Identification and localization of muscarinic acetylcholine receptors in the ocular tissues of the chick

Identification and Localizationof Muscarinic Acetylcholine Receptors

in the Ocular Tissues of the Chick

ANDY J. FISCHER,1* LISE A. MCKINNON,2 NEIL M. NATHANSON,2

AND WILLIAM K. STELL1

1Lions’ Sight Centre/Department of Anatomy, Faculty of Medicine, University of Calgary,Calgary, Alberta, Canada T2N 4N1

2Department of Pharmacology, University of Washington School of Medicine,Seattle, Washington, 98195

ABSTRACTThe purpose of this study was to characterize the distribution of muscarinic acetylcholine

receptors (mAChRs) in the ocular tissues of hatched chicks. In the chick, different isoforms ofthese receptors have been detected in the brain, heart, and retina, and mAChRs in oculartissues have been implicated in the pathogenesis of form-deprivation myopia. However, theprecise anatomical distribution of mAChRs within the retina, retinal pigment epithelium,choroid, ciliary body, and ciliary ganglion remains unknown. We used affinity-purified,type-specific antibodies directed to three different chick mAChR subtypes (cm2, cm3, and cm4)to detect receptor immunoreactivity in sections and extracts of these ocular tissues. We foundcm2, cm3, and cm4 in the retina, retinal pigment epithelium, choroid, and ciliary body. Withinthe retina, cm2 was expressed in numerous amacrine and ganglion cells; cm3 was expressedin many bipolar cells and small subsets of amacrine cells; and cm4 was found in most, if notall, amacrine and ganglion cells. Each mAChR was localized to distinct strata within the innerplexiform layer that cumulatively form three broad bands that closely match previouslydescribed localizations of subtype-nonspecific muscarinic ligand binding. Only cm3 wasdetected in the outer plexiform layer, and only cm4 was detected in the ciliary ganglion. Wepropose that each mAChR subtype has unique functions in each ocular tissue. J. Comp.Neurol. 392:273–284, 1998. r 1998 Wiley-Liss, Inc.

Indexing terms: retina; retinal pigment epithelium; choroid; ciliary body; ciliary ganglion;

immunocytochemistry

Cholinergic innervation to the eye is found in all verte-brate classes. Acetylcholine (ACh) is released from auto-nomic axon terminals on vascular smooth muscle and irisand ciliary muscles as well as from intrinsic interneuronsthat make synapses within the retina. Cholinergic innerva-tion to the choroid, iris, and ciliary muscles arises mainlyfrom distinct populations of parasympathetic neuronslocated in the ciliary ganglion. Parasympathetic activity inthe eye results in dilation of choroidal blood vessels,constriction of the pupil, and contraction of the ciliarymuscle. In the retina, cholinergic neurons have beenidentified as amacrine cells with somata located at theproximal margin of the inner nuclear layer (INL) anddisplaced to the ganglion cell layer (GCL) with processesconfined to two discrete strata in the inner plexiform layer(IPL; Eckenstein and Thoenen, 1982; Eckenstein et al.,1983; Famiglietti, 1983; Ma and Grant, 1984; Tauchi andMasland, 1984; Tumosa et al., 1984; Millar et al., 1985,

1987; Conley et al., 1986; Voigt, 1986; Spira et al., 1987); inthe avian retina, at least one additional subtype of cholin-ergic amacrine cells has been identified (Baughman andBader, 1977; Millar et al., 1987; Fischer et al., 1997). It iscurrently believed that these cells modulate receptive fieldproperties of ganglion cells (Ariel and Daw, 1982a,b;Masland et al., 1984; Schmidt and Vijayaraghavan, 1992;Kittila and Massey, 1997). To better understand the func-

Grant sponsor: Medical Research Council of Canada; Grant sponsor: TheRoy Allen Fund; Grant sponsor: The Marigold Foundation; Grant sponsor:The Edwin L. and John E. Gustus Endowment in Vision Disorders; Grantsponsor: National Institutes of Health; Grant number: HL30639.

*Correspondence to: Andy Fischer, Lions’ Sight Centre/Department ofAnatomy, Faculty of Medicine, University of Calgary, 3330 Hospital DriveN.W., Calgary, Alberta, Canada T2N 4N1. E-mail: [email protected]

Received 8 April 1997; Revised 11 September 1997; Accepted 13 October1997

THE JOURNAL OF COMPARATIVE NEUROLOGY 392:273–284 (1998)

r 1998 WILEY-LISS, INC.

tions of ocular cholinergic systems, it is necessary tocharacterize the postsynaptic targets for cholinergic inner-vation and to identify the specific ACh receptors (AChRs)that mediate specific postsynaptic responses.

