The Shapes and Numbers of AmacrineCells: Matching of Photofilled WithGolgi-Stained Cells in the Rabbit

Retina and ComparisonWith Other Mammalian Species

MARGARET A. MACNEIL,1 JOHN K. HEUSSY,2 RAMON F. DACHEUX2,3

ELIO RAVIOLA,2 AND RICHARD H. MASLAND1*1Howard Hughes Medical Institute, Massachusetts General Hospital,

Boston, Massachusetts 021142Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115

3Department of Ophthalmology, University of Alabama at Birmingham, Eye FoundationHospital, Birmingham, Alabama 35233

ABSTRACTAmacrine cells of the rabbit retina were studied by ‘‘photofilling,’’ a photochemical method

in which a fluorescent product is created within an individual cell by focal irradiation of thenucleus; and by Golgi impregnation. The photofilling method is quantitative, allowing anestimate of the frequency of the cells. The Golgi method shows their morphology in betterdetail. The photofilled sample consisted of 261 cells that were imaged digitally in through-focus series from a previous study (MacNeil and Masland [1998] Neuron 20:971–982). TheGolgi material consisted of 49 retinas that were stained as wholemounts. Eleven of thesesubsequently were cut in vertical section. Of the many hundreds of cells stained, digitalthrough-focus series were recorded for 208 of the Golgi-impregnated cells. The two methodswere found to confirm one another: Most cells revealed by photofilling were recognized easilyby Golgi staining, and vice versa. The greater resolution of the Golgi method allowed a moreprecise description of the cells and several types of amacrine cell were redefined. Two newtypes were identified. The two methods, taken together, provide an essentially completeaccounting of the populations of amacrine cells present in the rabbit retina. Many of themcorrespond to amacrine cells that have been described in other mammalian species, and thesehomologies are reviewed. J. Comp. Neurol. 413:305–326, 1999. r 1999 Wiley-Liss, Inc.

Indexing terms: fluorescence; morphology; classification; dendrites

Amacrine cells are major players in the retina’s process-ing of visual information. They make up approximately40% of all neurons in the inner nuclear layer of mamma-lian retinas (Strettoi and Masland, 1995; Jeon et al., 1998)and participate in 64–87%, depending on the species, of allsynapses in the inner plexiform layer (Dubin, 1970; Raviolaand Raviola, 1982). In the monkey, 50–70% of all synapsesupon retinal ganglion cells are made by amacrine cells(Cohen and Sterling, 1991; Jacoby et al., 1996).

Until recently, the only information about specific typesof amacrine cells came from Golgi sampling, staining witha few molecular probes, or microinjection. This recentlyhas been extended by the development of a fluorescence-based ‘‘photofilling’’ method (MacNeil and Masland, 1998).Microirradiation of a single cell by a spot of light causes the

oxidation within that cell of dihydrorhodamine 123(H2R123) to rhodamine 123, a fluorescent, positivelycharged molecule that is retained inside the cell anddiffuses within its dendrites. Because successful stainingof targeted cells is accomplished in a large fraction ofattempts, photofilling allowed quantitative sampling ofthe whole population of amacrine cells. The universe ofamacrine cells was partitioned into at least 26 distinct

Grant sponsor: National Institutes of Health; Grant numbers: EY-03011and EY-01344.

*Correspondence to: Dr. Richard Masland, Wellman 429, MassachusettsGeneral Hospital, Boston, MA 02114. E-mail: masland@helix.mgh.harvard.edu

Received 30 March 1999; Revised 21 June 1999; Accepted 28 June 1999

THE JOURNAL OF COMPARATIVE NEUROLOGY 413:305–326 (1999)

r 1999 WILEY-LISS, INC.

morphologic types; the most frequent, the AII amacrine,comprised 13% of the total population.

Although photofilling allows accurate sampling of thewhole population, images of the photofilled cells must becaptured from the living tissue and lack the contrast anddetail provided by the Golgi chromoargentic impregnation.Here, we have systematically matched the shapes of thecells revealed by photofilling to those seen by using theGolgi method, which stains neurons with still unparalleledresolution.

We had three goals. The first was to verify the complete-ness of the sample of photofilled amacrine cells and seewhether the Golgi technique would reveal additional types.The second was to refine our descriptions of the varioustypes of amacrines and clarify the differences among them.The third goal was to match these results, obtained in theretina of the rabbit, to descriptions of amacrine cells inother mammalian species. Many types of amacrines arewidely conserved among mammalian retinas (for reviews,see Masland, 1988; Vaney, 1990; Wassle and Boycott,1991). Wherever possible, we point out these correspon-dences. We describe our Golgi-stained cells, the matchwith photofilled cells, and the comparison with previouslypublished work together (see Results). Although conven-tion suggests that the last comparison should reside in ahistorically oriented discussion, this format seemed moredirect.

MATERIALS AND METHODS

We have studied the morphology of amacrine cells inrabbit retina by using two approaches: ‘‘photofilling’’ withH2R123 (MacNeil and Masland, 1998) and Golgi impregna-tion (Morest and Morest, 1966). The database of photo-filled cells consisted of 261 neurons that were digitallythrough-focused at 1-µm intervals in connection with aprevious study (MacNeil and Masland, 1998). The Golgi-stained cells were either imaged in wholemounts or drawnwith a camera lucida. Many cells were drawn first in awholemount; then, the retina was unmounted and sec-tioned and the cells were redrawn in a profile view. In thisway, the stratification of the processes could be correlatedwith the overall appearance of the dendritic tree. A fewcells were filled with horseradish peroxidase (HRP) afterintracellular recording of their light responses (Dacheuxand Raviola, 1995).

Photofilling

Photofilling begins with a photooxidation reaction insidea single cell; it generates a fluorescent product in a cell’ssoma that diffuses throughout the cell’s dendrites. Thenonfluorescent compound H2R123 is used as the precursormolecule. The rationale and experimental details havebeen described more fully elsewhere (MacNeil andMasland, 1998).All protocols were approved by the Subcom-mittee on Research Animal Care at the MassachusettsGeneral Hospital.

One day before experiments, the nuclei of retinal neu-rons in both eyes of anesthetized (intramuscular 50 mg/kgketamine and 10 mg/kg xylazine) adult New ZealandWhite rabbits (4.5–5.0 kg), were labeled nonspecifically byintraocular injection of the fluorescent molecule, 4,6-diamidino-2-phenylindole (DAPI; 10 µg; Sigma ChemicalCo., St. Louis, MO). The next day, the rabbits werereanesthetized by using the same agents, both eyes were

enucleated, and the animals were killed by a drug over-dose in accordance with institutional guidelines. Afterhemisection of the eyes, the retinas were teased away fromthe pigment epithelium and maintained in Ames medium(Sigma Chemical Co.) for the duration of the experiments,as described previously (Ames and Nesbett, 1981; Yangand Masland, 1994).

Retinas were rinsed for at least 3 hours to clear unboundDAPI from the tissue. After this period, a piece of ventralretina from the midperiphery (6–10 mm ventral to theoptic nerve head) was flatmounted ganglion cell side uponto a single piece of black, nonfluorescent filter mem-brane (type HABP filter; Millipore, Bedford, MA). Theretina-membrane preparation was moved to a Petri dishfilled with Ames medium positioned on the stage of a ZeissAxioplan microscope (Zeiss, Thornwood, NY), and thesurface was gassed with 75% N2, 20% O2, 5% CO2.

At this point, the retina was living and the nuclei of allretinal neurons were labeled with DAPI. H2R123 (Molecu-lar Probes, Eugene, OR) was then added to the bath toachieve a final concentration of 5–20 µM. Cells weretargeted randomly by using an indexed grid reticle in oneeyepiece and a table of random numbers, representingeach coordinate on the grid, to select the DAPI-stainednucleus to be irradiated. Evidence that this results in arepresentative sample of the actual cells present has beenreviewed elsewhere (MacNeil and Masland, 1998). A pin-hole 80 µm or 100 µm in diameter was placed in the backfocal plane of the epifluorescent light path to obscure alllight other than that needed to illuminate the nucleus.Irradiation with the microbeam of light (excitation, 340–380 nm; dichroic, 395 nm; emission, long pass 420 nm)through a Zeiss 340 Plan Neofluar water-immersion lenscaused the oxidation of the nonfluorescent H2R123 torhodamine 123, and the fluorescent oxidation productdiffused throughout the cytoplasm of the irradiated cell.After several cells in a piece of tissue were filled, theunfixed retina was rinsed, coverslipped in fresh Amesmedium, and imaged immediately.

