\\u003csup\\u003eVgat\\u003c/sup\\u003e-GFP dark vs SPZ\\u003csup\\u003eVgat\\u003c/sup\\u003e-DTA dark = ***p\\u0026lt;0.001) or subjective dark periods (\\u003cem\\u003eTwo-way ANOVA\\u003c/em\\u003e; \\u003cem\\u003eTukey's\\u003c/em\\u003e multiple comparisons: SPZ\\u003csup\\u003eVgat\\u003c/sup\\u003e-GFP subjective dark vs SPZ\\u003csup\\u003eVgat\\u003c/sup\\u003e-DTA subjective dark = ***p\\u0026lt;0.001). (\\u003cstrong\\u003eg\\u003c/strong\\u003e) The reduction of LMA during the dark or subjective dark period in SPZ\\u003csup\\u003eVgat\\u003c/sup\\u003e-ablated mice reduced circadian index (CI, for method of calculation of CI, see Statistical Analysis in the Materials and Methods) by 51.1 ±3.5% in LD (\\u003cem\\u003eUnpaired t-test\\u003c/em\\u003e: t=6.381,\\u0026nbsp;df=14, ***p\\u0026lt;0.001) and by 79.7 ±4.8% in DD (\\u003cem\\u003eUnpaired t-test\\u003c/em\\u003e: t=8.901,\\u0026nbsp;df=14, ***p\\u0026lt;0.001), (\\u003cstrong\\u003eh\\u003c/strong\\u003e) while the cosinor amplitude was also reduced in LD (\\u003cem\\u003eUnpaired t-test\\u003c/em\\u003e: t=6.142,\\u0026nbsp;df=14, ***p\\u0026lt;0.001) and DD (\\u003cem\\u003eUnpaired t-test\\u003c/em\\u003e: t=6.190,\\u0026nbsp;df=14, ***p\\u0026lt;0.001) (\\u003cstrong\\u003ei\\u003c/strong\\u003e) Representative LMA actograms, showing LD and DD recordings from SPZ\\u003csup\\u003eVgat\\u003c/sup\\u003e-GFP (blue) and SPZ\\u003csup\\u003eVgat\\u003c/sup\\u003e-DTA (red) mice. (\\u003cstrong\\u003ej-n\\u003c/strong\\u003e) SPZ\\u003csup\\u003eVgat\\u003c/sup\\u003e-ablated mice had a small increase in Tb during the light period and a small decrease during the dark period in both LD and DD, causing a significant change in the amplitude of the periodograms (LD: \\u003cem\\u003eTwo-way ANOVA\\u003c/em\\u003e; \\u003cem\\u003eŠídák's\\u003c/em\\u003e multiple comparisons test. SPZ\\u003csup\\u003eVgat\\u003c/sup\\u003e-GFP vs SPZ\\u003csup\\u003eVgat\\u003c/sup\\u003e-DTA: *p\\u0026lt;0.05; DD: \\u003cem\\u003eTwo-way ANOVA\\u003c/em\\u003e; \\u003cem\\u003eŠídák's\\u003c/em\\u003e multiple comparisons test. SPZ\\u003csup\\u003eVgat\\u003c/sup\\u003e-GFP vs SPZ\\u003csup\\u003eVgat\\u003c/sup\\u003e-DTA: *p\\u0026lt;0.05). However, (\\u003cstrong\\u003eo\\u003c/strong\\u003e) the CI was reduced by 50.4 ±5.6% in LD (\\u003cem\\u003eUnpaired t-test\\u003c/em\\u003e: t=6.551,\\u0026nbsp;df=14, ***p\\u0026lt;0.001) and by 58.3 ±6.5% in DD (\\u003cem\\u003eUnpaired t-test\\u003c/em\\u003e: t=5.228,\\u0026nbsp;df=14, ***p\\u0026lt;0.001), and (\\u003cstrong\\u003ep\\u003c/strong\\u003e) the cosinor amplitude of Tb was also reduced both in LD (Paired t-test: t=6.199,\\u0026nbsp;df=14, ***p\\u0026lt;0.001) and DD (\\u003cem\\u003eUnpaired t-test\\u003c/em\\u003e: t=4.885,\\u0026nbsp;df=14, ***p\\u0026lt;0.001). (\\u003cstrong\\u003eq\\u003c/strong\\u003e) Representative Tb actograms, red and blue lines as in panel \\u003cstrong\\u003ei\\u003c/strong\\u003e. LD, Light:Dark photoperiod; DD, Constant darkness.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"FigS1SPZDTA.tif\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4718850/v2/e2786ca66f8193a6f4ef298b.tif\"},{\"id\":84918280,\"identity\":\"828659c6-5ff5-4b1b-aefe-001b9062ab8e\",\"added_by\":\"auto\",\"created_at\":\"2025-06-18 19:22:56\",\"extension\":\"tif\",\"order_by\":2,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":139198855,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cem\\u003eExtended figure 2. Vgat gene deletion from SPZ neurons only partially reduces LMA and Tb during the dark period. \\u003c/em\\u003e(\\u003cstrong\\u003ea\\u003c/strong\\u003e) Representative micrograph of \\u003cem\\u003eVgat \\u003c/em\\u003emRNA expression (in red) from a control mouse (\\u003cem\\u003eleft\\u003c/em\\u003e panel) and an iCre-EGFP injected mouse (in green; \\u003cem\\u003eright\\u003c/em\\u003e panel). Notice the elimination of \\u003cem\\u003eVgat mRNA\\u003c/em\\u003e expression in the area where iCre-EGFP was expressed, whereas the \\u003cem\\u003eVgat \\u003c/em\\u003eexpression in the SCN was preserved. The EGFP signal was enhanced with immunofluorescence for EGFP. Density plots of the distribution of injections of AAV-EGFP-iCre in the SPZ of the \\u003cem\\u003eVgat\\u003c/em\\u003e\\u003csup\\u003e \\u003c/sup\\u003e\\u003csup\\u003e\\u003cem\\u003eloxP/loxP\\u003c/em\\u003e\\u003c/sup\\u003e\\u003csup\\u003e \\u003c/sup\\u003emice (\\u003cem\\u003en\\u003c/em\\u003e=7; \\u003cem\\u003eright\\u003c/em\\u003e panels). (\\u003cstrong\\u003eb\\u003c/strong\\u003e) Neither daily total LMA nor (\\u003cstrong\\u003ec\\u003c/strong\\u003e) the LMA periodogram was affected by the \\u003cem\\u003eVgat \\u003c/em\\u003edeletion in the SPZ when animals were in LD, but (\\u003cstrong\\u003ed\\u003c/strong\\u003e) LMA was reduced during the subjective dark period in DD (RM \\u003cem\\u003eTwo-way ANOVA\\u003c/em\\u003e; \\u003cem\\u003eŠídák's\\u003c/em\\u003e multiple comparisons test. SPZ\\u003csup\\u003eVgat/flox\\u003c/sup\\u003e-Control vs SPZ\\u003csup\\u003eVgat/flox\\u003c/sup\\u003e-EGFP-iCre: *p\\u0026lt;0.05), (\\u003cstrong\\u003ee\\u003c/strong\\u003e) with a reduction of the amplitude in the periodogram (\\u003cem\\u003eTwo-way ANOVA\\u003c/em\\u003e; \\u003cem\\u003eŠídák's\\u003c/em\\u003e multiple comparisons test. SPZ\\u003csup\\u003eVgat/flox\\u003c/sup\\u003e-Control vs SPZ\\u003csup\\u003eVgat/flox\\u003c/sup\\u003e-EGFP-iCre: *p\\u0026lt;0.05). (\\u003cstrong\\u003ef\\u003c/strong\\u003e) Total LMA counts in the light and dark periods from the SPZ\\u003csup\\u003eVgat/flox\\u003c/sup\\u003e-Control and SPZ\\u003csup\\u003eVgat/flox\\u003c/sup\\u003e-EGFP-iCre mice. \\u003cem\\u003eVgat\\u003c/em\\u003e ablation from SPZ neurons reduced the LMA during the subjective dark period under DD (\\u003cem\\u003eTwo-way ANOVA\\u003c/em\\u003e; \\u003cem\\u003eTukey's\\u003c/em\\u003e multiple comparisons: SPZ\\u003csup\\u003eVgat/flox\\u003c/sup\\u003e-Control subjective dark vs SPZ\\u003csup\\u003eVgat/flox\\u003c/sup\\u003e- EGFP-iCre subjective dark = **p=0.004), (\\u003cstrong\\u003eg\\u003c/strong\\u003e) resulting in a reduced circadian index (CI) by 39.7 ±4.8% in DD (\\u003cem\\u003eUnpaired t-test\\u003c/em\\u003e: t=4.674,\\u0026nbsp;df=10, ***p\\u0026lt;0.001), (\\u003cstrong\\u003eh\\u003c/strong\\u003e) and a similar reduction the cosinor amplitude in DD (\\u003cem\\u003eUnpaired t-test\\u003c/em\\u003e: t=9.399,\\u0026nbsp;df=12, ***p\\u0026lt;0.001) (\\u003cstrong\\u003ei\\u003c/strong\\u003e) Representative LMA actograms, showing LD and DD recordings from SPZ\\u003csup\\u003eVgat/flox\\u003c/sup\\u003e-Control (blue) and