Neuropeptide Y immunoreactivity identifies a group of gamma-type retinal ganglion cells in the cat

THE JOURNAL OF COMPARATIVE NEUROLOGY 336:468-480 (1993)

Neuropeptide Y Immunoreactivity Identifies a Group of Gamma-Type Retinal

Ganglion Cells in the Cat

JEFFREY J. HUTSLER, CHERYL A. WHITE, AND LEO M. CHALUPA Department of Psychology and the Center for Neuroscience, University of California, Davis

California 95616

ABSTRACT Ganglion cells within the cat retina have been traditionally grouped by morphological

criteria into three major classes: alpha, beta, and gamma. The gamma-type cells have been least well characterized, but the available evidence indicates that this class comprises a relatively heterogeneous population of neurons. In the present study we demonstrate that an antibody for neuropeptide Y (NPY) recognizes a subpopulation of about 2,000 gamma-type ganglion cells.

The NPY-immunoreactive (IR) neurons project to the superior colliculus and to the C layers of the lateral geniculate nucleus as demonstrated by retrograde labeling with fluorescent tracers (fluorogold or rhodamine latex microspheres). Virtually all of these cells disappear following lesions of the optic nerve. The NPY-IR ganglion cells were identified as gamma cells on the basis of soma size and dendritic branching patterns. The somas of these neurons are small (8-22 pm in diameter), and each cell is characterized by sparsely branching dendritic processes, usually extending into the middle third of the inner plexiform layer, the physiologically defined ON sublamina. These neurons are distributed across the entire retina, with the highest density at the area centralis. Within local regions of the retina, however, there was no indication that the NPY-IR gamma cells are arrayed in a regular mosaic pattern. These results provide the first evidence that the gamma class of ganglion cells of the cat retina can be subdivided on the basis of immunocytochemical properties. o 1993 WiIey-Liss, Inc.

Key words: immunohistochemistry, lateral geniculate nucleus, superior colliculus

The anatomical and functional diversity of retinal ganglion cells has been amply documented in numerous species. In the cat there is general agreement for two morphologically distinct ganglion cell classes: alpha and beta (Boycott and Wassle, '74), which correspond to the physiologically identified Y and X cells, respectively (Enroth- Cugell and Robson, '66; Saito, '83; Fukuda et al., '84; Stanford and Sherman, '84). These two classes combined account for about one-half of the entire population of ganglion cells. Most of the remaining cells have been classified morphologically as gamma cells (Boycott and Wassle, '741, which are thought to correspond functionally to the W cell class (Saito, '83). These neurons share the common property of a slow axonal conduction velocity (Levick and Thibos, '82; Stone, '831, but in other respects are quite heterogeneous (Stone and Fukuda, '74b; Rodieck, '79; Stone and Clarke, '80; Kolb et al., '81; Leventhal et al., '85; Stanford, '87). Indeed, Boycott and Wassle ('74), who originated the morphological classification of the three major ganglion cell classes in the cat retina, suggested the presence of a small group of morphologically distinct ganglion cells, termed delta cells. More recently, these neurons

have been identified on the basis of their uptake of indoleamine (Wassle et al., '871, and have been shown to stratify within the physiologically defined OFF sublamina of the inner plexiform layer (IPL; Dacey, '89). Additionally, on the basis of projection patterns and cell size, the further parceling of the gamma cell group into epsilon cells has been suggested by Leventhal et al. ('80).

We have been interested in establishing the cytochemical properties of ganglion cells in the cat retina. Recently, we have shown that somatostatin, or a somatostatin-like substance, is found in the alpha class of retinal ganglion cells (White and Chalupa, '91). Interestingly, not all alpha cells are immunoreactive (IR) for this neuropeptide. Rather, these somatostatin-IR cells are preferentially distributed in the inferior retina. The dendrites of most of these cells stratify in the OFF sublamina of the IPL.

Accepted May 28, 1993. Cheryl A. White's present address is Massachusetts Institute of Technol-

Address reprint requests to Jeffrey J. Hutsler, Center for Neuroscience, ogy, Department of Brain and Cognitive Sciences, Cambridge, MA 02139.

University of California, Davis, CA 95616.

O 1993 WILEY-LISS, INC.

NPY-IR GAMMA-TYPE RETINAL GANGLION CELLS 469

Fig. 1. A-E: The morphology of neuropeptide Y (NPY) immunoreactive (IR) neurons within the ganglion cell layer of the adult cat retina. Dendritic arbors of the neurons in these photomontages were revealed following colchicine treatment 16 hours prior to sacrifice. Although

dendritic arbors show some variability, each neuron has a small cell body and sparsely branching dendritic arbors. Arrows in A and D indicate the presence of a presumptive axon. Scale bar = 20 km.