Golgi method

Retinas from New Zealand White rabbits (2–6 kg) wereimpregnated by using a modified rapid-Golgi technique asdescribed by Morest and Morest (1966). The rabbits wereanaesthetized with 25% urethane, and the orbital areawas infiltrated locally with xylocaine. After the eyes wereenucleated and hemisected, the rabbits were killed by anoverdose of urethane. All protocols were approved by theAnimal Care and Use Committee at Harvard MedicalSchool. The vitreous was then removed from the eyecup.With sclera, choroid, and retina intact, each eyecup wastrimmed to a pentagonal shape, pinned against a flatsurface, and immersed in fixative (32 ml 3% potassiumdichromate and 0.25% osmium tetroxide, pH 5.8) at 25–30°C. Optimal fixation times ranged from 8 hours to 24hours.

After fixation, the retina and choroid were detachedfrom the sclera and placed choroid-side-up on a glass slide.The tissue was then covered with a glass coverslip, andthis was glued to the slide with a drop of 5-minute epoxy ateach corner. The preparations were then rinsed severaltimes with silver nitrate solution (0.75%) until the forma-tion of red-brown precipitate had stopped. These were thenplaced in 35–50 ml of fresh solution for periods of 12–36hours at room temperature with slow agitation. With

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this expedient, formation of a heavy precipitate of silverchromate over the retina was avoided.

After the silver impregnation, the retinas were removedfrom the glass slide, rinsed in double-distilled H2O, anddehydrated under small weights in 50%, 70%, 80%, and95% ethanol (10 minutes each), followed by absoluteethanol (changed every 30 minutes for 2 hours). Thedehydrated retinas were cleared in a 1:1 mixture ofcedarwood oil and a-terpineol for a 12–48 hour period.They were then cleared for 30 minutes in xylenes andmounted in Permount with the ganglion cell layer facingupward.

These flatmount preparations were examined with lightmicroscopy and some well-impregnated amacrine cellswere drawn by using a camera lucida. Other cells werephotographed by collecting through-focus series of imagesat 1-µm intervals by using brightfield differential interfer-ence contrast illumination (DIC; Fig. 1). The series ex-tended from the ganglion cell layer through the innernuclear layer, to delimit the borders of the inner plexiformlayer. The stratification patterns of nearby cells also couldbe used to assess the cells’ stratification.

Some of the Golgi-stained cells also were sectionedvertically. Flatmounted retinas were immersed in xylenesto dissolve the Permount and the tissue was rehydrated ina graded series of alcohol to 50% ethanol. The free-floatingretinas were then mounted in 4% agarose and sectionedserially at 80–100 µm in a tissue chopper. The sectionswere then dehydrated, cleared, and mounted in serialorder on glass slides, as described above. These cells wereeither drawn by using a camera lucida or imaged digitally.Most (but not all) of the cells studied were found in themidperipheral retina.

Data acquisition

Cells remained adequately photofilled for approximately1 hour as long as they were not excessively exposed tolight. The cells were studied with epifluorescence micros-copy by using a Zeiss Axioplan microscope and highnumerical aperture objectives (340 1.0 oil: Zeiss Plan-Apochromat; 340 and 363, 1.2 water: Zeiss C-Apochro-mat). Permanent records of each cell were made by using asensitive digital camera [MicroMax cooled CCD camerawith 4,096 gray levels (1,317 pixels 3 1,035 pixels);Princeton Instruments, Trenton, NJ] together with image-acquisition and processing software (MetaMorph, version2.5; Universal Imaging Corporation, West Chester, PA).MetaMorph software drove an electromagnetic shutter(Vincent Associates, Rochester, NY) to ensure short expo-sure times (< 400 msec) and a focus motor (Ludl ElectronicProducts, Hawthorn, NY) that advanced the microscopestage at regular, 1-µm intervals through the inner plexi-form layer. Two series of images were collected for eachcell. The first set used filters appropriate for rhodamine123 [either Zeiss (excitation, 450–590 nm; dichroic, 510nm; emission, band pass 515–565 nm) or Omega Optical(excitation, 488–512 nm; dichroic, 545 nm; emission, 526–562 nm] to reveal the shape and stratification of the filledcell. The second set, which was collected from the samefocal planes, used filters specific for viewing DAPI. Theseimages gave an independent measure of the thickness ofthe inner plexiform layer at that location (Fig. 1). Forillustration, Figure 2 shows four familiar amacrine cells(AII, starburst, DAPI-3, and indoleamine) that were im-aged in this way.

The quantitative results shown in Tables 1–4 includeevery cell that was photofilled. A less complete descriptionof these cells has been published elsewhere, and some ofthe cells shown here also were illustrated (in differentimages) in that paper (MacNeil and Masland, 1998). Thedigital images were adjusted for brightness and contrastand were unsharp masked to remove out-of-focus haze byusing MetaMorph or Adobe Photoshop (version 5.0.2;Adobe Systems, Inc., Mountain View, CA). The final fig-ures were assembled in CorelDraw (version 7.0; CorelCorporation, Dublin, Ireland). In Golgi-stained material,more than one cell was sometimes stained in a patch. Insuch cases (Figs. 11, 15–17, 19), nearby cells in the field ofview were cropped from the picture or removed withPhotoshop but without any alteration of the illustratedcell. No other digital processing was carried out.

RESULTS

The Golgi specimens were scanned at low power, andwell-stained cells were imaged digitally in through-focusseries or were drawn at higher power. In some instances,more than one example of a single type was stained in apatch of retina. These instances were used to verify levelsof stratification and to identify the features that wereconstant or variable in each cell type. Whenever possible,nearby cells (other amacrine, ganglion, or bipolar cells)were imaged in the same fields to aid in the analysis ofdendritic stratification. A total of 208 of the best stainedcells in wholemount and vertical section were recorded inthis way and then matched to the photofilled cells.

Our description of the cells will consist of four types ofimages: fluorescence images in wholemounts, DIC imagesof Golgi-stained cells in wholemounts, DIC images ofsections, and detailed drawings of the cells. The drawingsshow flat mounts and radial sections of the same cells. Inmost cases, we show images at several focal depths and/orimages of several cells.Although this creates many illustra-tions, it seemed more useful than the traditional, ‘‘typical’’instances of each cell. As a mnemonic, each cell type isaccompanied by a sketch of that cell’s major features.

We also list possible homologies to cells stained byothers. In some cases, these are tentative; most publisheddescriptions give only a single view of the cell, which oftencomes from an unknown retinal eccentricity. Such casesare indicated by question marks in Tables 1–4.

Amacrine cells were sorted into types by three majorcriteria: dendritic field size, the pattern and depth ofstratification within the inner plexiform layer, and theappearance of the dendrites. Generally, the size of thedendritic arbor and position of the dendrites in the innerplexiform layer were sufficient to separate cells unequivo-cally into types. These were supplemented by morphologiccriteria, which included the caliber of the dendrites, theircourse, and the size and distribution of their varicosities.In our combined photofilled and Golgi samples, we identi-fied 28 types of amacrine cells. Qualitatively, the twomethods provided similar results: All but one type of cellthat was identified by photofilling also was represented byat least a few examples in the Golgi-stained material.

Narrow-field amacrines

Narrow-field cells made up 55% of all photofilled ama-crine cells. They were divided into 13 types based on thestratification of their dendrites in the inner plexiform

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Fig. 1. Through-focus series of Golgi (left column), photofilled(middle column), and 4,6-diamidino-2-phenylindole (DAPI)-stainedcells (right column) in wholemounted retinas. Optical sections weremade of each cell at 1 µm intervals throughout its depth. Because thethickness of the inner plexiform layer (IPL) varies across the retina,stratification levels were measured as a fraction of the thickness of theinner plexiform layer. This thickness was measured from matchedimages of the DAPI staining (for photofilled cells) or from identifying

the amacrine and ganglion cell somata with brightfield differentialinterference contrast illumination (DIC; for Golgi stained cells). Bystandardizing the depth of the inner plexiform layer, it is possible tocompare cells from different eccentricities that were stained by usingdifferent methods. In this illustration, the Golgi-stained dendrites arecentered at 25% of the inner plexiform layer, whereas the photofilledcell has most of its dendrites between 25% and 50% of the innerplexiform layer. GC, ganglion cell layer. Scale bar 5 50 µm.

layer and in the richness and appearance of their den-drites. We have divided the narrow-field cells into twogroups: one with dendrites that stratify fairly sharplywithin one or more stratum (Table 1) and one with morediffuse arbors that cross several adjacent strata (Table 2).

Narrowly stratified narrow-field amacrine cells.

