Changes In Extracellular Ion Concentrations Research Articles

Serum Proteins Associated with Blood-Brain Barrier as Potential Biomarkers for Seizure Prediction.

As 30% of epileptic patients remain drug-resistant, seizure prediction is vital. Induction of epileptic seizure is a complex process that can depend on factors such as intrinsic neuronal excitability, changes in extracellular ion concentration, glial cell activity, presence of inflammation and activation of the blood−brain barrier (BBB). In this study, we aimed to assess if levels of serum proteins associated with BBB can predict seizures. Serum levels of MMP-9, MMP-2, TIMP-1, TIMP-2, S100B, CCL-2, ICAM-1, P-selectin, and TSP-2 were examined in a group of 49 patients with epilepsy who were seizure-free for a minimum of seven days and measured by ELISA. The examination was repeated after 12 months. An extensive medical history was taken, and patients were subjected to a follow-up, including a detailed history of seizures. Serum levels of MMP-2, MMP-9, TIMP-1, CCL-2, and P-selectin differed between the two time points (p < 0.0001, p < 0.0001, p < 0.0001, p < 0.0001, p = 0.0035, respectively). General linear model analyses determined the predictors of seizures. Levels of MMP-2, MMP-9, and CCL-2 were found to influence seizure count in 1, 3, 6, and 12 months of observation. Serum levels of MMP-2, MMP-9, and CCL-2 may be considered potential biomarkers for seizure prediction and may indicate BBB activation.

Chaotic Dynamics Mediate Brain State Transitions, Driven by Changes in Extracellular Ion Concentrations.

Previous studies have suggested that changes in extracellular ion concentrations initiate the transition from an activity state that characterizes sleep in cortical neurons to states that characterize wakefulness. However, because neuronal activity and extracellular ion concentrations are interdependent, isolating their unique roles during sleep-wake transitions is not possible invivo. Here, we extend the Averaged-Neuron model and demonstrate that, although changes in extracellular ion concentrations occur concurrently, decreasing the conductance of calcium-dependent potassium channels initiates the transition from sleep to wakefulness. We find that sleep is governed by stable, self-sustained oscillations in neuronal firing patterns, whereas the quiet awake state and active awake state are both governed by irregular oscillations and chaotic dynamics; transitions between these separable awake states are prompted by ionic changes. Although waking is indicative of a shift from stable to chaotic neuronal firing patterns, we illustrate that the properties of chaotic dynamics ensure that the transition between states is smooth and robust to noise.

Activity dependence of spreading depression in the locust CNS.

Spreading depression (SD) is associated with large changes in extracellular ion concentrations and can be induced by impairing mechanisms of K(+) ion homeostasis. We tested activity dependence of SD in the locust model of ouabain-induced SD in the metathoracic ganglion. Wind activation of thoracic circuitry resulted in small increases of K(+) concentration that took 5-10 s to be cleared from the extracellular space. In the presence of the Na(+)/K(+)-ATPase inhibitor ouabain, wind stimulation every 30 s halved the latency to the first SD event and increased its duration. Wind stimulation was able to trigger the first event, suggesting that local activity could determine the origin of successive SD events. Perfusion with calcium-free saline blocked neural activity in the ganglion and prevented the occurrence of ouabain-induced SD. We conclude that ouabain-induced SD in the locust CNS is strongly dependent on the existing level of neural activity.

A Kir6.2 pore mutation causes inactivation of ATP-sensitive potassium channels by disrupting PIP2-dependent gating.