The foregoing findings prompted us to search for other putative neuroactive substances that might be present within cat retinal ganglion cells. We were particularly interested in determining whether or not different neuroactive substances could be localized within particular classes of ganglion cells. In the present study we show that an antibody to neuropeptide Y (NPY) identifies a population of neurons within the ganglion cell layer of the adult cat, and that these are gamma cells that project to the superior colliculus (SC) and to the C layers of the lateral geniculate nucleus. An abstract summarizing some of these results has been published (Hutsler et al., '91).

MATERIALS AND METHODS A total of 17 adult pigmented cats of either sex was used

in this study. All surgical procedures were conducted under sterile conditions in accordance with a protocol approved by the Animals Use and Care Administrative Advisory Commit- tee of the University of California, Davis.

Retrograde labeling studies Ten cats received pressure injections of rhodamine latex

microspheres (Lumafluor, Inc.), or fluorogold (Fluoro- chrome, Inc.). After pretreatment with atropine (0.08 mgi kg), animals were anesthetized with 4% halothane gas in oxygen, and, following intubation, maintained with 2%

halothane in a mixture of 50% oxygen and 50% nitrous oxide.

Cats were placed in a stereotaxic instrument, small openings were made in the skull of each hemisphere overlying the area of interest, and bilateral injections of rhodamine latex microspheres (15-30 ~1 per hemisphere) or 2% fluorogold (5-7 ~1 per hemisphere) were made into the C layers of the lateral geniculate nucleus (two animals), the SC (seven animals), or optic tract (one animal) with a 26-gauge needle attached to a 5-p1 Hamilton syringe. In several cases, electrophysiological recordings were made prior to placing the deposits to confirm the location of these injections. Approximately 48 hours later, in the case of rhodamine latex microspheres, or 7 to 10 days later, in the case of fluorogold, each animal was administered a lethal dose of sodium pentobarbital.

Lesions of the optic nerve One animal sustained a unilateral lesion of the optic

nerve in order to induce retrograde degeneration of ganglion cells within the retina. The pattern of NPY-IR was compared between retinas to obtain an estimate of the number of immunoreactive cells within the ganglion cell layer that were ganglion cells rather than displaced amacrine cells. After the nerve was located stereotaxically, and its exact boundaries determined by electrophysiological recordings of light evoked responses, lesions were made

470 J.J. HUTSLER ET AL.

A

C

....

i

c

Fig. 2. A-E: Camera lucida drawings of NPY-IR cells within the GCL of the adult cat retina. Cells with round or ovoid somas and radially oriented dendrites were common (A and D). However, variations such as short interdendritic branching distances (B), elongated

I N = 833

8o 1 - Soma Area X = 189.78 S. D. = 54.74

3 60 Soma Diameter + X = 15.54 S. D. = 8.31 s

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W

40 e

20

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Fig. 3. Frequency histogram of NPY-IR cell areas. The equivalent soma1 diameter is indicated. Cell sizes were widely distributed, ranging from approximately 8 to 22 pm in diameter.

with radio frequencies at 70-75°C for 60 seconds (Radion- ics, Inc.). This animal was sacrificed 6 months after the surgery.

Colchicine pretreatment To enhance the immunoreactivity of dendrites, three

retinas were pretreated with colchicine. Animals were

cell bodies (C), a reduced number of primary dendrites (E), and large dendritic fields with large interdendritic branching distances (E), were - - also encountered. Scale bar = 500 pm.

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16 a,

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14 m

10 5 8

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0 2 5 10

Distance from AC (mm)

Fig. 4. Cell size as a function of retinal eccentricity from the area centralis (AC). Cell areas (left axis) and diameters (right axis) are given as a function of eccentricity from the AC (0, 2, 5, and 10 mm). The number of cells in each sample is shown at the top of the error bar, which depicts the interquartile range-the 25th and 75th percent i le for each cell group.


i b-

EL.. ............................................................................. INL

IPL INL .....................................................................................

Fig. 5. A,B: Camera lucida drawings of NPY-IR cells taken from the wholemount preparation, with the orthogonal reconstruction of each cell depicted below. In both of these cases dendrites arborized within the middle third of the inner plexiform layer (1PL)-the physiologically defined ON sublamina. The inset retinal outline indicates the position

of these two cells within the same retina. Both cells are oriented as they would appear in the retinal outline, and the arrows in each drawing indicate a process that entered the axonal fiber layer and extended toward the optic disk. INL, inner nuclear layer. Scale bars = 15 pm.

Normal Adult

N

Fig. 6. Retinal maps from an adult cat indicating the position of each NPY-IR cell within the ganglion cell layer of both retinas. Cell density was highest at the AC and, following an initially abrupt decrease, declined gradually toward the periphery. N, nasal; T, temporal. Scale bar = 3 mm.