These narrow-field cells have dendritic arbors that stratifynarrowly within one or more strata of the inner plexiformlayer. In some instances, such as the narrow S1 cells (Fig.3), a single arbor is confined rigorously to one stratum. Inother instances, such as amacrine cell AII, a cell has twoarbors at different depths.

The dendrites of the first type of narrow-field cellencountered when passing from the outer to the innerportion of the inner plexiform layer belong to the narrowS1 cells (Fig. 3). These amacrines, identified in Golgi-stained retinas, have a single, irregular dendritic arbor <45 µm in diameter that ramifies predominantly in stratum1 of the inner plexiform layer. In vertical section, thedendrites are seen to have large, closely spaced varicosi-ties. Narrow S1 cells were not identified in the photofilledsample; their density, size, and coverage has been derivedfrom a patch of cells in the Golgi material. The bright cellbodies of the photofilled cells create optical flare in alldimensions. Because the dendrites of narrow S1 cells areso short and lie so close to the soma, they are effectivelyhidden by flare.

AII amacrine cells comprise the most common amacrinecell type in our sample (13%) and have an easily recogniz-

able bistratified organization (Famiglietti and Kolb, 1975).Their narrow inner arbors originate from one or two largeprimary dendrites that exit from the vitreal pole of theperikaryon (Fig. 2). The dendrites that form the outer orlobular arbor arise from the cell body or primary dendrites;occasionally, they represent recurrent processes emergingfrom branches of the primary dendrites. These processesterminate in the outer 40% of the inner plexiform layer aslobular appendages. The inner dendrites branch from theprimary dendrites in the middle of the inner plexiformlayer and terminate in stratum 5, giving rise as a circulararborization, 55–70 µm in diameter.

AII cells are among the most studied amacrine cells ofmammalian retinas. They are glycinergic neurons that canbe labeled with antibodies against parvalbumin or calreti-nin (Pourcho and Goebel, 1985; Casini et al., 1995; Wassleet al., 1995). Their characteristic bistratified organizationcan be recognized in cat (Famiglietti and Kolb, 1975;McGuire et al., 1984), rabbit (Dacheux and Raviola, 1986;Vaney et al., 1991; Strettoi et al., 1992), rat (Perry andWalker, 1980; Menger et al., 1998), monkey (Polyak, 1941;Boycott and Dowling, 1969; Mariani, 1990), and humanretinas (Kolb et al., 1992).

A8 amacrine cells have a bistratified morphology similarto that of AII cells (Fig. 4) and make up a small fraction ofall amacrine cells (2.3%). A primary dendrite gives rise totwo separate arbors < 40 µm in diameter that stratify at30% and 50–65% of the inner plexiform layer. In contrastto AII cells, the outer arbor of A8 cells has smooth

Fig. 2. Four well-studied amacrine cells identified by Golgi impreg-nation (middle) and photofilling (bottom). Their distribution in thephotofilled sample nearly matches the values obtained by using othertechniques (Strettoi and Masland, 1996). Note that the appearance ofthe staining is similar regardless of the method used. All cells are

shown in wholemount views, except for the Golgi-stained AII cell,which is shown in vertical section. The plane of focus is indicated oneach photomicrograph, with 0% indicating the border of the innernuclear layer. The line drawings (top) represent schematically thevertical appearance of that cell type. Scale bars 5 50 µm.

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dendrites with few varicosities, and the inner arbor rami-fies narrowly in the middle of the inner plexiform layer(50–60%).

Even though A8 cells were identified in the photofilledsample, but not the Golgi sample, a similar cell that wascalled A8 by Kolb et al. (1981) was described in Golgi-stained cat retinas. In the cat, the vitreal arbor of A8 wasreported to occupy a larger fraction of the inner plexiformlayer (strata 3–5) than this cell (strata 3–4).

Flat bistratified amacrine cells, as seen in the rabbit(Fig. 5), have two narrowly stratified dendritic arborslocated at 20% and 60% of the inner plexiform layer. Thelarger, outer arbor originates from at least four primarydendrites arising from the cell body. These processesquickly branch to form an even and symmetric arbor aslarge as 120 µm in diameter. In Figure 5, all dendrites thatbelong to the outer arbor appear in focus simultaneously,and they are decorated with regularly spaced varicosities.The inner arbor is relatively less complex. It is made up ofshort and straight, spoke-like dendrites arising from acentral, vitreally directed process. This arbor is slightlysmaller, extending < 70 µm from the center. Similar celltypes have been identified in the ground squirrel (A4;Linberg, et al., 1996) and the monkey (knotty bistratified,type 1; Mariani, 1990) retinas.

Asymmetric bistratified amacrine cells (Fig. 6) also havebistratified dendritic arbors, but they arborize at 40% and60% of the inner plexiform layer. In contrast with the flatbistratified cells, the arbors are sparse; each is highlyasymmetric, and they lie out of register on opposite sides ofthe primary dendrite. Similar cells have been identified inmonkey (wavy multistratified, type 2; Mariani, 1990) andground squirrel (A22; Lindberg et al., 1996).

Narrow S3 amacrine cells accounted for nearly 3% of allamacrine cells. The bulk of the arbor originates from one tothree primary dendrites that spread in stratum 3 of theinner plexiform layer. The terminal branches often areshort and end with small terminal swellings. Four ex-amples are shown in Figure 7.

The narrow S3 type may be analogous to the phosphateNADPH-diaphorase-positive cells that have been stainedin rabbit retina (Sandell, 1985; Vaney and Young, 1988).Their dendritic spreads are similar in width and depth,and both have beaded processes. Cells with similar mor-phologies have been identified in the retinas of cats (A3;Kolb et al., 1981), rats (type 3; Menger et al., 1998), dogs(see plate V, Fig. 9c in Ramon y Cajal, 1972), and monkeys(unistratified; Boycott and Dowling, 1969).

Broadly stratified narrow-field amacrine cells.

Cells with thick, bushy dendritic trees that occupy two ormore adjacent strata and branch in at least 40% of theinner plexiform layer are termed ‘‘diffuse amacrine cells.’’

Fig. 3. Vertical (top photomicrograph) and flatmount (bottomphotomicrograph) views of the narrow S1 cell type. The bottomphotomicrograph shows a cluster of similar cells all with sparse,beaded dendrites located at 15% of the inner plexiform layer (IPL).The dendritic arbor is less than 50 µm in diameter yet is dense enoughto sample the retina uniformly. INL, inner nuclear layer; GC, ganglioncell layer. Scale bars 5 50 µm.

Fig. 4. Unlike an AII cell, the inner and outer dendrites of A8 cellsare similar in morphology, with small, unremarkable varicosities onboth arbors and no lobular appendages. Scale bar 5 50 µm.

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Altogether, 28% of narrow-field cells can be considereddiffuse by this definition. They can be divided into seventypes.

Flag cells are a common group of amacrines in rabbitretinas. This distinctive type comes in two varieties. Bothsend a single oblique process into the middle of the innerplexiform layer. Flag A cells spread in the space betweenthe plexus of the orthotopic and displaced starburst celldendrites, with a typically eccentric, narrow but profuseand varicose dendritic tree (Fig. 8). Its inner twin, the flagB cell (referred to as monostratified cell by MacNeil andMasland, 1998), has a similar eccentric arrangement ofdendrites. The dense bush of intricately woven den-drites, < 45 µm in diameter, occupies the inner half of theinner plexiform layer (Fig. 8). The two types are remark-ably similar in shape and size.

Cells with flag cell-like morphologies have been identi-fied in other mammalian retinas. Polyak (1941) drew threedifferent versions of such cells from rhesus monkey retinas

(‘‘knotty’’ cells; see Fig. 67A,D, ‘‘k’’ in Polyak, 1941; alsoredrawn in Fig. 54 in Boycott and Dowling, 1969). In eachinstance, the characteristic arbor originates from a singledendritic stalk that descends throughout the inner plexi-form layer. The arbors are compact and are < 45 µm indiameter. Although the three cells were not distinguishedby Polyak as belonging to different groups, clear differ-ences can be seen in their stratification patterns within theinner plexiform layer. We propose that these three cellsrepresent the narrow S1, flag A, and flag B cells. Mariani(1990) identified these same three cell types in his study ofthe rhesus retina. He noted that the cells each occupyapproximately one-third of the inner plexiform layer andoverlap one another only slightly.

