Potassium buffering in the central nervous system.

Rapid changes in extracellular K+ concentration ([K+](o)) in the mammalian CNS are counteracted by simple passive diffusion as well as by cellular mechanisms of K+ clearance. Buffering of [K+](o) can occur via glial or neuronal uptake of K+ ions through transporters or K+-selective channels. The best studied mechanism for [K+](o) buffering in the brain is called K+ spatial buffering, wherein the glial syncytium disperses local extracellular K+ increases by transferring K+ ions from sites of elevated [K+](o) to those with lower [K+](o). In recent years, K+ spatial buffering has been implicated or directly demonstrated by a variety of experimental approaches including electrophysiological and optical methods. A specialized form of spatial buffering named K+ siphoning takes place in the vertebrate retina, where glial Muller cells express inwardly rectifying K+ channels (Kir channels) positioned in the membrane domains near to the vitreous humor and blood vessels. This highly compartmentalized distribution of Kir channels in retinal glia directs K+ ions from the synaptic layers to the vitreous humor and blood vessels. Here, we review the principal mechanisms of [K+](o) buffering in the CNS and recent molecular studies on the structure and functions of glial Kir channels. We also discuss intriguing new data that suggest a close physical and functional relationship between Kir and water channels in glial cells.

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.

Propagation of intercellular calcium waves in retinal astrocytes and Müller cells.

Intercellular Ca(2+) waves are believed to propagate through networks of glial cells in culture in one of two ways: by diffusion of IP(3) between cells through gap junctions or by release of ATP, which functions as an extracellular messenger. Experiments were conducted to determine the mechanism of Ca(2+) wave propagation between glial cells in an intact CNS tissue. Calcium waves were imaged in the acutely isolated rat retina with the Ca(2+) indicator dye fluo-4. Mechanical stimulation of astrocyte somata evoked Ca(2+) waves that propagated through both astrocytes and Müller cells. Octanol (0.5 mm), which blocks coupling between astrocytes and Müller cells, did not reduce propagation into Müller cells. Purinergic receptor antagonists suramin (100 microm), PPADS (20-50 microm), and apyrase (80 U/ml), in contrast, substantially reduced wave propagation into Müller cells (wave radii reduced to 16-61% of control). Suramin also reduced wave propagation from Müller cell to Müller cell (51% of control). Purinergic antagonists reduced wave propagation through astrocytes to a lesser extent (64-81% of control). Mechanical stimulation evoked the release of ATP, imaged with the luciferin-luciferase bioluminescence assay. Peak ATP concentration at the surface of the retina averaged 78 microm at the stimulation site and 6.8 microm at a distance of 100 microm. ATP release propagated outward from the stimulation site with a velocity of 41 microm/sec, somewhat faster than the 28 microm/sec velocity of Ca(2+) waves. Ejection of 3 microm ATP onto the retinal surface evoked propagated glial Ca(2+) waves. Together, these results indicate that Ca(2+) waves are propagated through retinal glial cells by two mechanisms. Waves are propagated through astrocytes principally by diffusion of an internal messenger, whereas waves are propagated from astrocytes to Müller cells and from Müller cells to other Müller cells primarily by the release of ATP.

Genetic inactivation of an inwardly rectifying potassium channel (Kir4.1 subunit) in mice: phenotypic impact in retina.

The inwardly rectifying potassium channel Kir4.1 has been suggested to underlie the principal K(+) conductance of mammalian Müller cells and to participate in the generation of field potentials and regulation of extracellular K(+) in the retina. To further assess the role of Kir4.1 in the retina, we generated a mouse line with targeted disruption of the Kir4.1 gene (Kir4.1 -/-). Müller cells from Kir4.1 -/- mice were not labeled with an anti-Kir4.1 antibody, although they appeared morphologically normal when stained with an anti-glutamine synthetase antibody. In contrast, in Müller cells from wild-type littermate (Kir4.1 +/+) mice, Kir4.1 was present and localized to the proximal endfeet and perivascular processes. In situ whole-cell patch-clamp recordings showed a 10-fold increase in the input resistance and a large depolarization of Kir4.1 -/- Müller cells compared with Kir4.1 +/+ cells. The slow PIII response of the light-evoked electroretinogram (ERG), which is generated by K(+) fluxes through Müller cells, was totally absent in retinas from Kir4.1 -/- mice. The b-wave of the ERG, in contrast, was spared in the null mice. Overall, these results indicate that Kir4.1 is the principal K(+) channel subunit expressed in mouse Müller glial cells. The highly regulated localization and the functional properties of Kir4.1 in Müller cells suggest the involvement of this channel in the regulation of extracellular K(+) in the mouse retina.