AChRs can be segregated into two categories: 1) iono-tropic receptors that are selectively activated by nicotine-like ligands (nAChR) and 2) metabotropic receptors thatare selectively activated by muscarine-like ligands (musca-rinic ACh receptors; mAChRs). mAChRs belong to a familyof receptors that contain seven transmembrane domainsand elicit cellular responses via interactions with GTPbinding proteins (for review, see Nathanson, 1987). Inmammals, five distinct mAChR isoforms, representing theproducts of five separate genes, have been characterized byboth molecular biology (m1–m5; Bonner et al., 1987, 1988;Peralta et al., 1987, 1988) and pharmacology (M1–M5;Buckley et al., 1989; Dorje et al., 1991). Autoradiographicstudies using [3H]quinuclidinyl benzylate (QNB) or [3H]pro-pylbenzilylcholine mustard (PrBCM) have revealed musca-rinic binding sites, mainly in the IPL of human retina(Hutchins and Hollyfield, 1985; Zarbin et al., 1986), sala-manders (Polans et al., 1985; Townes-Anderson and Vogt,1989), ferrets (Hutchins, 1994), and monkeys (Zarbin etal., 1986).

In the chick, very little is known about the distributionof mAChR subtypes in the different tissues of the eye. Fouravian mAChR subtypes have been characterized andnamed according to sequence homology with their mamma-lian counterparts: cm2 (Tietje and Nathanson, 1991), cm3(Gadbut and Galper, 1994), cm4 (Tietje et al., 1990), andcm5 (Creason et al., 1995). Autoradiographic studies haverevealed muscarinic binding sites in the IPL (Sugiyama etal., 1977; Large et al., 1985), but the neurons that expressthese receptors remain unknown. In addition, cm2, cm3,and cm4 receptor mRNAs and proteins have been detectedby using solution hybridization, immunoblotting, and im-munoprecipitation techniques in retinal tissue extractsfrom embryonic and hatched chicks (McKinnon andNathanson, 1995), but the precise anatomical distributionof each isoform remains unknown. Pharmacological tech-niques have been used to detect muscarinic binding sitesin the chick ciliary ganglion (Schmidt and Vijayaragha-van, 1992; Sorimachi, 1993; Furukawa et al., 1994), irismuscles, ciliary body (Pilar et al., 1987), and choroid(Marwitt et al., 1971; Meriney and Pilar, 1987). Further-more, pharmacological studies have implicated mAChRsin the metabolism of retinal pigment epithelium (RPE) inhumans (Osborne et al., 1991) and rats (Salceda, 1994) andin the visual regulation of ocular growth in humans(Bedrossian, 1979), monkeys (Raviola and Weisel, 1985),tree shrews (McKanna and Casagrande, 1981), and chicks(Stone et al., 1991; McBrien et al., 1993; Leech et al., 1995).

Therefore, to better understand the roles of ocularmAChRs, we have characterized their distribution in theretina, RPE, choroid, ciliary body, and ciliary ganglion ofhatched chicks by using type-specific antibodies directedagainst cm2, cm3, and cm4. We found mAChRs distributedthroughout the eye, with unique patterns of distribution ineach tissue.

MATERIALS AND METHODS

Animals

Day-old male leghorn chickens (Gallus gallus domesti-cus) were obtained from Lillydale Hatchery (Linden, Al-

berta, Canada) and were kept under standard fluorescentroom lights on a cycle of 12 hours light, 12 hours dark(lights on at 7:00 AM). Chicks were held for 1 week in astainless-steel brooder at about 25°C prior to experimenta-tion. They received water and Purina chick starter adlibitum. The use of animals was conducted under guide-lines ascribed by the Canadian Council on Animal Care.

