Concentrations In Extracellular Space Research Articles

Role of the STING-IRF3 pathway in ambient GABA homeostasis and cognitive function.

Targeting altered expression and/or activity of GABA transporters (GATs) provide therapeutic benefit for age-related impairments, including cognitive dysfunction. However, the mechanisms underlying the transcriptional regulation of GATs are unknown. In the present study, we demonstrated that the stimulator of interferon genes (STING) upregulates GAT1 and GAT3 expression in the brain which resulted in cognitive dysfunction. Genetic and pharmacological intervention of STING suppressed the expression of both GAT1 and GAT3, increased the ambient GABA concentration, and therefore, enhanced tonic GABAA inhibition of principal hippocampal neurons, resulting in spatial learning and working memory deficits in mice in a type I interferon (IFN I)-independent manner. Stimulation of the STING-GAT pathway efficiently restored cognitive dysfunction in STING-deficient mice models. Our study uncovered for the first time that the STING signaling pathway regulates GATs expression in a cell autonomous manner and therefore could be a novel target for GABAergic cognitive deficits.Significance Statement GABA concentration in extracellular space is maintained by GABA release and clearance of GABA back to brain cells for degradation. GABA clearance from the synaptic cleft predominantly depends on level and activity of GABA transporters (GATs) in the brain. Insufficient GABA clearance resulted to an aberrant tonic GABAA inhibition in brain. In this study, we have identified an unusually high GABA content in brain of STING-deficient mice, resulting in cognitive impairment. Our results show that STING regulates GATs expression through STING-TBK1-IRF3 pathway and thus regulates GABAergic tone. This is the first study that indicates that the STING-TBK1-IRF3 signaling pathway maintains GABA homeostasis in brain, which may offer a novel therapeutic target for modulating GABAergic tone in cases of cognitive dysfunction.

New Pharmacokinetic Parameters of Imaging Substrates Quantified from Rat Liver Compartments.

Hepatobiliary imaging is increasingly used by pharmacologists to quantify liver concentrations of transporter-dependent drugs. However, liver imaging does not quantify concentrations in extracellular space, hepatocytes, and bile canaliculi. Our study compared the compartmental distribution of two hepatobiliary substrates gadobenate dimeglumine [BOPTA; 0.08 liver extraction ratio (ER)] and mebrofenin (MEB; 0.93 ER) in a model of perfused rat liver. A gamma counter placed over livers measured liver concentrations. Livers were preperfused with gadopentetate dimeglumine to measure extracellular concentrations. Concentrations coming from bile canaliculi and hepatocytes were calculated. Transporter activities were assessed by concentration ratios between compartments and pharmacokinetic parameters that describe the accumulation and decay profiles of hepatocyte concentrations. The high liver concentrations of MEB relied mainly on hepatocyte and bile canaliculi concentrations. In contrast, the three compartments contributed to the low liver concentrations obtained during BOPTA perfusion. Nonlinear regression analysis of substrate accumulation in hepatocytes revealed that cellular efflux is measurable ∼4 minutes after the start of perfusion. The hepatocyte-to-extracellular concentration ratio measured at this time point was much higher during MEB perfusion. BOPTA transport by multidrug resistance associated protein 2 induced an aquaporin-mediated water transport, whereas MEB transport did not. BOPTA clearance from hepatocytes to bile canaliculi was higher than MEB clearance. MEB did not efflux back to sinusoids, whereas BOPTA basolateral efflux contributed to the decrease in hepatocyte concentrations. In conclusion, our ex vivo model quantifies substrate compartmental distribution and transport across hepatocyte membranes and provides an additional understanding of substrate distribution in the liver. SIGNIFICANCE STATEMENT: When transporter-dependent drugs target hepatocytes, cellular concentrations are important to investigate. Low concentrations on cellular targets impair drug therapeutic effects, whereas excessive hepatocyte concentrations may induce cellular toxicity. With a gamma counter placed over rat perfused livers, we measured substrate concentrations in the extracellular space, hepatocytes, and bile canaliculi. Transport across hepatocyte membranes was calculated. The study provides an additional understanding of substrate distribution in the liver.

