Spatial Buffering Currents Research Articles

Glial K+ Clearance and Cell Swelling: Key Roles for Cotransporters and Pumps

An important feature of neuronal signalling is the increased concentration of K(+) in the extracellular space. The K(+) concentration is restored to its original basal level primarily by uptake into nearby glial cells. The molecular mechanisms by which K(+) is transferred from the extracellular space into the glial cell are debated. Although spatial buffer currents may occur, their quantitative contribution to K(+) clearance is uncertain. The concept of spatial buffering of K(+) precludes intracellular K(+) accumulation and is therefore (i) difficult to reconcile with the K(+) accumulation repeatedly observed in glial cells during K(+) clearance and (ii) incompatible with K(+)-dependent glial cell swelling. K(+) uptake into non-voltage clamped cultured glial cells is carried out by the Na(+)/K(+)-ATPase and the Na(+)/K(+)/Cl(-) cotransporter in combination. In brain slices and intact optic nerve, however, only the Na(+)/K(+)-ATPase has been demonstrated to be involved in stimulus-evoked K(+) clearance. The glial cell swelling associated with K(+) clearance is prevented under conditions that block the activity of the Na(+)/K(+)/Cl(-) cotransporter. The Na(+)/K(+)/Cl(-) cotransporter is activated by increased K(+) concentration and cotransports water along with its substrates. It thereby serves as a K(+)-dependent molecular water pump under conditions of increased extracellular K(+) load.

Switch of K + buffering conditions in rabbit retinal Müller glial cells during postnatal development

Although spatial buffering of excess extracellular K + by K + channels is a main function of retinal glial (Müller) cells, there are severe limitations to long distance K +-spatial buffering that have been predicted for (immature) glial cells: (i) a lack of inwardly rectifying K + (Kir) channels [Glia 21(1997) 46]; and (ii) high internal resistance of outgrowing (cable like) processes [W. Rall, Handbook of Physiology, Section 1, vol. 1, Part 1, American Physiological Society, Bethesda, 1977, pp. 39–97]. In order to determine if changes in developing Müller cells improve or worsen their capability of carrying K + spatial buffering currents, we compared the whole-cell currents of acutely isolated Müller cells at 5, 11 and 28 postnatal days of rabbits. Both K +-spatial buffer limitations described above were found in early postnatal stage (5 days), however, the cells overcome these limitations shortly after 11 days. During the period of 11–28 days, rabbit Müller cells simultaneously increase stalk axial conductance and express Kir channels. Both processes take place during the critical stage of retinal maturation, and should dramatically improve “cable” K +-spatial buffering.

Scotopic threshold response changes after vigabatrin therapy in a child without visual field defects: a new electroretinographic marker of early damage?

Vigabatrin (VGB) has been widely used in patients affected by drug-resistant epilepsy and West syndrome. Following reports of visual field loss associated with vigabatrin therapy, some authors have investigated retinal electrophysiologic variables to identify early electrophysiologic markers and pathogenetic mechanisms of retinal damage. There are no previous reports of a scotopic threshold response (STR) reduction associated with vigabatrin therapy. A 13-year-old male child was submitted to a complete electroretinographic study before and after the start of vigabatrin therapy. Of the electroretinographic responses analyzed, only the scotopic threshold response was altered. The scotopic threshold response is a corneal-negative wave in the electroretinogram (ERG) of a fully dark-adapted eye. In cat, this response has been shown to be mediated by K+ spatial buffer currents that flow from proximal to distal retina in retinal glia as a result of elevated concentration of K+ in proximal retina following depolarization of local neurons in response to light onset. The prospective nature of the study in a previously untreated patient on vigabatrin monotherapy allows us to speculate on the underlying pathogenetic mechanisms and level of action of vigabatrin therapy-related retinal damage. If the predictive value of the scotopic threshold response changes is documented, this ERG response could be used to perform a preliminary evaluation of drugs, which modify gamma-aminobutyric acid (GABA) receptors and/or GABA levels.

