Potassium-sensitive Microelectrodes Research Articles

Large-conductance Ca2+-activated potassium channels are potently involved in the inverse neurovascular response to spreading depolarization

Recurrent spreading depolarizations occur in the cerebral cortex from minutes up to weeks following acute brain injury. Clinical evidence suggests that the immediate reduction of cerebral blood flow in response to spreading depolarization importantly contributes to lesion progression as the wave propagates over vulnerable tissue zones, characterized by potassium concentration already elevated prior to the passage of spreading depolarization. Here we demonstrate with two-photon microscopy in anesthetized mice that initial vasoconstriction in response to SD triggered experimentally with 1 M KCl is coincident in space and time with the large extracellular accumulation of potassium, as shown with a potassium indicator fluorescent dye. Moreover, pharmacological manipulations in combination with the use of potassium-sensitive microelectrodes suggest that large-conductance Ca2+-activated potassium (BK) channels and L-type voltage-gated calcium channels play significant roles in the marked initial vasoconstriction under elevated baseline potassium. We propose that potassium efflux through BK channels is a central component in the devastating neurovascular effects of spreading depolarizations in tissue at risk.

Implication of Kir4.1 Channel in Excess Potassium Clearance: AnIn VivoStudy on Anesthetized Glial-Conditional Kir4.1 Knock-Out Mice

The K(ir)4.1 channel is crucial for the maintenance of the resting membrane potential of glial cells, and it is believed to play a main role in the homeostasis of extracellular potassium. To understand its importance in these two phenomena, we have measured in vivo the variations of extracellular potassium concentration ([K(+)](o)) (with potassium-sensitive microelectrodes) and membrane potential of glial cells (with sharp electrodes) during stimulations in wild-type (WT) mice and glial-conditional knock-out (cKO) K(ir)4.1 mice. The conditional knockout was driven by the human glial fibrillary acidic protein promoter, gfa2. Experiments were performed in the hippocampus of anesthetized mice (postnatal days 17-24). Low level stimulation (<20 stimuli, 10 Hz) induced a moderated increase of [K(+)](o) (<2 mm increase) in both WT and cKO mice. However, cKO mice exhibited slower recovery of [K(+)](o) levels. With long-lasting stimulation (300 stimuli, 10 Hz), [K(+)](o) in WT and cKO mice displayed characteristic ceiling level (>2 mm increase) and recovery undershoot, with a more pronounced and prolonged undershoot in cKO mice. In addition, cKO glial cells were more depolarized, and, in contrast to those from WT mice, their membrane potential did not follow the stimulation-induced [K(+)](o) changes, reflecting the loss of their high potassium permeability. Our in vivo results support the role of K(ir)4.1 in setting the membrane potential of glial cells and its contribution to the glial potassium permeability. In addition, our data confirm the necessity of the K(ir)4.1 channel for an efficient uptake of K(+) by glial cells.

Brain Water and Ion Fluxes: A Hard-to-Die Hypothesis to Explain Seizures

Increased Seizure Duration and Slowed Potassium Kinetics in Mice Lacking Aquaporin-4 Water Channels. Binder DK, Yao X, Zador Z, Sick TJ, Verkman AS, Manley GT. Glia 2006;53:631–636. The glial water channel aquaporin-4 (AQP4) has been hypothesized to modulate water and potassium fluxes associated with neuronal activity. In this study, we examined the seizure phenotype of AQP4 -/- mice using in vivo electrical stimulation and electroencephalographic (EEG) recording. AQP4 -/- mice were found to have dramatically prolonged stimulation-evoked seizures after hippocampal stimulation compared to wild-type controls (33 μ 2 s vs. 13 μ 2 s). In addition, AQP4 -/- mice were found to have a higher seizure threshold (167 μ 17 A vs. 114 μ 10 A). To assess a potential effect of AQP4 on potassium kinetics, we used in vivo recording with potassium-sensitive microelectrodes after direct cortical stimulation. Although there was no significant difference in baseline or peak [K+]o, the rise time to peak [K+]o ( t1/2, 2.3 μ 0.5 s) as well as the recovery to baseline [K+]o ( t1/2, 15.6 μ 1.5 s) were slowed in AQP4 -/- mice compared to WT mice ( t1/2, 0.5 μ 0.1 and 6.6 μ 0.7 s, respectively). These results implicate AQP4 in the expression and termination of seizure activity and support the hypothesis that AQP4 is coupled to potassium homeostasis in vivo.

