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Introduction The Salford Lung Studies (SLS) were real-world randomised controlled trials set within UK primary care that assessed the effectiveness and safety of initiating once-daily fluticasone furoate/vilanterol versus continuing usual care in patients with chronic obstructive pulmonary disease or asthma. Data were collected for a relatively short period, limiting the study of long-term outcomes. To broaden the capture of SLS patients’ data, we undertook the Extended SLS (Ext-SLS), aiming to better understand the patient disease journey and the effects of treatment in a real-world setting, through collection of patient-level data. Here, we present study design information and the challenges and learnings gathered in creating the Ext-SLS. Methods The Ext-SLS was intended to augment the SLS by collecting retrospective and prospective (up to 10 years from consent) primary and secondary care electronic health record (EHR) data and patient questionnaires. After ethics approval, general practitioners (GPs) obtained consent from SLS patients remotely (mean 3.2 years post-SLS completion). To facilitate GPs identifying eligible patients, a novel EHR-based approach flagged SLS patients who were alive and registered with their original GP. An automated system sent consent forms/questionnaires to patients. Medical data were collected via EHRs; primary care data were extracted from GPs’ systems whilst secondary care data were sourced from the UK NHS. Results Of the 75 GP sites from the SLS, 35 (47%) declined Ext-SLS participation leaving 4158 potentially eligible patients; 1169 (28%) patients were excluded as GPs could not confirm them as SLS participants or due to incapacity. Of 2989 patients invited, 1189 (40%) consented. Conclusions Developing an EHR-based trial extension was achieved, with reasonable consent rates amongst invited patients. The resulting Ext-SLS is a unique and valuable research resource. Leveraging EHRs and technology reduced GP burden, facilitating participation. Initiation of extension studies prior to study close-out may help increase GP and patient participation. Supplementary Information The online version contains supplementary material available at 10.1007/s12325-021-01827-2.

The Tele- Exercise and Multiple Sclerosis (TEAMS) study, funded by the Patient Centered Outcome Research Institute (PCORI), is a pragmatic, cluster randomized controlled trial aimed at comparing the effectiveness of a 12-week complementary and alternative medicine (CAM) program for people with multiple sclerosis (MS) delivered by a therapist at a clinic and the same program initiated by the participant at home using a tablet and pre-recorded videos. The 20-session CAM program consists of yoga, Pilates and dual tasking exercises. The study aimed to enroll 820 participants with MS living in Alabama, Mississippi and Tennessee.The information provided in this paper describes the strategies that led to the largest randomized controlled exercise trial ever conducted for people with multiple sclerosis. Specifically, the paper presents the result of incorporating stakeholder engagement, a novel participant recruitment method, to produce a successful recruitment outcome for a comparative effectiveness randomized controlled trial. This study used three tiers of engagement: panel members (9 members), clinical partners (88 occupational and physical therapists), and community organizations (6 non-profits).Engagement of the stakeholder panel, clinical partners and community organizations led to interest of over 1700 people with MS across three states in the Deep South (final enrollment was n = 837). The diversity of our stakeholder groups and their extensive reach into various communities were a critical aspect for achieving our target sample size. The recruitment numbers reflect the importance of involving multiple stakeholder groups at project inception, developing relationships over time, utilizing member strengths, and monitoring their engagement on a regular basis to ensure a meaningful experience for all involved.NCT03117881. Registered 18 April 2017, https://clinicaltrials.gov/ct2/show/NCT03117881?term=tele+rehabilitationcond=Multiple+Sclerosiscntry=USstate=US%3AALdraw=2rank=1 .The Tele Exercise and Multiple Sclerosis (TEAMS) study has been able to successfully screen over 1700 people with multiple sclerosis (MS) across three southern states (Alabama, Mississippi, Tenessee) largely due to the advice and input that the research team received from a stakeholder panel, clinic partners and community organizations. These groups met before the study was submitted for funding to the Patient-Centered Outcome Research Institute (PCORI), and was awarded in 2017. These engagement stages and framework established early in the study process were instrumental in generating strong enthusiasm for the study among various MS constituency groups. The feedback from our stakeholders, clinic partners, and community organizations led to the creation of a variety of recruitment methods (print material, email, social media, attendance at events, and health fairs) to connect with potential participants in a setting convenient within each location. In approximately 26 months, the study enrolled 837 participants with MS and baseline tested 759 individuals who participated in a rehabilitative exercise program at either a clinic site or in their home using a tablet that they were given (and kept) which included a set of preloaded videos. All milestones established by PCORI and research staff were met, leading to the largest exercise trail ever conducted with people with MS.

In this chapter, we review the extensive literature describing the roles of the subfornical organ (SFO), the organum vasculosum of the terminalis (OVLT), and the median preoptic nucleus (MnPO), comprising the lamina terminalis, in cardiovascular regulation and the control of fluid balance. We present this information in the context of both historical and technological developments which can effectively be overlaid upon each other. We describe intrinsic anatomy and connectivity and then discuss early work which described how circulating angiotensin II acts at the SFO to stimulate drinking and increase blood pressure. Extensive studies using direct administration and lesion approaches to highlight the roles of all regions of the lamina terminalis are then discussed. At the cellular level we describe c-Fos and electrophysiological work, which has highlighted an extensive group of circulating hormones which appear to influence the activity of specific neurons in the SFO, OVLT, and MnPO. We highlight optogenetic studies that have begun to unravel the complexities of circuitries underlying physiological outcomes, especially those related to different components of drinking. Finally, we describe the somewhat limited human literature supporting conclusions that these structures play similar and potentially important roles in human physiology.

