Rabbit retina contains connexin36 cDNA
RT-PCR of rabbit retina cDNA by using Cx36 primers amplified an 801-bp fragment. BLAST search revealed the 801-bp fragment was most similar to connexin36. The sequence was 93% and 89% identical to human and mouse Cx36, respectively, at the nucleotide level, and 96% and 95% similar to human and mouse Cx36, respectively, at the amino acid levels (Belluardo et al., 1999
). Rabbit Cx36 contained the glycine-rich region in the intracellular loop similar to mouse and human Cx36 but unlike the related perch and skate Cx35. This finding confirms that Cx36 is expressed in rabbit retina.
A polyclonal antibody to Cx36 at a dilution of 1:5,000 labeled a ~33-kDa band in Western blots of rabbit retinal homogenate. The antiserum also labeled a 39-kDa GST fusion protein containing the rabbit Cx36 intracellular loop domain (data not shown). This antibody produced distinct and repeatable staining in the rabbit retina at a dilution of 1:2000 (). The outer plexiform layer (OPL) contained very faint punctate labeling in sections. More prominent punctate labeling appeared throughout the inner plexiform layer (IPL), densely in sublamina b and sparsely in sublamina a. No Cx36 staining was visible in the ganglion cell layer.
Fig. 3 Cx36 immunoreactivity in oblique sections. a: Cx36 immunoreactivity appears as discrete puncta, densely in the ON region and more sparsely in the OFF region of the inner plexiform layer. Light labeling is also sometimes seen in the outer plexiform layer. (more ...)
We measured Cx36- and PKC-positive pixel densities across the breadth of the retina. The distribution of Cx36 revealed three distinct peaks, a large peak in sublamina b, a small peak in sublamina a, and a peak corresponding to rod bipolar somas in the outer nuclear layer. A smaller, less-reliable peak occurred at the OPL. The greatest concentration of Cx36 is in the lower IPL where the rod bipolar terminals and AII dendrites are located.
In some tissue sections, rod bipolar cells were diffusely stained along their axons and somas, but not on their terminals (, arrow). We suggest this staining is nonspecific because (1) rod bipolar staining appeared to be cytoplasmic, rather than in punctate membrane deposits, as it appeared in all other locations; and (2) rod bipolar staining weakened with dilution of the Cx36 antibody, whereas the punctate staining persisted. Cx36 antibody labeling of rod bipolar cells was confirmed by double immunolabeling with an antibody to PKC, which specifically labels rod bipolar cells in the rabbit retina ().
Because Cx36 faintly labeled rod bipolar cell somas and axons, but not their terminals, we investigated whether Cx36 gap junction plaques were localized on or near the rod bipolar cell terminals. We examined rabbit retinal whole-mounts labeled with PKC, calretinin, and Cx36 to determine the relative locations of rod bipolar terminals, calretinin-stained AII dendrites, and Cx36. The majority of Cx36 plaques near rod bipolar terminals were located a short distance from, rather than on, rod bipolar cell terminals. Colocalization software revealed a rim or caldera, rather than a peak, indicating Cx36 is adjacent to rod bipolar terminals. Gap junctions frequently occur between postsynaptic AII amacrine cell processes in this vicinity (Kolb, 1979
; Dacheux and Raviola, 1986
; Smith et al., 1986
; Strettoi et al., 1992
; Massey and Mills, 1999
). Although a few Cx36 plaques appeared to be located on rod bipolar cell terminals (cyan puncta in ), calretinin labeling of the same tissue revealed that overlying AII amacrine cell dendrites intersected at these points (). Based on these findings, we conclude that the cytoplasmic Cx36 labeling of rod bipolar cell somas and axons is nonspecific. Finally, there is no available evidence that rod bipolar cells make gap junctions. In fact, detailed electron microscopic observations reveal an absence of gap junctions on rod bipolar terminals (Kolb, 1979
; McGuire et al., 1984
; Freed et al., 1987
; Sterling et al., 1988
Fig. 4 Cx36 immunoreactivity in retinal whole-mounts. a: Rod bipolar cells, although sometimes stained diffusely by the Cx36 antibody in the somatic and axonal regions, are not stained at all at their terminals, which are stained by an antibody to protein kinase (more ...)
