Targeting aberrant dendritic integration to treat cognitive comorbidities of epilepsy.

Memory deficits are a debilitating symptom of epilepsy, but little is known about mechanisms underlying cognitive deficits. Here, we describe a Na+ channel-dependent mechanism underlying altered hippocampal dendritic integration, degraded place coding and deficits in spatial memory. Two-photon glutamate uncaging experiments revealed a marked increase in the fraction of hippocampal first-order CA1 pyramidal cell dendrites capable of generating dendritic spikes in the kainate model of chronic epilepsy. Moreover, in epileptic mice dendritic spikes were generated with lower input synchrony, and with a lower threshold. The Nav1.3/1.1 selective Na+ channel blocker ICA-121431 reversed dendritic hyperexcitability in epileptic mice, while the Nav1.2/1.6 preferring anticonvulsant S-Lic did not. We used in vivo two-photon imaging to determine if aberrant dendritic excitability is associated with altered place-related firing of CA1 neurons. We show that ICA-121431 improves degraded hippocampal spatial representations in epileptic mice. Finally, behavioural experiments show that reversing aberrant dendritic excitability with ICA-121431 reverses hippocampal memory deficits. Thus, a dendritic channelopathy may underlie cognitive deficits in epilepsy and targeting it pharmacologically may constitute a new avenue to enhance cognition.

Ste20-like Kinase Is Critical for Inhibitory Synapse Maintenance and Its Deficiency Confers a Developmental Dendritopathy.

The size and structure of the dendritic arbor play important roles in determining how synaptic inputs of neurons are converted to action potential output. The regulatory mechanisms governing the development of dendrites, however, are insufficiently understood. The evolutionary conserved Ste20/Hippo kinase pathway has been proposed to play an important role in regulating the formation and maintenance of dendritic architecture. A key element of this pathway, Ste20-like kinase (SLK), regulates cytoskeletal dynamics in non-neuronal cells and is strongly expressed throughout neuronal development. However, its function in neurons is unknown. We show that, during development of mouse cortical neurons, SLK has a surprisingly specific role for proper elaboration of higher, ≥ third-order dendrites both in male and in female mice. Moreover, we demonstrate that SLK is required to maintain excitation-inhibition balance. Specifically, SLK knockdown caused a selective loss of inhibitory synapses and functional inhibition after postnatal day 15, whereas excitatory neurotransmission was unaffected. Finally, we show that this mechanism may be relevant for human disease, as dysmorphic neurons within human cortical malformations revealed significant loss of SLK expression. Overall, the present data identify SLK as a key regulator of both dendritic complexity during development and inhibitory synapse maintenance.SIGNIFICANCE STATEMENT We show that dysmorphic neurons of human epileptogenic brain lesions have decreased levels of the Ste20-like kinase (SLK). Decreasing SLK expression in mouse neurons revealed that SLK has essential functions in forming the neuronal dendritic tree and in maintaining inhibitory connections with neighboring neurons.

Functional properties of granule cells with hilar basal dendrites in the epileptic dentate gyrus.

OBJECTIVE: The maturation of adult-born granule cells and their functional integration into the network is thought to play a key role in the proper functioning of the dentate gyrus. In temporal lobe epilepsy, adult-born granule cells in the dentate gyrus develop abnormally and possess a hilar basal dendrite (HBD). Although morphological studies have shown that these HBDs have synapses, little is known about the functional properties of these HBDs or the intrinsic and network properties of the granule cells that possess these aberrant dendrites. METHODS: We performed patch-clamp recordings of granule cells within the granule cell layer "normotopic" from sham-control and status epilepticus (SE) animals. Normotopic granule cells from SE animals possessed an HBD (SE+ HBD+ cells) or not (SE+ HBD- cells). Apical and basal dendrites were stimulated using multiphoton uncaging of glutamate. Two-photon Ca2+ imaging was used to measure Ca2+ transients associated with back-propagating action potentials (bAPs). RESULTS: Near-synchronous synaptic input integrated linearly in apical dendrites from sham-control animals and was not significantly different in apical dendrites of SE+ HBD- cells. The majority of HBDs integrated input linearly, similar to apical dendrites. However, 2 of 11 HBDs were capable of supralinear integration mediated by a dendritic spike. Furthermore, the bAP-evoked Ca2+ transients were relatively well maintained along HBDs, compared with apical dendrites. This further suggests an enhanced electrogenesis in HBDs. In addition, the output of granule cells from epileptic tissue was enhanced, with both SE+ HBD- and SE+ HBD+ cells displaying increased high-frequency (>100 Hz) burst-firing. Finally, both SE+ HBD- and SE+ HBD+ cells received recurrent excitatory input that was capable of generating APs, especially in the absence of feedback inhibition. SIGNIFICANCE: Taken together, these data suggest that the enhanced excitability of HBDs combined with the altered intrinsic and network properties of granule cells collude to promote excitability and synchrony in the epileptic dentate gyrus.