Antisera

Antisera and their working dilutions included anti-cm2at 1:200, anti-cm3 at 1:1,000, and anti-cm4 at 1:500[affinity-purified rabbit polyclonal immunoglobulins (IgGs);McKinnon and Nathanson, 1995]; antisomatostatin at1:300 (rat monoclonal IgG S-10; gift from Dr. A. Buchan,University of British Columbia); antivasoactive intestinalpolypeptide (anti-VIP) at 1:80 (rat monoclonal IgG VP31;gift from Dr. A. Buchan, University of British Columbia);antityrosine hydroxylase (anti-TH) at 1:50 (mouse monoclo-nal IgG; Developmental Studies Hybridoma Bank, Univer-sity of Iowa, Iowa City, IA); anticholine acetyltransferase(anti-ChAT) at 1:1,000 (rabbit polyclonal 1465; gift fromDr. M. Epstein, University of Wisconsin); and antiproteinkinase C (a and b isoforms) at 1:50 (mouse monoclonalRPN 536; Amersham, Oakville, Ontario). Antisera di-rected against the chick mAChRs were the same as thoseused previously by McKinnon and Nathanson (1995).Crude sera from immunized rabbits were affinity purified,first through a recombinant glutathione-S-transferase(GST)-conjugated affinity column and then through arecombinant GST-mAChR fusion protein affinity column.The fractions that bound to GST alone have been shown toproduce no immunolabelling of cultured retinal cells fromthe chick embryo (McKinnon and Nathanson, unpublishedobservations). Controls were treated identically, exceptthat the primary antiserum was excluded from the secondincubation step. The specificity of the antisera raisedagainst the chick mAChRs was tested by preabsorptionwith recombinant GST overnight at 4°C. Preabsorption ofmAChR antibodies with the immunizing fusion proteinswas not performed, because these antisera had beenaffinity purified previously by binding to their respectiveimmunizing proteins.

Immunocytochemistry

Between 7 and 14 days after hatching, chicks were killedby chloroform inhalation. Eyes were enucleated and hemi-sectioned equatorially, the gel vitreous was removed, andtissues were placed into fixative (4% paraformaldehyde,2% sucrose in 0.1 M phosphate buffer, pH 7.4) at roomtemperature for 30 minutes. Tissues were washed threetimes in phosphate-buffered saline (PBS; 0.05 M phos-phate buffer, 195 mM NaCl, 3 mM NaN3, pH 7.4) andcryoprotected in PBS plus 30% sucrose for at least 4 hours.Samples were soaked in embedding medium (O.C.T. com-pound; Tissue-TEK, Miles Inc., Elkhart, IN) for 30 min-utes, frozen in liquid nitrogen, and mounted onto section-ing blocks. Sections 10–15 µm thick were melted ontogelatin-coated slides, air dried, ringed with rubber cement,and stored at 220°C until use. Each slide was washedthree times in PBS and covered with 150 µl 5% normalgoat serum (NGS) in PBS plus 0.3% Triton X-100 (TX-100)for 1 hour at room temperature. The NGS solution wasthen aspirated, and the primary antibody solution wasapplied (100–150 µl of antibody diluted in PBS plus 0.3%TX-100) for 24 hours at room temperature in a humidified

274 A.J. FISCHER ET AL.

chamber. Slides were then washed three times in PBS andcovered with the secondary antibody solution (100–150 µlof either 1:80 fluorescein isothiocyanate [FITC]-conju-gated goat-anti-rabbit IgG or anti-mouse IgG [Sigma,Oakville, Ontario] or 1:1,000 Cy3-conjugated goat-anti-rabbit IgG or anti-mouse IgG [Biological Detection Sys-tems Inc., Pittsburgh, PA]) diluted in PBS plus 0.3%TX-100 for 2 hours at room temperature in a humidifiedchamber. The slides were washed in PBS and mounted in4:1 (v/v) glycerol to water, and coverslips were added forobservation under an epifluorescence microscope. Photo-graphs were taken on TMAX 400 black-and-white film(Kodak, Rochester, NY) and developed in TMAX Developer(Kodak).

Tissue dissection for immunoblots

The eyes from four chicks (8 days after hatching) wereenucleated and hemisectioned equatorially, the gel vitre-ous was removed, and the remaining eye cup was placedinto ice-cold PBS. The lens, iris, and ciliary body wereremoved together from the front of the eye and placed intoice-cold extraction buffer (0.05 M Tris-HCl plus 1% [w/v]sodium dodecylsulfate; SDS). The pecten was then cut outof the eye cup, and the retina was removed with the RPE

still attached, leaving the choroid adherent to the sclera.The choroid was then dissected out and placed into 1.5 mlof ice-cold extraction buffer. The RPE was pulled awayfrom the retina by using fine forceps, and the isolatedretina was placed into extraction buffer. The fragments ofRPE were gathered with a Pasteur pipette into a conicalcentrifuge tube. The RPE suspension was then centrifugedfor 5 minutes at 31,000g, the supernatant was aspirated,and the fragments of RPE were resuspended in 1 ml ofextraction buffer. Samples were homogenized by using aTissue-Tearor (Biospec Products, Bartlesville, OK), soni-cated for 15 minutes, and centrifuged at 34,300g. Thesupernatant was then decanted, and the pellet was dis-carded. The supernatant was stored at 220°C until use.

