Acetylcholinesterase and choline acetyltransferase localization patterns do correspond in cat and rat retinas

Vision Res. Vol. 33, No. 13, pp. 1747-1753, 1993 Printed in Great Britain. All rights reserved

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0042-6989/93 $6.00 + 0.00 1993 Pergamon Press Ltd

Acetylcholinesterase and Choline Acetyltransferase Localization Patterns Do Correspond in Cat and Rat Retinas MARK H. CRISWELL,*t

CHRISTOPHER

BRANDON*

Received 24 June 1992; in revised form 22 December 1992

Is acetylcholinesterase (AChE) a reliable marker for cholinergic activity in the cat and rat retinas? To evaluate this question, radial sections, labeled for AChE, have been compared to sections labeled for choline acetyltransferase (CLAT). Within the inner plexiform layer (IPL) of each species, two lightly-stained AChE bands are revealed which correspond to the depths of ChAT immunoreactivity. Although retinal AChE is not limited exclusively to sites where ChAT is present, AChE and ChAT activity do occur in the same IPL sublaminae. Used with proper caution, AChE is a reliable secondary indicator of cholinergic activity. Retina

Choline acetyltransferase

Amacrine cells

Acetylcholinesterase

INTRODUCTION Acetylcholinesterase

(AChE)

is the hydrolyzing

enzyme

that terminates the activity of the neurotransmitter acetylcholine (ACh). Because AChE can be identified histochemically, it serves as an indirect marker for ACh activity in the nervous system. Similarly, choline acetyltransferase (ChAT), the biosynthesizing enzyme for ACh, can be identified immunocytochemically, and thus is also used to localize ACh in neurons [for a review of retinal ACh and AChE, see Brecha (1983), Hutchins (1987) Neal (1983) and Puro (1985)]. In the retinas of various vertebrate species, AChE and ChAT are typically localized in discrete sublaminae of the inner plexiform layer (IPL); these enzymes also are localized in cell somas in the inner nuclear layer (INL) and in the ganglion cell layer (GCL), (for AChE: see Nichols & Koelle, 1968; for ChAT: see Brandon, 1987a, b; Famiglietti & Tumosa, 1987; Millar, Ishimoto, Johnson, Epstein, Chubb & Morgan, 1985; Millar & Morgan, 1987; Pourcho & Osman, 1986b; Tumosa & Stell, 1986; Voigt, 1986). However, the localizations of these two substances are not exactly the same; AChE has been shown to occur in certain portions of the nervous system where ChAT is not present (see Silver, 1967, 1974) and thus may have physiological functions other than ACh hydrolysis. In the retina, AChE histochemistry normally identifies those neurons (cholinergic conventional and displaced amacrine cells) that also can be labeled by ChAT *Department of Cell Biology and Anatomy, University of Health Sciences, The Chicago Medical School, North Chicago, IL 600643095, U.S.A. tTo whom all correspondence should be addressed.

Cat

Rat

immunocytochemistry. In addition, AChE stains certain ganglion cells (for reviews of retinal ChAT and AChE, see Hutchins, 1987; Neal, 1983). In general, a population of AChE-labeled processes stratify in the IPL at either the same depth, or at depths in close proximity to those of ChAT-labeled processes (see Hutchins, 1987; Neal, 1983). However, this is not always the case; evidence from cat and rat retinas indicates that the depths at which AChE-labeled bands stratify in the IPL are distinctly different from those of ChAT-labeled bands (Pourcho & Osman, 1986a; Ross, Dunning, Juengel & Godfrey, 1985). From enzyme assays of the rat retina, Ross et al. (1985) reported that AChE activity peaks at IPL depths of 20 and 80%, while ChAT activity is greatest at depths of 33 and 66%. Employing immunocytochemical- and histochemical-labeling techniques in the cat retina, Pourcho and Osman (1986a) concluded that AChE activity is most concentrated in two bands (O-6% and 64-78%, IPL depth), but ChAT activity is localized at depths of 20 and 49%. As a consequence, Pourcho and Osman (1986a) have challenged the reliability of AChE as a marker of cholinergic neurons. To further evaluate this situation, we have compared an improved histochemical method for AChE localization to ChAT immunocytochemistry in retinas from common cats and albino rats. METHODS Preparation