Sodium-bicarbonate cotransport in retinal astrocytes and Müller cells of the rat.

Sodium-bicarbonate cotransport in retinal glial cells was studied in the everted eyecup preparation of the rat. Intracellular pH was monitored with the indicator dye BCPCF and fluorescence confocal microscopy. Raising the K+ concentration from 3 to 12 mM in HCO3- -buffered perfusate evoked an intracellular alkalinization in both astrocytes and Müller cells. The alkalinization developed more rapidly and was larger in astrocytes. The K+ -induced alkalinization was HCO3- -dependent; it was reduced by 33% in astrocytes and 71% in Müller cells when HCO3- was removed from the perfusate. The alkalinization was effectively blocked by addition of 0.5 mM 4,4"-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS). Removal of Na+ from the perfusate evoked a rapid acidification in both types of glial cells. The results indicate that astrocytes and Müller cells in situ in the rat retina possess an electrogenic Na+/HCO3- cotransporter.

Tail docking and beliefs about the practice in the Victorian dairy industry.

OBJECTIVE: To determine the occurrence of tail docking and beliefs about the practice in the Victorian dairy industry. DESIGN: Survey responses were analysed using chi-square tests and by correlation and regression analyses to determine associations between husbandry practices and beliefs and to identify possible predictive variables in relation to docking. PROCEDURE: A survey of the occurrence of docking and beliefs about the practice was conducted in 1997 using face-to-face interviews of 313 respondents at 234 Victorian dairy farms. RESULTS: On average, 35% of dairy farms routinely docked cattle. The practice varied from 11 to 63% in different regions and 12% of stud farms docked their cows. Rubber rings were used on 75% of farms and the average age of the cow at docking was 18 months. Twenty-two percent of cows were docked at a level above the top of the udder and 54% were docked level with the top of the udder. Respondents that docked believed that milking was finished quicker, the risks of leptospirosis for the operator and mastitis for the cow were reduced, the cows were easier to handle, fly numbers were reduced and milk quality was improved. There was a general belief that intact tails could cause significant discomfort to the operator and that docking resulted in acute but not chronic pain. CONCLUSIONS: Docking is an entrenched practice in the Victorian dairy industry. Those farmers who docked generally believed that it was a highly desirable farming practice with particular benefits for the operator.

An eyecup preparation for the rat and mouse.

The eyecup preparation has traditionally been used to study retinal physiology in lower vertebrates and in some mammals. The procedures for preparing eyecups of the rat and mouse have not been described, however. We now describe methods for preparing and maintaining viable eyecups for these two species. Eyecups were everted over a plastic dome and held in place between the two halves of a superfusion chamber. Fluid exchange in the chamber was rapid, with near total exchange occurring in 9 s. Eyecup viability was tested by monitoring light-evoked retinal responses as the preparation aged. In both rat and mouse, the amplitude of the electroretinogram (ERG) b-wave decreased slowly, declining to 1/2 maximal amplitude in approximately 70 min. Light-evoked spike activity of neurons in the ganglion cell layer remained stable for approximately 3 h and attenuated responses were recorded for an additional 1-2 h. Eyecups were able to dark adapt. Retinal sensitivity, tested by monitoring b-wave amplitude, recovered following exposure to an adapting light.

Modulation of neuronal activity by glial cells in the retina.

Glial-neuronal communication was studied by monitoring the effect of intercellular glial Ca2+ waves on the electrical activity of neighboring neurons in the eyecup preparation of the rat. Calcium waves in astrocytes and Müller cells were initiated with a mechanical stimulus applied to the retinal surface. Changes in the light-evoked spike activity of neurons within the ganglion cell layer occurred when, and only when, these Ca2+ waves reached the neurons. Inhibition of activity was observed in 25 of 53 neurons (mean decrease in spike frequency, 28 +/- 2%). Excitation occurred in another five neurons (mean increase, 27 +/- 5%). Larger amplitude Ca2+ waves were associated with greater modulation of neuronal activity. Thapsigargin, which reduced the amplitude of the glial Ca2+ increases, also reduced the magnitude of neuronal modulation. Bicuculline and strychnine, inhibitory neurotransmitter antagonists, as well as 6-Nitro-7-sulphamoylbenzo[f]quinoxaline-2,3-dione (NBQX) and D(-)-2-amino-7-phosphonoheptanoic acid (D-AP7), glutamate antagonists, reduced the inhibition of neuronal activity associated with glial Ca2+ waves, suggesting that inhibition is mediated by inhibitory interneurons stimulated by glutamate release from glial cells. The results suggest that glial cells are capable of modulating the electrical activity of neurons within the retina and thus, may directly participate in information processing in the CNS.

