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PubMed1998

Greatly increased mucosal nitric oxide in ulcerative colitis determined in situ by a novel nitric oxide-selective microelectrode.

Although current nitric oxide (NO) electrodes are simple, selective and sensitive, they are fragile and hard to use in clinical studies of patients. By preparing an improved NO electroneedle that overcomes these defects, we directly measured mucosal NO concentrations in 11 patients (six male, five female; mean 26.0 years old) with ulcerative colitis (UC) and five normal volunteers (three male, two female; mean 28.3 years old) in situ. An electroneedle was inserted into colonic mucosa through a biopsy channel during colonoscopy. The information concerning the concentration of NO generated and the appearances of the colonic mucosa at the same site were obtained simultaneously. In the ulcerative colitis patients, NO concentrations were significantly increased at all 24 mucosal sites tested. These included sites where: there was an absence of visible inflammation (five sites); the mucosa was mildly inflamed (eight sites); the mucosa was moderately inflamed (five sites); or severely inflamed (six sites). The NO concentrations in ulcerative colitis patients were 12-72 times higher than the NO levels in normal controls (10 sites). At the same 10 sites in four ulcerative colitis patients, the high NO concentrations were decreased by 53% after glucocorticoid treatment. These data are consistent with those of previous studies utilizing different NO electrodes. Excess mucosal NO is generated from inducible NO synthase in the inflamed mucosa itself and the invading inflammatory cells. Our results suggested that mucosal NO could be a marker for the extent of inflammation and its various actions correlated with the pathogenesis, natural history and prognosis of UC. Using the NO microelectrode system reported here, the concentration of NO generated can be monitored in real-time while observing the mucosal condition at the same site during endoscopy. This novel NO electrode may contribute to understanding the role of NO in colonic mucosal inflammation.

Adolescent↗
PubMed2006

Microelectrode array studies of basal and potassium-evoked release of L-glutamate in the anesthetized rat brain.

L-glutamate (Glu) is the predominant excitatory neurotransmitter in the mammalian central nervous system. It plays major roles in normal neurophysiology and many brain disorders by binding to membrane-bound Glu receptors. To overcome the spatial and temporal limitations encountered in previous in vivo extracellular Glu studies, we employed enzyme-coated microelectrode arrays to measure both basal and potassium-evoked release of Glu in the anesthetized rat brain. We also addressed the question of signal identity, which is the predominant criticism of these recording technologies. In vivo self-referencing recordings demonstrated that our Glu signals were both enzyme- and voltage-dependent, supporting the identity of L-glutamate. In addition, basal Glu was actively regulated, tetrodotoxin (TTX)-dependent, and measured in the low micromolar range (approximately 2 microm) using multiple self-referencing subtraction approaches for identification of Glu. Moreover, potassium-evoked Glu release exhibited fast kinetics that were concentration-dependent and reproducible. These data support the hypothesis that Glu release is highly regulated, requiring detection technologies that must be very close to the synapse and measure on a second-by-second basis to best characterize the dynamics of the Glu system.

Anesthetics↗
PubMed2006

Cortical choline transporter function measured in vivo using choline-sensitive microelectrodes: clearance of endogenous and exogenous choline and effects of removal of cholinergic terminals.

The capacity of the high-affinity choline transporter (CHT) to import choline into presynaptic terminals is essential for acetylcholine synthesis. Ceramic-based microelectrodes, coated at recording sites with choline oxidase to detect extracellular choline concentration changes, were attached to multibarrel glass micropipettes and implanted into the rat frontoparietal cortex. Pressure ejections of hemicholinium-3 (HC-3), a selective CHT blocker, dose-dependently reduced the uptake rate of exogenous choline as well as that of choline generated in response to terminal depolarization. Following the removal of CHTs, choline signal recordings confirmed that the demonstration of potassium-induced choline signals and HC-3-induced decreases in choline clearance require the presence of cholinergic terminals. The results obtained from lesioned animals also confirmed the selectivity of the effects of HC-3 on choline clearance in intact animals. Residual cortical choline clearance correlated significantly with CHT-immunoreactivity in lesioned and intact animals. Finally, synaptosomal choline uptake assays were conducted under conditions reflecting in vivo basal extracellular choline concentrations. Results from these assays confirmed the capacity of CHTs measured in vivo and indicated that diffusion of substrate away from the electrode did not confound the in vivo findings. Collectively, these results indicate that increases in extracellular choline concentrations, irrespective of source, are rapidly cleared by CHTs.

