Abnormal Cone Synapses in HumanCone–Rod Dystrophy

Kevin Gregory–Evans, MD,1 Robert N. Fariss, PhD,1 Daniel E. Possin, BS,1 Cheryl Y. Gregory–Evans, PhD,2

Ann H. Milam, PhD1,3

Objective: Little is known of the cytopathology of photoreceptors in human inherited retinal dystrophies thatinitially affect the central retina, including the macula. The current study sought to determine the cytologicfeatures of dysfunctional cone and rod photoreceptors, as well as the pattern of degeneration of the cells inrepresentative cases of central retinal dystrophy.

Study Design: Comparative human tissue study.Materials: Four human donor eyes with the following forms of central retinal dystrophy: cone–rod dystrophy

(CRD), central areolar choroidal dystrophy, Bardet–Biedl syndrome, and cone dystrophy–cerebellar ataxia. Thecytologic features of retinal photoreceptors in these eyes were compared with those in an eye with retinitispigmentosa and six normal human eyes.

Methods and Outcome Measures: Immunocytochemistry and electron microscopy were used to evaluatethe retinal histopathology in the donor eyes.

Results: Cone numbers were decreased in the case of CRD, particularly in the central and far peripheral retina,and both cone and rod outer segments were slightly shortened. Occasional degenerate cones had dense cytoplasmand pyknotic nuclei dislocated sclerad to the external-limiting membrane. The most prominent alteration in this retinawas marked enlargement and distortion of the cone photoreceptor pedicles, which contained reduced numbers ofsynaptic vesicles. The retina with central areolar choroidal dystrophy contained a few cones with similarly abnormalsynapses. However, comparable cone synapse abnormalities were not observed in the cases of Bardet–Biedlsyndrome, cone dystrophy–cerebellar ataxia, retinitis pigmentosa, or in the normal retinas.

Conclusions: The functional consequences of the cone synapse abnormalities in CRD are not known butmay correlate with the electroretinographic abnormalities documented in some cases of CRD. To our knowledge,comparable synapse changes have not been noted in either rods or cones in other forms of retinal dystrophy,including retinitis pigmentosa, suggesting that different cytopathologic mechanisms may be involved.Ophthalmology 1998;105:2306–2312

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A number of studies have documented histopathologic chanin retinas with different forms of retinitis pigmentosa (RP).1–6

In RP, initial degeneration typically occurs in rod photorecetors in the peripheral retina, only later affecting cones inperiphery, along with both rods and cones in the central retLess attention has been paid to photoreceptor changes indystrophies that initially affect the central retina, including tmacula. This latter group of disorders includes macular, coand cone–rod dystrophies (CRD).7–13 These conditions are

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Originally received: March 20, 1998.Revision accepted: June 22, 1998. Manuscript no. 981501 Department of Ophthalmology, University of Washington, Seattle, Washingto2 Department of Molecular Genetics, Imperial College School of MedicineSt. Mary’s Campus, London, England.3 Scheie Eye Institute, University of Pennsylvania, 51 North 39th StreePhiladelphia, PA 19104.

Supported by the Paul and Evanina Bell Mackall Foundation Trust, Foundtion Fighting Blindness, Hunt Valley, MD (KG-E and AHM), Research toPrevent Blindness, Inc., New York, NY (AHM), the Chatlos Foundation,Longwood, FL (AHM), the TFC Frost Charitable Trust, Claygate, EsherSurrey, UK (KG-E), and NIH grants EY0 1311 (AHM) and EY0 1730 (AHM).

The authors have no proprietary interests in any materials mentioned in thpaper.

Reprint requests to Ann H. Milam, PhD, Scheie Eye Institute, University oPennsylvania, 51 North 39th Street, Philadelphia, PA 19104.

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characterized by initial loss of central visual field, visual acuitand color vision and can lead to significant loss of visionchildren as well as adults.

We performed immunocytochemistry and electron mcroscopy on postmortem human donor globes with fodifferent forms of retinal dystrophy initially affecting thecentral retina. Particular attention was paid to cytopathlogic changes in the cone photoreceptors because abnormities of these cells cause significant visual handicap innumber of retinal dystrophies, including RP. DNA obtainefrom fixed extraocular muscle of the donor eyes wascreened to establish a genetic diagnosis.

