Identifying presynaptic mechanisms of general anesthetics is critical to understanding their effects on synaptic transmission. We show that the volatile anesthetic isoflurane inhibits synaptic vesicle (SV) exocytosis at nerve terminals in dissociated rat hippocampal neurons through inhibition of presynaptic Ca 2+ influx without significantly altering the Ca 2+ sensitivity of SV exocytosis. A clinically relevant concentration of isoflurane (0.7 mM) inhibited changes in [Ca 2+ ] i driven by single action potentials (APs) by 25 ± 3%, which in turn led to 62 ± 3% inhibition of single AP-triggered exocytosis at 4 mM extracellular Ca 2+ ([Ca 2+ ] e ). Lowering external Ca 2+ to match the isoflurane-induced reduction in Ca 2+ entry led to an equivalent reduction in exocytosis. These data thus indicate that anesthetic inhibition of neurotransmitter release from small SVs occurs primarily through reduced axon terminal Ca 2+ entry without significant direct effects on Ca 2+ -exocytosis coupling or on the SV fusion machinery. Isoflurane inhibition of exocytosis and Ca 2+ influx was greater in glutamatergic compared with GABAergic nerve terminals, consistent with selective inhibition of excitatory synaptic transmission. Such alteration in the balance of excitatory to inhibitory transmission could mediate reduced neuronal interactions and network-selective effects observed in the anesthetized central nervous system. GCaMP3 | pHlourin | mechanisms of anesthesia | live cell imaging | presynaptic T he molecular and cellular mechanisms of anesthetic-induced amnesia, unconsciousness and immobilization are incompletely understood, particularly for the modern halogenated ether derivatives like isoflurane. General anesthetics, which are essential to both medical practice and experimental neuroscience, have potent and selective effects on neurotransmission (1), including both presynaptic actions (reduced neurotransmitter release) and postsynaptic actions (modulation of receptor function). These effects contribute to anesthetic-induced reductions in neuronal interactions, which are critical to information processing and consciousness (2-4). Knowledge of the fundamental synaptic effects of anesthetics is therefore essential to a molecular and physiological understanding of anesthetic mechanisms, and to development of more selective and safer anesthetics.Although postsynaptic electrophysiological effects of anesthetics can be assessed directly using whole cell recordings and heterologous expression of putative molecular targets, their presynaptic actions have been difficult to resolve by conventional approaches that do not clearly discriminate between presynaptic and postsynaptic contributions. Direct evidence for presynaptic effects of volatile anesthetics includes selective inhibition of glutamate release from isolated nerve terminals (5, 6) and of synaptic vesicle (SV) exocytosis in intact hippocampal neurons (7). However, it remains controversial whether these effects involve direct inhibition of SV exocytosis itself or of upstrea...

The abnormal tumor microenvironment fuels tumor progression, metastasis, immune suppression, and treatment resistance. Over last several decades, developments in and applications of intravital microscopy have provided unprecedented insights into the dynamics of the tumor microenvironment. In particular, intravital multiphoton microscopy has revealed the abnormal structure and function of tumor-associated blood and lymphatic vessels, the role of aberrant tumor matrix in drug delivery, invasion and metastasis of tumor cells, the dynamics of immune cell trafficking to and within tumors, and gene expression in tumors. However, traditional multiphoton microscopy suffers from inherently slow imaging rates—only a few frames per second, thus unable to capture more rapid events such as blood flow, lymphatic flow, and cell movement within vessels. Here, we report the development and implementation of a video-rate multiphoton microscope (VR-MPLSM) based on resonant galvanometer mirror scanning that is capable of recording at 30 frames per second and acquiring intravital multispectral images. We show that the design of the system can be readily implemented and is adaptable to various experimental models. As examples, we demonstrate the utility of the system to directly measure flow within tumors, capture metastatic cancer cells moving within the brain vasculature and cells in lymphatic vessels, and image acute responses to changes in a vascular network. VR-MPLSM thus has the potential to further advance intravital imaging and provide new insight into the biology of the tumor microenvironment.

Background: P/Q-and N-type voltage-gated calcium channels (VGCC) are the principal subtypes mediating synaptic vesicle (SV) exocytosis. Both the degree of isoflurane inhibition of SV exocytosis and VGCC subtype expression vary between brain regions and neurotransmitter phenotype. We hypothesised that differences in VGCC subtype expression contribute to synapse-selective presynaptic effects of isoflurane. Methods: We used quantitative live-cell imaging to measure exocytosis in cultured rat hippocampal neurones after transfection of the fluorescent biosensor vGlut1-pHluorin. Selective inhibitors of P/Q-and N-type VGCCs were used to isolate subtype-specific effects of isoflurane. Results: Inhibition of N-type channels by 1 mM u-conotoxin GVIA reduced SV exocytosis to 81±5% of control (n¼10). Residual exocytosis mediated by P/Q-type channels was further inhibited by isoflurane to 42±4% of control (n¼10). The P/ Q-type channel inhibitor u-agatoxin IVA at 0.4 mM inhibited SV exocytosis to 29±3% of control (n¼10). Residual exocytosis mediated by N-type channels was further inhibited by isoflurane to 17±3% of control (n¼10). Analysis of isoflurane effects at the level of individual boutons revealed no difference in sensitivity to isoflurane between P/Q-or N-type channelmediated SV exocytosis (P¼0.35). There was no correlation between the effect of agatoxin (P¼0.91) or conotoxin (P¼0.15) and the effect of isoflurane on exocytosis. Conclusions: Sensitivity of SV exocytosis to isoflurane in rat hippocampal neurones is independent of the specific VGCC subtype coupled to exocytosis. The differential sensitivity of VGCC subtypes to isoflurane does not explain the observed neurotransmitter-selective effects of isoflurane in hippocampus.

