General phenotype of Kir4.1 null (Kir4.1 −/−) mice
Heterozygous animals (Kir4.1 +/−) were identified by Southern blots () and PCR analysis () and were bred with each other to obtain homozygous animals. Progeny of the heterozygous animals show no gross discernable phenotypic differences in the first postnatal week. Afterward, the homozygous animals were considerably smaller than their littermates and displayed a higher rate of mortality. In addition, they developed clear motor coordination deficits, which became obvious ~2 weeks after birth. The animals displayed awkward and jerky movements and loss of balance and occasionally fell on their side. The general phenotype of Kir4.1 −/− and its impact in the CNS will be described in a separate report. Most of the homozygous mice survived up to 3 weeks of age, which allowed us to study their retinal physiology after eyelid opening [postnatal day 13 (P13) and P14]. All of the experiments were performed with young mice between P16 and P21 unless otherwise noted.
Analysis of Kir4.1 mRNA in retina
Wild-type (Kir4.1 +/+), heterozygous (Kir4.1 +/−), and homozygous (Kir4.1 −/−) animals were identified by PCR analysis from tail biopsies. Mouse retinas were dissected, and total RNA was extracted and subjected to reverse transcriptase-PCR amplification. PCR using mouse-specific oligonucleotide primers for Kir2.1, Kir2.2, Kir2.3, Kir4.1, and Kir5.1 showed the amplification of products of expected sizes for retinas from Kir4.1 +/+ mice. Using brain RNA from Kir4.1 +/+ mice, we could also detect the expression for all tested Kir channel subunits (). In contrast, Kir4.1 mRNA was not detected in retinas from Kir4.1 −/− mice using this assay, although other Kir channel subunits were detected after amplification using other Kir-specific oligonucleotide primers (). Thus, mRNA analysis in the retina shows the expected lack of Kir4.1 mRNA expression in retinas from genotyped Kir4.1 −/− mice.
Figure 2 Analysis of Kir4.1 mRNA in retina. PCR analysis of total RNA extracted from P21 Kir4.1 +/+ and Kir4.1 −/− mice retinas and Kir4.1 +/+ brain. Kir-specific oligonucleotide pairs were used to determine the expression of the various Kir channels (more ...)
Cellular organization and distribution of Kir4.1 in retina
Cellular organization and general morphology were assessed in retinal sections from Kir4.1 −/− and Kir4.1 +/+ mice stained with hematoxylin and eosin. shows light micrographs of retinal sections from P11 Kir4.1 +/+ and Kir4.1−/− mice. The retinas of the Kir4.1 −/− mice appeared to be normally organized with no apparent disruption of the normal pattern of lamination. Comparable results were obtained with older mice (P18–P21).
Figure 3 Histological analyses of the retinas of the Kir4.1 +/+ and Kir4.1 −/− mice. Cross-sections of mouse retinas from +/+ and −/− mutant mice (P11) were stained with hematoxylin and eosin. GCL, Ganglion cell layer; OPL, outer (more ...)
We raised an antibody against a peptide corresponding to a sequence in the C terminus of Kir4.1 to determine the cellular and subcellular distribution of Kir4.1 in the retina. The specificity of the affinity-purified anti-Kir4.1 antibody was tested by transient transfection of COS cells with Kir2.1, Kir3.1, Kir4.1, and Kir6.2 cDNAs, followed by immunostaining using the anti-Kir4.1 antibody. As expected, only cells transfected with Kir4.1 cDNA showed immunostaining. Furthermore, this labeling was blocked upon preadsorption of the anti-Kir4.1 antibody with a large excess of the antigenic peptide (data not shown).
