The noradrenergic system is involved in recovery from midazolam administration.

(A) Number of LORR and no LORR induced by different doses of midazolam in C57BL/6J mice.

(B) Rate of LORR (%) in C57BL/6J mice at different doses of midazolam.

(C) Protocol for investigating changes in the content and activity of TH in the prosencephalon and brainstem of C57BL/6J mice by ELISA.

(D) Left: Content of TH in the prosencephalon and brainstem in the vehicle and midazolam (60mg/Kg, i.p.) groups. Right: Activity of TH in the prosencephalon and brainstem in the vehicle and midazolam (60mg/Kg, i.p.) groups.

(E) Protocol for exploring the influence of intraperitoneal injection of DSP-4 on the atomoxetine-mediated shortening of the recovery time from midazolam administration.

(F) Comparison of recovery time in the vehicle, Atomoxitine (10mg/Kg, i.p.), and Atomoxitine (20mg/Kg, i.p.) groups.

(G) Comparison of recovery time in the vehicle, DSP-4 (50 mg/kg, 3 days before, i.p.), and DSP-4 (50 mg/kg, 10 days before, i.p.) groups.

(H) Comparison of recovery time in the vehicle + vehicle, vehicle + Atomoxitine, DSP-4 (3 days before) + Atomoxitine, and DSP-4(10 days before) + Atomoxitine groups.

(I) Comparison of normalized TH (+) cell number in the LC in the vehicle, DSP-4 (3 days before), and DSP-4(10 days before).

(J) The quantification of c-Fos (+)/TH (+) cells with or without intraperitoneal injection of atomoxetine.

(K) Images of TH+ neurons in the LC after intraperitoneal injection of vehicle, DSP-4 (3 days), or DSP-4 (10 days) (panels on the lower show magnified images of the panels on the upper).

(L) Representative images showing the changes in TH (+) neuronal activity with or without intraperitoneal injection of atomoxetine.

Mida: midazolam; TH: Tyrosine hydroxylase; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001

LCNE neuronal activity is significantly reduced after midazolam administration and selective ablation of LCNE neurons hinders recovery from midazolam

(A) A schematic of the fiber optic recording of calcium signalings in the LC.

(B) A schematic of the calcium signaling recording device.

(C) A representative photomicrograph showing the microinjection and optical fiber locations and the co-expression of GCaMP6s and TH.

(D) Schematic diagram of the method of dividing the entire recording of the calcium signal at the BLA into four stages, and sources of heatmap and ΔF/F(%) statistical data.

(E) A heatmap of calcium signaling changes in bilateral LCNE neurons induced by midazolam. (Two representative mice were selected for the fiber optic recording of calcium signals, and the whole recording was divided into four stages, each stage as a group for statistical analysis of calcium signaling).

(F) A statistical diagram of calcium signaling changes in bilateral LCNE neurons induced by midazolam.

(G) The peak ΔF/F in the wakefulness, before LORR, LORR-RORR, and after RORR stages.

(H-I) Representative images showing the co-expression of c-Fos and TH in the LC without or with midazolam treatment.

(J) The quantification of c-Fos (+)/TH (+) cells in the LC with midazolam or without midazolam treatment.

(K) Protocol for exploring the influence of intra-LC microinjection DSP-4 on the atomoxetine-mediated shortening of the recovery time from midazolam administration.

(L) The representative photomicrograph shows the tracks of cannulas implanted into bilateral LC.

(M) Results showed the effects of microinjection of DSP-4 (10 days before) into bilateral LC on the recovery time of midazolam.

(N) Results showed the effects of vehicle + vehicle, vehicle + atomoxetine, and DSP-4 (10 days before) + atomoxetine on the recovery time of midazolam.

(O) Comparison of normalized TH (+) cell number in the LC with or without DSP-4 (10 days before) microinjected in the bilateral LC.

(P) Images of TH+ neurons in the LC after microinjection of DSP-4 or vehicle for 10 days (panels on the right show magnified images of the panels on the left).

