Distribution of Connexin43 immunoreactivity in the retinas of different vertebrates

Distribution of Connexin43Immunoreactivity in the Retinas

of Different Vertebrates

ULRIKE JANSSEN-BIENHOLD,1* ROLF DERMIETZEL,2 AND RETO WEILER1

1Department of Biology, FB7, Carl von Ossietzky Universitat, 26111 Oldenburg, Germany2Institute of Anatomy, Ruhr-Universitat, 44801 Bochum, Germany

ABSTRACTThe distribution of Connexin43 (Cx43) was examined by immunoblotting and immunoflu-

orescence microscopy in the retinas of five different vertebrates by using a C-terminal specificpeptide antibody. The specificity of the antibody was proved on immunoblots, in which itshowed cross reactivity with a 43-kDa protein in rat heart homogenates as well as in homogenatesof rabbit, rat, chicken, turtle, and fish (carp and zebrafish) retinas.

Immunofluorescence histochemistry with retinal cryosections revealed the presence ofCx43 in the retinal pigment epithelium cells of all tested species and in blood vessels ofvascularized retinas (fish and rat). Cx43 immunoreactivity was further localized in the striamedullaris of rabbit retina, in the nerve fiber layer of rat retina, most likely in astrocytes, andin the area of the outer limiting membrane of the fish retina, most likely representing Cx43 inMuller glia cells. A punctate Cx43-immunoreactive pattern consistent with gap junctions wasalso detected in the outer plexiform layer of carp and zebrafish retinas, and a specific amacrinecell type, which ramified in two layers of the inner plexiform layer, was labeled in the zebrafishretina. The present results are in accordance with previous findings showing the abundance ofCx43 in astrocytes, endothelium, and epithelial cells. However, the presence of Cx43immunoreactivity in a specific population of amacrine cells of the zebrafish retina mightindicate that a Cx43-like protein is also expressed in neurons. J. Comp. Neurol. 396:310–321,1998. r 1998 Wiley-Liss, Inc.

Indexing terms: gap junctions; connexins; vertebrate retina; coupling; neural network

The presence of gap junctions in neurons and supportingcells of the vertebrate retina has been well documented,and electrical coupling of retinal neurons is thought to becrucial for visual processing (for reviews, see Vaney, 1994;Cook and Becker, 1995; Sterling, 1995). Gap junctions areareas of membrane specializations that consist of clus-tered channels spanning the plasma membrane of twoadjacent cells. The channels are formed by apposed hemi-channels (connexons), which possess a central aqueouspore of varying diameters (8–24 Å) and exhibit a variabledegree of ionic selectivity and size-dependent permeabil-ity. They provide a low-resistance pathway between cellsfor current flow and the exchange of small cytoplasmicmolecules up to 1 kDa in size, such as second messengersand other metabolites.

Each connexon is formed by six transmembrane proteins(connexins), several subtypes of which have been character-ized in different tissues and species by means of biochemi-cal analysis of subcellular fractions enriched in gap junc-tions and by molecular cloning. All connexin proteins

characterized to date exhibit highly conserved transmem-brane and extracellular domains, but they differ in theircytoplasmic domains. This has lead to the hypothesis thatthe cytoplasmic domains may determine the differentphysiological properties of gap junctions, such as theirionic conductance and sensitivity to pH, transjunctionalvoltage, phosphorylation, or pharmacologic agents (forreviews, see Bennett et al., 1991; Beyer, 1993; Veenstra,1996; Bruzzone et al., 1996).

There is considerable information about the distributionand ultrastructure of gap junctions in the vertebrateretina, their electrical properties, and their modulation by

Grant sponsor: Deutsche Forschungsgemeinschaft; Grant number: WE849/12–2.

*Correspondence to: Dr. Ulrike Janssen-Bienhold, Department of Biol-ogy, FB7, Carl von Ossietzky Universitat Oldenburg, P.O. Box 2503, 26111Oldenburg, Germany. E-mail: [email protected]

Received 13 August 1996; Revised 20 February 1998; Accepted 23February 1998

THE JOURNAL OF COMPARATIVE NEUROLOGY 396:310–321 (1998)

r 1998 WILEY-LISS, INC.

neurotransmitters and second messengers (for reviews,see Vaney, 1994, 1996; Cook and Becker, 1995; Weiler,1996).

