Bioelectric Properties Research Articles

Comparison of the depolarization response of human mesenchymal stem cells from different donors.

Bioelectric signaling is currently being explored as a novel regulator of cell processes in non-excitable cells. In particular, stem cells have demonstrated increasing evidence of electrophysiology-mediated regulation of stemness acquisition, proliferation, differentiation, and migration. However, in light of many reports of primary stem cell heterogeneity, it is important to characterize the variability of stem cell response to biophysical manipulations in order to assess the utility of bioelectric modulation as a universal strategy for stem cell control. In this work, human mesenchymal stem cells (hMSCs) from five donors were evaluated for their response to membrane potential (Vmem) depolarization. We compared the inter-donor variability of their osteogenic and adipogenic differentiation potential, as well as their ability to maintain a differentiated phenotype after induction. We identified the markers that responded most consistently across donors and found that calcium deposition and gene expression of bone sialoprotein, lipoprotein lipase, and fatty acid binding protein 4 are the preferred markers for assessing differentiation response to Vmem depolarization. We also note that since there exists variability even among some of these markers, these assays should be performed on any newly acquired hMSC population if their bioelectric properties are to be studied further.

Remodeling of ECM patch into functional myocardium in an ovine model: A pilot study.

Previous studies have demonstrated that surgical patches comprised of small intestinal submucosa-derived extracellular matrix (ECM) have biological remodeling potential. This pilot study investigated histological, mechanical, and bioelectrical properties of an ECM patch implanted in the ovine right-ventricular outflow tract (RVOT). ECM patches (2 × 2 cm2 ) were implanted in four Western Range sheep (wether males, 37-49 kg, age <1 year) and explanted at 5 months (n = 2) and 8 months (n = 2). In vivo analysis included epicardial echocardiography and contact electrical mapping. Optical mapping was used to map electrical activity of two hearts on a Langendorff preparation. Mechanical testing quantified stiffness. Histological stains characterized structure, neovascularization, and calcification; immunohistochemistry (IHC) assessed cell phenotype. In vivo analysis showed that ECM patch tissue was contractile by M-mode and two-dimensional echocardiographic evaluation. In vivo electrical mapping, and optical mapping confirmed that ECM conducted an organized electrical signal. Mechanical testing of native and ECM patched RVOT tissue showed an elastic modulus of the implanted patch comparable to native tissue stiffness. At 5 and 8 months, the ECM had undergone extracellular matrix remodeling and neovascularization without calcification. The ECM was populated with locally aligned muscle cells positive for sarcomeric alpha-actinin, CD45, and troponin I and T. In sheep, the ECM patch appears to have the potential of remodeling to resemble native, functional ventricular tissue as evidenced by histological, mechanical, and electrical properties. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 1713-1720, 2016.

Sensory transduction at the frog semicircular canal: how hair cell membrane potential controls junctional transmission.

At the frog semicircular canals, the afferent fibers display high spontaneous activity (mEPSPs), due to transmitter release from hair cells. mEPSP and spike frequencies are modulated by stimulation that activates the hair cell receptor conductance. The relation between receptor current and transmitter release cannot be studied at the intact semicircular canal. To circumvent the problem, we combined patch-clamp recordings at the isolated hair cell and electrophysiological recordings at the cytoneural junction in the intact preparation. At isolated hair cells, the K channel blocker tetraethylammonium (TEA) is shown to block a fraction of total voltage-dependent K-conductance (IKD) that depends on TEA concentration but not on membrane potential (Vm). Considering the bioelectric properties of the hair cell, as previously characterized by this lab, a fixed fractional block of IKD is shown to induce a relatively fixed shift in Vm, provided it lies in the range −30 to −10 mV. The same concentrations of TEA were applied to the intact labyrinth while recording from single afferent fibers of the posterior canal, at rest and during mechanical stimulation. At the peak of stimulation, TEA produced increases in mEPSP rate that were linearly related to the shifts produced by the same TEA concentrations (0.1–3 mM) in hair cell Vm (0.7–5 mV), with a slope of 29.8 Hz/mV. The membrane potential of the hair cell is not linearly related to receptor conductance, so that the slope of quantal release vs. receptor conductance depends on the prevailing Vm (19.8 Hz/nS at −20 mV; 11 Hz/nS at −10 mV). Changes in mEPSP peak size were negligible at rest as well as during stimulation. Since ample spatial summation of mEPSPs occurs at the afferent terminal and threshold-governed spike firing is intrinsically nonlinear, the observed increases in mEPSP frequency, though not very large, may suffice to trigger afferent spike discharge.