Cx36 immunoreactivity is primarily on puncta on AII dendrites
AII amacrine cells send a thick primary dendrite into the IPL before branching into many tapering dendrites in sublamina b. Because AII amacrine cells are well coupled to other AII amacrine cells and also to ON cone bipolar cells, the Cx36 punctate labeling in sublamina b was investigated to determine Cx36’s role in the gap junctions formed by AII amacrine cells. Calretinin antibody at a high dilution (1:50,000 in sections) specifically labels the AII amacrine cells (Massey and Mills, 1996
). Oblique Vibratome sections, double labeled for Cx36 and calretinin, revealed punctate Cx36 staining all along the AII amacrine cell dendrites (). Light Cx36 labeling appeared at the very top of the IPL but was not localized to AII amacrine cell somas. Again, no Cx36 staining was visible in the ganglion cell layer.
Cx36 immunoreactivity occurs at junctions between AII amacrine cells
Serial image scans were taken every 0.5 μm that encompassed the full span of the AII amacrine cell. Cx36 staining was anticorrelated with AII somas and lobules in sublamina a (). This finding suggests that the sparse Cx36-immunoreactive puncta in sublamina a are located on an unidentified amacrine or bipolar cell type. demonstrates that Cx36-containing plaques are colocalized with the AII dendritic matrix in sublamina b. A pixel scattergram confirmed colocalization in sublamina b and its lack in sublamina a by the presence or absence, respectively, of pixels containing both markers (data not shown). demonstrates at higher power the location of Cx36-immunoreactive puncta at the junctions between AII amacrine cells. There are very few isolated green pixels, where Cx36 immunoreactivity appears unassociated with AII amacrine cell processes.
Fig. 5 Cx36 puncta occur where AII amacrine cell dendrites cross. a: A high-power photomicrograph shows that almost all Cx36 immunoreactivity (green) in sublamina b is located on AII amacrine cells stained by anti-calretinin (CR, red) and that virtually every (more ...)
We estimated the percentage of Cx36 plaques that contact AII amacrine cells. Counting the Cx36 plaques in sublamina b in whole-mount revealed that 98% of the plaques occurred on AII amacrine dendrites. Of these, 84% of the Cx36 plaques on AII dendrites occurred where AII dendrites intersected and indicate homologous AII gap junction coupling. The remaining 16% of Cx36 plaques that are on AII dendrites do not intersect other AII dendrites. This finding suggests heterologous coupling of AII dendrites to ON-cone bipolar cells also occurs and places a lower limit on its relative frequency.
To examine the homologous junctions more closely, the apparent junction of two AII dendrites marked with Cx36 plaques was examined with serial confocal scans of 0.2 μm at high magnification. shows three narrow confocal images of two AII amacrine cell dendrites oriented at right angles to one another when viewed en face but at slightly different planes of focus. When the vertical AII dendrite is in its focal plane, weak Cx36 punctate labeling begins. The Cx36 signal occurs strongly only in the middle panel, where the vertical and horizontal dendrites abut. In the third panel, when the horizontal dendrite is in focus, the dendrites have diverged and no Cx36 immunoreactivity is found. This illustrates the general finding that Cx36 puncta appear only on contact points between dendrites, within the limits of confocal resolution.
An individual AII amacrine cell makes numerous Cx36-containing gap junctions with neighboring AII amacrine cells
We investigated the extent to which a single AII amacrine cell is coupled to other AII amacrine cells by measuring the number of contacts between a Neurobiotin-injected AII amacrine cell and the surrounding calretininstained AII amacrine cells. shows the lobular appendages in sublamina a of the injected cell (blue) and the surrounding AII lobules (red). The coverage factor of the lobules is near 1 (Mills and Massey, 1991
). This lack of overlap allows little opportunity for gap junctional coupling.