472 JJ. HUTSLER ET AL.

Area Centralis Ganglion Cells

25 1 ?O 4 I w

Random

Actual

Mean = 52.57 S. D. = 26.77 N = 116

Fig. 7. The distribution of distances to the nearest neighboring NPY-IR cell taken from a region near the area centralis as compared with a random distribution of cell distances generated for the same area. The location of cells within the sample area is shown in the lower right. Scale bar = 300 pm.

anesthetized with 2% halothane in oxygen 14 to 18 hours prior to sacrifice and injected with 30-40 p1 of a 1 pgl l p1 solution of colchicine (Sigma Labs) into the posterior cavity. Colchicine pretreatment did not alter the density or distribution of immunoreactive cell bodies.

Tissue fixation and preparation Following a lethal dose of sodium pentobarbital, all

animals were perfused through the heart with 0.1 M phosphate-buffered saline containing 0.1% heparin followed by 4% paraformaldehyde. During the saline rinse each eye was removed, hemisected, and deposited into 4% paraformaldehyde for 10 minutes. Retinas were later dis- sected and then postfixed further for 2 hours. Brains were removed following perfusion with 4% paraformaldehyde and allowed to sink for 24 to 72 hours in 30% sucrose prior to sectioning or freezing. Retinas processed for cross sections were cut at 15-30 pm on a cryostat and brain sections were cut at 25-50 pm on a sliding freezing microtome.

Immunocytochemistry Wholemounted retinas were frozen and thawed three

times, rinsed in three changes of 0.25 M Tris buffer (pH 7.4) for 30 minutes, pretreated for 10 minutes with 0.1 M sodium periodate, rinsed in three changes of 0.25 M Tris buffer, and then treated with 0.026 M sodium borohydride

for 10 minutes. To prevent nonspecific binding, retinas were again rinsed and transferred into a solution of 10% normal goat serum and 1% Triton X-100 diluted in Tris buffer for 1 hour. Subsequently, retinas were incubated for 5-7 days a t 4°C in an antibody to NPY (1:1,000 to 1:5,000) raised in the rabbit (Cambridge Research Biochemicals) and diluted in Tris buffer with 1% Triton X-100. After rinsing in four changes of Tris buffer for 1 hour, retinas were incubated for 3-5 days at 4°C in a biotinylated antibody raised in the goat against rabbit IgG (Vector Labs). This solution was diluted 1:200 in Tris buffer with 1% Triton-X-100. Retinas were again rinsed in four changes of Tris buffer for 1 hour and then transferred into an ABC solution (Vector Labs) for 2 days. Following another rinsing, retinas were incubated for 20 minutes in 3',3' diamino- benzidine (DAB) at 50 mg/100 ml of Tris buffer. Three milliliters of 0.3% hydrogen peroxide were added to this solution and the incubation continued for 5-10 minutes depending upon the rate of precipitation of the reaction product. Retinas were mounted ganglion cell layer up, fixed with 4% paraformaldehyde, and allowed to air dry before being coverslipped with Depex (BDH Laboratory Supplies).

Whole retinas utilized for double labeling with retrograde tracers were processed as above with several modifications. Prior to tissue processing, retinas were mounted onto cellophane-wrapped glass coverslips and transferred ganglion cell layer down onto a flat stage of ice for microtome sectioning. Approximately 100-125 pm of tissue were then shaved off the photoreceptor side so as to reduce the overall background fluorescence. Additionally, the ABC solution was replaced with either a Texas red avidin or fluorescein avidin solution diluted 1 : l O O and incubated for 12-24 hours. Following this period, retinas were immediately rinsed, mounted onto slides, allowed to air dry, and then coverslipped with Krystalon (EM Diagnostic Systems, Inc.).

Retinal and brain sections were processed as above; however, the following incubation times were used: primary antibody-12-18 hours; secondary antibody-1-2 hours; ABC solution-1 hour; Texas red or fluorescein avidin solution-1 hour; DAB preincubation-10 minutes. Fluorescent sections were coverslipped with Krystalon, and all other sections were coverslipped with Depex.

Controls Three types of controls were utilized to demonstrate the

specificity of the antibody staining procedure: preadsorption of the primary antibody in a 10 pM solution of the NPY antigen (Bachem Chemicals, Inc.); elimination of the primary antibody; and elimination of the secondary antibody. All control sections were processed in conjunction with sections treated with the standard immunostaining protocol.

Data analysis The positions of NPY-IR cells were mapped at 161 x onto

a projected enlargement of the retinal outline. For retinas in which cell counts were made, every cell was counted within adjacent 600 pm2 areas, and these counts were recorded onto an outline of the retina. Individual cells were drawn at a final magnification of either 4 0 0 ~ or 640x with the aid of a camera lucida attachment. Dendritic depths


Fig. 8. A,B: Two NPY-IR cells seen within the ganglion cell layer. The focus is on the optic nerve fiber layer. In each case a single process can be seen to extend initially away from the cell body. After this extension an abrupt turn of the axon toward the distant optic disk is observed. Arrows in B indicate the direction of axon extension. Scale bar = 10 Fm.

were assessed by reading from the calibrated focus adjustment of a Zeiss Universal microscope under oil immersion at a final magnification of 1,612X. Soma1 areas of individual cells were measured from the camera lucida drawings by means of a Macintosh I1 computer equipped with a digitiz- ing tablet.

