NE contribution to rebooting unconsciousness caused by midazolam
- Department of Anesthesiology, Zhejiang University School of Medicine, China
- Department of Anesthesiology, the Fourth Clinical School of Medicine, Zhejiang Chinese Medical University, China
- Department of Anesthesiology, Affiliated Hangzhou First People's Hospital, Westlake University School of Medicine, China
- Medical College of Jining Medical University, China
- Westlake Laboratory of Life Sciences and Biomedicine, China
Figures
Figure 1
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 of NE in the prosencephalon and brainstem of C57BL/6J mice by ELISA. (D) Content of NE in the prosencephalon and brainstem in the vehicle and midazolam (60 mg/kg, i.p.) groups. (E) Protocol for exploring the influence of i.p. injection of DSP-4 on the atomoxetine-mediated shortening of the recovery time from midazolam administration. (F) Comparison of recovery time in the vehicle, atomoxetine (10 mg/kg, i.p.), and atomoxetine (20 mg/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 + atomoxetine, DSP-4 (3 days before) + atomoxetine, and DSP-4 (10 days before) + atomoxetine 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 i.p. injection of atomoxetine. (K) Images of TH+ neurons in the LC after i.p. injection of vehicle, DSP-4 (3 days), or DSP-4 (10 days) (panels on the lower image show magnified images of the panels on the upper image). (L) Representative images showing the changes in TH (+) neuronal activity with or without i.p. injection of atomoxetine. Mida: midazolam; NE: norepinephrine; i.p.: intraperitoneal injection; LORR: loss of righting reflex; RORR: recovery of righting reflex; LC: locus coeruleus; TH: tyrosine hydroxylase; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.
Figure 2
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 signal 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) Quantification of the percentage of GCaMP6m (+)/TH (+) colocalization in all TH (+) neurons in LC. (E) Schematic diagram of the method of dividing the entire recording of the calcium signal at the LC into four stages, and sources of heatmap and ΔF/F(%) statistical data. (The time points for awake, midazolam injection, LORR, and RORR in mice were respectively selected as time = 0.The selected traces lasting for 20 s were based on the length of a complete Ca2+ signal.) (F) 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.) (G) A statistical diagram of calcium signaling changes in bilateral LCNE neurons induced by midazolam. (H) The peak ΔF/F in the wakefulness, before LORR, LORR-RORR, and after RORR stages. (I, J) Representative images showing the co-expression of c-Fos and TH in the LC without or with midazolam treatment. (K) The quantification of c-Fos (+)/TH (+) cells in the LC with midazolam or without midazolam treatment. (L) Protocol for exploring the influence of intra-LC microinjection DSP-4 on the atomoxetine-mediated shortening of the recovery time from midazolam administration. (M) The representative photomicrograph shows the tracks of cannulas implanted into bilateral LC. (N) Results show the effects of microinjection of DSP-4 (10 days before) into bilateral LC on the recovery time of midazolam. (O) Results show the effects of vehicle + vehicle, vehicle + atomoxetine, and DSP-4 (10 days before) + atomoxetine on the recovery time of midazolam. (P) Comparison of normalized TH (+) cell number in the LC with or without DSP-4 (10 days before) microinjected in the bilateral LC. (Q) 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). LC: locus coeruleus; i.p.: intraperitoneal injection; LORR: loss of righting reflex; RORR: recovery of righting reflex; TH: tyrosine hydroxylase; *p<0.05; **p<0.01; ****p<0.0001.
Figure 3
Optogenetic or chemogenetic activation of LCNE neurons promotes recovery from midazolam.
(A) Protocol for the effect of PS of LCNE neurons with different light parameters on the recovery time of midazolam. (B) A schematic of the optogenetic instrument. (C) A representative photomicrograph showing the locations of optical fiber and virus expression and the co-expression of hChR2 and TH. (D) Quantification of the percentage of eYFP (+)/TH (+) colocalization in all TH (+) neurons in LC. (E, F) Results show the effect of PS of LCNE neurons with different light parameters on the recovery time of midazolam. (G) Results show the effect of left, right, and bilateral PS of the LCNE neurons on the recovery time of midazolam. (H) The quantification of c-Fos (+)/TH (+) cells in the LC with or without PS. (I, J) Representative images showing the co-expression of c-Fos and TH cells in the LC with or without PS. (K, L) Protocol and instrument for the effect of chemogenetic activation of LCNE neurons on the recovery time of midazolam. (M) A representative photomicrograph showing the virus expression and the co-expression of mCherry and TH in the LC of male and female mice. (N, O) Quantification of the percentage of mCherry (+)/TH (+) colocalization in all TH (+) neurons in LC. (P, Q) Results show 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. (R, S) Results show the effect of left, right, and bilateral chemogenetic activation of the LCNE neurons on the recovery time of midazolam in male and female mice. (T) 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). (U, V) The quantification of c-Fos (+)/TH (+) cells in the LC with or without CS. LC: locus coeruleus; i.p.: intraperitoneal injection; LORR: loss of righting reflex; RORR: recovery of righting reflex; TH: tyrosine hydroxylase; PS: photostimulation; CS: chemogenetic stimulation; *p<0.05; ****p<0.0001.