The dendrites of narrow diffuse cells reside in all fivelayers of the inner plexiform layer. It can be seen readily invertical sections (Fig. 9) that the tiny arbor originates fromone or two, short primary dendrites that immediatelybranch to form a cylindrical bush no larger than 55 µm indiameter. Overall, the dendrites are so closely packed thatit is nearly impossible to follow single branches in singleimages for more that a few micrometers from their origin.Processes run both horizontally and radially in layersS1–S4, but they tend to be exclusively radial in stratum 5.In vertical sections, it is possible to appreciate the cylindri-cal shape of the dendritic arbor (Fig. 9). Cells with thistype of morphology have been identified in the groundsquirrel (A1; West, 1976; Linberg et al., 1996), human(small diffuse; Kolb et al., 1992), monkey (knotty; Polyak,1941; Boycott and Dowling, 1969; Mariani, 1990), and rat(type 5; Menger et al., 1998). In the rat, these cells wereshown to be glycinergic (Menger et al., 1998).

Narrow diffuse cells, like the narrow S1 cells, were notidentified in the photofilled sample, probably for the samereason: The narrow, vertically oriented dendritic arbor

Fig. 5. Two flat bistratified cells, one shown in vertical section(Golgi-stained cell; top photomicrograph) and the other in wholemount(photofilled cell; bottom photomicrographs). Scale bars 5 50 µm.

Fig. 6. Two asymmetric bistratified cells. This type is character-ized by two sparse, asymmetric dendritic arbors that stratify at < 40%and < 60% of the inner plexiform layer. Scale bar 5 50 µm.

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tends to be obscured by the bright soma. However, afterseeing these cells in Golgi specimens, it became clear that11 of the unclassified narrow-field cells reported earlier(MacNeil and Masland, 1998) had the same characteristicmorphology. In the photofills, these cells appeared as adense cloud of fluorescent processes spanning the entirethickness of the inner plexiform layer.

AB diffuse type 1 and 2 cells give rise to outwardlysloping primary and secondary dendrites in the outertwo-thirds of the inner plexiform layer (Figs. 10, 11). Type 1cells were very common in our sample and accounted for <7% of the total. They have rich, tent-shaped arbors madeup of squiggly and varicose dendrites that give off numer-ous branches throughout their length. A few branchesterminate in strata 1 and 2 of the inner plexiform layer,but most continue to descend toward the ganglion cells andterminate in stratum 4. Five examples of type 1 cells areshown in Figure 10. Type 2 cells occupy the same region ofthe inner plexiform layer but are less complex. Usually, asingle dendrite gives off three or more branches in stratum2 to form an oval arbor 70 µm in diameter situated instratum 3 and 4. At a depths of < 60%, type 2 cell dendritesassume a vertical course toward the ganglion cells and endabruptly with small swellings. Notice in Figure 11 how thedendritic endings are all in focus in the same optical plane.

It is likely that versions of these cells are present inother mammalian retinas. Ramon y Cajal (1972) identifieddiffuse amacrine cells with outwardly sloping dendritessimilar to type 1 in dog and ox retinas (see plate V, Figs. 2‘‘c’’ and 8 ‘‘D’’ and plate VII, Fig. 8 ‘‘f ’’ in Ramon y Cajal,1972). Kolb and colleagues (1981) describe two types ofnarrow-field cells in cat retina with dendrites in strata 2and 3 of the inner plexiform layer (types A3 and A4).Glycinergic cell types 3 and 6 in the rat (Menger et al.,1998) have morphologic features similar to type 1 cells andare distributed in the same general region of the innerplexiform layer.

Recurving diffuse cells have a complicated arborization.Dendrites descend to the middle of the inner plexiformlayer, where they give rise to a complex plexus of wavybranches (Fig. 12). Some of these dendrites curve back uptoward the amacrine cell bodies (Fig. 12,top), whereasothers descend to a depth of 85%. This cell is similar to thefountain cell shown in Figure 17, but it has a narrowerdendritic spread, more dendrites that slant, and fewerrecurrent dendrites. Cells with this curious stratificationpattern have not yet been identified in other species,probably because the recurving feature is easy to miss inwholemounts.

Spider cells initially were difficult to recognize in whole-mount preparations but became easier once their existencewas known. They have a single, short process that exitsfrom the inner pole of the cell body and branches immedi-ately to form a small horizontal arbor in the outer part ofthe inner plexiform layer. Extending from these processesare individual, vertical branches that terminate at 60% ofthe inner plexiform layer. The descending dendrites ap-pear as bright dots in Figure 13 (bottom). Vertical sectionsthrough spider cells confirm this pattern of branching.

Cells with similar (but not identical) morphologies havebeen identified in other species. Figure 67 ‘‘l’’ in Polyak

Fig. 7. Two Golgi-stained cells (drawing and brightfield; top) andtwo photofilled cells (fluorescence image; bottom) with the narrow S3morphology. Scale bars 5 50 µm.

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(1941) and Figure 67 in Boycott and Dowling (1969) bothshow a narrow-field amacrine cell with vertically orienteddendrites in monkey retina. However, in both instances,the vertically descending processes also have thin, short,horizontal branches that form a dense band in stratum 4. Asimilar cell type has been stained by expression of alkalinephosphatase under control of a Brn-3 promoter (Xiang etal., 1996). The type 7 amacrine cell in the rat retina(Menger et al., 1998) has an arbor more like the one shownhere; it has unbranched vertical dendrites that do not forman inner band.

Medium-field amacrine cells

We identified five types of medium-field amacrine cells;their frequencies ranged from 1.1% to 3.4% of the cellssampled. All of them were distributed over broad regions ofthe inner plexiform layer, possessed dendritic fields < 170µm in diameter, and had coverages between 3 and 12. Themost common type was the previously described DAPI-3amacrine cell (Wright et al., 1997), which made up 3% ofthe photofilled sample (Fig. 2). This value is consistentwith the values obtained by using other methods. Theseglycinergic cells have two separate arbors that are nearlyequal in size, one situated between 20% and 40% and theother between 55% and 70% of the inner plexiform layer inthe vicinity of the cholinergic bands. Each arbor is < 120µm in diameter and is made up of thin, loopy, repeatedlybranching dendrites dotted with an assortment of varicosi-ties. The ‘‘orphaned’’ dendrites surrounding the DAPI-3cell represented in Figure 2 originate from the inner arbor(the outer arbor is shown).

It has been suggested that the ‘‘type 2 wavy-multistrati-fied’’ cell in macaque retina (Mariani, 1990) and the ‘‘A14’’amacrine cell in human retina (Kolb et al., 1992) are likelyhomologues to the DAPI-3 cell in the rabbit, because all ofthem branch in strata 2 and 4–5 of the inner plexiformlayer (Wright et al., 1997). Other cell types, such as the‘‘A21’’ cell in ground squirrel (Linberg et al., 1996) and the‘‘tristratified’’ cell in humans (Kolb et al., 1992), havesimilar dendritic morphologies but do not stratify at thesame levels of the inner plexiform layer.

AB broad diffuse cells (Fig. 14) are a group of cells withmedium-sized arbors, < 180 µm in diameter, that occupythe middle strata of the inner plexiform layer. The den-drites run a long, meandering horizontal course, oftencross each other, and have small, irregularly spaced vari-cosities.

Diffuse multistratified cells (Fig. 15) have a dendriticarbor 175 µm in diameter at it widest location. Whenthrough focusing an example of this cell type in a flat-mount, one sees horizontal dendrites constantly going inand out of focus, as though the cell had multiple levels ofstratification. However, unlike true stratified cells, whichhave noticeable gaps between arbors, the dendrites of thistype appear throughout the inner plexiform layer untilthey end at depths of < 70%, avoiding the stratum

Fig. 8. Camera lucida drawings and photomicrographs of Golgi-stained flag A and flag B cell types (top) and flatmounted, photofilledcells (bottom). For both types, the dendritic arbor originates from asingle, obliquely directed process. However, each type branches atdifferent levels of the inner plexiform layer (IPL). The vertical sectionsbest illustrate the differences, showing type A cell dendrites positionedbetween 25% and 65% of the inner plexiform layer and type Bdendrites beginning to arborize at 50% and terminating close to theganglion cell (GC) layer. INL, inner nuclear layer. Scale bars 5 25 µm.

AMACRINE CELLS 313

occupied by the rod-bipolar endings. In human retina(Kolb et al., 1992), the ‘‘tristratified’’ cell has a pattern ofbranching similar to the diffuse multistratified cell. Themajor exception is that it extends into layer 5.

Wavy bistratified cells (Fig. 16) have a dendritic arbor<175 µm in diameter made out of numerous horizontalbranches that occupy most of the thickness of the innerplexiform layer. The interesting feature of this cell is thatit sends dendrites to S1, skips S2 and occupies the rest ofthe inner plexiform layer with numerous horizontal den-drites that branch very seldom. Another defining feature ofthis cell type is the prominent varicosities found along thelength and at the ends of all the dendrites. This cell typehas not been identified in other species with Golgi orimmunohistochemical techniques.

