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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. Herein, using pharmacology, optogenetics, chemogenetics, fiber photometry, and gene knockdown, this in vivo research revealed the role of locus coeruleus (LC)-ventrolateral preoptic nucleus noradrenergic 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 consciousness.

Abstract Background Desensitization of G protein–coupled receptors (GPCRs) refers to the attenuation of receptor responsiveness by prolonged or intermittent exposure to agonists. The binding of β-arrestin to the cytoplasmic cavity of the phosphorylated receptor, which competes with the G protein, has been widely accepted as an extensive model for explaining GPCRs desensitization. However, studies on various GPCRs, including dopamine D2-like receptors (D2R, D3R, D4R), have suggested the existence of other desensitization mechanisms. The present study employed D2R/D3R variants with different desensitization properties and utilized loss-of-function approaches to uncover the mechanisms underlying GPCRs homologous desensitization, focusing on the signaling cascade that regulates the ubiquitination of AKT. Results AKT undergoes K8/14 ubiquitination by TRAF6, which occurs in the nucleus and promotes its membrane recruitment, phosphorylation and activation under receptor desensitization conditions. The nuclear entry of TRAF6 relies on the presence of the importin complex. Src regulates the nuclear entry of TRAF6 by mediating the interaction between TRAF6 and importin β1. Ubiquitinated AKT translocates to the plasma membrane where it associates with Mdm2 to phosphorylate it at the S166 and S186 residues. Thereafter, phosphorylated Mdm2 is recruited to the nucleus, resulting in the deubiquitination of β-Arr2. The deubiquitinated β-Arr2 then forms a complex with Gβγ, which serves as a biomarker for GPCRs desensitization. Like in D3R, ubiquitination of AKT is also involved in the desensitization of β2 adrenoceptors. Conclusion Our study proposed that the property of a receptor that causes a change in the subcellular localization of TRAF6 from the cytoplasm to the nucleus to mediate AKT ubiquitination could initiate the desensitization of GPCRs.

The advent of general anesthesia (GA) has significant implications for clinical practice. However, the exact mechanisms underlying GA-induced transitions in consciousness remain elusive. Given some similarities between GA and sleep, the sleep-arousal neural nuclei and circuits involved in sleep-arousal, including the 5-HTergic system, could be implicated in GA. Herein, we utilized pharmacology, optogenetics, chemogenetics, fiber photometry, and retrograde tracing to demonstrate that both endogenous and exogenous activation of the 5-HTergic neural circuit between the dorsal raphe nucleus (DR) and basolateral amygdala (BLA) promotes arousal and facilitates recovery of consciousness from sevoflurane anesthesia. Notably, the 5-HT1A receptor within this pathway holds a pivotal role. Our findings will be conducive to substantially expanding our comprehension of the neural circuit mechanisms underlying sevoflurane anesthesia and provide a potential target for modulating consciousness, ultimately leading to a reduction in anesthetic dose requirements and side effects.

Summary: The locus coeruleus (LC) and noradrenergic neurotransmission are involved in the regulation of sudden unexpected death in epilepsy (SUDEP). Here, we present a protocol for modulating the noradrenergic pathway from LC to heart to prevent SUDEP in acoustic and pentylenetetrazole-induced DBA/1 mouse models of SUDEP. We describe steps for constructing SUDEP models, calcium signal recording, and electrocardiogram monitoring. We then detail measurement of tyrosine hydroxylase content and activity, β1 and p-β1-AR content, and destruction of LCNE neurons.For complete details on the use and execution of this protocol, please refer to Lian et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Sudden unexpected death in epilepsy (SUDEP) is the leading cause of death among epilepsy patients. However, the underlying mechanism remains elusive. Seizure-induced respiratory arrest (S-IRA) is recognized as a main cause of SUDEP, but the contribution of other factors such as cardiac arrhythmias cannot be excluded. Here, we found that both the locus coeruleus (LC) and peripheral noradrenergic neurotransmission were involved in S-IRA and the protective effect of atomoxetine in reducing the occurrence of S-IRA and SUDEP could be reversed by esmolol hydrochloride. Moreover, we investigated the connection between the LC and heart implicated in the modulation of SUDEP by fiber photometry. These data suggested that noradrenergic neurons in the LC might regulate the occurrence of SUDEP through β1-adrenergic receptors on cardiomyocytes. Overall, our findings indicate the involvement of the brain-heart axis in modulating S-IRA and SUDEP and, therefore, will open a new perspective on decoding SUDEP.

