Andreas Reichenbach

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.

Morphometric parameters of Müller (glial) cells dependent on their topographic localization in the nonmyelinated part of the rabbit retina. A consideration of functional aspects of radial glia

SummaryMorphometric parameters of Müller cells were evaluated by light microscopy both in whole retinae and in enzymatically isolated cells from adult pigmented rabbits. In spite of the marked decrease in cell densities from visual streak to far periphery, a constant glia-neuron ratio of about 1∶15 was found in all regions. The volume of individual Müller cells was found to increase strongly when the cells become shorter, i.e. when the retinal centre was compared to the retinal periphery. The contribution of Müller cell volume to the total retinal volume, however, was shown to be constant at about 6%. Long Müller cells have a thin vitreal process and a small vitreal endfoot surface. The consequences of this rule for the proposed function of Müller cells in retinal K+ clearance are discussed with respect to general features of radial glia. It is suggested that foetal radial glial cells too long to perform sufficient K+ clearance are destined to be transformed into ‘adult’ multipolar glia by mitotic cell division.

The structure of rabbit retinal Müller (glial) cells is adapted to the surrounding retinal layers

SummaryRadial glial (Müller) cells of the rabbit retina were studied by various techniques including Golgi impregnation, scanning electron microscopy, horseradish peroxidase application, and staining of enzymatically isolated cells. This combination of methods produced detailed information on the specialized morphology of the Müller cells within the different topographical regions of the retina, and of the Müller cell processes within the various retinal layers. As a general rule, the retinal periphery contains short thick Müller cells with big endfeet, whereas the thick central retina is occupied by long slender cells with small endfeet. Independent of their location within the retina, Müller cell processes were found to be adapted to the structure of the surrounding retinal layers. Within the outer and inner nuclear layers, Müller cell processes (and somata) extend thin cytoplasmic “bubbles” ensheathing the neuronal somata, as do the “velate” astrocytes in the brain. In the plexiform layers, Müller cells extend many fine side branches between the neuropil, comparable to the protoplasmic astrocytes of the brain. In the thick myelinated nerve fibre layer of the central retina the Müller cell processes are rather smooth, similar to those of fibrous astrocytes. It is concluded that the neuronal microenvironment determines the morphology of a given glial process, or even of a part of a glial process running through a specialized neuronal compartment.

Potassium as a signal for both proliferation and differentiation of rabbit retinal (Müller) glia growing in cell culture

Retinal glial (Müller) cells were grown from explants of early postnatal rabbit retinae. The resulting monolayers of flat cells were exposed to control media (containing 5.85 mM K+), and to media with enhanced K+ concentrations (10 and 20 mM) or arginine-vasopressin (AVP, 20 micrograms/ml) or epithelial growth factor (EGF, 10 ng/ml). Autoradiographically, protein synthesis was quantified as L-[3H]-lysine incorporation, and DNA synthesis as [3H]-thymidine incorporation. Furthermore, the activity of Na+,K(+)-ATPase was measured radiochemically. Short exposure to either moderately enhanced K+ concentrations (10 mM) or to AVP, stimulated L-[3H]-lysine incorporation into the cells. Long-lasting exposure to either high K+ concentrations (20 mM) or to EGF stimulated [3H]-uptake. The Na+,K(+)-ATPase activity of cell cultures increased with increasing K+ concentration of the media. It is suggested that release of K+ by active neuronal compartments stimulates local protein synthesis of glial cells, resulting in the formation of glial sheaths with active K+ uptake capacity. Strong K+ release may even induce glial proliferation.

Postnatal development of radial glial (Müller) cells of the rabbit retina

Radial glial (Müller) cells were isolated from postnatal rabbit retinae by enzymatic dissociation in papain-containing solution, air-dried, and submitted to Pappenheims panoptic stain. Morphometric data of these cells were evaluated by light microscopy. During postnatal development, the cells become substantially thicker and shorter, their nuclei lose the rod shape and move more toward scleral layers, and the nucleus-cytoplasm volume relation decreases. Whereas the cell volume increases from birth on, substantial outgrowth of fine side branches within the plexiform layers fails to occur before electrical activity is established there, i.e. after postnatal day 9. A model is proposed relating the growth of sheath-bearing glial processes to local protein synthesis stimulated by external K+ accumulation due to neuronal activity. Early myelinated nerve fibers are suggested to bear mechanical resistance to growing radial glial processes thus causing a splitting of these processes when they enter developing nerve fiber layers.

