There is a dense serotonergic projection from nucleus raphe pallidus and nucleus raphe obscurus to the trigeminal motor nucleus and serotonin exerts a strong facilitatory action on the trigeminal motoneurons. Some serotonergic neurons in these caudal raphe nuclei increase their discharge during feeding. The objective of the present study was to investigate the possibility that the activity of these serotonergic neurons is related to activity of masticatory muscles. Cats were implanted with microelectrodes and gross electrodes. Caudal raphe single neuron activity, electrocorticographic activity, and splenius, digastric and masseter electromyographic activities were recorded during active behaviors (feeding and grooming), during quiet waking and during sleep. Seven presumed serotonergic neurons were identified. These neurons showed a long duration action potential (>2.0 ms), and discharged slowly (2-7 Hz) and very regularly (interspike interval coefficient of variation <0.3) during quiet waking. The activity of these neurons decreased remarkably during fast wave sleep (78-100%). Six of these neurons showed tonic changes in their activity positively related to digastric and/or masseter muscle activity but not to splenius muscle activity during waking. These data are consistent with the hypothesis that serotonergic neurons in the caudal raphe nuclei play an important role in the control of jaw movements
(ANP, 1 µM) on the kinetics of bicarbonate reabsorption in the rat middle proximal tubule, we performed in vivo experiments using a stopped-flow microperfusion technique with the determination of lumen pH by Sb microelectrodes. These studies confirmed that ANG II added to the luminal or peritubular capillary perfusion fluid stimulates proximal bicarbonate reabsorption and showed that ANP alone does not affect this process, but impairs the stimulation caused by ANG II. We also studied the effects and the interaction of these hormones in cortical distal nephron acidification. Bicarbonate reabsorption was evaluated by the acidification kinetic technique in early (ED) and late (LD) distal tubules in rats during in vivo stopped-flow microperfusion experiments. The intratubular pH was measured with a double-barreled microelectrode with H+-sensitive resin. The results indicate that ANG II acted by stimulating Na+/H+ exchange in ED (81%) and LD (54%) segments via activation of AT1 receptors, as well as vacuolar H+-ATPase in LD segments (33%). ANP did not affect bicarbonate reabsorption in either segment and, as opposed to what was seen in the proximal tubule, did not impair the stimulation caused by ANG II. To investigate the mechanism of action of these hormones in more detail...
The present paper reviews work from our laboratories evaluating the importance of adrenal cortical hormones in acidification by proximal and cortical distal tubules. Proximal acidification was determined by stationary microperfusion, and measurement of bicarbonate reabsorption using luminal pH determination was performed with H+-ion-sensitive microelectrodes. Rats were adrenalectomized (ADX) 48 h before the experiments, and corticosteroids (aldosterone (A), corticosterone (B), and 18-OH corticosterone (18-OH-B)) were injected intramuscularly 100 and 40 min before the experiments. In ADX rats stationary pH increased significantly to 7.03 as compared to sham-operated rats (6.78). Bicarbonate reabsorption decreased from 2.65 ± 0.18 in sham-operated rats to 0.50 ± 0.07 nmol cm-2 s-1 after ADX. The administration of the three hormones stimulated proximal tubule acidification, reaching, however, only 47.2% of the sham values in aldosterone-treated rats. Distal nephron acidification was studied by measuring urine minus blood pCO2 differences (U-B pCO2) in bicarbonate-loaded rats treated as above. This pCO2 difference is used as a measure of the distal nephron ability to secrete H+ ions into an alkaline urine. U-B pCO2 decreased significantly from 39.9 ± 1.26 to 11.9 ± 1.99 mmHg in ADX rats. When corticosteroids were given to ADX rats before the experiment...
The striatum, the largest component of the basal ganglia, is usually subdivided into associative, motor and limbic components. However, the electrophysiological interactions between these three subsystems during behavior remain largely unknown. We hypothesized that the striatum might be particularly active during exploratory behavior, which is presumably associated with increased attention. We investigated the modulation of local field potentials (LFPs) in the striatum during attentive wakefulness in freely moving rats. To this end, we implanted microelectrodes into different parts of the striatum of Wistar rats, as well as into the motor, associative and limbic cortices. We then used electromyograms to identify motor activity and analyzed the instantaneous frequency, power spectra and partial directed coherence during exploratory behavior. We observed fine modulation in the theta frequency range of striatal LFPs in 92.5 ± 2.5% of all epochs of exploratory behavior. Concomitantly, the theta power spectrum increased in all striatal channels (P < 0.001), and coherence analysis revealed strong connectivity (coefficients >0.7) between the primary motor cortex and the rostral part of the caudatoputamen nucleus, as well as among all striatal channels (P < 0.001). Conclusively...
