Mapping electric currents around skeletal muscle with a vibrating probe

A vibrating microelectrode, or vibrating probe (Jaffe and Nuccitelli, 1974), was used to map the pattern of artificially created electric currents flowing around single muscle fibers at the edge of frog cutaneous pectoris muscles. When a muscle fiber was impaled with a micropipette, a "point sink" of current was often created at the site of impalement because of injury to the cell membrane. Current, being drawn from the flanking membrane, flowed into the cell only at this point. This defined current allowed us to map the spatial resolving power of the vibrating probe by moving to different positions near the impalement site. The results suggest that under our experimental conditions the limit of resolution is a few tens of micrometers. The results were fit reasonably well by a computer model. Current was also passed through a micropipette and mapped at various positions with the vibrating probe. In this case, the current flowed to a remote reference electrode. With the current electrode in the extracellular fluid, the probe signal decayed as the inverse square of the distance, as expected. With the current electrode placed intracellularly, current was funneled along the muscle fiber axis, reflecting its cable-like properties. The signal recorded by the vibrating probe was altered accordingly, and the results could be well fit by a simple model.     

Advances in vibrating probe techniques

Summary This paper summarizes the fundamentals and describes advances in the vibrating probe, a tool that was developed and introduced into biological research byJaffe andNuccitelli in 1974. The improvements reported are concerned on the one hand with the construction, micropositioning and maintenance of the probe itself and, on the other hand, with the origin of artifacts and possible ways to avoid or, at least, to detect and monitor them. The advances in construction and the elucidation of possible artifacts may help extend the usefulness of this unique tool in investigations of ionic currents which cells and tissues drive through themselves during development and movement.     

Endogenous bioelectric currents promote differentiation of the mammalian lens

The functional roles of bioelectrical signals (ES) created by the flow of specific ions at the mammalian lens equator are poorly understood. We detected that mature, denucleated lens fibers expressed high levels of the α1 and β1 subunits of Na⁺/K⁺‐ATPase (ATP1A1 and ATP1B1 of the sodium pump) and had a hyperpolarized membrane potential difference (V_mem). In contrast, differentiating, nucleated lens fiber cells had little ATP1A1 and ATP1B1 and a depolarized V_mem. Mimicking the natural equatorial ES with an applied electrical field (EF) induced a striking reorientation of lens epithelial cells to lie perpendicular to the direction of the EF. An EF also promoted the expression of β‐crystallin, aquaporin‐0 (AQP0) and the Beaded Filament Structural Protein 2 (BFSP2) in lens epithelial cells (LECs), all of which are hallmarks of differentiation. In addition, applied EF activated the AKT and CDC2 and inhibition of AKT reduced the activation of CDC2. Our results indicate that the endogenous bioelectrical signal at the lens equator promotes differentiation of LECs into denucleated lens fiber cells via depolarization of V_mem. Development of methods and devices of EF application or amplification in vivo may supply a novel treatment for lens diseases and even promote regeneration of a complete new lens following cataract surgery.     

Ion transport and the vibrating probe.

The theory of ion transport in the vicinity of a vibrating probe is developed. It is shown that the convection loops produced by the probe will not affect the electrical current density, assuming that the action of the probe does not affect the sources of the current in the biological system. However, the convection loops will significantly alter the ion concentration gradients in the unstirred layer near a tissue or cell surface. The concentration gradients within each convection loop will be reduced, while the concentration gradients between the loops and outside of the loops will be increased relative to the gradients existing without the probe. As a consequence, the electrical potential gradients can be changed relative to the potential gradients existing in the absence of the convection caused by the probe. If the mobility of the ion species carrying the electrical current is greater than the average ion mobility in the medium, then a decrease in ion concentration gradient will be accompanied by an increase in electrical potential gradient, while an increase in concentration gradient will be accompanied by a decrease or even a reversal of electrical potential gradient. Thus, the electrical potential gradient measured by the probe will depend on the concentration gradient in the vicinity of the probe, which will depend in turn on the spatial relation of the convection loops to the probe. An example of the effect of the convection loops on ion concentration and electrical potential is obtained from the theory via a numerical computer calculation. Experimental tests of this theory are discussed.     