A novel neurotransmitter‐independent communication pathway between axons and glial cells

Recent studies have provided evidence that transmitters released by neurons can activate glial receptors and stimulate calcium signalling in glial cells. Glial calcium signalling, in turn, may affect neuronal performance such as long-term changes in synaptic efficacy. Olfactory ensheathing cells (OECs) are a special glial cell type in vertebrates and insects and promote axon growth in the developing and mature nervous system. Physiological properties of OECs, however, have not been studied so far in detail. We measured changes in the calcium concentration in OECs of the moth Manduca sexta, in situ and in vivo. Electrical stimulation of olfactory receptor neurons in pupae or odour stimulation of receptor neurons in adults resulted in calcium transients in OECs. Olfactory receptor axons release acetylcholine; however, application of acetylcholine or other transmitters such as glutamate, GABA or nitric oxide did not induce calcium transients in OECs. Upon nerve stimulation, extracellular potassium rose by several millimolar as measured with potassium-sensitive microelectrodes. When potassium in the perfusion saline was increased from 4 to 10 mM or higher, voltage-dependent calcium transients in OECs that resembled stimulation-induced calcium transients were evoked. Blocking neuronal potassium channels with TEA reduced both the stimulation-induced increases in extracellular potassium and the calcium transients in OECs, whereas calcium transients in receptor axons were augmented. Our results show for the first time that accumulation of potassium, released by electrically active axons, is sufficient to evoke voltage-dependent calcium influx into glial cells, whereas neurotransmitters appear not to be involved in this neuron-glia communication in Manduca.

Increased seizure duration and slowed potassium kinetics in mice lacking aquaporin‐4 water channels

The glial water channel aquaporin-4 (AQP4) has been hypothesized to modulate water and potassium fluxes associated with neuronal activity. In this study, we examined the seizure phenotype of AQP4 -/- mice using in vivo electrical stimulation and electroencephalographic (EEG) recording. AQP4 -/- mice were found to have dramatically prolonged stimulation-evoked seizures after hippocampal stimulation compared to wild-type controls (33 +/- 2 s vs. 13 +/- 2 s). In addition, AQP4 -/- mice were found to have a higher seizure threshold (167 +/- 17 microA vs. 114 +/- 10 microA). To assess a potential effect of AQP4 on potassium kinetics, we used in vivo recording with potassium-sensitive microelectrodes after direct cortical stimulation. Although there was no significant difference in baseline or peak [K(+)](o), the rise time to peak [K(+)](o) (t(1/2), 2.3 +/- 0.5 s) as well as the recovery to baseline [K(+)](o) (t(1/2), 15.6 +/- 1.5 s) were slowed in AQP4 -/- mice compared to WT mice (t(1/2), 0.5 +/- 0.1 and 6.6 +/- 0.7 s, respectively). These results implicate AQP4 in the expression and termination of seizure activity and support the hypothesis that AQP4 is coupled to potassium homeostasis in vivo.