Animals↗
PubMed2006

Non-invasive microelectrode ion flux measurements to study adaptive responses of microorganisms to the environment.

The regulation of membrane-transport activity is crucial for intracellular pH homeostasis, maintenance of cell osmotic potential, nutrient acquisition, signalling, and adaptation of bacterial cells. The non-invasive microelectrode ion flux estimation (MIFE) technique is a powerful tool for kinetic studies of membrane-transport processes across cellular membranes. Since 2001, when this technique was first applied to the study of membrane-transport processes in bacterial cells (J Microbiol Methods 46, 119-129), a large amount of information has been accumulated. This review summarizes some of these findings and discusses the advantages and applicability of this technique in studying bacterial adaptive responses to adverse environmental conditions. First, various methodological aspects of the application of this novel technique in microbiology are discussed. Then, several practical examples ('case studies') are described. The latter include changes in membrane-transport activity in response to various stresses (acidic, osmotic, and temperature stresses) as well as flux changes as a function of bacterial growth stage and nutrient availability. It is shown that non-invasive ion flux measurements may provide a significant conceptual advance in our understanding of adaptive responses in bacteria, fungi and biofilms to a variety of environmental conditions. The technique can also be used for the rapid assessment of food-processing treatments aimed at reducing bacterial contamination of food and for the development of strategies to assess the resistance of organisms to antimicrobial agents.

Adaptation, Physiological↗
PubMed1997

Depression of neuromuscular transmission in methylmercury-poisoned rats: a glass microelectrode and single fiber electromyography study.

Changes in neuromuscular transmission were examined in methylmercury (MeHg)-poisoned rats, given a total oral dose of 60 mg CH3HgCl at 5 mg/kg/day. A microelectrode study was done on the 21st day. The mean quantal content and mean values of the immediately available pool of ACh in the MeHg-poisoned rats were reduced as compared to those in the control rats, but the mean values for the release probability of ACh did not differ significantly. Stimulation single fiber electromyography (SFEMG) was done on the biceps femoris muscle at 1, 5, 10 and 20 Hz on the 28th day. Both a significant and consistent increase in jitter were found at increasing stimulation rates in the MeHg-poisoned rats. The SFEMG findings suggest presynaptic involvement due to accelerated depletion of ACh. We confirmed that neuromuscular transmission is depressed in MeHg-poisoned rats in vivo and in vitro.

Animals↗
PubMed1977

K+-permeability of the blood-brain barrier, investigated by aid of a K+-sensitive microelectrode.

The K+-permeability of the blood-brain barrier at the capillary level was estimated from determinations of brain extracellular K+-concentration in response to an isotonic bolus containing KCl injected into the carotid artery. A very low permeability appeared from the fact that the extracellular K+-concentration--measured by aid of K+-sensitive microelectrodes--remained unchanged during the passage of the bolus. An upper limit for the blood-brain barrier K+-permeability in the rat was estimated to be 2.8 X 10(-7) cm. with s-1.

Animals↗
PubMed1990

Intracellular free magnesium and its regulation, studied in isolated ferret ventricular muscle with ion-selective microelectrodes.