Excitability and propagation of the electrical impulse in Venus flytrap; a comparative electrophysiological study of unipolar electrograms with myocardial tissue

Mammalian heart cells and cells of leaves of Dionaea muscipula share the ability to generate propagated action potentials, because the excitable cells are electrically coupled. In the heart the propagated action potential causes synchronized contraction of the heart muscle after automatic generation of the impulse in the sinus node. In Dionaea propagation results in closure of the trap after activation of trigger hairs by an insect. The electrical activity can be recorded in the extracellular space as an extracellular electrogram, resulting from transmembrane currents. Although the underlying physiological mechanism that causes the electrogram is similar for heart and Dionaea cells, the contribution of the various ions to the transmembrane current is different. We recorded extracellular electrograms from Dionaea leaves and compared the recorded signals with those known from the heart. The morphology of the electrograms differed considerably. In comparison to activation in mammalian myocardium, electrograms of Dionaea are more temporally and spatially variable. Whereas electrograms in healthy myocardium recorded at some distance from the site of activation reveal a simple biphasic pattern, Dionaea activation showed positive, negative or biphasic deflections. Comparison of patch clamp data from plant cells and cardiomyocytes suggests a role of temperature and ion concentrations in extracellular space for the diversity of morphologies of the Dionaea electrograms.

Spatiotemporal regulation of GABA concentration in extracellular space by gliotransmission crucial for extrasynaptic receptor-mediated improvement of sensory tuning performance in schizophrenia.

In schizophrenic patients, sensory tuning performance tends to be deteriorated (i.e., flattened sensory tuning), for which impaired intracortical tonic inhibition arising from a reduction in GABA concentration in extracellular space might be responsible. The δ subunit-containing GABAA receptor, located on extrasynaptic sites, is known to be involved in mediating tonic inhibitory currents in cortical pyramidal cells and is considered to be one of the beneficial therapeutic targets for the treatment of schizophrenia. The transporter GAT-1 in glial (astrocytic) membrane controls concentration of GABA molecules by removing them from extracellular space. We speculated that the upregulation of extrasynaptic receptors might compensate for the impaired tonic inhibition and thus improve their sensory tuning performance, in which the astrocytic GABA transporter might play an important role. To test our hypothesis, we simulated a schizophrenic neural network model with a GABAergic gliotransmission (i.e., GABA transport by transporters embedded in astrocytic membranes) mechanism that modulates local ambient (extracellular) GABA levels in a neuronal activity-dependent manner. Upregulating extrasynaptic GABA receptors compensated the impaired tonic inhibition and sharpened the sensory tuning, provided that ambient GABA molecules around stimulus-sensitive pyramidal cells were actively removed during sensory stimulation. We suggest that the upregulation of extrasynaptic GABA receptors can improve the performance of sensory tuning in schizophrenic patients, for which spatiotemporal regulation of ambient GABA concentration by gliotransmission may be crucial.

Metabolism plays a central role in the cortical spreading depression: Evidence from a mathematical model

The slow propagating waves of strong depolarization of neural cells characterizing cortical spreading depression, or depolarization, (SD) are known to break cerebral homeostasis and induce significant hemodynamic and electro-metabolic alterations. Mathematical models of cortical spreading depression found in the literature tend to focus on the changes occurring at the electrophysiological level rather than on the ensuing metabolic changes. In this paper, we propose a novel mathematical model which is able to simulate the coupled electrophysiology and metabolism dynamics of SD events, including the swelling of neurons and astrocytes and the concomitant shrinkage of extracellular space. The simulations show that the metabolic coupling leads to spontaneous repetitions of the SD events, which the electrophysiological model alone is not capable to produce. The model predictions, which corroborate experimental findings from the literature, show a strong disruption in metabolism accompanying each wave of spreading depression in the form of a sharp decrease of glucose and oxygen concentrations, with a simultaneous increase in lactate concentration which, in turn, delays the clearing of excess potassium in extracellular space. Our model suggests that the depletion of glucose and oxygen concentration is more pronounced in astrocyte than neuron, in line with the partitioning of the energetic cost of potassium clearing. The model suggests that the repeated SD events are electro-metabolic oscillations that cannot be explained by the electrophysiology alone. The model highlights the crucial role of astrocytes in cleaning the excess potassium flooding extracellular space during a spreading depression event: further, if the ratio of glial/neuron density increases, the frequency of cortical SD events decreases, and the peak potassium concentration in extracellular space is lower than with equal volume fractions.