Figure 4
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) Quantification of the percentage of eYFP (+)/TH (+) colocalization in all TH (+) neurons in LC. (F) Quantification of the percentage of GCaMP6m (+)/TH (+) colocalization in all TH (+) neurons in VLPO. (G) Results show the effects of optogenetic activation of LCNE neurons on the recovery time of midazolam. (H, I) A representative image showing the co-expression of hM3Dq and TH in the LC of male and female mice. (J) Quantification of the percentage of mCherry (+)/TH (+) colocalization in all TH (+) neurons in LC. (K) A representative image showing the co-expression of GCaMP6s and TH in the VLPO in female mice. (L) Quantification of the percentage of GCaMP6m (+)/TH (+) colocalization in all TH (+) neurons in VLPO. (M, N) Results show the effects of chemogenetic activation of LCNE neurons on the recovery time of midazolam in male and female mice. (O) Schematic diagram of the method of recording the calcium signal at the VLPO, and sources of statistical data. (P, Q) 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). (The time points for RORR in mice were respectively selected as time = 0. The selected traces lasting for 20 s were based on the length of a complete Ca2+ signal.) (R) The peak ΔF/F in the VLPO with or without activation of LCNE neurons using optogenetics. (S–T, V–W) 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). (U, X) The peak ΔF/F in the VLPO with or without activation of LCNE neurons using chemogenetics in male and female mice. LC: locus coeruleus; VLPO: ventrolateral preoptic nucleus; i.p.: intraperitoneal injection; LORR: loss of righting reflex; RORR: recovery of righting reflex; TH: tyrosine hydroxylase; PS: photostimulation; *p<0.05; **p<0.
Figure 5
Activation of LC-VLPO NEergic neural circuit promotes recovery from midazolam.
(A) Protocol for intra-LC/VLPO 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) Representative images showing the co-expression of hChR2 and TH in the LC. (E) Quantification of the percentage of eYFP (+)/TH (+) colocalization in all TH (+) neurons in LC. (F) Distributions of hChR2-eYFP terminals from LC-NA neurons and DAPI labeling in the VLPO. (G) Results show the effect of LC-VLPO long-range optogenetic activation on the recovery time of midazolam. (H) Protocol for intra-LC/VLPO microinjection of the virus and chemogenetic activation of VLPO. (I, J) Schematic of LC-VLPO long-range chemogenetic activation and the location of chemogenetic virus microinjection and cannula implantation. (K) Representative images showing the co-expression of mCherry and TH in the LC. (L) Quantification of the percentage of mCherry (+)/TH (+) colocalization in all TH (+) neurons in LC. (M) Distributions of hM3Dq-mCherry terminals from LC-NA neurons and DAPI labeling in the VLPO. (N) Results show the effect of LC-VLPO long-range chemogenetic activation on the recovery time of midazolam in male and female mice. LC: locus coeruleus; VLPO: ventrolateral preoptic nucleus; i.p.: intraperitoneal injection; LORR: loss of righting reflex; RORR: recovery of righting reflex; TH: tyrosine hydroxylase; PS: photostimulation; *p<0.05; **p<0.01.
Figure 6
ICV injection or intra-VLPO microinjection of α1-R antagonist reverses the pro-recovery effect of optogenetic or chemogenetic activation of LCNE neurons.
(A) A schematic of ICV 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 show the effects of ICV injection of different doses of phenylephrine, prazosin, clonidine, yohimbine, isoprenaline, and propranolol on the recovery time of midazolam. (I) Results show the effects of ICV injection of phenylephrine (20 mg/mL) and different doses of propranolol on the recovery time of midazolam. (J) Protocol for PS of LCNE neurons and ICV injection or intra-VLPO microinjection of α1-R antagonist. (K) Schematic of the brief process of optogenetic activation. (L) The representative photomicrograph showing microinjection and optical fiber locations of the co-expression of hChR2 and TH in the LC. Moreover, quantification of the percentage of eYFP (+)/TH (+) colocalization in all TH (+) neurons in LC is shown. (M, O) A representative photomicrograph showing the tracks of cannulas implanted into the lateral ventricle and bilateral VLPO. (N, P) Results show the effects of PS of LCNE neurons and ICV injection or intra-VLPO microinjection of prazosin on the recovery time of midazolam. (Q) Protocol for chemogenetic activation of LCNE neurons and ICV injection or intra-VLPO microinjection of an α1-R antagonist. (R) Schematic of the location of virus injection. (S) The representative photomicrograph showing the co-expression of hM3Dq and TH in the LC of male mice. Moreover, quantification of the percentage of mCherry (+)/TH (+) colocalization in all TH (+) neurons in LC is shown. (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 show the effects of chemogenetic activation of LCNE neurons and ICV injection or intra-VLPO microinjection of prazosin on the recovery time of midazolam in male mice. (X) A representative photomicrograph showing the co-expression of hM3Dq and TH in the LC. Moreover, quantification of the percentage of mCherry (+)/TH (+) colocalization in all TH (+) neurons in LC is shown. (Y) The tracks of cannulas implanted into the bilateral VLPO in the female mice. (Z) Results show the effects of chemogenetic activation of LCNE neurons and intra-VLPO microinjection of prazosin on the recovery time of midazolam in female mice. LC: locus coeruleus; VLPO: ventrolateral preoptic nucleus; i.p.: intraperitoneal injection; LORR: loss of righting reflex; RORR: recovery of righting reflex; TH: tyrosine hydroxylase; ICV: intracerebroventricular; PS: photostimulation; *p<0.05; **p<0.01; ***p<0.001.
