Evaluation of commercially available electrodes and gels for recording of slow EEG potentials.

OBJECTIVE: To test the applicability of different types of commercially available electrodes and electrode gels or pastes for recording of slow EEG potentials. METHODS: Experiments were carried out on six types of reusable electrodes (silver, tin and gold cup electrodes, sintered silver-silver chloride (Ag|AgCl), platinum, stainless steel), six disposable Ag|AgCl electrode models, and nine gels or pastes. We studied the parameters, which are critical in slow-potential recording, such as polarization, initial and long-term stability and low-frequency noise. RESULTS: The best results were obtained with the reusable sintered Ag|AgCl electrodes. The six disposable Ag|AgCl electrode models also proved to have appropriate electrical properties. Other types of reusable electrodes suffered from diverse degrees of polarization, baseline drift, low-frequency noise, high resistance, and changes in properties due to wear and tear. Seven out of nine gels or pastes contained a significant amount of chloride, which is a prerequisite for DC stability of Ag|AgCl electrodes, whereas the absolute concentration of chloride had little effect. CONCLUSIONS: Direct current (DC) coupled recording of EEG is critically dependent on the choice of electrode and gel. SIGNIFICANCE: Our results provide rigorous criteria for choosing DC-stable electrodes and gels for DC-coupled or long time-constant AC-coupled recordings of slow EEG potentials.

Infraslow oscillations modulate excitability and interictal epileptic activity in the human cortex during sleep.

Human cortical activity has been intensively examined at frequencies ranging from 0.5 Hz to several hundred Hz. Recent studies have, however, reported also infraslow fluctuations in neuronal population activity, magnitude of electroencephalographic oscillations, discrete sleep events, as well as in the occurrence of interictal events. Here we use direct current electroencephalography to demonstrate large-scale infraslow oscillations in the human cortex at frequencies ranging from 0.02 to 0.2 Hz. These oscillations, which are not detectable in conventional electroencephalography because of its limited recording bandwidth (typical lower limit 0.5 Hz), were observed in widespread cortical regions. Notably, the infraslow oscillations were strongly synchronized with faster activities, as well as with the interictal epileptic events and K complexes. Our findings suggest that the infraslow oscillations represent a slow, cyclic modulation of cortical gross excitability, providing also a putative mechanism for the as yet enigmatic aggravation of epileptic activity during sleep.

Topography and elimination of slow EEG responses related to tongue movements.

This study aimed to characterize the topography and mechanism of slow EEG potentials related to tongue movement (TMP), and to devise a method for their elimination. Eight adult subjects were recorded with 8-channel direct current (DC) EEG (n = 5) or with a 256-channel, dense array EEG (n = 4). Subjects were requested to push their tongue toward the incisors in a closed mouth. We examined the time course and topography of the ensuing TMP. The underlying mechanism and elimination of TMP were studied by electrical isolation of the tongue with a latex sheet, and by short-circuiting of tongue to gingival pouch. Forward movement of the tongue caused global changes in scalp potentials, with frontal areas more positive, and the strongest gradients between the mastoid region and other scalp areas. Scalp current source density was highest near ear canals and orbital fossae. Electrical isolation of the tongue tip with a latex sheet resulted in a near-complete elimination of TMPs, while short-circuiting resulted in high amplitude responses, suggesting that TMPs are caused by a change in the geometry of conductive pathways. Our results indicate that significant scalp potentials are caused by even modest tongue movements, which may occur subconsciously during various cognitive tasks. The topography and small amplitude of TMPs make their off-line rejection difficult, while an isolation method of the kind used here may substantially diminish TMP artifacts in studies on slow cognitive potentials.

Scalp-recorded slow EEG responses generated in response to hemodynamic changes in the human brain.

