NE contribution to rebooting unconsciousness caused by midazolam

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.

Methods
Evaluation of recovery time

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.

Statistical analysis

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. P < 0.05 indicated statistical significance.