The Functions Of Medial Parabrachial Nucleus Neurons On CO₂-induced Microarousal

Background:Sleep apnea was defined as some repeated airflow stopping and that resulted in hypoxia and hypercapnic carbogen.Both hypoxia and hypercapnic carbogen could induce microarousal to terminate apnea through stimulating the central nervous system.There are many studies on intermittent hypoxia inducing microarousal.However,there are relatively few researches on the neural mechanism of CO₂-induced microarousal.The brain circuit related to arousal is very complex.Recently some studies have demonstrated that parabrachial nucleus（PB）participate in the arousal,however,the function of parabrachial nucleus neurons on CO₂-induced microarousal remains poorly understood.In the present study,the microarousal was induced by administration of higher CO₂concentration during sleep in mice to simulate the effects of hypercapnia in clinical sleep apnea disease.Then the effects of hypercapnic carbogen on PB neurons was observed in vivo and in vitro.Method:First,recording electrodes were placed in the frontal cortex and hippocampus and silver wire electrodes were placed in the cervical muscles of mice in a stereotaxic apparatus to record cortical EEG and hippocampal LFP during sleep.High CO₂concentration was administered to induce microarousal when the sleep shift to the slow-wave sleep,and the power and power density were analyzed in the cortical EEG and hippocampal LFP frequency bands.Secondly,the number of c-fos positive cells was observed by immunohistochemical staining in the LPB,MPB and KF neurons of the control group and the high CO₂concentration group.Finally,whole-cell patch-clamp was used to record AP and s EPSCs of the MPB neurons in current clamp and voltage clamp modes respectively.Results:First,we observed the effects of hypercapnic air on cortical EEG and hippocampal LFP during slow-wave sleep in the microarousal mice model induced by administration of hypercapnic air.The hypercapnic air was administered slowly when the sleep shift to the slow-wave sleep in mice.When CO₂concentration was increased to 6.81±2.38%in the cage,the microarousal was induced.That was indicated by the amplitudes shifting to low-amplitude fast wave in cortical EEG and hippocampal LFP and EMG was activated briefly.The power and power density ofδ,θ,αandβband on cortical EEG andα,β,γand ripples on hippocampal LFP were reduced significantly during CO₂-induced microarousal.Secondly,the results of immunohistochemical staining showed that the number of c-fos positive cells in the MPB and KF increased significantly in the hypercapnic air mice compared with normal air mice,while the expression of c-fos protein in the LPB showed an increasing trend but no statistical significance.Finally,whole cell current clamp results showed that hypercapnia increased the frequency of the evoked firing in the MPB neurons and decreased the peak amplitude of action potential.When glutamate receptors were blocked,hypercapnia did not increase the frequency of the evoked firing in the MPB neurons,suggesting that glutamate receptors might mediate hypercapnia induced changes in the electrophysiological activity of the MPB neurons in brain slices of mice.In addition,whole cell voltage clamp results showed that hypercapnia increased the s EPSCs frequency in the MPB neurons in brain slices of mice,indicating that hypercapnia could increase the synaptic activity of the MPB neurons in brain slices of mice.Conclusion:High CO₂concentration can induce microarousal and activate neurons in the MPB,increasing MPB neuronal excitability and synaptic transmission mediated by glutamate receptors.