OBJECTIVE: To study whether hemodynamic changes in human brain generate scalp-EEG responses. METHODS: Direct current EEG (DC-EEG) was recorded from 12 subjects during 5 non-invasive manipulations that affect intracranial hemodynamics by different mechanisms: bilateral jugular vein compression (JVC), head-up tilt (HUT), head-down tilt (HDT), Valsalva maneuver (VM), and Mueller maneuver (MM). DC shifts were compared to changes in cerebral blood volume (CBV) measured by near-infrared spectroscopy (NIRS). RESULTS: DC shifts were observed during all manipulations with highest amplitudes (up to 250 microV) at the midline electrodes, and the most pronounced changes (up to 15 microV/cm) in the DC voltage gradient around vertex. In spite of inter-individual variation in both amplitude and polarity, the DC shifts were consistent and reproducible for each subject and they showed a clear temporal correlation with changes in CBV. CONCLUSIONS: Our results indicate that hemodynamic changes in human brain are associated with marked DC shifts that cannot be accounted for by intracortical neuronal or glial currents. Instead, the data are consistent with a non-neuronal generator mechanism that is associated with the blood-brain barrier. SIGNIFICANCE: These findings have direct implications for mechanistic interpretation of slow EEG responses in various experimental paradigms.

Very slow EEG responses lateralize temporal lobe seizures: an evaluation of non-invasive DC-EEG.

BACKGROUND: This study tested the idea that very slow EEG responses (direct current [DC] potential shifts) could be detected noninvasively during temporal lobe (TL) seizures, and that these shifts give lateralizing information consistent with that obtained by other methods. METHODS: Seven patients with TL epilepsy (TLE) were recorded with scalp DC-EEG technique at bedside. All recordings were performed simultaneously with conventional EEG (scalp in five, and intracranially in two; two patients with scalp recordings were recorded intracranially later). Seizures in five patients originated in the mesial TL. Ictal DC shifts were evaluated by comparing them to the temporal evolution of ictal discharges, and by comparing the laterality of these shifts to the side of seizure onset defined by routine EEG and other presurgical diagnostic tests. RESULTS: All seizures (35/35) were associated with negative DC shifts at temporal derivations (30 to 150 micro V relative to vertex), beginning at the electrical seizure onset, and lasting for the whole seizure. In eight seizures (five patients) with documented mesial TL onset, the polarity of the DC shift was initially positive followed by a negative one after lateral spread of seizure activity. In all cases, the side of the EEG shift agreed with other diagnostic tests, and, at times, was more clearly lateralized than the conventional scalp EEG. CONCLUSIONS: DC-EEG recordings are practical and achievable at the bedside. Ictal DC shifts are consistently observed in scalp recordings in TL seizures, and reliably lateralize them. This method may hold promise in reducing the need for invasive monitoring in patients with TLE where other noninvasive tests are equivocal.

DC-EEG discloses prominent, very slow activity patterns during sleep in preterm infants.

OBJECTIVES: The objective of this study is to test the hypothesis that the immature human brain exhibits slow electrical activity that is not detected by conventional (i.e. high-pass filtered) electroencephalography (EEG). METHODS: Six healthy preterm infants (conceptional age 33-37 weeks) were recorded bedside with direct current (DC) EEG during sleep. Epochs with quiet sleep were selected to study the delta frequency bursts during discontinuous EEG patterns (trace discontinu or trace alternant), and we compared the waveforms obtained without filtering (i.e. genuine DC-EEG) to those seen after high pass filtering of the same traces. RESULTS: In all infants, DC-EEG demonstrated that the typical delta frequency bursts are consistently embedded in very large amplitude (200-700 microV) and long lasting (1-5s) occipitally negative transients, which are not seen in conventional EEG. CONCLUSIONS AND SIGNIFICANCE: Our study demonstrates that (i) the most prominent spontaneous EEG activity of a sleeping preterm infant consists of very slow, large amplitude transients, and (ii) the most salient features of these transients are not seen in conventional EEG. Proper recording of this type of brain activity by DC-EEG provides a novel way for non-invasive assessment of neonatal brain function.

Extracellular carbonic anhydrase activity facilitates lactic acid transport in rat skeletal muscle fibres.