Nose-to-Brain Delivery: Investigation of the Transport of Nanoparticles with Different Surface Characteristics and Sizes in Excised Porcine Olfactory Epithelium.

The ability to deliver therapeutically relevant amounts of drugs directly from the nasal cavity to the central nervous system to treat neurological diseases is dependent on the availability of efficient drug delivery systems. Increased delivery and/or therapeutic effect has been shown for drugs encapsulated in nanoparticles; however, the factors governing the transport of the drugs and/or the nanoparticles from the nasal cavity to the brain are not clear. The present study evaluates the potential transport of nanoparticles across the olfactory epithelium in relation to nanoparticle characteristics. Model systems, 20, 100, and 200 nm fluorescent carboxylated polystyrene (PS) nanoparticles that were nonmodified or surface modified with polysorbate 80 (P80-PS) or chitosan (C-PS), were assessed for transport across excised porcine olfactory epithelium mounted in a vertical Franz diffusion cell. Assessment of the nanoparticle content in the donor chamber of the diffusion cell, accompanied by fluorescence microscopy of dismounted tissues, revealed a loss of nanoparticle content from the donor suspension and their association with the excised tissue, depending on the surface properties and particle size. Chitosan surface modification of PS nanoparticles resulted in the highest tissue association among the tested systems, with the associated nanoparticles primarily located in the mucus, whereas the polysorbate 80-modified nanoparticles showed some penetration into the epithelial cell layer. Assessment of the bioelectrical properties, metabolic activity, and histology of the excised olfactory epithelium showed that C-PS nanoparticles applied in pH 6.0 buffer produced a damaging effect on the epithelial cell layer in a size-dependent manner, with fine 20 nm sized nanoparticles causing substantial tissue damage relative to that with the 100 and 200 nm counterparts. Although histology showed that the olfactory tissue was affected by the application of citrate buffer that was augmented by addition of chitosan in solution, this was not reflected in the bioelectrical parameters and the metabolic activity of the tissue. Regarding transport across the excised olfactory tissue, none of the nanoparticle systems tested, irrespective of particle size or surface modification, was transported across the epithelium to appear in measurable amounts in the receiver chamber.

A highly polarized excitable cell separates sodium channels from sodium-activated potassium channels by more than a millimeter.

The bioelectrical properties and resulting metabolic demands of electrogenic cells are determined by their morphology and the subcellular localization of ion channels. The electric organ cells (electrocytes) of the electric fish Eigenmannia virescens generate action potentials (APs) with Na(+) currents >10 μA and repolarize the AP with Na(+)-activated K(+) (KNa) channels. To better understand the role of morphology and ion channel localization in determining the metabolic cost of electrocyte APs, we used two-photon three-dimensional imaging to determine the fine cellular morphology and immunohistochemistry to localize the electrocytes' ion channels, ionotropic receptors, and Na(+)-K(+)-ATPases. We found that electrocytes are highly polarized cells ∼ 1.5 mm in anterior-posterior length and ∼ 0.6 mm in diameter, containing ∼ 30,000 nuclei along the cell periphery. The cell's innervated posterior region is deeply invaginated and vascularized with complex ultrastructural features, whereas the anterior region is relatively smooth. Cholinergic receptors and Na(+) channels are restricted to the innervated posterior region, whereas inward rectifier K(+) channels and the KNa channels that terminate the electrocyte AP are localized to the anterior region, separated by >1 mm from the only sources of Na(+) influx. In other systems, submicrometer spatial coupling of Na(+) and KNa channels is necessary for KNa channel activation. However, our computational simulations showed that KNa channels at a great distance from Na(+) influx can still terminate the AP, suggesting that KNa channels can be activated by distant sources of Na(+) influx and overturning a long-standing assumption that AP-generating ion channels are restricted to the electrocyte's posterior face.