Localization of ionic pathways in the teleost opercular membrane by extracellular recording with a vibrating probe

We have adapted the vibrating probe extracellular recording technique to use on an epithelium under voltage clamp in an Ussing chamber. The vibrating probe allows very low drift measurements of current density immediately over the epithelial surface. These measurements allowed sites of electrogenic transport in the epithelium to be localized with a spatial resolution of 5 micrometers. The technique was applied to the opercular membrane of the teleost fish, the tilapia, Sarotherodon mossambicus. The mitochondrion-rich "chloride cells" were shown to be the only sites of electrogenic ion transport in this heterogeneous epithelium. Cell sampling experiments demonstrated variable negative short-circuit currents associated with nearly all of approximately 300 chloride cells examined, which appeared to account for all of the tissue short-circuit current. Current-voltage relations for individual cells were also measured. Conductance associated with chloride cells (i.e. cellular and junctional pathways) accounted for all but 0.5 mS/cm2 of the tissue conductance, with the balance apparently accounted for by leak pathways near the edge of the tissue. Current and conductance associated with other cell types was at least 50-fold smaller than for the chloride cell. Chloride-free solutions reduced chloride cell current and conductance by 98 and 95%, respectively.     

Localization of the photocurrent of Limulus ventral photoreceptors using a vibrating probe.

We have used a vibrating probe to determine the profile of electrical current density around ventral photoreceptors of the horseshoe crab following flashes of light that uniformly illuminated the entire surface of the photoreceptor's cell body. The vibrating probe signal indicated that the density of inward current was greatest at the distal region of the cell, the region that is expected to contain the light-sensitive rhabdom. The density of inward current typically declined at the midpoint of the cell body and then reversed to an outward current flow in the proximal region of the cell body, close to the axon. The profile of local sensitivity of the photoreceptor to light closely matched the profile of inward current density, suggesting that the light-activated conductance is localized to the light-sensitive region of the cell.     

Vibrating probe measurement of ionic currents around developing embryo of oil palm (Elaeis guineensis Jacq.)

The current patterns around zygotic embryos of the oil palmembryo (Elaeis guineensis Jacq.) were investigated. Ionic currentsaround the embryo were measured at different stages in its developmentusing a two-dimensional vibrating probe system. The averagecurrent density was found to be in the order of 1–µAcm^–2. In the early developmental stage, the current wasfound entering the cotyledon and leaving the radicle. Both themagnitude and direction of this current changed when embryosat different developmental stages were observed. The currentswere found to enter the region in the process of differentiationor elongation, and in some cases this could be the reverse ofthe current direction in the preceding stage. Within one ofthe stages of the embryos, current density was correlated withthe rate of elongation. A very large inward current (up to 20µAcm^–2) was also detected at the point where the shoot wasbreaking through the surface. This was probably caused by theso-called ‘injury current’. Key words: Ionic currents, vibrating probe, oil palm embryo, E. guineensis, injury current     