*p<0.05; **p<0.01; ****p<0.0001

Optogenetic or chemogentic activation of LCNE neurons promotes recovery from midazolam

(A) Protocol for the effect of photostimulation of LCNE neurons with different light parameters on the recovery time of midazolam.

(B) A schematic of the photogenetic instrument.

(C) A representative photomicrograph showing the locations of optical fiber and virus expression and the co-expression of hChR2 and TH.

(D-E) Results showed the effect of photostimulation of LCNE neurons with different light parameters on the recovery time of midazolam.

(F) Results showed the effect of left, right, and bilateral photostimulation of the LCNE neurons on the recovery time of midazolam.

(G) The quantification of c-Fos (+)/TH (+) cells in the LC with or without photostimulation.

(H-I) Representative images showing the co-expression of c-Fos and TH cells in the LC with or without photostimulation.

(J-K) Protocol and instrument for the effect of chemogenetic activation of LCNE neurons on the recovery time of midazolam.

(L) A representative photomicrograph showing the virus expression and the co-expression of mCherry and TH in the LC of male and female mice.

(M-N) Results showed the effect of chemogenetic activation of LCNE neurons with different doses of CNO (i.p.) on the recovery time of midazolam in male and female mice.

(O-P) Results showed the effect of left, right, and bilateral chemogenetic activation of the LCNE neurons on the recovery time of midazolam in male and female mice.

(Q) Representative images showing the co-expression of c-Fos and TH cells in the LC of male and female mice with or without intraperitoneal injection of CNO (0.2 mg/kg).

(R-S) The quantification of c-Fos (+)/TH (+) cells in the LC with or without chemogenetic stimulation.

PS: photostimulation; CS: chemogenetic stimulation; *p<0.05; ****p<0.0001

LC-VLPO NEergic neural circuit was involved in the recovery from midazolam

(A) Protocol for optogenetic or chemogenetic activation of LCNE neurons and recording of calcium signaling changes in VLPO.

(B) A schematic of the location of optogenetic or chemogenetic virus injection site in the LC and calcium signaling recording virus injection site in the VLPO.

(C) A representative image showing the co-expression of hChR2 and TH in the LC.

(D) A representative image showing the co-expression of GCaMP6s and TH in the VLPO in male mice.

(E) Results showed the effects of optogenetic activation of LCNE neurons on the recovery time of midazolam.

(F-G) A representative image showing the co-expression of hM3Dq and TH in the LC of male and female mice.

(H) A representative image showing the co-expression of GCaMP6s and TH in the VLPO in female mice.

(I-J) Results showed the effects of chemogenetic activation of LCNE neurons on the recovery time of midazolam in male and female mice.

(K) Schematic diagram of the method of recording the calcium signal at the VLPO, and sources of statistical data.

(L-M) Heatmap and statistical diagram of calcium signaling changes in bilateral VLPO at the after RORR stage with or without optogenetic activation of LCNE neurons. (Showing changes in calcium signaling during the RORR and the Recovery period; optogenetic activation group, n=3; no optogenetic activation group, n=3)

(N) The peak ΔF/F in the VLPO with or without activation of LCNE neurons using optogenetics.

(O-P, R-S) Heatmap and statistical diagram of calcium signaling changes in bilateral VLPO at the after RORR stage with or without chemogenetic activation of LCNE neurons in male and female mice. (Showing changes in calcium signaling during the RORR and the Recovery period; chemogenetic activation group, n=3; no chemogenetic activation group, n=3)

(Q, T) The peak ΔF/F in the VLPO with or without activation of LCNE neurons using chemogenetics in male and female mice.

PS: photostimulation; *p<0.05; **p<0. 01

Activation of LC-VLPO NEergic neural circuit promotes recovery from midazolam

(A) Protocol for intra-LC microinjection of the virus and optogenetic activation of VLPO.

(B-C) Schematic of LC–VLPO long-range optogenetic activation and the location of optogenetic virus microinjection and optic fiber implantation.

(D-E) Representative images showing the co-expression of hChR2 and TH in the LC and VLPO.

(F) Result showed the effect of LC–VLPO long-range optogenetic activation on the recovery time of midazolam.