Asymmetric gap junctional coupling between glial cells in the rat retina.

Gap junctional communication between glial cells is thought to play a role in K+ spatial buffering, in the propagation of inter-astrocytic Ca2+ waves, and in glial-neuronal signaling. In the present study, we characterize dye coupling between astrocytes, and between astrocytes and Müller cells, in the isolated rat retina. Whole-cell patch recordings were obtained from retinal astrocytes and Müller cells and the cells filled with Lucifer Yellow and neurobiotin. Spread of Lucifer Yellow to two to ten neighboring astrocytes occurred in 90% of the astrocyte recordings. After fixation and incubation of the retina with fluorescent conjugated streptavidin, neurobiotin was seen to label clusters of 13-88 astrocytes, as well as > 100 Müller cells. In contrast, when Müller cells were filled with Lucifer Yellow and neurobiotin, both tracers were confined solely to the recorded Müller cell. The uncoupling agents octanol, halothane, and doxyl-stearic acid were tested for their ability to uncouple retinal glia in situ. All three agents eliminated the visible spread of Lucifer Yellow from the injected astrocyte and the spread of neurobiotin into Müller cells. However, only doxyl-stearic acid combined with octanol eliminated the spread of neurobiotin between astrocytes. These results demonstrate that astrocytes in the rat retina are coupled to each other and to Müller cells. The astrocyte-to-Müller cell coupling is asymmetric, allowing transfer of the tracer in the forward direction only. In addition, astrocyte-to-Müller cell coupling is more sensitive to the uncoupling agents tested than is astrocyte-to-astrocyte coupling.

Calcium waves in retinal glial cells.

Calcium signals were recorded from glial cells in acutely isolated rat retina to determine whether Ca2+ waves occur in glial cells of intact central nervous system tissue. Chemical (adenosine triphosphate), electrical, and mechanical stimulation of astrocytes initiated increases in the intracellular concentration of Ca2+ that propagated at approximately 23 micrometers per second through astrocytes and Müller cells as intercellular waves. The Ca2+ waves persisted in the absence of extracellular Ca2+ but were largely abolished by thapsigargin and intracellular heparin, indicating that Ca2+ was released from intracellular stores. The waves did not evoke changes in cell membrane potential but traveled synchronously in astrocytes and Müller cells, suggesting a functional linkage between these two types of glial cells. Such glial Ca2+ waves may constitute an extraneuronal signaling pathway in the central nervous system.

Acid efflux from retinal glial cells generated by sodium bicarbonate cotransport.

Sodium bicarbonate cotransport was studied in freshly dissociated Müller cells of the salamander retina. Variations in intracellular and extracellular pH evoked extracellular potassium concentration ([K+]o were recorded. Intracellular pH was measured by standard ratio imaging of the pH-sensitive dye BCECF, whereas extracellular pH was monitored by imaging BCECF fixed to coverslips under dissociated cells. Increasing [K+]o from 2.5 to 50 mM resulted in an intracellular alkalinization. The rate of alkalinization, 0.047 pH units/min, was reduced to 42% of control when HEPES was substituted for HCO3- and was reduced to 36% of control by the addition of 0.5 mM DIDS, a Na+/HCO3- cotransport blocker. The K(+)-evoked alkalinization was Cl(-)-independent and was not substantially reduced by amiloride or bumetanide. Increasing [K+]o to 50 mM also produced a rapid extracellular acidification, 0.01 to 0.05 pH units in amplitude. HEPES substitution and addition of 0.5 mM DIDS reduced the acidification to 7-8% of control, respectively. These results confirm the presence of a Na+/HCO3- cotransport system in salamander Müller cells and provide definitive evidence that glial cells can generate an extracellular acidification when [K+]o is increased. The K(+)-evoked extracellular acidification measured beneath cell endfeet was 304% of the amplitude of the acidification beneath cell somata, confirming that cotransporter sites are preferentially localized to the endfoot. The carbonic anhydrase inhibitor benzolamide (2 x 10(-5) M), which is poorly membrane permeant, increased the K(+)-evoked extracellular acidification to 269% of control, demonstrating that salamander Müller cells possess extracellular carbonic anhydrase.