The impact of sphingosine kinase inhibitor-loaded nanoparticles on bioelectrical and biomechanical properties of cancer cells.

Cancer progression and physiological changes within the cells are accompanied by alterations in the biophysical properties. Therefore, the cell biophysical properties can serve as promising markers for cancer detection and physiological activities. To aid in the investigation of the biophysical markers of cells, a microfluidic chip has been developed which consists of a constriction channel and embedded microelectrodes. Single-cell impedance magnitudes at four frequencies and entry and travel times are measured simultaneously during their transit through the constriction channel. This microchip provides a high-throughput, label-free, automated assay to identify biophysical signatures of malignant cells and monitor the therapeutic efficacy of drugs. Here, we monitored the dynamic cellular biophysical properties in response to sphingosine kinase inhibitors (SphKIs), and compared the effectiveness of drug delivery using poly lactic-co-glycolic acid (PLGA) nanoparticles (NPs) loaded with SphKIs versus conventional delivery. Cells treated with SphKIs showed significantly higher impedance magnitudes at all four frequencies. The bioelectrical parameters extracted using a model also revealed that the highly aggressive breast cells treated with SphKIs shifted electrically towards that of a less malignant phenotype; SphKI-treated cells exhibited an increase in cell-channel interface resistance and a significant decrease in specific membrane capacitance. Furthermore, SphKI-treated cells became slightly more deformable as measured by a decrease in their channel entry and travel times. We observed no significant difference in the bioelectrical changes produced by SphKI delivered conventionally or with NPs. However, NPs-packaged delivery of SphKI decreased the cell deformability. In summary, this study showed that while the bioelectrical properties of the cells were dominantly affected by SphKIs, the biomechanical properties were mainly changed by the NPs.

Bioelectric patterning during oogenesis: stage-specific distribution of membrane potentials, intracellular pH and ion-transport mechanisms in Drosophila ovarian follicles.

BackgroundBioelectric phenomena have been found to exert influence on various developmental and regenerative processes. Little is known about their possible functions and the cellular mechanisms by which they might act during Drosophila oogenesis. In developing follicles, characteristic extracellular current patterns and membrane-potential changes in oocyte and nurse cells have been observed that partly depend on the exchange of protons, potassium ions and sodium ions. These bioelectric properties have been supposed to be related to various processes during oogenesis, e. g. pH-regulation, osmoregulation, cell communication, cell migration, cell proliferation, cell death, vitellogenesis and follicle growth. Analysing in detail the spatial distribution and activity of the relevant ion-transport mechanisms is expected to elucidate the roles that bioelectric phenomena play during oogenesis.ResultsTo obtain an overview of bioelectric patterning along the longitudinal and transversal axes of the developing follicle, the spatial distributions of membrane potentials (Vmem), intracellular pH (pHi) and various membrane-channel proteins were studied systematically using fluorescent indicators, fluorescent inhibitors and antisera. During mid-vitellogenic stages 9 to 10B, characteristic, stage-specific Vmem-patterns in the follicle-cell epithelium as well as anteroposterior pHi-gradients in follicle cells and nurse cells were observed. Corresponding distribution patterns of proton pumps (V-ATPases), voltage-dependent L-type Ca2+-channels, amiloride-sensitive Na+-channels and Na+,H+-exchangers (NHE) and gap-junction proteins (innexin 3) were detected. In particular, six morphologically distinguishable follicle-cell types are characterized on the bioelectric level by differences concerning Vmem and pHi as well as specific compositions of ion channels and carriers. Striking similarities between Vmem-patterns and activity patterns of voltage-dependent Ca2+-channels were found, suggesting a mechanism for transducing bioelectric signals into cellular responses. Moreover, gradients of electrical potential and pH were observed within single cells.ConclusionsOur data suggest that spatial patterning of Vmem, pHi and specific membrane-channel proteins results in bioelectric signals that are supposed to play important roles during oogenesis, e. g. by influencing spatial coordinates, regulating migration processes or modifying the cytoskeletal organization. Characteristic stage-specific changes of bioelectric activity in specialized cell types are correlated with various developmental processes.