Recurrent spreading depression (SD) causes early opening of the blood-brain barrier (BBB)

SD is a depolarization wave propagating at a rate of about 3 mm/min in the cerebral cortex. It is characterized by an amplitude reduction of the electrocorticographic activity, a rise of the extracellular potassium to 60 mM and a reduction of the extracellular volume. Typical changes of cerebral blood flow (CBF) accompany SD, which consist of a short lasting spreading hyperemia followed by a long lasting spreading oligemia. SD is triggered by electrical, mechanical or toxic factors and is probably involved in the pathogenesis of migraine, focal ischemia, head trauma and cerebral hemorrhage. SD is assumed to be the pathophysiological correlate of the migraine aura. We have recently observed a patient with familial hemiplegic migraine (FHM) type II and a long-lasting migraine aura with fluctuating neurological symptoms. Quantitative analysis of early gadolinium-enhanced MR images revealed a left-hemispheric, cortical blood brain-barrier (BBB) disruption preceding delayed cortical edema in this ATP1A2 mutation carrier. As we know from the work by Strong et al. repetitive SDs can occur in the human brain tissue, which could explain the fluctuating symptoms in our FHM patient. Additionally, a recent publication by Gursoy-Ozdemir et al. showed a delayed enhancement of BBB permeability and subsequent cortical oedema following a single SD. Based on these observations, we studied here whether recurrent SD causes early BBB disruption in the rat cortex in vivo. We triggered SD with artificial cerebrospinal fluid (ACSF) containing a potassium concentration ([K+]ACSF) of 130 mM at an open parietal cranial window. SDs were monitored with an intracortical potassium sensitive microelectrode and laser-Doppler flowmetry. We also recorded the propagation of SDs to a distant closed frontal cranial window using an epidural Ag-AgCl electrode. BBB integrity was evaluated quantitatively by analyzing fluorescence in brain sections following the peripheral injection of Evan's Blue or Lucifer yellow. At the occipital window, [K+]ACSF evoked several SDs in all animals. In the first group (n = 10), one or more SDs propagated to the frontal window. In contrast, in the second group (n = 9), pretreatment with MK-801 (5 mg/kg BW i.v.), abolished SD propagation. Within two hours after the onset of the first SD, significantly enhanced fluorescence intensity at the frontal cortex was only measured in animals from group 1 with multiple propagated SDs. In conclusion, we provide evidence that repetitive SDs significantly increase blood-brain barrier permeability within two hours after the onset.

Alcohol Exposure During the Brain Growth Spurt Promotes Hippocampal Seizures, Rapid Kindling, and Spreading Depression

Epilepsy is a prominent sign of brain dysfunction and a cause of substantial disability in some children with fetal alcohol syndrome. The hippocampal formation is vulnerable to alcohol-induced pathologic changes and is the source of seizure activity in a variety of epileptic conditions. This study tests the hypothesis that developmental alcohol exposure facilitates epileptic activity and promotes kindling within hippocampal circuitry. Rat pups received either a moderate dose (2.0 g/kg) or a high dose (3.75 g/kg) of alcohol via intragastric intubation over postnatal days 4 to 10. Intubated control and suckle control groups were also included. Upon reaching adulthood (postnatal days 85-100), the rats underwent electrophysiologic testing. A double-barrel potassium-sensitive microelectrode was placed into the right dentate gyrus stratum granulosum for the recording of extracellular field potential and extracellular potassium concentration. Stimuli were delivered via an electrode positioned in the CA3 subregion of the left hippocampus. To assess whether alcohol promotes hippocampal seizures and rapid kindling, the parameters of maximal dentate activation (MDA) were measured before, during, and after a series of stimulation-induced seizures. Developmental exposure to the high dose of alcohol permanently altered several parameters of MDA. Time to onset of MDA and stimulus threshold for afterdischarge production were both decreased, whereas the duration of the afterdischarge was increased. Although the moderate alcohol dose reduced time to onset of MDA, it did not affect any other MDA parameters. Over the course of the repeated induced seizures, spreading depression occurred more often and with fewer stimuli in the high-dose alcohol group than in any other group. The series of repeated electrographic seizures induced rapid kindling in all of the treatment groups. However, the kindling effect was enhanced in a dose-response manner by the previous alcohol exposures. These findings demonstrate that exposure to alcohol during brain development can permanently alter the physiology of the hippocampal formation, thus promoting epileptic activity, enhancing kindling, and facilitating spreading depression. The relative roles of alcohol intoxication and withdrawal in these abnormal physiologic responses remain unknown.