Gap junctions mediating homologous electrical couplinghave been demonstrated between photoreceptors, horizon-tal cells, bipolar cells, and amacrine cells, whereas heter-ologous electrical coupling has been observed between AIIamacrine cells and cone bipolar cells (Baylor et al., 1971;Kaneko, 1971; Schwartz, 1975; Copenhagen and Owen,1976, Burkhardt, 1977; Naka and Christensen, 1981;Bloomfield and Miller, 1982; Dacheux and Raviola, 1982,1986; Kujiraoka and Saito, 1986; Saito and Kujiraoka,1988; Bloomfield, 1992; Umino et al., 1994; Bloomfield etal., 1995). Tracer coupling between retinal cells is consis-tent with most of the above mentioned examples of electri-cal coupling and was also observed between certain typesof ganglion cells and between amacrine cells and ganglioncells, although the presence of gap junctions betweenganglion cells has not yet been confirmed by electronmicroscopy (Teranishi et al., 1984; Rodieck and Haun,1991; Vaney, 1991, 1993; Dacey and Brace, 1992; Hidaka etal., 1993; Hitchcock, 1993; Penn et al., 1994; Umino et al.,1994; Bloomfield et al., 1995; Mills and Massey, 1995; forreview, see Vaney, 1994, 1996).

Homologous and heterologous tracer couplings have alsobeen found between retinal glia cells, such as astrocytes,oligodendrocytes, and Muller cells (MCs; Mobbs et al.,1988; Robinson et al., 1993), and electron microscopicstudies have revealed the presence of gap junctions be-tween MCs of the vertebrate retina (Gold and Dowling,1979; Wolburg et al., 1990).

The identity of all of the connexin proteins that formhomologous or heterologous gap junctions between thedifferent retinal neurons and glia cells is largely unknown.There are only preliminary reports from immunohisto-chemical studies (Finch and Paul, 1989; Vardi et al., 1990;Janssen-Bienhold et al., 1995, 1996), from one recentstudy identifying a retinal connexin35 in the skate retina(O’Brien et al., 1996), and from a second showing thedistribution of Cx43 immunoreactivity in the catfish retina(Giblin and Christensen, 1997). In the present compara-tive study, we have used a polyclonal antibody directedagainst an oligopeptide corresponding to a C terminal-specific sequence of rat Cx43 to examine the occurrenceand distribution of Cx43 in retinal cells of five differentvertebrates.

MATERIALS AND METHODS

Experiments were carried out with zebrafish (Brachy-danio rerio), carp (Cyprinus carpio), turtle (Pseudemysscripta elegans), chicken (Gallus domesticus; 23 days old),female Sprague-Dawley rats (250–350 g), and rabbits(wild type). Animal use was in accordance with Germanlegislation regulating the use of animals in research.

Preparations

After killing each animal, one eye was prepared forimmunohistochemistry, and the other eye was preparedfor immunobiochemistry. Enucleation was carried out at4°C. The anterior portion of the eye was cut away with arazor blade or scissors, the lens and as much as possible ofthe vitreous humor were removed, and the eye cups werefixed in ice-cold ethanol (increasing/decreasing concentra-

tions of 30%, 50%, 75%, and 100% ethanol/15 minuteseach), or in 4% paraformaldehyde in 0.1 M sodium phos-phate buffer (PB), pH 7.4, at 4°C for 4 hours. Followingthree washes in 0.1 M PB, pH 7.4, the eye cups wereimmersed in 30% sucrose in 0.1 M PB, pH 7.4, overnight at4°C for cryoprotection. For control experiments, pieces ofrat heart were quick frozen in liquid nitrogen withoutfixation. Frozen sections of 15–20 µm thickness were cuton a Reichert Jung cryostat (Nussloch, Germany), mountedon gelatine-coated microscope slides, and either useddirectly or stored dessicated at 220°C.