Kir potassium channel subunit expression in retinal glial cells: implications for spatial potassium buffering.

To understand the role of different K(+) channel subtypes in glial cell-mediated spatial buffering of extracellular K(+), immunohistochemical localization of inwardly rectifying K(+) channel subunits (Kir2.1, Kir2.2, Kir2.3, Kir4.1, and Kir5.1) was performed in the retina of the mouse. Stainings were found for the weakly inward-rectifying K(+) channel subunit Kir4.1 and for the strongly inward-rectifying K(+) channel subunit Kir2.1. The most prominent labeling of the Kir4.1 protein was found in the endfoot membranes of Müller glial cells facing the vitreous body and surrounding retinal blood vessels. Discrete punctate label was observed throughout all retinal layers and at the outer limiting membrane. By contrast, Kir2.1 immunoreactivity was located predominantly in the membrane domains of Müller cells that contact retinal neurons, i.e., along the two stem processes, over the soma, and in the side branches extending into the synaptic layers. The results suggest a model in which the glial cell-mediated transport of extracellular K(+) away from excited neurons is mediated by the cooperation of different Kir channel subtypes. Weakly rectifying Kir channels (Kir4.1) are expressed predominantly in membrane domains where K(+) currents leave the glial cells and enter extracellular "sinks," whereas K(+) influxes from neuronal "sources" into glial cells are mediated mainly by strongly rectifying Kir channels (Kir 2.1). The expression of strongly rectifying Kir channels along the "cables" for spatial buffering currents may prevent an unwarranted outward leak of K(+), and, thus, avoid disturbances of neuronal information processing.

Electrical coupling between glial cells in the rat retina.

The strength of electrical coupling between retinal glial cells was quantified with simultaneous whole-cell current-clamp recordings from astrocyte-astrocyte, astrocyte-Müller cell, and Müller cell-Müller cell pairs in the acutely isolated rat retina. Experimental results were fit and space constants determined using a resistive model of the glial cell network that assumed a homogeneous two-dimensional glial syncytium. The effective space constant (the distance from the point of stimulation to where the voltage falls to 1/e) equaled 12.9, 6.2, and 3.7 microm, respectively for astrocyte-astrocyte, astrocyte-Müller cell, and Müller cell-Müller cell coupling. The addition of 1 mM Ba(2+) had little effect on network space constants, while 0.5 mM octanol shortened the space constants to 4.7, 4.4, and 2.6 microm for the three types of coupling. For a given distance separating cell pairs, the strength of coupling showed considerable variability. This variability in coupling strength was reproduced accurately by a second resistive model of the glial cell network (incorporating discrete astrocytes spaced at varying distances from each other), demonstrating that the variability was an intrinsic property of the glial cell network. Coupling between glial cells in the retina may permit the intercellular spread of ions and small molecules, including messengers mediating Ca(2+) wave propagation, but it is too weak to carry significant K(+) spatial buffer currents.

Current source density analysis of the electroretinographic d wave of frog retina

1. Dark flashes were used to evoke spatiotemporal profiles of the d wave of the electroretinogram in frog retina. The current source density technique was used to compute the extracellular current sinks and sources underlying the d wave. 2. The largest d wave current was a sink at the outer plexiform layer (OPL sink) and a source at the inner plexiform layer (IPL source). Several properties of this sink/source, including its relative insensitivity to Ba2+, suggest that it arises from hyperpolarizing bipolar cells. 3. There was a slower IPL sink, along with a source at the inner limiting membrane (ILM source). Its properties, along with its sensitivity to Ba2+ and enhancement by picrotoxin, suggest it arises from the K+ spatial buffer currents of Müller cells. This IPL sink/ILM source underlies the M wave. 4. A small photoreceptor sink/source also contributes to the d wave. This is most significant during the very beginning of the d wave. 5. Our results indicate that in frog retina and at the stimulus conditions used in this paper, the d wave originates primarily and directly from the hyperpolarizing bipolar cells. Photoreceptors likely contribute a small amount, especially during the initial portion of the d wave. There is possibly a small Müller cell contribution.