We investigated the GABA-induced inactivation of V2 neurons and terminals on the receptive field properties of this area in an anesthetized and paralyzed Cebus apella monkey. Extracellular single-unit activity was recorded using tungsten microelectrodes in a monkey before and after pressure-injection of a 0.25 or 0.5 M GABA solution. The visual stimulus consisted of a bar moving in 8 possible directions. In total, 24 V2 neurons were studied before and after blocker injections in 4 experimental sessions following GABA injection into area V2. A group of 10 neurons were studied over a short period. An additional 6 neurons were investigated over a long period after the GABA injection. A third group of 8 neurons were studied over a very long period. Overall, these 24 neurons displayed an early (1-20 min) significant general decrease in excitability with concomitant changes in orientation or direction selectivity. GABA inactivation in area V2 produced robust inhibition in 80% and a significant change in directional selectivity in 60% of the neurons examined. These GABA projections are capable of modulating not only levels of spontaneous and driven activity of V2 neurons but also receptive field properties such as direction selectivity.
The voltammetry at three micro-mesh electrodes is explored. It is found that at sufficiently short experimental durations, the micro-mesh working electrode first behaves as an ensemble of microband electrodes, then follows the behaviour anticipated for an array of diffusion-independent micro-ring electrodes of the same perimeter as individual grid-squares within the mesh. During prolonged electrolysis, the micro-mesh electrode follows that behaviour anticipated theoretically for a cubically-packed partially-blocked electrode. Application of the micro-mesh electrode for the electrochemical determination of carbon dioxide in DMSO electrolyte solutions is further illustrated.
Interactions of four aromatic nitrogen-heterocyclic herbicides (atrazine, imazaquin, metribuzin and paraquat) with the conductive polymer poly(o-ethoxyaniline)-POEA, were studied with atomic force microscopy (AFM), UV-visible spectrophotometry (UV-Vis) and electrochemical impedance spectroscopy. AFM profiles of self-assembled (SA) films of POEA revealed that the polymer surface became rougher (on the nanoscale) when exposed to atrazine, imazaquin and metribuzin, but slightly smoother in contact with paraquat. This suggested that specific chemical interactions, possibly electroscopic, have occurred between nitrogen atoms in the polymer chain and the dissimilar groups in the various herbicide molecules, during adsorption of the latter onto the film. The UV-Vis analyses indicated a stronger interaction of POEA in solution with imazaquin, that has a special importance for the intended application. Sensors produced by coating microelectrodes with SA films of POEA were used to perform impedance spectroscopy in aqueous solutions of each herbicide. With the resulting data, it was possible to distinguish and set detection limits for each herbicide in water, corroborating AFM and UV-Vis results.
In this work, the electrochemical behavior of Mg and Fe in ionic liquids (IL) were studied. We performed a series of cyclic voltammetry experiments to improve the understanding of Mg behavior in an IL containing the bis(trifluoromethanesulfonylimide) ([Tf2N]) anion. The results show an irreversible deposition/dissolution of Mg at a high water concentration (ca. 1300 ppm, 50 mmol L-1) and very low reversibility (7.3%) at a moderate water concentration (ca. 65 ppm, 5 mmol L-1). The formation of a film on the electrode surface and the presence of Mg were confirmed by scanning electron microscopy and energy-dispersive X-ray spectroscopy (SEM-EDS). The process irreversibility indicates the formation of a passivating film. Because the presence of water affects the reversibility of the process, studies of Fe deposition/dissolution were conducted in two different ILs and with microelectrodes to evaluate how the water modifies the reversibility and the diffusion of ions. Water plays an important role in the reversibility of Fe deposition/dissolution being that deposition is less reversible when water is absent. The Fe diffusion is also modified because the Fe ion coordination sphere is strongly affected by the presence or absence of water; the Fe diffusion was also shown to depend on the coordination ability of the cation.
Physiological evidence indicates that the resting tremor of
Parkinson’s disease originates in oscillatory neural activity in the
forebrain, but it is unknown whether that activity is globally
synchronized or consists of parallel, independently oscillating
circuits. In the present study, we used dual microelectrodes to record
tremor-related neuronal activity from eight sites in the internal
segment of the globus pallidus (GPi) from an awake Parkinson’s disease
patient undergoing stereotaxic pallidotomy. We utilized spectral
analysis to evaluate the temporal correlations between multiunit
activity at spatially separated sites and between neural and limb
electromyographic activity. We observed that some GPi neural pairs
oscillated synchronously at the tremor frequency, whereas other neural
pairs oscillated independently. Additionally, we found that GPi
tremor-related activity at a given site could fluctuate between states
of synchronization and independence with respect to upper limb tremor.