Downregulation of Spermine Augments Dendritic Persistent Sodium Currents and Synaptic Integration after Status Epilepticus.

Dendritic voltage-gated ion channels profoundly shape the integrative properties of neuronal dendrites. In epilepsy, numerous changes in dendritic ion channels have been described, all of them due to either their altered transcription or phosphorylation. In pilocarpine-treated chronically epileptic rats, we describe a novel mechanism that causes an increased proximal dendritic persistent Na(+) current (INaP). We demonstrate using a combination of electrophysiology and molecular approaches that the upregulation of dendritic INaP is due to a relief from polyamine-dependent inhibition. The polyamine deficit in hippocampal neurons is likely caused by an upregulation of the degrading enzyme spermidine/spermine acetyltransferase. Multiphoton glutamate uncaging experiments revealed that the increase in dendritic INaP causes augmented dendritic summation of excitatory inputs. These results establish a novel post-transcriptional modification of ion channels in chronic epilepsy and may provide a novel avenue for treatment of temporal lobe epilepsy. SIGNIFICANCE STATEMENT: In this paper, we describe a novel mechanism that causes increased dendritic persistent Na(+) current. We demonstrate using a combination of electrophysiology and molecular approaches that the upregulation of persistent Na(+) currents is due to a relief from polyamine-dependent inhibition. The polyamine deficit in hippocampal neurons is likely caused by an upregulation of the degrading enzyme spermidine/spermine acetyltransferase. Multiphoton glutamate uncaging experiments revealed that the increase in dendritic persistent Na current causes augmented dendritic summation of excitatory inputs. We believe that these results establish a novel post-transcriptional modification of ion channels in chronic epilepsy.

Selective aptamer-based control of intraneuronal signaling.

Cellular behavior is orchestrated by the complex interactions of a myriad of intracellular signal transduction pathways. To understand and investigate the role of individual components in such signaling networks, the availability of specific inhibitors is of paramount importance. We report the generation and validation of a novel variant of an RNA aptamer that selectively inhibits the mitogen-activated kinase pathway in neurons. We demonstrate that the aptamer retains function under intracellular conditions and that application of the aptamer through the patch-clamp pipette efficiently inhibits mitogen-activated kinase-dependent synaptic plasticity. This approach introduces synthetic aptamers as generic tools, readily applicable to inhibit different components of intraneuronal signaling networks with utmost specificity.

Culture change, leadership and the grass-roots workforce.

The NHS is arguably entering its most challenging era. It is being asked to do more for less and, in parallel, a cultural shift in response to its described weaknesses has been prescribed. The definition of culture, the form this change should take and the mechanism to achieve it are not well understood. The complexity of modern healthcare requires that we evolve our approach to the workforce and enhance our understanding of the styles of leadership that are required in order to bring about this cultural change. Identification of leaders within the workforce and dissemination of a purposeful and strategic quality improvement agenda, in part defined by the general workforce, are important components in establishing the change that the organisation currently requires. We are implementing this approach locally by identifying and developing grassroots networks linked to a portfolio of safety and quality projects.

Development of a national maternity early warning score: centile based score development and Delphi informed escalation pathways.

