Anna De Simoni

Development of Rat CA1 Neurones in Acute Versus Organotypic Slices: Role of Experience in Synaptic Morphology and Activity

Despite their wide use, the physiological relevance of organotypic slices remains controversial. Such cultures are prepared at 5 days postnatal. Although some local circuitry remains intact, they develop subsequently in isolation from the animal and hence without plasticity due to experience. Development of synaptic connectivity and morphology might be expected to proceed differently under these conditions than in a behaving animal. To address these questions, patch‐clamp techniques and confocal microscopy were used in the CA1 region of the rat hippocampus to compare acute slices from the third postnatal week with various stages of organotypic slices. Acute slices prepared at postnatal days (P) 14, 17 and 21 were found to be developmentally equivalent to organotypic slices cultured for 1, 2 and 3 weeks, respectively, in terms of development of synaptic transmission and dendritic morphology. The frequency of inhibitory and excitatory miniature synaptic currents increased in parallel. Development of dendritic length and primary branching as well as spine density and proportions of different spine types were also similar in both preparations, at these corresponding stages. The most notable difference between organotypic and acute slices was a four‐ to five‐fold increase in the absolute frequency of glutamatergic (but not GABAergic) miniature postsynaptic currents in organotypic slices. This was probably related to an increase in complexity of higher order dendritic branching in organotypic slices, as measured by fractal analysis, resulting in an increased total synapse number. Both increased excitatory miniature synaptic current frequency and dendritic complexity were already established during the first week in culture. The level of complexity then stayed constant in both preparations over subsequent stages, with synaptic frequency increasing in parallel. Thus, although connectivity was greater in organotypic slices, once this was established, development continued in both preparations at a remarkably similar rate. We conclude that, for the parameters studied, changes seem to be preprogrammed by 5 days and their subsequent development is largely independent of environment.

Preparation of organotypic hippocampal slice cultures: interface method

This protocol describes a method for making and culturing rat hippocampal organotypic slices on membrane inserts. Supplementary videos are included to demonstrate visually the different steps of the procedure. Cultured hippocampal slices has been increasingly used as a model for synaptic studies of the brain as they allow examination of mid to long term manipulations in a preparation where the gross cytoarchitecture of the hippocampus is preserved. Combining techniques such as molecular biology, electrophysiology and immunohistochemistry to study physiological or pathological processes can easily be applied to organotypic slices. The technique described here can be used to make organotypic slices from other parts of the brain, other rodent species and from a range of ages. This protocol can be completed in 3 h.This protocol describes a method for making and culturing rat hippocampal organotypic slices on membrane inserts. Supplementary videos are included to demonstrate visually the different steps of the procedure. Cultured hippocampal slices has been increasingly used as a model for synaptic studies of the brain as they allow examination of mid to long term manipulations in a preparation where the gross cytoarchitecture of the hippocampus is preserved. Combining techniques such as molecular biology, electrophysiology and immunohistochemistry to study physiological or pathological processes can easily be applied to organotypic slices. The technique described here can be used to make organotypic slices from other parts of the brain, other rodent species and from a range of ages. This protocol can be completed in 3 h.

Corticosterone reduces dendritic complexity in developing hippocampal CA1 neurons

Although prolonged stress and corticosteroid exposure induce morphological changes in the hippocampal CA3 area, the adult CA1 area is quite resistant to such changes. Here we addressed the question whether elevated corticosteroid hormone levels change dendritic complexity in young, developing CA1 cells. In organotypic cultures (prepared from P5 rats) that were 14–21 days cultured in vitro, two doses of corticosterone (30 and 100 nM) were tested. Dendritic morphology of CA1 neurons was established by imaging neurons filled with the fluorescent dye Alexa. Application of 100 nM corticosterone for 20 minutes induced atrophy of the apical dendritic tree 1–4 hours later. Fractal analysis showed that total neuronal complexity was reduced twofold when compared with vehicle‐treated neurons. Exposing organotypic slices to 30 nM corticosterone reduced apical length in a more delayed manner: only neurons examined more than 2 hours after exposure to corticosterone showed atrophy of the apical dendritic tree. Neither dose of corticosterone affected the length of basal dendrites or spine density. Corticosterone was ineffective in changing morphology of the apical dendrites when tested in the presence of the glucocorticoid receptor antagonist RU38486. These results suggest that high physiological levels of corticosterone, via activation of the glucocorticoid receptor, can, during the course of only a few hours, reduce the dendritic complexity of CA1 pyramidal neurons in young, developing hippocampal tissue. These findings suggest that it is relevant to maintain plasma corticosterone levels low during hippocampal development.