Immunohistochemistry

Sections were first rinsed three times (15 minutes each)in phosphate-buffered saline (PBS; 0.1 M PB, 0.1 M NaCl,pH 7.4) to wash out the cryomatrix. Nonspecific bindingwas blocked by incubating the sections for 1 hour at roomtemperature in PBS/0.3% Triton X-100 (TX-100) contain-ing 10% normal goat serum (NGS). Sections were thenincubated overnight at 4°C with the affinity-purified pri-mary antibody (generated in rabbit against an ovalbumin-conjugated synthetic peptide corresponding to amino acids359–381 of rat Cx43; pAB2; Hofer et al., 1996) diluted1:200 to 1:1,000 in PBS/0.3% TX-100.

Amino acid homologies of the rat Cx43 peptide used forantibody production were about 95% and 80% comparedwith the same chicken and fish sequence, respectively. Infish, 6 of 23 amino acids were exchanged, most of themshowing conservative exchanges. The fish sequence ofCx43 was taken from a carp Cx43 cDNA that was cloned inour laboratory (Dermietzel, Bochum) and will soon bepublished.

After three washes in PBS (10 minutes each), Cx43-IRwas visualized by immunofluorescence with Fluorescein(DTAF)-conjugated donkey anti-rabbit immunoglobulin(IgG; Jackson Immunoresearch Laboratories, West Grove,PA; diluted 1:300 in PBS/0.3% TX-100/2% NGS). All incu-bations with the secondary antibody were carried out atroom temperature for 2 hours. In control experiments,nonspecific binding was tested by omitting the primaryantibody.

For immunocytochemistry with dissociated and platedcarp retinal cells, retinas from dark-adapted animals weredissected and incubated in oxygenized Leibowitz (L-15)medium (Sigma, Deisenhofen, Germany) containing 1.5mg/ml papain (Sigma) for 15 minutes to induce chemicaldissociation. Subsequently, the retinas were washed threetimes in Leibowitz (L-15) medium, including 5% fetal calfserum to stop enzyme activity, and they were dissociatedmechanically by trituration with fire-polished Pasteurpipettes. Aliquots (500 µl) of the cell suspension wereplated onto poly-L-lysine-coated slides (Sigma), allowed tosettle for 1 hour at 18°C, and then fixed and furtherhandled for immunocytochemistry as described above.

Immunoblotting

Retinas were removed from the eyecups and homog-enized in ice-cold homogenization buffer (20 mM Tris/Cl,pH 7.4; 10 mM MgCl2; 0.6 mM CaCl2; 0.5 mM EGTA;0.005% TX-100; 1 mM phenylmethylsulfonyl fluoride(PMSF); 2 µg/ml of leupeptin; and 5 µg/ml of aprotinin). In

CONNEXINS IN THE VERTEBRATE RETINA 311

addition to the retinas, small pieces of rat heart werehomogenized by sonication in the same buffer. Proteinconcentrations of all samples were determined accordingto the method of Bradford (1976) by using bovine albuminas a standard.