Polyacrylamide gel electrophoresis

Tissue extracts were assessed for protein content byusing a modified Bradford assay. In short, 2 µl of seriallydiluted (1:2) tissue extract were blotted and dried on filterpaper in parallel with 2 µl of bovine serum albuminstandards (1.0, 0.1, 0.01, and 0.001 µg/µl). The filter paperwas stained in Coomassie blue (0.1% w/v Coomassie blueR-250; Bio-Rad, Oakville, Ontario, Canada; 40% methanoland 10% acetic acid in dH2O) for about 5 minutes on a

Fig. 1. Vertical sections of retina stained with antibodies to muscarinic acetylcholine receptor(mAChR) subtypes cm2 (a), cm3 (b), and cm4 (c) and with choline acetyltransferase (ChAT; d). IPL, innerplexiform layer; INL, inner nuclear layer; GCL, ganglion cell layer; OPL, outer plexiform layer. Scalebar 5 50 µm.

MUSCARINIC ACETYLCHOLINE RECEPTORS IN CHICK EYE 275

rotary shaker. The filter paper was then destained (in 40%methanol and 10% acetic acid). The staining intensities ofsamples and standards were compared by visual inspec-tion to estimate the total protein content of samples to the

nearest standard. Tissue extracts were diluted in samplebuffer (0.6 M Tris-HCl, pH 6.8; 4.8% v/v b-mercaptoetha-nol; 9.6% v/v glycerol; 1.9% w/v SDS; 0.0024% w/v bromo-phenol blue) to 1 µg/µl to allow equal amounts of protein

Fig. 2. Schematic diagram of the distribution of cholinergic innervation (Millar et al., 1985; Watt andFlorak, 1994; Fischer et al., 1997), muscarinic acetylcholine receptors (mAChRs), and nicotinic acetylcho-line receptors (nAChRs; Hamassaki-Britto et al., 1994) in the IPL. IPL, inner plexiform layer; INL, innernuclear layer; GCL, ganglion cell layer.


per sample to be loaded onto a gel. Finally, tubes contain-ing diluted samples were bathed in boiling water for 5minutes prior to loading onto a gel.

Twenty microliters of sample (equivalent to 20 µg ofprotein) per lane were loaded and run through a stackinggel (3.9% acrylamide, pH 6.8) at 100 V and a separating gel(7.5% acrylamide, pH 8.8) at 80 V. Prestained molecularweight standards (Bio-Rad) were run in lanes adjacent totissue samples. The contents of the gel were then blottedonto nitrocellulose membrane (NCM; Bio-Rad) overnightat 30 V. Membranes were washed three times in PBS, pH7.4; soaked in 5% (w/v) skim milk powder in 2.5 mMTris-HCl and 14 mM NaCl, pH 7.4 (buffer B), for 1 hour atroom temperature; and cut into separate lanes. The NCMlanes were added to the primary antibody solution (700 µl1:200 anti-cm2, 1:1,000 anti-cm3, or 1:500 anti-cm4 di-luted in buffer B) and incubated overnight in a humidifiedchamber at room temperature. The membranes werewashed three times in buffer B plus 1% BSA followed byone wash in buffer B, incubated in the secondary antibodysolution (5 ml of 1:1,000 alkaline phosphatase-conjugatedgoat-anti-rabbit IgG; Sigma) for at least 1 hour at roomtemperature, and then washed once in buffer B, once in 1%(v/v) TX-100 and 5 mM EDTA in buffer B, and twice inbuffer B. The membranes were covered with detectionsolution (100 mM Tris-HCl; 100 mM NaCl; 5 mM MgCl2,pH 9.5, plus 0.033% [w/v] nitroblue tetrazolium; 0.017%[w/v] 3-bromo-4-chloro-indolyl phosphate; and 0.69% [v/v]N,N-dimethylformamide) and were reacted for 5–30 min-utes. The color reaction was stopped by washing the NCMlanes in PBS.

Statistics and Measurements

All sample errors are given as the standard deviation ofthe mean. To express the distribution of cm3-immunoreac-tive amacrine cells, an index of regularity was calculatedas the sample mean of the distance to the nearest neighbordivided by the standard deviation (Wassle and Riemann,1978): The higher this ratio, the more regular the patternof distribution (values greater than 7 represent a highlyregular distribution, whereas values less than 3 representa random distribution). The distance between cells wasmeasured as the distance from the center of one cell to thecenter of the next nearest cell in horizontal sections(parallel to the plane of the retina). Sections were thickenough to include all cells of the type being analyzed.Percentage IPL depth was calculated as the distance fromthe IPL/INL border divided by the total thickness of theIPL multiplied by 100. All measurements were made fromphotomicrographs.

