Li Chen

Background: Glutamatergic neurons in the supramammillary nucleus (SuM) have been recently identified as a key node in arousal system, yet their role in regulating general anesthesia remains unclear. The aim of the current study is to examine the role of the glutamatergic supramammillary neurons and their projections to the medial septum in mediating the effects of isoflurane anesthesia. Methods: Fiber photometry recording was used to determine the changes in calcium signals of glutamatergic neurons in the SuM during isoflurane anesthesia. Optogenetic and chemogenetic approaches were employed to manipulate SuM glutamatergic neuron activity, and the effects on cortical activity, behavioral responses, and physiologic parameters—including pupil diameter, respiratory rate, and blood pressure—were examined in anesthetized mice. Both male and female mice were used in this study. Results: The activities of SuM glutamatergic neurons decreased during isoflurane anesthesia and recovered after the emergence. Optogenetic activation of these neurons enhanced cortical activity, decreasing electroencephalogram delta power (mean ± SD, prestimulation vs. stimulation: 51.35 ± 7.26% vs. 32.08 ± 10.48%, n = 8, P = 0.002) and burst suppression ratio (81.82 ± 7.83% vs. 44.53 ± 28.62%, n = 8, P = 0.002). Furthermore, optogenetic activation altered physiologic parameters including enlarged pupil diameter (prestimulation vs. stimulation: 1.05 ± 0.08% vs. 1.95 ± 0.46%, n = 8, P < 0.001), increased respiratory rate (0.98 ± 0.08% vs. 1.57 ± 0.39%, n = 10, P < 0.001) and elevated blood pressure and induced behavioral responses including increased arousal scores and accelerated emergence (light off vs. light on, 171.40 ± 56.39 s to 59.88 ± 27.18 s, n = 8, P = 0.007). Moreover, chemogenetic activation produced similar effects, whereas inhibition led to opposite effects. Finally, optogenetically activating SuM glutamatergic terminals projecting to the medial septum mimicked the effects of activating SuM glutamatergic soma and increased the activity of medial septum glutamatergic neurons. Conclusions: This study identifies glutamatergic neurons of the SuM as key neural substrates regulating isoflurane anesthesia and facilitating emergence through their projections to the medial septum.

Sleep-onset insomnia, characterized by difficulty falling asleep, is linked to increased health risks. Previous studies have shown that the central amygdala (CeA) plays a crucial role in stress regulation, with the somatostatin neurons in the CeA (CeA^SST+) involved in adaptive stress responses. However, the role of CeA^SST+ neurons in stress-induced sleep-onset insomnia remains unclear. In this study, we found that the activity of CeA^SST+ neurons is closely associated with stressful events using fiber photometry in mice. Acute optogenetic activation of CeA^SST+ neurons induced a rapid transition from non-rapid eye movement (NREM) sleep to wakefulness. Semi-chronic optogenetic and chemogenetic activation of CeA^SST+ neurons led to prolonged sleep-onset latency and increased wakefulness. Chemogenetic inhibition of these neurons ameliorated sleep-onset insomnia induced by stressful stimuli, but did not affect sleep-wake behavior under physiological conditions. Collectively, our results suggested that CeA^SST+ neurons are a key neural substrate for modulating stress-induced sleep-onset insomnia, without influencing physiological sleep. These findings highlight CeA^SST+ neurons as a promising target for treating stress-related sleep-onset insomnia in clinical practice.

Diabetic neuropathic pain (DNP) is a common chronic complication of diabetes mellitus and a clinically common form of neuropathic pain. The thalamus is an important center for the conduction and modulation of nociceptive signals. The paraventricular thalamic nucleus (PVT) is an important midline nucleus of the thalamus involved in sensory processing, but the specific role of PVT astrocytes and GABAergic neurons in DNP remains unclear. Here, we examined the activity of PVT astrocytes and neurons at various time points during the development of DNP by fluorescence immunohistochemistry and found that the activity of PVT astrocytes was significantly increased while that of PVT neurons was significantly decreased 14 d after streptozotocin injection in male rats. The inhibition of PVT astrocytes by chemogenetic manipulation relieved mechanical allodynia in male DNP model rats, whereas the activation of PVT astrocytes induced mechanical allodynia in normal male rats. Interestingly, chemogenetic activation of GABAergic neurons in the PVT alleviated mechanical allodynia in male DNP model rats, whereas chemogenetic inhibition of GABAergic neurons in the PVT induced mechanical allodynia in normal male rats. These data demonstrate the distinct roles of PVT astrocytes and GABAergic neurons in modulating DNP, revealing the mechanism of DNP pathogenesis and the role of the PVT in pain modulation.