Cat retinal tissue came from two adult males (approx. 3.6 kg body wt). They were anesthetized by the university veterinarian with an i.m. injection (consisting of ketamine hydrochloride, 80 mg; and xylazine, 4 mg, mixed

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H. CRlSWELL

and CHRISTOPHER

in the same syringe) and, following surgery, were killed with a barbiturate overdose (i.v. injection of pentobarbital, 400 mg). Rat retinal tissue was obtained from three adult females (approx. 300 g body wt). Rats were anesthetized with the same agents and procedures employed for cats, except that lower dosages were used. For both species, intact eyes were enucleated and their anterior segments and vitreous were dissected away. The remaining eyecups were immersed in a prefixative solution containing 0.15 M sodium phosphate and 0.2 mM CaCl, (pH 7.42 at 4°C). To this solution, concentrated formaldehyde (freshly prepared from paraformaldehyde) was added over a 45 min period, bringing the final concentration to 2% (at 20°C). Fixation continued overnight. Eyecups then were transferred gradually (in seven equimolar steps) to phosphate-buffered saline (PBS). Retinas were carefully dissected from the eyecup and peripheral regions were cut into small pieces (cat, 3 x 3 mm; rat, 2 x 2 mm). Retinal pieces were gradually transferred into a PBS solution containing 20% dimethylsulfoxide (DMSO), frozen in liquid nitrogen, and stored at -70°C. As needed, individual pieces were thawed and were gradually transferred back into PBS. For each comparative study, a single 3 x 3 mm piece of retina was mounted in PBS containing 4% low-gelling temperature agarose, and was cut into 50 pm radial sections using a vibrating microtome. Alternating 50 pm sections from the piece were processed either: (a) histochemically for AChE, or (b) immunocytochemically for ChAT. Therefore, all of the stained sections originated from the same, small region of peripheral retina. All incubations were carried out at room temperature. Histochemical

staining for AChE

For labeling AChE activity in the retina, we developed our own variation of the Hedreen, Bacon and Price (1985) technique. Their histochemical procedure was derived from Koelle and Friedenwald’s (1949) original AChE-labeling method, and from the Karnovsky and Roots’ (1964) “direct-coloring” modification of that method (which involves acetylthiocholine iodide and potassium ferricyanide to intensify the precipitated copper thiocholine substrate). In Hedreen et al’s technique, the reaction product is further intensified by treating the stained retinal tissue with ammonium sulfide, followed by silver nitrate. The resulting reaction product gives a clear indication of AChE activity against relatively light background staining. Occasionally, we also used the earlier Karnovsky and Roots (1964) procedure for comparative purposes. Using either method, tissue pieces first had to be gradually transferred into a 0.1 M sodium acetate buffer at a low pH (6.0) in order for them to be successfully stained. Because we used retinal tissue, we changed certain concentration amounts and incubation/wash times from the original procedure of Hedreen et al. For optimal labeling of 50 pm pieces of retinal tissue, incubation for times up to 75 min in the primary labeling medium intensified staining; after that, non-specific staining of

BRANDON

background tissues became a problem. The primary incubation medium consisted of: 25 mg acetylthiocholine iodide, 32.5 ml of 0.1 M sodium acetate buffer, 2 ml of 0.1 M sodium citrate, 5 ml of 0.03 M cupric sulfate (24 mg/5 ml H,O), 9.5 ml of distilled H,O, 1 ml of 5 mM potassium ferricyanide (1.6 mg/ 1 ml). Following two I-min washes in the sodium acetate buffer, tissue pieces were treated with 1% ammonium sulfide solution for 1 min and then two I-min washes in a 0.1 M sodium nitrate buffer. Staining was then intensified in a 0.1% silver nitrate solution for five minutes. Sections received two I-min washes in 0.1 M sodium nitrate, one 5-min wash in 0.1 M acetate buffer, and were transferred into 0.1 M sodium cacodylate buffer for final processing. Some retinal sections were post-stained with Azure II dye. Immunocytochemical

