Sodium Leak Channel in Glutamatergic Neurons of the Lateral Parabrachial Nucleus Helps Maintain Respiratory Rate Under Sevoflurane Anesthesia

Background

Respiration is a core function for maintaining life activities. General anesthetics and/or opioids often suppress respiratory function. However, the respiratory suppression caused by the intravenous anesthetic propofol is more severe, yet its molecular mechanisms are not fully elucidated. Therefore, studying the impact of general anesthetics on respiratory function has important significance. This study explores the role of glutamatergic neurons in the lateral parabrachial nucleus (PBL) in regulating respiratory rate under sevoflurane anesthesia.

Study Origins

This paper is written by six scientists: Lin Wu, Donghang Zhang, Yujie Wu, Jin Liu, Jingyao Jiang, and Cheng Zhou, affiliated with the Department of Anesthesiology at West China Hospital, Sichuan University, and the National-Local Joint Engineering Research Center of Anesthetic Translational Medicine Laboratory. The paper was accepted by the journal Neuroscience Bulletin on January 15, 2024.

Research Process

Experimental Animal Selection

The study used 8-week-old male C57BL/6J mice weighing 20-22 grams. All animals were housed under standard conditions and strictly followed the guidelines of the Animal Research: Reporting of In Vivo Experiments (ARRIVE).

Virus Injection

Mice were randomly divided into experimental and control groups and injected with viruses containing chemically controlled activation and inhibition receptors. The PBL area was targeted for virus injection, and the expression of the virus was verified through immunofluorescence staining.

In Vivo Chemogenetic Manipulation

Three weeks after virus injection, CNO (clozapine-N-oxide) was injected to activate or inhibit glutamatergic neurons. Subsequent respiratory activity of the mice was recorded using whole-body plethysmography.

Whole-Body Plethysmography

Mouse respiratory activity was measured using a whole-body plethysmography system, recording respiratory rate, tidal volume, and minute ventilation. All mice were acclimated in the testing room for one hour before the experiment began.

Tissue Collection and Immunofluorescence Staining

After the experiment, brainstem tissue sections were analyzed using immunofluorescence staining to identify the expression of glutamatergic neurons and sodium leak channels (NALCN).

In Situ Hybridization and Multichannel Recording

In situ hybridization was used to analyze the expression level of the NALCN gene in glutamatergic neurons. Multichannel recording technology was used to analyze the firing frequency of neurons in the PBL area under different experimental conditions.

Animal Behavioral Experiments and Blood Gas Analysis

Various experiments were conducted to detect the impact of NALCN gene knockout on mouse respiratory activity, along with blood gas analysis to assess oxygen content and partial pressure of carbon dioxide in the mice.

Pain Stimulation

The study also used acute inflammation and tail clip models to simulate pain stimulation, examining the role of NALCN in regulating respiratory rate under pain conditions.

Main Research Results

Chemogenetic Inhibition of PBL Glutamatergic Neurons Reduces Respiratory Rate

Inhibiting PBL glutamatergic neurons resulted in a significant reduction in C-Fos positive cells, indicating marked suppression of glutamatergic neuron activity. Whole-body plethysmography demonstrated that inhibiting glutamatergic neurons significantly reduced the respiratory rate of mice, accompanied by a compensatory increase in tidal volume.

Chemogenetic Activation of PBL Glutamatergic Neurons Increases Respiratory Rate

Activating glutamatergic neurons led to a significant increase in the number of C-Fos positive cells, indicating successful activation of glutamatergic neurons. Whole-body plethysmography showed that activating glutamatergic neurons significantly increased the respiratory rate of mice, with a corresponding reduction in tidal volume.

NALCN Knockout Reduces the Respiratory Regulation Function of PBL Glutamatergic Neurons

Using gene knockout technology to specifically downregulate NALCN expression in PBL glutamatergic neurons resulted in a significant reduction in the respiratory rate of mice, with an increase in tidal volume and no significant change in minute ventilation. Moreover, the firing frequency of PBL glutamatergic neurons significantly decreased, but blood gas analysis indicated that the mice did not experience hypoxia.

NALCN Knockout in PBL GABAergic Neurons Shows No Significant Impact

Knocking out NALCN in PBL GABAergic neurons showed no significant impact, suggesting that the regulatory function of NALCN may be limited to glutamatergic neurons.

NALCN Regulates Respiratory Rate Under Sevoflurane Anesthesia, But Not Under Propofol or Morphine

Further studies found that mice with NALCN knockout exhibited more severe respiratory suppression under sevoflurane anesthesia, but there was no significant impact on respiratory suppression caused by propofol or morphine. The changes in neuronal firing frequency also supported this conclusion.

NALCN Shows No Significant Impact on Pain-Induced Respiratory Response Under Sevoflurane Anesthesia

Under sevoflurane anesthesia, pain stimulation simulated by the tail clip model led to a rapid increase in respiratory rate in both control and NALCN knockout groups, with no significant differences in respiratory rate changes, indicating that NALCN does not play a significant role in the pain-induced respiratory response circuit.

Further Confirmation of NALCN’s Key Role in Respiratory Regulation

Additionally, the study further confirmed that the reduction in respiratory rate caused by NALCN gene knockout can be reversed by activating PBL glutamatergic neurons.

Conclusions and Significance

The study confirms that NALCN is a key ion channel in PBL glutamatergic neurons for regulating respiratory rate under sevoflurane anesthesia but has no significant impact under propofol or morphine anesthesia. This research provides new insights into the mechanisms of sevoflurane anesthesia’s effects on respiratory function and highlights NALCN as a potential target for controlling respiratory activity during sevoflurane anesthesia. This could lay the foundation for developing novel related drugs.

Research Highlights

1. Important Findings: Identification of NALCN as a key ion channel in PBL glutamatergic neurons for regulating respiratory rate.
2. Novel Methods: Using chemogenetic manipulation and gene knockout technology combined with whole-body plethysmography, in situ hybridization, and multichannel recording to comprehensively analyze NALCN’s role.
3. Practical Applications: The research outcomes could aid in developing new drugs to address respiratory suppression issues under sevoflurane anesthesia.

Additional Valuable Information

The study was funded by the National Natural Science Foundation of China and other institutions. All data can be accessed through reasonable requests to the corresponding author. The research conclusions expand our understanding of respiratory regulation mechanisms and have implications for clinical anesthetic practice.