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Anesthesiologists are increasingly encountering sicker patients that require potentially life-saving surgical interventions, and assess risk using the American Society of Anesthesiology Physical Status (ASA PS) classification system. Here, we examined long-term mortality along with hospital length of stay (LoS) and discharge disposition for survivors in ASA PS 5 and 5E patients.Adult surgeries were extracted from New York-Presbyterian Hospital/Weill Cornell Medical Center's Electronic Medical Record (EMR) for cases between January 1, 2013 and December 31, 2017; outcomes were collected from EMRs and the Social Security Death Index Master File.194,947 cases were identified. Mortality correlated with increasing ASA PS; the same trend was observed within both emergent and non-emergent sub-populations. Two hundred seventy-six cases were identified as 5/5E. This patient population had a higher rate of mortality at 30 days than at 48 hours (25.9% vs. 13.4%, respectively, p0.01); there was no difference between survivor functions at 30 or 90 days (p = 0.63, p = 0.09, respectively). Survivors within the 5 or 5E subpopulations did not have significantly different LoSs. Further, survivors after 90 days typically had a disposition of hospice, long-term facilities, inpatient rehabilitation, or self-discharged.Mortality increases with increases in ASA PS classifications. There is no difference in outcomes for 5 vs 5E at 30- or 90-day postoperatively. Similarly, emergency status did not play a role in LoS. Most 5 or 5E patients are not discharged home but to another facility. These outcomes should be considered during the informed consent process in this high-risk surgical population.

The pace of human brain development is highly protracted compared to most other species. The maturation of cortical neurons is particularly slow, extending over months to years to reach adult-like functions. Remarkably, such protracted timing is retained in cortical neurons derived from human pluripotent stem cell (hPSC) during in vitro differentiation or upon transplantation into the murine brain in vivo. Those findings suggest the presence of a cell intrinsic clock that sets the pace of neuronal maturation, though the molecular nature of such a clock has remained elusive.Here, we identify an epigenetic developmental program which sets the timing of human neuronal maturation. First, we developed a human PSC-based approach to synchronize the birth of cortical neurons in vitro which allowed us to define a detailed atlas of progressive morphological, functional, and molecular maturation in human cortical neurons. Interestingly, we observed a slow, temporal unfolding of maturation programs that is limited by the retention of a specific set of epigenetic factors. Loss-of-function studies for several of those factors in cortical neurons enables precocious molecular and physiological maturation. Remarkably, transient inhibition of EZH2, EHMT1/2 or DOT1L, at the progenitor stage primes newly born neurons to rapidly acquire mature properties upon differentiation. Therefore, our findings reveal that the rate at which human neurons mature is set well before neurogenesis through the establishment of an “epigenetic barrier” in progenitor cells. Mechanistically, this barrier acts by holding transcriptional maturation programs in a poised state that gets gradually released during neuronal differentiation to ensure the prolonged timeline characteristic of human cortical neuron maturation.

BACKGROUND: High intraoperative PEEP with recruitment manoeuvres may improve perioperative outcomes. We re-examined this question by conducting a patient-level meta-analysis of three clinical trials in adult patients at increased risk for postoperative pulmonary complications who underwent non-cardiothoracic and non-neurological surgery. METHODS: The three trials enrolled patients at 128 hospitals in 24 countries from February 2011 to February 2018. All patients received volume-controlled ventilation with low tidal volume. Analyses were performed using one-stage, two-level, mixed modelling (site as a random effect; trial as a fixed effect). The primary outcome was a composite of postoperative pulmonary complications within the first week, analysed using mixed-effect logistic regression. Pre-specified subgroup analyses of nine patient characteristics and seven procedure and care-delivery characteristics were also performed. RESULTS: Complete datasets were available for 1913 participants ventilated with high PEEP and recruitment manoeuvres, compared with 1924 participants who received low PEEP. The primary outcome occurred in 562/1913 (29.4%) participants randomised to high PEEP, compared with 620/1924 (32.2%) participants randomised to low PEEP (unadjusted odds ratio [OR]=0.87; 95% confidence interval [95% CI], 0.75-1.01; P=0.06). Higher PEEP resulted in 87/1913 (4.5%) participants requiring interventions for desaturation, compared with 216/1924 (11.2%) participants randomised to low PEEP (OR=0.34; 95% CI, 0.26-0.45). Intraoperative hypotension was associated more frequently (784/1913 [41.0%]) with high PEEP, compared with low PEEP (579/1924 [30.1%]; OR=1.87; 95% CI, 1.60-2.17). CONCLUSIONS: High PEEP combined with recruitment manoeuvres during low tidal volume ventilation in patients undergoing major surgery did not reduce postoperative pulmonary complications. CLINICAL TRIAL REGISTRATION: NCT03937375 (Clinicaltrials.gov).