A Kirchhoff-Nernst-Planck framework for modeling large scale extracellular electrodiffusion surrounding morphologically detailed neurons.

Many pathological conditions, such as seizures, stroke, and spreading depression, are associated with substantial changes in ion concentrations in the extracellular space (ECS) of the brain. An understanding of the mechanisms that govern ECS concentration dynamics may be a prerequisite for understanding such pathologies. To estimate the transport of ions due to electrodiffusive effects, one must keep track of both the ion concentrations and the electric potential simultaneously in the relevant regions of the brain. Although this is currently unfeasible experimentally, it is in principle achievable with computational models based on biophysical principles and constraints. Previous computational models of extracellular ion-concentration dynamics have required extensive computing power, and therefore have been limited to either phenomena on very small spatiotemporal scales (micrometers and milliseconds), or simplified and idealized 1-dimensional (1-D) transport processes on a larger scale. Here, we present the 3-D Kirchhoff-Nernst-Planck (KNP) framework, tailored to explore electrodiffusive effects on large spatiotemporal scales. By assuming electroneutrality, the KNP-framework circumvents charge-relaxation processes on the spatiotemporal scales of nanometers and nanoseconds, and makes it feasible to run simulations on the spatiotemporal scales of millimeters and seconds on a standard desktop computer. In the present work, we use the 3-D KNP framework to simulate the dynamics of ion concentrations and the electrical potential surrounding a morphologically detailed pyramidal cell. In addition to elucidating the single neuron contribution to electrodiffusive effects in the ECS, the simulation demonstrates the efficiency of the 3-D KNP framework. We envision that future applications of the framework to more complex and biologically realistic systems will be useful in exploring pathological conditions associated with large concentration variations in the ECS.

Astrocytic Mechanisms Explaining Neural-Activity-Induced Shrinkage of Extraneuronal Space

Neuronal stimulation causes ∼30% shrinkage of the extracellular space (ECS) between neurons and surrounding astrocytes in grey and white matter under experimental conditions. Despite its possible implications for a proper understanding of basic aspects of potassium clearance and astrocyte function, the phenomenon remains unexplained. Here we present a dynamic model that accounts for current experimental data related to the shrinkage phenomenon in wild-type as well as in gene knockout individuals. We find that neuronal release of potassium and uptake of sodium during stimulation, astrocyte uptake of potassium, sodium, and chloride in passive channels, action of the Na/K/ATPase pump, and osmotically driven transport of water through the astrocyte membrane together seem sufficient for generating ECS shrinkage as such. However, when taking into account ECS and astrocyte ion concentrations observed in connection with neuronal stimulation, the actions of the Na+/K+/Cl− (NKCC1) and the Na+/HCO3 − (NBC) cotransporters appear to be critical determinants for achieving observed quantitative levels of ECS shrinkage. Considering the current state of knowledge, the model framework appears sufficiently detailed and constrained to guide future key experiments and pave the way for more comprehensive astroglia–neuron interaction models for normal as well as pathophysiological situations.