In this chapter, we review the extensive literature describing the roles of the subfornical organ (SFO), the organum vasculosum of the terminalis (OVLT), and the median preoptic nucleus (MnPO), comprising the lamina terminalis, in cardiovascular regulation and the control of fluid balance. We present this information in the context of both historical and technological developments which can effectively be overlaid upon each other. We describe intrinsic anatomy and connectivity and then discuss early work which described how circulating angiotensin II acts at the SFO to stimulate drinking and increase blood pressure. Extensive studies using direct administration and lesion approaches to highlight the roles of all regions of the lamina terminalis are then discussed. At the cellular level we describe c-Fos and electrophysiological work, which has highlighted an extensive group of circulating hormones which appear to influence the activity of specific neurons in the SFO, OVLT, and MnPO. We highlight optogenetic studies that have begun to unravel the complexities of circuitries underlying physiological outcomes, especially those related to different components of drinking. Finally, we describe the somewhat limited human literature supporting conclusions that these structures play similar and potentially important roles in human physiology.

The Salford Lung Studies (SLS) were real-world effectiveness RCTs of initiating FF/VI vs. usual care (1:1) in asthma and COPD. Data were only collected in SLS for a relatively short period, limiting study of long-term outcomes. The Extended-SLS aims to augment the SLS, collecting 1° and 2° care electronic health record (EHR) data and patient questionnaires. After receiving Ethics approval, GPs remotely consented SLS subjects (mean 3.2 yrs post-completion). To facilitate GPs in identifying eligible patients, a novel EHR-based approach was used to flag subjects who completed the SLS, were still alive and registered with the same GP. An automated system (Docmail) was used to send consent forms/questionnaires direct to patients. Medical data were collected solely via EHRs; a trusted 3rd party extracted 1°-care data from GPs systems and NHS Digital provided linked 2°-care data. Of the 75 GP sites from the original SLS, 35 did not participate, resulting in 4,158 potentially eligible subjects from 40 practices; 18% were excluded as could not be confirmed as SLS subjects or incapacity. Of 2,989 invited, 39% consented to the study (see Figure). We demonstrate that developing an EHR-based, trial extension is achievable with good consent rates. Leveraging of EHRs and technology reduced GP burden, facilitating participation. Initiation of extension studies prior to study close out may help in reducing patient attrition.

Following the ban of many historically-used flame retardants (FRs), numerous replacement chemicals have been produced and used in products, with some being identified as environmental contaminants. One of these replacement flame retardants is 1,2-dibromo-4-(1,2-dibromoethyl)-cyclohexane (DBE-DBCH; formerly abbreviated as TBECH), which to date has not been identified for risk assessment and potential regulation. DBE-DBCH technical mixtures consist largely of α- and β-diastereomers with trace amounts of γ- and δ-DBE-DBCH. The α- and β-isomers are known contaminants in various environmental media. While current global use and production volumes of DBE-DBCH are unknown, recent studies identified that DBE-DBCH concentrations were among the highest of the measured bromine-based FRs in indoor and urban air in Europe. Yet our mass balance fugacity model and modeling of the physical-chemical properties of DBE-DBCH estimated only 1% partitioning to air with a half-life of 2.2 d atmospherically. In contrast, our modeling characterized DBE-DBCH adsorbing strongly to suspended particulates in the water column (~12%), settling onto sediment (2.5%) with minimal volatilization, but with most partitioning and adsorbing strongly to soil (~85%) with negligible volatilization and slow biodegradation. Our modeling further predicted that organisms would be exposed to DBE-DBCH through partitioning from the dissolved aquatic phase, soil, and by diet, and given its estimated logKow (5.24) and a half-life of 1.7 d in fish, DBE-DBCH is expected to bioaccumulate into lipophilic tissues. Low concentrations of DBE-DBCH are commonly measured in biota and humans, possibly because evidence suggests rapid metabolism. Yet toxicological effects are evident at low exposure concentrations: DBE-DBCH is a proven endocrine disruptor of sex and thyroid hormone pathways, with in vivo toxic effects on reproductive, metabolic, and other endpoints. The objectives of this review are to identify the current state of knowledge concerning DBE-DBCH through an evaluation of its persistence, potential for bioaccumulation, and characterization of its toxicity, while identifying areas for future research.

Halogenated polycyclic aromatic hydrocarbons (HPAHs) were identified in biological samples from the Alberta Oil-Sands Region (AOSR) using gas chromatography coupled with high-resolution time-of-flight mass spectrometry (GC-HRTOF-MS) at a resolving power of 25,000. Knowledge of the electron ionization (EI) fragmentation behavior of individual HPAH isomers, achieved by injecting authentic standards in full-scan MS mode, was paramount in identifying a suite of HPAHs in samples from the AOSR. Confirmation of compounds in biological samples was based on the measured mass accuracy (±3 ppm) of 2 characteristic ions prominent in the EI mass spectra of each compound. Numerous compounds were detected in the high resolution total ion chromatogram in liver extracts of 4 biological species from the AOSR: river otter (Lontra Canadensis), northern pike (Esox lucius), lake whitefish (Coregonus clupeaformis) and snails (Gastropod sp.) many of which remain unidentified. Careful examination of the high-resolution accurate mass data suggests that dichloro-anthracene/phenanthrene, bromo-anthracene/phenanthrene and dibromo-fluorene were present in the biological samples. Lipid corrected concentrations of dichloro-PAHs were estimated to be 16.3 ± 11.4 (n = 4) and 5.5 (n = 1) ng/g in lake whitefish and river otter, respectively. Concentrations of mono-bromo-PAHs were an order of magnitude greater in snails (170.5 ng/g) than in northern pike (12.5 ng/g) while concentrations of dibromo-PAHs were 4 times greater in snails than in northern pike. The detection of these compounds in biota implies that these compounds are bioaccumulative. The liver-based biomagnification factor of the dichloro-PAH congener in the river otter/lake whitefish feeding relationship is much smaller than 1 implying that this compound does not biomagnify.