Consistent with this finding, some paired recording sites within GPi
showed periods of transient synchronization. These observations support
the hypothesis of independent tremor-generating circuits whose coupling
can fluctuate over time.
Spines are specialized neuronal membrane structures, often localized at sites where synaptic information is relayed from one cell to another in the central nervous system. By electron immunomicroscopy we have found that the mammalian Shaw family potassium channel Kv3.1 is localized on spine-like protrusions, adjacent to postsynaptic membranes of bushy cells in the cochlear nucleus. As direct characterization of the electrophysiological behavior of ion channels in such structures is difficult, we have used Kv3.1-transfected CHO cells to create artificial spine-like membrane compartments. Membrane patches were sucked into microelectrodes to form small, cell-attached vesicles with dimensions comparable to those of the neuronal structures. Currents mediated by the Kv3.1 channel in these vesicles undergo rapid and complete inactivation, in contrast to their noninactivating behavior in whole-cell recordings. This apparent inactivation is caused by the rapid depletion of K+ from the vesicle and the slow refilling of K+ into the vesicle compartment from the bulk cytoplasm. Our data provide evidence that compartmentalized ionic transients can be generated in spine-like membrane structures and support the view that the localization of ion channels in spine-like structures may influence responses to synaptic stimulation.
The recent availability of mice lacking the neuronal form of the
vesicular monoamine transporter 2 (VMAT2) affords the opportunity to
study its roles in storage and release. Carbon fiber microelectrodes
were used to measure individual secretory events of histamine and
5-hydroxytryptamine (5-HT) from VMAT2-expressing mast cells as a model
system for quantal release. VMAT2 is indispensable for monoamine
storage because mast cells from homozygous (VMAT2−/−)
mice, while undergoing granule-cell fusion, do not release monoamines.
Cells from heterozygous animals (VMAT2+/−) secrete lower
amounts of monoamine per granule than cells from wild-type controls.
Investigation of corelease of histamine and 5-HT from granules in
VMAT2+/− cells revealed 5-HT quantal size was reduced more
than that of histamine. Thus, although vesicular transport is the
limiting factor determining quantal size of 5-HT and histamine release,
intragranular association with the heparin matrix also plays a
We previously have demonstrated that the colonic P-ATPase α subunit cDNA encodes an H,K-ATPase when expressed in Xenopus laevis oocytes. Besides its high level of amino acid homology (75%) with the Na,K-ATPase, the colonic H,K-ATPase also shares a common pharmacological profile with Na,K-ATPase, because both are ouabain-sensitive and Sch 28080-insensitive. These features raise the possibility that an unrecognized property of the colonic H,K-ATPase would be Na+ translocation. To test this hypothesis, ion-selective microelectrodes were used to measure the intracellular Na+ activity of X. laevis oocytes expressing various combinations of P-ATPase subunits. The results show that expression in oocytes of the colonic H,K-ATPase affects intracellular Na+ homeostasis in a way similar to the expression of the Bufo marinus Na,K-ATPase; intracellular Na+ activity is lower in oocytes expressing the colonic H,K-ATPase or the B. marinus Na,K-ATPase than in oocytes expressing the gastric H,K-ATPase or a β subunit alone. In oocytes expressing the colonic H,K-ATPase, the decrease in intracellular Na+ activity persists when diffusive Na+ influx is enhanced by functional expression of the amiloride-sensitive epithelial Na+ channel, suggesting that the decrease is related to increased active Na+ efflux. The Na+ decrease depends on the presence of K+ in the external medium and is inhibited by 2 mM ouabain...
We describe an efficient technique for the selective chemical and biological manipulation of the contents of individual cells. This technique is based on the electric-field-induced permeabilization (electroporation) in biological membranes using a low-voltage pulse generator and microelectrodes. A spatially highly focused electric field allows introduction of polar cell-impermeant solutes such as fluorescent dyes, fluorogenic reagents, and DNA into single cells. The high spatial resolution of the technique allows for design of, for example, cellular network constructions in which cells in close contact with each other can be made to possess different biochemical, biophysical, and morphological properties. Fluorescein, and fluo-3 (a calcium-sensitive fluorophore), are electroporated into the soma of cultured single progenitor cells derived from adult rat hippocampus. Fluo-3 also is introduced into individual submicrometer diameter processes of thapsigargin-treated progenitor cells, and a plasmid vector cDNA construct (pRAY 1), expressing the green fluorescent protein, is electroporated into cultured single COS 7 cells. At high electric field strengths, observations of dye-transfer into organelles are proposed.