Objective: To derive a new maternity early warning score (MEWS) from prospectively collected data on maternity vital signs and to design clinical response pathways with a Delphi consensus exercise. Design: Centile based score development and Delphi informed escalation pathways. Setting: Pregnancy Physiology Pattern Prediction (4P) prospective UK cohort study, 1 August 2012 to 28 December 2016. Participants: Pregnant people from the 4P study, recruited before 20 weeks' gestation at three UK maternity centres (Oxford, Newcastle, and London). 841, 998, and 889 women provided data in the early antenatal, antenatal, and postnatal periods. Main outcome measures: Development of a new national MEWS, assigning numerical weights to measurements in the lower and upper extremes of distributions of individual vital signs from the 4P prospective cohort study. Comparison of escalation rates of the new national MEWS with the Scottish and Irish MEWS systems from 18 to 40 weeks' gestation. Delphi consensus exercise to agree clinical responses to raised scores. Results: A new national MEWS was developed by assigning numerical weights to measurements in the lower and upper extremes (5%, 1%) of distributions of vital signs, except for oxygen saturation where lower centiles (10%, 2%) were used. For the new national MEWS, in a healthy population, 56% of observation sets resulted in a total score of 0 points, 26% a score of 1 point, 12% a score of 2 points, and 18% a score of ≥2 points (escalation of care is triggered at a total score of ≥2 points). Corresponding values for the Irish MEWS were 37%, 25%, 22%, and 38%, respectively; and for the Scottish MEWS, 50%, 18%, 21%, and 32%, respectively. All three MEWS were similar at the beginning of pregnancy, averaging 0.7-0.9 points. The new national MEWS had a lower mean score for the rest of pregnancy, with the mean score broadly constant (0.6-0.8 points). The new national MEWS had an even distribution of healthy population alerts across the antenatal period. In the postnatal period, heart rate threshold values were adjusted to align with postnatal changes. The centile based score derivation approach meant that each vital sign component in the new national MEWS had a similar alert rate. Suggested clinical responses to different MEWS values were agreed by consensus of an independent expert panel. Conclusions: The centile based MEWS alerted escalation of care evenly across the antenatal period in a healthy population, while reducing alerts in healthy women compared with other MEWS systems. How well the tool predicted adverse outcomes, however, was not assessed and therefore external validation studies in large datasets are needed. Unlike other MEWS systems, the new national MEWS was developed with prospectively collected data on vital signs and used a systematic, expert informed process to design an associated escalation protocol.

Aberrant hippocampal Ca2+ micro-waves following synapsin-dependent adeno-associated viral expression of Ca2+ indicators.

Genetically encoded calcium indicators (GECIs) such as GCaMP are invaluable tools in neuroscience to monitor neuronal activity using optical imaging. The viral transduction of GECIs is commonly used to target expression to specific brain regions, can be conveniently used with any mouse strain of interest without the need for prior crossing with a GECI mouse line and avoids potential hazards due to the chronic expression of GECIs during development. A key requirement for monitoring neuronal activity with an indicator is that the indicator itself minimally affects activity. Here, using common adeno-associated viral (AAV) transduction procedures, we describe spatially confined aberrant Ca2+ micro-waves slowly travelling through the hippocampus following expression of GCaMP6, GCaMP7 or R-CaMP1.07 driven by the synapsin promoter with AAV-dependent gene transfer, in a titre-dependent fashion. Ca2+ micro-waves developed in hippocampal CA1 and CA3, but not dentate gyrus (DG) nor neocortex, were typically first observed at 4 weeks after viral transduction, and persisted up to at least 8 weeks. The phenomenon was robust, observed across laboratories with various experimenters and setups. Our results indicate that aberrant hippocampal Ca2+ micro-waves depend on the promoter and viral titre of the GECI, density of expression as well as the targeted brain region. We used an alternative viral transduction method of GCaMP which avoids this artifact. The results show that commonly used Ca2+-indicator AAV transduction procedures can produce artefactual Ca2+ responses. Our aim is to raise awareness in the field of these artefactual transduction-induced Ca2+ micro-waves and we provide a potential solution.

Understanding the inclusion and participation of adults from Black African Diaspora Communities (BAFDC) in health and care research in the UK: a realist review protocol.