1. In skeletal muscle an extracellular sarcolemmal carbonic anhydrase (CA) has been demonstrated. We speculate that this CA accelerates the interstitial CO2/HCO3- buffer system so that H+ ions can be rapidly delivered or buffered in the interstitial fluid. Because > 80 % of the lactate which crosses the sarcolemmal membrane is transported by the H+-lactate cotransporter, we examined the contributions of extracellular and intracellular CA to lactic acid transport, using ion-selective microelectrodes for measurements of intracellular pH (pHi) and fibre surface pH (pHs) in rat extensor digitorum longus (EDL) and soleus fibres. 2. Muscle fibres were exposed to 20 mM sodium lactate in the absence and presence of the CA inhibitors benzolamide (BZ), acetazolamide (AZ), chlorzolamide (CZ) and ethoxzolamide (EZ). The initial slopes (dpHs/dt, dpHi/dt) and the amplitudes (DeltapHs, DeltapHi) of pH changes were quantified. From dpHi/dt, DeltapHi and the total buffer factor (BFtot) the lactate fluxes (mM min-1) and intracellular lactate concentrations ([lactate]i) were estimated. 3. BFtot was obtained as the sum of the non-HCO3- buffer factor (BFnon-HCO3) and the HCO3- buffer factor (BFHCO3). BFnon-HCO3 was 35 +/- 4 mM pH-1 for the EDL (n = 14) and 86 /- 16 mM pH-1 for the soleus (n = 14). 4. In soleus, 10 mM cinnamate inhibited lactate influx by 44 % and efflux by 30 %; in EDL, it inhibited lactate influx by 37 % and efflux by 20 %. Cinnamate decreased [lactate]i, in soleus by 36 % and in EDL by 45 %. In soleus, 1 mM DIDS reduced lactate influx by 18 % and efflux by 16 %. In EDL, DIDS lowered the influx by 27 % but had almost no effect on efflux. DIDS reduced [lactate]i by 20 % in soleus and by 26 % in EDL. 5. BZ (0.01 mM) and AZ (0.1 mM), which inhibit only the extracellular sarcolemmal CA, led to a significant increase in dpHs/dt and pHs by about 40 %-150 % in soleus and EDL. BZ and AZ inhibited the influx and efflux of lactate by 25 %-50 % and reduced [lactate]i by about 40 %. The membrane-permeable CA inhibitors CZ (0.5 mM) and EZ (0.1 mM), which inhibit the extracellular as well as the intracellular CAs, exerted no greater effects than the poorly permeable inhibitors BZ and AZ did. 6. In soleus, 10 mM cinnamate inhibited the lactate influx by 47 %. Addition of 0.01 mM BZ led to a further inhibition by only 10 %. BZ alone reduced the influx by 37 %. 7. BZ (0.01 mM) had no influence on the Km value of the lactate transport, but led to a decrease in maximal transport rate (Vmax). In EDL, BZ reduced Vmax by 50 % and in soleus by about 25 %. 8. We conclude that the extracellular sarcolemmal CA plays an important role in lactic acid transport, while internal CA has no effect, a difference most likely attributable to the high internal vs. low extracellular BF(non-HCO3). The fact that the effects of cinnamate and BZ are not additive indicates that the two inhibitors act at distinct sites on the same transport pathway for lactic acid.

Arterial responses in vitro and plasma digoxin immunoreactivity after losartan and enalapril treatments in experimental hypertension.

Treatment with the angiotensin-converting enzyme inhibitor, quinapril, has been shown to normalize increased dihydropyridine sensitivity and impaired potassium relaxation, characteristic features of arterial smooth muscle in spontaneously hypertensive rats, and also reduce the concentration of plasma digoxin-like immunoreactivity in these animals. However, whether angiotensin II receptor blocker therapy can beneficially influence these variables is not known. Therefore, we compared the effects of 10-week losartan and enalapril treatments (15 and 4 mg/kg/day, respectively) on functional responses of mesenteric arterial rings in spontaneously hypertensive rats and Wistar-Kyoto rats. Both losartan and enalapril normalized blood pressure, cardiac mass, and media to lumen ratio without significantly changing the media cross-sectional area in the mesenteric artery of spontaneously hypertensive rats (i.e. induced outward remodelling). The inhibitory effect of the calcium entry blocker nifedipine on calcium-evoked contractions was similar and less marked in arterial preparations from Wistar-Kyoto rats and losartan- and enalapril-treated spontaneously hypertensive rats than in those from untreated spontaneously hypertensive rats. Furthermore, the relaxations of arterial rings induced by the return of potassium to the organ bath (upon precontractions elicited by potassium-free solution) were used to evaluate the function of vascular Na+,K+-ATPase. The rate of potassium relaxation was faster in losartan- and enalapril-treated spontaneously hypertensive rats and all Wistar-Kyoto groups than in untreated spontaneously hypertensive rats, and the response was effectively inhibited by the sodium pump inhibitor ouabain. Both treatments especially augmented the ouabain-sensitive part of the potassium-relaxation in spontaneously hypertensive rats, indicating the involvement of the sodium pump in this response. However, no significant changes in plasma digoxin-like immunoreactivity were observed. In conclusion, the outward remodelling following long-term AT1-receptor blockade and angiotensin-converting enzyme inhibition in spontaneously hypertensive rats was associated with normalization of the increased dihydropyridine sensitivity of arteries. Both losartan and enalapril treatments also augmented arterial potassium relaxation in spontaneously hypertensive rats, suggesting enhanced function of Na+,K+-ATPase, but this effect could not be attributed to changes in circulating sodium pump inhibitor concentration.