Cell length to diameter relation of rat fetal radial glia — Does impaired K+ transport capacity of long thin cells cause their perinatal transformation into multipolar astrocytes?

In thick sections of Golgi-impregnated late fetal rat brains, radial glial cells were measured for both length and diameter of their main (basal) processes. The process diameter was found to decrease proportionally to the square root of cell length; thus, the cytoplasm volume remained fairly constant for cells in the range of lengths studied (100-2500 microns). The measured data were used for calculation of the cells space constant lambda in order to estimate their capability to carry spatial buffering K+ currents. These calculations show that long and slender cells are unable to perform sufficient K+ clearance by such currents. This supports the hypothesis that perinatally when the maturing neurons release enhanced K+ during electrical activity, such long thin cells are subject to long-lasting depolarizations and, thereby, forced to undergo mitotic cell division transforming them into multipolar astrocytes.

Cell type-specific distribution of cathepsin B and D immunoreactivity within the rabbit retina

The cellular localization of cathepsin B and D immunoreactivity was demonstrated at the light microscopic level in the retina of adult rabbits by use of the peroxidase-antiperoxidase technique. Antisera were raised against rat liver enzymes. Whereas cathepsin D immunoreactivity was confined to Müller (glial) cells, cathepsin B was demonstrated in some, but not all, neuronal cell types. It is proposed that the two enzymes might carry different functions within the neuronal versus glial compartment.

Quantitative-morphometric aspects of bergmann glial (Golgi epithelial) cell development in rats

SummaryBergmann glial (Golgi epithelial) cells in the cerebella of rats of various ages were stained by the rapid Golgi technique, and their radial stem processes were measured for length and diameter. Additionally, the average number of such processes per cell was counted, and the development of bushy lateral protrusions was quantified. The length of radial processes—depending on the thickness of the molecular layer—was found to increase up to the end of the 2nd year of life. This elongation was accompanied by a reduction of the mean process diameter which was, however, not sufficient to prevent an increase in the cytoplasmic volume of the elongating cells. A marked outgrowth of lateral protrusions was observed up to at least the 5th month of life. These data are compared with earlier findings on the development of rat brain stem fetal radial glia, and of rabbit retinal Müller cells. Common mechanisms of glial cell development are discussed.

Morphological variability, lectin binding and Na+,K+-activated adenosine triphosphatase activity of isolated müller (glial) cells from the rabbit retina

Rabbit retinal Müller cells were isolated by means of papaine and mechanical dissociation. These cells were shown to have a well preserved morphology and to preserve viability for many hours. Intense wheat germ agglutinin binding occurs on the photoreceptor side of Müller cells, especially in the microvillous region. Rabbit retinal Müller cells have a Na+,K+-activated adenosine triphosphatase activity in the same order of magnitude as brain astroglial cells.

Potassium accumulation by the glial membrane pump as revealed by membrane potential recording from isolated rabbit retinal Müller cells

Müller (glial) cells were isolated from rabbit retinae by papaine and mechanical dissociation. In a special perfusion chamber, the cells were penetrated with a recording electrode. When high-K+ solutions were applied into the environment of the cells by means of a second micropipette, the cell membrane depolarized strongly. During prolonged application of high-K+ solutions, however, there occurred a marked repolarization, and after cessation of high-K+ application, a strong hyperpolarization was observed. Both effects disappeared under the influence of ouabain, suggesting the accumulation of intracellular K+ by an active membrane pump. The data were used for calculation of the membranes Na+:K+ permeability ratio, the intracellular K+ concentration, the pump rate and the mean pump site density. The calculated values are in good agreement with published data from mammalian astrocytes and are compared with those from amphibian Müller cells.