A Multiple Baseline Trial of an Electronic ICU Discharge Summary Tool for Improving Quality of Care.

OBJECTIVE: Effective communication between clinicians is essential for seamless discharge of patients between care settings. Yet, discharge summaries are commonly not available and incomplete. We implemented and evaluated a structured electronic health record-embedded electronic discharge (eDischarge) summary tool for patients discharged from the ICU to a hospital ward. DESIGN: Multiple baseline trial with randomized and staggered implementation. SETTING: Adult medical-surgical ICUs at four acute care hospitals serving a single Canadian city. PATIENTS: Health records of patients 18 years old or older, in the ICU 24 hours or longer, and discharged from the ICU to an in-hospital patient ward between February 12, 2018, and June 30, 2019. INTERVENTION: A structured electronic note (ICU eDischarge tool) with predefined fields (e.g., diagnosis) embedded in the hospital-wide electronic health information system. MEASUREMENTS AND MAIN RESULTS: We compared the percent of timely (available at discharge) and complete (included goals of care designation, diagnosis, list of active issues, active medications) discharge summaries pre and post implementation using mixed effects logistic regression models. After implementing the ICU eDischarge tool, there was an immediate and sustained increase in the proportion of patients discharged from ICU with timely and complete discharge summaries from 10.8% (preimplementation period) to 71.1% (postimplementation period) (adjusted odds ratio, 32.43; 95% CI, 18.22-57.73). No significant changes were observed in rapid response activation, cardiopulmonary arrest, death in hospital, ICU readmission, and hospital length of stay following ICU discharge. Preventable (60.1 vs 5.7 per 1,000 d; p = 0.023), but not nonpreventable (27.3 vs 40.2 per 1,000d; p = 0.54), adverse events decreased post implementation. Clinicians perceived the eDischarge tool to produce a higher quality discharge process. CONCLUSIONS: Implementation of an electronic tool was associated with more timely and complete discharge summaries for patients discharged from the ICU to a hospital ward.

Delirium diagnosis without a gold standard: Evaluating diagnostic accuracy of combined delirium assessment tools.

BACKGROUND: Fluctuating course of delirium and complexities of ICU care mean delirium symptoms are hard to identify or commonly confused with other disorders. Delirium is difficult to diagnose, and clinicians and researchers may combine assessments from multiple tools. We evaluated diagnostic accuracy of different combinations of delirium assessments performed in each enrolled patient. METHODS: Data were obtained from a previously conducted cross-sectional study. Eligible adult patients who remained admitted to ICU for >24 hours with at least one family member present were consecutively enrolled as patient-family dyads. Clinical delirium assessments (Intensive Care Delirium Screening Checklist [ICSDC] and Confusion Assessment Method-ICU [CAM-ICU]) were completed twice daily by bedside nurse or trained research assistant, respectively. Family delirium assessments (Family Confusion Assessment Method and Sour Seven) were completed once daily by family members. We pooled all delirium assessment tools in a single two-class latent model and pairwise (i.e., combined, clinical or family assessments) Bayesian analyses. RESULTS: Seventy-three patient-family dyads were included. Among clinical delirium assessments, the ICDSC had lower sensitivity (0.72; 95% Bayesian Credible [BC] interval 0.54-0.92) and higher specificity (0.90; 95%BC, 0.82-0.97) using Bayesian analyses compared to pooled latent class analysis and CAM-ICU had higher sensitivity (0.90; 95%BC, 0.70-1.00) and higher specificity (0.94; 95%BC, 0.80-1.00). Among family delirium assessments, the Family Confusion Assessment Method had higher sensitivity (0.83; 95%BC, 0.71-0.92) and higher specificity (0.93; 95%BC, 0.84-0.98) using Bayesian analyses compared to pooled latent class analysis and the Sour Seven had higher specificity (0.85; 95%BC, 0.67-0.99) but lower sensitivity (0.64; 95%BC 0.47-0.82). CONCLUSIONS: Results from delirium assessment tools are often combined owing to imperfect reference standards for delirium measurement. Pairwise Bayesian analyses that explicitly accounted for each tool's (performed within same patient) prior sensitivity and specificity indicate that two combined clinical or two combined family delirium assessment tools have fair diagnostic accuracy.

Identification and Assessment of Strategies to Address Gender Inequity in the Specialty of Critical Care Medicine: A Scoping Review, Modified Consensus Process, and Stakeholder Meeting.