Zinc Supplementation or Regulation of its Homeostasis: Advantages and Threats

To accomplish its multifunctional biological roles, zinc requires precise homeostatic mechanisms. There are efficient mechanisms that regulate zinc absorption from the alimentary tract and its excretion by the kidney depending on the organism demands. The regulatory mechanisms of cellular zinc inflow, distribution, and zinc outflow are so efficient that symptoms of zinc deficiency are rare, and symptoms connected with its massive accumulation are even more rare. The efficiency of homeostatic mechanisms that prevent zinc deficiency or excessive zinc accumulation in the organism is genetically conditioned. It seems that an essential element of zinc homeostasis is the efficiency of zinc transmembrane exchange mechanisms. Intracellular free zinc concentration is higher than in extracellular space. Physiologically, the active outflow of zinc ions from the cell depends on the increase of its concentration in extracellular space. The ion pumps activity depends on the efficiency by which the cell manages energy. Considering the fact that zinc deficiency accelerates apoptosis and that excessive zinc accumulation inside cells results in a toxic effect that forces its death brings about several questions: Is intensification and acceleration of changes in zinc metabolism with age meaningful? Is there a real zinc deficiency occurring with age or in connection with the aforementioned pathological processes, or is it just a case of tissue and cell redistribution? When discussing factors that influence zinc homeostasis, can we consider zinc supplementation or regulation of zinc balance in the area of its redistribution? To clarify these aspects, an essential element will also be the clear understanding of the nomenclature used to describe changes in zinc balance. Zinc homeostasis can be different in different age groups and depends on sex, thus zinc dyshomeostasis refers to changes in its metabolism that deviate from the normal rates for a particular age group and sex. This concept is very ample and implies that zinc deficiency may result from a low-zinc diet, poor absorption, excessive loss of zinc, zinc redistribution in intra- and extracellular compartments, or a combination of these factors that is inadequate for the given age and sex group. Such factor or factors need to be considered for preventing particular homeostasis disorders (or dyshomeostasis). Regulation of zinc metabolism by influencing reversal of redistribution processes ought to be the main point of pharmacologic and nonpharmacologic actions to reestablish zinc homeostasis. Supplementation and chelation are of marginal importance and can be used to correct long-term dietary zinc deficiency or zinc poisoning or in some cases in therapeutic interventions. In view of its biological importance, the problem posed by the influence of zinc metabolism requires further investigation. To date, one cannot consider, for example, routine zinc supplementation in old age, because changes of metabolism with age are not necessarily a cause of zinc deficiency. Supplementation is warranted only in cases in which deficiency has been established unambiguously. An essential element is to prevent sudden changes in zinc metabolism, which lead to dyshomeostasis in the terms defined here. The primary prophylaxes, regular physical activity, efficient treatment of chronic diseases, are all elements of such prevention.

Comparison of Brain Extracellular Fluid, Brain Tissue, Cerebrospinal Fluid, and Serum Concentrations of Antiepileptic Drugs Measured Intraoperatively in Patients with Intractable Epilepsy