Patch-clamp recordings from Müller (glial) cell endfeet in the intact isolated retina and acutely isolated Müller cells of mouse and guinea-pig

Müller cells span through the entire retina and terminate with the formation of endfeet at the vitreous body. These endfeet are thought to be specialized for maintaining the K + homeostasis in the retina based on the assumption that voltage signals can passively spread from the cell body to the endfeet. We employed the patch-clamp technique to study the physiological properties of these endfeet in a retinal wholemount preparation from guinea-pig or mouse. After assessing one endfoot with the patch pipette and establishing the whole cell recording configuration, a membrane area which approximately matched the size of one endfoot and proximal process could be voltage-clamped. This morphological correlation could be established by filling the cytoplasm with the fluorescent dye Lucifer Yellow via the patch-pipette. The morphological, immunocytochemical and ultrastructural inspection of the recorded cells revealed that mouse Müller cell endfeet were connected by only a thin stalk to the proximal process. In contrast, guinea-pig endfeet were connected by thick stalks. The endfoot current in the mouse was dominated by a voltage and time-independent K + conductance. In contrast, in some of the recordings from guinea-pig, delayed and inwardly rectifying K + currents were observed. These voltage-gated currents were more frequently observed or were facilitated when the membrane area under voltage clamp was increased, blocking the passive K + currents by Ba 2+ in both, mouse and guinea-pig. We thus assume that the voltage-gated currents were not in the endfeet membrane, but rather in the proximal process and could thus be better activated in the guinea-pig with its thicker stalk or after increasing the membrane area under voltage clamp control. Similar results were obtained in freshly isolated Müller cells; in contrast to the cells from the wholemount the voltage-gated currents were more frequently observed. These studies demonstrate that the Müller cell endfoot of the mouse with its vascularized retina is an electrically isolated unit and that voltage signals do not spread to the proximal process. Such a property would, however, be required for the redistribution of K + via spatial buffer currents. In contrast, guinea-pig Müller glial cells with their stout morphological connection between endfoot and proximal process are better suited to fulfil this task.

What do retinal Müller (glial) cells do for their neuronal ‘small siblings’?

Müller (radial glial) cells are the predominant glia of the vertebrate retina. They arise, together with rod photoreceptor cells, bipolar cells, and a subset of amacrine cells, from common precursor cells during a late proliferative phase. One Müller cell and a species-specific number of such neurons seem to form a columnar unit within the retinal tissue. In contrast, ‘extracolumnar neurons’ (ganglion cells, cone photoreceptor cells, horizontal cells, and another subset of amacrine cells) are born and start differentiation before most Müller cells are generated. It may be essential for such neurons to develop metabolic capacities sufficient to support their own survival, whereas late-born (‘columnar’) neurons seem to depend on a nursing function of their ‘sisterly’ Müller cell. Thus, out of the cell types within a retinal column it is exclusively the Müller cell that possesses the enzymes for glycogen metabolism. We present evidence that Müller cells express functional insulin receptors. Furthermore, isolated Müller cells rapidly hydrolyse glycogen when they are exposed to an elevated extracellular K +ion concentration, a signal that is involved in the regulation of neuronal-glial metabolic cooperation in the brain. Müller cells are also thought to be essential for rapid and effective retinal K + homeostasis. We present patch-clamp measurements on Müller cells of various vertebrate species that all demonstrate inwardly rectifying K + channels; this type of channel is well-suited to mediate spatial buffering currents. A mathematical model is presented that allows estimation of Müller cell-mediated K + currents. A simulation analysis shows that these currents greatly limit lateral spread of excitation beyond the borders of light-stimulated retinal columns, and thus help to maintain visual acuity.