Alcohol Exposure During the Brain Growth Spurt Promotes Hippocampal Seizures, Rapid Kindling, and Spreading Depression

Background: Epilepsy is a prominent sign of brain dysfunction and a cause of substantial disability in some children with fetal alcohol syndrome. The hippocampal formation is vulnerable to alcohol-induced pathologic changes and is the source of seizure activity in a variety of epileptic conditions. This study tests the hypothesis that developmental alcohol exposure facilitates epileptic activity and promotes kindling within hippocampal circuitry. Methods: Rat pups received either a moderate dose (2.0 g/kg) or a high dose (3.75 g/kg) of alcohol via intragastric intubation over postnatal days 4 to 10. Intubated control and suckle control groups were also included. Upon reaching adulthood (postnatal days 85–100), the rats underwent electrophysiologic testing. A double-barrel potassium-sensitive microelectrode was placed into the right dentate gyrus stratum granulosum for the recording of extracellular field potential and extracellular potassium concentration. Stimuli were delivered via an electrode positioned in the CA3 subregion of the left hippocampus. To assess whether alcohol promotes hippocampal seizures and rapid kindling, the parameters of maximal dentate activation (MDA) were measured before, during, and after a series of stimulation-induced seizures. Results: Developmental exposure to the high dose of alcohol permanently altered several parameters of MDA. Time to onset of MDA and stimulus threshold for afterdischarge production were both decreased, whereas the duration of the afterdischarge was increased. Although the moderate alcohol dose reduced time to onset of MDA, it did not affect any other MDA parameters. Over the course of the repeated induced seizures, spreading depression occurred more often and with fewer stimuli in the high-dose alcohol group than in any other group. The series of repeated electrographic seizures induced rapid kindling in all of the treatment groups. However, the kindling effect was enhanced in a dose-response manner by the previous alcohol exposures. Conclusions: These findings demonstrate that exposure to alcohol during brain development can permanently alter the physiology of the hippocampal formation, thus promoting epileptic activity, enhancing kindling, and facilitating spreading depression. The relative roles of alcohol intoxication and withdrawal in these abnormal physiologic responses remain unknown.

Suppression of epileptiform activity by high frequency sinusoidal fields in rat hippocampal slices.

1. Sinusoidal high frequency (20-50 Hz) electric fields induced across rat hippocampal slices were found to suppress zero-Ca2+, low-Ca2+, picrotoxin, and high-K+ epileptiform activity for the duration of the stimulus and for up to several minutes following the stimulus. 2. Suppression of spontaneous activity by high frequency stimulation was found to be frequency (< 500 Hz) but not orientation or waveform dependent. 3. Potassium-sensitive microelectrodes showed that block of epileptiform activity was always coincident with a stimulus-induced rise in extracellular potassium concentration during stimulation. Post-stimulus inhibition was always associated with a decrease in extracellular potassium activity below baseline levels. 4. Intracellular recordings and optical imaging with voltage-sensitive dyes showed that during suppression neurons were depolarized yet did not fire action potentials. 5. Direct injection of sinusoidal current into individual pyramidal cells did not result in a tonic depolarization. Injection of large direct current (DC) depolarized neurons and suppressed action potential generation. 6. These findings suggest that high frequency stimulation suppresses epileptiform activity by inducing potassium efflux and depolarization block.

Cesium induces spontaneous epileptiform activity without changing extracellular potassium regulation in rat hippocampus.

