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1.  Chemoreception and asphyxia-induced arousal 
Arousal protects against the adverse and potentially fatal effects of asphyxia during sleep. Asphyxia stimulates the carotid bodies and central chemoreceptors but the sequence of events leading to arousal is uncertain. In this review, the theoretical mechanisms leading to arousal from sleep are briefly summarized and the issue of whether central respiratory chemoreceptors (CRCs) or other types of CO2-responsive CNS neurons contribute to asphyxia-induced arousal is discussed. We focus on the role of the retrotrapezoid nucleus, the raphe and the locus coeruleus and emphasize the anatomical and neurophysiological evidence which suggests that these putative central chemoreceptors could contribute to arousal independently of their effects on breathing. Finally, we describe recent attempts to test the contribution of specific brainstem pathways to asphyxia-induced arousal using optogenetic and other tools and the possible contribution of a group of hypoxia-sensitive brainstem neurons (the C1 cells) to breathing and arousal.
doi:10.1016/j.resp.2013.04.011
PMCID: PMC3749262  PMID: 23608705
retrotrapezoid nucleus; raphe; locus coerruleus; optogenetics; sleep apnea
2.  The respiratory chemoreception conundrum: light at the end of the tunnel? 
Brain research  2012;1511:126-137.
Summary
Arterial PCO2 is tightly regulated via changes in breathing. A rise in PCO2 activates the carotid bodies and exerts additional effects on neurons located within the CNS, causing an increase in lung ventilation. Central respiratory chemoreception refers to the component of this homeostatic reflex that is triggered by activation of receptors located within the brain (central chemoreceptors). Throughout the body, CO2 generally operates via the proxy of pH. Since countless proteins, ion channels and neurons display some degree of pH-sensitivity, the notion that central respiratory chemoreception could rely on a few specialized neurons seems a priori counter-intuitive. Yet, two types of neurons currently stand out as critically important for breathing regulation by CO2: the retrotrapezoid nucleus (RTN) and the raphe. RTN neurons are glutamatergic, strongly activated by hypercapnia in vivo and by CO2 or protons in slices. These neurons target selectively the pontomedullary regions implicated in generating the respiratory rhythm and pattern. Their response to CO2 seems to involve both cell-autonomous and paracrine effects of CO2, the latter presumably mediated by the surrounding glia. The specific connections that these excitatory neurons establish with the rest of the breathing network are likely to be the main explanation of their importance to respiratory chemoreception. Serotonergic neurons have a powerful stimulatory effect on breathing, they facilitate the chemoreflexes and a subset of them likely function as CO2 sensors.
Opto- and pharmacogenetic methods have played an important role in assessing the contribution of RTN and serotonergic neurons as well as glial cells to respiration. These particular experiments are emphasized here for thematic reasons although the current perception of the importance of the RTN and serotonergic cells to respiratory chemoreception also relies on many other types of evidence. A small portion of this evidence is presented as background.
doi:10.1016/j.brainres.2012.10.028
PMCID: PMC3570739  PMID: 23088963
3.  OPTOGENETIC STIMULATION OF C1 AND RETROTRAPEZOID NUCLEUS NEURONS CAUSES SLEEP STATE DEPENDENT CARDIORESPIRATORY STIMULATION AND AROUSAL IN RATS 
Hypertension  2013;61(4):835-841.
C1 catecholaminergic neurons and neurons of the retrotrapezoid nucleus are integrative nodes within the brainstem network regulating cardiorespiratory reflexes elicited by hypoxia and hypercapnia, stimuli that also produce arousal from sleep. In the present study, Channelrhodopsin2 was selectively introduced into these neurons with a lentiviral vector in order to determine if their selective activation also produces arousal in sleeping rats. Sleep stages were identified from electroencephalographic and neck muscle electromyographic recordings. Breathing was measured using unrestrained whole body plethysmography and blood pressure by telemetry. During non-rapid eye movement sleep, unilateral photostimulation of the C1 region caused arousal in 83.0 ± 14.7% of trials and immediate and intense cardiorespiratory activation. Arousal during photostimulation was also observed during rapid eye movement sleep (41.9 ± 5.6% of trials), but less reliably than during non-rapid eye movement sleep. The cardiorespiratory responses elicited by photostimulation were dramatically smaller during rapid eye movement sleep than non-rapid eye movement sleep or wakefulness. Systemic alpha1-adrenoreceptor blockade reduced the cardiorespiratory effects of photostimulation, but had no effect on the arousal caused by photostimulation during non-rapid eye movement sleep. Postmortem histology showed that neurons expressing Channelrhodopsin2-mCherry were predominantly catecholaminergic (81%). These results show that selective activation of C1 and retrotrapezoid nucleus neurons produces state dependent arousal and cardiorespiratory stimulation. These neurons, which are powerfully activated by chemoreceptor stimulation, may contribute to the sleep disruption associated with obstructive sleep apnea.