Fountain cells are bistratified, medium-field amacrinecells that link the inner and outer portions of the innerplexiform layer in a unique way. The cell body gives rise toa single, vitreally directed process that passes through theentire depth of the inner plexiform layer to layer 5 andforms a broad horizontal arbor that measures 110–150 µmin diameter (Fig. 17). Originating from this inner arbor area number of thin processes (.10) that follow a recurrentcourse back to the outer two strata of the inner plexiformlayer. These branches spread horizontally within strata 1and 2 to form a second arbor 150–180 µm in diameter.Unlike the dendrites of the inner arbor, which have manyspine-like protrusions, the outer dendrites are dotted withvariable-sized varicosities and end with prominent append-ages. This cell has been described previously only inabstracts (Heussy et al., 1988; Wright and Vaney, 1995).Because of the distribution of its processes, this cell typewas interpreted as a wide-field equivalent of amacrine AII(Strettoi et al., 1990).

Wide-field cells

Wide-field cells as a group accounted for < 24% of allamacrine cells and could be subdivided into ten typesbased on stratification within the inner plexiform layerand morphologic criteria. Starburst amacrine cells werethe most frequently encountered wide-field cell type in theinner nuclear layer: Nearly 1 cell in every 20 filledamacrines had a starburst morphology. These cells havebeen classified historically as medium-field cells. Ourreasons for changing this categorization are providedbelow (see Discussion). Their frequency is slightly greaterthan one would expect from the densities sampled by usingDAPI or choline acetyltransferase-stained retinas (5%versus 3%), but this is within the variability expected fromthe small size of our sample. Stained examples wereidentified with both techniques and had identical morpho-logic characteristics (Fig. 2). They had a broad radialarbor, 300–400 µm in diameter, that was narrowly strati-fied at 24% of the inner plexiform layer. The peripheralone-third of the dendritic arbor contained numerous vari-cosities.

These cells were first stained as a population by usingthe presence of their neurotransmitter, acetylcholine, as amarker (Masland and Mills, 1979). Their shape was seen

Fig. 9. Three narrow, diffuse cells identified in Golgi preparations.The dendrites in the vertically sectioned cells (top) branch throughoutthe entire depth of the inner plexiform layer. The narrow arbormaintains the same diameter throughout the depth, and thus hascylindrical appearance. In optical sections of the wholemounted cell(bottom), one can appreciate the high density and complex arrange-ment of the dendrites. Scale bars 5 25 µm.

314 M.A. MACNEIL ET AL.

in Golgi-stained rat retina by Perry (1979). They werenamed by Famiglietti (1983), who likened their appear-ance in rabbit retina to that of a firework. Since then,starburst cells have been identified in most vertebrateretinas (for review, see Masland and Tauchi, 1986).

Other wide-field cells appear similar to one another:They have thin, straight, unbranched dendrites with fewdistinctive features, such as spines or varicosities. Mostdendrites of each cell are confined rigorously to a singlestratum of the inner plexiform layer. These cells (WF1–1,WF2, WF3–1, and WF4) were named for the stratum thatcontains their dendrites (Figs. 18–20). They comprise 34%of all wide-field cells and appear similar morphologically.

Many of these cells have been stained previously byusing other methods. Cell WF1–1 is remarkably similar tothe type I tyrosine hydroxylase immunoreactive cells inrabbit (Brecha et al., 1984; Mitrofanis et al., 1988; Tauchiet al., 1990), the type 1CA cells and dopaminergic cells inmonkey (Hokoc and Mariani, 1987; Dacey, 1990), and theA18 cells in cat (Kolb et al., 1981). Similar cells have beennoted in the rat (Mitrofanis et al., 1988; Nguyen-Legros,1988) and mouse (Gustincich et al., 1997). Their dendritesspread horizontally at the scleral border of the innerplexiform layer and extend for hundreds of micrometersfrom the cell soma.

Also in stratum 1 were two other wide-field types thathad a dendritic pattern unlike the stellate shape of theWF1–1 cell (Fig. 18). WF1–2 had two primary dendritesthat exited from the cell body at 180° from one another.These processes branched once at 50 µm from the somaand then continued in layer 1 for many hundreds ofmicrometers. This type shows morphologic similarities tothe ‘‘semilunar type 1’’ cells identified in human retina(Kolb et al., 1992), the ‘‘wiry-type 1’’ cells identified in

monkey retina (Mariani, 1990), and the orientation-biasedamacrine cells in the rabbit (Bloomfield, 1994).

The third cell type (WF1–3) in stratum 1 was found nearthe border of layers S1 and S2. It is an asymmetric cell thatgives rise to four to six dendrites that form a bundle andproject in a single direction. In instances in which den-drites exited from the ‘‘wrong’’ side of the cell soma, theyreversed their course within 50 µm to join the rest of thedendrites. Their morphology is similar to that of the‘‘dorsally directed’’ amacrine cells described by Famiglietti(1989). Again, these are among the orientation-biased cellsdescribed by Bloomfield (1994).

In stratum 2 of the inner plexiform layer, a single celltype was identified based on its morphology. However,WF2 amacrines may include more than one cell popula-tion. The neurofibrillar, long-range cells in rabbit de-scribed by Vaney and colleagues (1988) ramify in stratum2 but are very rare, making up only 0.2% of all amacrinecells. Wide-field photofilled cells of stratum 2 represented3.1% of all amacrines in our sample, and other types mayaccount for the discrepancy. Cell types analogous to WF2have been described in the cat (A19 and A20; Kolb et al.,1981) and the ground squirrel (A25a; Linberg et al., 1996).

Stratum 3 of the inner plexiform layer has two mainwide-field cell types (Fig. 19). Type WF3–1 was describedafter intracellular recording and injection of horseradishperoxidase (Dacheux and Raviola, 1995). It depolarizes atboth ON and OFF and has straight dendrites that irradi-ate in all directions and exhibit sparse dendritic spines. Itsstellate branching pattern and location at the borderbetween A and B sublaminae suggest that this type isanalogous to the type I NADPH-diaphorase cell (Sandell,1985; Sagar, 1986, 1990; Vaney and Young, 1988) identifiedin rabbits. The second type of wide-field amacrine cell of

Fig. 10. Vertical (top) and wholemount (bottom) views of AB diffuse-1 cells. They have a tent-shaped,varicose dendritic arbor. This cell type was common in our sample and made up 7.3% of all amacrine cells.Scale bars 5 50 µm.

AMACRINE CELLS 315

stratum 3 was relatively common in our sample (2.3%) andwas distinguished by the unusually large numbers ofprocesses crossing one another but remaining confined tothe same level. Dendritic arbors of these WF3–2 cellsresemble those of ‘‘polyaxonal-1’’ cells (Famiglietti, 1992a)and the axon-bearing amacrine cells of the macaque(Dacey, 1989); however, our techniques did not reveal anyaxons.

WF4 is another wide-field amacrine cell with straight,nonbranching dendrites that radiate in all directions,possess sparse varicosities, and remain confined to a singlelevel (Fig. 20). In stratum 5, we identified type 1 and type 2indoleamine-accumulating cells (Sandell and Masland,1986; Vaney, 1986) by their characteristic morphologies: Atype 1 cell is shown in Figure 2. Both are characterized bytheir varicose, outwardly sloping dendrites that form adense plexus just scleral to the perikarya of ganglion cells.Equivalent types exist in cat (A17; Kolb et al., 1981), ox(semilunar spongioblasts; see plate V, Fig. 7 ‘‘C’’ and plateVII, Fig. 8 ‘‘d’’ in Ramon y Cajal, 1972), monkey (wide-fielddiffuse; see Figs. 55, 57, and 61 in Boycott and Dowling,1969; spidery 1, Mariani, 1990), and humans (A17 spiderydiffuse; Kolb et al., 1992).

Our visualization of the wide-field cells was the mostlimited of all the amacrine cell types. In some cases, theextent of the dendritic field could not be measured becausethe dendrites were too faint or faded too quickly. Long-range axons that have been identified on several wide-field

types (Dacey, 1989; Famiglietti, 1992a; Taylor, 1996) werenot seen in any of our material.