Cesium has been widely used to study the roles of the hyperpolarization-activated (I(h)) and inwardly rectifying potassium (K(IR)) channels in many neuronal and nonneuronal cell types. Recently, extracellular application of cesium has been shown to produce epileptiform activity in brain slices, but the mechanisms for this are not known. It has been proposed that cesium blocks the K(IR) in glia, resulting in an abnormal accumulation of potassium in the extracellular space and inducing epileptiform activity. This hypothesis has been tested in hippocampal slices and cultured hippocampal neurons using potassium-sensitive microelectrodes. In the present study, application of cesium produced spontaneous epileptiform discharges at physiological extracellular potassium concentration ([K(+)](o)) in the CA1 and CA3 regions of hippocampal slices. This epileptiform activity was not mimicked by increasing the [K(+)](o). The epileptiform discharges induced by cesium were not blocked by the N-methyl-D- aspartate (NMDA) receptor antagonist AP-5, but were blocked by the non-NMDA receptor antagonist CNQX. In the dentate gyrus, cesium induced the appearance of spontaneous nonsynaptic field bursts in 0 added calcium and 3 mM potassium. Moreover, cesium increased the frequency of field bursts already present. In contrast, ZD-7288, a specific I(h) blocker, did not cause spontaneous epileptiform activity in CA1 and CA3, nor did it affect the field bursts in the dentate gyrus, suggesting that cesium induced epileptiform activity is not directly related to blockade of the I(h). When potassium-sensitive microelectrodes were used to measure [K(+)](o), there was no significant increase in [K(+)](o) in CA1 and CA3 after cesium application. In the dentate gyrus, cesium did not change the baseline level of [K(+)](o) or the rate of [K(+)](o) clearance after the field bursts. In cultured hippocampal neurons, which have a large and relatively unrestricted extracellular space, cesium also produced cellular burst activity without significantly changing the resting membrane potential, which might indicate an increase in [K(+)](o). Our results suggest that cesium causes epileptiform activity by a mechanism unrelated to an alteration in [K(+)](o) regulation.

Axonal and glial currents activated during the post-tetanic hyperpolarization in non-myelinated nerve.

Changes in membrane potential and potassium concentration in the extracellular space ([K+]e) of rabbit vagus nerve were measured simultaneously during electrical activity and during the period of recovery using a modified sucrose-gap method and potassium-sensitive microelectrodes. After stimulation for 15 s at 15 Hz the main activity-induced increase in [K+]e reached 16.9 mM. This increase in [K+]e was paralleled by a depolarization of the preparation. The period of activity was followed by a post-tetanic hyperpolarization (PTH) lasting tens of seconds, generated by the axonal electrogenic Na+-K+ pump and to a lesser extent by the pump of the surrounding Schwann cells. The amplitude of the PTH dramatically increased in experiments in which inward currents were blocked by removal of Cl– or after application of Cs+ or Ba2+, indicating that under normal conditions the current generated by the Na+-K+ pump is strongly short-circuited. A pharmacological and kinetic study showed that these currents are: (1) the hyperpolarization-activated current I h, and (2) the inwardly rectifying I KIR current. The results show that the latter originates from Schwann cells. Our data indicate that in non-myelinated nerves there is a subtle association of inward ionic channels which (1) helps the cell to maintain an optimal membrane potential after a period of activity, and (2) contributes to the removal of excess K+ from the extracellular space.

Uptake of potassium by nonmyelinating Schwann cells induced by axonal activity.