doi:10.1161/HYPERTENSIONAHA.111.00860
PMCID: PMC3666866  PMID: 23438930
Sympathetic nervous system; chemoreception; Phox2b; asphyxia; sleep apnea
4.  Medical students’ perceptions and understanding of their specific learning difficulties 
Objectives
The purpose of this study is to explore how medical students with Specific Learning Difficulties perceive and understand their Specific Learning Difficulty and how it has impacted on their experience of medical training.
Method
A purposive sample of fifteen students from one medical school was interviewed. Framework Analysis was used to identify and organise themes emerging from the data. An interpretation of the data was made capturing the essence of what had been learned. The concept of ‘reframing’ was then used to re-analyse and organise the data.
Results
Students reported having found ways to cope with their Specific Leaning Difficulty in the past, some of which proved inadequate to deal with the pressures of medical school. Diagnosis was a mixed experience: many felt relieved to understand their difficulties better, but some feared discrimination. Practical support was available in university but not in placement. Students focused on the impact of their Specific Learning Difficulty on their ability to pass undergraduate exams. Most did not contemplate difficulties post-qualification.
Conclusions
The rigours of the undergraduate medical course may reveal undisclosed Specific Learning Difficulties. Students need help to cope with such challenges, psychologically and practically in both classroom and clinical practice. University services for students with Specific Learning Difficulties should become familiar with the challenges of clinical placements, and ensure that academic staff has access to information about the needs of these students and how these can be met.
doi:10.5116/ijme.524f.cd3f
PMCID: PMC4205534
Specific learning difficulties; academic support; medical students
5.  Selective optogenetic activation of rostral ventrolateral medullary catecholaminergic neurons produces cardiorespiratory stimulationin conscious mice 
Activation of rostral ventrolateral medullary catecholaminergic (RVLM-CA) neurons e.g. by hypoxia is thought to increase sympathetic outflow thereby raising blood pressure (BP). Here we test whether these neurons also regulate breathing and cardiovascular variables other than BP. Selective expression of ChR2-mCherry by RVLM-CA neurons was achieved by injecting Cre-dependent vector AAV2-EF1α-DIO-ChR2-mCherry unilaterally into RVLM of dopamine-beta-hydroxylaseCre/0 (DβHCre/0) mice. Photostimulation of RVLM-CA neurons increased breathing in anesthetized and conscious mice. In conscious mice, photostimulation primarily increased breathing frequency and this effect was fully occluded by hypoxia (10% O2). In contrast, the effects of photostimulation were largely unaffected by hypercapnia (3 and 6% CO2). The associated cardiovascular effects were complex (slight bradycardia and hypotension) and, using selective autonomic blockers, could be explained by co-activation of the sympathetic and cardiovagal outflows. ChR2-positive RVLM-CA neurons expressed VGLUT2 and their projections were mapped. Their complex cardiorespiratory effects are presumably mediated by their extensive projections to supraspinal sites such as the ventrolateral medulla, the dorsal vagal complex, the dorsolateral pons, and selected hypothalamic nuclei (dorsomedial, lateral, paraventricular nuclei).
In sum, selective optogenetic activation of RVLM-CA neurons in conscious mice revealed two important novel functions of these neurons, namely breathing stimulation and cardiovagal outflow control, effects that are attenuated or absent under anesthesia and are presumably mediated by the numerous supraspinal projections of these neurons. The results also suggest that RVLM-CA neurons may underlie some of the acute respiratory response elicited by carotid body stimulation but contribute little to the central respiratory chemoreflex.
doi:10.1523/JNEUROSCI.1046-12.2013
PMCID: PMC3596815  PMID: 23407970
adrenergic neurons; breathing; optogenetics; hypoxia; central respiratory chemoreceptors
6.  Wild type microglia arrest pathology in a mouse model of Rett syndrome 
Nature  2012;484(7392):105-109.