SPZ\\u003csup\\u003eVgat/flox\\u003c/sup\\u003e-EGFP-iCre (red). (\\u003cstrong\\u003ej\\u003c/strong\\u003e) The Vgat deletion from SPZ slightly reduced the Tb toward the end of the dark period in LD \\u003cem\\u003e(RM\\u003c/em\\u003e \\u003cem\\u003eTwo-way ANOVA\\u003c/em\\u003e; Šídák's multiple comparisons test. SPZ\\u003csup\\u003eVgat/flox\\u003c/sup\\u003e-Control vs SPZ\\u003csup\\u003eVgat/flox\\u003c/sup\\u003e-EGFP-iCre: *p\\u0026lt;0.05), (\\u003cstrong\\u003ek\\u003c/strong\\u003e) reducing the amplitude in the periodogram (\\u003cem\\u003eTwo-way ANOVA\\u003c/em\\u003e; \\u003cem\\u003eŠídák's\\u003c/em\\u003e multiple comparisons test. SPZ\\u003csup\\u003eVgat/flox\\u003c/sup\\u003e-Control vs SPZ\\u003csup\\u003eVgat/flox\\u003c/sup\\u003e- EGFP-iCre: *p\\u0026lt;0.05). (\\u003cstrong\\u003el\\u003c/strong\\u003e) Similar reduction was observed during the subjective dark period in DD in the SPZ\\u003csup\\u003eVgat/flox\\u003c/sup\\u003e-EGFP-iCre mice \\u003cem\\u003e(RM\\u003c/em\\u003e \\u003cem\\u003eTwo-way ANOVA\\u003c/em\\u003e; Šídák's multiple comparisons test. SPZ\\u003csup\\u003eVgat/flox\\u003c/sup\\u003e-Control vs SPZ\\u003csup\\u003eVgat/flox\\u003c/sup\\u003e-EGFP-iCre: *p\\u0026lt;0.05) (\\u003cstrong\\u003em\\u003c/strong\\u003e) and in the periodogram (\\u003cem\\u003eTwo-way ANOVA\\u003c/em\\u003e; \\u003cem\\u003eŠídák's\\u003c/em\\u003e multiple comparisons test. SPZ\\u003csup\\u003eVgat/flox\\u003c/sup\\u003e-Control vs SPZ\\u003csup\\u003eVgat/flox\\u003c/sup\\u003e-EGFP-iCre: *p\\u0026lt;0.05). (\\u003cstrong\\u003en\\u003c/strong\\u003e) The mean Tb was significantly reduced during the dark or subjective dark period in the SPZ\\u003csup\\u003eVgat/flox\\u003c/sup\\u003e-EGFP-iCre mice either in LD (\\u003cem\\u003eTwo-way ANOVA\\u003c/em\\u003e; \\u003cem\\u003eTukey's\\u003c/em\\u003e multiple comparisons: SPZ\\u003csup\\u003eVgat/flox\\u003c/sup\\u003e-Control vs SPZ\\u003csup\\u003eVgat/flox\\u003c/sup\\u003e-EGFP-iCre dark = *p=0.021) or DD, (\\u003cem\\u003eTwo-way ANOVA\\u003c/em\\u003e; \\u003cem\\u003eTukey's\\u003c/em\\u003e multiple comparisons: SPZ\\u003csup\\u003eVgat/flox\\u003c/sup\\u003e-Control subjective dark vs SPZ\\u003csup\\u003eVgat/flox\\u003c/sup\\u003e-EGFP-iCre subjective dark = ***p\\u0026lt;0.001). (\\u003cstrong\\u003eo\\u003c/strong\\u003e) The CI of Tb was reduced by 25.7 ±4.6% in LD (\\u003cem\\u003eUnpaired t-test:\\u003c/em\\u003e t=3.300,\\u0026nbsp;df=12, ***p=0.006) and by 38.6 ±7.3% in DD (\\u003cem\\u003eUnpaired t-test\\u003c/em\\u003e: t=4.689,\\u0026nbsp;df=12, ***p\\u0026lt;0.001), (\\u003cstrong\\u003ep\\u003c/strong\\u003e) similar to the cosinor amplitude in LD (\\u003cem\\u003eUnpaired t-test\\u003c/em\\u003e: t=2.864,\\u0026nbsp;df=12, **p=0.014) and DD (\\u003cem\\u003eUnpaired t-test\\u003c/em\\u003e: t=5.318,\\u0026nbsp;df=12, ***p\\u0026lt;0.001). (\\u003cstrong\\u003eq\\u003c/strong\\u003e) Representative Tb actograms, red and blue lines as in panel \\u003cstrong\\u003ei\\u003c/strong\\u003e.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"FigS2SPZFlFl.tif\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4718850/v2/28406d8688bdf718e4d42dc6.tif\"},{\"id\":84918274,\"identity\":\"ff4e277d-78d5-4756-ba7d-d65764ff47ea\",\"added_by\":\"auto\",\"created_at\":\"2025-06-18 19:22:54\",\"extension\":\"tif\",\"order_by\":3,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":139198855,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cem\\u003eExtended figure 3. DMH\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eVglut2\\u003c/em\\u003e\\u003c/sup\\u003e\\u003cem\\u003e neuron ablation reduces LMA during the subjective night and decreases Tb across the day. \\u003c/em\\u003e(\\u003cstrong\\u003ea\\u003c/strong\\u003e) \\u0026nbsp;Density plots of the distribution of injections of AAV-mCherry-DIO-DTA in the DMH of the \\u003cem\\u003eVglut2-ires-cre\\u003c/em\\u003e mice (\\u003cem\\u003en\\u003c/em\\u003e=8). (\\u003cstrong\\u003eb\\u003c/strong\\u003e) No statistically significant change was detected in the Cort levels after the restraint stress protocol. (\\u003cstrong\\u003ec\\u003c/strong\\u003e) Daily LMA was reduced during the dark period in LD after DMH\\u003csup\\u003eVglut2\\u003c/sup\\u003e neuron ablation (RM \\u003cem\\u003eTwo-way ANOVA\\u003c/em\\u003e; \\u003cem\\u003eŠídák's\\u003c/em\\u003e multiple comparisons test. Pre-DTA vs DMH\\u003csup\\u003eVglut2\\u003c/sup\\u003e-DTA: *p\\u0026lt;0.05), but (\\u003cstrong\\u003ed\\u003c/strong\\u003e) the periodogram showed no change. (\\u003cstrong\\u003ee\\u003c/strong\\u003e) DMH\\u003csup\\u003eVglut2 \\u003c/sup\\u003eneuron ablation reduced LMA during the subjective dark in DD (RM \\u003cem\\u003eTwo-way ANOVA\\u003c/em\\u003e; \\u003cem\\u003eŠídák's\\u003c/em\\u003e multiple comparisons test. Pre-DTA vs DMH\\u003csup\\u003eVglut2\\u003c/sup\\u003e-DTA: *p\\u0026lt;0.05), (\\u003cstrong\\u003ef\\u003c/strong\\u003e) reducing the amplitude of the periodogram (\\u003cem\\u003eTwo-way ANOVA\\u003c/em\\u003e; \\u003cem\\u003eŠídák's\\u003c/em\\u003e multiple comparisons test. SPZ\\u003csup\\u003eVgat\\u003c/sup\\u003e-GFP vs SPZ\\u003csup\\u003eVgat\\u003c/sup\\u003e-DTA: *p\\u0026lt;0.05). (\\u003cstrong\\u003eg\\u003c/strong\\u003e) Bar graphs showing total LMA counts in the light and dark periods before and after ablation of DMH\\u003csup\\u003eVglut2\\u003c/sup\\u003e neurons, during LD (left) and presumptive light and dark periods in DD (right). (\\u003cstrong\\u003eh\\u003c/strong\\u003e) The reduction of LMA during the dark period after DTA was sufficiently small that the circadian index (CI) was not significantly different. (\\u003cstrong\\u003ei\\u003c/strong\\u003e) However, the amplitude of the circadian rhythm of LMA as measured by cosinor analysis was reduced in DD after ablation (\\u003cem\\u003ePaired t-test\\u003c/em\\u003e: t=3.611, df=14, **p=0.002). (\\u003cstrong\\u003ej\\u003c/strong\\u003e) Representative LMA actograms, showing LD and DD recordings before (blue) and after (red) DTA. (k) Tb was reduced across the light phase and in the middle of the dark phase after DMH\\u003csup\\u003eVglut2 \\u003c/sup\\u003eneuron ablation in LD (\\u003cem\\u003eRepeated Measures [RM]\\u003c/em\\u003e \\u003cem\\u003eTwo-way ANOVA\\u003c/em\\u003e; Šídák's multiple comparisons test. Pre-DTA vs DMH\\u003csup\\u003eVglut2\\u003c/sup\\u003e-DTA: *p\\u0026lt;0.05), while (\\u003cstrong\\u003el\\u003c/strong\\u003e) the periodogram showed no change. (\\u003cstrong\\u003em\\u003c/strong\\u003e) The reduction in the daily Tb was larger in both the presumptive light and dark periods in DD after DMH\\u003csup\\u003eVglut2 \\u003c/sup\\u003eneuron ablation (RM \\u003cem\\u003eTwo-way ANOVA\\u003c/em\\u003e; \\u003cem\\u003eŠídák’s\\u003c/em\\u003e multiple comparisons test. Pre-DTA vs DMH\\u003csup\\u003eVglut2\\u003c/sup\\u003e-DTA: *p\\u0026lt;0.05), (\\u003cstrong\\u003en\\u003c/strong\\u003e) with no change in the periodogram. (\\u003cstrong\\u003eo\\u003c/strong\\u003e) The mean Tb was significantly reduced by DMH\\u003csup\\u003eVglut2\\u003c/sup\\u003e ablation for the light but not the dark period in LD, and for both in DD (\\u003cem\\u003eTwo-way ANOVA\\u003c/em\\u003e; \\u003cem\\u003eTukey's\\u003c/em\\u003e multiple comparisons. ** p\\u0026lt;0.01, ***p\\u0026lt;0.001). However, (\\u003cstrong\\u003ep\\u003c/strong\\u003e) the CI and (\\u003cstrong\\u003eq\\u003c/strong\\u003e) the cosinor amplitude of Tb was increased after ablation (by 46.65 ±12.8%) only during LD (\\u003cem\\u003ePaired\\u003c/em\\u003e \\u003cem\\u003et-test for CI\\u003c/em\\u003e: t=3.075, df=14, df=14, *p=0.008; \\u003cem\\u003efor cosinor amplitude\\u003c/em\\u003e: t=2.415, df=14, *p=0.03). (\\u003cstrong\\u003er\\u003c/strong\\u003e) Representative Tb actograms, red and blue lines as in panel \\u003cstrong\\u003ej\\u003c/strong\\u003e. LD, Light:Dark photoperiod; DD, Constant darkness.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"FigS3VglutDMHDTA.tif\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4718850/v2/1bfa55338315a7a098e1fd4b.tif\"},{\"id\":84918276,\"identity\":\"5e049d3f-1e35-4bcd-9054-d2735b774dab\",\"added_by\":\"auto\",\"created_at\":\"2025-06-18 19:22:54\",\"extension\":\"tif\",\"order_by\":4,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":139198855,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cem\\u003eExtended figure 4. Vglut2 gene deletion from DMH neurons reduces the peak of LMA and Tb during the transition from the dark to the light period. \\u003c/em\\u003e(\\u003cstrong\\u003ea\\u003c/strong\\u003e) Density plots of the injections of AAV-EGFP-iCre in the DMH of Vglut2\\u003csup\\u003eloxP/loxP \\u003c/sup\\u003emice (\\u003cem\\u003en\\u003c/em\\u003e=7). (\\u003cstrong\\u003eb\\u003c/strong\\u003e) Daily distribution of LMA in LD (\\u003cem\\u003eRM\\u003c/em\\u003e \\u003cem\\u003eTwo-way ANOVA\\u003c/em\\u003e; Šídák's multiple comparisons test. DMH\\u003csup\\u003eVglut2/flox\\u003c/sup\\u003e-Control vs DMH\\u003csup\\u003eVglut2/flox\\u003c/sup\\u003e-EGFP-iCre: *p\\u0026lt;0.05). \\u003cem\\u003eVglut2 \\u003c/em\\u003egene deletion from DMH neurons reduced LMA during the transition from the dark to the light period, and to a lesser extend in the early dark period, (\\u003cstrong\\u003ec\\u003c/strong\\u003e) reducing the amplitude of the periodogram peak at 24h (\\u003cem\\u003eTwo-way ANOVA\\u003c/em\\u003e; \\u003cem\\u003eŠídák's\\u003c/em\\u003e multiple comparisons test. DMH\\u003csup\\u003eVglut2/flox\\u003c/sup\\u003e-Control vs DMH\\u003csup\\u003eVglut2/flox\\u003c/sup\\u003e-EGFP-iCre: *p\\u0026lt;0.05). (\\u003cstrong\\u003ed\\u003c/strong\\u003e) In DD, the reduction in LMA during the transitions between the presumptive light and dark periods was similar to LD (RM \\u003cem\\u003eTwo-way ANOVA\\u003c/em\\u003e; \\u003cem\\u003eŠídák's\\u003c/em\\u003e multiple comparisons test. DMH\\u003csup\\u003eVglut2/flox\\u003c/sup\\u003e-Control vs DMH\\u003csup\\u003eVglut2/flox\\u003c/sup\\u003e-EGFP-iCre: *p\\u0026lt;0.05), (\\u003cstrong\\u003ee\\u003c/strong\\u003e) with similar reduction in the amplitude of the periodogram (\\u003cem\\u003eTwo-way ANOVA\\u003c/em\\u003e; \\u003cem\\u003eŠídák's\\u003c/em\\u003e multiple comparisons test. DMH\\u003csup\\u003eVglut2/flox\\u003c/sup\\u003e-Control vs DMH\\u003csup\\u003eVglut2/flox\\u003c/sup\\u003e-EGFP-iCre: *p\\u0026lt;0.05). (\\u003cstrong\\u003ef\\u003c/strong\\u003e) However, for the entire light and dark periods in LD and presumptive light and dark periods in DD, the small changes in LMA did not reach statistical significance for total LMA counts, (\\u003cstrong\\u003eg\\u003c/strong\\u003e) CI or (\\u003cstrong\\u003eh\\u003c/strong\\u003e) cosinor amplitude. (\\u003cstrong\\u003ei\\u003c/strong\\u003e) Representative LMA actograms from the Control and DMH \\u003cem\\u003eVglut2 \\u003c/em\\u003egene\\u003cem\\u003e-\\u003c/em\\u003edeleted mice. (\\u003cstrong\\u003ej\\u003c/strong\\u003e) Tb was also reduced in LD during the transition from the dark to the light phase and to a lesser extent in the early dark phase in DMH \\u003cem\\u003eVglut2 \\u003c/em\\u003egene-deleted mice (RM \\u003cem\\u003eTwo-way ANOVA\\u003c/em\\u003e; \\u003cem\\u003eŠídák's\\u003c/em\\u003e multiple comparisons test. DMH\\u003csup\\u003eVglut2/flox\\u003c/sup\\u003e-Control vs DMH\\u003csup\\u003eVglut2/flox\\u003c/sup\\u003e-EGFP-iCre: *p\\u0026lt;0.05), (\\u003cstrong\\u003ek\\u003c/strong\\u003e) reducing the amplitude in the periodogram around the 24 period (\\u003cem\\u003eTwo-way ANOVA\\u003c/em\\u003e; \\u003cem\\u003eŠídák's\\u003c/em\\u003e multiple comparisons test. DMH\\u003csup\\u003eVglut2/flox\\u003c/sup\\u003e-Control vs DMH\\u003csup\\u003eVglut2/flox\\u003c/sup\\u003e-EGFP-iCre: *p\\u0026lt;0.05). (\\u003cstrong\\u003el\\u003c/strong\\u003e) A similar pattern in Tb was seen during DD (RM \\u003cem\\u003eTwo-way ANOVA\\u003c/em\\u003e; \\u003cem\\u003eŠídák’s\\u003c/em\\u003e multiple comparisons test. DMH\\u003csup\\u003eVglut2/flox\\u003c/sup\\u003e-Control vs DMH\\u003csup\\u003eVglut2/flox\\u003c/sup\\u003e-EGFP-iCre: *p\\u0026lt;0.05), (\\u003cstrong\\u003em\\u003c/strong\\u003e) with similar reduction in the amplitude of the periodogram (\\u003cem\\u003eTwo-way ANOVA\\u003c/em\\u003e; \\u003cem\\u003eŠídák's\\u003c/em\\u003e multiple comparisons test. DMH\\u003csup\\u003eVglut2/flox\\u003c/sup\\u003e-Control vs DMH\\u003csup\\u003eVglut2/flox\\u003c/sup\\u003e-EGFP-iCre: *p\\u0026lt;0.05). (\\u003cstrong\\u003en\\u003c/strong\\u003e) However, as for LMA, these changes were not large enough to produce a statistically significant difference in mean Tb, (\\u003cstrong\\u003eo\\u003c/strong\\u003e) CI or (\\u003cstrong\\u003ep\\u003c/strong\\u003e) cosinor amplitude of the circadian rhythm of Tb. (\\u003cstrong\\u003eq\\u003c/strong\\u003e) Tb actograms from the DMH \\u003cem\\u003eVglut2 \\u003c/em\\u003egene-deleted mice included in this study. LD, Light:Dark photoperiod; DD, Constant darkness.