Non-invasive individual methane measurement in dairy cows

Attempts to lower the environmental footprint of milk production needs a sound understanding of the genetic and nutritional basis of methane (CH₄) emissions from the dairy production systems. This in turn requires accurate and reliable techniques for the measurement of CH₄ output from individual cows. Many of the available measurement techniques so far are either slow, expensive, labor intensive and are unsuitable for large-scale individual animal measurements. The main objectives of this study were to examine and validate a non-invasive individual cow CH₄ measurement system that is based on photoacoustic IR spectroscopy (PAS) technique implemented in a portable gas analysis equipment (F10), referred to as PAS-F10 method and to estimate the magnitude of between-animal variations in CH₄ output traits. Data were collected from 115 Nordic Red cows of the Minkiö experimental dairy farm, at the Natural Resources Institute Finland (Luke). Records on continuous daily measurements of CH₄, milk yield, feed intake and BW measurements over 2 years period were compiled for data analysis. The daily CH₄ output was calculated using carbon dioxide as a tracer method. Estimates from the non-invasive PAS-F10 technique were then tested against open-circuit indirect respiration calorimetric chamber measurements and against estimates from other widely used prediction models. Concordance analysis was used to establish agreement between the chamber and PAS-F10 methods. A linear mixed model was used for the analysis of the large continuous data. The daily CH₄ output of cows was 555 l/day and ranged from 330 to 800 l/day. Dry matter intake, level of milk production, lactation stage and diurnal variation had significant effects on daily CH₄ output. Estimates of the daily CH₄ output from PAS-F10 technique compared relatively well with the other techniques. The concordance correlation coefficient between combined weekly CH₄ output estimates of PAS-F10 and chamber was 0.84 with lower and upper confidence limits of 0.65 and 0.93, respectively. Similarly, when chamber CH₄ measurements were predicted from PAS-F10 measurements, the mean of two separate weekly PAS-F10 measurements gave the lowest prediction error variance than either of the separate weekly PAS-F10 measurements alone. This suggests that every other week PAS-F10 measurements when combined would improve the estimation of CH₄ output with PAS-F10 technique. The repeatability of daily CH₄ output from PAS-F10 technique ranged from 0.40 to 0.46 indicating that some between-animal variation exist in CH₄ output traits.     

Non-invasive measurement of brain damage in a primate model of multiple sclerosis

Early recognition of whether a product has potential as a new therapy for treating multiple sclerosis (MS) relies upon the quality of the animal models used in the preclinical trials. The promising effects of new treatments in rodent models of experimental autoimmune encephalomyelitis (EAE) have rarely been reproduced in patients suffering from MS. EAE in outbred marmoset monkeys, Callithrix jacchus, is a valid new model, and might provide an experimental link between EAE in rodent models and human MS. Using magnetic resonance imaging techniques similar to those used in patients suffering from MS pathological abnormalities in the brain, white matter of the animal can be visualized and quantified. Moreover, NMR spectroscopy, in combination with pattern recognition, offers an advanced uroscopic technique for the identification of biomarkers of inflammatory demyelination.     

Non-invasive imaging of cysteine cathepsin activity in solid tumors using a 64Cu-labeled activity-based probe

The papain family of cysteine cathepsins are actively involved in multiple stages of tumorigenesis. Because elevated cathepsin activity can be found in many types of human cancers, they are promising biomarkers that can be used to target radiological contrast agents for tumor detection. However, currently there are no radiological imaging agents available for these important molecular targets. We report here the development of positron emission tomography (PET) radionuclide-labeled probes that target the cysteine cathepsins by formation of an enzyme activity-dependent bond with the active site cysteine. These probes contain an acyloxymethyl ketone (AOMK) functional group that irreversibly labels the active site cysteine of papain family proteases attached to a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) tag for labeling with (64)Cu for PET imaging studies. We performed biodistribution and microPET imaging studies in nude mice bearing subcutaneous tumors expressing various levels of cysteine cathepsin activity and found that the extent of probe uptake by tumors correlated with overall protease activity as measured by biochemical methods. Furthermore, probe signals could be reduced by pre-treatment with a general cathepsin inhibitor. We also found that inclusion of a Cy5 tag on the probe increased tumor uptake relative to probes lacking this fluorogenic dye. Overall, these results demonstrate that small molecule activity-based probes carrying radio-tracers can be used to image protease activity in living subjects.     

Non-invasive measurement of human fetal circulation using ultrasound: a new method.