The mechanisms of drug resistance in epilepsy are only incompletely understood. According to a current concept, overexpression of drug efflux transporters at the blood-brain barrier may reduce levels of antiepileptic drugs (AEDs) in epileptogenic brain tissue. Increased expression of drug efflux transporters such as P-glycoprotein has been found in brain tissue surgically resected from patients with medically intractable epilepsy, but it is not known whether this leads to decreased extracellular (interstitial) AED concentrations in affected brain regions. This prompted us to measure concentrations of AEDs in the extracellular space of human neocortical tissue by using intraoperative microdialysis (IOMD) in those parts of the brain that had to be removed for therapeutic reasons. For comparison, AED levels were determined in brain tissue, subarachnoid CSF, and serum. Concentrations of carbamazepine (CBZ), 10-hydroxy-carbazepine (10-OH-CZ, metabolite of oxcarbazepine), lamotrigine (LTG), levetiracetam (LEV), topiramate, or phenytoin were determined by using one to four catheters during IOMD in the medial temporal gyrus. Furthermore, to calculate the individual recovery of every catheter, an in vitro microdialysis was performed with ultrafiltrate of serum concurrently obtained from the respective patient. In addition, AED levels were determined in the resected brain tissue, CSF, and serum of the same patients. Altogether 22 pharmacoresistant epilepsy patients (nine male, 13 female patients; age 15-54 years) with complex partial seizures or secondarily generalized seizures were involved. In a first series, IOMD samples 40 min after beginning of the microdialysis (flow rate, 1 microl/min), and in a second series, continuous measurements 25, 30, 35, and 40 min from the beginning were evaluated (flow rate, 2 microl/min). With in vitro recovery data of the individual catheters, the concentration in the extracellular space (ECS) was estimated. AED concentrations in the ECS of the cortex measured by catheters located at a distance of 0.6 cm differed markedly in some patients, whereas concentrations in the ultrafiltrate of the serum of the respective patients measured with the same catheters varied only slightly. Furthermore, ECS concentrations related to the ultrafiltrate of serum showed considerable interindividual variations. The high intra- and interindividual variation of ECS concentrations is demonstrated by the low correlation between concentrations in ECS and the ultrafiltrate of serum (CBZ, r= 0.41; 10-OH-CZ, r= 0.42; LTG, r= 0.27) in contrast to the high correlation between brain tissue concentration and the ultrafiltrate of serum (CBZ, r= 0.97; 10-OH-CZ, r= 0.88; LTG, r= 0.98) in the same group of patients. When comparing AED concentrations in the ECS with those in the CSF, ECS concentrations were significantly lower for CBZ, 10-OH-CZ, LTG, and LEV. The data demonstrate that AED concentrations show a considerable intraindividual and interindividual variation in the ECS of cortical regions. Furthermore, the ECS concentration of several AEDs is significantly lower than their CSF concentration in patients with intractable epilepsy. However, in the absence of data from nonepileptic tissues, it is not possible to judge whether the present findings relate to overexpression of multidrug transporters in the brain. Instead, the present study illustrates the methodologic difficulties involved in performing IOMD studies in patients and may thus be helpful for future approaches aimed at elucidating the role of multidrug transporters in epilepsy.

Temporal profile of changes in brain tissue extracellular space and extracellular ion (Na(+), K(+)) concentrations after cerebral ischemia and the effects of mild cerebral hypothermia.

Cerebral ischemic cellular swelling occurs primarily in astrocytes. This water influx into the intracellular space is believed to result from osmotic water movement after disruption of membrane ionic homeostasis. However, cellular swelling occurs earlier than expected after ischemia and new ionic and water channels have been discovered. This study examined the temporal profile of the water and ionic movement across the cell membrane after global ischemia by measuring the changes in extracellular space (ECS), extracellular K(+) and Na(+) ion concentrations ([K(+)](e) and [Na(+)](e)) using a high resolution tissue impedance probe and ion selective micropipettes in the rat cortex. The effect of mild cerebral hypothermia (31.5 +/- 2.6 degrees C brain temperature) on these parameters was also examined. The ECS started to decrease at 34 +/- 8 sec after global ischemia and reached half the maximum change at 61 +/- 17 sec. [K(+)](e) started to increase initially at 33 +/- 11 sec (phase 1) and then increased rapidly at 62 +/- 25 sec (phase 2), and [Na(+)](e) started to decrease at 88 +/- 27 sec after ischemia. With mild hypothermia, the ECS started to decrease at 75 +/- 35 sec after ischemia and reached half the maximum change at 123 +/- 44 sec, [K(+)](e) started to increase initially at 80 +/- 24 sec (phase 1) and then increased rapidly at 120 +/- 32 sec (phase 2), and [Na(+)](e) started to decrease at 172 +/- 70 sec. The present study shows that ischemic cellular swelling (decreased ECS) occurs concomitantly with the phase 1 increase of [K(+)](e) but precedes the disruption of ionic membrane homeostasis (phase 2). Mild hypothermia prolongs the onset of these phenomena but does not affect the magnitude of the changes in ECS and ion concentrations.