In the absence of intracellular nucleotides, ATP-sensitive potassium (KATP) channels exhibit spontaneous activity via a phosphatidylinositol-4,5-bisphosphate (PIP2)-dependent gating process. Previous studies show that stability of this activity requires subunit-subunit interactions in the cytoplasmic domain of Kir6.2; selective mutagenesis and disease mutations at the subunit interface result in time-dependent channel inactivation. Here, we report that mutation of the central glycine in the pore-lining second transmembrane segment (TM2) to proline in Kir6.2 causes KATP channel inactivation. Unlike C-type inactivation, a consequence of selectivity filter closure, in many K+ channels, the rate of inactivation in G156P channels was insensitive to changes in extracellular ion concentrations or ion species fluxing through the pore. Instead, the rate of G156P inactivation decreased with exogenous application of PIP2 and increased when PIP2-channel interaction was inhibited with neomycin or poly-L-lysine. These findings indicate the G156P mutation reduces the ability of PIP2 to stabilize the open state of KATP channels, similar to mutations in the cytoplasmic domain that produce inactivation. Consistent with this notion, when PIP2-dependent open state stability was substantially increased by addition of a second gain-of-function mutation, G156P inactivation was abolished. Importantly, bath application and removal of Mg2+-free ATP or a nonhydrolyzable analog of ATP, which binds to the cytoplasmic domain of Kir6.2 and causes channel closure, recover G156P channel from inactivation, indicating crosstalk between cytoplasmic and transmembrane domains. The G156P mutation provides mechanistic insight into the structural and functional interactions between the pore and cytoplasmic domains of Kir6.2 during gating.

Identification of critical residues within the extracellular loop of the Epithelial Sodium Channel (ENaC)

The epithelial Na+ channel (ENaC) plays a pivotal role in fluid homeostasis through its function in sodium reabsorption. Malfunction of this channel can lead to salt sensitive hypertension or hypotension. Although passage specific and constitutively active for the transport of Na+ ions, ENaC has exhibited self‐inhibition in the presence of excess Na+ as well as altered regulation from other ions such as Cl−. ENaC's structure is likely heterotrimeric and composed of α, β, and γ‐subunits, all contributing to proper function. In an effort to elucidate structural features of mouse αENaC, critical residues, E361 and K487, were identified using a functional yeast screen. Also, the effects of specific mutations within the extracellular region (K189 to A491) of mENaC were studied under various pH levels and in the presence of excess Cl−, K+ or Na+. Variations in the growth of yeast cells expressing wild‐type and mutant ENaC were correlated to changes in extracellular ion concentration. Residues involved in potential interactions with ions that contribute to ENaC regulation in yeast will be further characterized in mammalian cells. (Supported by NIH‐NIGMS R15GM086798)

A bistable computational model of recurring epileptiform activity as observed in rodent slice preparations

We describe a computational model of epileptiform activity mimicking the activity exhibited by an animal model of epilepsy in vitro. The computational model permits generation of synthetic data to assist in the evaluation of new algorithms for epilepsy treatment via adaptive neurostimulation. The model implements both single-compartment pyramidal neurons and fast-spiking interneurons, arranged in a one-dimensional network using both excitatory and inhibitory synapses. The model tracks changes in extracellular ion concentrations, which determine the reversal potentials of membrane currents. Changes in simulated ion concentration provide positive feedback which drives the system towards the epileptiform state. One mechanism of positive feedback explored by this model is the conversion of pyramidal cells from regular spiking to intrinsic bursting as extracellular potassium concentration increases. One of the main contributions of this work is the development of a slow depression mechanism that enforces seizure termination. The network spontaneously leaves the seizure-like state as the slow depression variable decreases. This is one of the first detailed computational models of epileptiform activity, which exhibits realistic transitions between inter-seizure and seizure states, and back, with state durations similar to the in vitro model. We validate the computational model by comparing its state durations to those of the biological model. We also show that electrical stimulation of the computational model achieves seizure suppression comparable to that observed in the in vitro model.

Glial modulation of neural excitability mediated by extracellular pH: a hypothesis revisited

This chapter discusses an update on a model interaction between astrocytes and neurons based on activity-induced changes in extracellular ion concentrations, K + and H +, mainly, the nature of the astrocyte-induced extracellular acidification, and the manner in which pH changes can alter neural excitability. The proposed interaction represented an example of volume transmission mediated by ions. It was viewed as a negative feedback loop whereby active neurons would signal to nearby astrocytes the extent of their current activity, in the form of graded changes in extracellular K+ concentration, and astrocytes would respond by producing graded degrees of extracellular acidification. The acidification would, in turn, dampen neuronal excitability. It is argued that this feedback system could function to adjust local brain excitability in relationship to ongoing activity; the more intense the activity, the greater the resulting acidification and reduction in excitability.