Intracellular free magnesium ([Mg2+]i) was measured in isolated ferret papillary muscles using ion-selective microelectrodes filled with the new magnesium sensor ETH 5214. This new sensor, unlike its predecessor ETH 1117, does not react to marked changes in K+, Na+ or pH. Reducing Ca2+ from 20 microM to around 10 nM also did not affect the response so these electrodes are ideally suited to study intracellular Mg2+ and its regulation. The mean value for the [Mg2+]i from thirty-two experiments (forty-two impalements) was 0.85 mM, confirming previous estimates from this laboratory. Intracellular Mg2+ is not passively distributed and the possibility that Mg2+ is transported out of the cell by a Na(+)-Mg2+ exchanger was investigated. An increase in [Mg2+]o caused an increase in [Mg2+]i, as did stepwise reduction in the [Na+]o. However, this increase in [Mg2+]i on Na+ reduction also occurred in Mg2(+)-free solution suggesting that the increase in [Mg2+]i was due to the increase in intracellular Ca2+ on Na+ reduction. Moreover, increasing [Na+]i by strophanthidin did not change the [Mg2+]i and on increasing [Mg2+]o there was no reduction in the [Na+]i. Blocking ATP production lead to small increases in the [Mg2+]i. These results are not consistent with a Na(+)-Mg2+ exchanger as being the main outward transport mechanism for Mg2+ in this tissue.

Adenosine Triphosphate↗
PubMed1988

A microelectrode study of the mechanisms of L-lactate entry into and release from frog sartorius muscle.

1. Changes in intracellular pH and intracellular anion levels were monitored in frog sartorius muscle fibres during exposure to extracellular L-lactate, using ion-sensitive microelectrodes. 2. Resting intracellular pH (pHi) in 20 mmol l-1 HEPES buffer was 7.18 +/- 0.015 (S.E. of mean, n = 62). Exposure to an extracellular solution at pH 6.5 buffered with 20 mmol l-1 3-(N-morpholino)propanesulphonic acid (MOPS) resulted in a slow intracellular acidification. 3. A reversible decrease in pHi and an increase in intracellular anion levels was observed when L-lactate replaced chloride in equimolar amounts. The increase in intracellular anion level is consistent with intracellular accumulation of L-lactate ion. 4. The rate and steady-state change in pHi and anion level was a function of both extracellular pH and L-lactate concentration, providing evidence for the coupled movement of lactate and proton equivalents. 5. The initial rate of uptake of L-lactate, as measured by the change of pHi, was a non-linear function of the extracellular L-lactate concentration at extracellular pH 6.8 and 7.35. 6. No saturation was observed with concentrations of L-lactate between 5 and 60 mmol l-1 at pH 7.35 and 2.5 and 40 mmol l-1 at pH 6.8. 7. The non-linear relationship between the initial rate of change in pHi and extracellular L-lactate was well fitted by a curve defining uptake as the sum of a carrier process displaying Michaelis-Menten kinetics and a passive diffusion component. The apparent Km of the carrier was 10 mmol l-1 at pHo 7.35 and 4 mmol l-1 at pHo 6.8. 8. The initial rate of change of pHi in the presence of L-lactate was significantly inhibited 39.1 +/- 6.2% by 2-5 mmol l-1 alpha-cyano-4-hydroxycinnamate (n = 9; P less than 0.05, paired t test). 9. alpha-Cyano-4-hydroxycinnamate had no detectable effect on the initial rate of change of pHi induced by propionate exposure. 10. The initial rate of change of pHi induced by L-lactate was not affected by 20-100 mumol l-1 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid (SITS). 11. We conclude that L-lactate crosses the membrane of the frog sartorius muscle with proton equivalents via (1) a carrier-mediated process, and (2) passive diffusion of lactic acid. In the physiological range of L-lactate concentrations and pH the transport process dominates.

Animals↗
PubMed2004

Microelectrode array recordings of cultured hippocampal networks reveal a simple model for transcription and protein synthesis-dependent plasticity.