Early postnatal overnutrition in humans is associated with long-term negative outcomes including obesity, increased risk of type-II diabetes, and cardiovascular disease. Hypothalamic neurons from rodents exposed to early postnatal overnutrition show altered expression of satiety signals and receptors, and exhibit altered responses to many satiety signals, suggesting a hypothalamic link between early overnutrition and development of these sequelae. Importantly, several hypothalamic nuclei receive information regarding circulating hormones (such as insulin, leptin and ghrelin) from the subfornical organ (SFO), a forebrain sensory circumventricular organ which lacks a blood brain barrier. Previous transcriptomic studies indicate that challenges to energy balance and hydration status stimulate changes in gene expression within the SFO, including genes encoding ion channels and receptors. In order to determine if early postnatal overnutrition also causes changes in SFO gene expression which may be associated with homeostatic dysregulation, we performed whole transcriptome sequencing on SFO tissue from rats raised in small (4 pups), or control (large, 12 pups) litters. Illumina RNA sequencing was performed on SFO tissue from rats raised from small and large litters, and read sequences were aligned to the Rat Rnor_6.0 genome. Control data were further compared to previously published microarray data set for validation. We found statistically significant (p

The sodium leak channel NALCN is poorly understood, but is reported as a Na+-permeable, nonselective cation leak channel which regulates resting membrane potential and electrical excitability. Previous work has indicated that NALCN currents can be stimulated by activation of several G protein coupled receptors, including the M3 muscarinic receptor. We undertook a study using voltage clamp electrophysiology to investigate NALCN currents. We compared currents elicited from untransfected control HEK239 cells in response to M3R agonists muscarine or Oxotremorine M to currents elicited from cells transfected with M3R only or the M3R plus NALCN and cDNA encoding accessory proteins UNC-80 and Src. Currents with similar properties were observed in all three groups of cells in response to muscarine agonists, in similar proportions of cells tested, from all three groups of cells. Our findings do not support previous electrophysiological studies suggesting that heterologously expressed NALCN functions as a Na+ leak channel in HEK293 cells. More research will be required to determine the molecular requirements for successful expression of the NALCN channel., Highlights • NALCN (sodium leak channel, non-selective) is a poorly understood ion channel. • Several reports indicate that NALCN current can be recorded from transfected cells. • Conflicting reports indicate NALCN currents are simply leaky patch clamp seals. • We were unable to record currents attributable to NALCN in transfected HEK293 cells. • Our experiments suggest that NALCN does not form channels in HEK293 cells.

Brominated flame-retardants are environmentally pervasive and persistent synthetic chemicals, some of which have been demonstrated to disrupt neuroendocrine signaling and electrical activity of neurons. 1,2-dibromo-4-(1,2-dibromoethyl)-cyclohexane (TBECH) lacks the toxicity of other classes of BFRs, however its safety is still questioned, as little is known of its neurological effects. Therefore, we sought to determine if TBECH could acutely alter the electrical activity of Purkinje neurons maintained in vitro. Briefly, cerebella from gestational day 20 rats were dissociated and maintained for up to three weeks in culture. Action potentials of Purkinje neurons were detected by cell-attached patch clamp before, during, and after application of β-TBECH. β-TBECH decreased action potential activity in a dose-dependent manner with an apparent EC50 of 396 nM. β-TBECH did not significantly alter the coefficient of variation, a measure of the regularity of firing, suggesting that the mechanism of β-TBECH's effects on firing frequency may be independent of Purkinje neuron intracellular calcium handling. Because levels of β-TBECH in exposed individuals may not approach the EC50, these data suggest that any abnormal neurodevelopment or behavior linked with β-TBECH exposure may result from endocrinological effects as opposed to direct disruption of electrical activity.

Record keeping within research animal care facilities is a key part of the guidelines set forth by national regulatory bodies and mandated by federal laws. Research facilities must maintain records of animal health issues, procedures and usage. Facilities are also required to maintain records regarding regular husbandry such as general animal checks, feeding and watering. The level of record keeping has the potential to generate excessive amounts of paper which must be retained in a fashion as to be accessible. In addition it is preferable not to retain within administrative areas any paper records which may have been in contact with animal rooms. Here, we present a flexible, simple and inexpensive process for the generation and storage of electronic animal husbandry records using smartphone technology over a WiFi or cellular network.

Novel techniques involving high-speed impact (~500–1500 m/s) of laser generated micro-fliers with thin metallic targets having a layer of reactive solid deposited on their back side ( ~ 10 – 100 μ m ) are being developed to interrogate its shock-induced chemistry. Because mass spectrometry is performed in vacuo on the reactive side of the target to identify the chemical species produced, it is important to initiate chemistry without perforation of the target plate by the flier. An analytically tractable model is formulated in this paper to guide experimental development by predicting the ballistic response of large numbers of micro-flier–target configurations in a computationally inexpensive manner. The model is posed in terms of multi-component conservation principles and interaction terms that account for important features of both the early-time wave mechanics and the longer-time target deformation mechanics including flier–target adhesion. The model, validated using published data for larger scale inert flier–target configurations, is used to predict the response of micro-scale configurations consisting of aluminum fliers and steel targets. Scanning Electron Microscopy (SEM) of post-impact, inert flier–target micro-scale configurations is used to both highlight target deformation and damage and to motivate modeling simplifications. To illustrate the model, configurations that result in initiation (detonation) of the high-explosive PETN (C5H8N4O12) without perforation of the steel substrate are parametrically characterized in the form of ballistic initiation maps based on its empirical critical shock energy.

