High potassium conductance in astrocyte endfeet.

The distribution of potassium conductance over the surface of freshly dissociated salamander astrocytes was determined by monitoring cell depolarizations evoked by focal increases in the extracellular potassium concentration. The specific potassium conductance of the endfoot processes of these cells was approximately tenfold higher than the conductance of other cell regions. This dramatically nonuniform conductance distribution may play an important role in the regulation of extracellular potassium levels by glia in the brain.

Does the release of potassium from astrocyte endfeet regulate cerebral blood flow?

Local increases in neuronal activity within the brain lead to dilation of blood vessels and to increased regional cerebral blood flow. Increases in extracellular potassium concentration are known to dilate cerebral arterioles. Recent studies have suggested that the potassium released by active neurons is transported through astrocytic glial cells and released from their endfeet onto blood vessels. The results of computer simulations of potassium dynamics in the brain indicate that the release of potassium from astrocyte endfeet raises perivascular potassium concentration much more rapidly and to higher levels than does diffusion of potassium through extracellular space, particularly when the site of a potassium increase is some distance from the vessel wall. On the basis of this finding, it is proposed that the release of potassium from astrocyte endfeet plays an important role in regulating regional cerebral blood flow in response to changes in neuronal activity.

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.

Integration of visual and infrared information in bimodal neurons in the rattlesnake optic tectum.

Bimodal neurons in the rattlesnake tectum, which receive sensory input from the retina and from the infrared-sensing pit organ, exhibit novel, highly nonlinear cross-modality interactions. Some units respond only to simultaneous bimodal stimulation. Others respond to only one of the two modalities, but show greatly enhanced or depressed responses when stimulated simultaneously in the second modality. These cross-modality interactions may play an important role in recognizing and orienting toward biologically important objects.

Control of extracellular potassium levels by retinal glial cell K+ siphoning.

Efflux of K+ from dissociated salamander Müller cells was measured with ion-selective microelectrodes. When the distal end of an isolated cell was exposed to high concentrations of extracellular K+, efflux occurred primarily from the endfoot, a cell process previously shown to contain most of the K+ conductance of the cell membrane. Computer simulations of K+ dynamics in the retina indicate that shunting ions through the Müller cell endfoot process is more effective in clearing local increases in extracellular K+ from the retina than is diffusion through extracellular space.

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.

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.

Light-evoked increases in extracellular K+ in the plexiform layers of amphibian retinas.

Recordings of light-evoked changes in extracellular K+ concentration (delta[K+]o) were obtained in the retinas of frog and mudpuppy. In eyecup preparations, various recording approaches were used and provided evidence for a K increase near the outer plexiform layer (distal K increase). This distal K increase could be pharmacologically dissociated from the well-known, large K increase in the proximal retina by the application of ethanol and gamma-aminobutyric acid. The distal K increase also often showed surround antagonism. A retinal slice preparation was used to permit electrode placement into the desired retinal layers under direct visual control and without the risk of electrode damage to adjacent layers. In the slice, a distinct distal K increase was found in the outer plexiform layer, in addition to the prominent K increase in the inner plexiform layer. Compared with eyecups, only weak K increases were found in the nuclear layers of the slice. This suggests that the K responses observed in the nuclear layers of eyecups may be generated by K+ diffusing along the electrode track from the plexiform layers. In the context of current models of ERG b-wave generation, the magnitude of the recorded distal K increase, compared with the proximal K increase, seems too small to give rise to the b-wave. However, the distal K increase may be differentially depressed by electrode dead space. It is also possible that if certain aspects of the models of b-wave generation were modified, then the observed distal K increase could give rise to the b-wave.

2-Deoxyglucose labelling of the infrared sensory system in the rattlesnake, Crotalus viridis.