The study for bioelectric properties continuous measurement by electrical impedance

The study for bioelectric properties continuous measurement by electrical impedance

Dose dependent effect of Iso-Octane on HaCaT: A model study

Objective:Improved understanding of cytotoxicity under chemical assaults may be achieved by multimodal analysis of cellular morphology, viability, molecular expressions, and biophysical properties.Materials and Methods:In this study dose-dependent effects of an organic solvent (OS), iso-octane (IO), known to cause skin irritation, has been explored multimodally for understanding its effect on structural and functional profile of normal epithelial cell population in vitro.Results:Under IO exposures, after 5 h there was a sharp decrease in viability of HaCaT with increasing doses which may be due to disruption in cellular association noted via immunocytochemical study and was further supported by the decreased expression of E-cadherin at transcriptomic level. Dislocation of E-cadherin from membrane to the cytoplasm occurred with increasing doses. The dose-dependent changes in varied aspects of bioelectrical properties, having plausible correlation with cellular viability, association, and adherence were noteworthy at 5 h of IO exposure. Evaluation of biomechanical properties by micropipette aspiration showed a distinct change in cellular stiffness in terms of increase in suction force and post-suction alteration in cellular shape. The cells became stiffer and fragile with increasing IO doses.Conclusion:Present study explicated dose–dependent cytotoxicity of IO on HaCaT and explored the usefulness of this approach to develop in vitro model system to evaluate epithelial toxicity with level-free markers.

Ethnic variability in body size, proportions and composition in children aged 5 to 11 years: is ethnic-specific calibration of bioelectrical impedance required?

BackgroundBioelectrical Impedance Analysis (BIA) has the potential to be used widely as a method of assessing body fatness and composition, both in clinical and community settings. BIA provides bioelectrical properties, such as whole-body impedance which ideally needs to be calibrated against a gold-standard method in order to provide accurate estimates of fat-free mass. UK studies in older children and adolescents have shown that, when used in multi-ethnic populations, calibration equations need to include ethnic-specific terms, but whether this holds true for younger children remains to be elucidated. The aims of this study were to examine ethnic differences in body size, proportions and composition in children aged 5 to 11 years, and to establish the extent to which such differences could influence BIA calibration.MethodsIn a multi-ethnic population of 2171 London primary school-children (47% boys; 34% White, 29% Black African/Caribbean, 25% South Asian, 12% Other) detailed anthropometric measurements were performed and ethnic differences in body size and proportion were assessed. Ethnic differences in fat-free mass, derived by deuterium dilution, were further evaluated in a subsample of the population (n = 698). Multiple linear regression models were used to calibrate BIA against deuterium dilution.ResultsIn children <11 years of age, Black African/Caribbean children were significantly taller, heavier and had larger body size than children of other ethnicities. They also had larger waist and limb girths and relatively longer legs. Despite these differences, ethnic-specific terms did not contribute significantly to the BIA calibration equation (Fat-free mass = 1.12+0.71*(height2/impedance)+0.18*weight).ConclusionAlthough clear ethnic differences in body size, proportions and composition were evident in this population of young children aged 5 to 11 years, an ethnic-specific BIA calibration equation was not required.

Cell Death Parameters as Revealed by Whole-Cell Patch-Clamp and Interval Weighted Spectra Averaging: Changes in Membrane Properties and Current Frequency of Cultured Mouse Microglial Cells Induced by Glutaraldehyde

The physiological and biochemical factors that lead to cell death have not been recognized completely. To our knowledge, there are no data on the bioelectric parameters that characterize early period of cell death, as well as on the appearance of related membrane current frequencies. We studied early parameters of glutaraldehyde (GA)-induced cell death, by examining the membrane properties of mouse microglia using the whole-cell patch-clamp technique. In addition, we investigated the GA-induced changes in the membrane current frequency, to see if characteristic frequencies would appear in dying cell. For data analysis, we applied a new approach, an improved multiple moving window length analysis and interval weighted spectra averaging (IWSA). We chose GA for its ability to induce almost instantaneous cell death. The 0.6% GA did not induce changes in the bioelectric membrane properties of microglia. However, the 3% GA caused significant decrease of membrane capacitance and resistance accompanied by the prominent increase in the membrane currents and nearly ohmic current response of microglial cells. These data indicate that 3% GA caused complete loss of the membrane function consequently inducing instantaneous cell death. The membrane function loss was characterized by appearance of the 1.26-4.62 Hz frequency peak in the IWSA spectra, while no significant increase of amplitudes could be observed for cells treated with 0.6% GA. To our knowledge, this is the first record of a frequency associated with complete loss of the membrane function and thus can be considered as an early indicator of cell death.