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"FigS4VglutDMHFl.tif\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4718850/v2/270561d6b788cdb5b6695dbf.tif\"},{\"id\":84919069,\"identity\":\"590a1f78-cd7d-4e4c-87cd-94991a9938b3\",\"added_by\":\"auto\",\"created_at\":\"2025-06-18 19:30:54\",\"extension\":\"tif\",\"order_by\":5,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":139198855,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cem\\u003eExtended figure 5. Chemogenetic inhibition of the DMH\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eVglut2\\u003c/em\\u003e\\u003c/sup\\u003e\\u003cem\\u003e neurons decreases the circadian index of LMA and reduces Tb. \\u003c/em\\u003e(\\u003cstrong\\u003ea\\u003c/strong\\u003e) Density plots of the distribution of injections of AAV-DIO-hGlyR-mCherry in the DMH of the \\u003cem\\u003eVglut2-ires-Cre\\u003c/em\\u003e mice (\\u003cem\\u003en\\u003c/em\\u003e=5). (\\u003cstrong\\u003eb-d\\u003c/strong\\u003e) Chemo-inhibitions with IVM in LD induced higher LMA at the transition from the dark to the light phase and reduced the peak during the first hours of the dark phase (\\u003cstrong\\u003eb\\u003c/strong\\u003e: \\u003cem\\u003eRM Two-way ANOVA\\u003c/em\\u003e; \\u003cem\\u003eTukey's\\u003c/em\\u003e multiple comparisons test p\\u0026lt;0.05. Line above the 24h graphs represent significative differences in Baseline vs IVM in green, and VEH vs IVM in blue; \\u003cstrong\\u003ec\\u003c/strong\\u003e: \\u003cem\\u003eTwo-way ANOVA\\u003c/em\\u003e; \\u003cem\\u003eTukey's\\u003c/em\\u003e multiple comparisons test. light Baseline vs light IVM: **p=0.001, light VEH vs light IVM: *p=0.042, dark Baseline vs dark IVM: *p=0.03), reducing the CI (\\u003cstrong\\u003ed\\u003c/strong\\u003e: \\u003cem\\u003eOne-way ANOVA\\u003c/em\\u003e; \\u003cem\\u003eTukey's\\u003c/em\\u003e multiple comparisons test. Baseline vs IVM: **p=0.001, VEH vs IVM: *p=0.016). (\\u003cstrong\\u003ee-g\\u003c/strong\\u003e) In DD, the daily increase in LMA at the beginning of the dark period was reduced (\\u003cstrong\\u003ee\\u003c/strong\\u003e: \\u003cem\\u003eRM Two-way ANOVA\\u003c/em\\u003e; \\u003cem\\u003eTukey's\\u003c/em\\u003e multiple comparisons test p\\u0026lt;0.05. Lines above same as in B. \\u003cstrong\\u003ef\\u003c/strong\\u003e: \\u003cem\\u003eTwo-way ANOVA\\u003c/em\\u003e; \\u003cem\\u003eTukey's\\u003c/em\\u003e multiple comparisons test. dark Baseline vs dark IVM: *p=0.036), reducing also the LMA CI (\\u003cstrong\\u003eg\\u003c/strong\\u003e: \\u003cem\\u003eOne-way ANOVA\\u003c/em\\u003e; \\u003cem\\u003eTukey's\\u003c/em\\u003e multiple comparisons test. Baseline vs IVM: ***p\\u0026lt;0.001, VEH vs IVM: *p=0.01). (\\u003cstrong\\u003eh-m\\u003c/strong\\u003e) Tb during the dark and presumptive dark period was reduced between 24-48 hr after IVM (\\u003cstrong\\u003eh\\u003c/strong\\u003e and \\u003cstrong\\u003ek\\u003c/strong\\u003e: \\u003cem\\u003eRM Two-way ANOVA\\u003c/em\\u003e; \\u003cem\\u003eTukey's\\u003c/em\\u003e multiple comparisons test p\\u0026lt;0.05. Line above same as in B. I: \\u003cem\\u003eTwo-way ANOVA\\u003c/em\\u003e; \\u003cem\\u003eTukey's\\u003c/em\\u003e multiple comparisons test. dark Baseline vs dark IVM: *p=0.01, dark VEH vs dark IVM: *p=0.024. \\u003cstrong\\u003el\\u003c/strong\\u003e: \\u003cem\\u003eTwo-way ANOVA\\u003c/em\\u003e; \\u003cem\\u003eTukey's\\u003c/em\\u003e multiple comparisons test. dark Baseline vs dark IVM: *p=0.01, dark VEH vs dark IVM: *p=0.014) resulting in a roughly 50% decrease in the CI of Tb during this same time period in LD and DD, although this only reached statistical significance in LD (\\u003cem\\u003eOne-way ANOVA\\u003c/em\\u003e; \\u003cem\\u003eTukey's\\u003c/em\\u003e multiple comparisons test. Baseline vs IVM: *p=0.015, VEH vs IVM: *p=0.024).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"FigS5VglutDMHhGlyR.tif\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4718850/v2/a034d1a7fbea4caf1594d39b.tif\"},{\"id\":84918281,\"identity\":\"55ed9cae-7ef6-474d-a9f6-2bc4559ebbac\",\"added_by\":\"auto\",\"created_at\":\"2025-06-18 19:22:57\",\"extension\":\"tif\",\"order_by\":6,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":556791615,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cem\\u003eExtended figure 6. Presumed monosynaptic inputs to the PVH\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eCRH\\u003c/em\\u003e\\u003c/sup\\u003e\\u003cem\\u003e neurons based on conditional rabies virus tracing. \\u003c/em\\u003e(\\u003cstrong\\u003ea\\u003c/strong\\u003e) Schematic of the rabies infection of PVH\\u003csup\\u003eCRH \\u003c/sup\\u003eneurons to map their inputs. (\\u003cstrong\\u003eb\\u003c/strong\\u003e) Expression of both TVA (in red) and rabies EnvA (in green) marks doubly-transfected PVH\\u003csup\\u003eCRH\\u003c/sup\\u003e neurons (yellow) as “starter cells” to which neurons labeled only with green are presumed to project. (\\u003cstrong\\u003ec\\u003c/strong\\u003e) Representative micrographs of areas with retrogradely labeled neurons. (\\u003cstrong\\u003ed\\u003c/strong\\u003e) Total counts of the EnvA-rabies transfected neurons through the brain. The hypothalamus represents the most important source of inputs to the PVH\\u003csup\\u003eCRH\\u003c/sup\\u003e neurons. (\\u003cstrong\\u003ee\\u003c/strong\\u003e) Schematic of the EnvA-rabies experiment to map the monosynaptic input from the DMH\\u003csup\\u003eVgat\\u003c/sup\\u003e neurons to PVH\\u003csup\\u003eCRH \\u003c/sup\\u003eneurons. (\\u003cstrong\\u003ef\\u003c/strong\\u003e) Mapping of the rabies-\\u003cem\\u003eVgat\\u003c/em\\u003e co-labeling distribution in the DMH at different rostro-caudal levels (\\u003cem\\u003eleft\\u003c/em\\u003e panel), and representative images showing \\u003cem\\u003eVgat mRNA\\u003c/em\\u003e expression (in magenta, f’ and f’’) and rabies expression (in green; f’ and f’’’) within the DMH (\\u003cem\\u003eright\\u003c/em\\u003e panels). Neurons without Vgat mRNA (presumably glutamatergic) are shown by arrowheads and a doubly labeled cell indicated by the arrow is shown in a magnified inset at the lower right of each panel. The green signal from the Rabies infected cells was enhanced with immunofluorescence for EGFP. Reference scale bar: in \\u003cstrong\\u003ec\\u003c/strong\\u003e = 200µm, in \\u003cstrong\\u003eb and f’-f’’’\\u003c/strong\\u003e= 50µm, in \\u003cstrong\\u003ef’-f’’’\\u003c/strong\\u003e insets = 10µm. 3V, third ventricle; AC, Anterior commissure; F, Fornix; OC, Optic Chiasm; SCP, Superior Cerebellar Peduncle; AHA, Anterior Hypothalamic Area; Arc, Arcuate Nucleus; BNST, Bed Nucleus of the Stria Terminalis; DMH, Dorsomedial Hypothalamus; LH, Lateral Hypothalamus; LPB, Lateral Parabrachial; LPO, Lateral Preoptic Area; MnPO, Median Preoptic Nucleus; MPA, Medial Preoptic Area; NTS, Nucleus of the Tractus Solitarius; PAG, Periaqueductal Gray Area; PE, Periventricular hypothalamic nucleus; POA, Preoptic Area; PVH, Paraventricular Hypothalamic nucleus ; RCh, Retrochiasmatic Nucleus; SCN, Suprachiasmatic Nucleus; SFO, Subfornical organ; SON, Supraoptic Nucleus; SPZ, Subparaventricular Zone; VLPO, Ventrolateral Preoptic Area; VMH, Ventromedial Hypothalamus; VMPO, Ventromedial Preoptic Area; VOLT, Vascular Organ of Lamina Terminalis; VP, Ventral Pallidum. Neuroanatomical regions and names were based on the Paxinos \\u0026amp; Franklin Atlas \\u003csup\\u003e47\\u003c/sup\\u003e.