Nitric oxide (NO) plays a critical role in vascular endothelial
growth factor (VEGF)-induced angiogenesis and vascular
hyperpermeability. However, the relative contribution of different NO
synthase (NOS) isoforms to these processes is not known. Here, we
evaluated the relative contributions of endothelial and inducible NOS
(eNOS and iNOS, respectively) to angiogenesis and permeability of
VEGF-induced angiogenic vessels. The contribution of eNOS was assessed
by using an eNOS-deficient mouse, and iNOS contribution
was assessed by using a selective inhibitor
l-NIL] and an iNOS-deficient mouse.
Angiogenesis was induced by VEGF in type I collagen gels placed in the
mouse cranial window. Angiogenesis, vessel diameter, blood flow rate,
and vascular permeability were proportional to NO levels measured with
microelectrodes: Wild-type (WT) ≥ WT with l-NIL or
eNOS−/− with l-NIL. The role
of NOS in VEGF-induced acute vascular permeability increase in
quiescent vessels also was determined by using eNOS- and
iNOS-deficient mice. VEGF superfusion significantly
increased permeability in both WT and
iNOS−/− mice but not in
eNOS−/− mice. These findings suggest that
eNOS plays a predominant role in VEGF-induced angiogenesis and vascular
Noninvasive, ion-selective vibrating
microelectrodes were used to measure the kinetics of H+,
Ca2+, K+, and Cl− fluxes and the
changes in their concentrations caused by illumination near the
mesophyll and attached epidermis of bean (Vicia faba
L.). These flux measurements were related to light-induced changes in
the plasma membrane potential. The influx of Ca2+ was the
main depolarizing agent in electrical responses to light in the
mesophyll. Changes in the net fluxes of H+, K+,
and Cl− occurred only after a significant delay of about 2
min, whereas light-stimulated influx of Ca2+ began within
the time resolution of our measurements (5 s). In the absence of
H+ flux, light caused an initial quick rise of external pH
near the mesophyll and epidermal tissues. In the mesophyll this fast
alkalinization was followed by slower, oscillatory pH changes (5–15
min); in the epidermis the external pH increased steadily and reached a
plateau 3 min later. We explain the initial alkalinization of the
medium as a result of CO2 uptake by photosynthesizing
tissue, whereas activation of the plasma membrane H+ pump
occurred 1.5 to 2 min later. The epidermal layer seems to be a
substantial barrier for ion fluxes but not for CO2
diffusion into the leaf.
In an earlier paper we showed that in
fully developed barley (Hordeum vulgare L.) root
epidermal cells a decrease in cytosolic K+ was associated
with an acidification of the cytosol (D.J. Walker, R.A. Leigh, A.J.
Miller  Proc Natl Acad Sci USA 93: 10510–10514). To show that
these changes in cytosolic ion concentrations contributed to the
decreased growth of K+-starved roots, we first measured
whether similar changes occurred in cells of the growing zone.
Triple-barreled ion-selective microelectrodes were used to measure
cytosolic K+ activity and pH in cells 0.5 to 1.0 mm from
the root tip. In plants growing from 7 to 21 d after germination
under K+-replete conditions, the mean values did not change
significantly, with values ranging from 80 to 84 mm for
K+ and 7.3 to 7.4 for pH. However, in
K+-starved plants (external [K+], 2
μm), the mean cytosolic K+ activity and pH
had declined to 44 mm and 7.0, respectively, after 14
d. For whole roots, sap osmolality was always lower in
K+-starved than in K+-replete plants, whereas
elongation rate and dry matter accumulation were significantly
decreased after 14 and 16 d of K+ starvation. The rate
of protein synthesis in root tips did not change for
K+-replete plants but declined significantly with age in
K+-starved plants. Butyrate treatment decreased cytosolic
pH and diminished the rate of protein synthesis in
K+-replete roots. Procaine treatment of
K+-starved roots gave an alkalinization of the cytosol and
increased protein synthesis rate. These results show that changes in
both cytosolic pH and K+ can be significant factors in
inhibiting protein synthesis and root growth during K+
Plant cells contain two major pools of K+, one in the vacuole and one in the cytosol. The behavior of K+ concentrations in these pools is fundamental to understanding the way this nutrient affects plant growth. Triple-barreled microelectrodes have been used to obtain the first fully quantitative measurements of the changes in K+ activity (aK) in the vacuole and cytosol of barley (Hordeum vulgare L.) root cells grown in different K+ concentrations. The electrodes incorporate a pH-selective barrel allowing each measurement to be assigned to either the cytosol or vacuole. The measurements revealed that vacuolar aK declined linearly with decreases in tissue K+ concentration, whereas cytosolic aK initially remained constant in both epidermal and cortical cells but then declined at different rates in each cell type. An unexpected finding was that cytoplasmic pH declined in parallel with cytosolic aK, but acidification of the cytosol with butyrate did not reveal any short-term link between these two parameters. These measurements show the very different responses of the vacuolar and cytosolic K+ pools to changes in K+ availability and also show that cytosolic K+ homeostasis differs quantitatively in different cell types. The data have been used in thermodynamic calculations to predict the need for...