RESULTS

Localization of mAChRs in retina

Anti-cm2 weakly labelled cells in the amacrine cell layerof the INL and in the GCL (Fig. 1a). Cm2 immunoreactiv-ity was localized to the outer plexiform layer (OPL) and tofour thick strata in the IPL (Fig. 1a) at 0–15%, 40–60%,70–85%, and 95–100% IPL depths (Fig. 2). Superimposedupon the stratum at 40–60% IPL depth was a moreintensely labelled layer at about 50% IPL depth (Fig. 2).No detectable staining was produced by the anti-cm2 inciliary body, choroid, or ciliary ganglion.

Cm3 immunoreactivity was localized to about two-thirds of all bipolar cells (i.e., cells between amacrine andhorizontal cell layers), sparsely distributed amacrine cells,and a few cells in the GCL (Figs. 1b, 3a,b). The amacrinecells labelled with anti-cm3 in the central retina were6.7 6 0.2 µm (n 5 20) in horizontal diameter and weredistributed at about 1,000 cells per mm2 with a regularityindex of 3.88. These cells appeared to contribute theirprocesses to an intensely stained stratum at 20–30% IPLdepth. The cells immunoreactive for cm3 in the GCLappeared to be displaced amacrine cells, because theirsomata were relatively small (6.9 6 0.5 µm in diameter;n 5 10). They were distributed randomly, with a regularityindex of only 2.31. Anti-cm3 produced punctate labelling inthe OPL but did not stain horizontal or photoreceptor cells.Anti-cm3 also labelled three strata at IPL depths of0–30%, 45–60%, and 70–95% (Fig. 2). Superimposed uponthe two distal strata were two more intensely stainedstrata at 20–30% and 50–55% IPL depths (Fig. 2). Inaddition, anti-cm3 diffusely labelled the muscles andepithelium of the ciliary body (Fig. 4a,b) and the RPE (Fig.5a) and robustly labelled the walls of most blood vessels inthe choroid (Fig. 6a). No cm3 immunoreactivity was seenin the ciliary ganglion.

Fig. 3. Horizontal sections of retina at the level of amacrine cells inthe inner plexiform layer (IPL; a) and the ganglion cell layer (GCL; b)stained with antibody directed against cm3. Scale bar 5 50 µm.


Anti-cm4 robustly labelled most, if not all, amacrinecells in the INL and most cells in the GCL as well as onethin stratum at about 60% IPL depth (Figs. 1c, 2). Inaddition, immunoreactive cm4 was detected in the ciliaryepithelium (Fig. 4c,d), the RPE (Fig. 5b), and the walls ofmost choroidal blood vessels (Fig. 6b). Anti-cm4 was theonly antiserum that labelled cell bodies in the ciliaryganglion (Fig. 7).

Double labelling of retinal neurons

Anti-cm2 was not used in double-labelling experiments,because its weak labelling of cell bodies would have madeit difficult to identify double-labelled cells. Double-labelling experiments were performed to characterize fur-ther the cm3- and cm4-immunoreactive cells by usingantisera known to label well-characterized subsets ofamacrine and bipolar cells that might be involved in theregulation of ocular growth. The bipolar cells labelled byanti-cm3 did not contain protein kinase C (PKC) immuno-reactivity (Fig. 8). The subset of amacrine cells labelledwith anti-cm3 did not express any of the other markers forwhich we probed.

Some amacrine cells labelled by anti-cm4 were alsodouble-labelled with other antibodies. Cm4 immunoreactiv-ity was found in all TH-immunoreactive amacrine cells(n 5 75) as well as all amacrine cells containing somatosta-

tin immunoreactivity (n 5 134) and VIP immunoreactivity(n 5 100).

Immunoblot analysis of mAChRs in theocular tissue of the chick

Cm2 immunoreactivity was detected in tissue extractsof retina, RPE, choroid, and ciliary body at an apparentmolecular mass of about 65 kDa (Fig. 9a). Cm3 immunore-activity was detected in the retina, RPE, and ciliary bodyat an apparent molecular mass of about 90 kDa (Fig. 9b),but, in the choroid, it was detected at about 60 and 70 kDa(Fig. 9b). Immunoreactive cm4 with an apparent molecu-lar mass of about 95 kDa was detected in the retina, RPE,choroid, and ciliary body (Fig. 9c).