Aliquots (30–100 µg of protein) of retinal and rat hearthomogenates were prepared for sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) by mixingtwo volumes of the samples with one volume of a concen-trated SDS-sample buffer (30% glycerol, 3% 2-mercaptoeth-anol, 9% SDS, and 187.5 mM Tris/Cl, pH 6.8) and allowingthem to react at room temperature for 30 minutes (noboiling). The samples were then separated on 10% lineargels at constant voltage (160 V) according to the method ofLaemmli (1970), and proteins were blotted onto nitrocellu-lose (Optitran BA-S 85; Schleicher and Schuell, Dassel,Germany) according to Towbin et al. (1979). Followingprotein transfer (1 hour/40 V, 2 hours/70 V), the blots werewashed in Tris-buffered saline (TBS; 50 mM Tris/Cl, pH7.4; 100 mM NaCl), and nonspecific binding was blockedby incubation in TBS containing 5% nonfat powdered milkat 37°C for 1 hour. The incubation with the preimmuneserum (diluted 1:5,000 in TBS) or with the primaryantibodies (diluted 1:2,000 in TBS) was carried out at 4°Covernight. Subsequently, the blots were washed threetimes (10 minutes each) in TBS, incubated with horserad-ish peroxidase-conjugated goat anti-rabbit IgG (BioradLaboratories, Munchen, Germany; diluted 1:3,000 inTBS/2% powdered milk) at room temperature for 2 hours,and washed again. Immunoreactive proteins were de-tected by using the chemiluminescence method (ECL-system; Amersham Buchler, Braunschweig, Germany).Incubations with the preimmune serum and the Cx43antibody were carried out with the same blots, one afterthe other. The blots were first incubated with the preim-mune serum to detect nonspecific immunoreactive pro-teins. Afterward, bound antibodies were stripped off theblots by washing the blots in two different strip-buffers at37°C for 1 hour each (strip-buffer 1: 0.01 M Tris/Cl, pH 8.8,1% SDS, 10 mM 2-mercaptoethanol; strip-buffer 2: 0.1 Msodium citrate, pH 3.0, 1% SDS, 10 mM 2-mercaptoetha-nol) and in TBS three times for 10 minutes each. The blotswere then incubated with the Cx43 antibodies to detectCx43-immunoreactive proteins.

RESULTS

The specificity of the antibody was analyzed by immuno-blotting using samples of rat heart homogenates, whichshowed two broad bands of 43–45 kDa and 84–86 kDa,representing the monomeric and dimeric forms of Cx43,respectively (Fig. 1B, RH). In addition, minor proteinsabove 125 kDa, most likely representing oligomeric formsof Cx43, showed cross reactivity with this antibody. NoCx43-immunoreactive proteins were detected in homoge-nates of rat liver (data not shown). The incubation of theblots with the preimmune serum (Fig. 1A) revealed immu-noreactive proteins only in rat heart homogenates (140kDa) and rabbit retinal homogenates (76–120 kDa), whichwere not found on immunoblots incubated with the affinity-purified Cx43 antibody (compare Fig. 1A with Fig. 1B), afinding that verifies the specifity of the antibody used inthis study. In longitudinal cryosections of rat heart ven-tricular muscle, this antibody further revealed the charac-

teristic Cx43-IR localized to the intercalated discs and thelateral margins of apposed myocytes (Fig. 2).

In retinal homogenates of rat (RR), rabbit (RbR), chicken(CR), turtle (TR), zebrafish (ZFR), and carp (FR), theantibody mainly bound to a broad protein component of43–45 kDa, whereas the mouse retina showed a strongerimmunoreactivity with a 46-kDa component (Fig. 1B). Insome experiments, immunolabeling of a 41-kDa proteinwas observed (data not shown), which might represent theinitial posttranslational form of Cx43 (Yamamoto et al.,1990). The immunoblots also showed substantial immuno-reactivity with the dimer component (84–86 kDa) in rat(RR), mouse (MR), and rabbit (RbR) retinal homogenates,whereas this component was labeled only sometimes inchicken (CR), turtle (TR), and fish (ZFR, FR) retinalhomogenates, the latter indicating a lower abundance ofthe Cx43 dimer (Fig. 1). In control experiments in whichthe primary antibody was omitted, no immunoreactiveproteins were observed on the blots of rat heart and retinalhomogenates (data not shown).