RESULTS We shall first describe the morphology and distribution of

NPY-IR cells within the ganglion cell layer of the cat retina. It will then be established that the majority of these cells are projection neurons on the basis of the loss of NPY-IR profiles within the ganglion cell layer following severance of the optic nerve. We shall further demonstrate that these neurons project to the SC and the C layers of the lateral geniculate nucleus.

Control experiments indicated that our labeling was specific for NPY. This was demonstrated by an absence of staining following elimination of the primary or the secondary antibody, and by preadsorption of the primary antibody with NPY antigens prior to tissue incubation. We cannot, however, rule out the possibility that the antibody is recognizing a substance similar to NPY. Thus, the staining we obtained should be interpreted as NPY-like. Neverthe- less, for brevity we shall use the term NPY-IR.

Morphology of NPY-IR neurons in the ganglion cell layer

In all cases incubation of whole retinas or cross sections in the antibody to NPY resulted in the labeling of cell profiles within the inner nuclear layer and the ganglion cell layer. Numerous NPY-IR profiles were evident within the amacrine cell layer of the inner nuclear layer (Hutsler and Chalupa, in preparation); however, the present study is concerned only with NPY-IR neurons within the ganglion cell layer (GCL). Although NPY-IR cells within the GCL usually appeared only as soma1 profiles in the wholemounted retina, colchicine treatment 16 hours prior to sacrifice dramatically improved dendritic staining without altering the number and distribution of these immunoreactive profiles. Photomicrographs of colchicine treated cells are shown in Figure 1, while camera lucida drawings of these neurons appear in Figures 2 and 5.

All NPY-IR cells examined shared two characteristics: 1) a small cell body; and 2) sparsely branching dendritic arborizations. Several generalizations concerning the most frequently encountered cell morphologies can be noted. Most of these cells had a round or oval soma, three to five primary dendrites with an interbranching distance of approximately 20 to 30 bm, and a radially extending dendritic tree (Figs. 1A,C-E, 2A,D). A substantial number of cells (approximately 10-20% in all regions of the retina) deviated from this general scheme, showing characteristics such as

4 74 J.J. HUTSLER ET AL.

Fig. 9. Examples of NPY-IR neurons projecting to the superior colliculus (SC). A$: NPY-IR cell bodies within the ganglion cell layer labeled with Texas Red, viewed under fluorescent illumination. A presumptive axonal process appears in C. B,D: The same fields as in A and C viewed with UV fluorescence to reveal retinal ganglion cells

retrogradely laheled after fluorogold deposits into the SC (see Fig. 10A). Arrows in C and D indicate the same cell body. In C the level of focus is on the optic nerve fiber layer, while in D the inner plexiform layer is focused to reveal the cell's dendritic arbors. Scale bar = 20 Fm.

elongated somas (Fig. 2C), only one or two primary dendrites (Figs. lB, 2E), larger dendritic fields (Fig. 2E), and either shorter (Fig. 2B) or longer (Fig. 2E) interdendritic branching distances. None of these variations clustered together to indicate different morphological groupings, nor did analyses of cell body shapes indicate separate groupings. Although some of these variations could be due to incomplete cell staining, retrograde labeling in conjunction with immunocytochemistry confirmed the morphological characteristics described above (see Fig. 9B,D).

Soma sizes (n = 833) ranged in area from 50 to 390 km2 (diameter: 8-22 km; see Fig. 3). The mean area was 189.78 pm2 (diameter: 15.54 km). Variations in cell sizes across the retina were examined by measuring cell sizes at the area centralis (AC) and within 1 mm concentric rings located 2, 5, and 10 mm from the AC (Fig. 4). The mean cell size at each location remained relatively constant except at the AC, where cells were significantly smaller than those at each of the three peripheral locations (omnibus F, method of least squares = 17.179, 3 df, P < 0.0001; post hoc comparisons using the Scheffe F-test: AC vs. 2 mm = 11.83, P < 0.025; AC vs. 5 mm = 11.704618, P < 0.025; AC vs. 10 mm = 15.93, P < 0.01). Additionally, comparisons between temporal and nasal cell sizes, and inferior and

superior cell sizes, showed no appreciable differences at any of the eccentricities examined.