labeling for ChAT

Instead of AChE labeling, alternating 50 pm retinal sections, cut from each tissue piece, were processed for the immunocytochemical localization of ChAT. We used a well-characterized antiserum to chick brain ChAT (Johnson & Epstein, 1986), and an immunocytochemical method based on Fab fragments (Brandon, 1985). After overnight incubation, the sections were incubated in goat anti-rabbit serum (Fab-specific), and were finally incubated in FabPAP (Slemmon, Salvaterra & Saito, 1980). Sections were then transferred to a 0.1 M sodium phosphate buffer and incubated for 25 min in diaminobenzidine (DAB) staining solutions containing 0.06% DAB with cobalt chloride and nickel ammonium sulfate (Adams, 1981). After 25 min, hydrogen peroxide was added to a concentration of 0.012% to obtain a peroxidase reaction product, and the sections were incubated for an additional 15 min. After staining, ChAT- and AChE-labeled sections were postfixed with 1% glutaraldehyde, osmicated [1 % 0~0, and 1.5% K,Fe(CN),], and were stained in 1% tannic acid, all in sodium cacodylate buffer. Tissues were then dehydrated in an ascending methanol series followed by dioxane, infiltrated by epoxy resin, and flatembedded. The resulting tissue/epoxy blocks were cut into serial sections 5 pm in thickness for light microscopic analysis. For determinations of relative depth within the inner plexiform layer, the INL-IPL border was designated as 0% depth and the IPL-GCL border was designated as 100% depth.

RESULTS

Proximal

cat retina

Labeling for AChE is confined almost exclusively to the IPL, with some light outline staining of certain somas in the INL and GCL, and some staining of blood vessels. Figure 1 shows that light background staining is evident throughout the IPL. Beyond this, four definite IPL bands contain AChE activity. Two thick and heavilylabeled bands are most prominent; the most distal band occurs at a relative IPL depth of &lo%, and the most

CAT AND RAT RETINAL AChE AND ChAT

proximal band at 65587%. These darker bands are analogous to the two AChE bands reported previously by Pourcho and Osman (1986a). Between these two prominent bands are two distinct, but more lightlylabeled, bands that have not been reported previously (Fig. 1). One of these two bands occurs more distally at an IPL depth of 25-30%, and the other more proximally at a depth of 45-50%. In agreement with earlier studies (Dann, 1989; Mitrofanis & Stone, 1988; Pourcho & Osman, 1986a, b; Schmidt, Wassle & Humphrey, 1985; Schmidt, Humphrey & WLsle, 1987; Vardi, Masarachia & Sterling, 1989), immunocytochemical labeling for ChAT reveals two bands within the IPL (Fig. 2). A distal band occurs at 20-28% depth and a proximal band occurs at 45-52% depth. Occasionally, somas of conventional amacrine cells also are labeled along the INL-IPL border, and somas of displaced amacrine cells along the IPL-GCL border. The proximal cat retina contains a small population of ChAT-immunoreactive neurons (see Schmidt er al., 1985; Pourcho & Osman, 1986b). As a result, the ChAT labeled sublaminae in the IPL are

ChAT

1749

intermittent and are relatively faint. Like Pourcho and Osman (1986a), we observe that the ChAT-labeled processes do not correspond to the two most densely labeled AChE bands. However, the two fainter AChE bands and the two ChAT bands lie at virtually identical depths. Unfortunately, our data do not tell us if these AChElabeled and ChAT-labeled processes belong to the same cell populations. Thus, it remains uncertain whether the cell processes which constitute these two AChE bands arise from cholinergic neurons, cholinoceptive neurons, or perhaps a mixture of both. We also stained retinal sections using an earlier AChE-labeling technique (Karnovsky & Roots, 1964), and found that while the two darker bands (at O-10% and at 65-87%) are quite apparent, the two fainter bands (at 20-28% and at 45-52%) are not discernible, even when staining times are increased. At the point where the Karnovsky and Roots method labels the two dark bands as strongly as the method of Hedreen et al., nonspecific staining of the entire retina becomes a problem. Therefore, silver nitrate intensification of labeled AChE substrates is not only necessary for viewing all