The immunocytological distribution patterns of Cx43-IRin rabbit, rat, chicken, turtle, and fish retinas revealed onesimilarity as well as some differences, the latter indicatinga species-specific distribution of Cx43 in the vertebrateretina. All tested retinas showed a substantial punctateimmunolabeling of the margins of neighboring pigmentepithelium cells. This pattern was most obvious in thechicken retina, in which it revealed a prominent hexagonalarray of pigment epithelium cells (Fig. 3B).Indeed, the pigment epithelium was the only structure inthe chicken and turtle retina that showed Cx43-IR (Figs. 3,4). In cryosections of the rabbit retina, Cx43-IR revealedan uneven distribution. Except for the pigment epithelium(Fig. 5F, arrowhead), punctate labeling was found only inthe region of the stria medullaris, which appears as anelliptic structure extending nasotemporally from the opticdisc in rabbit retinal wholemount preparations. The striamedullaris is formed by myelinated ganglion cell axonsand is known to contain oligodendrocytes and astrocytes(Schnitzer, 1988). Cx43-IR was always prominent in thecentre of the stria medullaris, close to the optic disc, and itdecreased toward its edges (Fig. 5). The rat retina showedpunctate Cx43-IR only in the area of the optic nerve fiberlayer (NFL), which might be related to astroglia present inthe NFL (Fig. 6B). In addition, arterial blood vessels in therat retina (Fig. 6B, asterisk) and fish retina (Fig. 8D,arrowhead) were strongly labeled by the antibody.

Compared with rabbit, rat, chicken, and turtle retinas,the fish retinas, especially in the zebrafish, showed a morecomplex pattern of Cx43-IR. In addition to the pigmentepithelium (see Fig. 8C), both fish retinas showed intenseimmunolabeling forming a line of punctate clusters at thearea of the outer limiting membrane (OLM; Figs. 7B-D,8B-D). Immunocytochemistry on dissociated retinal cellsfrom the carp retina indicated that this labeling patternwas due to the presence of Cx43-immunoreactive struc-tures at the distal ends of MCs, which form the OLM.Figure 7F,H, shows that strong immunolabeling was pres-ent around the photoreceptor inner segment (Fig. 7F,arrowhead) and at the distal end of an MC (Fig. 7H,arrowhead). Neither cells showed immunostaining when

312 U. JANSSEN-BIENHOLD ET AL.

Fig. 1. A,B: Immunoblots of 10% sodium dodecyl sulfate polyacryl-amide gel electrophoresis (SDS-PAGE) gels. The preimmune serumshows cross reactivity with a component of 140 kDa in rat heart homoge-nates (RH) and several proteins between 76 kDa and 120 kDa inhomogenates of the rabbit retina (RbR). In homogenates of rat (RR),mouse (MR), chicken (CR), turtle (TR), zebrafish (ZFR), and carp

retina (FR), no cross reactivity with the preimmune serum is present(A). In B, connexin43 (Cx43)-immunoreactive proteins are marked byarrowheads, representing the monomeric (43 kDa) and dimeric (86kDa) forms of Cx43. The amounts of protein loaded onto the gel varybetween 30 µg and 100 µg (30 µg: RH, MR, RbR; 50 µg: RH, RR, TR,ZFR, FR; 100 µg: CR).


the primary antibody was omitted (see Fig. 7E and G). Intransverse sections of both fish retinas, Cx43-IR was seenat the level of the outer plexiform layer (OPL; Figs. 7C,D,8B). At higher magification, it appeared that the punctatestaining was distributed around the photoreceptor termi-nals (Fig. 7D, arrowheads), and this finding was strength-ened by a weak immunolabeling of the terminals of singlecarp photoreceptor cells (see Fig. 7F, asterisk).

When cryosections of the carp retina were fixed for 4hours in 4% paraformaldehyde, in addition to the punctatelabeling of the OLM, a strong but diffuse immunolabelingwas sometimes found in horizontal cells and in their axonterminals projecting into the inner nuclear layer (Fig. 7B).This labeling pattern did not occur when the primaryantibody was omitted (compare Fig. 7A with Fig. 7B).

In zebrafish, but not in any of the other species tested,there was prominent Cx43-IR in some amacrine cells,independent of the type of fixation used (Fig. 8B,D,F). Theimmunolabeling was diffuse in the cytoplasm (Fig. 8D) andin a few primary processes extending into the IPL, whereasthe punctate staining that is characteristic of connexinsappeared in the more distal processes (Fig. 8F). TheCx43-immunoreactive amacrine cell formed a sparse butevenly distributed population (Fig. 8B, asterisks). Thesecells were bistratified, sending their processes into sublami-nae a and b of the IPL (Fig. 8E,F). A summary of thedistribution patterns of Cx43-IR in the retinas of thevertebrates tested here is provided in Table 1.