As a measure of dynamical structure, short-term fluctuations of coherence between 0.3 and 100 Hz in the electroencephalogram (EEG) of humans were studied from recordings made by chronic subdural macroelectrodes 5-10 mm apart, on temporal, frontal, and parietal lobes, and from intracranial probes deep in the temporal lobe, including the hippocampus, during sleep, alert, and seizure states. The time series of coherence between adjacent sites calculated every second or less often varies widely in stability over time; sometimes it is stable for half a minute or more. Within 2-min samples, coherence commonly fluctuates by a factor up to 2-3, in all bands, within the time scale of seconds to tens of seconds. The power spectrum of the time series of these fluctuations is broad, extending to 0.02 Hz or slower, and is weighted toward the slower frequencies; little power is faster than 0.5 Hz. Some records show conspicuous swings with a preferred duration of 5-15s, either irregularly or quasirhythmically with a broad peak around 0.1 Hz. Periodicity is not statistically significant in most records. In our sampling, we have not found a consistent difference between lobes of the brain, subdural and depth electrodes, or sleeping and waking states. Seizures generally raise the mean coherence in all frequencies and may reduce the fluctuations by a ceiling effect. The coherence time series of different bands is positively correlated (0.45 overall); significant nonindependence extends for at least two octaves. Coherence fluctuations are quite local; the time series of adjacent electrodes is correlated with that of the nearest neighbor pairs (10 mm) to a coefficient averaging approximately 0.4...
Solid tumors have been observed to develop an acidic extracellular environment, which is believed to occur as a result of lactic acid accumulation produced during aerobic and anaerobic glycolysis. Experiments using glycolysis-deficient ras-transfected Chinese hamster lung fibroblasts have been performed to test the hypothesis that lactic acid production within solid tumors is responsible for the development of tumor acidity. The variant cells have defects in glucose transport and in the glycolytic enzyme phosphoglucose isomerase with 1% activity compared to parental cells. Consequently, the in vitro rate of lactic acid production by variant cells was < 4% compared to parental cells. An in vitro correlation between lactic acid production and acidification of exposure medium was observed for parental and variant cells. Implantation of both cell lines into nude mice led to tumors with minimal difference in growth rate. As expected, variant cells died when exposed to hypoxic conditions in culture, and parental tumors were observed to have a larger fraction of cells resistant to radiation due to hypoxia (27%) than variant tumors (2%). Using pH microelectrodes, parental (n = 12) and variant (n = 12) tumors were observed to have extracellular pH (pHe) values of 6.65 +/- 0.07 and 6.78 +/- 0.04 (mean +/- SE...
Electrogenic cotransport of Na+ with HCO3- has been reported in numerous tissues. It has always been shown with a net transfer of negative charge, but in some situations achieves net outward transport of both species with a stoichiometry of at least three HCO3- ions per Na+ ion (3:1), and in other situations achieves net inward transport of both species and has a stoichiometry of at most two HCO3- ions per Na+ ion (2:1). This suggests either that there may be more than one protein responsible for Na(+)-HCO3- cotransport in different tissues or that if there is a single protein, its stoichiometry may differ depending on the orientation of net transport. The present study, using conventional or double-barreled ion-selective microelectrodes to follow basolateral membrane potential and intracellular pH or Na+ activity in Necturus proximal convoluted tubule in vivo, shows that the orientation of the basolateral Na(+)-HCO3- cotransporter can be reversed upon switching from a perfusate simulating normal acid-base conditions to one that imposes peritubular isohydric hypercapnia. Moreover, accompanying the reversal of orientation is a change of apparent stoichiometry from 3:1 to 2:1. Given that the observed change of orientation and accompanying change of apparent stoichiometry occur within seconds and in the same preparation...