The dendritic arbors of colchicine-treated neurons always displayed sparsely branching dendritic trees usually with three to five primary dendrites. This is similar to what has been described previously for ganglion cells of the gamma class (Boycott and Wassle, '74; Kolb et al., '81). Although colchicine treatments revealed only a portion of the dendritic field, these branches could often be followed for 200 km within the IPL of the GCL. The level of branching within the IPL of these processes was examined in wholemount retinas by taking measurements under 1 0 0 ~ oil immersion of cells with well-stained processes using the fine focus adjustment of a Zeiss Universal microscope. Figure 5 shows two examples of cells drawn with a camera lucida attachment. Below each cell is an orthogonal reconstruction of the depth of each dendrite, which is seen to extend into the middle one-third of the IPL, the physiologically defined ON sublamina (Famiglietti and Kolb, '76; Nelson et al., '78; W a d e and Boycott, '91). Cells were examined within 5 mm in the nasal and temporal direction of the area centralis. Of 40 cells sampled that exhibited extensive dendritic staining, 35 branched within the middle one-third of the IPL, that is, within the ON sublamina.


nasal and temporal edge of the retina, respectively). Addi- tionally, the distribution of NPY-IR cells in the ganglion cell layer shows a distinct horizontal streak, which is more apparent in the nasal than the temporal retina. Cell counts in a third retina (n = 2,315) confirmed these observations.

Several types of functionally distinct cell groups (ON alpha, OFF alpha, ON beta, OFF beta) have been shown to be regularly distributed across the retina as assessed by the distance to the nearest neighboring cell (Wassle and Rie- mann, '78; Wassle et al., '81a,b). Figure 7 shows the position of NPY cells within a 1,050 pm2 field centered at the AC. The distribution of the nearest neighbors did not differ significantly from a random distribution of distances ( x 2 = 6.202, df = 10, P > 0.25) generated for the same number of cells within a matching area (Wassle and Rie- mann, '78). Seven additional fields at various distances and directions from the AC were also analyzed ( 5 mm temporal, 5 mm inferior, 5 mm superior, 10 mm nasal, 10 mm temporal, 10 mm superior, and 10 mm inferior to the AC). These analyses also indicated that NPY-IR cells are not distributed in a regular fashion.

Neuropeptide Y-immunoreactive ganglion cells

As stated above, the morphology of the NPY-IR cells, as well as their distribution across the retina, is similar to that of the gamma cell class; however, positive identification of a neuron within the GCL as a ganglion cell is dependent upon the presence of a centrally projecting axon. Three lines of evidence indicate that the vast majority of these cells are ganglion cells: 1) a large number of cells in colchicine treated retinas exhibited a single axon-like process within the fiber layer that extended towards the optic nerve head; 2) virtually all NPY-IR cells of the GCL disappeared following retrograde degeneration induced by lesions of the optic nerve; and 3) a substantial number of immunoreactive cells could be labeled following central injections of retrograde tracers.

Examination of NPY-IR neurons in colchicine treated retinas often revealed a single process that could be followed within the optic fiber layer for several hundred microns (Figs. lA,D, 5 , 9C). As can be seen by comparing the direction of axon extension (Fig. 5A,B) with the cell location in the retina (inset of Fig. 51, these processes always turn toward the optic disk. In some cells in which an axon could be clearly visualized, there was a short initial extension in a direction opposite to the optic disk, followed by a sweeping, or sometimes abrupt, turn towards the distant optic disk (Fig. 8).

In addition to the appearance of a presumptive axon, many NPY-IR cells could be labeled with retrograde tracers (rhodamine latex microspheres or fluorogold) deposited into the optic tract, SC, or the lateral geniculate nucleus. Figure 9 shows two such cells in which the NPY antibody was visualized with Texas Red fluorescence (Fig. 9A,C). These same neurons were retrogradely labeled by fluorogold (Fig. 9B,D) injections into the SC (Fig. 10). In two animals that received bilateral injections of rhodamine latex microspheres into the lateral geniculate nucleus (LGN), double labeled cells were also found (not illustrated). In both cases, these injections were largely confined to the C layers of the geniculate (Fig. 111, which are known to be innervated by the gamma class of ganglion cells. There was some leakage of rhodamine microspheres along the elec- trode tract into the A layers of the geniculate; however,

Lateral *

Medial - /

Fig. 10. Camera lucida reconstructions of a fluorogold injection site within the superior colliculus. All sections were drawn in the parasagittal plane, and the hatched area delineates the borders of the injection site. ML, medialilateral coordinate in millimeters; IC, inferior colliculus; ICC, commissure of the inferior colliculus; MA, medial area pretectalis; PAG, periaqueductal gray; PC, posterior commissure; SCC, commissure of the superior colliculus; SCI, intermediate layers of the superior colliculus; SCS, superficial layers of the superior colliculus. Scale bar = 2 mm.

Four cells exhibited dendrites that clearly branched within the OFF sublamina, and one cell showed multiple levels of stratification with dendrites terminating within both the ON and OFF sublamina. Although immunoreactive dendrites did not commonly overlap, the dendrites of adjacent pairs of cells (no more than 30 km apart) appeared to stratify within the same layer of the IPL.