A simplified cell culture system was developed to study neuronal plasticity. As changes in synaptic strength may alter network activity patterns, we grew hippocampal neurones on a microelectrode array (MEA) and monitored their collective behaviour with 60 electrodes simultaneously. We found that exposure of the network for 15 min to the GABA(A) receptor antagonist bicuculline induced an increase in synaptic efficacy at excitatory synapses that was associated with an increase in the frequency of miniature AMPA receptor-mediated EPSCs and a change in network activity from uncoordinated firing of neurones (lacking any recognizable pattern) to a highly organized, periodic and synchronous burst pattern. Induction of recurrent synchronous bursting was dependent on NMDA receptor activation and required extracellular signal-regulated kinase (ERK)1/2 signalling and translation of pre-existing mRNAs. Once induced, the burst pattern persisted for several days; its maintenance phase (> 4 h) was dependent on gene transcription taking place in a critical period of 120 min following induction. Thus, cultured hippocampal neurones display a simple, transcription and protein synthesis-dependent form of plasticity. The non-invasive nature of MEA recordings provides a significant advantage over traditional assays for synaptic connectivity (i.e. long-term potentiation in brain slices) and facilitates the search for activity-regulated genes critical for late-phase plasticity.

Animals↗
PubMed2006

Pacemaker phase shift in the absence of neural activity in guinea-pig stomach: a microelectrode array study.

Gastrointestinal (GI) motility is well organized. GI muscles act as a functional syncytium to achieve physiological functions under the control of neurones and pacemaker cells, which generate basal spontaneous pacemaker electrical activity. To date, it is unclear how spontaneous electrical activities are coupled, especially within a micrometre range. Here, using a microelectrode array, we show a spatio-temporal analysis of GI spontaneous electrical activity. The muscle preparations were isolated from guinea-pig stomach, and fixed in a chamber with an array of 8 x 8 planar multielectrodes (with 300 microm in interpolar distance). The electrical activities (field potentials) were simultaneously recorded through a multichannel amplifier system after high-pass filtering at 0.1 Hz. Dihydropyridine Ca(2+) channel antagonists are known to differentiate the electrical pacemaker activity of interstitial cells of Cajal (ICCs) by suppressing smooth muscle activity. In the presence of nifedipine, we observed spontaneous electrical activities that were well synchronized over the array area, but had a clear phase shift depending on the distance. The additional application of tetrodotoxin (TTX) had little effect on the properties of the electrical activity. Furthermore, by constructing field potential images, we visualized the synchronization of pacemaker electrical activities resolving phase shifts that were measurable over several hundred micrometres. The results imply a phase modulation mechanism other than neural activity, and we postulate that this mechanism enables smooth GI motility. In addition, some preparations clearly showed plasticity of the pacemaker phase shift.

Action Potentials↗
PubMed1977

Membrane potential of mitochondrial measured with microelectrodes.

The membrane potentials of giant mitochondria from cuprizone-fed mice were found to be independent of metabolic state. Experiments are described in which the presence of the microelectrodes in the inner mitochondrial space, and the metabolic viability of the impaled mitochonidra, are validated.

Animals↗
PubMed1993

Distribution of sulfate-reducing bacteria, O2, and H2S in photosynthetic biofilms determined by oligonucleotide probes and microelectrodes.

The vertical distribution of sulfate-reducing bacteria (SRB) in photosynthetic biofilms from the trickling filter of a sewage treatment plant was investigated with oligonucleotide probes binding to 16S rRNA. To demonstrate the effect of daylight and photosynthesis and thereby of increased oxygen penetration, we incubated two 4-mm-thick biofilm samples in darkness or exposed to light at natural intensity. Gradients of O2, H2S, and pH were examined with microelectrodes during incubation. The samples were subsequently frozen with liquid nitrogen and sliced on a cryomicrotome in 20-microns vertical slices. Fluorescent-dye-conjugated oligonucleotides were used as "phylogenetic" probes to identify single cells in the slices. Oligonucleotide sequences were selected which were complementary to short sequence elements (16 to 20 nucleotides) within the 16S rRNA of sulfate-reducing bacteria. The probes were labeled with fluorescein or rhodamine derivatives for subsequent visualization by epifluorescence microscopy. Five probes were synthesized for eukaryotes, eubacteria, SRB (including most species of the delta group of purple bacteria), Desulfobacter spp., and a nonhybridizing control. The SRB were unevenly distributed in the biofilm, being present in all states from single scattered cells to dense clusters of several thousand cells. To quantify the vertical distribution of SRB, we counted cells along vertical transects through the biofilm. This was done in a blind experiment to ascertain the reliability of the staining. A negative correlation between the vertical distribution of positively stained SRB cells and the measured O2 profiles was found. The distribution differed in light- and dark-incubated samples presumably because of the different extensions of the oxic surface layer. In both cases the SRB were largely restricted to anoxic layers.