DISCUSSION

The data presented here indicate a species-specific distri-bution of Cx43 in the vertebrate retina. In all testedspecies, characteristic punctate Cx43-IR was localized inthe retinal pigment epithelium. Retinal pigment epithelialcells (RPECs) form junctional complexes along their apico-lateral borders, which consist of a zonula occludens, zonulaadherens, and gap junctions (Freddo, 1984), and culturedRPECs of the chick embryo show dye coupling, when theyare injected with Lucifer yellow (Kodama and Eguchi,

1994). Furthermore, a recent study showed that culturedrat RPECs propagate Ca21 waves, which could be blockedby the gap junction blocker halothane (Stalmans andHimpens, 1997). This indicates that RPECs express func-tional gap junctions that allow for ionic and metaboliccontinuity between the cells. In addition, we suggest thatthe junctionally coupled epithelial syncytium may alsofunction as a spatial buffering system for extracellularpotassium in the outer retina, because RPECs, like MCs,are sensitive to changes in extracellular potassium concen-trations and behave like potassium electrodes (Dowling,1987). Cx43 seems to represent the major connexin thatforms gap junctional channels between RPECs and otherepithelial cells of the eye, because it was localized in theretinal pigment epithelium of all species tested here, inRPECs of human and bovine eyes (Coca-Prados et al.,1992), and in lens and corneal epithelial cells (Beyer et al.,1989, Musil et al., 1990). The finding of Cx43-IR in bloodvessels of the vascularized retinas (fish and rat) is consis-tent with the fact that Cx43 is present in the endotheliumand smooth muscles of the cardiovascular system (Larsonet al., 1989), which further confirms the specificity of theantibody used in this study.

Although we have not analyzed immunolabeling at theelectron microscopic level, the punctate Cx43-IR in thestria medullaris of the rabbit retina, in the NFL of the ratretina, and in the OLM of the fish retina suggests that theantibody shows cross reactivity with Cx43 present inretinal glia cells, such as astrocytes and MCs. In previousstudies, Cx43 was characterized as the major gap junctionprotein in astrocytes of the central nervous system byimmunological, electrophysiological, and molecular biologi-cal techniques (Dermietzel et al., 1989, 1991; Micevychand Abelson, 1991; for reviews, see Dermietzel, 1996;Giaume and McCarthy, 1996). The presence of astrocytesin the stria medullaris of the rabbit retina, as well as in theNFL of other mammals, is known from studies usingglia-specific markers (Schnitzer, 1988). In the NFL ofvascularized retinas, astrocytes form a network of cellscoupled by prominent gap junctions (Bussow, 1980; Hol-

Fig. 2. Immunohistochemical localization of Cx43 in cryosections of rat heart ventricular muscle.Specific punctate, anti-Cx43 immunolabeling in particular is present at the intercalated discs (arrow-heads). No such staining is visible in the control sections, in which the primary antibody was omitted(left). Scale bar 5 50 µm.


lander et al., 1991) and exhibit dye coupling (Robinson etal., 1993). The fact that the Cx43-IR in the rabbit andrat retina was restricted to the stria medullaris and

to the NFL supports the idea that Cx43 also represents themajor connexin protein of astrocytes in the mammalianretina.

Fig. 3. A–C: Immunolocalization of Cx43 in ethanol-fixed cryosec-tions of chicken retina. C shows a brightfield photomicrograph of thechicken retina cryosection shown in B. PE, Pigment epithelial cells;OS, outer segment; OPL, outer plexiform layer. Scale bar 5 50 µm.

Fig. 4. A,B: Immunolocalization of Cx43 in ethanol-fixed cryosec-tions of turtle retina. The anti-Cx43 antibody revealed a prominent

cross reactivity with the pigment epithelial cells (PE; B). No otherimmunostaining was found. Staining in the area of photoreceptorouter segments (OS) and in the outer plexiform layer (OPL) is due to thenonspecific reactivity of the secondary antibody, as shown in controlsections (Figs. 3A and 4A). Scale bar 5 50 µm.