We sought to identify and prioritize improvement strategies that Critical Care Medicine (CCM) programs could use to inform and advance gender equity among physicians in CCM. DESIGN: This study involved three sequential phases: 1) scoping review that identified strategies to improve gender equity in all medical specialties; 2) modified consensus process with 48 CCM stakeholders to rate and rank identified strategies; and 3) in-person stakeholder meeting to refine strategies and discuss facilitators and barriers to their implementation. SETTING: CCM. SUBJECTS: CCM stakeholders (physicians, researchers, and decision-makers; mutually inclusive). INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: We identified 190 unique strategies from 416 articles. Strategies were grouped thematically into 20 categories across four overarching pillars of equity: access, participation, reimbursement, and culture. Participants prioritized 22 improvement strategies for implementation in CCM. The top-rated strategy from each pillar included: 1) nominate gender diverse candidates for faculty positions and prestigious opportunities (equitable access); 2) mandate training in unconscious bias and equitable treatment for committee (e.g., hiring, promotion) members (equitable participation); 3) ensure equitable starting salaries regardless of sex or gender (equitable reimbursement); and, 4) conduct 360° evaluations of leaders (including their direct work circle of supervisors, peers, and subordinates) through a diversity lens (equitable culture). Interprofessional collaboration, leadership, and local champions were identified as key enablers for implementation. CONCLUSIONS: We identified stakeholder-prioritized strategies that can be used to inform and enhance gender equity among physicians in CCM under four overarching equity pillars: access, participation, reimbursement, and culture. Implementation approaches should include education, policy creation, and measurement, and reporting.

Evolving Factors in Hospital Safety: A Systematic Review and Meta-Analysis of Hospital Adverse Events.

OBJECTIVE: This study aimed to estimate the frequency of hospital adverse events (AEs) and explore the rate of AEs over time, and across and within hospital populations. METHODS: Validated search terms were run in MEDLINE and EMBASE; gray literature and references of included studies were also searched. Studies of any design or language providing an estimate of AEs within the hospital were eligible. Studies were excluded if they only provided an estimate for a specific AE, a subgroup of hospital patients or children. Data were abstracted in duplicate using a standardized data abstraction form. Study quality was assessed using the Newcastle-Ottawa Scale. A random-effects meta-analysis estimated the occurrence of hospital AEs, and meta-regression explored the association between hospital AEs, and patient and hospital characteristics. RESULTS: A total of 45,426 unique references were identified; 1,265 full-texts were reviewed and 94 studies representing 590 million admissions from 25 countries from 1961 to 2014 were included. The incidence of hospital AEs was 8.6 per 100 patient admissions (95% confidence interval [CI], 8.3 to 8.9; I2 = 100%, P < 0.001). Half of the AEs were preventable (52.6%), and a third resulted in moderate/significant harm (39.7%). The most evaluated AEs were surgical AEs, drug-related AEs, and nosocomial infections. The occurrence of AEs increased by year (95% CI, -0.05 to -0.04; P < 0.001) and patient age (95% CI = -0.15 to -0.14; P < 0.001), and varied by country income level and study characteristics. Patient sex, hospital type, hospital service, and geographical location were not associated with AEs. CONCLUSIONS: Hospital AEs are common, and reported rates are increasing in the literature. Given the increase in AEs over time, hospitals should reinvest in improving hospital safety with a focus on interventions targeted toward the more than half of AEs that are preventable.

Scanned optogenetic control of mammalian somatosensory input to map input-specific behavioral outputs.

Somatosensory stimuli guide and shape behavior, from immediate protective reflexes to longer-term learning and higher-order processes related to pain and touch. However, somatosensory inputs are challenging to control in awake mammals due to the diversity and nature of contact stimuli. Application of cutaneous stimuli is currently limited to relatively imprecise methods as well as subjective behavioral measures. The strategy we present here overcomes these difficulties, achieving 'remote touch' with spatiotemporally precise and dynamic optogenetic stimulation by projecting light to a small defined area of skin. We mapped behavioral responses in freely behaving mice with specific nociceptor and low-threshold mechanoreceptor inputs. In nociceptors, sparse recruitment of single-action potentials shapes rapid protective pain-related behaviors, including coordinated head orientation and body repositioning that depend on the initial body pose. In contrast, activation of low-threshold mechanoreceptors elicited slow-onset behaviors and more subtle whole-body behaviors. The strategy can be used to define specific behavioral repertoires, examine the timing and nature of reflexes, and dissect sensory, motor, cognitive, and motivational processes guiding behavior.

