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 theKir4.1gene (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 situwhole-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.

Alzheimer’s disease (AD), the most common cause of dementia among the elderly, may either represent the far end of a continuum that begins with age-related memory decline or a distinct pathobiological process. Although mice that faithfully model all aspects of AD do not yet exist, current mouse models have provided valuable insights into specific aspects of AD pathogenesis. We will argue that transgenic mice expressing amyloid precursor protein should be considered models of accelerated brain aging or asymptomatic AD, and the results of interventional studies in these mice should be considered in the context of primary prevention. Studies in mice have pointed to the roles of soluble beta amyloid (Aβ) oligomers and soluble tau in disease pathogenesis, and support a model in which soluble Aβ oligomers trigger synaptic dysfunction, but formation of abnormal tau species leads to neuron death and cognitive decline severe enough to warrant a dementia diagnosis.

Calcium signals were recorded from glial cells in acutely isolated rat retina to determine whether Ca 2+ 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 Ca 2+ that propagated at ~23 micrometers per second through astrocytes and Müller cells as intercellular waves. The Ca 2+ waves persisted in the absence of extracellular Ca 2+ but were largely abolished by thapsigargin and intracellular heparin, indicating that Ca 2+ 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 Ca 2+ waves may constitute an extraneuronal signaling pathway in the central nervous system. Glial cells, long considered to be passive elements in the central nervous system (CNS), are now known to generate active responses (1), including intracellular Ca 2+ signals (2). Stimulation of astrocytes triggers increases in the intracellular Ca 2+ concentration ([Ca 2+ ] i ) that can propagate as waves between cells coupled by gap junctions (3,4). These glial Ca 2+ waves have been observed only in dissociated cell (3-7) and organotypic (8) culture preparations, which differ from cells in situ in several respects (9). Because these waves may represent a form of intercellular signaling in the CNS (5) and can potentially modulate neuronal activity (10,11), we tested whether Ca 2+ waves occur in situ in glial cells of acutely isolated rat retina.The rat retina contains two types of macroglial cells: astrocytes, which form a two-dimensional syncytium at the vitreal surface of the retina, and Müller cells, which are radial glial cells whose end feet terminate at the vitreal surface and whose trunks project downward into the retina (12). We detected [Ca 2+ ] i in these cells with the fluorescent Ca 2+ indicator dye Calcium Green-1 (13). The vitreal surfaces of flat-mounted retinas were imaged with video-rate confocal microscopy (14). Both astrocytes and Müller cells incorporated the dye and were identified by their morphology (Fig. 1A).Stimulation of a single astrocyte evoked increases in [Ca 2+ ] i in the simulated cell and in neighboring astrocytes and Müller cells. This Ca 2+ response propagated outward from the site of stimulation as a wave across the retinal surface (Fig. 1, B to D). Chemical, electrical, and mechanical stimuli were all effective in initiating Ca 2+ waves. Pressure ejection of adenosine triphosphate (ATP) (200 μM), carbachol (1 mM), or phenylephrine (100 μM) from micropipettes onto astrocyte somata initiated Ca 2+ waves. In contrast to findings in cultured cells (2,5,15), local ejection of glutamate (2 mM) or its application in the bath (0.3 to 1 mM) was ineffective at evoking [Ca 2+ ] i increases, but bath application (10 to 100 μM) did potentiate the Ca 2+ responses initiated by other s...