Rett syndrome is an X-linked autism spectrum disorder. The disease is characterized in the majority of cases by mutation of the MECP2 gene, which encodes a methyl-CpG-binding protein 1–5. Although MeCP2 is expressed in many tissues, the disease is generally attributed to a primary neuronal dysfunction 6. However, as shown recently, glia, specifically astrocytes, also contribute to Rett pathophysiology. Here we examined the role of another form of glia, microglia, in a murine model of Rett syndrome. Transplantation of wild type bone marrow into irradiation-conditioned Mecp2-null hosts resulted in engraftment of brain parenchyma by bone marrow-derived myeloid cells of microglial phenotype, and arrest of disease development. However, when cranial irradiation was blocked by lead shield, and microglial engraftment was prevented, disease was not arrested. Similarly, targeted expression of Mecp2 in myeloid cells, driven by Lysmcre on an Mecp2-null background, dramatically attenuated disease symptoms. Thus, via multiple approaches, wild type Mecp2-expressing microglia within the context of an Mecp2-null male mouse arrested numerous facets of disease pathology; lifespan was increased; breathing patterns were normalized; apneas were reduced; body weight was increased to near wild type, and locomotor activity was improved. Mecp2+/− females also exhibited significant improvements as a result of wild type microglial engraftment. These benefits mediated by wild type microglia, however, were diminished when phagocytic activity was inhibited pharmacologically using annexin V to block phosphatydilserine residues on apoptotic targets, thus preventing recognition and engulfment by tissue-resident phagocytes. These results suggest the importance of microglial phagocytic activity in Rett syndrome. Our data implicate microglia as major players in Rett pathophysiology, and suggest that bone marrow transplantation might offer a feasible therapeutic approach for this devastating disorder.
doi:10.1038/nature10907
PMCID: PMC3321067  PMID: 22425995
7.  Phox2b-expressing neurons of the parafacial region regulate breathing rate, inspiration and expiration in conscious rats 
The retrotrapezoid nucleus contains Phox2b-expressing glutamatergic neurons (RTN-Phox2b neurons) that regulate breathing in a CO2-dependent manner. Here we use channelrhodopsin-based optogenetics to explore how these neurons control breathing in conscious and anesthetized adult rats. Respiratory entrainment (pacing) of breathing frequency (fR) was produced over 57% (anesthetized) and 28% (conscious) of the natural frequency range by burst activation of RTN-Phox2b neurons (3–8 × 0.5–20 ms pulses at 20 Hz). In conscious rats, pacing under normocapnic conditions increased tidal volume (VT) and each inspiration was preceded by phase 2 (active) expiration, denoting abdominal muscle contraction. During long-term pacing, VT returned to pre-stimulation levels suggesting that central chemoreceptors such as RTN-Phox2b neurons regulate VT partly independently of their effect on fR. Randomly applied light-trains reset the respiratory rhythm and shortened the expiratory phase (TE) when the stimulus coincided with late-inspiration or early-expiration. Importantly, continuous (20 Hz) photostimulation of the RTN-Phox2b neurons and a saturating CO2 concentration produced similar effects on breathing that were much larger than those elicited by phasic RTN stimulation.
In sum, consistent with their anatomical projections, RTN-Phox2b neurons regulate lung ventilation by controlling breathing frequency, inspiration and active expiration. Adult RTN-Phox2b neurons can entrain the respiratory rhythm if their discharge is artificially synchronized but continuous activation of these neurons is much more effective at increasing lung ventilation. These results suggest that RTN-Phox2b neurons are no longer rhythmogenic in adulthood and that their average discharge rate may be far more important than their discharge pattern in driving lung ventilation.