General anesthesia has been administered for over 150 years, and in that time, has become progressively safer. Improvements in outcomes have been driven by multiple advances, including the use of non-invasive monitors to assess cardiovascular and respiratory status. More recent advances have included the development and use of monitors to measure neurologic status by means of “processed” electroencephalography (pEEG), wherein the frontal EEG signal is analyzed by proprietary algorithms to produce a dimensionless number (scaled from 0 to 100), wherein low values are associated with deepening levels of sedation that progresses to loss of consciousness. Such monitors have been shown to enable anesthetic titration so as to expedite emergence and early recovery, and their use is advocated for the prevention of intraoperative awareness in the setting of administration of total intravenous anesthesia and neuromuscular blockade. Whether their use can minimize, or prevent, longer term adverse events is a matter of debate. In this narrative review of the most recent literature, we provide an assessment on the use of pEEG monitors in the prevention of a notable, and important, postoperative adverse outcome – delirium – in elderly patients. As we will discuss, the existing data do not support its routine use for the prevention of postoperative delirium in this, or any other, patient population.

Pain is a prevalent biopsychosocial condition that poses a significant challenge to healthcare providers, contributes substantially to a 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 to 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.

Summary Clinical research involving human subjects and quality improvement (QI) projects share a common goal of seeking to improve human health, whether by directly changing the standard of care (research) or by improving the process(es) by which that care is delivered (QI). Whether a QI project requires informed consent (written or oral) is a function of the risk–benefit analysis of the study; such a determination should not be at the sole discretion of the investigators, but should come from an appropriately constituted review board with expertise in the ethics of biomedical research.

The mechanistic basis of gliogenesis, which occurs late in human development, is poorly understood. Here we identify nuclear factor IA (NFIA) as a molecular switch inducing human glial competency. Transient expression of NFIA is sufficient to trigger glial competency of human pluripotent stem cell-derived neural stem cells within 5 days and to convert these cells into astrocytes in the presence of glial-promoting factors, as compared to 3–6 months using current protocols. NFIA-induced astrocytes promote synaptogenesis, exhibit neuroprotective properties, display calcium transients in response to appropriate stimuli and engraft in the adult mouse brain. Differentiation involves rapid but reversible chromatin remodeling, glial fibrillary acidic protein (GFAP) promoter demethylation and a striking lengthening of the G1 cell cycle phase. Genetic or pharmacological manipulation of G1 length partially mimics NFIA function. We used the approach to generate astrocytes with region-specific or reactive features. Our study defines key mechanisms of the gliogenic switch and enables the rapid production of human astrocytes for disease modeling and regenerative medicine. A new method generates astrocytes from human pluripotent stem cells in 5 days.

Purpose of review Various neurologically focused monitoring modalities such as processed electroencephalography (pEEG), tissue/brain oxygenation monitors (SbO2), and even somatosensory evoked responses have been suggested as having the potential to improve the well tolerated and effective delivery of care in the setting of outpatient surgery. The present article will discuss the pros and cons of such monitors in this environment. Recent findings There is a paucity of evidence from rigorous, well designed clinical trials demonstrating that the routine use of any neuromonitoring technique in an ambulatory surgery setting leads to meaningful cost savings or a reduction in morbidity or mortality. Summary The use of advanced neuromonitoring techniques (primarily pEEG) may be considered reasonable in two instances: for the prevention of intraoperative awareness during the administration of total intravenous anesthesia coupled with the use of a neuromuscular blocking drug, and for the prevention of relative drug overdose (and possibly postoperative delirium) in the elderly.

This study sought to determine if the ability of sodium bicarbonate and ammonium bicarbonate to inhibit aflatoxigenesis in microbiological media could be extended to corn systems. A method for applying bicarbonates evenly to corn was developed. Aflatoxin levels of sodium bicarbonate-treated (0.17 % wt/wt) corn were reduced to one-third those of untreated corn when both were inoculated with Aspergillus parasiticus spores and incubated at 30°C for three weeks. While both sodium and ammonium bicarbonate reduced the amount of fungal growth, only sodium bicarbonate reduced aflatoxin production.

Parvalbumin-expressing, fast spiking interneurons have high-energy demands, which make them particularly susceptible to energy impairment. Recent evidence suggests a link between mitochondrial dysfunction in fast spiking cortical interneurons and neuropsychiatric disorders. However, the effect of mitochondrial dysfunction restricted to parvalbumin interneurons has not been directly addressed in vivo. To investigate the consequences of mitochondrial dysfunction in parvalbumin interneurons in vivo, we generated conditional knockout mice with a progressive decline in oxidative phosphorylation by deleting cox10 gene selectively in parvalbumin neurons (PV-Cox10 CKO). Cox10 ablation results in defective assembly of cytochrome oxidase, the terminal enzyme of the electron transfer chain, and leads to mitochondrial bioenergetic dysfunction. PV-Cox10 CKO mice showed a progressive loss of cytochrome oxidase in cortical parvalbumin interneurons. Cytochrome oxidase protein levels were significantly reduced starting at postnatal day 60, and this was not associated with a change in parvalbumin interneuron density. Analyses of intrinsic electrophysiological properties in layer 5 primary somatosensory cortex revealed that parvalbumin interneurons could not sustain their typical high frequency firing, and their overall excitability was enhanced. An increase in both excitatory and inhibitory input onto parvalbumin interneurons was observed in PV-Cox10 CKO mice, resulting in a disinhibited network with an imbalance of excitation/inhibition. Investigation of network oscillations in PV-Cox10 CKO mice, using local field potential recordings in anesthetized mice, revealed significantly increased gamma and theta frequency oscillation power in both medial prefrontal cortex and hippocampus. PV-Cox10 CKO mice did not exhibit muscle strength or gross motor activity deficits in the time frame of the experiments, but displayed impaired sensory gating and sociability. Taken together, these data reveal that mitochondrial dysfunction in parvalbumin interneurons can alter their intrinsic physiology and network connectivity, resulting in behavioral alterations similar to those observed in neuropsychiatric disorders, such as schizophrenia and autism.