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"FigS6CRHRabiesnew1k.tif\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4718850/v2/1b36ea4b8d058e0a469b3300.tif\"},{\"id\":84918283,\"identity\":\"194d45c5-4190-41fe-ad9c-fd19d256b218\",\"added_by\":\"auto\",\"created_at\":\"2025-06-18 19:22:58\",\"extension\":\"tif\",\"order_by\":7,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":139198855,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cem\\u003eExtended figure 7. \\u003c/em\\u003eAblation of \\u003cem\\u003eDMH\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eVgat \\u003c/em\\u003e\\u003c/sup\\u003e\\u003cem\\u003eneurons dramatically reduces the total amount and circadian rhythm of LMA, but only reduces the daily level of Tb with little effect on its circadian rhythm. \\u003c/em\\u003e(\\u003cstrong\\u003ea\\u003c/strong\\u003e) Density plots of the distribution of injections of AAV-mCherry-DIO-DTA in the DMH of Vgat-ires-cre mice (\\u003cem\\u003en\\u003c/em\\u003e=8). (\\u003cstrong\\u003eb\\u003c/strong\\u003e) Magnification of the representative micrograph showed in \\u003cstrong\\u003eFig 5b\\u003c/strong\\u003e, showing few if any remaining Vgat-expressing neurons (green, native signal) within the area of the injection site (red, native signal). (\\u003cstrong\\u003ec\\u003c/strong\\u003e) The Cort levels were similar before and after the DMH\\u003csup\\u003eVgat\\u003c/sup\\u003e ablation in the restraint stress protocol. (\\u003cstrong\\u003ed-j\\u003c/strong\\u003e) LMA was reduced during the dark phase after DMH\\u003csup\\u003eVgat\\u003c/sup\\u003e neuron ablation in both LD (\\u003cem\\u003eRM\\u003c/em\\u003e \\u003cem\\u003eTwo-way ANOVA\\u003c/em\\u003e; \\u003cem\\u003eŠídák's\\u003c/em\\u003e multiple comparisons test. Pre-DTA vs DMH\\u003csup\\u003eVgat\\u003c/sup\\u003e-DTA: *p\\u0026lt;0.05) and DD (\\u003cem\\u003eRM Two-way ANOVA\\u003c/em\\u003e; \\u003cem\\u003eŠídák's\\u003c/em\\u003e multiple comparisons test. Pre-DTA vs DMH\\u003csup\\u003eVgat\\u003c/sup\\u003e-DTA: *p\\u0026lt;0.05), resulting in a reduced circadian index by 38.23 ±9.2% in LD (Paired t-test: t=3.252, df=14, ***p=0.005) and by 50.61 ±12.9% in DD (\\u003cem\\u003ePaired t-test\\u003c/em\\u003e: t=3.605, df=14, ***p=0.003) and similar reductions in cosinor amplitude in LD (\\u003cem\\u003ePaired\\u003c/em\\u003e \\u003cem\\u003et-test\\u003c/em\\u003e: t=5.138, df=14, ***p\\u0026lt;0.001) and DD (\\u003cem\\u003ePaired t-test\\u003c/em\\u003e: t=5.503, df=14, ***p\\u0026lt;0.001), with a reduction in peak amplitude but no change in tau in the periodogram (LD and DD: \\u003cem\\u003eTwo-way ANOVA\\u003c/em\\u003e; \\u003cem\\u003eŠídák's\\u003c/em\\u003e multiple comparisons test. SPZ\\u003csup\\u003eVgat\\u003c/sup\\u003e-GFP vs SPZ\\u003csup\\u003eVgat\\u003c/sup\\u003e-DTA: *p\\u0026lt;0.05). (\\u003cstrong\\u003ek\\u003c/strong\\u003e) Representative LMA actograms before and after DMH\\u003csup\\u003eVgat \\u003c/sup\\u003eneuron ablation (blue lines represents pre-ablation and red lines is post-ablation). (\\u003cstrong\\u003el\\u003c/strong\\u003e) Tb was reduced by about 0.3\\u003csup\\u003eo\\u003c/sup\\u003e C in LD, but (\\u003cstrong\\u003em\\u003c/strong\\u003e) there was no change in the amplitude or period of the rhythm in periodogram analysis. (\\u003cstrong\\u003en\\u003c/strong\\u003e) In DD, only the Tb during the presumptive dark period was reduced, but (\\u003cstrong\\u003eo\\u003c/strong\\u003e) this reduced the amplitude of the mean periodogram peak at 24h (\\u003cem\\u003eTwo-way ANOVA\\u003c/em\\u003e; \\u003cem\\u003eŠídák's\\u003c/em\\u003e multiple comparisons test. SPZ\\u003csup\\u003eVgat\\u003c/sup\\u003e-GFP vs SPZ\\u003csup\\u003eVgat\\u003c/sup\\u003e-DTA: *p\\u0026lt;0.05). (\\u003cstrong\\u003ep\\u003c/strong\\u003e) The reduction in mean Tb during the dark period is statistically significant in both LD and DD (LD: \\u003cem\\u003eTwo-way ANOVA\\u003c/em\\u003e; \\u003cem\\u003eTukey's\\u003c/em\\u003e multiple comparisons. Dark Pre-DTA vs DMH\\u003csup\\u003eVgat\\u003c/sup\\u003e-DTA: *p=0.032; DD: Dark Pre-DTA vs DMH\\u003csup\\u003eVgat\\u003c/sup\\u003e-DTA: *p=0.012), but (\\u003cstrong\\u003eq-r\\u003c/strong\\u003e) there is no significant difference in the CI or cosinor amplitude of Tb. (\\u003cstrong\\u003es\\u003c/strong\\u003e) Representative Tb actograms before and after DMH\\u003csup\\u003eVgat\\u003c/sup\\u003e neuron ablation.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"FigS7VgatDMHDTAopt2.tif\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4718850/v2/1aa49742d7bf615ee4eec7b6.tif\"},{\"id\":84919070,\"identity\":\"46e01e18-edc3-4b2c-836d-6e59fddf452a\",\"added_by\":\"auto\",\"created_at\":\"2025-06-18 19:30:57\",\"extension\":\"tif\",\"order_by\":8,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":139198855,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cem\\u003eExtended figure 8. Vgat gene deletion in DMH neurons reduces the elevation of LMA and Tb during the middle of the dark and presumptive dark periods and the amplitude of their circadian rhythms. \\u003c/em\\u003e(\\u003cstrong\\u003ea\\u003c/strong\\u003e) Density plots of the injections of AAV-EGFP-iCre in the DMH of \\u003cem\\u003eVgat\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eloxP/loxP\\u003c/em\\u003e\\u003c/sup\\u003e\\u003csup\\u003e \\u003c/sup\\u003emice (\\u003cem\\u003en\\u003c/em\\u003e=7). (\\u003cstrong\\u003eb-e\\u003c/strong\\u003e) \\u003cem\\u003eVgat\\u003c/em\\u003e gene deletion in the DMH caused lower LMA during the middle of the dark phase in LD and presumptive dark phase in DD (LD: \\u003cem\\u003eRM\\u003c/em\\u003e \\u003cem\\u003eTwo-way ANOVA\\u003c/em\\u003e; \\u003cem\\u003eŠídák's\\u003c/em\\u003e multiple comparisons test. DMH\\u003csup\\u003eVgat/flox\\u003c/sup\\u003e-Control vs DMH\\u003csup\\u003eVgat/flox\\u003c/sup\\u003e-EGFP-iCre: *p\\u0026lt;0.05; DD: \\u003cem\\u003eRM Two-way ANOVA\\u003c/em\\u003e; \\u003cem\\u003eŠídák's\\u003c/em\\u003e multiple comparisons test. DMH\\u003csup\\u003eVgat/flox\\u003c/sup\\u003e-Control vs DMH\\u003csup\\u003eVgat/flox\\u003c/sup\\u003e-EGFP-iCre: *p\\u0026lt;0.05) with a reduction in the amplitude of in the mean periodogram peak at 24h (LD: \\u003cem\\u003eTwo-way