1. The electrophysiological properties of the rabbit vagus nerve (membrane potential, compound action potentials, and afterpotentials) and potassium accumulation were measured simultaneously during low-frequency stimulation (LFS) (0.5 and 1 Hz) by using a modified sucrose-gap apparatus and potassium-sensitive microelectrodes (KSM). 2. During LFS at 0.5 and 1 Hz, the concentration of K+ in the extracellular space ([K+]c) increased in approximately 30 s to a maximal level that was 0.6 and 1.5 mM, respectively, above the resting concentration. Concomitantly the preparation developed an ouabain-sensitive hyperpolarization. 3. The compound action potential (CAP) was followed by a fast hyperpolarizing afterpotential (fHAP), a depolarizing afterpotential (DAP), and a slow hyperpolarizing afterpotential (sHAP). During LFS the characteristics of all these afterpotentials were profoundly modified. In parallel to the increase in [K+]e, the fHAP was decreased and the amplitude of the DAP was dramatically enhanced. Furthermore, the sHAP which had a duration of < 1 s when it followed a single CAP, turned into a ouabain-sensitive hyperpolarization (indicating that it was generated by the electrogenic Na(+)-K+ pump) that lasted several minutes. 4. The application of external Ba2+ produced a hyperpolarizing sag on the sHAP following a single isolated CAP. During LFS, Ba2+ enhanced the build-up of the DAP, raised the maximal level of [K+]e, and increased the activity-induced ouabain-sensitive hyperpolarization. 5. The increase by Ba2+ of the activity-induced hyperpolarization shifted the spikes from both myelinated and nonmyelinated fibers toward a more negative potential but did not increase their amplitude, indicating that this Ba(2+)-induced hyperpolarization originated from an extra-axonal source, presumably the Schwann cells. 6. It is proposed that the electrogenic activity of the Na(+)-K+ pump was enhanced in Schwann cells situated near active axons. This hyperpolarization was, however, not recorded in normal conditions because it was fully short-circuited by a K+ influx through Ba(2+)-sensitive channels. 7. Our results lead to the hypothesis that the Na(+)-K+ pump of the nonmyelinating Schwann cells is important in the mechanisms maintaining the homeostasis of K+ in the axonal microenvironment. They show that the Na(+)-K+ pump contributes to the K+ buffering not only by actively pumping K+ but also by generating a hyperpolarization that drives a passive K+ influx through Ba(2+)-sensitive K+ channels.

Measurements of the extracellular potassium concentrations in the isolated rabbit retina with different kinds of potassium-sensitive microelectrodes

When the dark-adapted isolated rabbit retina has been superfused from one side with a plasma-saline mixture containing 3.5 mM potassium, the extracellular potassium concentration within the dark-adapted retina was significantly higher (4.5–6.1 mM) than the potassium of the superfusate, when measured with an electrode filled with Corning 477317. The substantial difference between the [K +] 0 of the perfusate and the [K +]0 within the retina is difficult to explain and could be an instrumental artefact. To probe this possibility measurements have now been repeated using 2 different K + electrodes, one filled with Corning 477317 and the other filled with a valinomycin-based ion exchanger. With the latter electrodes there was practically no gradient between the [K +]0 in the retina (3.2–4.0 mM) and the potassium concentration of the superfusate. It is thus evident that the earlier enigmatic values for [K +] 0 relate to the use of Corning 477317-filled K + electrodes. When, however, changes in [K +] 0 were induced by a light stimulus, the measured magnitudes of change in [K +] 0 were the same with the 2 types of electrode.

Extracellular potassium changes in the rat neurohypophysis during activation of the magnocellular neurosecretory system.

1. Potassium-sensitive microelectrodes were used to measure extracellular [K+] in the isolated rat neurohypophysis maintained in vitro. Electrical stimulation of the neurohypophysial stalk (20 Hz 5 s) increased the inferred extracellular [K+] by 9.2 +/- 0.4 mM (mean +/- S.E. of mean; n = 21). 2. Veratridine (10 microM) enhanced the response to stalk stimulation, and at a higher concentration (50 microM) increased extracellular [K+] in the absence of stimulation. By contrast, tetrodotoxin (1 microM) blocked the [K+] increase completely and reversibly in each of five experiments, indicating that the increase was a consequence of action potential generation. 3. At the end of brief periods of stimulation, the raised extracellular [K+] returned to pre-stimulation levels within 30 s. In the presence of ouabain (100 microM), the recovery was slower: the half-decay time was extended by 150-300% in each of three experiments. 4. Replacement of calcium in the medium with cobalt, cadmium or magnesium reduced the amplitude of the [K+] increase by 26-30%, indicating that the [K+] increase was largely independent of events subsequent to evoked release of hormone and/or transmitters. 5. Potassium-sensitive microelectrodes were placed in the neurohypophysis of rats anaesthetized with urethane. Electrical stimulation of the pituitary stalk (50 Hz, 5 s) produced transient voltage increases of 7.6 +/- 0.9 mV (mean +/- S.E. of mean of seven experiments). These voltage increases were similar in magnitude to the response of the electrodes to the addition of 7.6 +/- 1.0 mM-K+ to rat plasma. 6. In seven lactating rats, the suckling of a litter of hungry pups evoked periodic reflex milk ejections, as detected by increases in intramammary pressure. Potassium-sensitive microelectrodes in the neurohypophysis recorded transient voltage increases prior to each milk ejection (0.4-5.5 mV). Each increase preceded an increase in intramammary pressure by 12-30 s. 7. Thus synchronized high-frequency activation of magnocellular neurones can produce large changes in extracellular [K+]. The implications of these findings for stimulus-secretion coupling in the neurohypophysis are discussed in the light of previous reports that hormone release from the neurohypophysis is highly dependent on the frequency and pattern of electrical stimulation.