Figure 7
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. LC: locus coeruleus; LORR: loss of righting reflex; RORR: recovery of righting reflex; PS: photostimulation. *p<0.05; **p<0.01; ***p<0.001.
Figure 8
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 ICV 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 show 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) Quantification of the percentage of GCaMP6m (+)/TH (+) colocalization in all TH (+) neurons in LC. (H) Schematic diagram of the method of recording the calcium signal at the LC and sources of statistical data. (I) A heatmap of calcium signaling changes in bilateral LCNE neurons induced by midazolam with or without ICV 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. The time points for midazolam injection and RORR in mice were respectively selected as time = 0. The selected traces lasting for 20 s were based on the length of a complete Ca2+ signal.) (J) A statistical diagram of calcium signaling changes in bilateral LC with or without ICV injection of gabazine. (K) The peak ΔF/F in the Before RORR and RORR-Recovery stages with or without ICV injection of gabazine. (L) Protocol for intra-LC microinjection of GABAA-R antagonist and intra-VLPO microinjection of α1-R antagonist. (M) A schematic of the position of cannula implantation. (N) A representative photomicrograph showing the tracks of cannulas implanted into the bilateral VLPO. (O) Results show the effects of intra-LC injection of gabazine and intra-VLPO injection of prazosin on the recovery time of midazolam. LC: locus coeruleus; VLPO: ventrolateral preoptic nucleus; i.p.: intraperitoneal injection; LORR: loss of righting reflex; RORR: recovery of righting reflex; TH: tyrosine hydroxylase; ICV.: intracerebroventricular; *p<0.05; **p<0.01; ***p<0.001.
Figure 9
GABAergic and NEergic 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) 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) Quantification of the percentage of GCaMP6m (+)/TH (+) colocalization in all TH (+) neurons in VLPO (Q) Schematic diagram of the method of recording the calcium signal at the VLPO and sources of statistical data. (R, S) 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). (The time points for RORR in mice were respectively selected as time = 0. The selected traces lasting for 20 s were based on the length of a complete Ca2+ signal.) (T) The peak ΔF/F in the VLPO with or without knocking down GABAA-R on LCNE neurons. (U) 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. (V, W) Schematic diagram and the representative photomicrograph showing the positions of cannula implantation. (X, Y) 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. (Z) Results show recovery time between sham, shRNA + vehicle, and shRNA + prazosin. LC: locus coeruleus; VLPO: ventrolateral preoptic nucleus; i.p.: intraperitoneal injection; LORR: loss of righting reflex; RORR: recovery of righting reflex; TH: tyrosine hydroxylase; *p<0.05; ***p<0.001; ****p<0.0001.
Figure 10
Summary of neural mechanism of rebooting the unconsciousness caused by midazolam.
By using pharmacology, optogenetics, chemogenetics, fiber photometry, and gene knockdown, we revealed the role of LC-VLPO noradrenergic neural circuit in regulating midazolam-induced altered consciousness. This effect was mediated by α1 adrenergic receptors. Moreover, gamma-aminobutyric acid receptor type A (GABAA-R) is a mechanistically important binding site in the LC for midazolam. RISC: RNA-induced silencing complex; LC: locus coeruleus; VLPO: ventrolateral preoptic nucleus; NE: norepinephrine.
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Supplementary file 1
Summary table of statistical analysis and reagents conducted throughout the study.
- https://cdn.elifesciences.org/articles/97954/elife-97954-supp1-v1.docx
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- https://cdn.elifesciences.org/articles/97954/elife-97954-mdarchecklist1-v1.pdf
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NE contribution to rebooting unconsciousness caused by midazolam
eLife 13:RP97954.
https://doi.org/10.7554/eLife.97954.3