INTRODUCTION: People from Black African Diaspora Communities (BAFDC) experience poorer health outcomes, have many long-term conditions and are persistently under-represented in health and care research. There is limited focus on programmes, or interventions that support inclusion and participation of people from BAFDC in research. Through coproduction, this realist review seeks to provide a programme theory explaining what context and mechanisms may be required, to produce outcomes that facilitate inclusion and participation for people from BAFDC in health and care research, in the UK. METHODS AND ANALYSIS: A group of people from BAFDC with lived and professional experience, representing all levels of the health and care research system, will coproduce a realist review with a team of African-Caribbean, white British and white British of Polish origin health and care researchers. They will follow Pawson's five steps: (1) shaping the scope of the review; (2) searching for evidence; (3) document selection and appraisal; (4) data extraction and (5) data synthesis. The coproduction group will help to map the current landscape, identifying key issues that may inhibit or facilitate inclusion. Data will be extracted, analysed and synthesised following realist logic analysis, identifying and explaining how context and mechanisms are conceptualised in the literature and the types of contextual factors that exist and impact on inclusion and participation. Findings will be reported in accordance with Realist and Meta-narrative Evidence Synthesis Evolving Standards . ETHICS AND DISSEMINATION: The coproduction group will agree an ethical approach considering accountability, responsibility and power dynamics, by establishing a terms of reference, taking a reflexive approach and coproducing an ethical framework. Findings will be disseminated to BAFDC and the research community through arts-based methods, peer-reviewed publications and conference presentations, agreeing a coproduced strategy for dissemination. Ethical review is not required. PROSPERO REGISTRATION NUMBER: CRD42024517124.

The impact of patient and public involvement on COVID-19 immunology research: experiences from the UK Coronavirus Immunology Consortium.

BACKGROUND: Patient and Public Involvement (PPI) in clinical trial research is recognised as relevant but the active involvement of patients and the public in basic science or laboratory-based research is seen as more challenging and not often reported. PPI within the UK Coronavirus Immunology Consortium (UK-CIC), a translational research project aimed at tackling some of the key questions about the immune system's response to SARS-CoV-2, is an example of overcoming negative perceptions and obstacles. Given the widespread impact of COVID-19, it was important to consider the impact of UK-CIC research on patients and the public throughout, and the PPI panel were an integral part of the consortium. FINDINGS: Building in funding for a PPI panel to value involvement and ensuring effective expert administrative support and management of PPI were crucial to success. Facilitating relationships and quality interactions between public contributors and researchers required time and commitment to the project from all parties. Through creating a platform and open space to explore diverse views and a wide range of perspectives, PPI was able to influence researchers' ways of thinking about their research and impact future research questions about COVID-19 immunology. Moreover, there was long-term impact from the involvement of the PPI panel in COVID-19 research and their value was reflected in invitations to contribute to additional immunology projects. CONCLUSION: The ability to conduct meaningful PPI with basic immunology research has been shown possible through the UK-CIC in the context of the fast-moving COVID-19 pandemic. The UK-CIC project has laid the foundations for PPI in immunology and this should now be built upon for the advantage of future basic scientific research; PPI can impact greatly on laboratory-based research when given the opportunity to do so.

Serum Metabolome Analysis Identified Amino-Acid Metabolism Associated With Pain in People With Symptomatic Knee Osteoarthritis - A Cross-Sectional Study.

Osteoarthritis (OA) is the most common arthritis affecting synovial joints such as knees and hips of millions of people globally. Usage-related joint pain and reduced function are the most common symptoms experienced by people with OA. To improve pain management, there is a need to identify validated biomarkers predicting therapeutic responses in targeted clinical trials. Our study aimed to identify the metabolic biomarkers for pain and pressure pain detection thresholds (PPTs) in participants with knee pain and symptomatic OA using metabolic phenotyping. Metabolite and cytokine measurements were done on serum samples using LC-MS/MS (liquid gas chromatography integrated magnetic resonance mass spectrometry) and Human Proinflammatory panel 1 kit respectively. Regression analysis was done in a test (n = 75) and replication study (n = 79) to investigate the metabolites associated with current knee pain scores and pressure pain detection thresholds (PPTs). Meta-analysis and correlation were done estimating precision of associated metabolites and identifying relationship between significant metabolites and cytokines respectively. Acyl ornithine, carnosine, cortisol, cortisone, cystine, DOPA, glycolithocholic acid sulphate (GLCAS), phenylethylamine (PEA) and succinic acid were found to be significantly (FDR <.1) associated with pain scores in meta-analysis of both studies. IL-10, IL-13, IL-1ß, IL2, IL8 and TNF-α were also found to be associated with the significant metabolites. Significant associations of these metabolites and inflammatory markers with knee pain suggests that targeting relevant pathways of amino acid and cholesterol metabolism may modulate cytokines and these could be targeted as novel therapeutics development to improve knee pain and OA management. PERSPECTIVE: Foreseeing the global burden of knee pain in Osteoarthritis (OA) and adverse effects of current pharmacological therapies, this study is envisaged to investigate serum metabolites and molecular pathways involved in knee pain. The replicated metabolites in this study suggests targeting amino-acid pathways for better management of OA knee pain.

