Journal: bioRxiv

Article Title: Pharmacological recruitment of a VTA glutamatergic arousal circuit by the natural product BN3 drives broad-spectrum emergence from general anesthesia

doi: 10.64898/2026.02.17.706054

Figure Lengend Snippet: (A) Representative confocal microscopy images showing c-Fos immunoreactivity (green) in coronal brain sections from mice treated with vehicle (top) or BN3 (bottom). Scale bar: 100 µm. mPFC (Medial prefrontal cortex), PVT (Paraventricular thalamus), VTA (Ventral tegmental area), ACC (Anterior cingulate cortex), BLA (Basolateral amygdala), LC (Locus coeruleus), HDB (Horizontal limb of the diagonal band), VLPAG (Ventrolateral periaqueductal gray), DRN (Dorsal raphe nucleus), LHb (Lateral habenula), LH (Lateral hypothalamus). (B) Quantification of c-Fos + cells in the brain regions shown in (A). BN3 significantly increased neuronal activation in the mPFC, PVT and VTA. Data are represented as mean number of c-Fos + cells averaged across three slices from each mouse, n = 3 mice per group. Unpaired two-tailed t -test, * p < 0.05, *** p < 0.001. (C) Schematic of the chemogenetic inhibition protocol. Mice were injected with inhibitory DREADDs (hM4Di) in targeted brain areas 21 days before the experiment. At the day of experiment, CNO was intraperitonially injected 30 minutes prior to propofol anesthesia and BN3 administration. (D) Chemogenetic inhibition of VTA blunted the emergence-accelerating effect of BN3. Return of the righting reflex (RORR) times are shown for control (Saline + BN3) and inhibition (CNO + BN3) groups. n = 5 mice per group. Unpaired two-tailed t -test, * p < 0.05.

Article Snippet: On day 21 post-AAV microinjection, CNO (MedChemExpress, HY-17366, 3 mg/kg) was administered intraperitoneally 30 minutes before propofol and BN3 to activate the DREADDs, with an equal volume of saline serving as the control.

Techniques: Confocal Microscopy, Activation Assay, Two Tailed Test, Inhibition, Injection, Control, Saline

Journal: bioRxiv

Article Title: Pharmacological recruitment of a VTA glutamatergic arousal circuit by the natural product BN3 drives broad-spectrum emergence from general anesthesia

doi: 10.64898/2026.02.17.706054

Figure Lengend Snippet: (A) Schematic illustrating of the fiber photometry protocol. (B) Population heatmap of normalized GCaMp6s fluorescence signals in the VTA, aligned to BN3 administration (time = 0). Each row represents one mouse. BN3 evoked a rapid and sustained increase in calcium activity. (C) Representative averaged ΔF/F0 traces of VTA GCaMp6s signals from individual mice in the vehicle (blue) and BN3 (red) groups. (D) Quantification the area under the curve (AUC) for the 60 seconds period following BN3/vehicle administration; n = 5 mice per group. Unpaired two-tailed t -test, *** p < 0.001. (E–H) Chemogenetic inhibition of VTA neurons attenuated the BN3-induced calcium response. (E) Schematic illustrating experimental protocol. (F) Population heatmap, (G) representative traces, and (H) AUC quantification for mice expressing hM4Di in VTA neurons; n = 5 mice per group. Unpaired two-tailed t -test, * p < 0.05. (I–L) BN3 altered cortical EEG signatures toward an awake-like state. (I) Time-frequency spectrogram (heatmap) of cortical EEG power before and after BN3 administration. (J) Representative raw EEG traces under deep propofol anesthesia (top) and after BN3 administration (bottom). (K) Normalized power plots for the pre- and post-BN3 periods. (L) Quantification of the relative power in Delta (0.5–4 Hz), Theta (4–8 Hz), Alfa (8-15 Hz), Beta (15-25 Hz), and Gamma (25-50 Hz) frequency bands. BN3 significantly decreased delta power and increased beta and gamma power; n = 3 mice. Paired two-tailed t -test, * p < 0.05. (M–P) Chemogenetic inhibition of VTA neurons blunted BN3-induced EEG changes. (M– P) Parallel analyses to panels I-L in mice with VTA inhibition (CNO + BN3 group). The spectral shift toward wakefulness was abolished; n = 3 mice. Paired two-tailed t -test, * p < 0.05.