(G, L) Protocols for exploring the influence of optogenetic or chemogentic activation of NEergic terminals in the VLPO on the recovery time from midazolam.

(H, M) Schematic of the brief process of optogenetic or chemogenetic activation.

(I, N) Schematic of the location of optogenetic or chemogenetic virus microinjection and the location of optic fiber implantation.

(J) A representative photomicrograph showing the co-expression of eYFP and TH in the VLPO.

(K) Results showed the effect of Optogenetic activation of NEergic terminals in the VLPO could reduce the recovery time from midazolam.

(O, Q) A representative photomicrograph showing the co-expression of hM3Dq and TH in the VLPO.

(P, R) Results showed the effect of chemogenetic activation of NEergic terminals in the VLPO could reduce the recovery time from midazolam in both male and female mice.

PS: photostimulation; *p<0.05

ICV injection or intra-VLPO microinjection of α1-R antagonist reverses the pro-recovery effect of optogenetic or chemogenetic of activation LCNE neurons

(A) A schematic of intracerebroventricular injection of different noradrenergic receptor agonists and antagonists.

(B) A representative photomicrograph showing the tracks of cannulas implanted into the lateral ventricle.

(C-H) Results showed the effects of intracerebroventricular injection of different doses of phenylephrine, prazosin, clonidine, yohimbine, isoprenaline, and propranolol on the recovery time of midazolam.

(I) Results showed the effects of intracerebroventricular injection of phenylephrine (20mg/mL) and different doses of propranolol on the recovery time of midazolam.

(J) Protocol for photostimulation of LCNE neurons and intracerebroventricular injection or intra-VLPO microinjection of α1-R antagonist.

(K, L) Schematic of the brief process of optogenetic activation and the representative photomicrograph showing microinjection and optical fiber locations of the co-expression of hChR2 and TH in the LC.

(M, O) A representative photomicrograph showing the tracks of cannulas implanted into the lateral ventricle and bilateral VLPO.

(N, P) Results showed the effects of photostimulation of LCNE neurons and intracerebroventricular injection or intra-VLPO microinjection of prazosin on the recovery time of midazolam.

(Q) Protocol for chemogenetic activation of LCNE neurons and intracerebroventricular injection or intra-VLPO microinjection of an α1-R antagonist.

(R, S) Schematic of the location of virus injection and the representative photomicrograph showing the co-expression of hM3Dq and TH in the LC of male mice.

(T, V) A representative photomicrograph showing the tracks of cannulas implanted into the lateral ventricle and bilateral VLPO in the male mice.

(U, W) Results showed the effects of chemogenetic activation of LCNE neurons and intracerebroventricular injection or intra-VLPO microinjection of prazosin on the recovery time of midazolam in male mice.

(X, Y) A representative photomicrograph showing the co-expression of hM3Dq and TH in the LC and the tracks of cannulas implanted into the bilateral VLPO in the female mice.

(Z) Results showed the effects of chemogenetic activation of LCNE neurons and intra-VLPO microinjection of prazosin on the recovery time of midazolam in female mice.

icv: intracerebroventricular; PS: photostimulation; *p<0.05; **p<0.01; ***p<0.001

Effect of ICV injection of α1-R antagonist and activation of LCNE neurons on EEG activity

(A, D) Schematic diagram of EEG recording method.

(B) EEG and spectrum of mice in vehicle+no PS, vehicle+PS, Prazosin+no PS, and Prazosin+PS at the Before LORR, LORR-RORR, and After RORR stages.

(C) Alpha, Beta, Theta, Gamma, and Delta wave proportion of EEG in four groups of mice in different stages.

(E) EEG and spectrum of mice in vehicle+no CNO, vehicle+ CNO, Prazosin+no CNO, and Prazosin+ CNO at the Before LORR, LORR-RORR, and After RORR stages.

(F) Alpha, Beta, Theta, Gamma, and Delta wave proportion of EEG in four groups of mice in different stages.