Differential Effect of Isooctane Doses on HaCaT and HeLa: A Multimodal Analysis

A multimodal approach is effective in analyzing biological problems critically and thus also useful in assessing cytotoxicity under chemicals assaults. In this study effects of isooctane, an organic solvent and component of gasoline produced in petroleum industries, have been explored on normal (HaCaT) and cancerous (HeLa) epithelial cells. Besides morphological alterations, impacts on viability, prime molecular expressions, and bioelectrical properties on exposure to different doses of isooctane were noted. Scanning electron microscopy and viability assay demonstrated remarkable structural alterations and cell death, respectively, in HaCaT but not in HeLa. Transcriptomic and immunocytochemical studies on E-cadherin expression also elucidated pronounced toxic effects on HaCaT. Remarkable changes on the bioelectrical properties (e.g., impedance and phase angle) of the HaCaT, in contrast to HeLa, at different temporal points on isooctane exposure also indicated cytotoxic effects in the former. Hence this study illustrated cytotoxicity of isooctane on HaCaT multidimensionally which was evaded by HeLa.

Permeability changes in bladder cancer cell gemcitabine due to electromagnetic field.

e15524 Background: Improving the efficiency of cancer chemotherapy is largely due to the detailed study of the transport of drugs across the membranes of tumor cells, bioelectric properties that ar...

HEMODYNAMIC AND BIOELECTRICAL PROPERTIES OF THE BRAIN IN YOUNGER ADULTS WITH NONSTABLE BLOOD PRESSURE

Aim.To study macro- and microhemodynamics and bioelectrical properties of the brain in younger adults with nonstable blood pressure (BP).Material and methods.A population of 79 adults at the age of 19–24 (average 19,9±0,9 years) was examined. According to the BP level they were divided into two groups: 1–46 (58,5%) with stable normal BP (SNBP), and 2–33 (41,5%) with high normal BP inside the range for 1stlevel arterial hypertension, whose BP was not stable (UsBP), but changing from normal values to 1st level hypertension. All patients undergone investigation of brachiocephal and intracerebral vessels with blood flow velocity measurement along the vessel direction, the blood sectional volumetric flow and specific kinetic energy. Microcirculation in the brain was measured by rheoencephalography including parameters of the velocity, time and volumetric parameters. Bioelectricity studied by electroencephalography.Results.In patients with UsBP we found the increase of blood flow velocity, blood volume and specific kinetic flow energy in vertebral and general carotid arteries as in basilar and middle cerebral arteries to. In the area of microcirculation in patients with UsBP the blood filling of frontal lobes was decreased comparing to SNBP patients in whom blood filling showed “mosaic” pattern. In UsBP patients we found changes of bioelectrical activity which are related to the functioning of cortical and subcortical portions of the brain.Conclusion.In younger patients with UsBP there is developed discirculatory encephalopathy with damage of cortex and subcortical portions of the brain due to impaired hemodynamic and microcirculation.

An overview of the history of electro-vectorcardiography. Tribute to the memory of the unforgettable Dr. Gustavo A. Medrano Castro

The history of the investigations about of the so-called irritability of animal tissues showed by English physician Francis Glisson in the 17th century, is summarized. During the 18th century, reliable studies on the bioelectric properties of these tissues began, due to the Swiss scientist Albrecht von Haller and continuated by the Italian naturalist Felice Fontana. In the second half of this century, multiple controversies of the partisans of the animal electricity against the partisans of the contact electricity took place. The Danish scientist Oersted in 1820 proved the close relation of magnetism to electricity, which led to construction of electrometers. These instruments allowed to register and measure record of the electric current. On this way, at middle 21st century, the true animal electricity was identified as the injury current. Later it was possible to record the electric current, risen in the myocardium, out the thorax first by means of the Lippmann' capillary electrometer and later thanks to the Einthoven's string galvanometer at the beginning of the 20th century. So the modern electro-vectorcardiography took off, due to English Thomas Lewis, the North-American Frank N. Wilson and the Mexican Demetrio Sodi Pallares. The last one allowed to rationalize the electro-vectorcardiographic exploration on experimental bases.