Bacteria↗
PubMed1999

In situ analysis of nitrifying biofilms as determined by in situ hybridization and the use of microelectrodes.

We investigated the in situ spatial organization of ammonia-oxidizing and nitrite-oxidizing bacteria in domestic wastewater biofilms and autotrophic nitrifying biofilms by using microsensors and fluorescent in situ hybridization (FISH) performed with 16S rRNA-targeted oligonucleotide probes. The combination of these techniques made it possible to relate in situ microbial activity directly to the occurrence of nitrifying bacterial populations. In situ hybridization revealed that bacteria belonging to the genus Nitrosomonas were the numerically dominant ammonia-oxidizing bacteria in both types of biofilms. Bacteria belonging to the genus Nitrobacter were not detected; instead, Nitrospira-like bacteria were the main nitrite-oxidizing bacteria in both types of biofilms. Nitrospira-like cells formed irregularly shaped aggregates consisting of small microcolonies, which clustered around the clusters of ammonia oxidizers. Whereas most of the ammonia-oxidizing bacteria were present throughout the biofilms, the nitrite-oxidizing bacteria were restricted to the active nitrite-oxidizing zones, which were in the inner parts of the biofilms. Microelectrode measurements showed that the active ammonia-oxidizing zone was located in the outer part of a biofilm, whereas the active nitrite-oxidizing zone was located just below the ammonia-oxidizing zone and overlapped the location of nitrite-oxidizing bacteria, as determined by FISH.

Ammonia↗
PubMed2002

Responses of Listeria monocytogenes to acid stress and glucose availability revealed by a novel combination of fluorescence microscopy and microelectrode ion-selective techniques.

Fluorescence ratio imaging microscopy and microelectrode ion flux estimation techniques were combined to study mechanisms of pH homeostasis in Listeria monocytogenes subjected to acid stress at different levels of glucose availability. This novel combination provided a unique opportunity to measure changes in H(+) at either side of the bacterial membrane in real time and therefore to evaluate the rate of H(+) flux across the bacterial plasma membrane and its contribution to bacterial pH homeostasis. Responses were assessed at external pHs (pH(o)) between 3.0 and 6.0 for three levels of glucose (0, 1, and 10 mM) in the medium. Both the intracellular pH (pH(i)) and net H(+) fluxes were affected by the glucose concentration in the medium, with the highest absolute values corresponding to the highest glucose concentration. In the presence of glucose, the pH(i) remained above 7.0 within a pH(o) range of 4 to 6 and decreased below pH(o) 4. Above pH(o) 4, H(+) extrusion increased correspondingly, with the maximum value at pH(o) 5.5, and below pH(o) 4, a net H(+) influx was observed. Without glucose in the medium, the pH(i) decreased, and a net H(+) influx was observed below pH(o) 5.5. A high correlation (R = 0.75 to 0.92) between the pH(i) and net H(+) flux changes is reported, indicating that the two processes are complementary. The results obtained support other reports indicating that membrane transport processes are the main contributors to the process of pH(i) homeostasis in L. monocytogenes subjected to acid stress.

Cells, Immobilized↗
PubMed1987

The mechanism of ryanodine action in rabbit ventricular muscle evaluated with Ca-selective microelectrodes and rapid cooling contractures.