DISCUSSION

Labelling of cell bodies with antibodiesdirected against mAChRs

The antibodies directed against mAChRs labelled cellbodies in both the INL and the GCL. Similarly, antibodiesraised to different subunits of the nAChR (Keyser et al.,1988; Hamassaki-Britto et al., 1991, 1994) and the g-aminobutyric acid (GABA)A receptor (Hughes et al., 1989)have also been reported to label cell bodies in addition to

Fig. 4. a–d: Transverse sections through the ciliary body at the lens (a,c) and just lateral to the lens(b,d). These sections have been stained for cm3 immunoreactivity (a,b) and cm4 immunoreactivity (c,d).L, lens. Scale bar 5 50 µm.


processes in synaptic layers of the retina. The significanceof receptor immunoreactivity in neural somata remainsuncertain. Such immunoreactivity may represent 1) label-ling of cytoplasmic nonreceptor proteins sharing homologywith antigenic epitopes of the receptor, 2) newly synthe-sized receptor molecules en route to peripheral synapses,3) degradation products or internalized receptors beingrecycled or down-regulated, or 4) functionally expressedreceptors at the surface of the cell body. The fact that somemuscarinic binding sites have been detected in the INL(Sugiyama et al., 1977) suggests that functional mAChRsmay be expressed at the surface of the cell body.

Muscarinic AChRs have been detected on isolated reti-nal cells, including ganglion, amacrine, bipolar, and hori-zontal cells, from the tiger salamander (Townes-Andersonand Vogt, 1989). Similarly, we have shown that all of thesecell types, except horizontal cells, express mAChR-immunoreactive proteins in the chick retina. In situ hybrid-ization of mAChR mRNAs will be required to confirm thatthese cells do synthesize mAChRs.

Distribution of muscarinic receptorsin the IPL and OPL

Previous reports of muscarinic ligand binding within theretina of chicks described three distinct bands of mAChRswithin the IPL (Sugiyama et al., 1977). The results pre-sented here indicate that cm2 and cm3 are distributed intothree broad bands within the IPL, with cm4-like immuno-

reactivity contributing to the most proximal stratum. Thecomposite distribution of all three immunoreactive mAChRsubtypes in the IPL falls into three broad bands at 0–30%,40–60%, and 70–95% IPL depths (Fig. 2). Because thebinding affinity of [3H]QNB for each isoform of chickenmAChR is roughly equivalent (Tietje et al., 1990; Tietjeand Nathanson, 1991; Gadbut and Galper, 1994), it islikely that the autoradiographic localization of mAChRswithin the IPL represents binding to at least these threedifferent isoforms. Furthermore, the distribution ofmAChRs within the IPL closely matches cholinergic inner-vation. In the chick retina, cholinergic innervation of theIPL is supplied by four different subtypes of cholinergicamacrine cell. Millar et al. (1987) identified three subtypesof ChAT-immunoreactive amacrine cells in the chick retina:type I cholinergic amacrine cells, with cell bodies at theIPL/INL border and neurites stratified at 10–20% IPLdepth; type II cholinergic amacrine cells, with cell bodiesin the GCL and processes at 55–65% IPL depth; and typeIII cholinergic amacrine cells, with cell bodies near themiddle of the INL and processes diffusely distributed at0–10% and 50–95% IPL depths (Fig. 2). Furthermore, typeIII cholinergic amacrine cells can be roughly divided inhalf into two subtypes: type IIIa, including cells thatcontain enkephalin, neurotensin, and somatostatin-likeimmunoreactivity, and type IIIb cells, which do not (Fischeret al., 1998). In addition, type III cholinergic amacrinecells ramify more densely in the proximal IPL at 50–55%,75–85%, and 90–95% IPL depths (Fig. 2; Millar et al.,1987; Watt and Florack, 1994).

Assuming that cholinergic synaptic transmission occurswithin well-defined IPL strata where neurites of choliner-gic amacrine cells ramify, then several conclusions can bedrawn by comparing the distributions of mAChRs andcholinergic innervation in the IPL. First, cm3 is the onlymAChR subtype that can be directly postsynaptic to type Icholinergic amacrine cells, whereas cm2, cm3, and cm4 areall potentially postsynaptic to type II cholinergic amacrinecells. Second, in the distal IPL, type III cholinergic ama-crine cells can be presynaptic only to the cm2 and cm3isoforms of mAChR, whereas, in the proximal IPL, type IIIcells could form synapses involving all three types ofmAChR. However, it is also likely that some ACh escapeshydrolysis by ACh esterase and diffuses away from itspoint of release to activate ectopic mAChRs.