Cerebellar subregions are recognized as having specialized roles, with lateral cerebellum considered crucial for cognitive processing, whereas vermal cerebellum is more strongly associated with motor control. In human Duchenne muscular dystrophy, loss of the cytoskeletal protein dystrophin is thought to cause impairments in cognition, including learning and memory. Previous studies demonstrate that loss of dystrophin causes dysfunctional signaling at γ-aminobutyric acid (GABA) synapses on Purkinje neurons, presumably by destabilization of GABAA receptors. However, potential differences in the intrinsic electrophysiological properties of Purkinje neurons, including membrane potential and action potential firing rates, have not been investigated. Here, using a 2×2 analysis of variance (ANOVA) experimental design, we employed patch clamp analysis to compare membrane properties and action potentials generated by acutely dissociated Purkinje neurons from vermal and lateral cerebellum in wild-type (WT) mice and mdx dystrophin-deficient mice. Compared to Purkinje neurons from WT mice, neurons from mdx mice exhibited more irregular action potential firing and a hyperpolarization of the membrane potential. Firing frequency was also lower in Purkinje neurons from the lateral cerebellum of mdx mice relative to those from WT mice. Several action potential waveform parameters differed between vermal and lateral Purkinje neurons, irrespective of dystrophin status, including action potential amplitude, slope (both larger in the vermal region), and duration (shorter in the vermal region). Moreover, the membrane potential of Purkinje neurons from the vermal region of WT mice exhibited a significant hyperpolarization and concurrent reduction in the frequency of spontaneous action potentials compared to Purkinje neurons from the lateral region. This regional hyperpolarization and reduction in spontaneous action potential frequency was abolished in mdx mice. These results from mice demonstrate the presence of differential electrophysiological properties between Purkinje neurons from different regions of the WT mouse cerebellum and altered intrinsic membrane properties in the absence of dystrophin. These findings provide a possible mechanism for the observations that absence of cerebellar dystrophin contributes to deficits in mental function observed in humans and mouse models of muscular dystrophy. Moreover, these results highlight the importance of distinguishing functional zones of the cerebellum in future work characterizing Purkinje neuron electrophysiology and studies using the model of dissociated Purkinje neurons from mice.

We investigated the mechanisms responsible for increased blood pressure and sympathetic nerve activity (SNA) caused by 2-3 days dehydration (DH) both in vivo and in situ preparations. In euhydrated (EH) rats, systemic application of the AT(1) receptor antagonist Losartan and subsequent pre-collicular transection (to remove the hypothalamus) significantly reduced thoracic (t)SNA. In contrast, in DH rats, Losartan, followed by pre-collicular and pontine transections, failed to reduce tSNA, whereas transection at the medulla-spinal cord junction massively reduced tSNA. In DH but not EH rats, selective inhibition of the commissural nucleus tractus solitarii (cNTS) significantly reduced tSNA. Comparable data were obtained in both in situ and in vivo (anaesthetized/conscious) rats and suggest that following chronic dehydration, the control of tSNA transfers from supra-brainstem structures (e.g. hypothalamus) to the medulla oblongata, particularly the cNTS. As microarray analysis revealed up-regulation of AP1 transcription factor JunD in the dehydrated cNTS, we tested the hypothesis that AP1 transcription factor activity is responsible for dehydration-induced functional plasticity. When AP1 activity was blocked in the cNTS using a viral vector expressing a dominant negative FosB, cNTS inactivation was ineffective. However, tSNA was decreased after pre-collicular transection, a response similar to that seen in EH rats. Thus, the dehydration-induced switch in control of tSNA from hypothalamus to cNTS seems to be mediated via activation of AP1 transcription factors in the cNTS. If AP1 activity is blocked in the cNTS during dehydration, sympathetic activity control reverts back to forebrain regions. This unique reciprocating neural structure-switching plasticity between brain centres emphasizes the multiple mechanisms available for the adaptive response to dehydration.

The area postrema (AP) is a sensory circumventricular organ characterized by extensive fenestrated vasculature and neurons which are capable of detecting circulating signals of osmotic, cardiovascular, immune and metabolic status. The AP can communicate these messages via efferent projections to brainstem and hypothalamic structures that are able to orchestrate an appropriate response. We have used microarrays to profile the transcriptome of the AP in the Sprague-Dawley (SD) and Wistar-Kyoto rat and present here a comprehensive catalogue of gene expression, focusing specifically on the population of ion channels, receptors and G protein-coupled receptors expressed in this sensory tissue; of the G protein-coupled receptors expressed in the rat AP, we identified ∼36% that are orphans, having no established ligand. We have also looked at the ways in which the AP transcriptome responds to the physiological stressors of 72 h dehydration (DSD) and 48 h fasting (FSD) and have performed microarrays in these conditions. Comparison between the DSD and SD or between FSD and SD revealed only a modest number of AP genes that are regulated by these homeostatic challenges. The expression levels of a much larger number of genes are altered in the spontaneously hypertensive rat AP compared with the normotensive Wistar-Kyoto control rat, however. Finally, analysis of these 'hypertension-related' elements revealed genes that are involved in the regulation of both blood pressure and immune function and as such are excellent targets for further study.

Prokineticin 2 (PK2) is a neuropeptide that acts as a signaling molecule regulating circadian rhythms in mammals. We have previously reported PK2 actions on subfornical organ (SFO) neurons, identifying this circumventricular organ as a target at which PK2 acts to influence autonomic control (Cottrell GT, and Ferguson AV. J. Neurosci. 24: 2375–2379, 2004). In this study, we have examined the cellular mechanisms by which PK2 increases the excitability of SFO neurons. Whole cell patch recordings from dissociated rat SFO neurons demonstrated that the mitogen-activated protein (MAP) kinase inhibitor PD-98059 prevented PK2-induced depolarization and decreases in delayed rectifier K+ current. PK2 also increased intracellular Ca2+ concentration ([Ca2+]i) in 39% of dissociated SFO neurons (mean increase = 20.8 ± 5.5%), effects that were maintained in the presence of thapsigargin but abolished by both nifedipine, or the absence of extracellular Ca2+, suggesting that PK2-induced [Ca2+]i transients resulted from Ca2+ entry through voltage-gated Ca2+ channels. Voltage-clamp recordings showed that PK2 was without effects on Ca2+ currents evoked by voltage ramps, suggesting that PK2-induced Ca2+ influx was secondary to PK2-induced increases in action potential frequency, an hypothesis supported by data showing that tetrodotoxin abolished effects of PK2 on [Ca2+]i. These observations suggested PK2 modulation of voltage-gated Na+ currents, a possibility confirmed by voltage-clamp experiments showing that PK2 increased the amplitude of both transient and persistent Na+ currents in 29% of SFO neurons (by 34 and 38%, respectively). These data indicate that PK2 influences SFO neurons through the activation of a MAP kinase cascade, which, in turn, modulates Na+ and K+ conductances.