ANOVA\\u003c/em\\u003e; \\u003cem\\u003eŠídák's\\u003c/em\\u003e multiple comparisons test. DMH\\u003csup\\u003eVgat/flox\\u003c/sup\\u003e-Control vs DMH\\u003csup\\u003eVgat/flox\\u003c/sup\\u003e-EGFP-iCre: *p\\u0026lt;0.05; DD: \\u003cem\\u003eTwo-way ANOVA\\u003c/em\\u003e; \\u003cem\\u003eŠídák's\\u003c/em\\u003e multiple comparisons test. DMH\\u003csup\\u003eVgat/flox\\u003c/sup\\u003e-Control vs DMH\\u003csup\\u003eVgat/flox\\u003c/sup\\u003e-EGFP-iCre: *p\\u0026lt;0.05). (\\u003cstrong\\u003ef\\u003c/strong\\u003e) Deletion of the \\u003cem\\u003eVgat\\u003c/em\\u003e gene in the DMH reduced movement during the dark or subjective dark period (\\u003cem\\u003eTwo-way ANOVA\\u003c/em\\u003e; \\u003cem\\u003eTukey's\\u003c/em\\u003e multiple comparisons test. LD DMH\\u003csup\\u003eVgat/flox\\u003c/sup\\u003e-Control vs DMH\\u003csup\\u003eVgat/flox\\u003c/sup\\u003e-EGFP-iCre: ***p\\u0026lt;0.001; DD Dark DMH\\u003csup\\u003eVgat/flox\\u003c/sup\\u003e-Control vs DMH\\u003csup\\u003eVgat/flox\\u003c/sup\\u003e-EGFP-iCre: ***p\\u0026lt;0.001). (\\u003cstrong\\u003eg\\u003c/strong\\u003e) The CI of LMA rhythm was reduced by 25.73 ±6.01% in LD (\\u003cem\\u003eUnpaired\\u003c/em\\u003e t-test t=4.275 df=12, **p=0.001) and 33.35 ± 12.63% in DD (\\u003cem\\u003eUnpaired\\u003c/em\\u003e t-test t=2.642 df=12, *p=0.021) and (\\u003cstrong\\u003eh\\u003c/strong\\u003e) the cosinor amplitude of LMA was similarly reduced in LD (\\u003cem\\u003eUnpaired\\u003c/em\\u003e t-test: t=5.194, df=12, ***p\\u0026lt;0.001) and DD (\\u003cem\\u003eUnpaired\\u003c/em\\u003e t-test: t=5.232, df=12, ***p\\u0026lt;0.001). (\\u003cstrong\\u003ei\\u003c/strong\\u003e) Representative LMA actograms from AAV-DIO-GFP and AAV-DIO-EGFP-iCre injected mice. (\\u003cstrong\\u003ej-m\\u003c/strong\\u003e) Tb is reduced during the middle of the dark phase in DMH \\u003cem\\u003eVgat\\u003c/em\\u003e gene-deleted mice in LD (\\u003cem\\u003eRM Two-way ANOVA\\u003c/em\\u003e; \\u003cem\\u003eŠídák's\\u003c/em\\u003e multiple comparisons test. DMH\\u003csup\\u003eVgat/flox\\u003c/sup\\u003e-Control vs DMH\\u003csup\\u003eVgat/flox\\u003c/sup\\u003e-EGFP-iCre: *p\\u0026lt;0.05) and the presumptive dark phase in DD (\\u003cem\\u003eRM\\u003c/em\\u003e \\u003cem\\u003eTwo-way ANOVA\\u003c/em\\u003e; \\u003cem\\u003eŠídák's\\u003c/em\\u003e multiple comparisons test. DMH\\u003csup\\u003eVgat/flox\\u003c/sup\\u003e-Control vs DMH\\u003csup\\u003eVgat/flox\\u003c/sup\\u003e-EGFP-iCre: *p\\u0026lt;0.05), with no change in the mean periodogram. \\u0026nbsp;(\\u003cstrong\\u003en\\u003c/strong\\u003e) The reduction in mean Tb during the dark and presumptive dark phase in the DMH \\u003cem\\u003eVgat\\u003c/em\\u003e-gene deleted mice was statistically significant (\\u003cem\\u003eTwo-way ANOVA\\u003c/em\\u003e; \\u003cem\\u003eTukey’s\\u003c/em\\u003e multiple comparisons test. LD Dark DMH\\u003csup\\u003eVgat/flox\\u003c/sup\\u003e-Control vs DMH\\u003csup\\u003eVgat/flox\\u003c/sup\\u003e-EGFP-iCre: **p=0.001; DD Dark DMH\\u003csup\\u003eVgat/flox\\u003c/sup\\u003e-Control vs DMH\\u003csup\\u003eVgat/flox\\u003c/sup\\u003e-EGFP-iCre: ***p\\u0026lt;0.001). (\\u003cstrong\\u003eo\\u003c/strong\\u003e) As a result, the CI of Tb was also reduced in the DMH \\u003cem\\u003eVgat\\u003c/em\\u003e-gene deleted mice by 20.84 ±8.8% in LD (\\u003cem\\u003eUnpaired\\u003c/em\\u003e t-test t=2.342 df=12, *p=0.037) and by 26.6 ±8.9% under constant dark (\\u003cem\\u003eUnpaired\\u003c/em\\u003e t-test t=2.958 df=12, *p=0.012), and (\\u003cstrong\\u003ep\\u003c/strong\\u003e) the cosinor amplitude was lower in DMH \\u003cem\\u003eVgat\\u003c/em\\u003e gene-deleted mice in LD (\\u003cem\\u003eUnpaired\\u003c/em\\u003e t-test: t=2.482, df=12, *p= 0.028) and DD (\\u003cem\\u003eUnpaired\\u003c/em\\u003e t-test: t=3.902, df=12, **p=0.002). (\\u003cstrong\\u003eq\\u003c/strong\\u003e) Representative Tb actograms from AAV-DIO-GFP and AAV-DIO-EGFP-iCre injected mice.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"FigS8VgatDMHFl.tif\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4718850/v2/7d564cf33cde3e594ccd484d.tif\"},{\"id\":84918278,\"identity\":\"31e8ef0a-806f-43d7-84dc-8fa535f0ad4a\",\"added_by\":\"auto\",\"created_at\":\"2025-06-18 19:22:55\",\"extension\":\"tif\",\"order_by\":9,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":139198855,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cem\\u003eExtended figure 9. Chemogenetic inhibition of DMH\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eVgat\\u003c/em\\u003e\\u003c/sup\\u003e\\u003cem\\u003e neurons flattens the circadian rhythm of LMA and Tb. \\u003c/em\\u003e(\\u003cstrong\\u003ea\\u003c/strong\\u003e) Density plots of the AAV-DIO-hGlyR-mCherry injection sites in the DMH of \\u003cem\\u003eVgat-ires-Cre\\u003c/em\\u003e mice (\\u003cem\\u003en\\u003c/em\\u003e=5). (\\u003cstrong\\u003eb-g\\u003c/strong\\u003e) There was a reduction in the amount of LMA during the dark and presumptive dark periods between 24-48 hr after IVM injection (\\u003cstrong\\u003eb\\u003c/strong\\u003e and \\u003cstrong\\u003ee\\u003c/strong\\u003e: \\u003cem\\u003eRM Two-way ANOVA\\u003c/em\\u003e; \\u003cem\\u003eTukey's\\u003c/em\\u003e multiple comparisons test p\\u0026lt;0.05. Line above the 24h graphs represent significative differences in Baseline vs IVM in green, and VEH vs IVM in blue; \\u003cstrong\\u003ec\\u003c/strong\\u003e: \\u003cem\\u003eTwo-way ANOVA\\u003c/em\\u003e; \\u003cem\\u003eTukey's\\u003c/em\\u003e multiple comparisons test. dark Baseline vs dark IVM: ***p\\u0026lt;0.001, dark VEH vs dark IVM: **p=0.001. F: \\u003cem\\u003eTwo-way ANOVA\\u003c/em\\u003e; \\u003cem\\u003eTukey's\\u003c/em\\u003e multiple comparisons test. dark VEH vs dark IVM: *p=0.04), resulting in a dramatic reduction in CI during the same time period (\\u003cstrong\\u003ed\\u003c/strong\\u003e: \\u003cem\\u003eOne-way ANOVA\\u003c/em\\u003e; \\u003cem\\u003eTukey's\\u003c/em\\u003e multiple comparisons test. Baseline vs IVM: ***p\\u0026lt;0.001, VEH vs IVM: **p=0.001. \\u003cstrong\\u003eg\\u003c/strong\\u003e: \\u003cem\\u003eOne-way ANOVA\\u003c/em\\u003e; \\u003cem\\u003eTukey's\\u003c/em\\u003e multiple comparisons test. Baseline vs IVM: ***p\\u0026lt;0.001, VEH vs IVM: **p=0.002). (\\u003cstrong\\u003eh-m\\u003c/strong\\u003e) Reductions in mean Tb after IVM administration were observed during the dark and subjective dark periods (\\u003cstrong\\u003eh\\u003c/strong\\u003e and \\u003cstrong\\u003ek\\u003c/strong\\u003e: \\u003cem\\u003eRM Two-way ANOVA\\u003c/em\\u003e; \\u003cem\\u003eTukey's\\u003c/em\\u003e multiple comparisons test p\\u0026lt;0.05. \\u003cstrong\\u003ei\\u003c/strong\\u003e: \\u003cem\\u003eTwo-way ANOVA\\u003c/em\\u003e; \\u003cem\\u003eTukey's\\u003c/em\\u003e multiple comparisons test. dark Baseline vs dark IVM: *p=0.041. \\u003cstrong\\u003el\\u003c/strong\\u003e: \\u003cem\\u003eTwo-way ANOVA\\u003c/em\\u003e; \\u003cem\\u003eTukey's\\u003c/em\\u003e multiple comparisons test. dark Baseline vs dark IVM: *p=0.038), driving a reduction in CI in both LD and DD (\\u003cstrong\\u003ej\\u003c/strong\\u003e: \\u003cem\\u003eOne-way ANOVA\\u003c/em\\u003e; \\u003cem\\u003eTukey's\\u003c/em\\u003e multiple comparisons test. Baseline vs IVM: ***p\\u0026lt;0.001, VEH vs IVM: **p=0.005. \\u003cstrong\\u003em\\u003c/strong\\u003e: \\u003cem\\u003eOne-way ANOVA\\u003c/em\\u003e; \\u003cem\\u003eTukey's\\u003c/em\\u003e multiple comparisons test. Baseline vs IVM: ***p\\u0026lt;0.001, VEH vs IVM: **p=0.004).