Pharmacological isolation of the synaptic and nonsynaptic components of the GABA-mediated biphasic response in rat CA1 hippocampal pyramidal cells.

High-frequency stimulation (HFS) applied to stratum radiatum of a rat hippocampal slice in the presence of ionotropic glutamate receptor antagonists evokes a biphasic GABA(A) receptor-dependent response in CA1 pyramidal neurons, with a brief hyperpolarizing IPSP (hIPSP) followed by a long-lasting depolarization. We show now that it is possible to pharmacologically separate the hIPSP and late depolarization from one another. In neurons intracellularly perfused for 1-2 hr with F(-) as the major anion and no ATP, the hIPSP (and the corresponding current, hIPSC) evoked by HFS was blocked, whereas neither the late depolarization nor its underlying current was attenuated. In contrast, internal perfusion with a high concentration (5 mM) of the impermeant lidocaine derivative QX-314 selectively abolished the depolarizing component of the biphasic response and also strongly reduced depolarizations evoked by extracellular microinjection of K(+). Bath application of quinine (0. 2-0.5 mM) or quinidine (0.1 mM) resulted in a pronounced inhibition of the HFS-induced extracellular K(+) concentration ([K(+)](o)) transient but not of the bicarbonate-dependent alkaline shift in extracellular pH. The attenuation of the [K(+)](o) transient was closely paralleled by a suppression of the HFS-evoked depolarization but not of the hIPSP. Quini(di)ne did not affect depolarizations induced by exogenous K(+) either. These data provide direct pharmacological evidence for the view that the HFS-induced biphasic response of the pyramidal neuron is composed of mechanistically distinct components: a direct GABA(A) receptor-mediated phase, which is followed by a slow, nonsynaptic [K(+)](o)-mediated depolarization. The bicarbonate-dependent, activity-induced [K(+)](o) transient can be blocked by quini(di)ne, whereas its depolarizing action in the pyramidal neuron is inhibited by internal QX-314. The presence of fundamentally distinct components in GABA(A) receptor-mediated actions evoked by HFS calls for further investigations of their functional role(s) in standard experimental maneuvers, such as those used in studies of synaptic plasticity and induction of gamma oscillations.

Effects of voluntary hyperventilation on cortical sensory responses. Electroencephalographic and magnetoencephalographic studies.

It is well established that voluntary hyperventilation (HV) slows down electroencephalographic (EEG) rhythms. Little information is available, however, on the effects of HV on cortical responses elicited by sensory stimulation. In the present study, we recorded auditory evoked potentials (AEPs) and magnetic fields (AEFs), and somatosensory evoked magnetic fields (SEFs) from healthy subjects before, during, and after a 3- to 5-min period of voluntary HV. The effectiveness of HV was verified by measuring the end-tidal CO2 levels. Long-latency (100-200 ms) AEPs and long-latency AEFs originating at the supratemporal auditory cortex, as well as long-latency SEFs from the primary somatosensory cortex (SI) and from the opercular somatosensory cortex (OC), were all reduced during HV. The short-latency SEFs from SI were clearly less modified, there being, however, a slight reduction of the earliest cortical excitatory response, the N20m deflection. A middle-latency SEF deflection from SI at about 60 ms (P60 m) was slightly increased. For AEFs and SEFs, the center-of-gravity locations of the activated neuronal populations were not changed during HV. All amplitude changes returned to baseline levels within 10 min after the end of HV. The AEPs were not altered when the subjects breathed 5% CO2 in air in a hyperventilation-like manner, which prevented the development of hypocapnia. We conclude that moderate HV suppresses long-latency evoked responses from the primary projection cortices, while the early responses are less reduced. The reduction of long-latency responses is probably mediated by hypocapnia rather than by other nonspecific effects of HV. It is suggested that increased neuronal excitability caused by HV-induced hypocapnia leads to spontaneous and/or asynchronous firing of cortical neurones, which in turn reduces stimulus-locked synaptic events.