To safely navigate their world, animals need to be able to tell apart a gentle touch from an eye-watering pinch, detect cold water or sense the throbbing pain stemming from an infected cut. These 'somatic' sensations are relayed through thousands of nerve endings embedded in the skin and other tissues. Yet the neurological mechanisms that underpin these abilities are complex and still poorly understood. Indeed, these nerve endings can be stimulated by extreme temperatures, harmful chemicals, friction or even internal signals such as inflammation. One event can also recruit many different types of endings: a cut for example, will involve responses to mechanical pressure, tissue damage and local immune response. To disentangle these different actors and how they affect behavior, scientists need to develop approaches that allow them to deliver specific stimuli with increased precision, and to monitor the impact on an animal. To achieve this goal, Schorscher-Petcu et al. used mice in which blue light could trigger specific types of nerve endings. For instance, depending on the genetic background of the animals, a laser could either activate nerve endings involved in pain or gentle touch. Crucially, this could be done from a distance by beaming light with exquisite precision onto the paws of the mice without physically touching or disturbing the animals. How the mice responded could then be observed without any interference. Their behavior was analyzed using a combination of high-speed videos, computer-driven recording systems, and machine learning. This revealed subtle changes in behavior that had not been detected before, spotting microscopic movements of the stimulated paw and mapping simultaneous whole-body movements such as changes in posture or head orientation. The approach therefore allows scientists to assess the impact of touch, pain or temperature sensation in freely behaving mice. It could also be harnessed to develop much needed treatments against chronic pain.

Effectiveness of an Electronic Communication Tool on Transitions in Care From the Intensive Care Unit: Protocol for a Cluster-Specific Pre-Post Trial.

BACKGROUND: Transitions in care are vulnerable periods in health care that can expose patients to preventable errors due to incomplete or delayed communication between health care providers. Transitioning critically ill patients from intensive care units (ICUs) to other patient care units (PCUs) is particularly risky, due to the high acuity of the patients and the diversity of health care providers involved in their care. Instituting structured documentation to standardize written communication between health care providers during transitions has been identified as a promising means to reduce communication breakdowns. We developed an evidence-informed, computer-enabled, ICU-specific structured tool-an electronic transfer (e-transfer) tool-to facilitate and standardize the composition of written transfer summaries in the ICUs of one Canadian city. The tool consisted of 10 primary sections with a user interface combination of structured, automated, and free-text fields. OBJECTIVE: Our overarching goal is to evaluate whether implementation of our e-transfer tool will improve the completeness and timeliness of transfer summaries and streamline communications between health care providers during high-risk transitions. METHODS: This study is a cluster-specific pre-post trial, with randomized and staggered implementation of the e-transfer tool in four hospitals in Calgary, Alberta. Hospitals (ie, clusters) were allocated randomly to cross over every 2 months from control (ie, dictation only) to intervention (ie, e-transfer tool). Implementation at each site was facilitated with user education, point-of-care support, and audit and feedback. We will compare transfer summaries randomly sampled over 6 months postimplementation to summaries randomly sampled over 6 months preimplementation. The primary outcome will be a binary composite measure of the timeliness and completeness of transfer summaries. Secondary measures will include overall completeness, timeliness, and provider ratings of transfer summaries; hospital and ICU lengths of stay; and post-ICU patient outcomes, including ICU readmission, adverse events, cardiac arrest, rapid response team activation, and mortality. We will use descriptive statistics (ie, medians and means) to describe demographic characteristics. The primary outcome will be compared within each hospital pre- and postimplementation using separate logistic regression models for each hospital, with adjustment for patient characteristics. RESULTS: Participating hospitals were cluster randomized to the intervention between July 2018 and January 2019. Preliminary extraction of ICU patient admission lists was completed in September 2019. We anticipate that evaluation data collection will be completed by early 2021, with first results ready for publication in spring or summer 2021. CONCLUSIONS: This study will report the impact of implementing an evidence-informed, computer-enabled, ICU-specific structured transfer tool on communication and preventable medical errors among patients transferred from the ICU to other hospital care units. TRIAL REGISTRATION: ClinicalTrials.gov NCT03590002; https://www.clinicaltrials.gov/ct2/show/NCT03590002. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/18675.