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"FigS9VgatDMHhGlyR.tif\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4718850/v2/6c665b34803ac10855b7814f.tif\"},{\"id\":84918279,\"identity\":\"908af097-d8b9-4619-9fdd-b09dc4a0772d\",\"added_by\":\"auto\",\"created_at\":\"2025-06-18 19:22:55\",\"extension\":\"tif\",\"order_by\":10,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":139198855,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cem\\u003eExtended figure 10. cvPVH\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eVgat \\u003c/em\\u003e\\u003c/sup\\u003e\\u003cem\\u003eneuron ablation increases the Cort response to restraint stress, but does not affect the circadian rhythm of LMA or Tb. \\u003c/em\\u003e(\\u003cstrong\\u003ea\\u003c/strong\\u003e) Density plots showing the distribution of injections of AAV-mCherry-DIO-DTA in the cvPVH of Vgat-ires-Cre mice (\\u003cem\\u003en\\u003c/em\\u003e=5). (\\u003cstrong\\u003eb\\u003c/strong\\u003e) After 1 hour of movement restraint, mice with cvPVH\\u003csup\\u003eVgat\\u003c/sup\\u003e ablation had higher levels of Cort (Unpaired t test: t=2.782, df=8, p= 0.047). (\\u003cstrong\\u003ec\\u003c/strong\\u003e) The 24h LMA counts and (\\u003cstrong\\u003ed\\u003c/strong\\u003e) mean periodogram under LD, as well as under DD (\\u003cstrong\\u003ee, f\\u003c/strong\\u003e) did not differ from control mice. (\\u003cstrong\\u003eg-i\\u003c/strong\\u003e) As a result, no differences were detected in the circadian rhythms of LMA after cvPVH\\u003csup\\u003eVgat\\u003c/sup\\u003e neuron ablation (\\u003cstrong\\u003ej\\u003c/strong\\u003e) Representative LMA actograms from both controls and mice with ablation of cvPVH\\u003csup\\u003eVgat\\u003c/sup\\u003e neurons. (\\u003cstrong\\u003ek-n\\u003c/strong\\u003e) There were no significant changes in mean Tb levels or their periodograms under either LD or DD. (\\u003cstrong\\u003eo-q\\u003c/strong\\u003e) Likewise, the circadian rhythm of Tb was undisturbed by the cvPVH\\u003csup\\u003eVgat\\u003c/sup\\u003e ablation. (\\u003cstrong\\u003er\\u003c/strong\\u003e) Representative Tb actograms from Vgat-Cre animals with injections of AAV-DIO-GFP and AAV-mCherrry-DIO-DTA the cvPVH. LD, Light:Dark photoperiod; DD, Constant darkness.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"FigS10VgatPVHDTA.tif\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4718850/v2/21dba88f355b705d242a40b2.tif\"},{\"id\":84918285,\"identity\":\"2d81f523-2259-4c89-be40-5bc08fd1a536\",\"added_by\":\"auto\",\"created_at\":\"2025-06-18 19:23:01\",\"extension\":\"tif\",\"order_by\":11,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":313195879,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cem\\u003eExtended Figure 11.\\u003c/em\\u003e \\u003cem\\u003eIn vitro\\u003c/em\\u003e \\u003cem\\u003eoptogenetic stimulation of the GABAergic input from the caudal DMH inhibits PVH\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003eCRH\\u003c/em\\u003e\\u003c/sup\\u003e\\u003cem\\u003e neurons. \\u003c/em\\u003e(\\u003cstrong\\u003ea\\u003c/strong\\u003e) A schematic of the experiment demonstrating connectivity between the caudal DMH (cDMH) Vgat neurons and ipsilateral PVH\\u003csup\\u003eCRH\\u003c/sup\\u003e neurons (cDMH\\u003csup\\u003eVgat \\u003c/sup\\u003e→ PVH\\u003csup\\u003eCRH\\u003c/sup\\u003e; the DMH is shown on the opposite side of the brain to ease illustration). \\u003cem\\u003eVgat-ires-Cre::CRH-Venus \\u003c/em\\u003emice were injected with \\u003cem\\u003eAAV-DIO-ChR2-mCherry\\u003c/em\\u003e in the cDMH, and recordings were conducted in brain slices from Venus-labeled PVH\\u003csup\\u003eCRH\\u003c/sup\\u003e neurons while photostimulating the cDMH\\u003csup\\u003eVgat\\u003c/sup\\u003e input. (\\u003cstrong\\u003eb\\u003c/strong\\u003e) An example of ChR2-mCherry expression in the cDMH (\\u003cem\\u003etop left, \\u003c/em\\u003enative signal) and density plots of the \\u003cem\\u003eAAV-DIO-ChR2-mCherry\\u003c/em\\u003e injection sites (\\u003cem\\u003en\\u003c/em\\u003e = 4 mice; \\u003cem\\u003eright\\u003c/em\\u003e and \\u003cem\\u003ebottom\\u003c/em\\u003e). (\\u003cstrong\\u003ec\\u003c/strong\\u003e) Opto-evoked inhibitory post-synaptic currents (oIPSCs) recorded in the PVH\\u003csup\\u003eCRH\\u003c/sup\\u003e neurons. (\\u003cstrong\\u003ed\\u003c/strong\\u003e) Percentages of PVH\\u003csup\\u003eCRH\\u003c/sup\\u003e neurons responding (Connected) and not responding (Not Connected) to photostimulation of the cDMH\\u003csup\\u003eVgat\\u003c/sup\\u003e input (\\u003cem\\u003en\\u003c/em\\u003e= 23 PVH\\u003csup\\u003eCRH\\u003c/sup\\u003e recorded neurons from 4 mice). (\\u003cstrong\\u003ee\\u003c/strong\\u003e) Amplitude (\\u003cem\\u003eleft\\u003c/em\\u003e; filled markers, cells responding to photostimulation, \\u003cem\\u003en\\u003c/em\\u003e=22, open markers, cells not responding to photostimulation, \\u003cem\\u003en\\u003c/em\\u003e=1 neurons, from 4 mice; mean and ± SEM of responding neurons) and latency (\\u003cem\\u003eright\\u003c/em\\u003e) of oIPSCs in PVH\\u003csup\\u003eCRH\\u003c/sup\\u003e neurons in response to photostimulation of the cDMH\\u003csup\\u003eVgat\\u003c/sup\\u003e input (mean and ± SEM;\\u003cem\\u003e n\\u003c/em\\u003e=22 from 4 mice). (\\u003cstrong\\u003ef\\u003c/strong\\u003e) Raster plot of IPSCs in a representative PVH\\u003csup\\u003eCRH\\u003c/sup\\u003e neurons with photostimulation of the cDMH\\u003csup\\u003eVgat\\u003c/sup\\u003e → PVH\\u003csup\\u003eCRH\\u003c/sup\\u003e input (bin duration: 50ms). (\\u003cstrong\\u003eg\\u003c/strong\\u003e) IPSC probability in response to photostimulation of the cDMH\\u003csup\\u003eVgat \\u003c/sup\\u003e→ PVH\\u003csup\\u003eCRH\\u003c/sup\\u003e input (black, \\u003cem\\u003en\\u003c/em\\u003e = 23). Reference scale bar: in (\\u003cstrong\\u003eb\\u003c/strong\\u003e) = 250 µm. f, fornix; 3V, third ventricle.