Changes in extracellular Ca2+ can affect the pattern of discharge in rat thalamic neurons

1 The aim of this study was to investigate some of the cellular mechanisms involved in the effects caused by changes in extracellular Ca2+ concentration ([Ca2+]o). 2 Current‐ and voltage‐clamp experiments were carried out on acutely isolated thalamic neurons of rats. 3 Increasing [Ca2+]o alone induced a transition of the discharge from single spike to burst mode in isolated current‐clamped neurons. 4 Increasing [Ca2+]o caused the voltage‐dependent characteristics of the low voltage‐activated (LVA) transient Ca2+ currents to shift towards positive values on the voltage axis. Changing [Ca2+]o from 0.5 to 5 mM caused the inactivation curve to shift by 21 mV. 5 Extracellular Ca2+ blocked a steady cationic current. This current reversed at ‐35 mV, was scarcely affected by Mg2+ and was completely blocked by the non‐selective cation channel inhibitor gadolinium (10 μM). The effect of [Ca2+]o was mimicked by 500 μM spermine, a polyamine which acts as an agonist for the Ca2+‐sensing receptor, and was modulated by intracellular GTP‐γ‐S. 6 At the resting potential, both the voltage shift and the block of the inward current removed the inactivation of LVA calcium channels and, together with the increase in the Ca2+ driving force, favoured a rise in the low threshold Ca2+ spikes, causing the thalamic firing to change to the oscillatory mode. 7 Our data indicate that [Ca2+]o is involved in multiple mechanisms of control of the thalamic relay and pacemaker activity. These findings shed light on the correlation between hypercalcaemia, low frequency EEG activity and symptoms such as sleepiness and lethargy described in many clinical papers.

Charge compensation for NADPH oxidase activity in microglia in rat brain slices does not involve a proton current

The membrane properties of isolated cultured microglia have been extensively studied but it is important to understand their properties in situ, where they protect the brain against infection, but also contribute to neurodegenerative diseases. Microglia and macrophages attack bacteria by generating reactive oxygen species, a process which involves NADPH oxidase pumping electrons out across the cell membrane. The resulting inward current evokes a depolarization, which would inhibit the activity of the NADPH oxidase if there were no charge‐compensating current which moves positive charge out across the membrane. The mechanism of this charge compensation is controversial. In neutrophils and in cultured microglia a depolarization‐activated H+ conductance has been proposed to provide charge compensation, and also to remove protons generated intracellularly by the NADPH oxidase. Alternatively, a depolarization‐activated K+ conductance has been proposed to mediate charge compensation. Here we show that in microglia, either in the resting state or when activated by the bacterial coat component lipopolysaccharide, both in acute and in cultured hippocampal slices, no significant H+ current is detectable. This implies that the membrane properties of microglia in their normal cellular environment differ from those of cultured microglia (similarly, microglia generated a current in response to ATP but, unlike in culture, not to glutamate or GABA). Furthermore, the K+ current (Kv1.3) that is activated by lipopolysaccharide is inactivated by depolarization, making it unsuitable for mediating charge compensation on a long time scale at positive voltages. Instead, charge compensation may be mediated by a previously undescribed non‐selective cation current.

Trials to improve blood pressure through adherence to antihypertensives in stroke/TIA: systematic review and meta-analysis.