A comparison between the stimulated and paroxysmal release of endogenous amino acids from rat cerebellar, striatal and hippocampal slices: a manifestation of spreading depression?

Spreading depression, which can be evoked by a variety of stimuli both in vitro and in vivo, is associated with profound changes in extracellular ion concentrations and enhanced release of neurotransmitter amino acids. We have observed a transient spontaneous release of amino acids in slice preparations obtained from rat cerebellum, striatum and hippocampus; this phenomenon has similar properties to stimulus-evoked spreading depression. Aspartate, glutamate, glutamine, serine, glycine and γ-aminobutyric acid (GABA) release were potentiated during these episodes in all three brain regions, with a variable effect upon taurine release. When compared to glutamate release, a consistently high release of aspartate, glycine and serine was observed. Amino acid release, evoked by whole slice depolarization using veratridine (10–25 μM) or elevated potassium (35–60 mM) consistently enhanced glutamate release, and to a lesser extent aspartate release, but had negligible effect upon the other amino acids. Thus, the release profiles for spontaneous and depolarization-evoked release are markedly different. We suggest that the spontaneous release observed in brain slices represents a spreading depression-like phenomenon; the putative roles of the amino acids are discussed.

Pharmacological and electrographic properties of epileptiform activity induced by elevated K+ and lowered Ca2+ and Mg2+ concentration in rat hippocampal slices.

We studied some of the physiological and pharmacological properties of an in vitro model of epileptic seizures induced by elevation of [K+]0 (to 8 mM and 10 mM) in combination with lowering of [Mg2+]0 (to 1.4 mM and 1.6 mM) and [Ca2+]0 (to 0.7 mM and 1 mM) in rat hippocampal slices. These concentrations correspond to the ionic constitution of the extracellular microenvironment during seizures in vivo. The resulting activity was rather variable in appearance. In area CA3 recurrent discharges were observed which resulted in seizure-like events with either clonic-like or tonic-clonic-like ictaform events in area CA1. With ion-sensitive electrodes, we measured the field potential and the changes in extracellular ion concentrations which accompany this activity. The recurrent discharges in area CA3 were accompanied by small fluctuations in [K+]0 and [Ca2+]0. The grouped clonic-like discharges in area CA1 were associated with moderate increases in [K+]0 and small decreases in [Ca2+]0 in the order of 2 mM and 0.2 mM, respectively. Large, negative field-potential shifts and increases in [K+]0 to 13 mM, as well as decreases in [Ca2+]0 by up to 0.4 mM, accompanied the tonic phase of ictaform events. The ictaform events were not blocked by D-2-aminophosphonovalerate (2-APV) but were sensitive to 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) alone and in combination with 2-APV and ketamine. In order to determine the pharmacological characteristics of the ictaform events we bath-applied most clinically employed anticonvulsants (carbamazepine, phenytoin, valproate, phenobarbital, ethosuximide, trimethadione) and some experimental anticonvulsants (losigamone, vinpocetine, and apovincaminic acid). Carbamazepine, phenytoin, valproate, and phenobarbital were effective at clinically relevant doses. The data suggest that the high-K+ model of epileptiform activity is a good model of focal convulsant activity.

Continuous determination of extracellular space and changes of K +, Na +, Ca 2+, and H + during global ischaemia in isolated rat hearts