Article Snippet: On day 21 post-AAV microinjection, CNO (MedChemExpress, HY-17366, 3 mg/kg) was administered intraperitoneally 30 minutes before propofol and BN3 to activate the DREADDs, with an equal volume of saline serving as the control.

Techniques: Fluorescence, Activity Assay, Two Tailed Test, Inhibition, Expressing

Journal: bioRxiv

Article Title: Pharmacological recruitment of a VTA glutamatergic arousal circuit by the natural product BN3 drives broad-spectrum emergence from general anesthesia

doi: 10.64898/2026.02.17.706054

Figure Lengend Snippet: (A) Schematic of the experimental timeline. (B) Representative fluorescence in situ hybridization (FISH) images of coronal VTA sections from BN3-treated mice, showing c-Fos mRNA (green) with mRNA markers for dopaminergic ( TH , purple), glutamatergic ( Vglut2 , yellow), GABAergic ( GABA , red) neurons. Merged images showing co-localization of Vglut2 with c-Fos . Scale bar: 200 µm, 50 µm in zoom in images. (C) Quantification of activated neuronal subtypes. The number of Vglut2 + neurons that were c-fos + was significantly higher in BN3 group than vehicle group; n = 6 mice. Unpaired two-tailed t -test, ** p < 0.01. (D) Schematic of AAV virus injection and optic fiber implantation in the VTA of Vglut2 -Cre mice to express GCaMP6s selectively in VTA glutamatergic neurons. Right: Representative histology confirming targeted virus expression. (E) Population heatmap of normalized GCaMP6s signals from VTA glutamatergic neurons, aligned to BN3 administration. (F) Representative calcium traces from individual mice. (G) Quantification of the neural response as the area under the curve (AUC); n = 5 mice per group. Unpaired two-tailed t -test, *** p < 0.001. (H, I) Chemogenetic inhibition of VTA glutamatergic neurons partially reversed BN3’s effect. (H) Schematic of AAV-mediated expression of the inhibitory DREADD hM4Di in VTA glutamatergic neurons of Vglut2 -Cre mice. Right: Representative image confirming viral expression. (I) Inhibition (via CNO) of VTA glutamatergic neurons significantly delayed RORR time caused by BN3; n = 6 mice per group. Unpaired two-tailed t -test, * p < 0.05. (J–K) Chemogenetic inhibition of VTA glutamatergic neurons blunted BN3-induced EEG changes.

Article Snippet: On day 21 post-AAV microinjection, CNO (MedChemExpress, HY-17366, 3 mg/kg) was administered intraperitoneally 30 minutes before propofol and BN3 to activate the DREADDs, with an equal volume of saline serving as the control.

Techniques: Fluorescence, In Situ Hybridization, Two Tailed Test, Virus, Injection, Expressing, Inhibition

Journal: bioRxiv

Article Title: Pharmacological recruitment of a VTA glutamatergic arousal circuit by the natural product BN3 drives broad-spectrum emergence from general anesthesia