Adiponectin is an adipocyte derived hormone which acts in the brain to modulate energy homeostasis and autonomic function. The paraventricular nucleus of the hypothalamus (PVN) which plays a key role in controlling pituitary hormone secretion has been suggested to be a central target for adiponectin actions. A number of hormones produced by PVN neurons have been implicated in the regulation of energy homeostasis including oxytocin, corticotropin releasing hormone and thyrotropin releasing hormone. In the present study we investigated the role of adiponectin in controlling the excitability of magnocellular (MNC ‐ oxytocin or vasopressin secreting) neurons within the PVN. Using RT-PCR techniques we have shown expression of both adiponectin receptors in the PVN. Patch clamp recordings from MNC neurons in hypothalamic slices have also identified mixed (27% hyperpolarization, 42% depolarization) effects of adiponectin in modulating the excitability of the majority of MNC neurons tested. These effects are maintained when cells are placed in synaptic isolation using tetrodotoxin. Additionally we combined electrophysiological recordings with single cell RT-PCR to examine the actions of adiponectin on MNC neurons which expressed oxytocin only, vasopressin only, or both oxytocin and vasopressin mRNA and assess the profile of receptor expression in these subgroups. Adiponectin was found to hyperpolarize 100% of oxytocin neurons tested (n = 6), while vasopressin cells, while all affected (n = 6), showed mixed responses. Further analysis indicates oxytocin neurons express both receptors (6/7) while vasopressin neurons express either both receptors (3/8) or one receptor (5/8). In contrast 6/6 oxytocin/vasopressin neurons were unaffected by adiponectin. Co-expressing oxytocin and vasopressin neurons express neither receptor (4/6). The results presented in this study suggest that adiponectin plays specific roles in controlling the excitability oxytocin secreting neurons, actions which correlate with the current literature showing increased oxytocin secretion in the obese population.

Cerebellar Purkinje neurons in vivo exhibit high frequency and multi-spike action potentials with transient (INaT), resurgent (INaR) and persistent (INaP) Na+ currents arising from voltage-gated Na+ channels, which play important roles in shaping the action potentials and electrical activity of these cells. However, little is known about Na+ channel expression in cultured Purkinje neurons despite the use of in vitro approaches to study these cells. Therefore, GFP-expressing Purkinje neurons isolated from transgenic mice were analysed after four weeks in culture, when, coincident with distinct axonal and dendritic morphologies, cultured Purkinje neurons exhibited dendrite-specific MAP2 expression characteristic of polarized neurons. In cell-attached patch clamp recordings, Na+ currents occurred at significantly higher frequencies and amplitudes in patches from the soma and axon than from dendrites, similar to the polarized distribution observed in vivo. INaT, INaR and INaP Na+ currents with properties similar to those observed in acutely isolated Purkinje neurons were detected in nucleated outside-out patches from cultured Purkinje cells. RT-PCR analysis detected Nav1.1, Nav1.2 and Nav1.6, but not Nav1.3, Nav1.4, Nav 1.5 or Nav1.8 Na+ channel alpha subunit gene expression in cultured Purkinje neurons, as observed in vivo. Together, the results indicate that key aspects of Na+ channel expression in mature Purkinje neurons in vivo occur in vitro.

While the appetite-stimulating hormone ghrelin can act to acutely modulate electrical activity of neurons in the appetite regulating network, it also has a role in regulating neuronal outgrowth, synaptic connectivity and intrinsic electrophysiological properties. In this study, we investigated whether ghrelin may cause alteration in neurite outgrowth and electrophysiological properties of tyrosine hydroxylase (TH) neurons from the ventrolateral arcuate nucleus (VL-ARC), which are thought to contribute to regulation of energy balance. We prepared dissociated neuronal cultures from the VL-ARC of transgenic mice expressing EGFP under control of the tyrosine hydroxylase (TH) promoter, thus allowing visual identification of putative catecholaminergic (TH-EGFP) neurons. After five days of treatment with 100 nM ghrelin, TH-EGFP neurons exhibited significantly more and longer neurites than control treated neurons, and the effects of ghrelin were abolished by 100 μM ghrelin antagonist, D-Lys-GHRP-6. To investigate whether ghrelin altered electrophysiological properties of TH-EGFP neurons, we carried out patch clamp experiments measuring electrophysiological properties. No significant differences were identified for resting membrane potential or spontaneous action potential frequency, however we observed a hyperpolarization of threshold for action potentials and increased input resistance, indicating increased excitability. This increased excitability is consistent with an observed hyperpolarizing shift in the activation of voltage-gated Na(+) currents. These data indicate that the hunger signal ghrelin induces plastic changes in TH-neurons from VL-ARC.

As Purkinje neurons mature during postnatal development, they change from electrically quiescent to active and exhibit high frequency spontaneous action potentials. This change in electrical activity is determined by both alteration in ion channel expression and the acquisition of synaptic input. To gain a better understanding of the development the intrinsic electrical properties of these neurons, acutely isolated Purkinje neurons from mice aged postnatal day 4 (P4) to P18 were examined. This included recording action potential frequency, threshold, height and slope, and input resistance and capacitance. Changes in a number of these properties were observed, suggesting significant changes in voltage-gated Na(+) currents. Because voltage-gated Na(+) currents, including the transient, resurgent and persistent currents, are known to play important roles in generating spontaneous action potentials, the developmental changes in these currents were examined. A large increase in the density of transient current, resurgent current and persistent current was observed at times corresponding with changes in action potential properties. Interestingly, the developmental up-regulation of the persistent current and resurgent current occurred at rate which was faster than the up-regulation of the transient current. Moreover, the relative amplitudes of the persistent and resurgent currents increased in parallel, suggesting that they share a common basis. The data indicate that developmental up-regulation of Na(+) currents plays a key role in the acquisition of Purkinje neuron excitability.