*

FIGURE 1. Radial sections of the common cat retina (5 pm thickness). The retinal section on the left is labeled for choline acetyltransferase (ChAT) and the section on the right is labeled for acetylcholinesterase (AChE). Both sections originated from the same 3 x 3 mm piece of peripheral retina. In cat, four AChE-labeled bands are evident in the inner plexiform layer (marked by white arrows on the right side). The middle two AChE bands occur at approximately the same depths (25-30% and 45-50%) as do the two intermittent ChAT bands (at 2&28% and 45-52%; marked by white arrows on left side). ChAT somal staining in the inner nuclear layer (INL) and in the ganglion cell layer (GCL) is also apparent. Bar = 25 pm.

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MARK H. CRISWELL and CHRISTOPHER

ChAT

BRANDON

_

,-

FIGURE 2. Radial sections of the albino rat retina (5 pm thickness). The section on the left is labeled for ChAT and the section on the right is labeled for AChE. Both retinal sections originated from the same 2 x 2 mm piece of peripheral retina. In rat, five AChE-labeled bands are evident in the inner plexiform layer (marked by white arrows on the right side). The second and fourth AChE bands occur at approximately the same depths (21-27% and 54460%) as do the two ChAT bands (at 23-29% and 5762%; marked by white arrows on left side). Like in the cat, ChAT somal staining can be observed in the INL and in the GCL of the rat retina. Bar = 25 pm.

four AChE bands in the IPL, it may also be better for accurate labeling of the tissue. Unlike earlier studies (Nichols & Koelle, 1968; Pourcho & Osman, 1986a), somal staining for AChE appears,

CAT 0

0

ChAT

AChE

80

80 loo'

RAT AChE

ChAT

I

1 100

FIGURE 3. These two schematic diagrams depict the relative IPL depths at which labeled bands of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activity occur in the cat retina and in the rat retina. In each species, there is a close correspondence of ChAT and AChE activity at two IPL depths. There are also two additional AChE-labeled bands in the cat IPL and three additional AChE-labeled bands in the rat retina which probably are not associated with cholinergic activity (see Discussion).

at best, very faint. Some membrane staining occasionally can be noted in INL and GCL cells adjacent to the IPL. Although we could not identify them, these cells may be cholinergic Al4 conventional and dA14 displaced starburst amacrines, respectively (see Nichols & Koelle, 1968; Pourcho 8 Osman, 1986a, b; Schmidt et al., 1987; Vardi et al., 1989). We did not observe AChE-labeled ganglion cells, although others have (Nichols & Koelle, 1968; Pourcho & Osman, 1986a). In experiments where we lengthened tissue incubation time in the primary (acetylthiocholine iodide) staining solution, we determined that somas are better labeled for AChE; however, in exchange, distinct AChE banding in the IPL is lost because the nonspecific background staining is increased. Likewise, in an experiment where we fixed a retina in a solution consisting of 2.25% formaldehyde and 0.25% glutaraldehyde, which is similar to that used by others (Pourcho & Osman, 1986a; Reale, Luciano & Spitznas, 1971), intensified somal staining occurs in the INL and GCL; but again, this happens at the cost of sacrificing clearly-labeled bands in the IPL, even when primary incubation times are brief (30 min). Proximal

rat retina

Labeling for AChE in the rat retina is similar to that in the cat retina. As seen in Fig. 2, some faint outline