Glial-neuronal communication was studied by monitoring the effect of intercellular glial Ca 2ϩ waves on the electrical activity of neighboring neurons in the eyecup preparation of the rat. Calcium waves in astrocytes and Mü ller 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 Ca 2ϩ waves reached the neurons. Inhibition of activity was observed in 25 of 53 neurons (mean decrease in spike frequency, 28 Ϯ 2%). Excitation occurred in another five neurons (mean increase, 27 Ϯ 5%). Larger amplitude Ca 2ϩ waves were associated with greater modulation of neuronal activity. Thapsigargin, which reduced the amplitude of the glial Ca 2ϩ 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(Ϫ)-2-amino-7-phosphonoheptanoic acid (D-AP7), glutamate antagonists, reduced the inhibition of neuronal activity associated with glial Ca 2ϩ 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. Key words: calcium waves; glial cells; astrocytes; Mü ller cells; neurons; ganglion cells; retina; modulation; glial-neuronal interactionIntercellular C a 2ϩ waves have been observed in various cell types, including glial cells of the C NS (Finkbeiner, 1993). These Ca 2ϩ waves are transient increases in intracellular C a 2ϩ concentration ([C a 2ϩ ] i ) that propagate through networks of cells coupled together by gap junctions. They are initiated by various types of focal stimuli.Intercellular C a 2ϩ waves in glial cells have been observed in a number of in vitro preparations (Finkbeiner, 1993). They have been recorded in syncytia of cultured astrocytes (Cornell-Bell et al., 1990;Charles et al., 1991;Cornell-Bell and Finkbeiner, 1991;Enkvist and McCarthy, 1992) and in glial cells of organotypic hippocampal slices (Dani et al., 1992). The waves can be initiated by mechanical stimuli or application of neurotransmitters (Finkbeiner, 1993).Recently, we demonstrated that intercellular C a 2ϩ waves can also be propagated through networks of glial cells in situ in the freshly isolated mammalian retina (Newman and Z ahs, 1997). These waves are initiated by electrical or mechanical stimuli as well as focal application of neurotransmitters. The waves travel out concentrically across the retinal surface and travel synchronously in astrocytes and Müller cells, the two principal glial cells of the mammalian retina.These glial C a 2ϩ waves could have several possible functions. The waves could serve as a signaling mechanism permitting glial cells to communicate with each other over long distances. Such communic...

A lthough bacteria and many invertebrate species use both Dand L-enantiomers of amino acids for cellular functions, it was generally believed that higher organisms had a more restricted stereospecificity and were confined to the use of L-amino acids. Thus, the D-amino acids detected in vertebrates were generally assumed to come from ingested material or intestinal flora (1). Indeed, the enzyme D-amino acid oxidase (D-AAO), which degrades many D-amino acids, is found in vertebrate tissues, but it was assumed to exist for degrading D-amino acids from external sources. However, in the past decade, it has become clear that some D-amino (6) proposed that D-serine released by glia plays a regulatory role as a necessary coagonist for the glutamate activation of NMDA receptors. Thus, an expanded model for glutamate synapses emerged in which perisynaptic glial processes respond to glutamate released from the presynaptic neuron by releasing D-serine, which, in turn, facilitates the activation of NMDA receptors on the postsynaptic cell. The functional importance of D-serine was further supported by the more recent discovery that the enzyme serine racemase, which converts L-serine to D-serine, is localized to astrocytes in the nervous system (7). The discovery of this mechanism adds to the accumulating evidence that glial cells dynamically influence neuronal activity.Although D-serine and serine racemase have been found in many regions of the brain, no study has yet been carried out in the vertebrate retina, a tissue that offers several advantages for studying the actions of D-serine. The retinal network is a well-characterized region of the central nervous system that can be studied in an intact preparation, permitting the use of light stimulation to provide natural activation of the neural circuitry and test more directly the physiological significance of D-serine. Virtually all retinal ganglion cells have AMPA and NMDA receptors, which both contribute to light-evoked excitation (8-12), and NMDA receptor currents can be enhanced through pharmacological techniques and studied in relative isolation from AMPA receptor contributions (13).In the present study, we have used immunohistochemistry, immunoblotting, analytical chemistry (HPLC), and electrophysiology to evaluate the presence of D-serine and serine racemase in the retinas of several vertebrate species. Our findings indicate that D-serine and serine racemase are present in the retina and seem to be localized to Müller glial cells and astrocytes. In addition, we have demonstrated that exogenous D-serine can enhance NMDA receptor responses and modulate light-evoked activity in retinal ganglion cells. The enhancement of NMDA receptor function seems to involve both tonic and phasic components of glutamatergic input. When the D-serine-degrading enzyme D-AAO (14) is added to the bathing medium, currents mediated by NMDA receptors are reduced, suggesting that these responses depend on endogenous D-serine as a coagonist for the glutamate activation of NMDA receptors. Method...