The significance of cm3-like immunoreactivity in theOPL remains uncertain. This localization is reminiscent ofthe finding that nAChRs have been detected in bipolarcells and in the OPL of the chick retina (Yazulla andSchmidt, 1977; Hamassaki-Britto et al., 1994). However,no source of ACh in the OPL is known, because ChAT hasnot been detected in this layer or in photoreceptors or inhorizontal or bipolar cells. Although some evidence sug-gests that photoreceptors in turtle retina might synthesizeACh (Lam, 1972; Ross and McDougal, 1976; Sarthy andLam, 1979) possibly to modulate the activity of bipolarcells (James and Klein, 1985), there is no evidence thatouter retinal neurons release ACh in the chick. Therefore,cm3 immunoreactivity in the OPL may represent 1) func-tional nonsynaptic receptors that are activated at a dis-tance by ACh released from cholinergic amacrine cells,2) synaptic ACh receptors that are activated by an uniden-tified local source of ACh or other unknown mAChR ligand,3) nonfunctional receptors that have been transported orotherwise allocated to noncholinoceptive sites, or 4) a cm3

Fig. 5. Horizontal sections through the retinal pigment epitheliumthat were stained for cm3 immunoreactivity (a) and cm4 immunoreac-tivity (b). Scale bar 5 50 µm.


cross-reactive protein that is not a receptor. With regard tothe second point, it has been proposed that dopaminereleased from neurites in the IPL may escape reuptakeand diffuse, in substantial amounts, through the INL toactivate receptors in the OPL (Witkovsky and Schutte,1991). The diffusion of ACh out of the IPL may be largelyprevented by ACh esterase, but the distributions of ChATand ACh esterase in the IPL do not entirely overlap (Millaret al., 1985). Therefore, if ChAT-immunoreactive amacrinecells are the only retinal source of ACh, then activation ofmAChRs in the OPL may require ectopic release ortransport of ACh into areas devoid of ACh esterase or mayrequire pharmacologically significant amounts of ACh toescape hydrolysis and diffuse across the retina. Alterna-tively, ACh may be produced by a ChAT-independent

process in outer retinal neurons and may be released fromthem to act upon AChRs nearby in the OPL.

Significance of mAChRs in RPE, choroid,and ciliary body

In the choroid, cm3 immunoreactivity was detected at alower molecular weight than in other tissues in proteinblots and appeared as intense staining of the walls of bloodvessels in sections of the choroid. Cm3 is 639 amino acidslong (Gabut and Galper, 1994), which is equivalent toabout 70 kDa. Therefore, the cm3-immunoreactive band inblotted RPE and choroidal extracts (Fig. 9b) may representa functional but nonglycosylated form of the receptor.Pharmacological evidence suggests that mAChRs are ex-

Fig. 6. Horizontal section through the choroid stained with antibody directed to cm3 (a) and cm4 (b).L, lumen of blood vessel. Scale bar 5 50 µm.


pressed in the RPE of rats (Salceda, 1994) and humans(Osborne et al., 1991), but, to the best of our knowledge,there have been no prior reports of mAChR proteins,mRNAs, or mAChR-mediated functions in the RPE ofchickens.

It has been reported that mAChR antagonists do notinterfere with the normal functioning of the accommoda-tive mechanisms in the anterior of the chick eye and thatonly nAChRs participate in accommodation (McBrien etal., 1993). Therefore, it is likely that mAChRs in the ciliary

Fig. 7. Longitudinal section through the ciliary ganglion stained with antibody to cm4. Scale bar 550 µm.

Fig. 8. Horizontal section of the retina at the level of bipolar cells inthe inner plexiform layer that was double-stained for cm3 immunore-activity (red) and protein kinase C (PKC) immunoreactivity (green).The negatives of separate images were digitized, assigned color,

enhanced for contrast, and overlayed by aligning fiduciaries usingAdobe Photoshop 4.0 (Adobe Systems, Inc., Mountain View, CA). Theabsence of yellow structures (red 1 green) indicates that cm3 and PKCimmunoreactivities were not colocalized. Scale bar 5 50 µm.


body have some function other than evoking the contrac-tion of ciliary and iris muscles. It has also been reportedthat mAChRs participate in the development of form-deprivation myopia in chicks (Stone et al., 1991; McBrienet al., 1993; Leech et al., 1995). However, the location of themAChRs that participate in growth-regulating pathwaysin the eye remains unknown. It is possible that one or moremAChR isoform(s) in the retina, RPE, choroid, or ciliarybody may be involved in form-deprivation myopia and thevisual regulation of ocular growth.