Spatial buffering of extracellular potassium by Müller (glial) cells in the toad retina

We examined the role of Müller (glial) cells in buffering light-evoked changes in extracellular K+ concentration, [K+]o, in the isolated retina of the toad, Bufo marinus. We found evidence for two opposing Müller cell current loops that are generated by a light-evoked increase in [K+]o in the inner plexiform layer. These current loops, which are involved in the generation of the M-wave of the electroretinogram (ERG), prevent the accumulation of K+ in the inner plexiform layer by transporting K+ both to vitreous and to distal retina. In addition, under dark-adapted conditions, we found evidence for a Müller cell current loop that is generated by a light-evoked decrease in [K+]o in the receptor layer. This current loop, which is involved in the generation of the slow PIII component of the ERG, helps to buffer the light-evoked decrease in [K+]o throughout distal retina by transporting K+ from vitreous. The spatial buffering fluxes of K+ can be abolished by blocking Müller cell K+ conductance with 200 microM Ba2+. The separate contributions of the M-wave and slow PIII currents to Müller cell spatial buffering were isolated by various pharmacological treatments that were designed to enhance or suppress light-evoked activity in specific retinal neurons. Our results show that Müller cell K+ currents not only buffer light-evoked increases in [K+]o, but also buffer light-evoked decreases in [K+]o, and thereby diminish any deleterious effects upon neuronal function that could arise in response to large changes in [K+]o in the plexiform layers. Moreover, our results emphasize that spatial buffering currents generate many components of the electroretinogram.

K+ ion regulation in retina.

During onset and offset of illumination, considerable changes in extracellular K+ concentration ([K+]c) occur within particular retinal layers. There are two ways in which glial cells may control [K+]c: (1) by space-independent processes, for example, by K+ uptake due to the Na(+)-K+ ATPase, and (2) by space-dependent processes, that is, by spatial buffering currents flowing through K+ channels. Rabbit retinal Müller (glial) cells were studied for expression of mechanisms supporting both kinds of processes. This review demonstrates that rabbit Müller cells have Na-K pumps whose distribution and properties are highly adapted to meet the needs of efficient K+ clearance. Furthermore, spatial buffering currents through specialized K+ channels of Müller cells greatly accelerate retinal K+ clearance during and after stimulation.

Spatial buffering of light-evoked potassium increases by retinal Müller (glial) cells.

Activity-dependent variations in extracellular potassium concentration in the central nervous system may be regulated, in part, by potassium spatial buffering currents in glial cells. The role of spatial buffering in the retina was assessed by measuring light-evoked potassium changes in amphibian eyecups. The amplitude of potassium increases in the vitreous humor was reduced to approximately 10 percent by 50 micromolar barium, while potassium increases in the inner plexiform layer were largely unchanged. The decrease in the vitreal potassium response was accurately simulated with a numerical model of potassium current flow through Müller cells, the principal glial cells of the retina. Barium also substantially increased the input resistance of Müller cells and blocked the Müller cell-generated M-wave, indicating that barium blocks the potassium channels of Müller cells. Thus, after a light-evoked potassium increase within the retina, there is a substantial transfer of potassium from the retina to the vitreous humor by potassium current flow through Müller cells.

Relations between slow extracellular potential changes, glial potassium buffering, and electrolyte and cellular volume changes during neuronal hyperactivity in cat brain