Adiponectin is an adipocyte-derived peptide hormone involved in energy homeostasis and the pathogenesis of obesity, including hypertension. Area postrema (AP) lacks a blood–brain barrier and is a critical homeostatic integration center for humoral and neural signals. Here we investigate the role of AP in adiponectin signaling. We show that rat AP expresses AdipoR1 and AdipoR2 adiponectin receptor mRNA. We used current-clamp electrophysiology to investigate whether adiponectin influenced membrane properties of AP neurons and found that ∼60% of rat AP neurons tested were sensitive to adiponectin. Additional electrophysiology experiments coupled with single-cell reverse transcription-PCR indicated that all neurons that expressed both subtypes of receptor were sensitive to adiponectin, whereas neurons expressing only one subtype were predominantly insensitive. Last, microinjection of adiponectin into AP caused significant increases in arterial blood pressure, with no change in heart rate, suggesting that adiponectin acts at AP to provide a possible link between control of energy homeostasis and cardiovascular function.

Voltage-gated K+ channels play important roles in shaping the characteristics of action potentials and electrical activity. In a previous study, we isolated cDNAs encoding several distinct K+ channel isoforms, including a novel isoform (XKv1.10) expressed in Xenopus laevis spinal cord neurons and myocytes. Here, we report the biophysical characterization of XKv1.10 expressed in transiently transfected HEK293 cells. Whole cell patch clamp recordings revealed a voltage-gated, rapidly activating and inactivating K+ current. Interestingly, the rate of inactivation of XKv1.10 channels showed apparent voltage dependence, with time constants between 77.7–213.3 ms. The predicted protein sequence of XKv1.10 does not appear to encode an N-terminal inactivating “ball and chain” domain, and instead these channels may inactivate via a C/P-type mechanism. Consistent with this, either increasing the external concentration of K+ or external application of tetraethylammonium caused a decrease in the rate of inactivation. Pharmacologically, XKv1.10 K+ channels were sensitive to 4-aminopyridine and tetraethylammonium with apparent IC50 values of 68.5 μM and 17.1 mM, respectively. When simulated action potentials were used as a voltage command, XKv1.10 was similar to XKv1.4 in that it carried more repolarizing current during the action potential than XKv1.2. However, while XKv1.4 was active during the interspike interval, XKv1.10 and XKv1.2 were not. Overall, the data suggest that XKv1.10 channels make a unique contribution to the developmental maturation of electrical signaling in Xenopus laevis. © 2004 Wiley Periodicals, Inc. J Neurobiol 60: 227–235, 2004

Embryonic Xenopus muscle cells grown in culture express voltage-gated K+ currents with inactivating and non-inactivating kinetics. Here we report the cloning of three K+ channel cDNAs, designated XKv1.2', XKv1.4 and XKv1.10, from muscle which may underlie these currents. XKv1.2' cDNA appears to be an allelic variant of the XKv1.2 previously cloned from Xenopus. The second cDNA encodes a homologue of Kv1.4 that has not been previously cloned from Xenopus. The predicted XKv1.4 protein shows 73% overall similarity to mouse and chick Kv1.4, but shows significant divergence in the region corresponding to the chain of the inactivating 'ball and chain' domain. The third K+ channel cDNA isolated from Xenopus muscle is a novel Kv1 isoform designated XKv1.10. The predicted protein shares about 70% similarity with other members of the Kv1 subfamily, and about 40% with members of the Kv2, Kv3 and Kv4 subfamilies. XKv1.4 mRNA appears as early as stage 10.5 in whole embryos and is prominent in muscle throughout development from stage 14 to adult. XKv1.2' mRNA is detected by stage 11.5 in whole embryos, but remains at low levels in embryonic skeletal muscle (stages 14 and 21), and is absent from adult muscle. XKv1.10 mRNA is first detected at stage 21 in whole embryos, and is present in muscle from this stage onwards. All three transcripts are present in spinal cord at stage 21. The results support the notion that channels encoded by XKv1.4 contribute to the inactivating K+ current observed in embryonic muscle cells in culture.

Macro-patch current recordings were used to determine the spatial distribution of INa, inactivating (IIK) and non-inactivating (IK) outward potassium currents on nerve-contacted Xenopus muscle cells grown in culture. Sites of nerve contact were identified under 400x magnification and macroscopic currents recorded from cell-attached membrane patches ranging in diameter from 2 microm to 6 microm. Membrane patch (n=133) recordings revealed no detectable current, or INa and/or IIK, or just IK. More than 85% of the membrane patches from which INa (n=35) could be recorded occurred within 40 microm of the position of nerve contact. In addition, more than 90% of the membrane patches which contained INa also contained IIK. The density of IIK in these membrane patches that also contained INa was significantly higher than the density of IIK or IK in membrane patches without INa. Sites of ACh receptor localization at nerve-muscle contacts, identified by labelling with rhodamine-alpha-bungarotoxin, were also found to contain both INa (4/5 cells) and IIK (5/5 cells). These results suggest that in Xenopus muscle cells in culture the channels mediating IIK along with sodium channels are clustered at sites of nerve contact and synaptic specializations.