Infrared (IR) responsive nuclei in the rattlesnake Crotalus viridis were identified by using 14C-2-deoxyglucose (2DG) and autoradiography. Following 2DG intracardial injection, the IR-sensitive pit organ was stimulated periodically with an IR stimulus for 5 hours. The nucleus of the lateral descending trigeminal tract (LTTD, the primary IR sensory nucleus) was labelled heavily with 2DG. Labelling was bilateral, but somewhat heavier ipsilateral to the stimulated pit organ. The nucleus reticularis caloris (RC, the secondary nucleus of the IR system) was lightly labelled ipsilaterally. The middle laminae of the contralateral optic tectum (which contain IR-responsive units) were distinctly labelled; the corresponding layers of the ipsilateral tectum were lightly labelled. A subcerebellar nucleus not known to be part of the IR system was heavily labelled bilaterally. No consistent labelling was found in the diencephalon or telencephalon. Since units in the LTTD do not respond to stimulation of the contralateral pit yet the LTTD is labelled with 2DG when there is contralateral pit stimulation, several controls were carried out. Unilateral injection of 3H-proline into LTTD revealed no projection to the contralateral LTTD. In a monocularly, visually stimulated animal with both pits occluded, the LTTD still showed heavy but equal 2DG labelling bilaterally. In addition, the outer layers of the contralateral optic tectum were heavily labelled. No 2DG labelling of the LTTD was obtained when branches of the trigeminal nerve innervating the LTTD were previously cut. These results suggest that much of the 2DG labelling in the LTTD is due to spontaneous ongoing activity from the pit organ rather than from IR evoked activity.(ABSTRACT TRUNCATED AT 250 WORDS)

The infrared trigemino-tectal pathway in the rattlesnake and in the python.

We have studied the infrared trigemino-tectal pathway of the rattlesnake (Crotalus viridis) and the python (P. reticulatus). In the rattlesnake, horseradish perosidase (HRP) injections into the nucleus reticularis caloris (RC) result in retrograde filling of cells in the ipsilateral nucleus of the lateral descending trigeminal tract (LTTD) and in the anterograde labelling of terminal fields in the contralateral optic tectum, confirming our previous finding of an RC-tectal projection. The primary projection of the pit organ of the rattlesnake was traced by injecting cobalt chloride into the pit, demonstrating that the pit organ projects exclusively to the ipsilateral LTTD. Electrophysiological recording from single units in the RC shows that these cells respond to infrared stimulation. Taken together, these results demonstrate that the infrared pathway in the rattlesnake proceeds from the pit organ to the LTTD, to the RC, to the contralateral tectum. In contrast, HRP injection into the tectum of the python results in the retrograde filling of the large cells of the contralateral LTTD. Thus, a direct LTTD-tectal projection occurs in the python. The cells of the rattlesnake RC and the larger cells of the python LTTD stain heavily for acetylcholinesterase activity and have a similar multipolar appearance, suggesting that the tectal-projecting cells in the two species may have a common origin.

Connections of the tectum of the rattlesnake Crotalus viridis: an HRP study.

We have studied the connections of the tectum of the rattlesnake by tectal application of horseradish peroxidase. The tectum receives bilateral input from nucleus lentiformis mesencephali, posterolateral tegmental nuclei, anterior tegmental nuclei and periventricular nuclei; ipsilateral input from nucleus geniculatus pretectalis, and lateral geniculate nucleus pars dorsalis; and contralateral input from dorso-lateral posterior tegmental nucleus and the previously undescribed nucleus reticularis caloris (RC). RC is located on the ventro-lateral surface of the medulla and consists of large cells 25--45 micrometer in diameter. Efferent projections from the tectum can be traced to the ipsilateral nucleus lentiformis mesencephali, the ipsilateral lateral geniculate region, anterior tegmental region and a wide bilateral area of the neuropil of the ventral tegmentum and ventral medualla. We have not found any direct tectal projections from the sensory trigeminal nuclei including the nucleus of the lateral descending trigeminal tract (LTTD). We suggest that in the rattlesnake, RC is the intermediate link connecting LTTD to the tectum.

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.

Regional specialization of the membrane of retinal glial cells and its importance to K+ spatial buffering.

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.

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.

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.

Generation of the e-wave of the electroretinogram in the frog retina.

The e-wave and a delayed-OFF increase in extracellular K+ concentration are both maximum in the distal half of the inner plexiform layer. These responses also have similar latency, time-course, intensity-dependence, surround properties, and sensitivity to tetrodotoxin. Current source-density analysis of the e-wave reveals a current sink through the proximal retina, a source at the retinal surface, and, in some cases, a weaker source in the mid-retina. These results suggest a model for e-wave generation: delayed-OFF activity in proximal neurons releases K+, which enters Muller cells in the inner plexiform layer; a current exists Muller cells primarily via their endfeet, and the return flow through extracellular space produces the e-wave.

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.