containment of both droplets and aerosols, the ability to clear the bag of aerosolised particles with negative air flow, and the protection of the operator and environment. Airway devices are supported on hooks, which increases the working space available. During a simulated airway management training session of our COVID-19 intubation team, direct vision, communication, and manoeuverability were accomplished for 12 operators. Even though our results are preliminary and qualitative in nature, we demonstrate proof of concept for an additional physical barrier during aerosol-generating procedures. We believe that this easily constructed barrier, and others, have the potential to protect healthcare providers in caring for confirmed or suspected COVID-19 patients. These include patients undergoing monitored anaesthesia care or regional anaesthesia, treated with noninvasive ventilation, in overcrowded emergency department or wards, in endoscopy/bronchoscopy suites, in radiology suites, and during patient transportation. We acknowledge that further research and testing are necessary to quantify the contamination level within the multipurpose portable negative air flow protection isolation chamber and whether it is possible to inactivate any virus before removal and doffing of the plastic drape and frame. We remain concerned that healthcare providers may develop a false sense of security if these barriers are used, so we strongly emphasise that local guidelines for PPE be maintained, and that new devices being developed during this healthcare crisis are used mainly as an additional complementary resource, not a replacement, to face the undeniable scarcity of PPE.

Pain is a prevalent biopsychosocial condition that poses a significant challenge to healthcare providers, contributes substantially to disability, and is a major economic burden worldwide. An overreliance on opioid analgesics, which primarily target the μ-opioid receptor, has caused devastating morbidity and mortality in the form of misuse and overdose-related death. Thus, novel analgesic medications are needed that can effectively treat pain and provide an alternative to opioids. A variety of cellular ion channels contribute to nociception, the response of the sensory nervous system to a noxious stimulus that commonly leads to pain. Ion channels involved in nociception may provide a suitable target for pharmacologic modulation to achieve pain relief. This narrative review summarizes the evidence for two ion channels that merit consideration as targets for non-opioid pain medications: ryanodine receptors (RyRs), which are intracellular calcium channels, and hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which belong the superfamily of voltage-gated K+ channels. The role of these channels in nociception and neuropathic pain is discussed and suitability as targets for novel analgesics and antihyperalgesics is considered.

Control of neurotransmission efficacy is central to theories of how the brain computes and stores information. Presynaptic G-protein coupled receptors (GPCRs) are critical in this problem as they locally influence synaptic strength and can operate on a wide range of time scales. Among the mechanisms by which GPCRs impact neurotransmission is by inhibiting voltage-gated calcium (Ca2+) influx in the active zone. Here, using quantitative analysis of both single bouton Ca2+ influx and exocytosis, we uncovered an unexpected non-linear relationship between the magnitude of action potential driven Ca2+ influx and the concentration of external Ca2+ ([Ca2+]e). We find that this unexpected relationship is leveraged by GPCR signaling when operating at the nominal physiological set point for [Ca2+]e, 1.2 mM, to achieve complete silencing of nerve terminals. These data imply that the information throughput in neural circuits can be readily modulated in an all-or none fashion at the single synapse level when operating at the physiological set point.

increased and wake time after sleep onset was decreased during melatonin treatment (both P¼0.04). Latency and efficiency were not affected. Participants gave positive feedback on the acceptability of trial design and organisation. We report here that the trial design for MIDNIGHT was both feasible and acceptable and that melatonin treatment in healthy night shift workers was well tolerated. During the first two night shifts staff reported sleepiness and had slower reaction times but showed signs of adaptation by shift 3. The study was not powered to show effects of melatonin, but there was some evidence that mid shift sleepiness was decreased and subjects slept longer when they took melatonin. These data will inform a sample size calculation for a definitive trial.

Control of neurotransmission efficacy is central to theories of how the brain computes and stores information. Neuromodulators are critical in this problem as they locally influence synaptic strength and can operate on a wide range of time scales. Presynaptic function is heavily influenced by G-protein coupled receptors (GPCRs) that in part restrict voltage-gated calcium (Ca2+) influx in the active zone. Here, using quantitative analysis of both single bouton Ca2+ influx and exocytosis, we uncovered an unexpected non-linear relationship between the magnitude of action potential driven Ca2+ influx and the concentration of external Ca2+ ([Ca2+]e). We find that this unexpected relationship is leveraged by neuromodulator signaling when operating at the nominal physiological set point for [Ca2+]e, 1.2 mM, to achieve complete silencing of nerve terminals. These data imply that the information throughput in neural circuits can be readily modulated in an all-or none fashion at the single synapse level when operating at the physiological set point.