doi:10.1523/JNEUROSCI.3280-11.2011
PMCID: PMC3236529  PMID: 22072691
Central chemoreceptors; breathing; respiration; retrotrapezoid nucleus; lentivirus; medulla oblongata; optogenetics
8.  Retrotrapezoid nucleus, respiratory chemosensitivity and breathing automaticity 
SUMMARY
Breathing automaticity and CO2 regulation are inseparable neural processes. The retrotrapezoid nucleus (RTN), a group of glutamatergic neurons that express the transcription factor Phox2b, may be a crucial nodal point through which breathing automaticity is regulated to maintain CO2 constant. This review updates the analysis presented in prior publications. Additional evidence that RTN neurons have central respiratory chemoreceptor properties is presented but this is only one of many factors that determine their activity. The RTN is also regulated by powerful inputs from the carotid bodies and, at least in the adult, by many other synaptic inputs. We also analyze how RTN neurons may control the activity of the downstream central respiratory pattern generator. Specifically, we review the evidence which suggests that RTN neurons a) innervate the entire ventral respiratory column, and b) control both inspiration and expiration. Finally, we argue that the RTN neurons are the adult form of the parafacial respiratory group in neonate rats.
doi:10.1016/j.resp.2009.02.001
PMCID: PMC2734912  PMID: 19712903
Central chemoreceptors; breathing; Phox2b; retrotrapezoid nucleus; parafacial respiratory group
9.  Collaborative working within UK NHS secondary care and across sectors for COPD and the impact of peer review: qualitative findings from the UK National COPD Resources and Outcomes Project 
Introduction
We investigated the effects on collaborative work within the UK National Health Service (NHS) of an intervention for service quality improvement: informal, structured, reciprocated, multidisciplinary peer review with feedback and action plans. The setting was care for chronic obstructive pulmonary disease (COPD).
Theory and methods
We analysed semi-structured interviews with 43 hospital respiratory consultants, nurses and general managers at 24 intervention and 11 control sites, as part of a UK randomised controlled study, the National COPD Resources and Outcomes Project (NCROP), using Scott's conceptual framework for action (inter-organisational, intra-organisational, inter-professional and inter-individual). Three areas of care targeted by NCROP involved collaboration across primary and secondary care.
Results
Hospital respiratory department collaborations with commissioners and hospital managers varied. Analysis suggested that this is related to team responses to barriers. Clinicians in unsuccessful collaborations told ‘atrocity stories’ of organisational, structural and professional barriers to service improvement. The others removed barriers by working with government and commissioner agendas to ensure continued involvement in patients' care. Multidisciplinary peer review facilitated collaboration between participants, enabling them to meet, reconcile differences and exchange ideas across boundaries.
Conclusions
The data come from the first randomised controlled trial of organisational peer review, adding to research into UK health service collaborative work, which has had a more restricted focus on inter-professional relations. NCROP peer review may only modestly improve collaboration but these data suggest it might be more effective than top-down exhortations to change when collaboration both across and within organisations is required.
PMCID: PMC2948684  PMID: 20922063
collaboration; inter-organizational; inter-professional; quality improvement; respiratory disease
10.  Photostimulation of retrotrapezoid nucleus Phox2b-expressing neurons in vivo produces long-lasting activation of breathing in rats 
SUMMARY
The retrotrapezoid “nucleus” (RTN), located in the rostral ventrolateral medullary reticular formation, contains a bilateral cluster of about 1000 glutamatergic non catecholaminergic Phox2b-expressing propriobulbar neurons that are activated by CO2 in vivo and by acidification in vitro. These cells are thought to function as central respiratory chemoreceptors but this theory still lacks a crucial piece of evidence, namely that stimulating these particular neurons selectively in vivo increases breathing. The present study carried out in anesthetized rats seeks to test whether this expectation is correct. We injected into the left RTN a lentivirus that expresses the light-activated cationic channel channelrhodopsin-2 (ChR2, H134R mutation, fused to the fluorescent protein mCherry) under the control of the Phox2-responsive promoter PRSx8. Transgene expression was restricted to 423 ± 38 Phox2b-expressing neurons per rat consisting of non-catecholaminergic and C1 adrenergic neurons (3/2 ratio). Photostimulation delivered to the RTN region in vivo via a fiberoptic activated the CO2-sensitive neurons vigorously, produced a long-lasting (t1/2 = 11 s) increase in phrenic nerve activity and caused a small and short-lasting cardiovascular stimulation. Selective lesions of the C1 cells eliminated the cardiovascular response but left the respiratory stimulation intact. In rats with C1 cell lesions, the mCherry-labeled axon terminals originating from the transfected non-catecholaminergic neurons were present exclusively in the lower brainstem regions that contain the respiratory pattern generator.
