Journal of Neurochemistry, 1976. Vol. 26, pp. 521-526. Pergamon Press. Printed in Great Britain.
THE DISTRIBUTION OF CHOLINE ACETYLTRANSFERASE ACTIVITY IN VERTEBRATE RETINA' C. D. Ross2 and D. B. MCDOUGAL, JR. Department of Pharmacology, Washington University School of Medicine, St. Louis, M O 631 10, U.S.A. (Received 7 May 1975. Accepted 4 August 1975) Abstract-Choline acetyltransferase (ChAc) activity was determined in retinal layers from 10 vertebrates. In all animals, the highest activity was in the inner plexiform layer, intermediate activity in the inner nuclear and ganglion cell layers, and very low activity in the photoreceptor and outer plexiform layers and optic nerve. The pattern of distribution of enzyme activity within the inner nuclear layer corresponds quantitatively to the distribution of amacrine cells within that layer. A species difference of almost 90-fold was found between the lowest and highest values for ChAc activity in inner plexiform layer. The variation in enzyme activity found among homeotherms in inner nuclear and inner plexiform layers is related to the number ofamacrine cell synapses in the inner plexiform layer. But the differences in enzyme activity are generally greater than those which have been found in numbers of amacrine cell synapses between species. The data suggest that cholinergic neurons in retina are to be found predominantly among the amacrine cell types and that not all amacrine cells will bc found to be cholinergic.
IT HAS been suggested for a number of years that @LA, 1963; NICHOLS & KOELLE,1968; REALErt aE., acetylcholine might be involved in neural transmis- 1971; NICHOLSet al., 1972). Following complete inhision in the retina. Application of ACh evokes electri- bition of AChE by diisopropyl Auorophosphate, syncal responses from retinal cells, which can be blocked thesis of new cnzyme was observed first in amacrine by ACh antagonists (AMES & POLLEN, 1969; VON BRE- cells (NICHOLS & KOELLE,1968). DOW eta]., 1971). The retinas of most animals contain One possible interpretation of results from AChE significant amounts of ACh (FELDBERG histochemical studies is that some of the cells, most & MA", 1946; LINDEMAN,1947), choline acetyltransferase likely amacrine, which form synapses in the inner (ChAc, acetyl-CoA: choline 0-acetyltransferase, EC plexiform layer are cholinergic. However, conclusions 2.3.1.6; HEBB& SILVER, 1956), and acetylcholinester- that cells are cholinergic based only on the fact that ase (AChE, EC 3.1.1.7; ANFINSEN,1944; NACHMAN- they contain AChE must be tentative, since non-choSOHN, 1959). A review has appeared recently dealing linergic neurons may also contain this enzyme (HEBB, et al., 1972). Identificawith some comparative aspects of neurotransmitters, 1957; SILVER,1967; PARTLOW tion of the cholinergic neurons in retina as amacrine including ACh, in the retina (GRAHAM,1974). Most attempts to identify cholinergic cells in the cells is further obscured by evidence that other putaretina have involved the localization of cholinesterase tive neurotransmitters, such as dopamine (EHINGER 1972; activity by histochemical staining procedures. The & FALCK, 1969, 1971) and GABA (GRAHAM, & VOADEN,1975) have product of the histochemical reaction is consistently EHINGER,1972; MARSHALL associated with amacrine cells and ganglion cells also been attributed to amacrine cells. Since specific cell types and synaptic regions are and with their processes in the inner plexiform layer concentrated in strata or layers within the retina, the problem of localization of the enzymes of neurotransPreliminary reports of this study were presented to the mitter metabolism can be approached using the tech58th Annual Meeting of the Federation of American Socieniques of quantitative histochemistry ( L o m y et al., ties for Experimental Biology (Ross & M c D o u ~ u ,1974) 1956; MATSCHMSKY & MCDOUGAL, 1968). Of the two and to the 1974 Meeting of the Association for Research enzymes involved in the metabolism of ACh, ChAc in Vision and Ophthalmology. Supported by Grants NS06800, EY-00258, and NS-05221 of the National Institutes and AChE, ChAc is considered the more reliable indicator of the presence of cholinergic mechanisms of Health. 1945). * Present address: Department of Anatomy, Washington (HEBB,1957, 1963; SILVER,1967; FELDBERG, The purpose of this study was to determine the University School of Medicine, 660 So. Euclid Ave., St. activity of ChAc in layers of retina from various verteLouis, MO 63110, U.S.A. Abbreviations used: ChAc, choline acetyltransferase; Na brate species in an effort to determine which cell type(s) in the retina might be cholinergic. TPB, sodium tetraphenylboron. 52 1