The aim of this investigation is to estimate the contribution of spatial glial K+ buffer currents to extracellular K+ homeostasis during enhanced neuronal activity. Neuronal hyperactivity was induced by electrical stimulation of the cortical surface or the ventrobasal thalamic nuclei of cats (5-50 Hz, 0.1-0.2 ms, two to three times threshold stimulation intensity, 5-20 s). The accompanying slow field potential changes were recorded simultaneously across the grey matter with vertical assemblies of eight micropipettes glued 300 microns apart. Using the Poisson equation, the amplitudes of the underlying current sources and sinks were calculated. The current source densities depended on the depth of recording, frequency, strength, and duration of the stimulation. Current sinks, corresponding to a removal of 0.1-0.5 mmoles of monovalent cations per liter of brain tissue and second from the extracellular space, were observed in middle cortical layers, whereas sources appeared at superficial and deeper sites. These sinks and sources might represent K+ moved across glial membranes by spatial buffer currents. The consequences of glial buffer currents of this magnitude were investigated with model calculations. It turned out that measurements of electrolyte and volume changes of the extracellular space (Dietzel et al. Exp. Brain Res. 40:432-439, 1980; Exp. Brain Res. 46:73-84, 1982) could only partially be explained by spatial buffer currents of this magnitude. Comparison of the calculated values with intracellular measurements in neurons and glial cells (Coles et al. Ann. N.Y. Acad. Sci. 481:303-317, 1986; Ballanyi et al. J. Physiol. 382:159-174, 1987) suggests that spatial buffering combines with an approximately equimolar KCl transport and, depending on the preparation, also K+/Na+-exchange across glial membranes.

Efficient K+ buffering by mammalian retinal glial cells is due to cooperation of specialized ion channels

Radial glial (Müller) cells were isolated from rabbit retinae by papaine and mechanical dissociation. Regional membrane properties of these cells were studied by using the patch-clamp technique. In the course of our experiments, we found three distinct types of large K+ conducting channels. The vitread process membrane was dominated by high conductance inwardly rectifying (HCR) channels which carried, in the open state, inward currents along a conductance of about 105 pS (symmetrical solutions with 140 mM K+) but almost no outward currents. In the membrane of the soma and the proximal distal process, we found low conductance inwardly rectifying (LCR) channels which had an open state-conductance of about 60 pS and showed rather weak rectification. The endfoot membrane, on the other hand, was found to contain non-rectifying very high conductance (VHC) channels with an open state-conductance of about 360 pS (same solutions). These results suggest that mammalian Müller cells express regional membrane specializations which are optimized to carry spatial buffering currents of excess K+ ions.

Single voltage-dependent K+ and Cl− channels in cultured rat astrocytes

The kinetic reactions of a voltage-dependent K+ channel, which constituted about 14% of all the recorded K+ channels in the membrane of cultured rat astrocytes were studied in detail. A scheme of one open and three closed states is necessary to describe the kinetic reactions of this channel. The channel contributes little to the resting membrane potential. Its steady state open probability (Po) is 0.06 at -70 mV. When the cell is depolarized to O mV, Po approaches 1. This represents a 17-fold increase. Such channels could contribute to the potassium clearance by enhancing the effect of "spatial buffering." Additionally, single anion-selective channels with very high conductances were found in inside-out patches in approximately 15% of all recorded channels in the membrane of rat astrocytes. Channel openings are characterized by more than one conductance level; the main level showed a mean conductance of 400 pS. These channels are divided into two groups. Approximately 90% of the recorded chloride channels showed a strong voltage dependency of their current fluctuations. Within a relatively small potential range (+/- 15 mV) the channels have a high probability of being in the active state. After a voltage jump to varying testing potentials in the range of +/- 20 to +/- 50 mV the channels continued to be in the active state for some time and then closed to a shut state. If the testing potential persisted, the channels were not able to leave this shut state.(ABSTRACT TRUNCATED AT 250 WORDS)

Regional Specialization of the Membrane of Retinal Glial Cells and Its Importance to K+ Spatial Bufferinga

Electrophysiological experiments, obtained primarily from dissociated salamander cells, demonstrate that the K+ conductance of Müller cells is distributed in a highly nonuniform manner over the cell surface. A large fraction of the total cell conductance is localized to that portion of the endfoot process that faces the vitreous humor. Along the remainder of the cell, specific K+ conductance is larger in the outer plexiform layer than in neighboring regions. High-endfoot conductance directs K+ spatial buffering currents preferentially through the endfoot process, leading to an efficient form of spatial buffering termed K+ siphoning. Preliminary experiments suggest that the endfeet of astrocytes also have high K+ conductance.