Fig. 6 An individually injected AII amacrine cell is extensively coupled with its anti-calretinin stained neighbors. a: There is little spatial overlap in sublamina a, hence little opportunity for gap junctions, between the lobules of Neurobiotin-stained AII (more ...)
The fine dendrites in sublamina b of the injected cell are seen in isolation in , and with the calretinin-(red) and Cx36-(green) immunoreactivity in . The dendritic overlap is such that any point is within the dendritic field of three to eight different AII amacrine cells (Mills and Massey, 1991
). The dendrites of a single cell do not intersect one another. As before, the great majority of Cx36 puncta appear on AII amacrine cell processes that contact other AII processes. The extent of coupling between the injected cells and its neighbors is great, with over 100 puncta on the injected cell, 66% contacting calretinin-stained processes.
demonstrates that Cx36 immunoreactivity on the Neurobiotin-injected cell occurs where it intersects other AII processes stained with calretinin. Several 24 × 24 pixel squares were centered on intersections between the injected amacrine cell and calretinin-stained processes (). When these squares were averaged, a large peak of associated Cx36 immunoreactivity appears (). (In Duncan’s test, the central 1.8 μm, mean Cx36 intensity = 34.1 was greater [P < 0.05] than the peripheral portion of the clipped area, mean = 14.3.) If the image of the injected AII amacrine cell is rotated, and squares are centered on the new intersections of processes, no peaks appear at above chance levels (). (In Duncan’s test, no portions of the clipped area were significantly different, means = 15.5-22.4.)
Fig. 7 Association of Cx36 with AII amacrine cell processes is not a chance occurrence. a: Squares (36 × 36 pixels) are centered on crossings of AII amacrine cell processes. b: When all such squares are averaged, a distinct peak of Cx36 immunoreactivity (more ...)
Cx36 is contained in junctions between AII amacrine cells and ON cone bipolar cells
AII amacrine cells also form heterologous gap junctions with ON cone bipolar cells, as demonstrated by electron microscopy (Famiglietti and Kolb, 1975
; McGuire et al., 1984
; Dacheux and Raviola, 1986
; Freed et al., 1987
; Sterling et al., 1988
; Cohen and Sterling, 1990
; Strettoi et al., 1992
; Massey and Mills, 1999
), tracer coupling (Vaney, 1991
; Mills and Massey, 1995
; Bloomfield et al., 1997
) and physiological recording (Xin and Bloomfield, 1999
). To determine whether Cx36 forms gap junctions between AII and ON cone bipolar cells, retinal whole-mount tissue was labeled with antibodies to Cx36, calretinin, and calbindin. Calbindin antibodies label a single type of ON cone bipolar cell in the rabbit retina (Massey and Mills, 1996
). shows that some Cx36-immunoreactive puncta appear on calbindin-stained processes in sublamina b (arrows). When the calretinin image is observed, there are also AII amacrine cell processes at these sites (; arrows). Hence, Cx36 also participates in coupling between AII amacrine cells and an identified type of ON cone bipolar cell. Several types of ON cone bipolar cell are known to make gap junctions with the AII amacrine cell. We have also found Cx36 immunoreactivity on ON cone bipolar cells stained by Neurobiotin injection into AII amacrine cells, and which are not immunoreactive for calbindin (not shown). This finding indicates that gap junctions from AII amacrine cells to other types of ON cone bipolar cell can also contain Cx36.
Fig. 8 Cx36 immunoreactivity is also found at the intersections of AII amacrine cell processes with calbindin-positive ON cone bipolar cells. a: The arrows point to Cx36-immunoreactive puncta (green) that appear in conjunction with bipolar cell processes stained (more ...)