Epineural optogenetic activation of nociceptors initiates and amplifies inflammation.

Activation of nociceptor sensory neurons by noxious stimuli both triggers pain and increases capillary permeability and blood flow to produce neurogenic inflammation1,2, but whether nociceptors also interact with the immune system remains poorly understood. Here we report a neurotechnology for selective epineural optogenetic neuromodulation of nociceptors and demonstrate that nociceptor activation drives both protective pain behavior and inflammation. The wireless optoelectronic system consists of sub-millimeter-scale light-emitting diodes embedded in a soft, circumneural sciatic nerve implant, powered and driven by a miniaturized head-mounted control unit. Photostimulation of axons in freely moving mice that express channelrhodopsin only in nociceptors resulted in behaviors characteristic of pain, reflecting orthodromic input to the spinal cord. It also led to immune reactions in the skin in the absence of inflammation and potentiation of established inflammation, a consequence of the antidromic activation of nociceptor peripheral terminals. These results reveal a link between nociceptors and immune cells, which might have implications for the treatment of inflammation.

A national cross-sectional survey of public perceptions of the COVID-19 pandemic: Self-reported beliefs, knowledge, and behaviors.

INTRODUCTION: Efforts to mitigate the global spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing Corona Virus Disease-19 (COVID-19) have largely relied on broad compliance with public health recommendations yet navigating the high volume of evolving information can be challenging. We assessed self-reported public perceptions related to COVID-19 including, beliefs (e.g., severity, concerns, health), knowledge (e.g., transmission, information sources), and behaviors (e.g., physical distancing) to understand perspectives in Canada and to inform future public health initiatives. METHODS: We administered a national online survey aiming to obtain responses from 2000 adults in Canada. Respondent sampling was stratified by age, sex, and region. We used descriptive statistics to summarize responses and tested for regional differences using chi-squared tests, followed by weighted logistic regression. RESULTS: We collected 1,996 eligible questionnaires between April 26th and May 1st, 2020. One-fifth (20%) of respondents knew someone diagnosed with COVID-19, but few had tested positive themselves (0.6%). Negative impacts of pandemic conditions were evidenced in several areas, including concerns about healthcare (e.g. sufficient equipment, 52%), pandemic stress (45%), and worsening social (49%) and mental/emotional (39%) health. Most respondents (88%) felt they had good to excellent knowledge of virus transmission, and predominantly accessed (74%) and trusted (60%) Canadian news television, newspapers/magazines, or non-government news websites for COVID-19 information. We found high compliance with distancing measures (80% reported self-isolating or always physical distancing). We identified associations between region and self-reported beliefs, knowledge, and behaviors related to COVID-19. DISCUSSION: We found that information about COVID-19 is largely acquired through domestic news sources, which may explain high self-reported compliance with prevention measures. The results highlight the broader impact of a pandemic on the general public's overall health and wellbeing, outside of personal infection. The study findings should be used to inform public health communications during COVID-19 and future pandemics.

Engaging patients in de-implementation interventions to reduce low-value clinical care: a systematic review and meta-analysis.