Background: Recent evidence indicates that general anesthesia and sleep–wake behavior share some overlapping neural substrates. γ-Aminobutyric acid–mediated (GABAergic) neurons in the central amygdala have a high firing rate during wakefulness and play a role in regulating arousal-related behaviors. The objective of this study was to investigate whether central amygdala GABAergic neurons participate in the regulation of isoflurane general anesthesia and uncover the underlying neural circuitry. Methods: Fiber photometry recording was used to determine the changes in calcium signals of central amygdala GABAergic neurons during isoflurane anesthesia in Vgat-Cre mice. Chemogenetic and optogenetic approaches were used to manipulate the activity of central amygdala GABAergic neurons, and a righting reflex test was used to determine the induction and emergence from isoflurane anesthesia. Cortical electroencephalogram (EEG) recording was used to assess the changes in EEG spectral power and burst–suppression ratio during 0.8% and 1.4% isoflurane anesthesia, respectively. Both male and female mice were used in this study. Results: The calcium signals of central amygdala GABAergic neurons decreased during the induction of isoflurane anesthesia and were restored during the emergence. Chemogenetic activation of central amygdala GABAergic neurons delayed induction time (mean ± SD, vehicle vs. clozapine-N-oxide: 58.75 ± 5.42 s vs. 67.63 ± 5.01 s; n = 8; P = 0.0017) and shortened emergence time (385.50 ± 66.26 s vs. 214.60 ± 40.21 s; n = 8; P = 0.0017) from isoflurane anesthesia. Optogenetic activation of central amygdala GABAergic neurons produced a similar effect. Furthermore, optogenetic activation decreased EEG delta power (prestimulation vs. stimulation: 46.63 ± 4.40% vs. 34.16 ± 6.47%; n = 8; P = 0.0195) and burst–suppression ratio (83.39 ± 5.15% vs. 52.60 ± 12.98%; n = 8; P = 0.0003). Moreover, optogenetic stimulation of terminals of central amygdala GABAergic neurons in the basal forebrain also promoted cortical activation and accelerated behavioral emergence from isoflurane anesthesia. Conclusions: The results suggest that central amygdala GABAergic neurons play a role in general anesthesia regulation, which facilitates behavioral and cortical emergence from isoflurane anesthesia through the GABAergic central amygdala–basal forebrain pathway.

General anesthesia shares many similarities with natural sleep in behavior and electroencephalogram (EEG) patterns. The latest evidence suggests that general anesthesia and sleep–wake behavior may share overlapping neural substrates. The GABAergic neurons in the basal forebrain (BF) have recently been demonstrated to play a key role in controlling wakefulness. It was hypothesized that BF GABAergic neurons may participate in the regulation of general anesthesia. Here, usingin vivofiber photometry, we found that the activity of BF GABAergic neurons was generally inhibited during isoflurane anesthesia, having obviously decreased during the induction of anesthesia and being gradually restored during the emergence from anesthesia, in Vgat-Cre mice of both sexes. Activation of BF GABAergic neurons with chemogenetic and optogenetic approaches decreased sensitivity to isoflurane, delayed induction, and accelerated emergence from isoflurane anesthesia. Optogenetic activation of BF GABAergic neurons decreased EEG δ power and the burst suppression ratio (BSR) during 0.8% and 1.4% isoflurane anesthesia, respectively. Similar to the effects of activating BF GABAergic cell bodies, photostimulation of BF GABAergic terminals in the thalamic reticular nucleus (TRN) also strongly promoted cortical activation and behavioral emergence from isoflurane anesthesia. Collectively, these results showed that the GABAergic BF is a key neural substrate for general anesthesia regulation that facilitates behavioral and cortical emergence from general anesthesia via the GABAergic BF-TRN pathway. Our findings may provide a new target for attenuating the depth of anesthesia and accelerating emergence from general anesthesia.SIGNIFICANCE STATEMENTThe basal forebrain (BF) is a key brain region controlling sleep–wake behavior. Activation of GABAergic neurons in the BF potently promotes behavioral arousal and cortical activity. Recently, many sleep–wake-related brain structures have been reported to participate in the regulation of general anesthesia. However, it is still unclear what role BF GABAergic neurons play in general anesthesia. In this study, we aim to reveal the role of BF GABAergic neurons in behavioral and cortical emergence from isoflurane anesthesia and elucidate the underlying neural pathways. Understanding the specific role of BF GABAergic neurons in isoflurane anesthesia would improve our understanding of the mechanisms of general anesthesia and may provide a new strategy for accelerating emergence from general anesthesia.