Dendritic coverage

After (but only after) cells were partitioned among the28 types, we computed the coverage of the retina for eachof the narrow or medium-field cells, using the typology anddensities shown in Tables 1–3. For the narrow-field cells,the coverage was near a value of 1 (a possible exception wasthe narrow S3 type, with an apparent coverage of 3.9). Thecoverages of the medium-field cells were higher, averaging7. In general, the rarest wide-field cells have coverages ofabout 9 (Vaney, 1990). In our sample, WF4 was rare (0.4%),but its extremely wide arbor ensures a coverage of at least100. The more common indoleamine-accumulating cellshave coverage factors of 500–900 (Vaney, 1990). Note thatwe used the actual measured dendritic field area tocompute coverage rather than the more common mean ofthe field’s widest and narrowest dimensions. The lattermeasure yields slightly higher values for the coveragefactor.

DISCUSSION

Amacrine cells stained by using the Golgi method wereeasy to match to the photofilled cells, and, with the fewexceptions noted, most of the cells seen by photofillingwere immediately recognizable in the Golgi preparations:

Fig. 11. AB diffuse-2 cells have outwardly sloping dendrites that end abruptly with small swellings at60% of the inner plexiform layer. The bottom photomicrographs in each of the three examples illustratethese small terminal swellings. Scale bar 5 50 µm.

316 M.A. MACNEIL ET AL.

the two methods confirm each other. Both methods stain areliable likeness of a cell type; living photofilled cells werethe same size and had the same dendritic specializationsas those of Golgi-stained cells, confirming that the processof Golgi-impregnation does not alter normal cell morphol-ogy. Some cells were underfilled by both methods. Thesilver-chromate precipitate sometimes terminated abruptly,leaving an obviously truncated dendritic branch. With photo-filling, understaining usually was manifest as gradual

fading of the distal dendrites. However, when cells ap-peared completely filled, their morphologies in Golgi andphotofilling matched remarkably well. The major disadvan-tage of the Golgi technique, as has long been recognized, isthat the frequency of staining particular types is erratic;their numbers in the Golgi material were hugely variable.Although the cells are visualized less cleanly in the photofilledimages, that methodology complements the Golgi techniqueby providing an estimate of the cells’ actual frequencies.

Fig. 12. Recurving diffuse cells have a major arborization locatedat 50% of the inner plexiform layer. These processes give rise to anumber of branches that either ascend to the inner nuclear layer ordescend to < 70% of the inner plexiform layer. Shadows indicatedendrites outside the plane of focus. Scale bar 5 50 µm.

Fig. 13. Spider cells are characterized by the preponderance ofvertically oriented dendrites. In wholemount optical sections, theseappear as bright dots (bottom), indicating dendrites that were cut incross section. Scale bar 5 50 µm.

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Expansions and revisions of earlierdescriptions

The spread and thickness of the dendrites was easier toresolve in the Golgi-stained tissue and allowed us to refineprevious values (MacNeil and Masland, 1998) for thelevels of stratification. The data in Tables 1–4 and inFigure 21 reflect the new assessment. In addition, twoadditional cell types were identified in the Golgi-stainedretinas: narrow S1 and narrow-diffuse amacrine cells.Narrow-diffuse cells have a dense and compact arbor witha narrow spread. This arrangement makes their dendriteshard to resolve in any stain, but especially in fluorescence,where flare from the cell body obscures processes that arein close proximity. Cells with this morphology originallywere included among the ‘‘unclassified narrow-field’’ photo-

Fig. 14. Two photofilled AB broad diffuse cells. The arbors span <180 µm and are located in the middle one-third of the inner plexiformlayer. Scale bar 5 50 µm.

Fig. 15. Three diffuse multistratified amacrine cells. The imageswere selected to show the horizontal appearance of the dendrites.Scale bars 5 50 µm.

318 M.A. MACNEIL ET AL.

filled cells. Narrow S1 cells were not identified in thephotofilled sample, but their sparse and narrow dendriticarbor would be even harder to see in fluorescence: Theyalmost certainly were considered failed attempts at filling.

In three instances, the names of cells were changed.‘‘A2-like’’ was renamed A8 after a similar cell identified incat retina (Kolb et al., 1981). Cells termed ‘‘broad diffuse’’and ‘‘AB broad diffuse, type 2’’ initially were distinguishedbecause of apparent differences in field diameter. However,the dendrites in these two groups are now seen to havesimilar branching patterns, the same size varicosities, andthe same depth (stratum 3 of the inner plexiform layer).They are grouped together here as ‘‘narrow S3’’.

Finally, the amacrine cell called ‘‘monostratified’’ hasbeen renamed ‘‘flag B.’’ Its dendrites occupy the vitreal halfof the inner plexiform layer and have an appearanceremarkably similar to that of the original flag cells. Wehave not combined the flag B cells with the original ‘‘flag’’group, because A and B flag cells occupy different levels ofthe inner plexiform layer; therefore, they must havedifferent connections and functions.

Completeness of our sample

Cells were selected in a systematically random mannerfor photofilling, and the total of 261 includes every case inwhich a photofill was attempted. Of these, 15 cells werelost because the photofilling failed. Another group wasclassified only as narrow, medium, or wide-field cells(MacNeil and Masland, 1998), but the morphologic clarityof the Golgi specimens now allows us to assign several ofthose cells to specific categories.

The overall accuracy of the sampling is verified by thefact that four cells with frequencies that are knownindependently (AII, DAPI-3, indoleamine, and starburst)appear in our sample in the correct numbers. We probablyoversampled large cells; their larger somata give them ahigher probability of encountering an intersection on thesampling grid. However, the error introduced is probablyno greater than that inherent in our small sample of eachtype. We can classify 87% of all successfully photofilledcells into one of the types shown in Tables 1–4. Thus, only31 cells remain unclassified out of a total of 246 success-fully photofilled cells.

This of course pertains only to those amacrine cells withsomata that are located in the inner nuclear layer; thedisplaced amacrine cells were not targeted for photofilling.However, displaced amacrine cells make up a small frac-tion of the total amacrine cell population. In the rabbit, <80% of all displaced amacrine cells are starburst cells(Masland et al., 1984; Vaney, 1990). Because their numberclosely matches the number of orthotopic starburst cells,displaced starburst cells represent slightly less than 5% ofall amacrine cells. The remaining 20% of the displacedamacrine cells, a mixed group of types, thus comprise inaggregate less than 1% of all amacrine cells in the rabbit.They will not be considered further here.

Some of the 31 unclassified amacrine cells were stainedpoorly. For others, the dendritic morphology was clear, butthe cell could not be assigned comfortably to one of theexisting categories. At least one type of amacrine cell has

Fig. 16. The varicose processes of three wavy bistratified cells.This type is called bistratified because of a dendrite-free zone between20% and 30% of the inner plexiform layer. Otherwise, the outwardlysloping branches course throughout all depths. Scale bars 5 50 µm.

AMACRINE CELLS 319

been identified by immunohistochemical staining but wasnot observed by photofilling or Golgi impregnation. This isthe type 2 catecholamine-accumulating cell (Tauchi et al.,1990), a tristratified, wide-field cell. These are very rarecells (< 3,500 per rabbit retina) and other rare amacrinesmay have been missed, particularly among the wide-fieldcells. However, the unclassified amacrine cells were allstained well enough to convince us that they do not form asingle type. Although a few may represent new, rare types,the majority almost certainly are variants of existing ones.We therefore are quite confident that no common type ofamacrine cell present in the rabbit retina has been missed.

Different shapes imply different functions

We have followed the time-honored practice (Ramon yCajal, 1972) of denominating different types of amacrinecells on the basis of their morphology. Our implicit assump-tion was that a different shape reflects a different function.Four main reasons support such a conclusion: First, cellswith differing amounts of dendritic spread sample differ-ing areas of visual space—areas that vary by two orders ofmagnitude. Second, the cells’ processes occupy differentstrata of the inner plexiform layer and communicateamong the strata in different ways. Because each stratumis occupied by processes from a different set of bipolar andganglion cells, the functional properties of the amacrinecells connected with them must be different. Third, theamacrine cells have highly distinctive morphologic fea-tures, notably the caliber of the dendrites and the size,number, and shape of their varicosities. Because thevaricosities are the primary sites of output synapses, thisimplies different patterns of synaptic connections.

Finally, the cell types distinguished in this way haveappropriate coverage factors. The coverages of the narrow-field cells hovered around 1. The medium-field cells hadcoverages of < 7. We cannot compute coverage factors forall wide-field cells because of uncertainty about their fulldendritic spreads. For the Golgi-stained cells that we havedrawn with a camera lucida, the coverages ranged from 6to 100, depending on the cell’s frequency and area. Thus,dendrites of rare cell types still cover the retina multipletimes.