CAT AND RAT RETINAL AChE AND ChAT

staining along somal membranes in the INL and GCL is noticeable, but neither cell identification nor tracing of dendrites to specific sublaminae of the IPL is possible. Staining across the IPL is obvious from the distal to the proximal borders. Whereas four AChE bands occur in the cat’s IPL, there are five distinct bands which contain AChE activity in the rat’s IPL (Fig. 2). Two of these five bands are heavily stained and they occur along the distal and proximal borders of the IPL. A prominent distal band occurs at a relative IPL depth of O-9%, and a densely-labeled proximal band extends from 67 to 100%. Three lightly-labeled AChE bands occur between the two prominent bands at relative IPL depths of 21-27%, 38-46%, and 5460% (Fig. 2). Immunocytochemistry for ChAT in the rat retina labels two bands within the IPL, a distal band at a relative IPL depth of 23-29% and a proximal band at 5762%. These findings are similar to those values (25 and 55% respectively) reported by Voigt (1986). These ChAT-labeled bands are derived from processes of conventional and displaced amacrine cells, whose somas and principal dendrites are also ChAT labeled [Fig. 2; see also Kondo, Kuramoto, Wainer and Yanaihara (1985) and Mitrofanis and Stone (1988)]. We also wanted to demonstrate that the modified AChE labeling method of Hedreen et al. (1985) gives results that are qualitatively similar to earlier methods. To accomplish this, we used the newer AChE technique, and ChAT immunocytochemistry, to label rabbit retina (unpublished data). In the rabbit IPL, the four principal AChE-labeled bands correspond to those values which were published previously (Brandon, 1987b) and which were originally obtained using Karnovsky and Roots’ (1964) AChE-labeling method. Furthermore, in the turtle retina, the two ChAT-labeled bands occur at the same IPL depths as do the two principal AChE bands (Criswell & Brandon, 1992). These results confirm the validity of the newer AChE histochemical method. DISCUSSION

As a result of this investigation in cat and rat, we conclude that AChE is present in the immediate vicinity of cholinergic synapses (Fig. 3), where it can terminate the biological activity of ACh. We do not know if these AChE-labeled processes belong to the same cholinergic cells which were labeled for ChAT. According to Kuhar (1985) and Hutchins (1987), AChE and ACh do not need, nor should they really be expected, to colocalize precisely (as seen in Fig. 3). First, the different methods for localizing cholinergic activity may label pre- and/or post-synaptic (i.e. different) cell populations. Second, the labeling of ACh-related enzymes (AChE or ChAT) is an indirect approach for localizing acetylcholine. AChE labeling does not necessarily occur exclusively at cholinergic synapses; AChE may also be found in somas and processes of presynaptic cholinergic cells, as well as in postsynaptic neurons. The results from this investigation reaffirm our confidence in AChE as a marker of cholinergic activity.

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However, an important question still remains: why are there two additional AChE-labeled bands in the IPL of the cat retina and of the rat retina which are even more prominent than the bands which are associated with cholinergic activity? Evidence from several related investigations suggest a possible answer. First, the presence of two types of AChE bands (one type that is functionally related to ACh neurotransmission and a second type which is not) is not unique to cat. Millar, Ishimoto, Johnson, Epstein, Chubb and Morgan (1985) have reported that while overlap of ChAT and AChE occurs in the IPL of the chicken retina, additional AChE bands are labeled as well. They also reported that the best somal staining for AChE takes place in non-cholinergic neurons. Because the cat retina contains a relatively small population of cholinergic neurons, it is not surprising that the corresponding AChE-labeled bands are also less pronounced. Examples of non-cholinergic AChE-labeled neurons (and glia) exist in various parts of the central nervous system (see Silver, 1974). For this reason, Lehmann and Fibiger (1979), and Silver (1967) have developed a “necessary but not sufficient” rule: i.e. AChE is a necessary component for demonstrating the presence of ACh, but the occurrence of AChE is not, by itself, sufficient to prove that ACh is also present. Additional evidence, such as the presence of ChAT, is also required. Second, AChE may perform additional roles in the nervous system. Besides a cholinesterase, AChE may be a peptidase (Chubb, Hodgson & White, 1980; Small, 1988). According to Chubb et al. (1980), AChE consists of at least two active binding sites, one which hydrolyzes ACh, and another which operates as a peptidase. In the pigeon retina, supposedly eight neuropeptides exist as either neurotransmitters or as neuromodulators, and six of these peptides are localized in amacrine cells (Stell, Marshak, Yamada, Brecha & Karten, 1980). In chicken retina, AChE, acting as a hydrolyzing peptidase, may break down the necessary protein precursor molecules for the synthesis of substance P, enkephalins, and less probably, somatostatin. Based on immunocytochemical localizations in the retina of these same peptides (Chubb & Millar, 1984; Goebel & Pourcho, 1992a; Millar & Chubb, 1984; Morgan, Oliver & Chubb, 1981), and on actual measurements of peptide hydrolysis (Chubb et al., 1980; Chubb, Ranier, White & Hodgson, 1983; Goebel & Pourcho, 1992a, b), exogenous AChE may also, as a hydrolyzing peptidase, terminate (by direct or possibly indirect action), the biological activity of these same neuropeptides. In addition, Greenfield (1984) has suggested that exogenous AChE might itself function as a neuromodulator, but this has not been clearly demonstrated. Finally, data of Pourcho and Goebel (1988) and of Vaney, Whitington and Young (1989) support the hypothesis that the two prominent AChE-labeled bands in the IPL of the cat retina contain substance P. Conventional amacrine cells that contain substance P stratify within the proximal 20% of the IPL, and displaced amacrines that contain substance P project to an IPL