Cellular Ca uptake and efflux in rabbit ventricular muscle was measured using double-barreled Ca microelectrodes in the extracellular space. When repetitive stimulation was stopped there was a slow loss of cellular Ca. Upon resumption of stimulation Ca was taken up by the cells. These Ca movements are thought to represent the loss of Ca from the sarcoplasmic reticulum and the cell during rest and the refilling of the sarcoplasmic reticulum during stimulation. Ryanodine (100 nM) greatly enhanced both the efflux of Ca during rest and the uptake of Ca induced by stimulation. These results are consistent with the conclusions drawn below, but they are dependent upon the interpretation that these extracellular Ca depletions are indicative of sarcoplasmic reticulum Ca movements. To examine further this process, contractures induced by rapid cooling to 0 degrees C were used as an independent assay of sarcoplasmic reticulum Ca content. These rapid cooling contractures were smaller after longer rest intervals (declining with a half time of 1.5 min). In the presence of ryanodine, the rapid cooling contracture immediately after a contraction was greater than that seen under control conditions. However, in the presence of ryanodine these rapid cooling contractures decline as a function of rest duration with a half time of about 1 s. These results suggest that in the presence of ryanodine the sarcoplasmic reticulum can still take up Ca, but that it also loses this Ca very rapidly at the onset of rest.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaloids↗
PubMed1987

Tip size of ion-exchanger based K+-selective microelectrodes. I. Effects on selectivity.

Double-barreled ion-exchanger based K+-selective microelectrodes (K+ ISMs) of a variety of tip diameters were used to study the dependency of ion selectivity upon tip size. The selectivity of K+ ISMs depended on tip size and barrel configuration. Within the range of tip diameters tested (approximately 0.5-6 micron) all K+ ISMs constructed of two barrels glued side by side ("figure-eight glass") exhibited sensitivity to K+ and NH4+. Figure-eight K+ ISMs with tip diameters less than 1.5 micron were not sensitive to tetramethylammonium, tetraethylammonium, or choline, whereas K+ ISMs with tip diameters greater than or equal to 1.5 micron sensed all of the quaternary amines. Tip size dependent selectivity was not present in K+ ISMs made from thick septum theta glass. The explanation for tip size dependent changes in ion selectivity is unknown but a discussion of theoretical possibilities is given.

Amines↗
PubMed1987

Cationic membrane conductances induced by intracellularly elevated cAMP and Ca2+: measurements with ion-selective microelectrodes.

Adenosine 3',5'-cyclic monophosphate (cAMP) and CaCl2 were injected by a fast and quantitative pressure injection technique into voltage-clamped, identified Helix neurons. Intracellular elevation of cAMP as well as of Ca2+ activated an inward current (IcAMP and IN). To identify the ionic fluxes during IcAMP and IN changes in [Na+]i, [K+]o, [H+]i, and [Cl-]i were measured with ion-selective microelectrodes (ISMs). Near resting potential, Na+ was the main carrier of IcAMP. K+, and less effectively Ca2+, could substitute for Na+ in carrying IcAMP. H+ and Cl- were excluded as current carriers for IcAMP by means of ISMs. Simultaneous to this action, cAMP decreased a K+ conductance. This decrease was associated with a reduction of the K+ efflux activated by long-lasting depolarizing voltage steps, as directly measured with ISMs located near the external membrane surface. The nearly compensatory increase and decrease of two membrane conductances in the same neuron left the cell input resistance unchanged despite the considerable depolarizing action of intracellularly elevated cAMP. IN was also of nonspecific nature. However, our findings indicate less selectivity for the Ca2+-activated nonspecific channels. Large cations such as choline, TEA, and Tris passed nearly as well as Na+ through the channels. Measurements with ISMs showed that [H+]i and [Cl-]i were unchanged during IN. IN was largest in bursting pacemaker neurons compared with other cells of similar size. It was found to be essential for the burst production in these cells. IcAMP, on the other hand, might be involved in the presynaptic facilitatory action of cAMP, which as yet was attributed solely to a reduction of a K+ conductance.

Animals↗