Methods

Tissue PreparationPostmortem eyes were obtained through the donor programs ofFoundation Fighting Blindness (FFB, Hunt Valley, MD) and thUniversity of Washington Lions Eye Bank (Seattle, WA). Donoglobes with the following diagnoses were evaluated (Table 1multiplex CRD (Case FFB 482), presumed autosomal-recesscentral areolar choroidal dystrophy (Case FFB 531), autosomdominant cerebellar ataxia–cone dystrophy (Case FFB 416), aautosomal-recessive Bardet–Biedl syndrome (Case FFB 508).comparison, an eye with simplex RP (Case FFB 543) andnormal eyes matched roughly by age and postmortem interva

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Table 1. Clinical Features of Donor Eyes Used in the Study

Case No. Clinical History InheritanceAge(yrs)

Postmortem Intervalto Fixation (hrs)

FFB 482 Cone-rod dystrophy Multiplex 69 1.6FFB 531 Central areolar choroidal dystrophy Presumed AR 74 10.5FFB 416 Cerebellar ataxia/cone dystrophy AD 77 3.3FFB 508 Bardet-Biedl syndrome AR 42 4.5FFB 543 RP Simplex 63 8.5UW65496 Normal 30 3.25UW82395 Normal 53 8.0U5777036 Normal 53 1.0UW80295 Normal 54 4.0UW0220-95 Normal 83 6.0UW2896R Normal 84 1.5

FFB 5 Foundation Fighting Blindness; AR 5 autosomal recessive; AD 5 autosomal dominant; RP 5 retinitispigmentosa; UW 5 University of Washington.

Gregory–Evans et al z Abnormal Cone Synapses in CRD

fixation also were studied (Table 1). The diseased and normal ewere fixed in 4% paraformaldehyde and 0.5% glutaraldehyde0.13-mol/l phosphate buffer, pH 7.3, for several weeks and stothereafter in 2% paraformaldehyde in the same buffer.

ImmunocytochemistryFor immunofluorescence, retinal samples were rinsed in phphate-buffered saline, embedded in 5% agarose (Sigma ChemSt. Louis, MO), and sectioned at 90 to 100mm with a vibratingmicrotome (Leica, Deerfield, IL). To reduce nonspecific labelinthe sections were incubated for 4 hours at room temperaturebuffer solution (phosphate-buffered saline, 0.5% bovine seralbumin, 0.2% Triton X-100, 0.5% sodium azide) containing 2normal goat serum. The sections were incubated in primary abody in the same buffer solution for 14 hours at room temperaturinsed in buffer solution, and incubated for 14 hours at rootemperature in secondary antibodies (goat antirabbit or antimoimmunoglobulin G) labeled with fluorescein isothiocyana(green), Cy-2 (green), or Cy-3 (red) (Jackson ImmunoReseaLaboratories, Inc, West Grove, PA).

Cell-specific antibodies prepared in rabbits or mice were usedanalyze the various retinal cell types. Rods were identified wanti-rhodopsin: monoclonal antibody 4D2 (1:40) (from Dr. RMolday, University of British Columbia, Vancouver, Canada) anpolyclonal antibody (1:10,000) (from Dr. E. Kean, Case WesteReserve University, Cleveland, OH). Photoreceptor outer segmeand cytoplasm were labeled with polyclonal antibody anti-phosphoiesterase-gamma (1:1000–1:2000) (from Dr. B. K. Fung, UCLA, LAngeles, CA). Cone outer segments were identified with polyclonantibody anti-red/green or -blue cone opsin (1:5000) (from Dr.Nathans, Johns Hopkins University of Washington, Seattle, WA).

Immunolabeled retinal sections were examined with a Bio-RMRC-600 (Richmond, CA) laser scanning confocal microscopScanned images and confocal files were imported into a graphprogram (Photoshop 4.0; Adobe, San Jose, CA), and dye-sublition prints were generated.

Electron MicroscopyRetinal samples from Case FFB 482 (CRD) were embeddedMedcast (Ted Pella, Inc, Redding, CA). Ultrathin sections wecontrasted with uranyl acetate and lead citrate and examinedtransmission electron microscopy.