Background: The parabrachial nucleus (PBN) is an important structure regulating the sleep–wake behavior and general anesthesia. Astrocytes in the central nervous system modulate neuronal activity and consequential behavior. However, the specific role of the parabrachial nucleus astrocytes in regulating the sleep-wake behavior and general anesthesia remains unclear.Methods: We used chemogenetic approach to activate or inhibit the activity of PBN astrocytes by injecting AAV-GFAabc1d-hM3Dq-eGFP or AAV-GFAabc1d-hM4Di-eGFP into the PBN. We investigated the effects of intraperitoneal injection of CNO or vehicle on the amount of wakefulness, NREM sleep and REM sleep in sleep–wake behavior, and on the time of loss of righting reflex, time of recovery of righting reflex, sensitivity to isoflurane, electroencephalogram (EEG) power spectrum and burst suppression ratio (BSR) in isoflurane anesthesia.Results: The activation of PBN astrocytes increased wakefulness amount for 4 h, while the inhibition of PBN astrocytes decreased total amount of wakefulness during the 3-hour post-injection period. Chemogenetic activation of PBN astrocytes decreased isoflurane sensitivity and shortened the emergence time from isoflurane-induced general anesthesia. Cortical EEG recordings revealed that PBN astrocyte activation decreased the EEG delta power and BSR during isoflurane anesthesia. Chemogenetic Inhibition of PBN astrocytes increased the EEG delta power and BSR during isoflurane anesthesia.Conclusion: PBN astrocytes are a key neural substrate regulating wakefulness and emergence from isoflurane anesthesia.

Background and Purpose: New remedies are required for the treatment of diabetic neuropathic pain (DNP) due to insufficient efficacy of available therapies. Here, we used chemogenetic approaches combined with in vivo pharmacology to elucidate the role of basolateral amygdala (BLA) astrocytes in DNP pathogenesis and provide new insights into therapeutic strategies for DNP.Experimental Approach: A streptozotocin‐induced DNP model was established. Designer receptors exclusively activated by designer drugs (DREADDs) were used to regulate astrocyte activity. Mechanical hyperalgesia was assessed using the electronic von Frey test. Anxiety‐like behaviours were detected using open field and elevated plus maze tests. Astrocytic activity was detected by immunofluorescence, and cytokine content was determined by ELISA.Key Results: BLA astrocytes were regulated by DREADDs, and inhibition of BLA astrocytes attenuated mechanical allodynia and pain‐related negative emotions in DNP rats. In contrast, temporary activation of BLA astrocytes induced allodynia without anxious behaviours in naive rats. In addition, koumine (KM) alleviated mechanical allodynia and anxiety‐like behaviours in DNP rats, inhibited the activation of BLA astrocytes and suppressed the inflammatory response. Furthermore, persistent activation of BLA astrocytes through chemogenetics mimicked chronic pain, and KM alleviated the pain hypersensitivity and anxiety‐like behaviours.Conclusion and Implications: DREADDs bidirectionally regulate the activity of BLA astrocytes, which proves for the first time the role of BLA astrocyte activation in the pathogenesis of DNP and represents a novel therapeutic strategy for DNP. KM ameliorates DNP, perhaps by inhibiting the activation of BLA astrocytes and reveal KM as a potential candidate for treating DNP.