Fig. 5. A–G: Cx43 immunoreactivity (-IR) in paraformaldehyde-fixed cryosections of the rabbit retina. Punctate immunolabeling isvisible in the stria medullaris (SM; D) and in the pigment epithelialcells (PE; F, arrowhead), whereas the remaining retinal layers show nostaining. Control sections (B,G) show nonspecific staining of blood

vessels (B, asterisk). A,C,E are the corresponding brightfield photomi-crographs of B,D,F. ONL, outer nuclear layer; OPL, outer plexiformlayer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL,ganglion cell layer. Scale bar 5 50 µm.


The finding of Cx43-IR in MCs of the fish retina is notsurprising, because MCs and astrocytes are thought to berelated and express some of the same astrocyte-specificmarker proteins, such as glial filament acidic protein (forreview, see Schnitzer, 1988). MCs are specialized radialglia cells that form the external and internal limitingmembranes, they are distributed evenly over the verte-brate retina, and they are thought to represent the princi-pal glia cells of the retina (for review, see Newman andReichenbach, 1996). Compared with mammalian MCs,which show only insignificant dye coupling (Robinson etal., 1993), MCs of some lower vertebrates exhibit strongelectrical and dye coupling (Mobbs et al., 1988), and gapjunctions have been identified in the OLM of the cane toadretina (Gold and Dowling, 1979). The presence of Cx43-IRin the OLM of the fish retina (see also Giblin and Chris-tensen, 1997) is consistent with these findings and sug-gests that coupling between MCs, which has not yet beendemonstrated in the fish retina by means of dye and/orelectrical coupling, may involve gap-junction channelsformed by Cx43, as shown for astrocytes from other areasof the central nervous system. The significance of such afunctionally coupled MC syncytium could be to provide apathway for lateral buffering of extracellular potassium(Mobbs et al., 1988) in addition to the K1 siphoning(Newman et al., 1984) through the MC endfeet to thevitreous (see also Newman and Reichenbach, 1996; Vaney,1996).

A surprising finding was the presence of Cx43-IR in theOPL of both fish retinas and in a subpopulation of ama-crine cells in the zebrafish retina. Because this type ofimmunolabeling was a punctate staining, which is charac-teristic of connexins, and was not affected by the type offixation (see the discussion of Cx43-IR in fish horizontalcells, below), it rather suggests that the gap junctions ofthese neurons, which have been demonstrated by using ofelectron microscopy (Witkovsky et al., 1974; Marc et al.,1988), may be formed from Cx43-like protein components.In some characteristics-a large, prolate soma and a fewlarge-diameter proximal dendrites extending laterally andin an oblique fashion into the distal and proximal inner

plexiform layer-the Cx43-immunoreactive zebrafish ama-crine cell resembles the coupled transient amacrine celldescribed previously in the catfish (Naka and Christensen,1981), goldfish (Marc et al., 1988), and carp (Teranishi andNegishi, 1994) retina.

The unitary conductance of cardiac gap junctions, whichcontain Cx43, is around 50 pS, whereas that of channelsformed by Cx32 is between 120 pS and 150 pS (Veenstraand DeHaan, 1986; Burt and Spray, 1988; for review, seeBennett and Verselis, 1992). Gap-junction channels inhorizontal cells of the white perch and zebrafish retinahave unitary conductances of 50–60 pS (McMahon et al.,1989; McMahon and Brown, 1994), suggesting that thesechannels might be formed by Cx43 protein subunits.

The significance of Cx43-IR found in carp horizontalcells after fixation in paraformaldehyde is not clear. If aCx43-like protein is a major component of connexons formedby horizontal cells, then the diffuse rather than punctateimmunolabeling of horizontal cells suggests that the con-nexons were distributed evenly over the entire cell surface.