522
JR. C. D. Ross and D. B. MCDOUGAL,
METHODS Protein was determined by the Lowry method ( L O W Y Anulytical methods et al., 1951). Tissue handling. Histologically defined samples, ranging Experimental materials in dry weight from 0.03 to 2 pg, were dissected from frozenEyes from mice, turtles, goldfish, and pigeons were prodried sections cut tangentially to the eyeballs of several, poikilothermic and homeothermic vertebrates (LOWRY ' vided by Dr. Adolph Cohen, Department of Ophthalmology. Frozen-dried sections from monkey and rabbit a/., 1956) and weighed on a quartz-fiber balance (LOWRY retinas were provided by Dr. Oliver Lowry. Rats were ob& PASSONNEAU, 1972). tained from National Laboratories, St. Louis, Mo., frogs Et1zyrne and protein assays. The assay of ChAc was based on the radiometric method developed by MCCAMAN from J. R. Schettle Biologicals, Oshkosh, Wisconsin, and & HUNT(1965), using the sodium tetraphenylboron (Na garfish from E. Saeugling, Gutenberg, Iowa. Cats were obTPB) procedure of FONNUM(1969) for extraction of tained from a local supplier. [1-'4C]acety1-CoA (46-54 mCi/mmol) and [I-l4C]ace[1-14Clacetylcholine. Samples were incubated in oil tylcholine (2-5 mCi/mmol) were obtained from New Enget ul., 1968; LOWRY & PASSONNEAU, wells (MATSCHINSKY 1972) for 45-60 min at 25°C in 2 pl of a reagent containing land Nuclear. 75 p~-[l-~~c]acetyl-CoA, 6 mM-choline bromide, 250 m ~ - N a C l , 0.1% Triton x-100, 0.08 mM-neostigmine RESULTS methylsulphate, 0.1% bovine serum albumin, 110 mMThe patterns of distribution of ChAc activity sodium phosphate buffer, pH 7.6. After incubation, the aqueous droplet was removed and added to 5 pl 10mM- among retinal layers were similar for all vertebrates sodium phosphate pH 7.8 in a 400 pl plastic centrifuge tube, studied (Table la and b, Fig. 1). Almost all the enwhich was placed in an ice bath. Forty-five p1 of Na TPB zyme activity was confined to the inner retinal layers, (15 mg/ml) in 3-heptanone was added, the contents mixed with the highest activity being in the inner plexiform well,and layers separated by centrifugation (Beckman Micro- layer. The activity in the outer and inner segments fuge model 152). The organic phase was removed and and nuclei of the photoreceptors was very low comadded to another tube containing 1M)pl Na TPB pared to the value for inner plexiform layer. The ratio (0.5 mg/ml) in 10 mM-sodium phosphate buffer pH 7% of enzyme activity between inner plexiform and After mixing and centrifugation, a 38 p1 aliquot of the or- photoreceptor layers was at least loo0 in mouse, rat, ganic layer was removed and added to a scintillation vial cat, pigeon, garfish, and goldfish, about 500 in turtle, containing 0.4% 2,5-diphenyloxazole (PPO) and 001% p50 in frog. The activities rabbit, monkey, and about bis-[2-(4-methyl-5-phenyloxazoyl)] benzene (dimethyl of the in the outer plexiform layer, the outer third POPOP) in toluene with 0.1 ml hyaminebhydroxide added to each 8 ml volume. The blank, including background, for inner nuclear layer, and the optic nerve were also the ChAc assay was about 50 c.p.m. at 91% counting effi- very low relative to the inner plexiform layer. ChAc activities showed a graded increase within ciency, equivalent to 0.55pmol or 055 mmol/kg dry wt per h for a 1 pg sample incubated 1 h. The reaction rate the inner nuclear layer, with the lowest value being was linear for at least 1 h. Sample sizes and incubation in the outer third and highest value in the inner third times were adjusted with activity so that AcCoA concen- next to the inner plexiform layer. Because the retinas tration was not limiting. were cut tangentially, invaginations of inner plexiform The assay of ChAc activity in homogenates was similar layer into inner nuclear layer were easy to see in the except that the volumes used during incubation, extraction, and washing were increased, the incubation was done in frozen-dried sections and were dissected away from plastic tubes, and the incubation medium was not covered the inner nuclear samples. It is estimated that not more than 10% of the enzyme activity ascribed to with oil (Ross et al., 1975). No enzyme activity was detected when choline was omit- the inner third of inner nuclear was due to inner plexted from the incubation medium. The product formed dur- iform layer contamination. ing incubation with both substrates was demonstrated to Although the relative distribution of ChAc activity be ACh by the addition of AChE (electric eel), which re- between retinal layers was similar, a