Molecular Genetics

DNA was extracted from paraformaldehyde–glutaraldehyde-fixsamples of extraocular muscle by means of a DNA extraction

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(Qiagen, Cambridge, UK). Heteroduplex screening and directquencing14 were used to detect mutations in genes known to causecentral retinal dystrophies, including peripherin/RDS15–18 andCRX.19–21 RETGC1 also was screened because this retinal dystro-phy-associated gene is mapped within aCRD region (CORD6).22

Results

Case FFB 482 (CRD)

The patient donor reported night blindness from adolescencewas diagnosed with CRD at age 43. His brother and cousin hamore severe form of CRD, with night blindness and loss of cenacuity from early childhood. Electroretinographic responses ofpatient donor were recorded at age 56 at another institutDark-adapted responses were elicited using a Burian–Allen ctact lens electrode and a Ganzfeld stimulation system. Rod b-wresponses (blue flash stimulus) were mildly reduced, and cresponses (red flash stimulus) were undetectable, consistentthe diagnosis of CRD. At age 62, visual acuity of the patient donwas 20/30. He died of bronchial carcinoma at age 69.

The retina showed loss of cone photoreceptors throughmost pronounced in the macula (Fig 1A) and far periphery, withrelative preservation of cone numbers in the midperiphery.comparison with similar regions in normal human retina (Fig 1B),occasional cones in the CRD retina had abnormally dense cplasm and pyknotic nuclei dislocated sclerad to the externlimiting membrane (Fig 1A). Most cone and rod outer segmentswere slightly shortened, but rods were otherwise morphologicanormal throughout the retina.

The most striking change in the retina was the enlarged, dtorted shape of the cone pedicles, along with thickening of socone axons (Figs 1C–E). These abnormalities were most apparentin thecentral retina (Figs1C, D), where theenlarged conepediclesreached 15mm in diameter (normal,;8-mm diameter). Peripheralcone pedicles also were distorted but to a lesser extent (Fig 1E)compared with cone synapses in a normal retina (Fig 1F).

Electron microscopy of the CRD retina revealed normal fistructure of the outer and inner segments of the rods and mostphotoreceptors, although the outer segments were slightly short(Fig 2). Occasional cones (Fig 2, inset) showed cytoplasmic densifi-cation and outer segment fragmentation suggestive of ongoing clar degeneration. The most dramatic changes were found in thesynapses. While the adjacent rod spherules had near-normalstructure(Figs3A and 4A), theconepediclesweremarkedly enlarged(compare Fig 3A with Fig 3B, illustrating a cone pedicle from anormal human retina). Double-labeling experiments showed thacone types (red/green and blue) had abnormal synapses.

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Ophthalmology Volume 105, Number 12, December 1998

Normal cone synapses contain ribbons and numerous synand coated vesicles and have zones of membrane specializwith postsynaptic processes in the outer plexiform layer (Fig 3B).

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Figure 1. (A, B) Light microscopic comparison of retina from Case FFB 482 (cone–rod dystrophy [CRD]) (A) and a normal human retina (B). Onemicrometer Medcast sections stained with Richardson’s methylene blue/azure II mixture. Asterisk indicates external-limiting membrane; N, inner nuclearlayer; RPE, retinal pigment epithelium (X800) (A). The CRD retina contains reduced numbers of cones (arrows), but the rods appear normal. One cone(closed arrowhead) shows increased cytoplasmic density, abnormal location of its nucleus sclerad to the external limiting membrane (*), and fragmentationof its outer segment (open arrowhead). The cone outer segments are slightly shorter than normal (compare with B). (B) Cones (arrows) and rods in anormal human retina. (C–F) Confocal microscopy of retina from Case FFB 482 (CRD) processed by immunofluorescence. The sections have beendouble-labeled with anti-phosphodiesterase-gamma (red) and anti-rhodopsin (green). Asterisk indicates the external-limiting membrane. (C) Cones in thecentral retina show abnormally enlarged pedicles, visualized by immunolabeling for phosphodiesterase-gamma (red). The outer segments (open arrowhead)of the adjacent rods are rhodopsin-positive. G, ganglion cell layer; N, inner nuclear layer (X115). (D) Higher magnification of image (C). The cone somata(arrowheads) appear normal, but the cone pedicles (arrows) are markedly enlarged compared with those in a normal human retina (F) (X570). (E)Peripheral retina. The cone pedicles (arrows) are enlarged compared with those in a normal human retina (F) (X570). (F) Normal human retina forcomparison. The cone somata, axons, and pedicles are normal in size and shape (X570).