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depth of approx. 5&70%, leading Vaney et al. (1989) to propose that these two AChE bands most likely contain this particular neuropeptide. In conclusion, the results of our investigation demonstrate that AChE-activity can be specifically localized in the same IPL sublaminae where ChAT-activity is found. In this regard, the cat and rat retinas are not different from the retinas of other vertebrates. Evidence from various investigations suggests that the additional labeling of AChE in the IPL could represent localization of neuropeptides, the most likely one in this instance being substance P. If used in conjunction with other cholinergic labeling techniques, we are confident that AChEstaining can be used as a reliable secondary indicator for identifying sites of ACh activity.

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Karnovsky, M. J. & Roots, L. (1964). A “direct-coloring” thiocholine method for cholinesterases. Journal of Histochemistry and Cytochemistry, 12, 219-221. Koelle, G. B. & Friedenwald, J. S. (1949). A histochemical method for localizing cholinesterase activity. Proceedings of the Society for Experimental Biological Medicine, 70, 617622. Kondo, H., Kuramoto, H., Wainer, B. H. & Yanaihara, N. (1985). Discrete distribution of cholinergic and vasoactive intestinal polypeptidergic amacrine cells in the rat retina. Neuroscience Letters, 54, 213-218. Kuhar, M. J. (1985). The mismatch problem in receptor mapping studies. Trends in Neurosciences, 8, 190-191. Lehmann, J. & Fibiger, H. C. (1979). Acetylcholinesterase and the cholinergic neuron. f@ Sciences, 25, 1939-1947. Millar, T. J. & Chubb, 1. W. (1984). Treatment of sections of chick retina with acetylcholinesterase increases the enkephalin and substance P immunoreactivity. Neuroscience, 12, 44451. Millar, T. J. & Morgan, I. G. (1987). Cholinergic amacrine cells in the rabbit retina synapse onto other cholinergic amacrine cells. Neuroscience Letters, 74, 281-285. Millar, T., Ishimoto, I., Johnson, C. D., Epstein, M. L., Chubb, I. W. & Morgan, I. G. (1985). Cholinergic and acetylcholinesterase-containing neurons of the chicken retina. Neuroscience Letters, 61, 311 -316. Mitrofanis, J. & Stone, J. (1988). Distribution of cholinergic amacrine cells in the retinas of normally pigmented and hypopigmented strains of rat and cat. Visual Neuroscience, 1, 367-376. Morgan, I. G., Oliver, J. & Chubb, I. W. (1981). Discrete distributions of putative cholinergic and somatostatinergic amacrine cell dendrites in chicken retina. Neuroscience Letters. 27, 5560. Neal, M. J. (1983). Cholinergic mechanisms in the vertebrate retina. Progress in Retinal Research, 2, 19lk212. Nichols, C. W. & Koelle, G. B. (1968). Comparison of the localization of acetylcholinesterase and non-specific cholinesterase activities in mammalian and avian retinas. Journal of Comparative Neurology, 133, 1-16. Pourcho, R. G. & Goebel, D. J. (1988). Substance P-like immunoreactive amacrine cells in the cat retina. Journal of Comparative Neurology, 275, 542-552. Pourcho, R. G. & Osman. K. (1986a). Acetylcholinesterase localization in