These results provide strong evidence that the Phox2b-expressing non-catecholaminergic neurons of the RTN region function as central respiratory chemoreceptors.
doi:10.1523/JNEUROSCI.1106-09.2009
PMCID: PMC2696034  PMID: 19420248
Central chemoreceptors; breathing; respiration; Phox2b; retrotrapezoid nucleus; lentivirus; medulla oblongata; channel rhodopsin
11.  Systematic review of recent innovations in service provision to improve access to primary care 
Background: In England, there are particularly pressing problems concerning access to adequate primary care services. Consequently, innovative ways of delivering primary care have been introduced to facilitate and broaden access.
Aims: The aim of this study was to review the evidence of seven recent innovations in service provision to improve access or equity in access to primary care, by performing a systematic review of the literature.
Design of study: Systematic review.
Setting: Primary care in the United Kingdom (UK).
Method: Seven electronic databases were searched and key journals were hand-searched. Unpublished and ‘grey’ literature were sought via the Internet and through professional contacts. Intervention studies addressing one of seven recent innovations and conducted in the UK during the last 20 years were included. Two researchers independently assessed the quality of papers.
Results: Thirty studies (32 papers and two reports) were identified overall. Variation in study design and outcome measures made comparisons difficult. However, there was some evidence to suggest that access is improved by changing the ways in which primary care is delivered. First-wave personal medical services pilots facilitated improvements in access to primary care in previously under-served areas and/or populations. Walk-in centres and NHS Direct have provided additional access to primary care for white middle-class patients; there is some evidence suggesting that these innovations have increased access inequalities. There is some evidence that telephone consultations with GPs or nurses can safely substitute face-to-face consultations, although it is not clear that this reduces the number of face-to-face consultations over time. Nurse practitioners and community pharmacists can manage common conditions without the patient consulting a general practitioner.
Conclusion: The evidence is insufficient to make clear recommendations regarding ways to improve access to primary care. In the future, it is important that, as new initiatives are planned, well-designed evaluations are commissioned simultaneously.
PMCID: PMC1266174  PMID: 15113523
delivery of health care; health services accessibility; primary health care
13.  RecA Protein from the Extremely Radioresistant Bacterium Deinococcus radiodurans: Expression, Purification, and Characterization 
Journal of Bacteriology  2002;184(6):1649-1660.
The RecA protein of Deinococcus radiodurans (RecADr) is essential for the extreme radiation resistance of this organism. The RecADr protein has been cloned and expressed in Escherichia coli and purified from this host. In some respects, the RecADr protein and the E. coli RecA (RecAEc) proteins are close functional homologues. RecADr forms filaments on single-stranded DNA (ssDNA) that are similar to those formed by the RecAEc. The RecADr protein hydrolyzes ATP and dATP and promotes DNA strand exchange reactions. DNA strand exchange is greatly facilitated by the E. coli SSB protein. As is the case with the E. coli RecA protein, the use of dATP as a cofactor permits more facile displacement of bound SSB protein from ssDNA. However, there are important differences as well. The RecADr protein promotes ATP- and dATP-dependent reactions with distinctly different pH profiles. Although dATP is hydrolyzed at approximately the same rate at pHs 7.5 and 8.1, dATP supports an efficient DNA strand exchange only at pH 8.1. At both pHs, ATP supports efficient DNA strand exchange through heterologous insertions but dATP does not. Thus, dATP enhances the binding of RecADr protein to ssDNA and the displacement of ssDNA binding protein, but the hydrolysis of dATP is poorly coupled to DNA strand exchange. The RecADr protein thus may offer new insights into the role of ATP hydrolysis in the DNA strand exchange reactions promoted by the bacterial RecA proteins. In addition, the RecADr protein binds much better to duplex DNA than the RecAEc protein, binding preferentially to double-stranded DNA (dsDNA) even when ssDNA is present in the solutions. This may be of significance in the pathways for dsDNA break repair in Deinococcus.
doi:10.1128/JB.184.6.1649-1660.2002
PMCID: PMC134872  PMID: 11872716

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