Distribution of NPY-IR neurons within the ganglion cell layer

The distribution and density of NPY-IR neurons is shown in Figure 6 for two retinas taken from the same animal. In each case there were approximately 2,000 immunoreactive profiles (right eye, 1,960 cells; left eye, 2,064 cells), with the highest density of cells located at the AC (l10/mm2). Although both retinas were well stained, the absence of cells in small retinal segments (ventral region of left retina) may be due to tissue folding during immunocytochemical processing or incomplete penetration of antibod- ies due to vitreal obstruction. As can be seen, the density of NPY-IR cells drops abruptly about 1.5 mm from the AC, and then declines gradually towards the periphery (approximately 25 and 16 cells/mm2 a t locations 5 mm from the

476 J J . HUTSLER ET AL.

Fig. 11. Camera lucida reconstructions of a rhodamine latex micro- sphere injection site within the lateral geniculate nucleus. All sections were drawn in the parasagittal plane, and the hatched area delineates the borders of the injection site. ML, medialilateral coordinate in

0 . . 0

0 . 0 0 . . . .

0

0 *

2.. . . 0

0

0 00 . .

0 0

. O

0 *a*

millimeters; A, the A layer of the dorsal lateral geniculate nucleus; A*, the Al layer of the dorsal lateral geniculate nucleus; C, the C layer of the dorsal lateral geniculate nucleus; MG, medial geniculate; OR, optic radiations; OT, optic tract. Scale bar = 2 mm.

0 NPY immunoreactive cells n = 112

0 NPY immunoreactivity + retrograde label n = 84

Fig. 12. Camera lucida drawing of a retinal area, approximately 2 mm inferior to the AC, which contained a high density of retrogradely labeled cells. Circles indicate the position of NPY-IR cell bodies within the ganglion cell layer (GCL). Filled circles indicate those NPY-IR cells that also contained fluorogold following deposits of this tracer into the SC.

these layers do not receive projections from gamma cells (Stone, '83).

We were curious as to whether NPY-IR terminals could be visualized within the retinorecipient zone of the SC and/or the C layers of the LGN. NPY-IR fibers were seen within the stratum griseum superficiale of the SC; however, comparison of the two colliculi in animals that sustained lesions of the optic nerve did not reveal a significant decrease in the number of these fibers. NPY-IR fibers could

also occasionally be seen within the LGN, but again no difference in fiber density was apparent in animals that sustained lesions of the optic nerve.

To obtain an estimate of the percentage of NPY-IR cells that are ganglion cells, retinal regions containing a high density of retrogradely labeled cells were examined. Figure 12 shows such a region, located just nasal and inferior to the area centralis. As can be seen, of 112 NPY-IR cells in this region, 84 were also retrogradely labeled with fluoro-

NPY-IR GAMMA-TYPE RETINAL GANGLION CELLS

Lesioned Nerve 477

N

Intact Nerve

Fig. 13. Retinal maps showing the position of NPY-IR cells within the GCL from an animal that sustained a unilateral optic nerve lesion 6 months prior to sacrifice. N, nasal; T, temporal. Scale bar = 3 mm.

gold from the SC (filled circles). Thus, near the area centralis at least 75% of all NPY-IR neurons are ganglion cells projecting to the SC. Examination of a 1 mm square area within another retina containing a high density of retrogradely labeled cells from the LGN showed that of 35 NPY-IR labeled neurons, 18 (51%) also contained rhodamine latex microspheres. Because retrograde tracers are unlikely to label all cells, the percentage of immunoreactive ganglion cells identified in this manner is undoubtedly an underestimation.

In order to obtain a more accurate estimate of the number of NPY-IR cells that are ganglion cells, the optic nerve of one animal was lesioned at 1 month of age. Following retrograde degeneration (6 months), both eyes were processed for NPY-IR and the number of cells in each retina compared. Figure 13 shows the location of NPY-IR cells within both retinas of this animal. As can be seen, there was a dramatic reduction in the number of NPY-IR cells within the lesioned retina (a total of only 25 neurons remained). The pattern of NPY immunoreactivity within the inner nuclear layer was unaltered. This indicates that virtually all of the NPY-IR cells within the ganglion cell layer of the cat retina are ganglion cells.

DISCUSSION We have found that NPY immunoreactivity identifies a

subpopulation of ganglion cells in the adult cat retina. These NPY-IR neurons were shown to be gamma cells that project to the SC and the LGN. Most commonly, the dendrites of these neurons stratify within the functionally defined ON sublayer of the IPL. The NPY-IR gamma cells are scattered across the retina with the highest density at the area centralis. There was no evidence, however, for a regular mosaic pattern in the distribution of these neurons.