Double-immunofluorescence experiments were performed on retinal whole mounts from Kir4.1 +/+ mice. Labeling for Kir4.1 was revealed with secondary antibodies coupled to FITC. Müller cells were labeled with an antibody against GS and visualized with secondary antibodies coupled to Texas Red. shows confocal optical sections in several retinal layers. At the inner limiting membrane, staining for Kir4.1 was detected along with staining for GS (). Particularly intense staining for Kir4.1 was seen along the superficial blood vessels. In addition, the cell bodies of ganglion cells clearly showed a lack of labeling for both antibodies. In the IPL (), large punctate staining was apparent for Kir4.1 and GS. These puncta presumably reflects the expression of both proteins in the stalk of Müller cells. In the inner nuclear layer (INL) (), overlapping expression of Kir4.1 and GS was seen surrounding neuronal cell bodies and very prominent Kir4.1 clustering along the blood vessels. Finally, in the outer nuclear layer (ONL) (), Kir4.1 and GS were concentrated in Müller cell processes surrounding the photoreceptors. Thus, double-immunolabeling reveals the overlapping expression of GS and Kir4.1 proteins, indicating the expression of Kir4.1 in the Müller glial cell population. Kir4.1 labeling was particularly intense in Müller cell processes contacting blood vessels. The labeling pattern for Kir4.1 for the mouse retinas agrees closely with the results reported for rat retinas (Nagelhus et al., 1999
Figure 4 Immunohistochemistry of Kir4.1 in retinal whole mounts from Kir4.1 +/+ mouse (P18). Retinal whole mount stained with anti-Kir4.1 antibody ( green) and anti-GS antibody (red). Confocal images were obtained in the ganglion cell layer (A), inner plexiform (more ...)
Because amphibian astrocytes isolated from the optic nerve have K+
channels preferentially localized to their endfeet (Newman, 1986
), we also asked whether Kir4.1 is expressed in retinal astrocytes. In retinal whole mounts, immunofluorescence labeling for GFAP revealed astrocytes in the superficial layers of retina. However, we failed to detect the labeling of Kir4.1 in astrocytes somata and proximal processes (data not shown). We could not determine whether Kir4.1 is found on the astrocytic terminal processes adjacent to blood vessels given the strong Kir4.1 immunoreactivity of adjacent Müller cell processes.
Double-immunolabeling with the anti-Kir4.1 and anti-GS antibodies was also performed on cross-sections of Kir4.1 +/+ and Kir4.1 −/− retinas. In retinal sections from Kir4.1 +/+ mice (), both antisera labeled all parts of Müller cells, staining the outer limiting membrane, the vitreal endfeet, and the main processes spanning the retina from the outer to the inner limiting membrane. Ganglion cells were clearly immunonegative for both anti-GS and anti-Kir4.1 antibodies (). Strikingly intense signal for Kir4.1 was found at the inner limiting membrane and near blood vessels in the INL (), confirming the immunolabeling pattern obtained in retinal whole mounts. In contrast to reports for albino rats (Kusaka et al., 1999a
), we did not detect Kir4.1 in the retinal pigment epithelium (RPE) (data not shown).
Figure 5 Immunohistochemical analysis of Kir4.1 in retinal sections of Kir4.1 +/+ (+/+) (A, C, E) and Kir4.1 −/− (−/−) (B, D, F) P18 mice. Sections were double-stained with affinity-purified rabbit anti-rat Kir4.1 antibody, followed (more ...)
In age-matched Kir4.1 −/− retinas, there was no detectable labeling with the anti-Kir4.1 antibody (), although the Müller cells appeared morphologically normal as revealed by immunolabeling with the anti-GS antibody (). Because Müller cells express GFAP under some conditions, most notably in pathological states (Bignami and Dahl, 1979
), we also stained retinal sections with anti-GFAP antibody. For both the Kir4.1 +/+ and Kir4.1 −/− mouse retinas, the GFAP immunolabeling was confined to cells located in the superficial layers in which the retinal astrocytes are located (data not shown). Thus, the lack of Kir4.1 did not induce the upregulation of GFAP in the Kir4.1 −/− Müller cells.
Müller cell membrane potential and input resistance
The electrophysiological properties of Müller cells in Kir4.1 +/+, Kir4.1 +/−, and Kir−/− mice were measured using whole-cell recordings in current-clamp mode. The resting membrane potential and input resistance of cells are given in . In Kir4.1 +/+ and Kir4.1 +/− mice, the resting membrane potential (−85 and −88 mV, respectively) was in the normal range for Müller cells (Newman, 1987
). The resting membrane potential in Kir4.1 −/− mice was significantly depolarized, to −13 mV, indicating that Müller cell K+
conductance was substantially reduced in these animals. Input resistance measurements confirmed that the K+
conductance of Müller cells in mutant mice was reduced (, ). The input resistance in Kir4.1 +/− mice (47 MΩ) was nearly double that of Kir4.1 +/+ mice (25 MΩ), whereas the input resistance in Kir4.1 −/− mice (231 MΩ) was more than nine times that of Kir4.1 +/+ animals.