Summary: The dorsal raphe nucleus (DR) and the pre-Bötzinger complex (PBC) may play an important role in regulating seizure-induced respiratory arrest (S-IRA), the main contributor to sudden unexpected death in epilepsy. Here, we describe pharmacological, optogenetic, and retrograde labeling approaches to specifically modulate the DR to PBC serotonergic pathway. We detail steps for implanting optical fibers and viral infusion into DR and PBC regions and optogenetic techniques for exploring the role of 5-hydroxytryptophan (5-HT) neural circuit of DR-PBC in S-IRA.For complete details on the use and execution of this protocol, please refer to Ma et al. (2022).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Sudden unexpected death in epilepsy (SUDEP) is the leading cause of death among patients with epilepsy. However, the underlying mechanism of SUDEP remains elusive. Previous studies showed seizure-induced respiratory arrest (S-IRA) is the main factor in SUDEP, and that enhancement of serotonin (5-HT) function in the dorsal raphe nucleus (DR) can significantly reduce the incidence of S-IRA in the DBA/1 mouse model of SUDEP. The pre-Bötzinger complex (PBC), known for its role in regulating respiratory rhythm, can express the 5-HT2A receptor (5-HT2AR). Here, using the pharmacological and optogenetic methods, respectively, we observed that the serotonergic neural circuit between DR and PBC was involved in S-IRA evoked by either acoustic stimulation or pentylenetetrazole (PTZ) injection in the DBA/1 mice, and found 5-HT2AR located in PBC plays an important role in it. Our findings will further significantly improve our understanding of SUDEP and provide a promising therapeutic target for SUDEP prevention.

Vesicular glutamate transporter type 2 (VGLUT2) is known to play an important role in mediating heat hyperalgesia induced by inflammation. However, the underlying mechanism for this activity is poorly understood. Cyclin-dependent kinase 5 (Cdk5), serving as a key regulator in modulating release of glutamate, acted a key player in the formation of heat hyperalgesia of inflammatory pain. However, it remains unknown whether there is a bridge between Cdk5 and VGLUT2 for mediating inflammatory pain. Therefore, we designed the experiment to determine whether VGLUT2 signaling pathway is involved in inflammatory pain mediated by Cdk5 in the inflammatory pain model induced by complete Freund’s adjuvant (CFA). Our results showed that the coexpression of Cdk5/VGLUT2 in small- and medium-sized neuronal cells of the dorsal root ganglion (DRG) and spinal cord between days 1 and 3 following subcutaneous injection of CFA was significantly increased. Moreover, our study revealed that the expression of VGLUT2 protein in the DRG and spinal cord was remarkably increased between days 1 and 3 following CFA injection and was significantly reduced by roscovitine, a selective antagonist of Cdk5. Additionally, p25 but not p35, an activator of Cdk5, protein was significantly increased by CFA and reduced by roscovitine. Our findings suggested that VGLUT2/Cdk5 signaling pathway contributes to inflammatory pain mediated by Cdk5/p25.

We report on a novel optical vortex, named as centrosymmetric optical vortex (CSOV), which is constructed via four conventional optical vortices (OVs) with different topological charges (TCs). The orbital angular momentum (OAM) density satisfies centrosymmetric distribution. Meanwhile, it is confined within a single ring whose radius is determined by the cone angle of an axicon. Furthermore, its magnitude and distribution are modulated by a parameter determined via the TCs of the four OVs, named as phase reconstruction factor. Our work provides a novel detached asymmetric light field, which possesses the potential application in macro-particle manipulation, especially separating cells.