Interpretation of immunoblots

The apparent molecular masses of the chick mAChRsclosely match those revealed in retina by previous affinityalkylation and immunoblotting analyses (Large et al.,1985; McKinnon and Nathanson, 1995). We detected immu-noreactive cm2 in tissue extracts of RPE, choroid, andciliary body despite the fact that we were unable to detectit in tissue sections. This likely resulted in part from thegreater inherent sensitivity of the immunoblot assay andfrom having concentrated the antigen through the tissueextraction and blotting procedures used in this study. Wealso detected additional immunoreactive bands in blottedextracts of choroid, RPE, and ciliary body. These bandsmay represent proteins that contain mAChR-like epitopesor, in some cases, the degradation products of proteolyticcleavage. It is also possible that these bands resulted fromnonspecific binding of antibody with proteins that werepresent in high amounts in these concentrated, whole-tissue extracts. It is noteworthy that the specificity ofthese antibodies has been tested previously by McKinnonand Nathanson (1995), who only reported immunoblots forextracts of membrane from retina but from not choroid,RPE, or ciliary body; whereas, in the present study, wholecell extracts were used. Differences between the apparentmolecular masses of chick mAChRs and banding patternsreported previously and those detected in our experimentswere likely due to variations in procedures for electropho-resis and detection of blotted proteins. Despite the pres-ence of extraneous bands on immunoblots, it can still beconcluded that immunoreactive mAChRs are present atthe appropriate molecular masses in extracts of severalocular tissues.

Reports of previous studies in which mAChRs wereassayed by PrBCM binding have also described a shift inthe relative abundance of mAChR isoforms in the retinabetween embryonic and adult chicks. Large et al. (1985)reported that the lower molecular mass mAChR isoform(likely to be cm2) was less abundant than the largerisoforms (likely to be the sum of cm3 and cm4) in the retinaof embryonic chicks, whereas the smaller mAChR isoformwas more abundant than the larger isoform in hatchedchicks. In contrast, McKinnon and Nathanson (1995)reported that cm4 mRNA and protein were more abundantthan those of other isoforms in both embryonic and hatchedchick retina and that expression levels decreased as devel-opment progressed. However, we detected substantial

c

b

a

Fig. 9. Protein blots labelled with antibodies to cm2 (a), cm3 (b),and cm4 (c). Lanes were loaded with 20 µg of protein extracted fromretina (lane I), retinal pigment epithelium (lane II), choroid (laneIII), and ciliary body (lane IV). The molecular masses on the left ofeach blot indicate the positions of molecular mass standards on thesame gel.


amounts of cm4 protein in the retina of hatched chicks.This discrepancy may have resulted from differences intissue extraction, antigen concentration, and immunoblot-ting techniques employed in these two studies. In addition,both cm2 and cm3 mRNAs in the retina were expressed atlow levels early in development but at higher levels afterhatching (McKinnon and Nathanson, 1995). These find-ings suggest that the higher molecular weight isoforms ofmAChRs are more abundant than the lower molecularweight isoform in the retina of hatched chicks. This is inagreement with our immunocytochemical findings in retinaof hatched chicks that the apparent amounts of bothhigher molecular mass mAChR isoforms are greater thanthe apparent amount of the lower molecular mass form.The discrepancy between the results of these two methodsfor receptor localization may have been due to differencesin the relative binding affinity of PrBCM or antibodies foreach mAChR subtype or may have been due to thepresence and developmental regulation of additionalmAChRs for which we do not yet have antisera.

CONCLUSIONS

We have reported here the localization of three differentsubtypes of mAChRs in the retina, choroid, ciliary body,RPE, and ciliary ganglion of the chick. The results indicatethat all three isoforms of chick mAChR for which we wereable to test are present in the RPE, ciliary body, and retina.Although different mAChR isoforms may be present in oneocular tissue, their distribution within that tissue isvariable. For example, in the retina, immunoreactivity fordifferent mAChR subtypes appeared in unique layers ofthe IPL. In addition, cm3 was expressed by distinctsubsets of amacrine, ganglion, and bipolar cells, whereascm2 and cm4 appeared in the vast majority of amacrineand ganglion cells but were absent from bipolar cells. Theexpression of different mAChR isoforms is tissue-specific,because cm3 was not detectable in the choroid and neithercm3 nor cm2 was detected in the ciliary ganglion, whereascm4 was found in all tissues tested.

ACKNOWLEDGMENTS

We thank Dr. A. Buchan and Dr. M. Epstein for kindlyproviding antisera. This work was supported by a studentfellowship in memory of James Thurber from the Fight ForSight research division of Prevent Blindness America andby studentships from the Neuroscience Research Group atthe University of Calgary, the Pharmaceutical and MedicalAssociation of Canada-Medical Research Council ofCanada, and the Alberta Heritage Foundation for MedicalResearch to A.J.F.; by grants from the Medical ResearchCouncil of Canada, the Roy Allen Fund, the MarigoldFoundation, and the Edwin L. and John E. Gustus Endow-ment in Vision Disorders at the University of Calgary toW.K.S.; and by NIH grant HL30639 to N.M.N.

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Identification and localization of muscarinic acetylcholine receptors in the ocular tissues of the chick