An important implication of this finding is that we havenot inflated the number of amacrine cell types artificially.If that were the case, then the apparent coverages of many‘‘types’’ would necessarily be less than 1. Although somevariability is evident, this clearly is not the case. In thefuture, some refinement of these values surely will bemade, especially because our sample includes a group ofunclassified cells; some of these probably are atypicalmembers of one of the existing classes and this would raisethe densities for those cell types and thus their coverages.

Classification of the cells

Our names for the cells are simple, descriptive onesaimed at conveying one or more features of the cells’dendritic arborization. A more elegant and possibly moreefficient conceptualization of amacrine cell typology maybecome possible as the microcircuitry and functional rolesof the cells become better understood. We have divided the

Fig. 17. Two fountain cells. The inner arbor arises from a single,inward-directed process from the soma. From this arbor, sclerallydirected processes branch and terminate in layers 1 and 2. Scalebars 5 50 µm.

320 M.A. MACNEIL ET AL.

cells into four groups. The distinction between diffuse andstratified narrow-field cells has already been presented.This distinction should not be misconstrued: almost allnarrow-field amacrine cell types span different strata ofthe inner plexiform layer. Perhaps the most strikingdifference between the two narrow-field groups is that thedendrites of diffuse cells occupy adjacent strata of theinner plexiform layer, whereas many of the stratified cellshave widely separated dendritic arbors (Fig. 21). Thefunctional significance of this difference remains to bedetermined.

The medium-field amacrine cells were the most coherentgrouping in our sample. They had a remarkably uniformdendritic field diameter (171.5 µm 6 2.5 µm) and sharedthe property of having dendrites that spread at severaldifferent depths within the inner plexiform layer (Table 3).

Wide-field cells spread widely and remain narrowlystratified. The starburst amacrine cells traditionally havebeen termed medium-field cells, following the descriptionof Perry and Walker (1980). We have now included themamong the wide-field cells, because they have so manyproperties in common with the wide-field (and not with themedium-field) amacrine cells: 1) They have more than

twice the average dendritic field diameter of the amacrinecells grouped here as medium-field cells. In fact, theirdendritic spread is essentially the same as that of WF-1,which invariably is classified as wide-field. 2) They arestratified very narrowly, a characteristic of virtually all ofthe wide-field but none of the medium-field amacrine cells.3) Their coverage factor is approximately 100. Again, thisis characteristic of the wide-field group but not of themedium-field group, in which the average coverage factorwas < 7.

It is worth pointing out that the dendritic spread of thecells is correlated quite strongly with their degree ofstratification. Wide-field cells are highly stratified, medium-field cells are less so, and all but two narrow-field cellscommunicate vertically between two or more levels of theinner plexiform layer.

Functional considerations

Why are there 28 different types of amacrine cell?Although our classification may be imperfect, there is nodoubt that the shapes of the cells are the visible expressionof their connections and, thus, imply specificity of function.This means that large numbers of specialized tasks are

Fig. 18. Three types of wide-field cells with dendrites in stratum 1 of the inner plexiform layer. Scale bars 5 50 µm.

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carried out in the inner plexiform layer, with an amacrinecell type dedicated to each task (MacNeil and Masland,1998).

In fact, of the few amacrine cell types whose function isknown more or less securely, each carries out a differentcomputation. For example, starburst cells promote re-sponses to movement (He and Masland, 1997), and dopa-minergic amacrine cells modulate the retina’s responsive-ness to light (Dowling, 1986; Piccolino et al., 1987;Gustincich et al., 1997). Particularly specialized in termsof morphofunctional correlations are the AII amacrinecells, which distribute rod signals to cone-bipolar endingsand, thus, generate ON and OFF signals with increasingor decreasing scotopic illumination (Kolb and Famiglietti,1974; McGuire et al., 1984; Strettoi et al., 1990). It is worthnoting that AII cells are centrifugal amacrines, becausethey carry signals from their inner to their outer dendritictree. We would not be surprised if fountain cells andperhaps other cells also turn out to be centrifugal ama-crine cells.

Are there any simplifying concepts that would linkgroups of cells together? The easiest case is that of the

wide-field amacrine cells. Each stratum of the inner plexi-form layer contains at least one type of wide-field amacrinecell. Some of them are known to generate action potentials(Stafford and Dacey, 1997), and it is likely that theremainder do as well. Generically, the wide-field cells carryout global functions that cover wide areas of the retina.This does not mean that they are structurally minor.Although the wide-field cells, taken one at a time, appearsparse, their long dendrites can create a dense plexuswithin each sublamina of the inner plexiform layer (Vaneyet al., 1988; Famiglietti, 1992a,b). For example, dendritesof the WF1 type cell (the dopaminergic amacrine cell) inthe mouse fill a substantial fraction of the total volume inthe sublayer that they occupy (Gustincich et al., 1997).

The situation is more complicated for narrow-field cells.Their dendritic span roughly matches that of the axonalendings of bipolar cells, and one of their functions probablyis to modify the receptive field properties of bipolar cells ortheir action on ganglion cells. Many of such narrow-fieldcells are encompassed by the dendritic tree of each gan-glion cell, at least in a species like the rabbit, which doesnot possess midget cells; they carry out computations

Fig. 19. Two types of wide-field cell with dendrites in stratum 3 of the inner plexiform layer. Scale bars 5 50 µm.

322 M.A. MACNEIL ET AL.

Fig. 20. A wide-field cell (WF) with dendrites in layer 4 of the inner plexiform layer. Note that theextent of the dendrites is nearly twice that of the other wide-field types. Scale bars 5 50 µm.

Fig. 21. Stratification of dendrites in the inner plexiform layer(IPL). Amacrine cell types: a, narrow S1, b, AII; c, A8; d, flatbistratified; e, asymmetric bistratified; f, narrow S3; g, flag A; h, flag B;i, narrow diffuse; j, AB diffuse-1; k, AB diffuse-2; l, spider; m, recurving

diffuse; n, DAPI-3; o, diffuse multistratified; p, AB broad diffuse; q,fountain; r, wavy bistratified; s1, wide field WF1–1; s2, WF1–2; S3,WF1–3; t, starburst; u, WF2; v1, WF3–1; v2, WF3–2; w, WF4; x,indoleamine accumulating.

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TABLE 1. Narrowly Stratifying Narrow-Field Cells1

Celltype

Stratification(% IPL)

Sample size/frequency

(%)

Approx.density

(cells/mm2)

Arborarea(µm2)

Averagediameter

(µm)Coverage

factor Equivalent cells

Narrow S1 0–20 11 (4.2) 700 1,450 44 1.0 A1 (cat: Kolb et al., 1981; human: Kolb et al., 1992);knotty, type 1 (monkey: Polyak, 1941; Mariani,1990); type 1 (rat: Menger et al., 1998); neuropep-tide Y (cat: Hutsler and Chalupa, 1994)

AII 10–3555–95

34 (13.0) 2,301 7421,683

3549

1.73.9

AII (cat: Kolb et al., 1981; monkey: Mariani, 1990;human: Kolb et al., 1992; rabbit: Dacheux andRaviola, 1986; Strettoi et al., 1992; Vaney et al.,1991; rat: Menger et al., 1998); centripetal bipolar(monkey: Polyak, 1941); narrow field diffuse(monkey: Boycott and Dowling, 1969; narrow-fieldbistratified (rat: Perry and Walker 1980; dog:Ramon y Cajal, 1972)

A8 3050–65

6 (2.3) 397 1,0022,058

4258

0.40.8

A8 (cat: Kolb et al., 1981; human: Kolb et al., 1992)

Flat bistratified 2060

4 (1.5) 212 5,0342,696

8668

1.10.6

A4 (ground squirrel: Linberg et al., 1996); knotty bis-tratified, type 1 (monkey: Mariani, 1990)

Asymmetric bistratified 4060

3 (1.1) 211 5,3594,150

9183

1.10.9

Wavy multistratified, type 2 (monkey: Mariani,1990)?; A22 (ground squirrel: Linberg et al., 1996)

Narrow S3 40–60 7 (2.7) 415 9,450 116 3.9 NADPH diaphorase-2 (rabbit: Sandell, 1985; Sager,1986, 1990; Vaney and Young, 1988); Type 3 (rat:Menger et al., 1998)?; unistratified (monkey: Boy-cott and Dowling, 1969); A3 (cat: Kolb et al., 1981)?

Unclassified narrow-field — 5 (2.3) — — — — —Total — 70 (26.8) — — — — —

1In this and all other tables, stratification levels are mean values and have been rounded off to the nearest 5%. Total number of amacrine cells in photofilled sample were 261 cells. Ofthese, 15 cells could not be classified, even by their dendritic field size. INL, inner nuclear layer.