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Gregory–Evans et al z Abnormal Cone Synapses in CRD

in number (Fig 4B). The cone pedicles also contained sphericalinclusions filled with synaptic and coated vesicles (Fig 4B). Thesespherical structures were surrounded by several membranes,gesting that they represent invaginating postsynaptic procerather than organelles free within the cone synapse cytoplasm

The outer plexiform layer had abnormal fine structure, incluing the presence of numerous electron dense granules withinprocesses postsynaptic to the cones (Fig 3A). The Muller cellprocesses surrounding the abnormal cone pedicles were abmally pale and swollen (Figs 3A, 4A).

Case FFB 531 (Central Areolar Choroidal Dystrophy)

The patient’s history included parental consanguinity and tbrothers with the same retinal dystrophy. The visual history wincomplete, but funduscopically the central regions of both retinwere atrophic with prominent underlying choroidal blood vesseThe donor died at age 74 of a stroke.

The macula showed marked thinning and near-total loss of ptoreceptors, except at the edge, where rods and cones with

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short outer segments were retained. In the peripheral retina,and cones were reduced in number, and their outer segmentsvery short. Occasional cone pedicles were enlarged and abnoin shape, although most appeared normal.

Case FFB 416 (Cerebellar Ataxia–Cone Dystrophy)

The donor was an affected member (patient 3) of a family wautosomal-dominant cone dystrophy and cerebellar ataxia.23 Af-fected members of this family have a mutation in the SCA1 g(ataxin-1), which includes a trinucleotide repeat expansion (DrBird, Department of Neurology, University of Washington, pesonal communication, 1998). Gradually deteriorating visislurred speech, and swallowing difficulties had developed inpatient in her 60s. At age 70, her visual acuity was 20/200 in eeye, and she had a moderate, nonspecific color vision abnormShe died at age 77.

Rods and cones in the peripheral retina were present in nonumbers and had normal morphology. The macula was thin

Figure 2. Case FFB 482. Electron micrographs ofphotoreceptors (cone–rod dystrophy). N, cone nuclei;n, rod nuclei; * indicates external-limiting membrane(X1750). The rod (R) and cone (C) outer (arrow-heads) and inner segments have near-normal finestructure. Open arrows indicate melanin granules inprocesses of the retinal pigment epithelium (RPE)surrounding a cone outer segment. (Inset). An occa-sional cone (c) shows cytoplasmic densification andouter segment fragmentation (arrowheads).

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Figure 3. Electron micrographs of cone pedicles inretina of Case FFB 482 (cone–rod dystrophy [CRD])(A) and a normal human retina (B) (X 9750). (A)The rod spherules (R) in the CRD retina are near-normal in fine structure, but the cone pedicle (C) isincreased in size compared with a cone pedicle in anormal human retina (B). The Muller cell processes(M) surrounding the abnormal cone pedicle are ab-normally pale and swollen. Outer plexiform layer(OPL) processes postsynaptic to the abnormal conepedicle contain abnormal electron-dense granules(open arrows). (B) The rod spherules (R), cone pedi-cle (C), and outer plexiform layer (OPL) in a normalhuman retina display normal fine structure.

Ophthalmology Volume 105, Number 12, December 1998

and lacked foveal cones. The remaining perifoveal rods and cohad shortened outer segments, but their synapses appeared no

Case FFB 508 (Bardet–Biedl Syndrome)

The donor, who had multiple birth defects and was mentaretarded, died at age 42 of renal failure. The retina showed wispread loss of rods and cones, with retention of only a single laof cones in the macula. These cones had markedly shortened osegments but normal-appearing synapses.

Case FFB 543 (Simplex RP)

The patient donor had night blindness and loss of peripheral fisince age 21. His last recorded visual acuity was 20/200 in eeye. He died at age 63 of multisystem failure. Both retinas hextensive loss of photoreceptors, with retention in the maculaperiphery of only a few cones with short outer segments bnormal-appearing pedicles.

Molecular Genetics

No mutations were identified in peripherin–RDS, RETGC1, orthe three published exons of CRX in the samples screened.

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Discussion

Microscopic study of human retinal dystrophies has behampered by the relatively small number of well-preservdonor eyes available in early stages of disease.3 This isespecially true of the central retinal dystrophies becapathologic photoreceptor changes can occur at an earlyIn the current study, two cases of central retinal dystropdisplayed novel changes in the cone photoreceptorswere not present in the rods. These changes includedlargement of the cone axons and pedicles and reductiotheir content of synaptic vesicles.