A toolkit for capturing a representative and equitable sample in health research.

Research participants often do not represent the general population. Systematic exclusion of particular groups from research limits the generalizability of research findings and perpetuates health inequalities. Groups considered underserved by research include those whose inclusion is lower than expected based on population estimates, those with a high healthcare burden but limited research participation opportunities and those whose healthcare engagement is less than others. The REP-EQUITY toolkit guides representative and equitable inclusion in research. The toolkit was developed through a methodological systematic review and synthesis and finalized in a consensus workshop with 24 participants. The REP-EQUITY toolkit describes seven steps for investigators to consider in facilitating representative and equitable sample selection. This includes clearly defining (1) the relevant underserved groups, (2) the aims relating to equity and representativeness, (3) the sample proportion of individuals with characteristics associated with being underserved by research, (4) the recruitment goals, (5) the strategies by which external factors will be managed, (6) the methods by which representation in the final sample will be evaluated and (7) the legacy of having used the toolkit. Using the REP-EQUITY toolkit could promote trust between communities and research institutions, increase diverse participation in research and improve the generalizability of health research. National Institute for Health and Care Research PROSPERO identifier: CRD42022355391.

Improving teamwork in maternity services: A rapid review of interventions.

BACKGROUND: Teamwork is essential for providing safe, effective and women-centred maternity care and several high profile investigations have highlighted the adverse conseqences of dysfuntional teamwork. Maternity teams may need support to identify the most relevant intervention(s) for improving teamwork. OBJECTIVE: To identify and describe current 'off-the-shelf' teamwork interventions freely or commercially available to support improvements to teamworking in UK maternity services and conduct a gap analysis to identify areas for future development. DESIGN: Rapid scoping review METHODS: A multi-component search process was used to identify teamwork interventions, comprising: (1) bibliographic database search (Medline, PsycINFO, CINAHL, MIDRS, NICE evidence research database); (2) identification of relevant policies and UK reports; and (3) expert input from key stakeholders (e.g., maternity service clinicians, managers, policymakers, and report authors). Data were extracted including the scope and content of each intervention and a gap analysis used to map interventions to the integrated team effectiveness model (ITEM) and structure level (macro, meso, micro) and results presented narratively. FINDINGS: Ten interventions were identified. Interventions were heterogeneous in their purpose and scope; six were classified as training courses, three were tools involving observational or diagnostics instruments, and one was a programme involving training and organisational re-design. Interventions were focused on teamwork in obstetric emergencies (n = 5), enhancing routine care (n = 4) or understanding workplace cultures (n = 1). Users of interventions could vary, from whole organisations, to departments, to individual team members. All interventions focused on micro (e.g., team leadership, communication, decision-making, cohesion, and problem solving), with two also focused on meso aspects of teamwork (resources, organisational goals). Evidence for intervention effective on objective outcomes was limited. CONCLUSIONS: Interventions that address key aspects of teamworking are available, particularly for improving safety in obstetric emergency situations. Most interventions, however, are focused on micro features, ignoring the meso (organisational) and macro (systems) features that may also impact on team effectiveness. Evidence-based team improvement interventions that address these gaps are needed. Such interventions would support team ownership of quality improvement, leading to improvements in outcomes for service users, staff and organisations.

Clinical and Preclinical Evidence for Roles of Soluble Epoxide Hydrolase in Osteoarthritis Knee Pain.