Background The purpose of this study was to determine whether interventions including components to improve adherence to antihypertensive medications in patients after stroke/transient ischemic attack (TIA) improve adherence and blood pressure control. Methods and Results We searched MEDLINE, EMBASE, CINAHL, BNI, PsycINFO, and article reference lists to October 2012. Search terms included stroke/TIA, adherence/prevention, hypertension, and randomized controlled trial (RCT). Inclusion criteria were participants with stroke/TIA; interventions including a component to improve adherence to antihypertensive medications; and outcomes including blood pressure, antihypertensive adherence, or both. Two reviewers independently assessed studies to determine eligibility, validity, and quality. Seven RCTs were eligible (n=1591). Methodological quality varied. All trials tested multifactorial interventions. None targeted medication adherence alone. Six trials measured blood pressure and 3 adherence. Meta‐analysis of 6 trials showed that multifactorial programs were associated with improved blood pressure control. The difference between intervention versus control in mean improvement in systolic blood pressure was −5.3 mm Hg (95% CI, −10.2 to −0.4 mm Hg, P=0.035; I2=67% [21% to 86%]) and in diastolic blood pressure was −2.5 mm Hg (−5.0 to −0.1 mm Hg, P=0.046; I2=47% [0% to 79%]). There was no effect on medication adherence where measured. Conclusions Multifactorial interventions including a component to improve medication adherence can lower blood pressure after stroke/TIA. However, it is not possible to say whether or not this is achieved through better medication adherence. Trials are needed of well‐characterized interventions to improve medication adherence and clinical outcomes with measurement along the hypothesized causal pathway.

Barriers and facilitators to staying in work after stroke: insight from an online forum

Objective To explore barriers and facilitators to staying in work following stroke. Design Qualitative analysis of posts regarding staying in work following stroke using the archives of an online forum for stroke survivors. Participants 60 stroke survivors (29 male, 23 female, 8 not stated; mean age at stroke 44 years) who have returned to work, identified using terms ‘return to work’ and ‘back at work’. Setting Posts from UK stroke survivors and family members on Talkstroke, the forum of the Stroke Association, between 2004 and 2011. Results Stroke and transient ischaemic attack (TIA) survivors reported residual impairments that for many had impact on work. Most impairments were ‘invisible’, including fatigue, problems with concentration, memory and personality changes. Participants described positive (eg, back at work being better than expected) and negative work experiences, including being at risk of losing the job because of stroke-related impairments. Barriers to successfully staying in work included lack of understanding of stroke—in particular invisible impairments—of survivors, employers and general practitioners (GPs), and lack of support in terms of formal adjustments, and ‘feeling supported’. Stroke survivors described how they developed their own coping strategies, and how workplace and employer helped them to stay in work. Conclusions Despite having been able to return to work after a stroke, people may still experience difficulties in staying in work and risking losing their job. There is a need to improve awareness, in particular of invisible stroke-related impairments, among stroke survivors, work personnel and clinicians. This might be achieved through improved assessments of residual impairments in the workplace and in general practice. Future studies should investigate the effect of unrecognised fatigue and invisible impairments on staying in work following stroke, and explore the potential role for primary care in supporting stroke survivors who have returned to employment.

Making sense of patients’ internet forums: a systematic method using discourse analysis

A survey of access to the internet in the UK conducted in 2008 revealed that the internet is used by 79% of men and 75% of women of all ages including 72% of people aged 55–64 years and 32% of people aged ≥65 years.1 Internet data that is freely and publicly accessible are now being used for research purposes.2,3 Internet communities offer an increasingly important source of information expressed openly by individuals. In particular, the internet offers access to hard-to-reach groups who are often excluded (or exclude themselves) from traditional research studies. Discourse analysis (DA), an approach to analysing naturally occurring language, is a technique that is particularly suited to examining internet data.4,5 DA is pertinent to health care for it has the potential to reveal the dimensions of health beliefs, the doctor–patient relationships and the dissemination of health information. The focus of DA is on communicative behaviour.6 Within internet forums communicative behaviour is the manner in which individuals communicate through written text. At a basic level, interrogation with linguistic analysis software reveals word frequency. Frequency is a simple way to identify problems and issues. We can look at how patterns of words colocate together and uncover associations between words (that is, concordances) that may provide insights into people, groups, and ideas. With the development of computers, linguistics has become involved using concordancing where keywords from a body of text, often termed a corpus, are highlighted in their surrounding context. Search engines like Google and Yahoo are, at heart, simple concordancers in their browsing functions. They offer the casual user the opportunity to search a very large database …