Isolated rat hearts were perfused according to the Langendorff technique and prepared to allow the measurement of left ventricular pressure (LVP), the first derivative of LVP ( dP dt ), coronary perfusion pressure (CPP) and heart rate (HR). The hearts were perfused with a modified Krebs-Henseleit solution (KHS; control group) or KHS containing tetramethylammonium-chloride (TMA; 100 μ m), which did not influence heart haemodynamics or extracellular potassium accumulation during global ischaemia (GI). TMA was used as a marker to determine changes in the size of the extracellular space (ECS) during 60 min of GI. Extracellular concentrations of K +, Na +, Ca 2+, H + and TMA were measured using double-barreled polyvinyl chloride (PVC) mini-electrodes. Relative changes in the ECS size and net cation movements were calculated from the extracellular TMA and cation concentrations. After 60 min of GI ECS decreased by 74% due to a water shift into the intracellular space (ICS). Within 10 s after the onset of GI extracellular potassium concentration increased in a typical triphasic pattern. A biphasic net efflux of K + was maximal 2 and 15 min after the onset of ischaemia interrupted by a maximal uptake after 7 min, probably due to the stimulation of the Na + K + ATPase. The changes in extracellular sodium and calcium concentrations were biphasic; showing an initial increase occurring approximately during the first 20 min after the onset of GI followed by a decrease. Despite the initial increase of extracellular Na + and Ca 2+ maximal net influx of Na + and Ca 2+ were calculated after 2 and 19 min of GI. Accumulation of H + in the extracellular space occurred mainly during the first 20 min of GI. It is concluded that, (1) TMA is a useful marker for continuous measurement of changes in the size of the ECS during GI; (2) Changes in extracellular ion concentrations are influenced markedly by a water shift from the ECS into the ICS; (3) Stimulation of the Na + K + pump causes a transient net uptake of K + and reduces the net influx of Na +; (4) Ca 2+ uptake is decreased transiently probably by increased Ca 2+-ATPase activity; (5) Energy dependent protective mechanisms for the maintainance of intracellular ionic homeostasis are exhausted after approximately 15 to 20 min of GI in isolated working rat hearts.

Ion-selective microelectrodes suitable for recording rapid changes in extracellular ion concentration

A method for fabricating double-barrel, ion-selective microelectrodes with fine tips (0.5–1.5 μm) and rapid response times is described. When made into K +-selective microelectrodes, the electrodes respond to changes in [ K +] o with a time constant of 70–95 ms. The electrical response of these electrodes to common-mode voltages can be made to have a time constant of < 2 ms, which minimizes electrical artifacts from field potentials. The application of these microelectrodes to the measurement of rapid, transient changes in retinal [ K +] o is presented.

Alterations in the microenvironment during spreading depression associated with epileptiform activity in the immature neocortex

Local changes in extracellular ion concentrations were measured with ion-sensitive microelectrodes in slices of mature (>40 days of age) or immature (16–30 days of age) rat neocortex maintained in vitro. Repetitive stimulation resulted in increases in extracellular potassium ([K +] o) to levels of 8.85 ± 2.1 mM in slices from adult animals and 12.77 ± 1.8 mM in slices from immature animals. During exposure to picrotoxin, maximum levels were 11.3 ± 2.6 and 14.8 ± 2.5 mM in the mature and immature groups, respectively. Picrotoxin (50 μM) induced spontaneous bursts of repetitive spiking, followed by a slow, negative field potential, associated with spreading depression (SD), in slices from immature animals. [K +] o levels increased to 10.2 ± 3.9 mM during repetitive spike discharges and reached 30.3 ± 18.5 mM during SDs. Variations in the size of the extracellular space (ES) were examined during SD. The ES was found to reversibly decrease by 39 ± 4.5%. Clusters of repetitive spikes were associated with 0.1–0.2 mM decreases in [Ca 2+] o, whereas 1.12 ± 0.06 mM decreases were observed during SDs. Decreases in [Na +] o and [Cl - o of 56 ± 10 mM and 41 ± 9 mM, respectively, were observed during SDs suggesting that a net transmembrane movement of water occurred during SDs. These results indicate that changes in [K +] o associated with epileptiform activity in the immature nervous system are quantitatively different from those observed in the mature brain. These large increases in [K +] o may contribute to the prolonged nature of epileptiform discharges in the developing nervous system.

Hippocampal slices from kindled rats show an increased sensitivity for induction of epileptiform activity by changes in extracellular ion concentrations.