OBJECTIVE: Chronic pain due to osteoarthritis (OA) is a major clinical problem, and existing analgesics often have limited beneficial effects and/or adverse effects, necessitating the development of novel therapies. Epoxyeicosatrienoic acids (EETs) are endogenous antiinflammatory mediators, rapidly metabolized by soluble epoxide hydrolase (EH) to dihydroxyeicosatrienoic acids (DHETs). We undertook this study to assess whether soluble EH-driven metabolism of EETs to DHETs plays a critical role in chronic joint pain associated with OA and provides a new target for treatment. METHODS: Potential associations of chronic knee pain with single-nucleotide polymorphisms (SNPs) in the gene-encoding soluble EH and with circulating levels of EETs and DHETs were investigated in human subjects. A surgically induced murine model of OA was used to determine the effects of both acute and chronic selective inhibition of soluble EH by N-[1-(1-oxopropy)-4-piperidinyl]-N'-(trifluoromethoxy)phenyl]-urea (TPPU) on weight-bearing asymmetry, hind paw withdrawal thresholds, joint histology, and circulating concentrations of EETs and DHETs. RESULTS: In human subjects with chronic knee pain, 3 pain measures were associated with SNPs of the soluble EH gene EPHX2, and in 2 separate cohorts of subjects, circulating levels of EETs and DHETs were also associated with 3 pain measures. In the murine OA model, systemic administration of TPPU both acutely and chronically reversed established pain behaviors and decreased circulating levels of 8,9-DHET and 14,15-DHET. EET levels were unchanged by TPPU administration. CONCLUSION: Our novel findings support a role of soluble EH in OA pain and suggest that inhibition of soluble EH and protection of endogenous EETs from catabolism represents a potential new therapeutic target for OA pain.

Stable mossy fiber long-term potentiation requires calcium influx at the granule cell soma, protein synthesis, and microtubule-dependent axonal transport.

The synapses formed by the mossy fiber (MF) axons of hippocampal dentate gyrus granule neurons onto CA3 pyramidal neurons exhibit an intriguing form of experience-dependent synaptic plasticity that is induced and expressed presynaptically. In contrast to most other CNS synapses, long-term potentiation (LTP) at the MF-CA3 synapse is readily induced even during blockade of postsynaptic glutamate receptors. Furthermore, blocking voltage-gated Ca(2+) channels prevents MF-LTP, supporting an involvement of presynaptic Ca(2+) signaling via voltage-gated Ca(2+) channels in MF-LTP induction. We examined the contribution of activity in both dentate granule cell somata and MF terminals to MF-LTP. We found that the induction of stable MF-LTP requires tetanization-induced action potentials not only at MF boutons, but also at dentate granule cell somata. Similarly, blocking Ca(2+) influx via voltage-gated Ca(2+) channels only at the granule cell soma was sufficient to disrupt MF-LTP. Finally, blocking protein synthesis or blocking fast axonal transport mechanisms via disruption of axonal tubulin filaments resulted in decremental MF-LTP. Collectively, these data suggest that-in addition to Ca(2+) influx at the MF terminals-induction of MF synaptic plasticity requires action potential-dependent Ca(2+) signaling at granule cell somata, protein synthesis, and fast axonal transport along MFs. A parsimonious interpretation of these results is that somatic activity triggers protein synthesis at the soma; newly synthesized proteins are then transported to MF terminals, where they contribute to the stabilization of MF-LTP. Finally, the present data imply that synaptic plasticity at the MF-CA3 synapse can be affected by local modulation of somatic and presynaptic Ca(2+) channel activity.

Rapid realist review of the role of community pharmacy in the public health response to COVID-19.

INTRODUCTION: Community pharmacists and their teams have remained accessible to the public providing essential services despite immense pressures during the COVID-19 pandemic. They have successfully expanded the influenza vaccination programme and are now supporting the delivery of the COVID-19 vaccination roll-out. AIM: This rapid realist review aims to understand how community pharmacy can most effectively deliver essential and advanced services, with a focus on vaccination, during the pandemic and in the future. METHOD: An embryonic programme theory was generated using four diverse and complementary documents along with the expertise of the project team. Academic databases, preprint services and grey literature were searched and screened for documents meeting our inclusion criteria. The data were extracted from 103 documents to develop and refine a programme theory using a realist logic of analysis. Our analysis generated 13 context-mechanism-outcome configurations explaining when, why and how community pharmacy can support public health vaccination campaigns, maintain essential services during pandemics and capitalise on opportunities for expanded, sustainable public health service roles. The views of stakeholders including pharmacy users, pharmacists, pharmacy teams and other healthcare professionals were sought throughout to refine the 13 explanatory configurations. RESULTS: The 13 context-mechanism-outcome configurations are organised according to decision makers, community pharmacy teams and community pharmacy users as key actors. Review findings include: supporting a clear role for community pharmacies in public health; clarifying pharmacists' legal and professional liabilities; involving pharmacy teams in service specification design; providing suitable guidance, adequate compensation and resources; and leveraging accessible, convenient locations of community pharmacy. DISCUSSION: Community pharmacy has been able to offer key services during the pandemic. Decision makers must endorse, articulate and support a clear public health role for community pharmacy. We provide key recommendations for decision makers to optimise such a role during these unprecedented times and in the future.