Silicon nanograss based impedance biosensor for label free detection of rare metastatic cells among primary cancerous colon cells, suitable for more accurate cancer staging

Detection of rare metastatic cells within a benign tumor is a key challenge to diagnose the cancerous stage of the patients tested by clinical human biopsy or pap smear samples. We have fabricated and tested a nanograssed silicon based bioelectronic device with the ability of detecting a few human colon invasive cancer cells (SW48) in a mixed cell culture of primary cancerous colon cells (HT29) without any biochemical labels. A discernible impedance change was elicited after the presence of 5% metastatic cells in the whole benign sample. The electric field penetration as well as current flow to metastatic cells is different from benign ones due to their different membrane dielectric parameters. Beta dispersion as one of intrinsic bioelectrical properties of the cell membrane in blocking the stimulating current flow in the range of kHz is the specific parameter involved in our diagnosis approach. It can reflect in-depth information about the dielectric properties and the pathological condition of a cell before and after metastatic transformation. Electrically active doped silicon nanograss structures owing to their superior nanocontacts with cell membrane can detect any slight variations in current being originated from the presence of rare metastatic cells on the surface of the sensing electrode. The experimental results revealed that bare doped silicon microelectrodes are incapable of resolving different grades of attached cells.

Intracellular Gold Nanoparticles Increase Neuronal Excitability and Aggravate Seizure Activity in the Mouse Brain

Due to their inert property, gold nanoparticles (AuNPs) have drawn considerable attention; their biological application has recently expanded to include nanomedicine and neuroscience. However, the effect of AuNPs on the bioelectrical properties of a single neuron remains unknown. Here we present the effect of AuNPs on a single neuron under physiological and pathological conditions in vitro. AuNPs were intracellularly applied to hippocampal CA1 neurons from the mouse brain. The electrophysiological property of CA1 neurons treated with 5- or 40-nm AuNPs was assessed using the whole-cell patch-clamp technique. Intracellular application of AuNPs increased both the number of action potentials (APs) and input resistance. The threshold and duration of APs and the after hyperpolarization (AHP) were decreased by the intracellular AuNPs. In addition, intracellular AuNPs elicited paroxysmal depolarizing shift-like firing patterns during sustained repetitive firings (SRF) induced by prolonged depolarization (10 sec). Furthermore, low Mg2+-induced epileptiform activity was aggravated by the intracellular AuNPs. In this study, we demonstrated that intracellular AuNPs alter the intrinsic properties of neurons toward increasing their excitability, and may have deleterious effects on neurons under pathological conditions, such as seizure. These results provide some considerable direction on application of AuNPs into central nervous system (CNS).

Optogenetics in Developmental Biology: using light to control ion flux-dependent signals in Xenopus embryos.

Developmental bioelectricity, electrical signaling among non-excitable cells, is now known to regulate proliferation, apoptosis, gene expression, and patterning during development. The extraordinary temporal and spatial resolution offered by optogenetics could revolutionize the study of bioelectricity the same way it has revolutionized neuroscience. There is, however, no guide to adapting optogenetics to patterning systems. To fill this gap, we used optogenetic reagents, both proteins and photochemical switches, to vary steady-state bioelectrical properties of non-spiking embryonic cells in Xenopus laevis. We injected mRNA for various proteins, including Channelrhodopsins and Archaerhodopsin, into 1-8 cell embryos, or soaked embryos in media containing photochemical switches, then examined the effect of light and dark on membrane voltage (Vmem) using both electrodes and fluorescent membrane voltage reporters. We also scored tadpoles for known effects of varying Vmem, including left-right asymmetry disruption, hyperpigmentation, and craniofacial phenotypes. The majority of reagents we tested caused a significant increase in the percentage of light-exposed tadpoles showing relevant phenotypes; however, the majority of reagents also induced phenotypes in controls kept in the dark. Experiments on this "dark phenotype" yielded evidence that the direction of ion flux via common optogenetic reagents may be reversed, or unpredictable in non-neural cells. When used in combination with rigorous controls, optogenetics can be a powerful tool for investigating ion-flux based signaling in non-excitable systems. Nonetheless, it is crucial that new reagents be designed with these non-neural cell types in mind.