The ability of increases in extracellular potassium ([K+]o) and/or decreases in extracellular calcium ([Ca2+]o) to induce epileptiform activity in hippocampal slices was studied by systematically varying [K+]o and [Ca2+]o. Slices prepared from kindled rats, both 1 week and 1 month after the last kindled seizure, showed an increased sensitivity to perturbations of both ions. Stimulus-locked epileptiform discharges occurred with small displacements of [K+]o and/or [Ca2+]o. The ionic threshold for spontaneous epileptiform discharges was not significantly affected. This long-lasting change in sensitivity to the ionic environment produced by the kindling process had important implications for epileptogenesis in chronically epileptic tissue.

The Membrane Potential of Human Platelets

The membrane potential of the human platelet was investigated using the membrane potential probes 3,3'-dipropyl-2,2'-thiadicarbocyanine iodide and tritiated triphenylmethylphosphonium bromide. The membrane potential in physiologic buffer was estimated to be 52-60 mV inside negative. The membrane was depolarized when extracellular potassium or hydrogen ion concentrations were increased. Changes in extracellular sodium, chloride, or calcium ion concentration had no measurable effect on membrane potential. Elevated extracellular potassium has been shown to increase platelet sensitivity to the aggregating agent, adenosine diphosphate. Our results show that changes in extracellular ion concentrations that depolarize platelets increase platelet sensitivity to aggregating agents. These results suggest that membrane potential changes may play a role in modulating the response of platelets to aggregating agents.

The membrane potential of human platelets

The membrane potential of the human platelet was investigated using the membrane potential probes 3,3′-dipropyl-2,2′-thiadicarbocyanine iodide and tritiated triphenylmethylphosphonium bromide. The membrane potential in physiologic buffer was estimated to be 52–60 mV inside negative. The membrane was depolarized when extracellular potassium or hydrogen ion concentrations were increased. Changes in extracellular sodium, chloride, or calcium ion concentration had no measurable effect on membrane potential. Elevated extracellular potassium has been shown to increase platelet sensitivity to the aggregating agent, adenosine diphosphate. Our results show that changes in extracellular ion concentrations that depolarize platelets increase platelet sensitivity to aggregating agents. These results suggest that membrane potential changes may play a role in modulating the response of platelets to aggregating agents.

Cell membrane potential and resistance in liver.

1. Isolated segments of mouse liver were placed in a Perspex bath through which physiological saline solutions of varying composition were circulated. Two microelectrodes were inserted in different liver cells under microscopic control allowing measurement of distance between the two micro-electrode tips. Current pulses were injected through one of these electrodes, causing electrotonic potential changes in nearby cells by current spread through intercellular junctions. These electrotonic potential changes were recorded with the second micro-electrode. The spatial decrement of the amplitude of the electrotonzpotential changes and their dependence on extracellular ion concentrations were analysed by three-dimensional cable analysis, modified to account for the geometry of the tissue. 2. During exposure to control solution the mean resting cell membrane potential was -37 mV, the space constant for intracellular current spread (lambda3 = square root of Rm/chrRi) was 390 micron and Ri, a measure which includes the intracellular resistivity and the junctional resistances, was 1.4 komegacm. From these values, and an estimate of tissue cell membrane density (chi) obtained by others, the specific membrane resistance (Rm) was calculated to be 5.1 komegacm2. 3. Replacement of extracellular Na+ by K+ resulted in a large depolarization and a large decrease in the membrane resistance. Replacement of extracellular Na+ by choline resulted in a small transient hyperpolarization and a small increase in the membrane resistance. Replacement of extracellular Cl- by methylsulphate or sulphate or of NaCl by sucrose resulted in a small transient depolarization and a large increase in the membrane resistance. 4. Glucagon (10(-7) M) and adrenaline (10(-5) M) evoked membrane hyperpolarization and reduction of membrane resistance (Rm). 5. The resting membrane ion conductance can be considered to consist of three components, Cl conductance (GCl), GK and GNa. The results suggest that GCl greater than GK greater than GNa. Changes in extracellular ion concentrations specifically alter the permeability properties of the cell membrane. The glucagon action can be explained in part by an increase in GK.