cat retina: A comparison with choline acetyltransferase. Elxperimental Eye Research, 43, 585-594. Pourcho, R. G. & Osman, K. (1986b). Cytochemical identification of cholinergic amacrine cells in cat retina. Journal of Comparative Neurology, 247, 497-504. Pure, D. G. (1985). Cholinergic systems. In Morgan, W. W. (Ed.), Retinal transmitters and modulators: Models .for the brain (Vol. 1, pp. 63-91). Boca Raton, Fla: CRC Press. Reale, E., Luciano, L. & Spitznas, M. (1971). The fine structural localization of acetylcholinesterase activity in the retina and optic nerve of rabbits. Journal of Histochemistry and Cytochemistry, 19, 85596. Ross, C. D., Dunning, D. D., Juengel, L. I. & Godfrey, D. A. (1985). Laminar distributions of choline acetyltransferase and acetylcholinesterase activities in the inner plexiform layer of rat retina. Journal of Neurochemistry, 44, 1091-1099. Schmidt, M., Humphrey, M. F. & Wassle, H. (1987). Action and localization of acetylcholine in the cat retina. Journal of Neurophysiology, 58, 997-1015. Schmidt, M., Wiissle, H. & Humphrey, M. (1985). Number and distribution of putative cholinergic neurons in the cat retina. Neuroscience Letters, 59, 2355240. Silver, A. (1967). Cholinesterases of the central nervous system with special reference to the cerebellum. International Review of Neurobiology, IO, 57-109. Silver, A. (1974). The biology of cholinesterases. In Neuberger, A. & Tatum, E. L. (Eds), Frontiers of biology, (Vol. 36, pp. l-596). Amsterdam: North-Holland. Slemmon, J. R., Salvaterra, P. M. & Saito, K. (1980). Preparation and characterization of peroxidase: Antiperoxidase-Fab complex. Journal of Histochemistry and Cytochemistry, 28, 1O-l 5.

CAT AND RAT RETINAL AChE AND ChAT Small, D. H. (1988). Amino acid sequence similarity between Drosophila acetylcholinesterase and the active site region of trypsin. Neuroscience Letters, 94, 237-238. Stell, W., Marshak, D., Yamada, T., Brecha, N. & Karten, H. (1980). Peptides are in the eye of the beholder. Trends in Neurosciences, 12, 292-295. Tumosa, N. & Stell, W. K. (1986). Choline acetyltransferase immunoreactivity suggests that ganglion cells in the goldfish retina are not cholinergic. Journal of Comparative Neurology, 244, 261-275. Vaney, D. I., Whitington, G. E. & Young, H. M. (1989). The mo~hology and topographic dist~bution of subs~nce-P-like immunoreactive amacrine cells in the cat retina. Proceedings of the Royal Society of London B, 237, 471488.

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Vardi, N., Masarachia, P. J. & Sterling, P. (1989). Structure of the starburst amacrine network in the cat retina and its association with alpha ganglion cells. Journal of Comparative Neurolagy, 288, 60-611. Voigt, T. (1986). Cholinergic amacrine cells in the rat retina. Journaf of Comparative Neurology, 248, 19-35.

Acknowledgements-This research was supported by NIH grant EY03886 to CB. The anti-(choline acetyltransferase) serum used in this study was generously provided by Carl D. Johnson, Miles L. Epstein, and June Dahl, all of the University of Wisconsin, Madison. The authors also thank Elzbieta Indyk for her technical assistance.