The number of contacts of Cx36 puncta with calretininstained AII dendrites and calbindin-positive ON cone bipolar cells were counted. The majority (66%) of Cx36 puncta on calbindin-positive bipolar cells were located where two AII dendrites crossed. However, 34% of Cx36 puncta located on calbindin-positive bipolar cells contacted single, uncrossed AII dendrites. The lack of a second AII dendrite in the vicinity of the Cx36-containing calbindin bipolar cell process strongly suggests that AII-ON cone bipolar gap junctions also utilize Cx36.
Our signal colocalization software was used to measure the level of association of Cx36 plaques with AII-ON cone bipolar cell junctions. Cx36 plaques contacting a calbindin bipolar cell were selected, while the calretinin image was turned off to eliminate observer bias. The markers for Cx36 and calbindin bipolar cells were of course colocalized by the selection procedure. However, a large peak indicating the presence of calretinin-stained AII processes also appeared (). (In Duncan’s test, the central 1.8 μm, mean Cx36 intensity = 47.3 was greater [P < 0.05] than the peripheral portion of the clipped area, mean = 6.1.) This finding indicates that Cx36 immunoreactivity on calbindin-stained ON cone bipolar cells occurs where an AII amacrine cell contacts the bipolar cell. To verify this conclusion, all occurrences of Cx36 puncta contacting ON cone bipolar cells were reanalyzed after the Cx36 image was rotated 180 degrees. (In Duncan’s test, no portions of the clipped area were significantly different, means = 6.4 -9.9.) In this condition, AII dendrites were no longer colocalized with Cx36/calbindin (). A complementary analysis of junctions between calretinin- and calbindin-stained processes revealed a peak of Cx36 immunoreactivity. Both analyses support the idea that Cx36 forms gap junctional channels between AII amacrine cells and ON cone bipolar cells.
Many retinal gap junctions do not contain Cx36
A great many retinal neurons are coupled by gap junctions (Vaney, 1994
). Horizontal cells are perhaps the best-known example. Because very faint Cx36 labeling was detected in the OPL of Vibratome sections, the expression of Cx36 in the horizontal cell layer was examined. The antibody to calbindin also stains A-type horizontal cells (Röhrenbeck et al., 1987
; Massey and Mills, 1996
). However, Cx36 staining at the level of horizontal cell somas revealed only a faint diffuse staining of rod bipolar cell somas. Very faint punctate labeling was also observed in the OPL, compared with the intense Cx36 staining of inner plexiform layer in the same tissue region. Contrast the near-exclusive association of Cx36-immunoreactive puncta (green) with AII amacrine cell (red) and calbindin bipolar cells (blue) in , with the lack of association of the faint punctate labeling in the OPL with A-type horizontal cells ().
Fig. 9 Many retinal gap junctions are not stained with Cx36 antibody. (a,b) Labeling with antibodies to calbindin (blue), Cx36 (green), and calretinin (CR, red) show that Cx36 is intimately associated with AII amacrine cells and calbindin-stained bipolar cells (more ...)
Two closely related amacrine cells (S1/S2) that accumulate indolamines form a dense meshwork of highly overlapping dendrites. Like AII amacrine cells, they receive synaptic input from and make reciprocal synapses back onto rod bipolar terminals (Sandell et al., 1989
). They are also extensively tracer coupled. To determine whether Cx36 formed gap junctions in the S1/S2 network, tissue was labeled with antibodies to serotonin and Cx36. As S1/S2 and AII amacrine cell processes converge at the rod bipolar cell synapse, some association must occur. Nevertheless, when images () were analyzed with the signal colocalization software, Cx36 immunoreactivity () occurred at a hole in the S1/S2 signal (). (Duncan’s test found a significant dip, mean Cx36 intensity = 69.5 in the central 3.6 μm, compared with the peripheral regions, mean = 80.8, P
< 0.05.) This finding indicates that Cx36 does not form gap junctions between S1/S2 cells. These holes were also found to contain calretinin-labeled AII amacrine cell processes where the Cx36 immunoreactivity appears.