Activity-dependent changes in extracellular potassium and excitability in turtle olfactory nerve.

The excitability properties of turtle olfactory nerve (o.n.) were studied in vitro using potassium-sensitive microelectrodes (KSM), a modified sucrose gap chamber, and a standard nerve chamber to measure conduction velocity. A pronounced supernormal period (SNP), as indicated by increased conduction velocity of the o.n. fiber volley, lasting up to several seconds, was observed following a single stimulus. The compound action potential recorded in the sucrose gap chamber showed a prolonged depolarization with a similar time course to the SNP. When stimulation intensity was submaximal the response amplitude, and the extracellular potassium concentration [K+]o, continuously increased during repetitive stimulation. In contrast, when supramaximal stimuli were applied, the amplitude of the o.n. fiber volley was reduced during a high-frequency stimulus train for all responses after the initial one even though latency was maximally reduced, i.e., during supernormal conduction. Superfusion with various levels of K+ elicited changes in the excitability of the o.n. fibers. Small increases in [K+]o above the resting concentration of 2.6 mM led to an increase in resting excitability, whereas larger increases resulted in decreased excitability and conduction block. The SNP was eliminated when extracellular potassium was elevated between 3 and 4 mM above resting levels. Microstimulation of a small bundle of o.n. fibers led to an increase in [K+]o along the bundle but also around adjacent nonactivated fibers. The excitability of these neighboring nonactivated fibers was increased, further indicating the importance of activity-dependent changes in [K+]o in modulating axonal excitability. These results demonstrate the importance of activity-dependent increases in extracellular potassium in modulating nonmyelinated o.n. fiber excitability. They also indicate that increases in [K+]o and an associated membrane depolarization contribute to the increased excitability observed during fiber recruitment and the supernormal period.

Light-induced changes of extra- and intracellular potassium concentration in photoreceptors of the leech,Hirudo medicinalis

The potassium concentration was measured in the cytoplasm, perimicrovillar extracellular space (=‘vacuole’) and intercellular space of leech photoreceptors with double-barrelled potassium-sensitive microelectrodes in darkness and upon photostimulation. The mean intracellular potassium concentration in cells with membrane potentials >50 mV was 100±34 mmol/l. Photostimulation with 90 saturating 20 ms light flashes (1/s) evoked a potassium loss of 10.6±7.6 mmol/l. In the dark, there was no potassium concentration gradient between vacuole and intercellular space (K VAC + =4.5±0.9 mmol/l, K ECS + =4.5±0.5 mmol/l). In both compartments the potassium concentration increased upon repetitive photostimulation. Thus, the potassium loss from the cell is due to potassium movements across both the receptive and the non-receptive membrane domains.