We combined two ultrasound techniques to develop a safe, non-invasive, transcutaneous method of observing the circulation in the umbilical arteries and vein in the fetus. The umbilical cord can be located by standard echo ultrasound procedures, and this information can be used to direct a Doppler ultrasound beam on to the vessels in the cord. The signals can be heard through audio headphones or recorded on a tape recorded and spectrum-analysed. The method was successful in each of 20 patients examined, whose pregnancies ranged from 12 to 40 weeks'' gestational age, and was suitable for outpatient use. It should be useful in assessing such conditions as pre-eclampsia and intrauterine growth retardation.     

Optical measurement of presynaptic calcium currents.

Measurements of presynaptic calcium currents are vital to understanding the control of transmitter release. However, most presynaptic boutons in the vertebrate central nervous system are too small to allow electrical recordings of presynaptic calcium currents (I(Ca)pre). We therefore tested the possibility of measuring I(Ca)pre optically in boutons loaded with calcium-sensitive fluorophores. From a theoretical treatment of a system containing an endogenous buffer and an indicator, we determined the conditions necessary for the derivative of the stimulus-evoked change in indicator fluorescence to report I(Ca)pre accurately. Matching the calcium dissociation rates of the endogenous buffer and indicator allows the most precise optical measurements of I(Ca)pre. We tested our ability to measure I(Ca)pre in granule cells in rat cerebellar slices. The derivatives of stimulus-evoked fluorescence transients from slices loaded with the low-affinity calcium indicators magnesium green and mag-fura-5 had the same time courses and were unaffected by changes in calcium influx or indicator concentration. Thus both of these indicators were well suited to measuring I(Ca)pre. In contrast, the high-affinity indicator fura-2 distorted I(Ca)pre. The optically determined I(Ca)pre was well approximated by a Gaussian with a half-width of 650 micros at 24 degrees C and 340 micros at 34 degrees C.     

Potassium secretion by vestibular dark cell epithelium demonstrated by vibrating probe.

Detection of motion and position by the vestibular labyrinth depends on the accumulation of potassium within a central compartment of the inner ear as a source of energy to drive the transduction process. Much circumstantial evidence points to the vestibular dark cell (VDC) epithelium as being responsible for concentrating K+ within the lumen. We have used the vibrating probe technique to directly observe voltage and ion gradients produced by this tissue to put this assumption on a solid experimental footing. Relative current density (Isc,probe) over the apical membrane of VDC epithelium was measured with the vibrating voltage-sensitive probe, and this technique was validated by performing maneuvers known to either stimulate or inhibit the transepithelial equivalent short circuit current. Basolateral bumetanide (5 x 10(-5) M) and ouabain (1 x 10(-3) M) caused a decrease in Isc,probe by 55 +/- 6% and 39 +/- 3%, respectively while raising the basolateral K+ concentration from 4 to 25 mM caused an increase by 35 +/- 8%. A K+ gradient directed toward the apical membrane was detected with the vibrating K(+)-selective electrode, demonstrating that, indeed, the VDC epithelium secretes K+ under control conditions. This secretion was inhibited by bumetanide (by 94 +/- 7%) and ouabain (by 52 +/- 8%). The results substantiate the supposition that dark cells produce a K+ flux and qualitatively support the correlation between this flux and the transepithelial current.     