A physiological measure of carbonic anhydrase in Müller cells.

Carbonic anhydrase activity was characterized in freshly dissociated Müller cells of the salamander retina. Intracellular pH was monitored using ratio imaging of the indicator dye BCECF as extracellular PCO2 was varied. The extracellular solution was switched rapidly (141 ms rise time) from a HEPES buffered to a CO2-HCO3- buffered solution (both pH 7.4). Introduction of CO2-HCO3- produced a rapid cell acidification. Cell pH dropped from a steady-state pH of 7.02 in HEPES solution to pH 6.81 in CO2-HCO3-. Methazolamide, a carbonic anhydrase inhibitor, dramatically reduced the initial rate of acidification, demonstrating that the acidification was produced by the carbonic anhydrase-catalyzed hydration of CO2. The initial rate of acidification, 52.6 pH units per min (0.88 pH units per s), was reduced approximately 150-fold to 0.36 pH units per min by 10(-3) M methazolamide. Half-maximal inhibition occurred at a methazolamide concentration of 5.6.10(-7) M. The carbonic anhydrase inhibitor acetazolamide (10(-3) M) also greatly reduced the rate of cell acidification. The latency to the onset of carbonic anhydrase inhibition was 660 ms for methazolamide and 7.5 s for acetazolamide. The carbonic anhydrase inhibitor benzolamide (10(-4) M, 4 min exposure), which is poorly membrane permeant, had little effect on the rate of cell acidification, indicating that the site of carbonic anhydrase action was intracellular. The activity of Müller cell carbonic anhydrase may help to buffer extracellular CO2 variations in the retina.

Inward-rectifying potassium channels in retinal glial (Müller) cells.

The voltage- and K(+)-dependent properties of Müller cell currents and channels were characterized in freshly dissociated salamander Müller cells. In whole-cell voltage-clamp experiments, cells with endfeet intact and cells missing endfeet both displayed strong inward rectification. The rectification was similar in shape in both groups of cells but currents were 9.2 times larger in cells with endfeet. Ba2+ at 100 microM reduced the inward current to 6.8% of control amplitude. Decreasing external K+ concentration shifted the cell current-voltage (I-V) relation in a hyperpolarizing direction and reduced current magnitude. In multichannel, cell-attached patch-clamp experiments, patches from both endfoot and soma membrane displayed strong inward rectification. Currents were 38 times larger in endfoot patches. In single-channel, cell-attached patch-clamp experiments, inward-rectifying K+ channels were, in almost all cases, the only channels present in patches of endfoot, proximal process, and soma membrane. Channel conductance was 27.8 pS in 98 mM external K+. Reducing external K+ shifted the channel reversal potential in a hyperpolarizing direction and reduced channel conductance. Channel open probability varied as a function of voltage, being reduced at more negative potentials. Together, these observations demonstrate that the principal ion channel in all Müller cell regions is an inward-rectifying K+ channel. Channel density is far higher on the cell endfoot than in other cell regions. Whole-cell I-V plots of cells bathed in 12, 7, 4, and 2.5 mM K+ were fit by an equation including Boltzmann relation terms representing channel rectification and channel open probability. This equation was incorporated into a model of K+ dynamics in the retina to evaluate the significance of inward-rectifying channels to the spatial buffering/K+ siphoning mechanism of K+ regulation. Compared with ohmic channels, inward-rectifying channels increased the rate of K+ clearance from the retina by 23% for a 1 mM K+ increase and by 137% for a 9.5 mM K+ increase, demonstrating that Müller cell inward-rectifying channels enhance K+ regulation in the retina.

Fear of humans and its relationships with productivity in laying hens at commercial farms.

1. The relationship between the behavioural responses of laying hens to humans and productivity was determined at 16 commercial sheds from 14 farms. 2. A number of behaviour variables were moderately to highly correlated with production variables; for example, the proportion of birds that moved away from an approaching experimenter in an unfamiliar environment ('shute test') was negatively correlated with peak hen day production, (PKHDP). 3. Behavioural responses to humans accounted for between 23 and 63% of the variation in a number of production variables, including PKHDP and the duration of a high level of production. 4. Inclusion of farm factor variables increased the amount of variation accounted for by the behaviour variables. For example, adding the variable 'time/day spent in the shed by stockpeople' to the behaviour variables 'the proportion of birds that moved away from an approaching human' in the shute test and 'the number of times birds in cages adopted an erect posture' in response to an approaching human increased the variation accounted for in PKHDP from 53 to 61%. 5. The results suggest that fear of humans may be a factor that limits the productivity of commercial laying hens.