Light scattering in rat neocortical slices differs during spreading depression and ischemia

Spreading depression (SD) and ischemia are different pathophysiological events but have similar characteristics. This study investigated whether similarity exists in the light scattering (LS) properties during SD and ischemia in rat neocortical slices. SD was induced by injection of K + while ischemia was simulated by removing oxygen and glucose. LS was simultaneously recorded with changes in extracellular direct current (DC) potential and extracellular space (ECS) volume. LS was measured using a photon counting fiber optic system and the ECS volume change was determined by measuring the ECS concentration of tetramethylammonium (TMA +). Slices maintained in normal artificial cerebrospinal fluid (ACSF) showed a consistent LS increase during SD, but exhibited two different LS behaviors during 6 min of ischemia. In eight slices, LS decreased and remained so until the end of the ischemic challenge. In another 10 slices, LS diminished initially but, after 2 min, suddenly reversed sign, accompanied by a rapid negative shift in extracellular DC potential. When 50 or 91% of Cl − in the ACSF was replaced by membrane-permeable propionate, LS retained its increase during SD, but always showed the sudden LS reversal during ischemia. In contrast, when Cl − was substituted with membrane-impermeant methylsulfate, the SD-induced LS increase was replaced by an LS decrease, and the sudden LS reversal during ischemia was absent. While the LS signal showed different characteristics during SD and ischemia, the DC potential always presented negative shifts and the ECS volume always exhibited similar decreases. These results suggest that the polarity of the LS signal is determined by the competition of at least two factors: cell swelling and anion influx.

Glucose concentration in subcutaneous extracellular space.

To compare the subcutaneous glucose sensor measurements with two reference methods. Previous studies provide conflicting findings about the real glucose concentrations in subcutaneous tissue. Some suggest substantially lower concentration, whereas others measure proportionally higher glucose concentrations compared with the blood compartment. Before these results can be taken seriously as an expression of the real glucose concentration in the extracellular space, the measurements must be validated by an independent method. We applied a microdialysis-based enzyme sensor to measure glucose concentration in subcutaneous tissue. We also developed two reference methods: subcutaneous filtrate collection and an equilibration method using ultrafiltration membranes to support the earlier findings. We provided an anatomical model to explain the results. The mean overall intercellular filtrate glucose concentration, sampled with the filtrate collector and taken after a 6-h stabilization time, including the values during the glucose clamp period, was 46 +/- 9%. The mean subcutaneous glucose concentration measured with the glucose sensor, calibrated in vitro, was 44 +/- 8% of the mean venous blood glucose concentration. Mean overall intercellular equilibrate glucose concentration, i.e., the mean glucose concentration in the subcutaneous extracellular space, taken after a 4-h stabilization time, was 46 +/- 15% of the mean venous blood glucose concentration. The close agreement between the mean values of subcutaneous glucose concentrations, obtained with three independent methods--filtration, equilibration, and dialysis (sensor)--shows the real glucose concentration in subcutaneous interstitial fluid is approximately 50% the blood glucose value in normal humans. Our results clarify some of the conflicting evidence presented in previous studies.

Mode d'action du pentetrazole au niveau cellulaire

The question of the origin of the paroxysmal depolarization shifts which can be recorded from neurones within an experimental epileptogenic focus is still controversial. In this paper, a special reference is made to data obtained in thin hippocampal slices in vitro during intra- and extracellular recordings from CA1 pyramidal cell somas before and after pentylenetetrazole (PTZ) application onto the slice surface. It is shown that PTZ can decrease inhibitory potentials, increase membrane input resistance and increase action potential duration. Using calcium-selective microelectrodes, it is shown that PTZ can enhance the lowering of calcium ion concentration in extracellular space, which is normally induced by repetitive stimulations. These data, as well as those from others, support the hypothesis that paroxysmal depolarization shifts seen in hippocampus could result from the facilitation of a slow depolarizing calcium current through the cell membrane uncovered by a depression of repolarizing potassium current produced by the convulsants. The observed depression of inhibitory potential would further facilitate the development of long-lasting depolarizing potentials.