The NPY-IR cells we studied exhibited a number of salient characteristics that have been described for the overall population of gamma cells, including: soma size, dendritic branching (Boycott and Wassle, '74; Stone and Clarke, '80; Kolb et al., '81; Leventhal et al., '85; Stanford, '87; Wassle and Boycott, '91), retinal distribution (Stone and Keens, '80; Stone, '831, innervation of the SC (Wassle and Illing, '80), and innervation of the C layers of the lateral geniculate nucleus (Itoh et al., '81; Leventhal et al., '85). Thus, it can be concluded, with considerable assurance, that NPY immunoreactivity identifies a subgroup of gamma cells in the cat retina. Of course, we cannot rule out the possibility that some of the NPY-IR neurons are displaced amacrine cells. This caveat is necessitated by the fact that optic nerve lesions did not entirely eliminate NPY-IR profiles in the GCL ipsilateral to the severed nerve. How- ever, such neurons were few in number, and we think that these cells remained either because the retrograde degeneration was incomplete (Hollander et al., '84; Scherer and Schnitzer, '911, or because the lesion spared some fine caliber axons within the optic nerve.

Although the number of NPY-IR gamma cells is relatively small (about 2,00O/retina), this is comparable to what has been reported for some other subgroups of ganglion cells in the cat retina. For instance, there are 2,500-3,000 ON alpha and an equivalent number of OFF alpha cells, and about 5,400 delta cells (Wassle et al., '81a; Dacey, '89). Additionally, epsilon cells comprise approximately 2% of all ganglion cells (Leventhal et al., '85). It should be noted that neither the delta nor the epsilon cell class appears to correspond to the NPY-IR gamma cells we have described. Delta cells somas (which are 13-21 pm in diameter) are similar in size to NPY-IR ganglion cells; however, delta cell dendrites stratify mainly in the OFF sublayer of the IPL, rather than in the ON sublayer, and

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have dendritic arbors that are readily distinguishable from the typical gamma cell. Furthermore, unlike NPY-IR ganglion cells, delta cells are distributed in a clearly defined mosaic pattern (Wassle et al., '87; Dacey, '89). To our knowledge, their central projections have yet to be documented. Epsilon cells are defined by their projection to the thalamus (pulvinar, medial intralaminar nucleus and the C laminae of the lateral geniculate body) as well as soma1 sizes in the range of beta cells (Leventhal et al., '85), appreciably greater than those of NPY-IR gamma cells.

Based upon cell body size, the majority of NPY-IR ganglion cells appear to correspond to what has been denoted as the G2 subgroup of gamma cells (Stone and Clarke, '80; Leventhal et al., '85). Stanford ('87) has shown that G2 gamma cells respond to light in a phasic manner, although in other respects their response properties appear to be quite diverse. It has been estimated that about 40% of the ganglion cells in the cat retina are G2 gamma cells (Leventhal et al., '85). Clearly, the subgroup of cells identified here on the basis of NPY immunoreactivity constitutes only a small fraction of the total G2 gamma cell population. Given approximately 63,000-75,000 gamma cells in the adult cat retina (Williams and Chalupa, '84), and that at least 80% of all gamma cells send an axon to the SC (Wassle and Illing, 'SO), the NPY-IR gamma-type ganglion cells identified in the present study comprise approximately 2.6-3.1% of all gamma-type cells and at least 2.5-2.9% of those innervating the SC. Approximately half of the gamma cells of the cat retina innervate the C layers of the lateral geniculate (Wassle and Illing, '81); thus, a t least 2.7-3.2% of these cells are NPY-IR. Many gamma cells bifurcate to innervate both the geniculate and the SC (Illing, '80; Illing and Wassle, '81; Leventhal et al., '85). The high proportion of NPY-IR cells we found to be retrogradely labeled following injections of either the SC or the LGN indicates that many NPY-IR gamma cells also have bifurcating axons.

NPY-IR retinal fibers did not appear to be significantly reduced within either the LGN or the SC following lesions of the optic nerve or tract. This observation does not rule out the presence of NPY in retinal terminals since a decrease in NPY-IR retinal fibers may have been masked by NPY-IR fibers arising from other sources. Additionally, the presence of NPY-IR within intraretinal axons directed towards the optic disk would seem to indicate that this substance is being transported centrally.

NPY immunoreactivity in the retina of other species

Previous studies have reported NPY-IR cells in the ganglion cell layer of the human (Tornqvist and Ehinger, '88; Straznicky and Hiscock, '891, rat (Ferriero and Sagar, '891, and frog (Bruun et al., '861, but it has not been established whether these are ganglion cells or displaced amacrine cells. Straznicky and Hiscock ('891, however, observed that some of the NPY-IR cells in the human retina exhibited a long process extending in the direction of the optic disk and, on this basis, suggested that these were ganglion cells. More commonly, NPY-IR amacrine cells have been noted in the inner nuclear layer of a variety of species including the human, cat, pig, guinea pig, mouse, pigeon, chicken, turtle, frog, carp, goldfish, skate, and mudpuppy (Bruun et al., '86; Isayama et al., '88; Hiscock and Straznicky, '89; Straznicky and Hiscock, '89). We have also identified NPY-IR amacrine cells (Hutsler and Cha- lupa, in preparation) and find that there are more than

J J . HUTSLER ET AL.