Insulin plays a crucial role in the regulation of energy balance. Within the central nervous system, hypothalamic nuclei such as the arcuate and ventromedial nuclei are targets of insulin; however, insulin may only access these nuclei after transport across the blood-brain barrier. Neurons of the subfornical organ are not protected by the blood-brain barrier and can rapidly detect and respond to circulating hormones such as leptin and ghrelin. Moreover, subfornical organ neurons form synaptic connections with hypothalamic control centers that regulate energy balance, including the arcuate and dorsomedial nuclei. However, it is unknown whether subfornical organ neurons respond to insulin. Using whole-cell current clamp, we examined the electrophysiological effects of insulin on rat subfornical organ neurons. Upon insulin application, 70% of neurons tested were responsive, with 33% of neurons tested (9/27) exhibiting hyperpolarization of membrane potential (-8.7 ± 1.7 mV) and 37% (10/27) exhibiting depolarization (10.5 ± 2.8 mV). Using pharmacological blockade, our data further indicate that the hyperpolarization was mediated by opening of KATP channels, whereas depolarization resulted from opening of Ih channels. These data are the first to show that insulin exerts a direct effect on the electrical activity of subfornical organ neurons and support the notion that the subfornical organ may act to communicate information on circulating satiety signals to homeostatic control centers.

The area postrema (AP) is a sensory circumventricular organ characterized by extensive fenestrated vasculature and neurons which are capable of detecting circulating signals of osmotic, cardiovascular, immune and metabolic status. The AP can communicate these messages via efferent projections to brainstem and hypothalamic structures that are able to orchestrate an appropriate response. We have used microarrays to profile the transcriptome of the AP in the Sprague-Dawley (SD) and Wistar-Kyoto rat and present here a comprehensive catalogue of gene expression, focusing specifically on the population of ion channels, receptors and G protein-coupled receptors expressed in this sensory tissue; of the G protein-coupled receptors expressed in the rat AP, we identified ∼36% that are orphans, having no established ligand. We have also looked at the ways in which the AP transcriptome responds to the physiological stressors of 72 h dehydration (DSD) and 48 h fasting (FSD) and have performed microarrays in these conditions. Comparison between the DSD and SD or between FSD and SD revealed only a modest number of AP genes that are regulated by these homeostatic challenges. The expression levels of a much larger number of genes are altered in the spontaneously hypertensive rat AP compared with the normotensive Wistar-Kyoto control rat, however. Finally, analysis of these 'hypertension-related' elements revealed genes that are involved in the regulation of both blood pressure and immune function and as such are excellent targets for further study.

Article history: Accepted 25 February 2010 Available online 4 March 2010 Adiponectin (ADP) is a peptide produced by adipose tissue, which acts as an insulin sensitizing hormone. Recent studies have shown that adiponectin receptors (AdipoR1 and AdipoR2) are present in the CNS, and although adiponectin does appear in both circulation and the cerebrospinal fluid there is still some debate as to whether or not ADP crosses the blood brain barrier (BBB). Circumventricular organs (CVO) are CNS sites which lack normal BBB, and thus represent sites at which circulating adiponectin may act to directly influence the CNS. The subfornical organ (SFO) is a CVO that has been implicated in the regulation of energy balance as a consequence of the ability of SFO neurons to respond to a number of different circulating satiety signals including amylin, CCK, PYY and ghrelin. Our recent microarray analysis suggested the presence of adiponectin receptors in the SFO. We report here that the SFO shows a high density of mRNA for both adiponectin receptors (AdipoR1 and AdipoR2), and that ADP influences the excitability of dissociated SFO neurons. Separate subpopulations of SFO neurons were either depolarized (8.9±0.9 mV, 21 of 97 cells), or hyperpolarized (�8.0±0.5 mV, 34 of 97 cells), by bath application of 10 nM ADP, effects which were concentration dependent and reversible. Our microarray analysis also suggested that 48 h of food deprivation resulted in specific increases in AdipoR2 mRNA expression (no effect on AdipoR1 mRNA), observations which we confirm here using real-time PCR techniques. The effects of food deprivation also resulted in a change in the responsiveness of SFO neurons to adiponectin with 77% (8/11) of cells tested responding to adiponectin with depolarization, while no hyperpolarizations were observed. These observations support the concept that the SFO may be a key player in sensing circulating ADP and transmitting such information to critical CNS sites involved in the regulation of energy balance.

Ghrelin, a peptide hormone secreted by the stomach, has been shown to regulate energy homeostasis by modulating electrical activity of neurons in the central nervous system (CNS). Like many circulating satiety signals, ghrelin is a peptide hormone and is unable to cross the blood-brain barrier without a transport mechanism. In this review, we address the notion that the arcuate nucleus of the hypothalamus is the only site in the CNS that detects circulating ghrelin to trigger orexigenic responses. We consider the roles of a specialized group of CNS structures called the sensory circumventricular organs (CVOs), which are not protected by the blood-brain barrier. These areas include the subfornical organ and the area postrema and are already well known to be key areas for detection of other circulating hormones such as angiotensin II, cholecystokinin, and amylin. A growing body of evidence indicates a key role for the sensory CVOs in the regulation of energy homeostasis.

Ghrelin, a peptide hormone secreted from the stomach, is known to have a potent appetite-stimulating activity. Recently, it has been shown that area postrema (AP), a caudal brain stem center that lacks a blood-brain barrier, is a key site of activity for ghrelin in stimulating appetite and regulating pancreatic protein secretion. In this study, we have examined the ability of ghrelin to regulate the electrical activity of area postrema neurons using patch-clamp electrophysiology. Using current-clamp configuration, we found that at a concentration of 10 nM, ghrelin caused inhibition in 19% of neurons tested, while a further 19% were excited by similar application of ghrelin. The remaining 62% of AP neurons were insensitive to ghrelin. These effects were concentration dependent, with an apparent EC50 of 1.9 nM. Voltage-clamp recordings revealed that ghrelin caused a potentiation of voltage-gated K+ currents in neurons that exhibited a hyperpolarization and a potentiation of a depolarizing nonspecific cation current (NSCC) in those neurons that exhibited a depolarization of membrane potential. These are the first data showing that ghrelin exerts a direct effect on electrical activity of AP neurons and supports the notion that ghrelin can act via the AP to regulate energy homeostasis.