Ammonium influx pathways into astrocytes and neurones of hippocampal slices.

Ammonium (NH(4) (+) ) is required to maintain pathways involved in shuttling metabolic precursors between astrocytes and neurones. Under hyperammonaemic conditions, increases in the cellular influx of NH(4) (+) , and accompanying changes in ion concentrations, may contribute to disruptions in metabolism and neurotransmission. We investigated mechanisms of cellular NH(4) (+) influx in hippocampal slices by measuring acute NH(4) (+) /NH(3) -evoked changes in intracellular pH (pH(i) ) and sodium ([Na(+) ](i) ). In both astrocytes and neurones, application of 5 mM NH(4) Cl for 30-45 min decreased pH(i) by 0.2-0.3 units, consistent with NH(4) (+) influx. In astrocytes, but not neurones, acidifications were accompanied by [Na(+) ](i) increases of 25-30 mM. Glial [Na(+) ](i) increases were blocked by bumetanide, suggesting that NH(4) (+) /NH(3) activated Na(+) -dependent, K(+) , Cl(-) cotransport. Bumetanide also reduced NH(4) (+) /NH(3) -evoked acidifications in astrocytes. Neuronal acidifications were insensitive to bumetanide and inhibition of Cl(-) -dependent transport and K(+) channels, but were prevented by inhibition of Na(+) ,K(+) -ATPase with ouabain. Furthermore, ouabain reduced astrocyte acidifications. Our results suggest that following rapid elevation of NH(4) (+) , Na(+) ,K(+) -ATPase is the major influx pathway for NH(4) (+) in neurones, whereas Na(+) ,K(+) -ATPase and Na(+) -dependent, K(+) , Cl(-) cotransport mediate NH(4) (+) transport into astrocytes. The different mechanisms of NH(4) (+) influx in astrocytes and neurones may contribute to the different susceptibility of both cell types to acute hyperammonaemic conditions.

Quantitative properties of a feedback circuit predict frequency-dependent pattern separation.

Feedback inhibitory motifs are thought to be important for pattern separation across species. How feedback circuits may implement pattern separation of biologically plausible, temporally structured input in mammals is, however, poorly understood. We have quantitatively determined key properties of netfeedback inhibition in the mouse dentate gyrus, a region critically involved in pattern separation. Feedback inhibition is recruited steeply with a low dynamic range (0% to 4% of active GCs), and with a non-uniform spatial profile. Additionally, net feedback inhibition shows frequency-dependent facilitation, driven by strongly facilitating mossy fiber inputs. Computational analyses show a significant contribution of the feedback circuit to pattern separation of theta modulated inputs, even within individual theta cycles. Moreover, pattern separation was selectively boosted at gamma frequencies, in particular for highly similar inputs. This effect was highly robust, suggesting that frequency-dependent pattern separation is a key feature of the feedback inhibitory microcircuit.