The K+/Cl- co-transporter KCC2 renders GABA hyperpolarizing during neuronal maturation.

GABA (gamma-aminobutyric acid) is the main inhibitory transmitter in the adult brain, and it exerts its fast hyperpolarizing effect through activation of anion (predominantly Cl-)-permeant GABA(A) receptors. However, during early neuronal development, GABA(A)-receptor-mediated responses are often depolarizing, which may be a key factor in the control of several Ca2+-dependent developmental phenomena, including neuronal proliferation, migration and targeting. To date, however, the molecular mechanism underlying this shift in neuronal electrophysiological phenotype is unknown. Here we show that, in pyramidal neurons of the rat hippocampus, the ontogenetic change in GABA(A)-mediated responses from depolarizing to hyperpolarizing is coupled to a developmental induction of the expression of the neuronal (Cl-)-extruding K+/Cl- co-transporter, KCC2. Antisense oligonucleotide inhibition of KCC2 expression produces a marked positive shift in the reversal potential of GABAA responses in functionally mature hippocampal pyramidal neurons. These data support the conclusion that KCC2 is the main Cl- extruder to promote fast hyperpolarizing postsynaptic inhibition in the brain.

Long-lasting GABA-mediated depolarization evoked by high-frequency stimulation in pyramidal neurons of rat hippocampal slice is attributable to a network-driven, bicarbonate-dependent K+ transient.

Biphasic GABAA-mediated postsynaptic responses can be readily evoked in CA1 pyramidal neurons of rat hippocampal slices by high-frequency stimulus (HFS) trains in the presence of ionotropic glutamate receptor antagonists. In the present experiments with sharp microelectrodes, whole-cell techniques, and K+-selective microelectrodes, an HFS train (40 pulses at 100 Hz) applied in stratum radiatum close to the recording site evoked a brief hyperpolarizing IPSP (hIPSP), which turned into a prolonged (2-3 sec) depolarization (GABA-mediated depolarizing postsynaptic potential; GDPSP). The I-V relationships of the postsynaptic currents (hIPSC and GDPSC) had distinct characteristics: the hIPSC and the early GDPSC showed outward rectification, whereas the late GDPSC was reduced with positive voltage steps to zero or beyond (inward rectification), but often no clear reversal was seen. That two distinct currents contribute to the generation of the GDPSP was also evident from the finding that a second HFS train at peak or late GDPSP induced a prompt GABAA-mediated hyperpolarization. The GDPSP/C was dependent on the availability of bicarbonate, but not on interstitial or intrapyramidal carbonic anhydrase activity. The HFS train evoked a rapid GABAA-mediated bicarbonate-dependent increase in the extracellular K+ concentration ([K+]o), and the GDPSP followed the K+ transient in a sub-Nernstian manner. The spatial and pharmacological characteristics of the [K+]o shift indicated that it is generated by a local network of GABAergic interneurons. The brief ascending phase of the GDPSP is linked to a K+-dependent accumulation of intracellular Cl-. Thereafter, a nonsynaptic mechanism, a direct depolarizing effect of the [K+]o shift, is responsible for the most conspicuous characteristics of the GDPSP: its large amplitude and prolonged duration.

Interstitial PCO2 and pH, and their role as chemostimulants in the isolated respiratory network of neonatal rats.