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"FigS11DMHVGATcaudalPVHCRH.tif\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4718850/v2/d0e2f034ce917a7e78cc5438.tif\"}],\"financialInterests\":\"The authors declare no competing interests.\",\"formattedTitle\":\"A hypothalamic circuit for circadian regulation of corticosterone secretion\",\"fulltext\":[],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[{\"identity\":\"1f4851f1-7271-4973-8c5d-466f9eb03446\",\"identifier\":\"10.13039/100000002\",\"name\":\"National Institutes of Health\",\"awardNumber\":\"P01HL149630-04\",\"order_by\":0},{\"identity\":\"4e816ca0-ab5c-4689-80a7-72765f24d234\",\"identifier\":\"10.13039/100000002\",\"name\":\"National Institutes of Health\",\"awardNumber\":\"R01 NS085477\",\"order_by\":1},{\"identity\":\"14bfea9a-46ab-443c-98fd-bf7b85aec159\",\"identifier\":\"10.13039/100000002\",\"name\":\"National Institutes of Health\",\"awardNumber\":\"R01 NS122589-03\",\"order_by\":2},{\"identity\":\"45cd2a9c-02db-4f8d-aed3-8cd82c87d944\",\"identifier\":\"10.13039/100000002\",\"name\":\"National Institutes of Health\",\"awardNumber\":\"P01HL149630-04\",\"order_by\":3},{\"identity\":\"67da4944-0e0c-4657-ad80-0a220dc7bd66\",\"identifier\":\"10.13039/100000002\",\"name\":\"National Institutes of Health\",\"awardNumber\":\"R03 NS128993-02\",\"order_by\":4}],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":false,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":true,\"hideJournal\":true,\"highlight\":\"\",\"institution\":\"Harvard Medical School\",\"isAcceptedByJournal\":false,\"isAuthorSuppliedPdf\":true,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":true,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true},\"keywords\":\"corticosterone, corticotropin-releasing hormone, dorsomedial hypothalamus and paraventricular hypothalamus\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-4718850/v2\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-4718850/v2\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eThe secretion of cortisol in humans and corticosterone (Cort) in rodents follows a daily rhythm which is important in readying the individual for daily activity. This rhythm is orchestrated by the suprachiasmatic nucleus (SCN), but how it ultimately regulates the circadian rhythm of activity of neurons in the paraventricular nucleus of the hypothalamus that produce corticotropin-releasing hormone (PVH\\u003csup\\u003eCRH\\u003c/sup\\u003e neurons) is not known. We hypothesized that the SCN may exert this influence by projections to the subparaventricular zone (SPZ), which in turn innervates neurons in the dorsomedial nucleus of the hypothalamus (DMH) that regulate PVH\\u003csup\\u003eCRH\\u003c/sup\\u003e neurons. First, we found that ablating SPZ\\u003csup\\u003eVgat\\u003c/sup\\u003e neurons eliminates the circadian rhythm of Cort secretion, but that deleting \\u003cem\\u003eVgat \\u003c/em\\u003efrom them does not, suggesting that they predominantly use some other transmitter. Next, we found that either ablating or acutely inhibiting the DMH glutamatergic (DMH\\u003csup\\u003eVglut2\\u003c/sup\\u003e) neurons resulted in a 40-70% reduction in the daily peak of Cort. Deletion of the \\u003cem\\u003eVglut2\\u003c/em\\u003e gene within the DMH produced a similar effect, highlighting the indispensable role of glutamatergic signaling. Chemogenetic stimulation of DMH\\u003csup\\u003eVglut2\\u003c/sup\\u003e neurons led to an increase of Cort levels, and optogenetic activation of their terminals in the PVH in hypothalamic slices directly activated PVH\\u003csup\\u003eCRH\\u003c/sup\\u003e neurons through glutamate action on AMPA receptors (the DMH\\u003csup\\u003eVglut2\\u003c/sup\\u003e → PVH\\u003csup\\u003eCRH\\u003c/sup\\u003e pathway). Similar to the disruption of DMH\\u003csup\\u003eVglut2\\u003c/sup\\u003e neurons, ablating, inhibiting, or disrupting GABA transmission by DMH GABAergic (DMH\\u003csup\\u003eVgat\\u003c/sup\\u003e) neurons diminished the circadian peak of Cort, particularly under constant darkness conditions. Chemogenetic stimulation of rostral DMH\\u003csup\\u003eVgat\\u003c/sup\\u003e neurons increased Cort, although with a lower magnitude compared to DMH\\u003csup\\u003eVglut2\\u003c/sup\\u003e neuron stimulation, suggesting a role in disinhibiting PVH\\u003csup\\u003eCRH\\u003c/sup\\u003e neurons. Supporting this hypothesis, we found that rostral DMH\\u003csup\\u003eVgat \\u003c/sup\\u003eneurons project directly to GABAergic neurons in the caudal ventral part of the PVH and adjacent peri-PVH area (cvPVH), which directly inhibit PVH\\u003csup\\u003eCRH\\u003c/sup\\u003e neurons, and that activating the rostral DMH\\u003csup\\u003eVgat\\u003c/sup\\u003e terminals in the cvPVH in brain slices reduced GABAergic afferent input onto the PVH\\u003csup\\u003eCRH\\u003c/sup\\u003e neurons. Finally, ablation of cvPVH\\u003csup\\u003eVgat\\u003c/sup\\u003e neurons resulted in increased Cort release at the onset of the active phase, affirming the pivotal role of the DMH\\u003csup\\u003eVgat \\u003c/sup\\u003e→ cvPVH\\u003csup\\u003eVgat\\u003c/sup\\u003e → PVH\\u003csup\\u003eCRH\\u003c/sup\\u003e pathway in Cort secretion. In summary, our study delineates two parallel pathways transmitting temporal information to PVH\\u003csup\\u003eCRH\\u003c/sup\\u003e neurons, collectively orchestrating the daily surge in Cort in anticipation of the active phase. These findings are crucial to understand the neural circuits regulating Cort secretion, shedding light on the mechanisms governing this physiological process and the coordinated interplay between the SCN, SPZ, DMH, and PVH.\\u003c/p\\u003e\",\"manuscriptTitle\":\"A hypothalamic circuit for circadian regulation of corticosterone secretion\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":2,\"date\":\"2025-06-18 19:22:48\",\"doi\":\"10.21203/rs.3.rs-4718850/v2\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true}},{\"code\":1,\"date\":\"2024-07-12 15:21:59\",\"doi\":\"10.21203/rs.3.rs-4718850/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"3408baeb-fcca-42e4-847b-064251c77487\",\"owner\":[],\"postedDate\":\"June 18th, 2025\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"posted\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2025-02-21T18:26:04+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2025-06-18 19:22:48\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v2\",\"identity\":\"rs-4718850\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-4718850\",\"identity\":\"rs-4718850\",\"version\":[\"v2\"]},\"buildId\":\"8U1c8b4HqxoKbykW_rLl7\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}