Extracellular Potassium Activity in Attached Leaves and its Relation to Stomatal Function

Apoplastic potassium activities (ak) in leaves of Commelina communis L., Vicia faba L. and Pisum sativum L. var. argenteum were recorded with neutral-carrier-based, potassium-sensitive micro-electrodes. Measurements were carried out in 0.3–1.4 nl volumes contiguous with the extracellular space of attached leaves and were held for periods of 7–68 min. Mean steady-state aK values recorded from all three species were below 50 μM. Similar potassium activities were attained, regardless of the initial values obtained after washing with distilled water or potassium additions, and the activities recorded showed only minimal dependence on the relative vapour pressure difference. Tissue capacity for K+ absorption was increased 15–30-fold in the presence of added Ca2+. By contrast, cyanide reduced both the initial rate of potassium absorption by the tissues and their apparent capacity for the cation. These observations indicate that the free potassium pool in the leaf apoplast is significantly smaller than has previously been assumed. The results contradict the notion that high concentrations of potassium accumulate locally as a result of transpiration, and may indicate the presence in the leaf tissues of potassium transport activity sensitive to Ca2+ and dependent on metabolism.

Phasic variations of extracellular potassium during fictive swimming in the lamprey spinal cord in vitro.

The lamprey spinal cord in vitro can generate the motor pattern underlying locomotion, which can be recorded with suction electrodes in the ventral roots. To test if the extracellular level of potassium changed during rhythmic activity, potassium-sensitive microelectrodes were used to systematically (every 25 micrometers) explore the level of extracellular potassium [K+]e in different loci in the transverse plane of the spinal cord. During fictive locomotion the baseline level of [K+] increased with 0.08-0.4 mM in the grey matter. As a rule phasic variations of up to 0.2 mM, correlated to each ventral root burst, were superimposed on the tonic deviation of [K+]e. Changes in this range may cause a moderate depolarization of the spinal neurones and might also affect other neuronal functions including the rhythm-generating circuits.

Ammonia does not selectively block IPSPs in rat hippocampal pyramidal cells.

Intracellular recordings from CA1 pyramidal cells in the rat hippocampal slice preparation have been used to study the action of ammonia on inhibitory postsynaptic potentials (IPSPs). Concentrations of ammonia less than 2 mM had little effect on IPSPs or the action of iontophoretically applied gamma-aminobutyric acid (GABA). This concentration has been reported to be fully effective in blocking hyperpolarizing IPSPs in spinal cord and neocortex. Concentrations above 2 mM did cause a depolarizing shift in the IPSP and GABA reversal potentials, but this effect was accompanied by several generalized effects. The conductance increase during the IPSP but not during the GABA response was depressed, indicating that ammonia has a presynaptic depressant effect on the IPSP. Ammonia also depressed excitatory postsynaptic potentials (EPSPs), presynaptic fiber potentials, and pyramidal cell population spikes. In addition, the calcium-dependent potassium response elicited by depolarizing current pulses was depressed. This depression was due, in part, to a depolarizing shift in the reversal potential for this response. Responses recorded with potassium-sensitive microelectrodes indicate that ammonia releases potassium into the extracellular space. The possibility is discussed that the shifts in IPSP reversal potential seen with high concentrations of ammonia are a consequence of generalized nonspecific effects. We conclude that the relative insensitivity of hippocampal IPSPs to blockade by ammonia suggests that a mechanism fundamentally unlike an ammonia-sensitive chloride pump must maintain the hippocampal IPSP gradient.

Activity-dependent K+ accumulation in the developing rat optic nerve.

Potassium-sensitive microelectrodes were used to study activity-dependent changes of extracellular potassium ion concentration ([K+]o) in rat optic nerves of different postnatal ages (1 day to adulthood). The maximum level to which [K+]o rose with optimal frequencies of stimulation depended on age: mean maximum evoked [K+]o was 17.2 microM in 1- to 3-day-old optic nerves and 9.8 microM in adult nerves. The ceiling [K+]o seen in immature optic nerves, which is uniquely large for a mammalian central nervous system structure, may result from a relatively enhanced rate of evoked K+ release.