You can probably recall where you left your car this morning without too much trouble. But assuming you use the same busy parking lot every day, can you remember which space you parked in yesterday? Or the day before that? Most people find this difficult not because they cannot remember what happened two or three days ago, but because it requires distinguishing between very similar memories. The car, the parking lot, and the time of day were the same on each occasion. So how do you remember where you parked this morning? This ability to distinguish between memories of similar events depends on a brain region called the hippocampus. A subregion of the hippocampus called the dentate gyrus generates different patterns of activity in response to events that are similar but distinct. This process is called pattern separation, and it helps ensure that you do not look for your car in yesterday's parking space. Pattern separation in the dentate gyrus is thought to involve a form of negative feedback called feedback inhibition, a phenomenon where the output of a process acts to limit or stop the same process. To test this idea, Braganza et al. studied feedback inhibition in the dentate gyrus of mice, before building a computer model simulating the inhibition process and supplying the model with two types of realistic input. The first consisted of low-frequency theta brainwaves, which occur, for instance, in the dentate gyrus when animals explore their environment. The second consisted of higher frequency gamma brainwaves, which occur, for example, when animals experience something new. Testing the model showed that feedback inhibition contributes to pattern separation with both theta and gamma inputs. However, pattern separation is stronger with gamma input. This suggests that high frequency brainwaves in the hippocampus could help animals distinguish new events from old ones by promoting pattern separation. Various brain disorders, including Alzheimer's disease, schizophrenia and epilepsy, involve changes in the dentate gyrus and altered brain rhythms. The current findings could help reveal how these changes contribute to memory impairments and to a reduced ability to distinguish similar experiences.

Ammonium-evoked alterations in intracellular sodium and pH reduce glial glutamate transport activity.

The clearance of extracellular glutamate is mainly mediated by pH- and sodium-dependent transport into astrocytes. During hepatic encephalopathy (HE), however, elevated extracellular glutamate concentrations are observed. The primary candidate responsible for the toxic effects observed during HE is ammonium (NH(4) (+)/NH(3)). Here, we examined the effects of NH(4) (+)/NH(3) on steady-state intracellular pH (pH(i)) and sodium concentration ([Na(+)](i)) in cultured astrocytes in two different age groups. Moreover, we assessed the influence of NH(4) (+)/NH(3) on glutamate transporter activity by measuring D-aspartate-induced pH(i) and [Na(+)](i) transients. In 20-34 days in vitro (DIV) astrocytes, NH(4) (+)/NH(3) decreased steady-state pH(i) by 0.19 pH units and increased [Na(+)](i) by 21 mM. D-Aspartate-induced pH(i) and [Na(+)](i) transients were reduced by 80-90% in the presence of NH(4) (+)/NH(3), indicating a dramatic reduction of glutamate uptake activity. In 9-16 DIV astrocytes, in contrast, pH(i) and [Na(+)](i) were minimally affected by NH(4) (+)/NH(3), and D-aspartate-induced pH(i) and [Na(+)](i) transients were reduced by only 30-40%. Next we determined the contribution of Na(+), K(+), Cl(-)-cotransport (NKCC). Immunocytochemical stainings indicated an increased expression of NKCC1 in 20-34 DIV astrocytes. Moreover, inhibition of NKCC with bumetanide prevented NH(4) (+)/NH(3)-evoked changes in steady-state pH(i) and [Na(+)](i) and attenuated the reduction of D-aspartate-induced pH(i) and [Na(+)](i) transients by NH(4) (+)/NH(3) to 30% in 20-34 DIV astrocytes. Our results suggest that NH(4) (+)/NH(3) decreases steady-state pH(i) and increases steady-state [Na(+)](i) in astrocytes by an age-dependent activation of NKCC. These NH(4) (+)/NH(3)-evoked changes in the transmembrane pH and sodium gradients directly reduce glutamate transport activity, and may, thus, contribute to elevated extracellular glutamate levels observed during HE.

Axon-carrying dendrites convey privileged synaptic input in hippocampal neurons.

Neuronal processing is classically conceptualized as dendritic input, somatic integration, and axonal output. The axon initial segment, the proposed site of action potential generation, usually emanates directly from the soma. However, we found that axons of hippocampal pyramidal cells frequently derive from a basal dendrite rather than from the soma. This morphology is particularly enriched in central CA1, the principal hippocampal output area. Multiphoton glutamate uncaging revealed that input onto the axon-carrying dendrites (AcDs) was more efficient in eliciting action potential output than input onto regular basal dendrites. First, synaptic input onto AcDs generates action potentials with lower activation thresholds compared with regular dendrites. Second, AcDs are intrinsically more excitable, generating dendritic spikes with higher probability and greater strength. Thus, axon-carrying dendrites constitute a privileged channel for excitatory synaptic input in a subset of cortical pyramidal cells.