BACKGROUND: Many decisions regarding health resource utilization flow through the patient-clinician interaction. Thus, it represents a place where de-implementation interventions may have considerable effect on reducing the use of clinical interventions that lack efficacy, have risks that outweigh benefits, or are not cost-effective (i.e., low-value care). The objective of this systematic review with meta-analysis was to determine the effect of de-implementation interventions that engage patients within the patient-clinician interaction on use of low-value care. METHODS: MEDLINE, EMBASE, and CINAHL were searched from inception to November 2019. Gray literature was searched using the CADTH tool. Studies were screened independently by two reviewers and were included if they (1) described an intervention that engaged patients in an initiative to reduce low-value care, (2) reported the use of low-value care with and without the intervention, and (3) were randomized clinical trials (RCTs) or quasi-experimental designs. Studies describing interventions solely focused on clinicians or published in a language other than English were excluded. Data was extracted independently in duplicate and pertained to the low-value clinical intervention of interest, components of the strategy for patient engagement, and study outcomes. Quality of included studies was assessed using the Cochrane Risk of Bias tool for RCTs and a modified Downs and Black checklist for quasi-experimental studies. Random effects meta-analysis (reported as risk ratio, RR) was used to examine the effect of de-implementation interventions on the use of low-value care. RESULTS: From 6736 unique citations, 9 RCTs and 13 quasi-experimental studies were included in the systematic review. Studies mostly originated from the USA (n = 13, 59%), targeted treatments (n = 17, 77%), and took place in primary care (n = 10, 45%). The most common intervention was patient-oriented educational material (n = 18, 82%), followed by tools for shared decision-making (n = 5, 23%). Random effects meta-analysis demonstrated that de-implementation interventions that engage patients within the patient-clinician interaction led to a significant reduction in low-value care in both RCTs (RR 0.74; 95% CI 0.66-0.84) and quasi-experimental studies (RR 0.61; 95% CI 0.43-0.87). There was significant inter-study heterogeneity; however, intervention effects were consistent across subgroups defined by low-value practice and patient-engagement strategy. CONCLUSIONS: De-implementation interventions that engage patients within the patient-clinician interaction through patient-targeted educational materials or shared decision-making tools are effective in decreasing the use of low-value care. Clinicians and policymakers should consider engaging patients within initiatives that seek to reduce low-value care. REGISTRATION: Open Science Framework (https://osf.io/6fsxm).

Understanding the public's role in reducing low-value care: a scoping review.

BACKGROUND: Low-value care initiatives are rapidly growing; however, it is not clear how members of the public should be involved. The objective of this scoping review was to systematically examine the literature describing public involvement in initatives to reduce low-value care. METHODS: Evidence sources included MEDLINE, EMBASE, and CINAHL databases from inception to November 26, 2019, grey literature (CADTH Tool), reference lists of included articles, and expert consultation. Citations were screened in duplicate and included if they referred to the public's perception and/or involvement in reducing low-value care. Public included patients or citizens without any advanced healthcare knowledge. Low-value care included medical tests or treatments that lack efficacy, have risks that exceed benefit, or are not cost-effective. Extracted data pertained to study characteristics, low-value practice, clinical setting, and level of public involvement (i.e., patient-clinician interaction, research, or policy-making). RESULTS: The 218 included citations were predominantly original research (n = 138, 63%), published since 2010 (n = 192, 88%), originating from North America (n = 146, 67%). Most citations focused on patient engagement within the patient-clinician interaction (n = 156, 72%), using tools that included shared decision-making (n = 66, 42%) and patient-targeted educational materials (n = 72, 46%), and reported both reductions in low-value care and improved patient perceptions regarding low-value care. Fewer citations examined public involvement in low-value care policy-making (n = 33, 15%). Among citations that examined perspectives regarding public involvement in initiatives to reduce low-value care (n = 10, 5%), there was consistent support for the utility of tools applied within the patient-clinician interaction and less consistent support for involvement in policy-making. CONCLUSIONS: Efforts examining public involvement in low-value care concentrate within the patient-clinician interaction, wherein patient-oriented educational materials and shared decision-making tools have been commonly studied and are associated with reductions in low-value care. This contrasts with inclusion of the public in low-value care policy decisions wherein tools to promote engagement are less well-developed and involvement not consistently viewed as valuable. TRIAL REGISTRATION: Open Science Framework (https://osf.io/6fsxm).

Controlling Engineered P2X Receptors with Light.

This chapter details methods to express and modify ATP-gated P2X receptor channels so that they can be controlled using light. Following expression in cells, a photoswitchable tool compound can be used to covalently modify mutant P2X receptors, as previously demonstrated for homomeric P2X2 and P2X3 receptors, and heteromeric P2X2/3 receptors. Engineered P2X receptors can be rapidly and reversibly opened and closed by different wavelengths of light. Light-activated P2X receptors can be mutated further to impart ATP-insensitivity if required. This method offers control of specific P2X receptor channels with high spatiotemporal precision to study their roles in physiology and pathophysiology.

Mapping structure, process and outcomes in the removal of low-value care practices in Canadian intensive care units: protocol for a mixed-methods exploratory study.