PS: photostimulation; *p<0.05; **p<0.01; ***p<0.001

GABAA-R is an important mechanical binding site for midazolam in the LC

(A) Protocol for the fiber optic recording of calcium signals in the LC after intracerebroventricular injection or intra-LC microinjection of different doses of gabazine.

(B, D) A representative photomicrograph showing the tracks of cannulas implanted into the lateral ventricle and the bilateral LC.

(C, E) Results showed the effects of ICV injection or intra-LC microinjection of different doses of gabazine on the recovery time of midazolam.

(F) A representative photomicrograph showing the microinjection and optical fiber locations and the co-expression of GCaMP6s and TH in the LC.

(G) Schematic diagram of the method of recording the calcium signal at the LC, and sources of statistical data.

(H) A heatmap of calcium signaling changes in bilateral LCNE neurons induced by midazolam with or without intracerebroventricular injection of gabazine. (vehicle group: n=2; gabazine group: n=2; Calcium signaling data from two representative mice per group. Calcium signaling information of each mouse in the stage Before RORR and R-Recovery were selected for presentation and further statistical analysis.)

(I) A statistical diagram of calcium signaling changes in bilateral LC with or without intracerebroventricular injection of gabazine.

(J) The peak ΔF/F in the Before RORR and RORR-Recovery stages with or without intracerebroventricular injection of gabazine.

(K) Propofol for intra-LC microinjection of GABAA-R antagonist and intra-VLPO microinjection of α1-R antagonist.

(L) A schematic of the position of cannula implantation.

(M) A representative photomicrograph showing the tracks of cannulas implanted into the bilateral VLPO.

(N) Results showed the effects of intra-LC injection of gabazine and intra-VLPO injection of prazosin on the recovery time of midazolam.

icv: intracerebroventricular; *p<0.05; **p<0.01; ***p<0.001

GABA-ergic and NE-ergic systems interact with each other, co-regulate the recovery from midazolam

(A) Protocol for exploring the influence of knocking down the GABAA-R on the recovery time from midazolam.

(B-C) Schematic diagram and the representative photomicrograph showing the position of virus injection.

(D) A representative photomicrograph showing the co-expression of shRNA, TH, and GABAA-R in the LC.

(E) Representative photomicrograph of the difference of GABAA-R expression in the LC after shRNA-mediated knockdown.

(F) The quantification of GABAA-R (+)/TH (+) cells in the LC between the shRNA group and the sham group.

(G) Number of LORR and no LORR induced by different doses of midazolam in GABAA-R knockdown mice.

(H) Rate of LORR (%) in GABAA-R knockdown mice at different doses of midazolam.

(I) The statistical bar shows shRNA-mediated GABAA-R knockdown effect on the recovery time from midazolam.

(J-K) Protocol for exploring the calcium signals changes in the VLPO after knocking down the GABAA-R.

(L-M, N-O) Schematic diagram and the representative photomicrograph showing the position of virus injection and the co-expression of shRNA, TH, and GABAA-R in the LC.

(N-O) Schematic diagram of the brief process of calcium signal recording and the representative photomicrograph showing the co-expression of GCaP6m and TH in the VLPO.

(P) Schematic diagram of the method of recording the calcium signal at the VLPO, and sources of statistical data.

(Q-R) Heatmap and statistical diagram of calcium signaling changes in bilateral VLPO at the after RORR stage with or without knocking down GABAA-R on LCNE neurons. (Showing changes in calcium signaling during the RORR and the Recovery period; shRNA group, n=3; sham group, n=3)

(S) The peak ΔF/F in the VLPO with or without knocking down GABAA-R on LCNE neurons.

(T) Protocol for exploring the effect of blocking α1-R in the VLPO and knocking down the GABAA-R in the LC on the recovery time from midazolam.

(U-V) Schematic diagram and the representative photomicrograph showing the positions of cannula implantation.

(W-X) Schematic diagram and the representative photomicrograph showing the position of virus injection and the co-expression of shRNA, TH, and GABAA-R in the LC.

(Y) Results showed recovery time between sham, shRNA + Vehicle, and shRNA + Prazosin.