Significant progress has been made in the 21st century towards a comprehensive understanding of the mechanisms of action of general anesthetics, coincident with progress in structural biology and molecular, cellular, and systems neuroscience. This review summarizes important new findings that include target identification through structural determination of anesthetic binding sites, details of receptors and ion channels involved in neurotransmission, and the critical roles of neuronal networks in anesthetic effects on memory and consciousness. These recent developments provide a comprehensive basis for conceptualizing pharmacological control of amnesia, unconsciousness, and immobility.

Background As minimally invasive spine surgery evolves, spine surgeons increasingly rely on advanced intraoperative computed tomography (iCT). iCT provides rapid acquisition of high-resolution images, reduces radiation exposure, improves surgical accuracy, and decreases operative time. However, all iCT systems currently available pose a patient safety risk as their physical space requirements limit patient access in the event of an emergency, particularly when patients are in the prone position. After a near-cardiac arrest at our institution during posterior cervical spine surgery, it was apparent that the presence of the iCT complicated the ability to rapidly reposition the patient in order to provide appropriate resuscitation. Methods To ensure our ability to provide timely care during an emergency, we determined that a process which included all members of the operating room (OR) team was required. We held an initial planning meeting where a detailed plan-of-action was created, reviewed, and revised in response to feedback from all stakeholders. We then simulated a cardiac arrest to test our resuscitation plan with all members of the neurosurgery team. A mannequin was positioned prone on an OR table within the iCT, and a resuscitation plan was created. Results The team orchestrated the mock resuscitation, and the time of cardiac arrest in the prone position to supine repositioning required 110 seconds. The simulation was recorded for post-“code” performance review. Application of the protocol during an actual cardiac arrest was associated with successful restoration of spontaneous circulation and full recovery. Conclusions The development and rehearsal of an emergency plan of action greatly facilitated the timely responsiveness of the neurosurgical OR team during a simulated cardiac arrest and was an effective way to identify and address key logistical issues regarding the use of an iCT system.

Retrospective studies indicate that acetaminophen iv administration reduces hospital length of stay (LoS) and opiate consumption in patients undergoing bariatric surgery.This study sought to determine whether using acetaminophen iv in morbidly obese subjects undergoing sleeve gastrectomy decreased LoS and total hospital charges as compared to patients receiving saline placebo.Single-center university hospital METHODS: Using a randomized, double-blind, placebo-controlled design, subjects were assigned to receive either acetaminophen iv (group A) or saline placebo iv (group P). Data were collected between Jan 1 and Dec 31, 2016. Group A received acetaminophen every 6 h for a total of four doses. The first dose was administered following the induction of general anesthesia; group P received saline iv on the same schedule. Anesthetic management and prophylactic antiemetic regimen were standardized in all subjects. Postoperative pain management consisted of hydromorphone via patient-controlled infusion pump. Primary outcomes include hospital LoS and associated hospital costs. Secondary outcomes include patient satisfaction and postoperative nausea and pain scores.Subject demographics (n = 127) and intraoperative management were similar in the two groups. Across all subjects, median hospital LoS in group A (n = 63) was 1.87 vs. 1.97 days in group P (n = 64) (p = 0.03, Wilcoxon rank-sum test). Postoperatively, daily quality-of-recovery (QoR-15) scores, narcotic consumption, and the use of rescue antiemetics were not significantly different between groups. Median hospital costs were as follows: group A, $12,885 vs. group P, $12,977 (n = 64).Acetaminophen iv may reduce hospital LoS in subjects undergoing sleeve gastrectomy.

The ability of a diverse group of agents to produce general anesthesia has long been an area of intense speculation and investigation. Over the past century, we have seen a paradigm shift from proposing that the anesthetized state arises from nonspecific interaction of anesthetics with the lipid membrane to the recognition that the function of distinct, and identifiable, membrane-embedded proteins is dramatically altered in the presence of intravenous and inhaled agents. Among proteinaceous targets, metabotropic and ionotropic receptors garnered much of the attention over the last 30 years, and it is only relatively recently that voltage-gated ion channels have clearly and rigorously been shown to be important molecular targets. In this review, we will consider the experimental issues relevant to two important ion channel anesthetic targets, HCN and K

Capturing the full potential of human pluripotent stem cell (PSC)-derived neurons in disease modeling and regenerative medicine requires analysis in complex functional systems. Here we establish optogenetic control in human PSC-derived spinal motorneurons and show that co-culture of these cells with human myoblast-derived skeletal muscle builds a functional all-human neuromuscular junction that can be triggered to twitch upon light stimulation. To model neuromuscular disease we incubated these co-cultures with IgG from myasthenia gravis patients and active complement. Myasthenia gravis is an autoimmune disorder that selectively targets neuromuscular junctions. We saw a reversible reduction in the amplitude of muscle contractions, representing a surrogate marker for the characteristic loss of muscle strength seen in this disease. The ability to recapitulate key aspects of disease pathology and its symptomatic treatment suggests that this neuromuscular junction assay has significant potential for modeling of neuromuscular disease and regeneration.

