![]() ![]() 7052 glass tubing (Gardner Glass Co., Claremont, CA, USA) using a multistage programmable puller (Sutter Instruments), coated with Sylgard No. Patch pipettes were pulled from Corning No. Unless noted otherwise, the bathing solution consisted of (mM): 133 NaCl, 10 KCl, 1.8 CaCl 2, 0.8 MgCl 2, 10 Na-Hepes and 20 glucose, at pH 7.4 with the osmolarity adjusted to 305–310 mosmol l −1. Whole-cell currents were monitored using the perforated-patch configuration of the patch clamp technique. A gravity-fed system with multiple reservoirs was used to continuously perfuse (1–2 ml min −1) the recording chamber (0.5 ml volume) with various solutions. Perforated-patch recordings from fresh Müller cells were made at room temperature (22–24☌). Experiments were performed within 3 h of cell dissociation. Müller cells were identified by their characteristic morphology ( Puro, Yuan & Sucher, 1996). Cells were examined at × 400 magnification with an inverted microscope equipped with phase-contrast optics. A suspension of cells (∼0.1 ml) was placed in a recording chamber and allowed to settle ∼15 min prior to the addition of bathing solution to the chamber. The piece of retina was then washed with the appropriate bathing solution, drawn up into a glass pipette and gently ejected back into a microcentrifuge tube. In brief, approximately 0.5 cm × 0.5 cm pieces of retina were incubated in Earle's balanced salt solution supplemented with 0.5 mM EDTA, 1.5 mM CaCl 2, 1 mM MgSO 4, 20 mM glucose, 26 mM sodium bicarbonate, 15 u papain (Worthington Biochemicals Co.), 0.04% DNase, 2 mM cysteine and 12% chicken serum for 40 min at 30☌, whilst 95% oxygen-5% CO 2 was bubbled through to maintain pH and oxygenation. Thus, when there is a breakdown in the blood-retinal barrier, this glycerophospholipid may be one of the serum-derived molecules that regulates ion channel activity in Müller cells.įreshly dissociated human and bovine Müller cells were prepared as detailed previously ( Kusaka, Dabin, Barnstable & Puro, 1996). We also found that lysophosphatidic acid (LPA), a component of serum, induces currents similar to those activated by whole serum. ![]() In addition, the electroretinograms (ERGs) from isolated retinas exposed to serum showed changes consistent with the idea that the serum-induced changes in the activity of ion channels reduces the role of Müller cells in the redistribution of K +. Another motivation for studying retinal cells is that a breakdown of the blood-retinal barrier is a frequently occurring, sight-threatening pathophysiological process ( Gass, 1997).īased on perforated-patch recordings from fresh bovine and human Müller cells, we now report that serum causes these glial cells to depolarize as a non-specific cation current and an outwardly rectifying K + current are activated. ![]() The extensive information concerning K + siphoning via Müller cells facilitates attempts to relate changes in ion channel activity to the function of these cells in regulating o. By a specialized mechanism of K + spatial buffering, termed K + siphoning (Newman, Frambach & Odette, 1994), K + enters a Müller cell where o is high and exits where o is lower. A reason for selecting these glial cells is that their role in K + redistribution has been particularly well studied ( Newman, 1995). To identify and characterize the effects of serum on glial channels, we chose to study Müller cells, the predominant glia of the retina. This redistribution via glial cells serves to limit wide swings in o which can alter neuronal excitability. Glial K + channels are pathways for the redistribution of excess potassium. We focused our study on the activity of ion channels, since they are involved in important glial functions such as the maintenance of K + homeostasis ( Newman & Reichenbach, 1996). One reason the glia are of interest is that serum leaking from the vascular system would almost certainly contact these cells, since they ensheath the blood vessels of the nervous system. In this study, we examined the effect of serum on the activity of ion channels in glial cells. We hypothesize that serum-derived molecules enter the nervous system, induce receptor-mediated changes in cell function and thereby alter the activity of neural circuits. Whilst gross tissue swelling and distortion due to an influx of fluid from the vascular compartment can cause damage, knowledge of more subtle mechanisms by which a breakdown of this barrier alters function is limited. When there is a breakdown of the barrier between the circulatory and nervous systems, the function of the CNS is compromised.
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