However, it is also possible that the diffuse Cx43-likeimmunolabeling of horizontal cells might be due to thebinding of the antibody to some cytoplasmic or more evenlydistributed membrane protein carrying a rat Cx43-relatedepitope rather than an actual connexin, and it is possiblethat this type of nonspecific binding is dependent onprotein fixation by paraformaldehyde. Another reason tosuspect that the Cx43-IR in paraformaldehyde-fixed carpretinas may be nonspecific is that rabbit, mouse, andmonkey horizontal cells contain Cx32 (Finch and Paul,1989), and Cx32-IR was also found in dispersed and platedhorizontal cells of the carp retina as well as on immuno-blots of horizontal cell homogenates (Janssen-Bienhold et.al., 1995; Weiler, 1996).

The present results demonstrate the presence of Cx43-IRin pigment epithelium cells of several vertebrate retinasand in glial elements, e.g., astrocytes in rat and rabbitretina and MCs in fish retina. Retinal neurons, with theexception of photoreceptors and one type of amacrine cellin the zebrafish retina, do not appear to express Cx43.

Fig. 6. A,B: Localization of Cx43-IR in ethanol-fixed cryosections of the rat retina. Characteristicpunctate labeling is obvious in the nerve fiber layer (NFL) and around a blood vessel (B, asterisk). No suchstaining occurs in the other retinal cell layers or in control sections (A). ONL, outer nuclear layer; INL,inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bar 5 50 µm.


Figure 7


ACKNOWLEDGMENTS

We thank Dr. D.I. Vaney for a critical reading of thepaper.

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Fig. 8. A–F: Cx43-IR in ethanol-fixed cryosections of the zebrafishretina. In addition to the staining of the outer limiting membrane(OLM; B,C), the outer plexiform layer (OPL; B), and the pigmentepithelium (PE; C), staining occurs in a subpopulation of amacrinecells (B and E, cell bodies marked by asterisks). In these cells, thewhole cell body appears to be stained as well as their large-caliber

processes, which ramify in sublayers a and b of the inner plexiformlayer (IPL; D–F). Punctate labeling characteristic of gap junctions isvisible in both the distal part and the proximal part of the IPL (E). ACx43-immunoreactive blood vessel is marked by an arrowhead in D.Scale bars 5 50 µm.

TABLE 1. Connexin43 Immunoreactivity in the Retinasof Different Vertebrates

Animal PE OLM OPL INL IPL GC NFL

Rabbit 1 2 2 2 2 2 1(SM)1

Rat 1 2 2 2 2 2 1Chicken 1 2 2 2 2 2 2Turtle 1 2 2 2 2 2 2Carp 1 1 1 1(HC)2 2 2 2Zebrafish 1 1 1 1/AC 1 2 2

1SM, stria medullaris. Area containing astrocytes and oligodendrocytes of the rabbitretina.2HC, horizontal cells. These cells reveal only a diffuse staining when the retinas arefixed in paraformaldehyde. PE, pigment epithelium; OLM, outer limiting membrane;OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GC,ganglion cell layer; NFL, nerve fiber layer; AC, amacrine cells.

Fig. 7. A–H: Cx43-IR in the carp retina. In paraformaldehyde-fixed cryosections (A,B), characteristic punctate immunolabeling isvisible only at the outer limiting membrane (OLM). The outerplexiform layer (OPL) and horizontal cells (HC) show prominent,diffuse staining of the entire cell bodies. In comparison, in ethanol-fixed cryosections (C,D), punctate immunolabeling is obvious only inthe OLM (C, arrowheads) and in the OPL (D, arrowheads). In the OPL,the staining occurs around photoreceptor terminal-like structures (D,arrowheads), whereas horizontal cells are not labeled. In dissociated,plated, and ethanol-fixed retinal cells, anti-Cx43 shows cross reactiv-ity only with an area at the level of the inner segment of photorecep-tors, where the OLM is formed (F, arrowhead), and at photoreceptorterminals (F, asterisk). The distal parts of Muller cells (H, arrowhead)are stained. No such immunolabeling was visible in the controls, inwhich the primary antibody was omitted (A,E,G). Scale bars 5 20 µm.


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Distribution of Connexin43 immunoreactivity in the retinas of different vertebrates

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