INTRODUCTION: The challenge of implementing best evidence into clinical practice is a major problem in modern healthcare that can result in ineffective, inefficient and unsafe care. There is a growing body of literature which suggests that the removal or reduction of low-value care practices (ie, deadoption) is integral to the delivery of high-quality care and the sustainability of our healthcare system. However, currently very little is known about deadoption practices in Canada. We propose to map the current state of deadoption in Canadian intensive care units (ICUs). A key deliverable of this work will include development of an inventory of barriers, facilitators and potential implementation strategies for guiding the deadoption efforts. METHODS AND ANALYSIS: We will use Canadian adult general systems ICUs as our laboratory of investigation and employ a two-phased sequential exploratory mixed-methods approach: (1) semi-structured interviews with critical care stakeholders to develop an understanding of the structure (ie, healthcare context), process (ie, actions and events in healthcare) and outcomes (ie, effects on health status, quality, knowledge or behaviour) of deadoption (phase I) and (2) surveys with a broader sample of critical care stakeholders to further identify important barriers and facilitators, as well as potential implementation strategies (phase II). Interview data will be analysed through qualitative content analysis and survey data will be analysed through quantitative analyses to identify top barriers and facilitators, as well as top rated strategies. ETHICS AND DISSEMINATION: Ethical approval has been obtained through the University of Calgary Research Ethics Board (REB 17-2153). Participants involved will have the opportunity to provide feedback on the final written reports to support accurate representation of the data. The findings of this study will be disseminated through peer-reviewed publications and oral presentations with critical care stakeholders across Canada. Patient and family partners will receive an executive summary of the findings.

Effect of GPR84 deletion on obesity and diabetes development in mice fed long chain or medium chain fatty acid rich diets.

PURPOSE: Although there is good evidence showing that diets rich in medium chain fatty acids (MCFAs) have less marked obesogenic and diabetogenic effects than diets rich in long chain fatty acids (LCFAs), the role of the pro-inflammatory, medium chain fatty acid receptor (GPR84) in the aetiology of obesity and glucose intolerance is not well characterised. We set out to determine whether GPR84 expression influences obesity and glucose intolerance susceptibility in MCFA and LCFA rich diet fed mice. METHODS: Wild type (WT) and GPR84 knockout (KO) mice were fed a control, MCFA or LCFA diet, and body mass, heart, liver and epididymal fat mass was assessed, as well as glucose tolerance and adipocyte size. RESULTS: LCFA diets increased body mass and decreased glucose tolerance in both WT and GPR84 KO animals while MCFA diets had no effect on these parameters. There were no differences in body weight when comparing WT and GPR84 KO mice on the respective diets. Glucose tolerance was also similar in WT and GPR84 KO mice irrespective of diet. Liver mass was increased following LCFA feeding in WT but not GPR84 KO mice. Hepatic triglyceride content was increased in GPR84 KO animals fed MCFA, and myocardial triglyceride content was increased in GPR84 KO animals fed LCFA. CONCLUSIONS: GPR84 deletion had no effects on body weight or glucose tolerance in mice fed either a high MCFA or LCFA diet. GPR84 may influence lipid metabolism, as GPR84 KO mice had smaller livers and increased myocardial triglyceride accumulation when fed LCFA diets, and increased liver triglyceride accumulation in responses to increased dietary MCFAs.

Optical cuff for optogenetic control of the peripheral nervous system.

OBJECTIVE: Nerves in the peripheral nervous system (PNS) contain axons with specific motor, somatosensory and autonomic functions. Optogenetics offers an efficient approach to selectively activate axons within the nerve. However, the heterogeneous nature of nerves and their tortuous route through the body create a challenging environment to reliably implant a light delivery interface. APPROACH: Here, we propose an optical peripheral nerve interface-an optocuff-, so that optogenetic modulation of peripheral nerves become possible in freely behaving mice. MAIN RESULTS: Using this optocuff, we demonstrate orderly recruitment of motor units with epineural optical stimulation of genetically targeted sciatic nerve axons, both in anaesthetized and in awake, freely behaving animals. Behavioural experiments and histology show the optocuff does not damage the nerve thus is suitable for long-term experiments. SIGNIFICANCE: These results suggest that the soft optocuff might be a straightforward and efficient tool to support more extensive study of the PNS using optogenetics.

Time-Resolved Fast Mammalian Behavior Reveals the Complexity of Protective Pain Responses.