1. CO2-H(+)-sensitive microelectrodes were used for simultaneous measurements of the partial pressure of CO2 (PCO2) and extracellular pH (pHo) in the ventral respiratory group (VRG) of the isolated brainstem-spinal cord of neonatal rats. Some of the data were analysed using diffusion equations. 2. With increasing recording depth within the boundaries of the VRG (300-600 microns below the tissue surface), PCO2 increased from 77 to 95 mmHg and pHo fell from 7.0 to 6.8 at steady state in standard saline equilibrated with 5% CO2 and 95% O2. 3. Elevating bath CO2 from 5 to 10-12.5% produced a mean increase in PCO2 of 18 mmHg, a fall in pHo of 0.13 pH units, and a 50-250% increase in the frequency of respiration-related spinal (C2) nerve bursts. Similar effects on C2 activity and pHo were observed upon lowering bath [HCO3-] from 25 to 10 mM, leading to a mean decrease in PCO2 of 4.4 mmHg in the VRG. 4. Raising bath [HCO3-] to 50 mM produced a substantial frequency decrease, a rise in pHo of 0.24 pH units and an elevation in PCO2 of 9.3 mmHg. C2 activity was not profoundly affected upon doubling the CO2-HCO3- content, leading to a mean increase in pHo of 0.13 pH units and elevation of PCO2 by 30 mmHg. 5. In a CO2-HCO3(-)-free, Hepes-buffered solution, PCO2 decreased to 18 mmHg in the VRG and pHo fell by 0.15 pH units with no major effect on rhythmic activity. Subsequent anoxic exposure for more than 15 min produced a further fall in PCO2 to below 1 mmHg, a decrease in pHo of 0.55 pH units, and blockade of respiration-related activity. In three out of the six preparations tested, C2 activity could be restored by reapplication of CO2-HCO3- in the absence of O2. 6. C2 activity persisted at a reduced frequency, even up to 30 min, during anoxia in the CO2-HCO(-)-buffered saline,leading to an elevation in PCO2 of 15 mmHg and a fall in pHo of 0.18 pH units. 7. The diffusion coefficient of CO2 in the tissue was found to be equal to that in saline. Two mean estimates for anoxic tissue of the function lambda 2/ alpha of tortuosity (lambda) and extracellular volume fraction (alpha), affecting extracellular diffusion of bicarbonate, were 4.7 and 4.1. The mean rate of acid production by anoxic tissue was 1.1 mequiv 1-1 min-1. 8. The results suggest that extracellular H+ is the primary stimulating factor in central chemosensitivity, which may often mask the less evident effects of CO2. A model of diffusion of acid equivalents in brain tissue is proposed.

Pharmacological characterization of extracellular pH transients evoked by selective synaptic and exogenous activation of AMPA, NMDA, and GABAA receptors in the rat hippocampal slice.

1. Inhibitors of extracellular carbonic anhydrase (CAo) offer much promise as diagnostic tools in the study of the synaptic basis of activity-induced alkaline transients in the brain. However, most of the present information related to the effects of CAo blockers in nervous tissue comes from experiments that involve simultaneous synaptic activation of various types of postsynaptic receptor channels. In the present work, double-barreled H(+)-selective microelectrodes were used to study alkaline shifts in extracellular pH (pHo) evoked by selective synaptic and pharmacological activation of glutamate and gamma-aminobutyric acid (GABA) receptors in the CA1 cell body layer in rat hippocampal slices. Inhibition of CAo was achieved with the use of the poorly permeant carbonic anhydrase inhibitor, benzolamide (10 microM; applied in the bath solution) or the impermeant macromolecular inhibitor, prontosil-dextran 5000 (PD 5000; applied in microdrops). 2. Alkaline transients that were exclusively attributable to synaptic activation of glutamate receptors were induced by stimulation of Schaffer collaterals in the presence of picrotoxin (PiTX, 100 microM). An enhancement by the CAo inhibitors of these alkaline transients took place at all stimulus frequencies (5-200 Hz) and stimulus train durations (0.5-20 s) examined. 3. Inhibition of CAo enhanced the alkaline transients evoked by selective synaptic activation of alpha-amino-3-hydroxy-5-methyli-oxazolate- 4-propionic acid (AMPA)/kainate receptors in experiments involving stimulation of Schaffer collaterals in the simultaneous presence of PiTX and D-2-amino-5-phosphonopentoate (AP5, 40-80 microM). 4. Alkaline shifts evoked by selective synaptic activation of N-methyl-D-aspartate (NMDA) receptors were enhanced after inhibition of CAo as seen in experiments where Schaffer collaterals were stimulated in the simultaneous presence of PiTX and 6-cyano-2,3-dihydroxy-7-nitroquinoxaline (CNQX, 20-40 microM) in an Mg(2+)-free solution. 5. Benzolamide and PD 5000 also enhanced the alkaline shifts seen upon pressure injection of glutamate, AMPA, or NMDA. The glutamate-induced alkaline shifts were inhibited by AP5+CNQX, suggesting that uptake of glutamate did not significantly contribute to their generation. 6. Stimuli applied at 5-10 Hz in stratum radiatum close (within 0.5 mm) to the recording site evoked alkaline shifts that were blocked by CNQX plus AP5. In the continuous presence of the two glutamate antagonists, PiTX-sensitive alkaline transients were observed in response to brief high-frequency (20-100 Hz) trains consisting of 100 stimuli. Upon application of pentobarbital sodium (100 microM), these apparently monosynaptically evoked GABAA receptor-mediated alkaline transients were evident also at low stimulation frequencies (5-10 Hz).(ABSTRACT TRUNCATED AT 400 WORDS)