The derivation of somatic motoneurons (MNs) from ES cells (ESCs) after exposure to sonic hedgehog (SHH) and retinoic acid (RA) is one of the best defined, directed differentiation strategies to specify fate in pluripotent lineages. In mouse ESCs, MN yield is particularly high after RA + SHH treatment, whereas human ESC (hESC) protocols have been generally less efficient. In an effort to optimize yield, we observe that functional MNs can be derived from hESCs at high efficiencies if treated with patterning molecules at very early differentiation steps before neural induction. Remarkably, under these conditions, equal numbers of human MNs were obtained in the presence or absence of SHH exposure. Using pharmacological and genetic strategies, we demonstrate that early RA treatment directs MN differentiation independently of extrinsic SHH activation by suppressing the induction of GLI3. We further demonstrate that neural induction triggers a switch from a poised to an active chromatin state at GLI3. Early RA treatment prevents this switch by direct binding of the RA receptor at the GLI3 promoter. Furthermore, GLI3 knock-out hESCs can bypass the requirement for early RA patterning to yield MNs efficiently. Our data demonstrate that RA-mediated suppression of GLI3 is sufficient to generate MNs in an SHH-independent manner and that temporal changes in exposure to patterning factors such as RA affect chromatin state and competency of hESC-derived lineages to adopt specific neuronal fates. Finally, our work presents a streamlined platform for the highly efficient derivation of human MNs from ESCs and induced pluripotent stem cells. SIGNIFICANCE STATEMENT Our study presents a rapid and efficient protocol to generate human motoneurons from embryonic and induced pluripotent stem cells. Surprisingly, and in contrast to previous work, motoneurons are generated in the presence of retinoic acid but in the absence of factors that activate sonic hedgehog signaling. We show that early exposure to retinoic acid modulates the chromatin state of cells to be permissive for motoneuron generation and directly suppresses the induction of GLI3, a negative regulator of SHH signaling. Therefore, our data point to a novel mechanism by which retinoic acid exposure can bypass the requirement for extrinsic SHH treatment during motoneuron induction.

Background: The thalamus is thought to be crucially involved in the anesthetic state. Here, we investigated the effect of the inhaled anesthetic xenon on stimulus-evoked thalamocortical network activity and on excitability of thalamocortical neurons. Because hyperpolarization-activated, cyclic nucleotide-gated cation (HCN) channels are key regulators of neuronal excitability in the thalamus, the effect of xenon on HCN channels was examined. Methods: The effects of xenon on thalamocortical network activity were investigated in acutely prepared brain slices from adult wild-type and HCN2 knockout mice by means of voltage-sensitive dye imaging. The influence of xenon on single-cell excitability in brain slices was investigated using the whole-cell patch-clamp technique. Effects of xenon on HCN channels were verified in human embryonic kidney cells expressing HCN2 channels. Results: Xenon concentration-dependently diminished thalamocortical signal propagation. In neurons, xenon reduced HCN channel-mediated Ih current amplitude by 33.4 ± 12.2% (at −133 mV; n = 7; P = 0.041) and caused a left-shift in the voltage of half-maximum activation (V1/2) from −98.8 ± 1.6 to −108.0 ± 4.2 mV (n = 8; P = 0.035). Similar effects were seen in human embryonic kidney cells. The impairment of HCN channel function was negligible when intracellular cyclic adenosine monophosphate level was increased. Using HCN2−/− mice, we could demonstrate that xenon did neither attenuate in vitro thalamocortical signal propagation nor did it show sedating effects in vivo. Conclusions: Here, we clearly showed that xenon impairs HCN2 channel function, and this impairment is dependent on intracellular cyclic adenosine monophosphate levels. We provide evidence that this effect reduces thalamocortical signal propagation and probably contributes to the hypnotic properties of xenon.

The ability of a diverse group of agents to produce general anesthesia has long been an area of intense speculation and investigation. Over the past century, we have seen a paradigm shift from proposing that the anesthetized state arises from nonspecific interaction of anesthetics with the lipid membrane to the recognition that the function of distinct, and identifiable, membrane-embedded proteins is dramatically altered in the presence of intravenous and inhaled agents. Among proteinaceous targets, metabotropic and ionotropic receptors garnered much of the attention over the last 30 years, and it is only relatively recently that voltage-gated ion channels have clearly and rigorously been shown to be important molecular targets. In this review, we will consider the experimental issues relevant to two important ion channel anesthetic targets, HCN and K2P.

SummaryHuman pluripotent stem cells are a powerful tool for modeling brain development and disease. The human cortex is composed of two major neuronal populations: projection neurons and local interneurons. Cortical interneurons comprise a diverse class of cell types expressing the neurotransmitter GABA. Dysfunction of cortical interneurons has been implicated in neuropsychiatric diseases, including schizophrenia, autism, and epilepsy. Here, we demonstrate the highly efficient derivation of human cortical interneurons in an NKX2.1::GFP human embryonic stem cell reporter line. Manipulating the timing of SHH activation yields three distinct GFP+ populations with specific transcriptional profiles, neurotransmitter phenotypes, and migratory behaviors. Further differentiation in a murine cortical environment yields parvalbumin- and somatostatin-expressing neurons that exhibit synaptic inputs and electrophysiological properties of cortical interneurons. Our study defines the signals sufficient for modeling human ventral forebrain development in vitro and lays the foundation for studying cortical interneuron involvement in human disease pathology.