Copper tailings are used as the main raw material, metakaolin is added as the silicon-aluminum correction raw material, and the copper tailings geopolymer is prepared under the action of alkali activator. The effects of different metakaolin content, alkali stimulant modulus, water-cement ratio and curing age on the properties of copper tailings geopolymers were studied, and the copper tailings geopolymer prepared under the optimal conditions were analyzed by X-ray diffraction, Fourier infrared spectroscopy and scanning electron microscope. The results showed that the compressive strength of geopolymer with 20% of metakaolin content, 1.4 alkali excitation method, 0.3 water-cement ratio and 28d curing age reached 88.8MPa, and its solidification efficiency to Cu2+ reached more than 99%. The microscopic morphology and structure analysis show that there are a large amount of N-A-S-H amorphous gel material and C-S-H gel phase in the copper tailings geopolymer. These gel materials are tightly cemented with the mineral crystals serving as the skeleton in the copper tailings, filling between the copper tailings and the metakaolin particles, a dense structure is formed, which ultimately further improves the performance of the geopolymer. [ABSTRACT FROM AUTHOR]

Hepatocellular carcinoma (HCC), ranking as the seventh most prevalent cancer worldwide, poses a significant health challenge. Actinidia chinensis Planch Root extracts (acRoots), a traditional Chinese medicine, has exhibited promising inhibitory effects on the proliferation, invasion, and migration of various cancer cell types. Nevertheless, its specific impact and underlying mechanisms concerning HCC remain unclear. This research aimed to elucidate the anticancer properties and potential molecular mechanisms of acRoots in the HepG2 and LM3 cell lines. Our findings demonstrate that acRoots effectively hampers the in vitro proliferation, migration, and invasion of HCC cells. Furthermore, acRoots induces apoptosis and autophagy by impeding the AKT/mTOR signaling pathway, with its inhibitory effects on cells being restored under AKT activator induction. This study, for the first time, elucidates that acRoots can suppress HepG2 and LM3 cell proliferation by blocking the Akt/mTOR pathway, thereby activating apoptosis and autophagy. These results underscore the potential of acRoots as a promising antitumor agent for HCC.

Under the background of global warming, the studies of quantitative water vapor transport characteristics in arid and semi-arid Central Asia are rare. We examine the spatiotemporal variation of precipitation in Central Asia from 1979 to 2017 based on Global Precipitation Climatology Project (GPCP) and circulation reanalysis data. We simulate the sources and transport of water vapor in different regions of Central Asia in winter and summer using the Hybrid Single-Particle Lagrangian Integrated Trajectory (HYSPLIT) model. The seasonal variation in precipitation over the whole region of Central Asia shows a single minimum in September, with little change in the amount of precipitation in other months; however, there are clear regional and seasonal differences in precipitation in western (region I) and eastern (region II) Central Asia. In region I, the maximum and minimum amounts of precipitation are observed in winter and summer, respectively, whereas in region II, the maximum amount of precipitation occurs in summer and the minimum in winter. The amount of precipitation in winter increased significantly more than the amount of precipitation in summer in both regions I and II from 1979 to 2017. Quantitative calculations of water vapor transport show that Europe and the North Atlantic Ocean are the largest sources of water vapor for regions I and II and contribute > 50% of the transport of water vapor in both winter and summer. The contribution of water vapor from other sources varies greatly in different seasons. For region I, the second highest contribution to water vapor in winter is transport from the South Atlantic Ocean, while in summer is from the Arctic Ocean and northern Asia. For region II, water vapor from the local area (Xinjiang) makes an important contribution in summer. These differences in the transport of water vapor are related to seasonal adjustments in the atmospheric circulation system. In winter, straight westerly winds can bring large amounts of water vapor from the Atlantic Ocean and the Mediterranean and Caspian seas. In summer, the northerly currents from the Arctic Ocean are strong due to the anticyclonic circulation maintained in Central Asia. The water vapor flux budget across each border and the net budget in region I are higher than those in region II in winter. The response of water vapor transport to global warming in Central Asia shows a north shift during past 40 years.