Resting membrane potential and input resistance of Müller cells
Figure 6 Input resistance of Müller cells in Kir4.1 +/+, Kir4.1 +/−, and Kir4.1 −/− mice. Traces show the displacement of the membrane potential produced by an injected current pulse. The traces are scaled so that the amplitudes (more ...)
The membrane potential of Kir4.1 −/− cells was substantially depolarized, and it is possible that the large input resistance measured in these cells is attributable to this depolarization. To avoid this complication, we measured the input resistance of Kir4.1 −/− cells that were artificially hyperpolarized by injection of a constant negative current. Kir4.1 −/− cells hyperpolarized to an average of −84 mV had an input resistance of 310 MΩ, >12 times that of Kir4.1 +/+ cells, demonstrating that the increased input resistance of Kir4.1 −/− cells was not attributable to cell depolarization.
Several components of the ERG, including the slow PIII response, are believed to be generated by K+
current flow through Müller cells (Witkovsky et al., 1975
; Bolnick et al., 1979
; Newman, 2000
). These ERG components should be absent or greatly reduced in mutants lacking the predominant Müller cell K+
channel. We tested this prediction by measuring the slow PIII response in Kir4.1 +/+ and Kir4.1 −/− mice. The slow PIII response was monitored with an intraretinal electrode positioned in the distal retina of the mouse eyecup. In Kir4.1 +/+ mice, a brief light flash evoked a transient negative intraretinal b-wave followed by a slower positive response, the slow PIII (, +/+). Addition of the K+
channel blocker Ba2+
(0.4 mM) eliminated the slow PIII, as expected, but spared the b-wave (, +/+plus Ba2+
). When the intraretinal ERG recorded in Ba2+
was subtracted from the control ERG (, +/+ difference
), the Ba2+
-sensitive component, the slow PIII, was revealed in isolation.
Figure 7 The slow PIII response of the ERG in Kir4.1 +/+ and Kir4.1 −/− mice. Traces show the intraretinal ERG recorded between an electrode in the distal retina and one in the vitreous humor. In a Kir4.1 +/+ mouse, a transient negative b-wave (more ...)
The intraretinal ERG recorded from Kir4.1 −/− mice differed qualitatively from that of Kir4.1 +/+ mice (, −/−). Although the b-wave was present, the slow PIII response was absent. After decay of the b-wave, the ERG did not rise above the level of the prestimulus baseline. Addition of Ba2+ (, −/− plus Ba2+) produced little change in the ERG, as shown in the difference trace (, −/− difference), which is flat after an initial transient reflecting a small Ba2+-induced change in the amplitude of the b-wave.
Amplitudes of the intraretinal slow PIII and b-wave, as well as the transretinal b-wave, are given in . The intraretinal slow PIII amplitude, measured from the prestimulus baseline to the peak of the response, was 283 μV in Kir4.1 +/+ mice and −34 μV in Kir4.1 −/− mice, demonstrating that the slow PIII response was completely absent in the mutant. A negative slow PIII amplitude indicates that the response is not present.
Amplitude of the slow PIII response and b-wave of the ERG
The b-wave response, in contrast, was not eliminated in the mutant (). Measured intraretinally, the b-wave was 418 μV in Kir4.1 +/+ mice and 629 μV in Kir4.1 −/− mice. The larger intraretinal b-wave measured in Kir4.1 −/− mice probably arose because of the absence in these animals of the positive slow PIII, which normally would offset the negative b-wave. This would explain why b-wave amplitude in −/− animals was similar to the amplitude in +/+ animals treated with Ba2+. Measured transretinally, the b-wave was 142 μV in Kir4.1 +/+ and 117 μV in Kir4.1 −/− mice. The smaller transretinal b-wave in Kir4.1 −/− mice most likely reflects the smaller size of the eyes in mutant animals.