In the course of primary infection with herpes simplex virus 1 (HSV-1), children with inborn errors of toll-like receptor 3 (TLR3) immunity are prone to HSV-1 encephalitis (HSE) 1–3 .W e tested the hypothesis that the pathogenesis of HSE involves nonhaematopoietic CNS-resident cells. We derived induced pluripotent stem cells (iPSCs) from the dermal fibroblasts of TLR3- and UNC-93B-deficient patients and from controls. These iPSCs were differentiated into highly purified populations of neural stem cells (NSCs), neurons, astrocytes and oligodendrocytes. The induction of interferon-b (IFN-b) and/or IFN-l1 in response to stimulation by the dsRNA analogue polyinosinic:polycytidylic acid (poly(I:C)) was dependent on TLR3 and UNC-93B in all cells tested. However, the induction of IFN-b and IFN-l1 in response to HSV-1 infection was impaired selectively in UNC-93B-deficient neurons and oligodendrocytes. These cells were also much more susceptible to HSV-1 infection than control cells, whereas UNC-93B-deficient NSCs and astrocytes were not. TLR3-deficient neurons were also found to be susceptible to HSV-1 infection. The rescue of UNC-93B- and TLR3deficient cells with the corresponding wild-type allele showed that the genetic defect was the cause of the poly(I:C) and HSV-1 phenotypes. The viral infection phenotype was rescued further by treatment with exogenous IFN-a or IFN-b ( IFN-a/b) but not IFN-l1. Thus, impaired TLR3- and UNC-93B-dependent IFN-a/b intrinsic immunity to HSV-1 in the CNS, in neurons and oligodendrocytes in particular, may underlie the pathogenesis of HSE in children with TLR3-pathway deficiencies. Childhood HSE is a rare, life-threatening, central nervous system (CNS)-restricted complication of primary infection with HSV-1, an almost ubiquitous virus that is typically innocuous 4 . Children with HSE are not unusually susceptible to other infectious agents, including viruses, or even to HSV-1-related diseases affecting sites other than the CNS 4,5 . HSV-1 reaches the CNS from the oral and nasal epithelium, via the cranial nerves 4 . We identified autosomal recessive UNC-93B deficiency as the first genetic aetiology of childhood HSE 1 . UNC-93B is required for TLR3, TLR7, TLR8 and TLR9 responses 1,6 . We then identified autosomal-recessive or autosomal-dominant deficiencies of TLR3 (refs 2 and 3), TRAF3 (ref. 7), TRIF 8 and TBK1 (ref. 9), revealing

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels generate a pacemaking current, I(h), which regulates neuronal excitability and oscillatory activity in the brain. Although all four HCN isoforms are expressed in the brain, the functional contribution of HCN3 is unknown. Using immunohistochemistry, confocal microscopy, and whole-cell patch-clamp recording techniques, we investigated HCN3 function in thalamic intergeniculate leaflet (IGL) neurons, as HCN3 is reportedly preferentially expressed in these cells. We observed that I(h) recorded from IGL, but not ventral geniculate nucleus, neurons in HCN2(+/+) mice and rats activated slowly and were cAMP insensitive, which are hallmarks of HCN3 channels. We also observed strong immunolabeling for HCN3, with no labeling for HCN1 and HCN4, and only very weak labeling for HCN2. Deletion of HCN2 did not alter I(h) characteristics in mouse IGL neurons. These data together indicate that the HCN3 channel isoform generated I(h) in IGL neurons. Intracellular phosphatidylinositol-4,5-bisphosphate (PIP(2)) shifted I(h) activation to more depolarized potentials and accelerated activation kinetics. Upregulation of HCN3 function by PIP(2) augmented low-threshold burst firing and spontaneous oscillations; conversely, depletion of PIP(2) or pharmacologic block of I(h) resulted in a profound inhibition of excitability. The results indicate that functional expression of HCN3 channels in IGL neurons is crucial for intrinsic excitability and rhythmic burst firing, and PIP(2) serves as a powerful modulator of I(h)-dependent properties via an effect on HCN3 channel gating. Since the IGL is a major input to the suprachiasmatic nucleus, regulation of pacemaking function by PIP(2) in the IGL may influence sleep and circadian rhythms.