We have employed microarray technology using Affymetrix 230 2.0 genome chips to initially catalog the transcriptome of the subfornical organ (SFO) under control conditions and to also evaluate the changes (common and differential) in gene expression induced by the challenges of fluid and food deprivation. We have identified a total of 17,293 genes tagged as present in one of our three experimental conditions, transcripts, which were then used as the basis for further filtering and statistical analysis. In total, the expression of 46 genes was changed in the SFO following dehydration compared with control animals (22 upregulated and 24 downregulated), with the largest change being the greater than fivefold increase in brain-derived neurotrophic factor (BDNF) expression, while significant changes in the expression of the calcium-sensing (upregulated) and apelin (downregulated) receptors were also reported. In contrast, food deprivation caused greater than twofold changes in a total of 687 transcripts (222 upregulated and 465 downregulated), including significant reductions in vasopressin, oxytocin, promelanin concentrating hormone, cocaine amphetamine-related transcript (CART), and the endothelin type B receptor, as well as increases in the expression of the GABAB receptor. Of these regulated transcripts, we identified 37 that are commonly regulated by fasting and dehydration, nine that were uniquely regulated by dehydration, and 650 that are uniquely regulated by fasting. We also found five transcripts that were differentially regulated by fasting and dehydration including BDNF and CART. In these studies we have for the first time described the transcriptome of the rat SFO and have in addition identified genes, the expression of which is significantly modified by either water or food deprivation.

Adiponectin is an adipocyte derived hormone which acts in the brain to modulate energy homeostasis and autonomic function. The paraventricular nucleus of the hypothalamus (PVN) which plays a key role in controlling pituitary hormone secretion has been suggested to be a central target for adiponectin actions. A number of hormones produced by PVN neurons have been implicated in the regulation of energy homeostasis including oxytocin, corticotropin releasing hormone and thyrotropin releasing hormone. In the present study we investigated the role of adiponectin in controlling the excitability of magnocellular (MNC – oxytocin or vasopressin secreting) neurons within the PVN. Using RT-PCR techniques we have shown expression of both adiponectin receptors in the PVN. Patch clamp recordings from MNC neurons in hypothalamic slices have also identified mixed (27% hyperpolarization, 42% depolarization) effects of adiponectin in modulating the excitability of the majority of MNC neurons tested. These effects are maintained when cells are placed in synaptic isolation using tetrodotoxin. Additionally we combined electrophysiological recordings with single cell RT-PCR to examine the actions of adiponectin on MNC neurons which expressed oxytocin only, vasopressin only, or both oxytocin and vasopressin mRNA and assess the profile of receptor expression in these subgroups. Adiponectin was found to hyperpolarize 100% of oxytocin neurons tested (n = 6), while vasopressin cells, while all affected (n = 6), showed mixed responses. Further analysis indicates oxytocin neurons express both receptors (6/7) while vasopressin neurons express either both receptors (3/8) or one receptor (5/8). In contrast 6/6 oxytocin/vasopressin neurons were unaffected by adiponectin. Co-expressing oxytocin and vasopressin neurons express neither receptor (4/6). The results presented in this study suggest that adiponectin plays specific roles in controlling the excitability oxytocin secreting neurons, actions which correlate with the current literature showing increased oxytocin secretion in the obese population.

Previous work has demonstrated that subfornical organ neurons exhibit spontaneous action potentials interspersed with periods of membrane potential oscillation.We used whole cell patch clamp recording to investigate properties of these oscillations. The amplitude, but not the frequency, of the oscillations exhibited voltage dependence. The oscillations were unaffected by application of blockers for γ-amino-n-butyric acid type A (GABA A ) and glutamate receptors, nor were they affected by application of Cd 2+ to block Ca 2+ channels, or Cs + to block hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels. The oscillations, however, were abolished by the application of tetro-dotoxin, indicating a role for voltage-gated Na + channels. Voltage clamp experiments demonstrated that the activation of persistent Na + current, but not transient Na + current, matched with the voltage dependence of activation of the oscillation.

Sensory circumventricular organs (CVOs) are specialized areas of the brain that lack a normal blood-brain barrier, and therefore are in constant contact with signaling molecules circulating in the bloodstream. Neurons of the CVOs are well endowed with a wide spectrum of receptors for hormones and other signaling molecules, and they have strong connections to hypothalamic and brainstem nuclei. Therefore, lying at the blood-brain interface, the sensory CVOs are in a unique position of being able to detect and integrate humoral and neural information and relay the resulting signals to autonomic control centers of the hypothalamus and medulla. This review focuses primarily on the roles played by the sensory CVOs in fluid balance and energy metabolism.

Cerebellar Purkinje neurons in vivo exhibit high frequency and multi-spike action potentials with transient (INaT), resurgent (INaR) and persistent (INaP) Na+ currents arising from voltage-gated Na+ channels, which play important roles in shaping the action potentials and electrical activity of these cells. However, little is known about Na+ channel expression in cultured Purkinje neurons despite the use of in vitro approaches to study these cells. Therefore, GFP-expressing Purkinje neurons isolated from transgenic mice were analysed after four weeks in culture, when, coincident with distinct axonal and dendritic morphologies, cultured Purkinje neurons exhibited dendrite-specific MAP2 expression characteristic of polarized neurons. In cell-attached patch clamp recordings, Na+ currents occurred at significantly higher frequencies and amplitudes in patches from the soma and axon than from dendrites, similar to the polarized distribution observed in vivo. INaT, INaR and INaP Na+ currents with properties similar to those observed in acutely isolated Purkinje neurons were detected in nucleated outside-out patches from cultured Purkinje cells. RT-PCR analysis detected Nav1.1, Nav1.2 and Nav1.6, but not Nav1.3, Nav1.4, Nav 1.5 or Nav1.8 Na+ channel alpha subunit gene expression in cultured Purkinje neurons, as observed in vivo. Together, the results indicate that key aspects of Na+ channel expression in mature Purkinje neurons in vivo occur in vitro.