Sodium-bicarbonate cotransport in retinal Müller (glial) cells of the salamander.

An electrogenic Na+/HCO3- cotransport system was studied in freshly dissociated Müller cells of the salamander retina. Cotransporter currents were recorded from isolated cells using the whole-cell, voltage-clamp technique following the block of K+ conductance with external Ba2+ and internal Cs+. At constant pHo, an outward current was evoked when extracellular HCO3- concentration was raised by pressure ejecting a HCO3(-)-buffered solution onto the surface of cells bathed in nominally HCO3(-)-free solution. The HCO3(-)-evoked outward current was reduced to 4.4% of control by 0.5 mM DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonate), to 28.8% of control by 2 mM DNDS (4,4'-dinitrostilbene-2,2'-disulfonate), and to 28.4% of control by 2 mM harmaline. Substitution of choline for Na+ in bath and ejection solutions reduced the response to 1.3% of control. Bicarbonate-evoked currents of normal magnitude were recorded when methane sulfonate was substituted for Cl- in bath, ejection, and intracellular solutions. Similarly, an outward current was evoked when extracellular Na+ concentration was raised in the presence of HCO3-. The Na(+)-evoked response was reduced to 16.2% of control by 2 mM DNDS and was abolished by removal of HCO3- from bath and ejection solutions. Taken together, these results (block by stilbenes and harmaline, HCO3- and Na+ dependence, Cl- independence) indicate that salamander Müller cells possess an electrogenic Na+/HCO3- cotransport system. Na+/HCO3- cotransporter sites were localized primarily at the endfoot region of Müller cells. Ejection of HCO3- onto the endfoot evoked outward currents 10 times larger than currents evoked by ejections onto the opposite (distal) end of the cell. The reversal potential of the cotransporter was determined by DNDS block of cotransport current. In the absence of a transmembrane HCO3- gradient, the reversal potential varied systematically as a function of the transmembrane Na+ gradient. The reversal potential was -0.1 mV for a [Na+]o:[Na+]i ratio of 1:1 and -25.2 mV for a Na+ gradient ratio of 7.4:1. Based on these values, the estimated stoichiometry of the cotransporter was 2.80 +/- 0.13:1 (HCO3-:Na+). Possible functions of the glial cell Na+/HCO3- cotransporter, including the regulation of CO2 in the retina and the regulation of cerebral blood flow, are discussed.

Localization and stoichiometry of electrogenic sodium bicarbonate cotransport in retinal glial cells.

An electrogenic Na+/HCO3- cotransport system was identified and characterized in freshly dissociated salamander Müller (glial) cells. Under voltage-clamp, these cells generated an outward current when external HCO3- concentration [( HCO3-]o) was raised. This current was Na(+)-dependent, Cl(-)-independent, and was blocked by the stilbenes 4,4'-diisothiocyanato-stilbene-2,2'-disulfonate (DIDS) and 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS), and by harmaline, demonstrating that the current was generated by a Na+/HCO3- cotransport system. Substantially larger currents were evoked when [HCO3-]o was raised at the Müller cell endfoot as compared to other cell regions, indicating that cotransporter sites are localized preferentially to the endfoot. The reversal potential of the current, which varied as a function of HCO3- and Na+ transmembrane gradients, indicated that the cotransporter has a HCO3-:Na+ stoichiometry of 3:1.

Potassium conductance block by barium in amphibian Müller cells.

The effect of barium on Müller cell K+ conductance was evaluated in the tiger salamander using enzymatically dissociated cells and cells in situ (retinal slice and isolated retina). Barium effects were similar in both cases. In dissociated cells, 50 microM Ba2+ depolarized cells 14.7 mV and raised cell input resistance from a control value of 16.0 to 133 M omega. For cells in situ, 50 microM Ba2+ depolarized cells 6.9 mV and raised cell resistance from 12.5 to 50.4 M omega. At corresponding Ba2+ concentrations, the resistance of cells in situ was somewhat lower than was the resistance of dissociated cells, a phenomenon that may be due to the small degree of electrical coupling present between Müller cells in situ. There was a similar positive correlation between the magnitude of Ba2+-induced depolarization and input resistance in both dissociated cells and in situ cells. The magnitude of depolarizations generated by localized K+ ejections onto Müller cells was reduced substantially by Ba2+. These observations indicate that Ba2+ is an effective K+ channel blocker in Müller cells in situ as well as in enzymatically dissociated cells.

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.