GABAergic neurons in the reticular thalamic nucleus (RTN) synapse onto thalamocortical neurons in the ventrobasal (VB) thalamus, and this reticulo-thalamocortical pathway is considered an anatomic target for general anesthetic-induced unconsciousness. A mutant mouse was engineered to harbor two amino acid substitutions (S270H, L277A) in the GABA(A) receptor (GABA(A)-R) alpha1 subunit; this mutation abolished sensitivity to the volatile anesthetic isoflurane in recombinant GABA(A)-Rs, and reduced in vivo sensitivity to isoflurane in the loss-of-righting-reflex assay. We examined the effects of the double mutation on GABA(A)-R-mediated synaptic currents and isoflurane sensitivity by recording from thalamic neurons in brain slices. The double mutation accelerated the decay, and decreased the (1/2) width of, evoked inhibitory postsynaptic currents (eIPSCs) in VB neurons and attenuated isoflurane-induced prolongation of the eIPSC. The hypnotic zolpidem, a selective modulator of GABA(A)-Rs containing the alpha1 subunit, prolonged eIPSC duration regardless of genotype, indicating that mutant mice incorporate alpha1 subunit-containing GABA(A)-Rs into synapses. In RTN neurons, which lack the alpha1 subunit, eIPSC duration was longer than in VB, regardless of genotype. Isoflurane reduced the efficacy of GABAergic transmission from RTN to VB, independent of genotype, suggesting a presynaptic action in RTN neurons. Consistent with this observation, isoflurane inhibited both tonic action potential and rebound burst firing in the presence of GABA(A)-R blockade. The suppressed excitability in RTN neurons is likely mediated by isoflurane-enhanced Ba(2+)-sensitive, but 4-aminopyridine-insenstive, potassium conductances. We conclude that isoflurane enhances inhibition of thalamic neurons in VB via GABA(A)-R-dependent, but in RTN via GABA(A)-R-independent, mechanisms.

Mutations in human KCNE2, which encodes the MiRP1 potassium channel ancillary subunit, associate with long QT syndrome (LQTS), a defect in ventricular repolarization. The precise cardiac role of MiRP1 remains controversial, in part, because it has marked functional promiscuity in vitro. Here, we disrupted the murine kcne2 gene to define the role of MiRP1 in murine ventricles. kcne2 disruption prolonged ventricular action potential duration (APD), suggestive of reduced repolarization capacity. Accordingly, kcne2 (−/−) ventricles exhibited a 50% reduction in IK,slow1, generated by Kv1.5—a previously unknown partner for MiRP1. Ito,f, generated by Kv4 α subunits, was also diminished, by ∼25%. Ventricular MiRP1 protein coimmunoprecipitated with native Kv1.5 and Kv4.2 but not Kv1.4 or Kv4.3. Unexpectedly, kcne2 (−/−) ventricular membrane fractions exhibited 50% less mature Kv1.5 protein than wild type, and disruption of Kv1.5 trafficking to the intercalated discs. Consistent with the reduction in ventricular K+ currents and prolonged ventricular APD, kcne2 deletion lengthened the QTc under sevoflurane anesthesia. Thus, targeted disruption of kcne2 has revealed a novel cardiac partner for MiRP1, a novel role for MiRPs in α subunit targeting in vivo, and a role for MiRP1 in murine ventricular repolarization with parallels to that proposed for the human heart.—Roepke, T. K., Kontogeorgis, A., Ovanez, C., Xu, X., Young, J. B., Purtell, K., Goldstein, P. A., Christini, D. J., Peters, N. S., Akar, F. G., Gutstein, D. E., Lerner, D. J., Abbott, G. W. Targeted deletion of kcne2 impairs ventricular repolarization via disruption of IK,slow1 and Ito,f.

Neuropathic pain arises from injury to the nervous system. Conditions associated with neuropathic pain are diverse, and lesions and/or pathological changes in the central nervous system (CNS) or peripheral nervous system (PNS) can frequently, but not always, be identified. It is difficult to treat, with patients often on multiple, different classes of medications, all with appreciable adverse side effect profiles. Consequently, there is a pressing need for the development of new medications. The development of such therapeutics is predicated on a clear understanding of the relevant molecular and cellular processes that contribute to the development, and maintenance, of the neuropathic pain state. One proposed mechanism thought to contribute to the ontogeny of neuropathic pain is altered expression, trafficking, and functioning of ion channels expressed by primary sensory neurons. Here, we will focus on three voltage-gated ion channel families, CaV, HCN, and NaV, first reviewing the preclinical data and then the human data where it exists.

Volatile anesthetics are used clinically to produce analgesia, amnesia, unconsciousness, blunted autonomic responsiveness, and immobility. Previous work has shown that the volatile anesthetic isoflurane, at concentrations that produce unconsciousness (250-500 microM), enhances fast synaptic inhibition in the brain mediated by GABA(A) receptors (GABA(A)-Rs). In addition, isoflurane causes sedation at concentrations lower than those required to produce unconsciousness or analgesia. In this study, we found that isoflurane, at low concentrations (25-85 microM) associated with its sedative actions, elicits a sustained current associated with a conductance increase in thalamocortical neurons in the mouse ventrobasal (VB) nucleus. These isoflurane-evoked currents reversed polarity close to the Cl(-) equilibrium potential and were totally blocked by the GABA(A)-R antagonist gabazine. Isoflurane (25-250 microM) produced no sustained current in VB neurons from GABA(A)-R alpha(4)-subunit knockout (Gabra4(-/-)) mice, although 250 microM isoflurane enhanced synaptic inhibition in VB neurons from both wild-type and Gabra4(-/-) mice. These data indicate an obligatory requirement for alpha(4)-subunit expression in the generation of the isoflurane-activated current. In addition, isoflurane directly activated alpha(4)beta(2)delta GABA(A)-Rs expressed in human embryonic kidney 293 cells, and it was more potent at alpha(4)beta(2)delta than at alpha(1)beta(2)gamma(2) receptors (the presumptive extrasynaptic and synaptic GABA(A)-R subtypes in VB neurons). We conclude that the extrasynaptic GABA(A)-Rs of thalamocortical neurons are sensitive to low concentrations of isoflurane. In view of the crucial role of the thalamus in sensory processing, sleep, and cognition, the modulation of these extrasynaptic GABA(A)-Rs by isoflurane may contribute to the sedation and hypnosis associated with low doses of this anesthetic agent.