Relative contributions of excitatory and inhibitory neuronal activity to alkaline transients evoked by stimulation of Schaffer collaterals in the rat hippocampal slice.

1. The neuronal basis of alkaline shifts in extracellular pH (pHo) evoked by stimulation of Schaffer collaterals was studied by means of double-barreled H(+)-selective microelectrodes in the area CA1 of rat hippocampal slices. 2. Alkaline transients in stratum pyramidale evoked by stimulation at a low frequency (5-10 Hz) were enhanced by pentobarbital sodium (100 microM). In the absence of the drug, inhibition of extracellular carbonic anhydrase (CAo) by benzolamide or by prontosildextran 5000 (PD 5000) resulted in an increase in the alkaline shifts. In contrast to this, alkaloses evoked by low-frequency stimulation in the presence of pentobarbital were attenuated by a subsequent inhibition of CAo. 3. Blockade of gamma-aminobutyric acid-A (GABAA) receptors with picrotoxin (PiTX; 100 microM) resulted in an enhancement of alkaline transients in s. pyramidale evoked by low-frequency stimulation (10 Hz) but suppressed alkaline shifts evoked by brief high-frequency (1 s, 100 Hz) trains of stimuli. 4. Application of trains of stimuli consisting of a constant number of pulses (50 or 100) revealed a striking dependence of the effect of benzolamide on stimulation frequency (10-200 Hz) in s. pyramidale: the enhancement of the alkaloses seen upon inhibition of CAo became progressively smaller with an increase in frequency, and at 100-200 Hz benzolamide produced a suppression or a complete block of the alkaline transients. However, alkaline transients evoked with the use of a constant train duration (5 s) were enhanced by benzolamide at all stimulation frequencies examined (5-200 Hz).(ABSTRACT TRUNCATED AT 250 WORDS)

Extracellular alkaline transients mediated by glutamate receptors in the rat hippocampal slice are not due to a proton conductance.

1. The ionic mechanism of extracellular alkaline transients mediated by alpha-amino-3-hydroxy-5-methylisoxazolate-4-propionic acid (AMPA) receptor channels was studied in the rat hippocampal slice (area CA1) by means of H(+)-, Ca(2+)- and Na(+)-selective microelectrodes. 2. Alkaline transients mediated by selective synaptic activation of AMPA receptors were coupled to a transient fall in extracellular Ca2+ ([Ca2+]o). 3. Removal of [Ca2+]o blocked the alkaline transient evoked by microinjection of AMPA, but had little effect on the simultaneous tetrodotoxin-resistant fall in extracellular Na+ ([Na+]o). 4. Alkaline transients evoked by microinjection of gamma-aminobutyric acid (GABA) were not affected by removal of [Ca2+]o. GABA had no effect on [Na+]o. 5. The present results indicate that activity-induced glutamatergic alkaline transients in the hippocampal slice are due to an influx of Ca2+, and not to a conductive movement of H+ ions across glutamate-gated cation channels.