Daily variation in the electrophysiological activity of mouse medial habenula neurones

Intrinsic daily or circadian rhythms arise through the outputs of the master circadian clock in the brain's suprachiasmatic nuclei (SCN) as well as circadian oscillators in other brain sites and peripheral tissues. SCN neurones contain an intracellular molecular clock that drives these neurones to exhibit pronounced day–night differences in their electrical properties. The epithalamic medial habenula (MHb) expresses clock genes, but little is known about the bioelectric properties of mouse MHb neurones and their potential circadian characteristics. Therefore, in this study we used a brain slice preparation containing the MHb to determine the basic electrical properties of mouse MHb neurones with whole-cell patch clamp electrophysiology, and investigated whether these vary across the day–night cycle. MHb neurones (n = 230) showed heterogeneity in electrophysiological state, ranging from highly depolarised cells (∼ −25 to −30 mV) that are silent with no membrane activity or display depolarised low-amplitude membrane oscillations, to neurones that were moderately hyperpolarised (∼40 mV) and spontaneously discharging action potentials. These electrical states were largely intrinsically regulated and were influenced by the activation of small-conductance calcium-activated potassium channels. When considered as one population, MHb neurones showed significant circadian variation in their spontaneous firing rate and resting membrane potential. However, in recordings of MHb neurones from mice lacking the core molecular circadian clock, these temporal differences in MHb activity were absent, indicating that circadian clock signals actively regulate the timing of MHb neuronal states. These observations add to the extracellularly recorded rhythms seen in other brain areas and establish that circadian mechanisms can influence the membrane properties of neurones in extra-SCN sites. Collectively, the results of this study indicate that the MHb may function as an intrinsic secondary circadian oscillator in the brain, which can shape daily information flow in key brain processes, such as reward and addiction.

Bioelectric Characterization of Epithelia from Neonatal CFTR Knockout Ferrets

Cystic fibrosis (CF) is a life-shortening, recessive, multiorgan genetic disorder caused by the loss of CF transmembrane conductance regulator (CFTR) chloride channel function found in many types of epithelia. Animal models that recapitulate the human disease phenotype are critical to understanding pathophysiology in CF and developing therapies. CFTR knockout ferrets manifest many of the phenotypes observed in the human disease, including lung infections, pancreatic disease and diabetes, liver disease, malnutrition, and meconium ileus. In the present study, we have characterized abnormalities in the bioelectric properties of the trachea, stomach, intestine, and gallbladder of newborn CF ferrets. Short-circuit current (ISC) analysis of CF and wild-type (WT) tracheas revealed the following similarities and differences: (1) amiloride-sensitive sodium currents were similar between genotypes; (2) responses to 4,4'-diisothiocyano-2,2'-stilbene disulphonic acid were 3.3-fold greater in CF animals, suggesting elevated baseline chloride transport through non-CFTR channels in a subset of CF animals; and (3) a lack of 3-isobutyl-1-methylxanthine (IBMX)/forskolin-stimulated and N-(2-Naphthalenyl)-((3,5-dibromo-2,4-dihydroxyphenyl)methylene)glycine hydrazide (GlyH-101)-inhibited currents in CF animals due to the lack of CFTR. CFTR mRNA was present throughout all levels of the WT ferret and IBMX/forskolin-inducible ISC was only observed in WT animals. However, despite the lack of CFTR function in the knockout ferret, the luminal pH of the CF ferret gallbladder, stomach, and intestines was not significantly changed relative to WT. The WT stomach and gallbladder exhibited significantly enhanced IBMX/forskolin ISC responses and inhibition by GlyH-101 relative to CF samples. These findings demonstrate that multiple organs affected by disease in the CF ferret have bioelectric abnormalities consistent with the lack of cAMP-mediated chloride transport.