doi: 10.64898/2026.02.17.706054

Figure Lengend Snippet: (A) Schematic for in vivo glutamate sensing in the NAc. (B) Population heatmap of normalized iGluSnFR3 fluorescence signals in the NAc, aligned to BN3 administration (time = 0). Each row represents one animal. (C) Left: Representative traces of glutamate transients in the NAc for vehicle (blue) and BN3 (red) groups. Right: Quantification the area under the curve (AUC) for the 30 seconds following BN3/vehicle administration. BN3 significantly increased glutamate release in the NAc; n = 6 mice per group. Unpaired two-tailed t -test, *** p < 0.001. (D–F) Chemogenetic inhibition of VTA glutamatergic neurons prevented BN3-evoked glutamate release. (D) Schematic for expressing the inhibitory hM4Di in VTA glutamatergic neurons and glutamate sensor iGluSnFR3 in the NAc. (E) Population heatmap and (F) representative traces with AUC quantification for mice treated with CNO (inhibition) or vehicle prior to 30 seconds following BN3 administration. Inhibition abolished the BN3-induced glutamate transient; n = 3 mice per group. Unpaired two-tailed t -test, ** p < 0.01. (G–I) Optogenetic inhibition of VTA→NAc projections blunted BN3’s behavioral effect. (G) Experimental timeline. Archaerhodopsin (Archt) or control (EYFP) was expressed in VTA glutamatergic neurons of Vglut2 -Cre mice, and an optic fiber was implanted in the NAc. Continuous yellow light (λ = 594 nm, 5-8mW) was delivered 5-min before propofol administration until emergence. (H) Optogenetic inhibition of the VTA→NAc pathway reversed the emergence-accelerating effect of BN3 in Archt-expressing mice. This was not observed in the control animals (I); n = 5 mice per group. Unpaired two-tailed t -test, * p < 0.05; ns, not significant.

Article Snippet: On day 21 post-AAV microinjection, CNO (MedChemExpress, HY-17366, 3 mg/kg) was administered intraperitoneally 30 minutes before propofol and BN3 to activate the DREADDs, with an equal volume of saline serving as the control.

Techniques: In Vivo, Fluorescence, Two Tailed Test, Inhibition, Expressing, Control

Journal: bioRxiv

Article Title: Pharmacological recruitment of a VTA glutamatergic arousal circuit by the natural product BN3 drives broad-spectrum emergence from general anesthesia

doi: 10.64898/2026.02.17.706054

Figure Lengend Snippet: (A) Schematic for in vivo glutamate sensing in the mPFC. (B) Population heatmap of normalized iGluSnFR3 fluorescence signals in the mPFC, aligned to BN3 administration (time = 0). Each row represents one animal. (C)Left: Representative traces of glutamate transients in the mPFC for vehicle (blue) and BN3 (red) groups. Right: Quantification of the area under the curve (AUC) for the 30 seconds period following BN3/vehicle administration. BN3 potently increased glutamate release in the mPFC; n = 6 mice per group. Unpaired two-tailed t -test, *** p < 0.001. (D–F) Chemogenetic inhibition of VTA glutamatergic neurons attenuated BN3-evoked glutamate release. (D) Schematic for expressing the inhibitory hM4Di in VTA glutamatergic neurons and glutamate sensor iGluSnFR3 in the mPFC. (E) Population heatmap and (F) Representative traces with AUC quantification for mice pre-treated with CNO (inhibition) or vehicle (control). Inhibition significantly reduced the glutamate transient; n = 3 mice per group. Unpaired two-tailed t -test, ** p < 0.01. (G–I) Optogenetic inhibition of VTA→mPFC projections partially reversed BN3’s behavioral effect. (G) Experimental timeline. Archaerhodopsin (Archt) or control (EYFP) was expressed in VTA glutamatergic neurons of Vglut2 -Cre mice, with an optic fiber in the mPFC. Continuous yellow light (λ = 594 nm, 5-8mW) was delivered 5-min before propofol administration until emergence. (H) Optogenetic inhibition of the VTA→mPFC pathway significantly attenuated the emergence-accelerating effect of BN3 in Archt-expressing mice, but not in control group (I); n = 6 mice per group. Unpaired two-tailed t -test, ** p < 0.01; ns, not significant.

Article Snippet: On day 21 post-AAV microinjection, CNO (MedChemExpress, HY-17366, 3 mg/kg) was administered intraperitoneally 30 minutes before propofol and BN3 to activate the DREADDs, with an equal volume of saline serving as the control.

Techniques: In Vivo, Fluorescence, Two Tailed Test, Inhibition, Expressing, Control