Hyperpolarization activated cyclic nucleotide (HCN) gated channels conduct a current,Ih; howIhinfluences excitability and spike firing depends primarily on channel distribution in subcellular compartments. For example, dendritic expression of HCN1 normalizes somatic voltage responses and spike output in hippocampal and cortical neurons. We reported previously that HCN2 is predominantly expressed in dendritic spines in reticular thalamic nucleus (RTN) neurons, but the functional impact of such nonsomatic HCN2 expression remains unknown. We examined the role of HCN2 expression in regulating RTN excitability and GABAergic output from RTN to thalamocortical relay neurons using wild-type and HCN2 knock-out mice. Pharmacological blockade ofIhsignificantly increased spike firing in RTN neurons and large spontaneous IPSC frequency in relay neurons; conversely, pharmacological enhancement of HCN channel function decreased spontaneous IPSC frequency. HCN2 deletion abolishedIhin RTN neurons and significantly decreased sensitivity to 8-bromo-cAMP and lamotrigine. Recapitulating the effects ofIhblock, HCN2 deletion increased both temporal summation of EPSPs in RTN neurons as well as GABAergic output to postsynaptic relay neurons. The enhanced excitability of RTN neurons afterIhblock required activation of ionotropic glutamate receptors; consistent with this was the colocalization of HCN2 and glutamate receptor 4 subunit immunoreactivities in dendritic spines of RTN neurons. The results indicate that, in mouse RTN neurons, HCN2 is the primary functional isoform underlyingIhand expression of HCN2 constrains excitatory synaptic integration.

The thalamus is thought to be crucially involved in the anesthetic state. Here, we investigated the effect of the inhaled anesthetic xenon on stimulus-evoked thalamocortical network activity and on excitability of thalamocortical neurons. Because hyperpolarization-activated, cyclic nucleotide-gated cation (HCN) channels are key regulators of neuronal excitability in the thalamus, the effect of xenon on HCN channels was examined.The effects of xenon on thalamocortical network activity were investigated in acutely prepared brain slices from adult wild-type and HCN2 knockout mice by means of voltage-sensitive dye imaging. The influence of xenon on single-cell excitability in brain slices was investigated using the whole-cell patch-clamp technique. Effects of xenon on HCN channels were verified in human embryonic kidney cells expressing HCN2 channels.Xenon concentration-dependently diminished thalamocortical signal propagation. In neurons, xenon reduced HCN channel-mediated Ih current amplitude by 33.4 ± 12.2% (at -133 mV; n = 7; P = 0.041) and caused a left-shift in the voltage of half-maximum activation (V1/2) from -98.8 ± 1.6 to -108.0 ± 4.2 mV (n = 8; P = 0.035). Similar effects were seen in human embryonic kidney cells. The impairment of HCN channel function was negligible when intracellular cyclic adenosine monophosphate level was increased. Using HCN2 mice, we could demonstrate that xenon did neither attenuate in vitro thalamocortical signal propagation nor did it show sedating effects in vivo.Here, we clearly showed that xenon impairs HCN2 channel function, and this impairment is dependent on intracellular cyclic adenosine monophosphate levels. We provide evidence that this effect reduces thalamocortical signal propagation and probably contributes to the hypnotic properties of xenon.

Although the depressant effects of the general anesthetic propofol on thalamocortical relay neurons clearly involve gamma-aminobutyric acid (GABA)(A) receptors, other mechanisms may be involved. The hyperpolarization-activated cation current (I(h)) regulates excitability and rhythmic firing in thalamocortical relay neurons in the ventrobasal (VB) complex of the thalamus. Here we investigated the effects of propofol on I(h)-related function in vitro and in vivo. In whole-cell current-clamp recordings from VB neurons in mouse (P23-35) brain slices, propofol markedly reduced the voltage sag and low-threshold rebound excitation that are characteristic of the activation of I(h). In whole-cell voltage-clamp recordings, propofol suppressed the I(h) conductance and slowed the kinetics of activation. The block of I(h) by propofol was associated with decreased regularity and frequency of delta-oscillations in VB neurons. The principal source of the I(h) current in these neurons is the hyperpolarization-activated cyclic nucleotide-gated (HCN) type 2 channel. In human embryonic kidney (HEK)293 cells expressing recombinant mouse HCN2 channels, propofol decreased I(h) and slowed the rate of channel activation. We also investigated whether propofol might have persistent effects on thalamic excitability in the mouse. Three hours following an injection of propofol sufficient to produce loss-of-righting reflex in mice (P35), I(h) was decreased, and this was accompanied by a corresponding decrease in HCN2 and HCN4 immunoreactivity in thalamocortical neurons in vivo. These results suggest that suppression of I(h) may contribute to the inhibition of thalamocortical activity during propofol anesthesia. Longer-term effects represent a novel form of propofol-mediated regulation of I(h).