Potentially harmful stimuli are detected at the skin by nociceptor sensory neurons that drive rapid protective withdrawal reflexes and pain. We set out to define, at a millisecond timescale, the relationship between the activity of these sensory neurons and the resultant behavioral output. Brief optogenetic activation of cutaneous nociceptors was found to activate only a single action potential in each fiber. This minimal input was used to determine high-speed behavioral responses in freely behaving mice. The localized stimulus generated widespread dynamic repositioning and alerting sub-second behaviors whose nature and timing depended on the context of the animal and its position, activity, and alertness. Our findings show that the primary response to injurious stimuli is not limited, fixed, or localized, but is dynamic, and that it involves recruitment and gating of multiple circuits distributed throughout the central nervous system at a sub-second timescale to effectively both alert to the presence of danger and minimize risk of harm.

Ectodomain movements of an ATP-gated ion channel (P2X2 receptor) probed by disulfide locking.

The ectodomain of the P2X receptor is formed mainly from two- or three-stranded ß-sheets provided symmetrically by each of the three subunits. These enclose a central cavity that is closed off furthest from the plasma membrane (the turret) and that joins with the transmembrane helices to form the ion permeation pathway. Comparison of closed and open crystal structures indicates that ATP binds in a pocket positioned between strands provided by different subunits and that this flexes the ß-sheets of the lower body and enlarges the central cavity: this pulls apart the outer ends of the transmembrane helices and thereby opens an aperture, or gate, where they intersect within the membrane bilayer. In the present work, we examined this opening model by introducing pairs of cysteines into the rat P2X2 receptor that might form disulfide bonds within or between subunits. Receptors were expressed in human embryonic kidney cells, and disulfide formation was assessed by observing the effect of dithiothreitol on currents evoked by ATP. Substitutions in the turret (P90C, P89C/S97C), body wall (S65C/S190C, S65C/D315C) and the transmembrane domains (V48C/I328C, V51C/I328C, S54C/I328C) strongly inhibited ATP-evoked currents prior to reduction with dithiothreitol. Western blotting showed that these channels also formed predominately as dimers and/or trimers rather than monomers. The results strongly support the channel opening mechanism proposed on the basis of available crystal structures.

Optical control of trimeric P2X receptors and acid-sensing ion channels.

P2X receptors are trimeric membrane proteins that function as ion channels gated by extracellular ATP. We have engineered a P2X2 receptor that opens within milliseconds by irradiation at 440 nm, and rapidly closes at 360 nm. This requires bridging receptor subunits via covalent attachment of 4,4'-bis(maleimido)azobenzene to a cysteine residue (P329C) introduced into each second transmembrane domain. The cis-trans isomerization of the azobenzene pushes apart the outer ends of the transmembrane helices and opens the channel in a light-dependent manner. Light-activated channels exhibited similar unitary currents, rectification, calcium permeability, and dye uptake as P2X2 receptors activated by ATP. P2X3 receptors with an equivalent mutation (P320C) were also light sensitive after chemical modification. They showed typical rapid desensitization, and they could coassemble with native P2X2 subunits in pheochromocytoma cells to form light-activated heteromeric P2X2/3 receptors. A similar approach was used to open and close human acid-sensing ion channels (ASICs), which are also trimers but are unrelated in sequence to P2X receptors. The experiments indicate that the opening of the permeation pathway requires similar and substantial movements of the transmembrane helices in both P2X receptors and ASICs, and the method will allow precise optical control of P2X receptors or ASICs in intact tissues.

P2X receptor intermediate activation states have altered nucleotide selectivity.

Purinergic P2X receptors are widely distributed in the nervous system and are known to play roles in primary afferent transmission and central respiratory regulation. They are trimeric membrane proteins, with the extracellular domain that provides three intersubunit ATP binding sites. We expressed the rat P2X7 receptor in human embryonic kidney cells and measured membrane currents before and after photo-affinity labeling with the agonist 2'(3')-O-(4-benzoylbenzoyl)-ATP (BzATP). After tethering BzATP with ultraviolet light, a persistent current remained after washing out the agonist. Additional current could now be elicited by other nucleotides (CTP and ADP) that are not normally effective as P2X receptor agonists. Similar results were obtained at P2X2 receptors even without previous agonist tethering: exposure to low concentrations of ATP caused the receptor to become sensitive to activation by CTP and ADP. The results show that ATP binding to the first of the three binding sites causes a conformational change to an intermediate closed state that shows increased effectiveness of pyrimidine and diphosphate nucleotide analogs.