A previous histopathologic study of a case of CRD8

demonstrated photoreceptor loss in the central and farripheral retinal regions, with relative preservation of photreceptors in the midperiphery. The main cytopathologchange noted in the rods and cones was shortened osegments. We identified similar pathologic changes inrods and cones in our case of CRD, plus the abnormal caxons and pedicles. A few cones had abnormal axonspedicles in a case of central areolar choroidal dystrop

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Figure 4. Case FFB 482 (cone rod dystrophy [CRD]).Low-magnification (A) and high-magnification (B)electron micrographs of abnormal cone pedicles inretina. (A) Cone pedicles in the CRD retina areexpanded in size, whereas rod spherules (R) havenear-normal fine structure. The Muller cell processes(M) are pale and swollen; (*) indicates unidentifiedprofile, possibly of a degenerating photoreceptor;OPL, outer plexiform layer (X 5500) (B). Highermagnification of cone pedicle to the right in (A). Thesynaptic ribbons (straight arrows) and membrane spe-cializations onto postsynaptic processes (P) appearnormal, but very few coated (open arrows) and syn-aptic vesicles (arrowheads) are present in the conepedicle. Two spherical inclusions (S), surrounded bymultiple membranes (curved arrows), are filled withsynaptic and coated vesicles (open arrows) (X52,500).

Gregory–Evans et al z Abnormal Cone Synapses in CRD

although most appeared normal. However, this novchange was not found in the other cases of central retidystrophy or reported in a case of X-linked cone degenation,13 suggesting that this is not a generalized feature ofcone degeneration per se.

The functional consequences of the abnormal copedicles are unknown but might manifest as inner retindysfunction by electrophysiologic testing. Limited electrodiagnostic information available for Case FFB 482 indcated a functional defect in the photoreceptors, especiathe cones, but information as to possible inner retinal anormalities was not available. However, several other stuies of CRD patients have shown electronegative ERGconsistent with inner retinal dysfunction.24,25

Recent studies showed mutations in the CRX genesome CRD patients.19–21 The CRX gene has been shown tobe expressed in rods, although cone cell expression conot be determined.19 Analysis of a transgenic mouse model

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of CRD caused by a dominant-negative mutation in tCRX gene demonstrated that rod terminals failed to form.21

Further studies are indicated to determine whether cterminals are abnormal, as found in the case of CRDported here, and, if so, to elucidate the functional conquences of this unusual synapse abnormality.

A different cone abnormality, axon elongation, was noteda case of RP caused by the glutamine-64-ter rhodopsin mtion.2 In that case, some cone axons terminated in the innerplexiform layer and the remaining cone synapses appeashrunken and dense by electron microscopy. Similar chanin cone synapses (e.g., shrinkage and densification) also wnoted in rhodopsin transgenic pigs26,27 and may represent thehistopathologic correlate of decreased cone function occursecondary to rod cell degeneration.

All retinal dystrophies studied here showed shorteningthe cone outer segments, as found in RP and other formretinal degeneration.3 Other cytopathologic features of the

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cones in the CRD retina, including cytoplasmic densifiction and nuclear migration, have been less frequentlyported.8,26–28 These features, along with our finding of conepedicle abnormalities in CRD and central areolar choroiddystrophy, suggest that the mechanism of cone degeneradiffers from that in rods, although the final common patway of cell death probably involves the rapid processapoptosis in both cell types.29,30 With regard to new thera-peutic strategies for retinal dystrophies, it is possible thmethods developed to preserve rods may not be as effecfor cones. Because the most important aim in the treatmof retinal dystrophies is preservation of central cones, dvelopment of effective therapies for the central retinal dytrophies will require additional insight into the mechanism(s) of cone cell degeneration.

Acknowledgments. The authors thank Ms. J. Chang and Ms.Klock for technical assistance, the scientists listed in the Methosection for providing antibodies, personnel of the UniversityWashington Lions Eye Bank for providing normal human eyeMs. P. Brunner of the University of Washington Wm. Keck Centfor assistance with confocal microscopy, and Drs. R. G. WelebS. G. Jacobson, and A. V. Cideciyan for critical review of thmanuscript. Legal requirements for use of human donor postmtem tissues were met (University of Washington Human SubjeApproval #28-0053-E, dated 12/12/97).

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