Implementation of a nurse-led behaviour change intervention to support medication taking in type 2 diabetes: beyond hypothesised active ingredients (SAMS Consultation Study)

BackgroundImplementation of trial interventions is rarely assessed, despite its effects on findings. We assessed the implementation of a nurse-led intervention to facilitate medication adherence in type 2 diabetes (SAMS) in a trial against standard care in general practice. The intervention increased adherence, but not through the hypothesised psychological mechanism. This study aimed to develop a reliable coding frame for tape-recorded consultations, assessing both a priori hypothesised and potential active ingredients observed during implementation, and to describe the delivery and receipt of intervention and standard care components to understand how the intervention might have worked.Methods211 patients were randomised to intervention or comparison groups and 194/211 consultations were tape-recorded. Practice nurses delivered standard care to all patients and motivational and action planning (implementation intention) techniques to intervention patients only. The coding frame was developed and piloted iteratively on selected tape recordings until a priori reliability thresholds were achieved. All tape-recorded consultations were coded and a random subsample double-coded.ResultsNurse communication, nurse-patient relationship and patient responses were identified as potential active ingredients over and above the a priori hypothesised techniques. The coding frame proved reliable. Intervention and standard care were clearly differentiated. Nurse protocol adherence was good (M (SD) = 3.95 (0.91)) and competence of intervention delivery moderate (M (SD) = 3.15 (1.01)). Nurses frequently reinforced positive beliefs about taking medication (e.g., 65% for advantages) but rarely prompted problem solving of negative beliefs (e.g., 21% for barriers). Patients’ action plans were virtually identical to current routines. Nurses showed significantly less patient-centred communication with the intervention than comparison group.ConclusionsIt is feasible to reliably assess the implementation of behaviour change interventions in clinical practice. The main study results could not be explained by poor delivery of motivational and action planning components, definition of new action plans, improved problem solving or patient-centred communication. Possible mechanisms of increased medication adherence include spending more time discussing it and mental rehearsal of successful performance of current routines, combined with action planning. Delivery of a new behaviour change intervention may lead to less patient-centred communication and possible reduction in overall trial effects.Trial registrationISRCTN30522359.

Medicines optimisation in primary care: can community pharmacies deliver?

One pound in every eight of NHS spending is on medicines, yet it is generally agreed that up to half of all the medicines prescribed are not used as the prescriber intended.1 The problem of sub-optimal use of medicines in chronic diseases has been recognised for many years, but only recently has ‘medicines optimisation’ been on the agenda of policy makers. Medicines optimisation refers to the process of making the use of medication by patients as safe, effective, and efficient as possible. A key part of the government’s strategy towards achieving this has been to extend the role of community pharmacies, and to make better use of pharmacists’ specific medicines-related skills and knowledge. This strategy for medicines optimisation began with the 2005 Medicines Use Review (MUR) service, and received another fillip in October last year with the launch of the New Medicines Service (NMS) in England.2 Whereas the focus of the MUR was on improving medicines use by patients already taking multiple medicines for a period of time, the NMS aims to provide early support to patients who are newly prescribed a medicine for a long-term condition. Similar services have been introduced in Scotland and Wales. Given the investment of scarce NHS resources (up to £55 million pounds per annum until 2013 in England for the NMS) what are the chances of success? Here we explore the potential, the evidence, and the challenges of the NMS service. The NMS seeks to improve medicines use in people with long-term conditions who are newly prescribed …