150,000 of such neurons. The high incidence of NPY-IR amacrine cells relative to the number of NPY-IR gamma cells, in conjunction with the cross-sectional retinal sampling technique used by Bruun et al. ('86), may explain why immunoreactive profiles in the ganglion cell layer of the cat retina have not been previously reported.

Neurochemical identification of cell groups Ganglion cell classes have been historically defined on the

basis of a cluster of common anatomical and physiological properties, although there is often disagreement as to which properties are salient for a given population of neurons (Rowe and Stone, '77; Rodieck and Brening, '83; Stone, '83). Although the localization of a neuroactive substance within a group of cells is not sufficient to invoke a new class, the chemical content of the cells reported here indicates that this group, as compared with other gamma- type cells, possesses at least one unique attribute.

I t has been stipulated that for a group of retinal ganglion cells to constitute a functionally meaningful class, the cells must provide complete coverage of the retinal surface and be distributed in an orderly mosaic pattern (e.g., Peichl, '91; Wassle and Boycott, '91). This viewpoint is based largely on what has been found to be the case for the most thoroughly studied groups of ganglion cells in the cat retina, the alpha and beta classes. The presence of a regular cell array, however, has not been documented for the overall gamma cell class, nor for any of the gamma cell class subgroups proposed by previous investigators (e.g., Stone and Clarke, '80; Stone and Keens, '80; Rowe and Dreher, '82; Leventhal et al., '85; Stanford, '87). Several of these studies provide pictorial representations of cell distributions either across the entire retina or within a limited area. This includes cells projecting to the SC (Wassle and Illing, '80, see small cells in their Figs. 4a, 5a, 6a, and 9a; Itoh et al., '81; see their Fig. 2b), the nucleus of the optic tract (Ballas et al., '81; see their Fig. 11, the pretectum (Koontz et al., '85, Fig. 71, the retinorecipient zone of the pulvinar (Leventhal et al., '80, Fig. 71, the accessory optic system (Farmer and Rodieck, '82, Fig. 21, and the suprachiasmatic nucleus (Murakami et al., '89, Fig. 4). In all cases there does not appear to be evidence of regular intercell spacing among the small ganglion cells. It could be argued that these distributions were the result of technical problems, such as the inability to label the entire cell group, or the intermingling of diverse cell subgroups. While it would be unwise to rule out such considerations, it is still the case that retinal mosaics remain to be established for cell classes other than the alpha, beta, and delta cells.

Possible functional implications The role of NPY in the subpopulation of gamma cells we

have described remains to be established, but several alternatives could be considered. First, these cells could release NPY, and such action could serve to modulate visual activity at the central target of these neurons or within the retina. From this perspective, it would be of interest to examine the effects of NPY on the functional properties of neurons receiving input from the group of gamma cells identified in the present study. In other systems NPY has been shown to reduce voltage-gated calcium currents (Ni- coll et al., '901, thus inhibiting the depolarization-evoked release of several neuroactive substances (Walker et al., '88;


Yokoo et al., ’87; Wiley and Owyang, ’87). NPY can also induce depolarization by increasing membrane conduc- tance (Brooks et al., ’87). Either of these actions would be consistent with a neuromodulatory role. Alternatively, the immunolabeling we observed could reflect the uptake of NPY, presumably from NPY-IR amacrine cells, which are substantial in number within the inner nuclear layer of the cat retina (Hutsler and Chalupa, in preparation). In this context it would be useful to know which retinal cells are capable of manufacturing NPY. Finally, it may also be the case that gamma cells produce NPY, but the peptide remains within the cell to serve either a structural or biochemical role. Whichever of these alternatives proves correct, the cytochemical diversity of the gamma cell class demonstrated here may be useful in further differentiating among specific subpopulations of these neurons.

It seems noteworthy that the two neuropeptides that have been identified in cat retinal ganglion cells to date, NPY in the present study and somatostatin in a previous study from our laboratory (White and Chalupa, ’91) are localized exclusively in two distinct morphological classes of cells: gamma and alpha, respectively. Thus, the neuropeptide content of cat retinal ganglion cells appears to be correlated with the salient properties established for the major ganglion cell classes. This could relate to the different functions subserved by these neurons, perhaps involving their neuromodulatory role in the processing of visual information. Whether or not other neuropeptides are also confined to distinct retinal ganglion cell classes remains to be established.

ACKNOWLEDGMENTS This research was supported by NIH grant EY-03991

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Neuropeptide Y immunoreactivity identifies a group of gamma-type retinal ganglion cells in the cat

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