Interstitial PCO2 and pH in rat hippocampal slices measured by means of a novel fast CO2/H(+)-sensitive microelectrode based on a PVC-gelled membrane.

We describe here the construction and properties of a double-barrelled microelectrode (tip diameter 4-10 microns) which permits simultaneous measurements of PCO2 and pH, and which has a 90% response time of only one or a few seconds for a step change in PCO2. The fast response of the CO2-sensitive barrel is due to (i) the use of a PVC-gelled (tridodecylamine-containing) membrane solution which enables the construction of extremely short (> or = 4 microns), yet mechanically stable, membrane columns, and (ii) the presence of carbonic anhydrase in the filling solution. Recordings made in the pyramidal layer of area CA1 in rat hippocampal slices showed that the deviation in the acid direction of the basal interstitial pH (pH0) from that of the perfusion solution was attributable to a higher PCO2 level within the tissue. Most of the late acid shift evoked by stimulation of the Schaffer collaterals (5- to 20-s trains at 10 Hz) could also be explained on the basis of an accumulation of interstitial CO2 at a constant HCO3- concentration. This conclusion was supported by the finding that inhibition of extracellular carbonic anhydrase activity by 10 microM benzolamide completely abolished the activity-induced fall in pH0, but not the increase in PCO2. The initial stimulus-induced alkalosis was accompanied by a slight decrease in PCO2 only, implying a parallel increase in the interstitial HCO3- concentration. Benzolamide produced a dramatic enhancement of the early alkaline shift as well as of the simultaneous fall in PCO2. The latter effect of the drug unmasks a cellular CO2 sink that is induced by neuronal activity.

The role of bicarbonate in GABAA receptor-mediated IPSPs of rat neocortical neurones.

1. The ionic mechanism underlying the fast, GABAA receptor-mediated inhibitory postsynaptic potential (IPSPA) was examined in rat neocortical neurones using intracellular recording techniques. Synaptic responses were evoked by orthodromic stimulation applied to the subcortical white matter or to the pial surface. All experiments were carried out at a constant extracellular Cl- concentration. 2. The resting membrane potential was -76.2 +/- 1.0 mV (mean +/- S.E.M., n = 32) and in most cells IPSPA was depolarizing. The reversal potential of IPSPA (EIPSP-A) was -70.2 +/- 0.9 mV (n = 32) and that of a more slowly developing hyperpolarizing response (IPSPB) was -91.4 +/- 1.3 mV (n = 28). 3. An examination of the temporal relationships between excitatory postsynaptic potentials (EPSPs) and IPSPAs in different cells suggested that, despite partial overlap of these responses, EPSPs had little influence on the measured values of EIPSP-A. 4. Application of 20 mM trimethylamine (TriMA), a membrane-permeant weak base which is expected to produce a rise in pHi (and hence in intracellular HCO3-), induced a reversible positive shift in EIPSP-A of up to +9.0 mV (mean + 4.2 mV) at an extracellular pH (pHo) of 7.4. In some experiments, the shift in reversal potential was associated with a change in the polarity of IPSPA from hyperpolarizing to depolarizing. 5. Application of 20 mM lactate (a membrane-permeant weak acid which is expected to produce a fall in pHi and hence in intracellular HCO3-) at pHo 7.0 produced a hyperpolarizing shift in EIPS-A of up to -7.5 mV (mean -5.6 mV). In some experiments, exposure to lactate changed the polarity of IPSPA from depolarizing to hyperpolarizing. 6. Changes in pHo from 7.4 to 7.0 reduced the effect of TriMA and augmented that of lactate on EIPSP-A, as could be expected on the basis of the pHo-dependent change in the fraction of membrane permeable non-charged weak base or acid. 7. Under control conditions, a change in pHo from 7.4 to 7.0 produced a slight positive shift (< +2 mV) in EIPSP-A. In the presence of TriMA, a similar change in pHo gave rise to a negative shift (-1.8 to -2.7 mV). 8. The results obtained indicate that HCO3- ions contribute significantly to the IPSPA, thereby making EIPSP-A more positive than the Cl- equilibrium potential.(ABSTRACT TRUNCATED AT 400 WORDS)