TABLE 2. Broadly Stratifying Narrow-Field Cells

Celltype

Stratification(% IPL)

Sample size/frequency

(%)

Approx.density

(cells/mm2)

Arborarea(µm2)

Averagediameter

(µm)Coverage

factor Equivalent cells

Flag A 20–60 14 (5.4) 837 1,518 46 1.3 A3 (ground squirrel: Linberg et al., 1996); knotty, type2 (monkey: Polyak, 1941; Mariani, 1990); stratifieddiffuse (monkey: Boycott and Dowling, 1969); A2(cat: Kolb et al., 1981); A2?, A4 (human: Kolb et al.,1992); type 2 (rat: Menger et al., 1998)

Flag B 50–90 9 (3.4) 585 1,454 43 0.9 A5, A6 (cat: Kolb et al., 1981); A5 (human: Kolb et al.,1992); knotty type 3 (monkey: Polyak, 1941;Mariani, 1990); stratified diffuse (monkey: Boycottand Dowling, 1969); type 4 (rat: Menger et al., 1998);stratified diffuse-b (rat: Perry and Walker, 1980)

Narrow diffuse 10–90 11 (4.2) 704 1,951 54 1.4 A1 (ground squirrel: West, 1976; Linberg et al., 1996);small diffuse (human: Kolb et al., 1992); knotty(Polyak, 1941); type 5 (rat: Menger et al., 1998); strati-fied diffuse-a (rat: Perry and Walker, 1980)

AB diffuse-1 15–75 19 (7.3) 1,257 2,495 60 3.1 A6 (ground squirrel: Linberg et al., 1996); A3 (human:Kolb et al., 1992)??; A9 (cat: Kolb et al., 1981); (monkey:see Fig. 67C, Polyak, 1941); types 3 and 6 (rat: Mengeret al., 1998?; dog: Ramon y Cajal, 1972).

AB diffuse-2 20–60 6 (2.3) 385 3,265 70 1.3 Unknown

Spider 5–60 11 (4.2) 747 1,844 52 1.4 Tassled amacrines (chimpanzee: see Fig. 68B-‘‘L’’,Polyak, 1941); stratified diffuse (monkey: Boycottand Dowling, 1969); type 7 (rat: Menger et al., 1998)

Recurving diffuse 0–85 3 (1.1) 207 11,257 118 2.3 Unknown

Total — 73 (28.0) — — — — —

TABLE 3. Medium-Field Cells

Celltype

Stratification(% IPL)

Sample size/frequency

(%)

Approx.density

(cells/mm2)

Arborarea(µm2)

Averagediameter

(µm)Coverage

factor Equivalent cells

DAPI-31 20–4055–70

9 (3.4) 525 23,07922,559

168161

12.111.8

DAPI-3 (rabbit: Wright et al., 1997); wavy-multistratified, type 2 (monkey: Mariani,1990); A14 (human: Kolb et al., 1992);also A21 (ground squirrel: Linberg et al.,1996)?

Diffuse multistratified 10–70 6 (2.3) 364 19,114 175 7.0 Tristratified (human: Kolb et al., 1992); A9(ground squirrel: Linberg et al., 1996)?;multistratified (monkey: Boycott andDowling, 1969)

AB broad diffuse 30–65 4 (1.5) 260 24,476 178 6.4 ‘‘Spiny’’ (monkey: Mariani, 1990); mul-tistratified (monkey: see Fig. 67E ‘‘d’’ inPolyak, 1941); substance P immunoreac-tive (cat: Vaney et al., 1989)?; A17a (Lin-berg et al., 1996)?

Fountain 0–85 4 (1.5) 184 21,356 173 3.9 Wide-field AII (rabbit: Heussy et al., 1988);retroflexive (rabbit: Wright and Vaney,1995)

Wavy bistratified 0–20 3 (1.1) 180 19,809 174 3.6 Unknown30–90 19,809 174 3.6

Unclassified — 15 (5.7) — — — — —Total — 41 (15.7) — — — — —

1DAPI, 4,6-diamidino-2-phenylindole.

within the center of ganglion cells’ receptive fields. Accord-ing to various estimates, there are seven or eight types ofcone bipolar cells (for review, see Sterling, 1998), and theiraxonal arborizations, as a rule, stratify at different levelsin the inner plexiform layer. If each bipolar type had itsown private narrow-field amacrine, returning reciprocalsynapses onto that bipolar cell’s axonal endings, then onewould expect as many unistratified amacrines as there arebipolar cells. This clearly is not the case: There are onlytwo types of unistratified narrow-field cells, namely, nar-row S1 and narrow S3. On the other hand, there is only onetype of amacrine cell, the narrow-diffuse cell, in which theprocesses span the entire thickness of the inner plexiformlayer; therefore, it has the opportunity to sample from andpossibly influence all types of bipolar cells.

Especially intriguing are the five types of medium-fieldcells, in which the dendritic arbors have an intermediatespan of < 175 µm—larger than the axonal arborizations ofbipolar cells but smaller than the arbors of many ganglioncells. Their significance remains a mystery. Now that theinventory of amacrine cells is nearing completion, the nextstep will be to learn their relationships with specific typesof bipolar and ganglion cells.

LITERATURE CITED

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TABLE 4. Wide-Field Cells

Cell type1Stratification

(% IPL)Sample

sizeFrequency

(%) Equivalent cells

WF1-1 0–20 4 1.5 Type I tyrosine hydroxylase (rabbit: Brecha et al., 1984; rat, guinea pig, cat, rabbit: Mitrofanis et al.,1988; rabbit: Tauchi et al., 1990, Taylor, 1996; mouse: Gustincich et al., 1997); type 1CA (monkey:Mariani, 1990); A16 (monkey: Hokoe and Mariani, 1988); dopaminergic (monkey: Dacey, 1990); A18(cat: Kolb et al., 1981; human: Kolb et al., 1992); A24, A27 (ground squirrel: Linberg et al., 1996)?

WF1-2 0–20 4 1.5 Somatostatin (rabbit: Sagar, 1987); semilunar type 1 (monkey: Mariani, 1990; human: Kolb et al., 1992);‘‘wiry’’-type 1 (monkey: Mariani, 1990); orientation-biased (rabbit: Bloomfield, 1994)

WF1-3 0–20 2 0.8 Dorsally directed amacrine, type d (rabbit: Famiglietti, 1989; Massey, personal communication); orienta-tion-biased (rabbit: Bloomfield, 1994)

Starburst 25 13 5.0 Type IV (rat: Perry, 1979); narrow-field unistratified (rat: Perry and Walker, 1980); starburst (rabbit:Famiglietti, 1983; Tauchi and Masland, 1984; cat: Schmidt et al., 1987); cholinergic (rat: Voigt, 1986);Sa (monkey: Rodieck, 1989; Mariani, 1990); ACh (human: Kolb et al., 1992); A5a (ground squirrel: Lin-berg et al., 1996)

WF2 20–40 8 3.1 A19, A20 (cat: Kolb et al., 1981); neurofibrillar long-range (rabbit and cat: Vaney et al., 1988); A25a(ground squirrel: Linberg et al., 1996); stellate-varicose (monkey: Mariani, 1990)

WF3-1 40–60 8 3.1 NADPH diaphorase (rabbit: Sandell, 1985; Sager 1986, 1990; Vaney and Young, 1988); ON-OFF (rabbit:Dacheux and Raviola, 1995); A22 (cat: Kolb et al., 1981); wispy, type 2 CA catecholaminergic; (monkey:Mariani, 1990); wiry type 2 (monkey: Mariani, 1990); semilunar-type 2 (monkey: Mariani, 1990)

WF3-2 40–60 6 2.3 Polyaxonal 1 (rabbit: Famiglietti, 1992a); axon bearing (monkey: Dacey, 1989; rabbit: Taylor, 1996)WF4 60–80 1 0.4 Stellate wavy (monkey: Mariani, 1990)Indoleamine accumulating

Type 1Type 2

80–10023

0.81.1

Indoleamine accumulating (rabbit: Ehinger and Floren, 1976); S1 and S2 (rabbit: Vaney, 1986); types 1and 2 (rabbit: Sandell and Masland, 1986; Sandell et al., 1989); A17 (cat: Kolb et al., 1981; human:Kolb et al., 1992); wide-field diffuse (monkey: Boycott and Dowling, 1969); semilunar spongeoblast (ox:Ramon y Cajal, 1972); spidery types 1 and 2 (Mariani, 1990)

Unclassified — 11 4.2 —Total — 62 23.8 —

1It is likely that some wide-field (WF) categories contain more than one cell type. ACh, acetylcholine.

AMACRINE CELLS 325

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