Non-invasive measurement of adrenocortical activity in male and female rats

Rats are widely used in biomedical research as animal models for human diseases. However, due to their small body size, blood sampling is complicated and invasive and thereby can seriously interfere with endocrine functions and possibly compromise the animals' welfare. Therefore, a non-invasive technique to monitor stress hormones in these animals is highly desired. Our study aimed to gain general information about corticosterone metabolism and excretion and to validate a 5alpha-pregnane-3beta,11beta,21-triol-20-one enzyme immunoassay (EIA) to reliably measure faecal corticosterone metabolites (CMs) in laboratory rats. In total, 18 rats were administered 2.3 MBq of (3)H-corticosterone intravenously and per os, respectively (intravenous: 6 males and 6 females; per os: 3 males and 3 females). Subsequently, all voided excreta were frequently collected for five days. About 75+/-9% of the recovered CMs were found in the faeces. Peak concentrations of radiolabelled steroids appeared in the urine after 1.7+/-0.6 h in males and after 6.0+/-3.5 h in females. In faeces, maxima were observed after 14.7+/-2.4 h in both sexes. In principle, the time course and delay for both routes of administration (intravenous or per os) were the same, except for a delay of peak concentrations in urine (4.5+/-2.1 h) in per os administered males. Using high-performance liquid chromatography (HPLC), faecal (3)H-CMs were characterized and differences were found between the sexes. In both sexes, corticosterone was extensively metabolized, but while males showed only minor variations in their CM patterns, those of females differed largely between individuals. To validate the mentioned EIA, we investigated the diurnal variation (DV) of glucocorticoids as well as effects of the injection procedure itself and conducted an adrenocorticotropic hormone challenge test and a dexamethasone suppression test, using six male and six female rats each. Our results demonstrated that pharmacological stimulation, suppression and DV of adrenocortical activity were accurately reflected by means of CM measurement in faeces. By successful physiological validation, we proved for the first time the suitability of an immunoassay to non-invasively monitor adrenocortical activity in rats of both sexes. This method opens up new perspectives for biomedical and pharmacological investigations as well as for animal welfare related issues.     

Non-invasive measurement of hemoglobin: assessment of two different point-of-care technologies

Background

Measurement of blood hemoglobin (Hb) concentration is a routine procedure. Using a non-invasive point-of-care device reduces pain and discomfort for the patient and allows time saving in patient care. The aims of the present study were to assess the concordance of Hb levels obtained non-invasively with the Pronto-7 monitor (version 2.1.9, Masimo Corporation, Irvine, USA) or with the NBM-200MP monitor (Orsense, Nes Ziona, Israel) and the values obtained from the usual colorimetric method using blood samples and to determine the source of discordance.

Methods and Findings

We conducted two consecutive prospective open trials enrolling patients presenting in the emergency department of a university hospital. The first was designed to assess Pronto-7™ and the second NBM-200MP™. In each study, the main outcome measure was the agreement between both methods. Independent factors associated with the bias were determined using multiple linear regression. Three hundred patients were prospectively enrolled in each study. For Pronto-7™, the absolute mean difference was 0.56 g.L⁻¹ (95% confidence interval [CI] 0.41 to 0.69) with an upper agreement limit at 2.94 g.L⁻¹ (95% CI [2.70;3.19]), a lower agreement limit at -1.84 g.L⁻¹ (95% CI [-2.08;-1.58]) and an intra-class correlation coefficient at 0.80 (95% CI [0.74;0.84]). The corresponding values for the NBM-200MP™ were 0.21 [0.02;0.39], 3.42 [3.10;3.74], -3.01 [-3.32;-2.69] and 0.69 [0.62;0.75]. Multivariate analysis showed that age and laboratory values of hemoglobin were independently associated with the bias when using Pronto-7™, while perfusion index and laboratory value of hemoglobin were independently associated with the bias when using NBM-200MP™.

Conclusion

Despite a relatively limited bias in both cases, the large limits of agreement found in both cases render the clinical usefulness of such devices debatable. For both devices, the bias is independently and inversely associated with the true value of hemoglobin.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01321580","term_id":"NCT01321580"}}NCT01321580 {"type":"clinical-trial","attrs":{"text":"NCT01321593","term_id":"NCT01321593"}}NCT01321593     

Access tube location within a simulated potato crop and the measurement of soil moisture with a neutron probe

Laboratory calibrations simulating different locations of access tubes in a potato crop, examined the possible influence of the water content of tubers on measurements of soil moisture made with a neutron probe. It was concluded that even when access tubes were located within the row, the presence of tubers did not significantly affect estimates of soil moisture made with the neutron probe.