DOI: 10.5061/dryad.2rbnzs7zs
title: NE contribution to rebooting unconsciousness caused by midazolam

description: The advent of midazolam holds profound implications for modern clinical
 practice. The hypnotic and sedative effects of midazolam afford it broad
 clinical applicability. However, the specific mechanisms underlying the
 modulation of altered consciousness by midazolam remain
 elusive.Therefore,we collated data on changes in time to consciousness
 recovery in mice through pharmacological, optogenetics,
 chemogenetics,fiber photometry, and gene knockout interventions to reveal
 the role of the locus cyanulus (LC) -ventrolateral preoptic nucleus (VLPO)
 noadrenergic 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)
 represents a mechanistically crucial binding site in the LC for midazolam.
 These findings will provide novel insights into the neural circuit
 mechanisms underlying the recovery of consciousness after midazolam
 administration and will help guide the timing of clinical dosing and
 propose effective intervention targets for timely recovery from
 midazolam-induced loss of consciousne.
description: <strong>Evaluation of recovery
 time</strong> We collated data on changes in time
 to consciousness recovery in mice through pharmacological, optogenetics,
 chemogenetics,fiber photometry, and gene knockout interventions to reveal
 the role of the locus cyanulus (LC) -ventrolateral preoptic nucleus (VLPO)
 noadrenergic neural circuit  in regulating midazolam-induced altered
 consciousness.Before the experiment, the bottom of the open box with no
 lid and air circulation was covered with cotton pads, and an electric
 blanket was placed on the lower side of the open box, and the temperature
 in the open box was preheated for 10 min before the experiment to keep the
 temperature in the open box suitable and constant during the experiment.
 Eight-week-old C57BL/6J mice were placed in the open box for 30 min, and
 then placed in the open box again after intraperitoneal injection of the
 appropriate dose of midazolam (60 mg/kg) to start the timer. At the same
 time, the open box was gently rotated every 15 s. When the mice were
 rotated to the dorsal recumbent position and could not be voluntarily
 turned over for more than 1 min, it was the loss of righting reflex
 (LORR), i.e., loss of consciousness from midazolam. Subsequently, the mice
 were placed in the dorsal recumbent position for continuous observation,
 and when the mice could autonomously return to the normal position from
 the dorsal recumbent position, they were considered as recovery of
 righting reflex (RORR). The time from the onset of LORR to RORR was
 recorded as the Recovery Time of midazolam.
 <strong>Statistical analysis</strong>
 All the experimental data were reported as mean ± SD. GraphPad
 Prism (GraphPad Software, Inc., San Diego, CA, USA) and SPSS (SPSS
 Software Inc., Chicago, IL, USA) were used for data visualization and
 statistical analysis, respectively. Before data analysis, all experimental
 data were subjected to the Shapiro-Wilk normality test. For comparative
 analyses of the two groups, if the data were normally distributed,
 Student's t-test, including independent samples t-test and paired
 samples t-test, was used. Conversely, if the data were non-normally
 distributed, the Mann–Whitney U test or Wilcoxon signed-rank test was
 used. Notably, the Levene test was used to evaluate the homogeneity of
 variances. After the data met the normal distribution and homogeneity of
 variances, a one-way analysis of variance followed by Bonferroni’s
 multiple comparison test was used for multiple comparisons.
 <em>P</em> &lt; 0.05 indicated statistical
 significance.
description: # NE contribution to rebooting unconsciousness caused by midazolam
 [https://doi.org/10.5061/dryad.2rbnzs7zs](https://doi.org/10.5061/dryad.2rbnzs7zs) ## Description of the data and file structure The advent of midazolam holds profound implications for modern clinical practice. The hypnotic and sedative effects of midazolam afford it broad clinical applicability. However, the specific mechanisms underlying the modulation of altered consciousness by midazolam remain elusive.Therefore,we collated data on changes in time to consciousness recovery in mice through pharmacological, optogenetics, chemogenetics,fiber photometry, and gene knockout interventions to reveal the role of the locus cyanulus (LC) -ventrolateral preoptic nucleus (VLPO) noadrenergic 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) represents a mechanistically crucial binding site in the LC for midazolam. These findings will provide novel insights into the neural circuit mechanisms underlying the recovery of consciousness after midazolam administration and will help guide the timing of clinical dosing and propose effective intervention targets for timely recovery from midazolam-induced loss of consciousne. ### Files and variables We have submitted our data  (**Recovery time_Data.xls**). ### **Recovery time\_Data** **Description:**  * **Strain,Sex,Age and Weight:** The experimental animals in this study were male and female wild-type C57BL/6J mice purchased from the Animal Experiment Center of Zhejiang University. All mice were housed and bred in the SPF-Class House in a standard condition (indoor temperature 25℃, ambient humidity 65%, 12-h/12-h light/dark cycle) with rodent food and water available *ad libitum*. All mice were aged 8 weeks and weighed 22–25 g at the start of the experiments. * **Group and Treatment**: We divided the recovery time of mice into different groups according to different treatments. *Group 1-5,18-24,30-32:* Different pharmacological experiments 。 IP= Intraperitoneal Injections 。LC MI= Microinjection into Locus Coeruleus 。DSP-4= N-(2-chloroethyl)-N-ethyl-2-bromoben-zylamine hydrochloride (DSP-4) (50 mg/kg), a highly selective NEergic neurotoxin, reduced the number of TH+ cells in the Locus Coeruleus. 。Atomoxetine=A central selective NE reuptake inhibitor (20 mg/kg, IP) 。ICV=Intra-cerebroventricular injection  。Phenylephrine=  The α1 receptor agonist  **。** Prazosin=The α1 receptor antagonist 。Clonidine=The α2 receptor agonist 。Yohimbine=The α2 receptor antagonist 。Isoprenaline=The β agonist 。Propranolol=The β receptor antagonist  。Gabazine=A GABAA receptor antagonist 。VLPO MI=Microinjection into Ventrolateral Preoptic Nucleus *Group 6-8,12,15,25-26: *Three weeks before the experiment began, the LC or VLPO was microinjected with optogenetic virus. One week before the experiment began, the LC or VLPO was implanted with optical fibers. The mice were intraperitoneally injected with midazolam, and 20 min later, they were given 465 nm blue light to activate neurons in the LC or VLPO. In this study, we used different parameters for optogenetic activation: 20 min/2mW; 20 min/4mW; 10 min/4mW; and 20 min/4mW. We finally identified 20 min/4mW as the optimal optogenetic activation parameter and used it in the subsequent experiments. 。LC= Locus Coeruleus 。PS= Photostimulation 。VLPO=Ventrolateral Preoptic Nucleus *Group 9-10,13-14,16-17,28-29*: Three weeks before starting the experiment, chemogenetic viruses were microinjected into the LC or VLPO. Clozapine-N-oxide (CNO) was dissolved in saline, and the mice were intraperitoneally injected with CNO 20 min after intraperitoneally injected with midazolam to activate neurons using the chemogenetics approach. In this study, we tested two concentrations of CNO: 0.1 mg/kg and 0.2 mg/kg. We concluded that 0.2 mg/kg was the optimal concentration and used this concentration for the subsequent intraperitoneal administration.  。CNO= Clozapine-N-oxide *Group 33*: We microinjected 80nL of rAAV-DBH-EGFP-S'miR-30a-shRNA (GABAA receptor)-3’-miR30a-WPRES into the LC. Four weeks later, these mice were sacrificed and perfused for counting the GABAA-R (+) and tyrosine hydroxylase-positive (TH+) cells to evaluate the effect of GABAA-R knockdown. 。Sham= Sham group was set up at the same time.