The GABAergic reticular thalamic nucleus (RTN) is a major source of inhibition for thalamocortical neurons in the ventrobasal complex (VB). Thalamic circuits are thought to be an important anatomic target for general anesthetics. We investigated presynaptic actions of the intravenous anesthetic propofol in RTN neurons, using RTN-retained and RTN-removed brain slices. In RTN-retained slices, focal and bath application of propofol increased intrinsic excitability, temporal summation, and spike firing rate in RTN neurons. Propofol-induced activation was associated with suppression of medium afterhyperpolarization potentials. This activation was mimicked and completely occluded by the small conductance calcium-activated potassium (SK) channel blocker apamin, indicating that propofol could enhance RTN excitability by blocking SK channels. Propofol increased GABAergic transmission at RTN-VB synapses, consistent with excitation of presynaptic RTN neurons. Stimulation of RTN resulted in synaptic inhibition in postsynaptic neurons in VB, and this inhibition was potentiated by propofol in a concentration-dependent manner. Removal of RTN resulted in a dramatic reduction of both spontaneous postsynaptic inhibitory current frequency and propofol-mediated inhibition of VB neurons. Thus the existence and activation of RTN input were essential for propofol to elicit thalamocortical suppression; such suppression resulted from shunting through the postsynaptic GABAA receptor-mediated chloride conductance. The results indicate that propofol enhancement of RTN-mediated inhibitory input via blockade of SK channels may play a critical role in “gating” spike firing in thalamocortical relay neurons.

Propofol is a widely used intravenous general anesthetic. Propofol-induced unconsciousness in humans is associated with inhibition of thalamic activity evoked by somatosensory stimuli. However, the cellular mechanisms underlying the effects of propofol in thalamic circuits are largely unknown. We investigated the influence of propofol on synaptic responsiveness of thalamocortical relay neurons in the ventrobasal complex (VB) to excitatory input in mouse brain slices, using both current- and voltage-clamp recording techniques. Excitatory responses including EPSP temporal summation and action potential firing were evoked in VB neurons by electrical stimulation of corticothalamic fibers or pharmacological activation of glutamate receptors. Propofol (0.6 – 3 μM) suppressed temporal summation and spike firing in a concentration-dependent manner. The thalamocortical suppression was accompanied by a marked decrease in both EPSP amplitude and input resistance, indicating that a shunting mechanism was involved. The propofol-mediated thalamocortical suppression could be blocked by a GABAA receptor antagonist or chloride channel blocker, suggesting that postsynaptic GABAA receptors in VB neurons were involved in the shunting inhibition. GABAA receptor-mediated inhibitory postsynaptic currents (IPSCs) were evoked in VB neurons by electrical stimulation of the reticular thalamic nucleus. Propofol markedly increased amplitude, decay time, and charge transfer of GABAA IPSCs. The results demonstrated that shunting inhibition of thalamic somatosensory relay neurons by propofol at clinically relevant concentrations is primarily mediated through the potentiation of the GABAA receptor chloride channel-mediated conductance, and such inhibition may contribute to the impaired thalamic responses to sensory stimuli seen during propofol-induced anesthesia.

Calling for major reform in medical education, the Carnegie Institute report ‘Educating Physicians’ espoused the importance of assisting student trainees in forming their professional identities. Here, we consider the question: At what educational stage should future physicians begin this process? The literature suggests that the process begins when students matriculate in medical school; we posit, however, that premedical students can begin their proto-professional development as college undergraduates. We describe here the pedagogy of Cornell University’s urban semester program (USP), which enables college students to participate in shadowing experiences as part of an integrated structured study programme. USP students report improved communicative competency, changes in their perceptions and attitudes toward medical practice, and powerful influences on their personal and professional development upon completion of the programme. We suggest the solution to the question of ‘When and under what conditions should shadowing take place?’ is to utilize a structure that combines the exposure of college students to the professional environment with a didactic and self-reflective curriculum, thereby supporting students in their early professional development. We conclude that educational efforts aimed at developing professional identity and behaviour can begin before students enter medical school.

Hyperpolarization-activated cyclic nucleotide-regulated HCN channels underlie the Na+-K+ permeable IH pacemaker current. As with other voltage-gated members of the 6-transmembrane KV channel superfamily, opening of HCN channels involves dilation of a helical bundle formed by the intracellular ends of S6 albeit this is promoted by inward, not outward, displacement of S4. Direct agonist binding to a ring of cyclic nucleotide-binding sites, one of which lies immediately distal to each S6 helix, imparts cAMP sensitivity to HCN channel opening. At depolarized potentials, HCN channels are further modulated by intracellular Mg2+ which blocks the open channel pore and blunts the inhibitory effect of outward K+ flux. Here, we show that cAMP binding to the gating ring enhances not only channel opening but also the kinetics of Mg2+ block. A combination of experimental and simulation studies demonstrates that agonist acceleration of block is mediated via acceleration of the blocking reaction itself rather than as a secondary consequence of the cAMP enhancement of channel opening. These results suggest that the activation status of the gating ring and the open state of the pore are not coupled in an obligate manner (as required by the often invoked Monod-Wyman-Changeux allosteric model) but couple more loosely (as envisioned in a modular model of protein activation). Importantly, the emergence of second messenger sensitivity of open channel rectification suggests that loose coupling may have an unexpected consequence: it may endow these erstwhile “slow” channels with an ability to exert voltage and ligand-modulated control over cellular excitability on the fastest of physiologically relevant time scales.