significant differduced the Na TPB-heptanone extractable radioactivity to ence was found in the absolute activities in inner nuG blank value. The possibility of synthesis of ACh by carni- lear and inner plexiform layers. The highest activity tine acetyltransferase, however, cannot be excluded (WHITE for inner plexiform layer among the homeotherms & WU, 1973). This enzyme could be significant in samples (Fig. 2) was found in pigeon, intermediate values were with low synthetic activity. found in rat, rabbit, and mouse, and the lowest activiNo significant differences were found among Km,AeCaA) values for ChAc in homogenates of frozen-dried tissues ties were in monkey and cat. The ChAc activity in of four species. Individual figures were 1 5 p ~(mouse pigeon inner plexiform layer was 35 times that in cat. retina), 12 p~ (goldfish retina), 14 p~ (turtle retina), and An even greater range in ChAc activity was found 17 p~ (rat cerebral cortex). These values are comparable among the poikilotherms (Fig. 3). Activity in goldfish values obtained for ChAc: 10 PM (bovine inner plexiform layer was the highest of all the anito other K,,,(A~C,,A) striate nuclei, GLOVER & POTTER,1972), 10-25 p~ (rabbit mals tested, almost 45 times the value for frog and & HUNT, 1965), and 8 p~ (cat brain, D. brain, MCCAMAN twice the value for pigeon. GODFREY, personal communication). The ChAc activity in homogenates of mouse retina inDISCUSSION creased between 25" and 38°C by almost 3-fold. No detectThe consistency in the pattern of distribution of able loss in enzyme activity occurred during the freeze-drying process. ChAc in the retinas of 10 species from 9 orders and
Choline acetyltransferase in vertebrate retina
TABLE la. ChAc ACTIVITY IN RETINAL
Outer and inner photoreceptor segments Photoreceptor nuclei Outer plexiform layer Inner nuclear layer (outer third)
1
LAYERS FROM FOUR HOMEOTHERMS
Cat
Monkey
0 (5)
0'06' (12)0'01
0 (2)
0.013 I 0.01 (10) 0.12 k 0.01 (8)
*
0 (1)
0.267
& 0.17
0 (6)
(middle third)
523
0438 i 0.3
}
Mouse
O.O43(&0Ql
Rabbit
0.121 i 0.09 0.012
0 (5)
*
(8)
0.01
(4) 0.39 (2)
0.256 f 0.08 (12) 6.93 1.0
(inner third) Inner plexiform layer Ganglion cell layer Fiber layer Optic nerve
(4) 3.61 t 0.52
(3) 9.53 i0.98
1.16
6.87 k 0.89 (5) 1.08 (2) -
0.23 (5) -
TABLElb. ChAc ACTIVITYIN
RETINAL LAYERS FROM POIKILOTHERMS
Frog Outer and inner photoreceptor segments Photoreceptor nuclei Outer plexiform layer Inner nuclear layer (outer third) (middle third)
(inner third)
(7) 40.4 k 1.4 (67) 23.9' +'13 (15) 0.36 k 0.5 (14)
Turtle
0.055
+ 0.02
1.90 k 0.44 (4) 5.78 f 037 (10) 21.6 f 4.7
0225 f 006 (7) 1.71 f 0.4 (6) 5.60 k 1.2
Garfish
Goldfish
0.01 i 0.033 .
13.2 & 3.9 (7) 53.8 k 4.5 (6) 125.0 k 12.0
Inner plexiform layer Ganglion cell layer Fiber layer Optic nerve
-+
Values are expressed in mmol/kg dry wt per h at 25°C S.E.M. Numbers in parentheses indicate number of samples assayed. Samples were dissected from frozen-dried sections from 2 eyes from 1 cat, 1 eye from 1 monkey, 12 eyes from 10 mice, 1 eye from 1 rabbit, 4 eyes from 2 frogs, 5 eyes from 3 turtles, 2 eyes from 2 garfish, and 3 eyes from 2 goldfish. Since no significant differences were found in ChAc activities between black Cg7BL(6J)and albino mice, the data for pigmented and nonpigmented animals were pooled. The photoreceptor layer was divided into segments (inner and outer) and nuclei in all species except frog and mouse. The inner nuclear layer was divided into sublayers in all species except garfish. ChAc activity in undivided inner nuclear layer of garfish (24% of inner plexiform layer) compares well with undivided inner nuclear layers from rat (21%) and from turtle (26%), and with the average of inner nuclear layer subdivisions in goldfish (20%).
6 classes of vertebrates is quite remarkable (Fig. 4), especially when it is considered that the absolute range of activities in inner plexiform layer is nearly 90-fold. This suggests that the distribution of a single cell type and its processes may be responsible for the major portion of the pattern seen. TWOlines of evidence from this study suggest that the cholinergic neurons in retina are predominantly
among the amacrine cell types. First, the pattern of increase in ChAc activity within the inner nuclear layer (Table 1, Fig. 1) follows the distribution of amacrine cell bodies found histologically (CAJAL, 1972). Amacrine cell terminals are distributed throughout the inner plexiform layer, and a few even extend into the ganglion cell layer. Second, the variation in ChAc activity among species resembles the variation
C. D. ROSSand D. B. MCDOUGAL,JR.
524
0
.= .
3 160-
h\q
f 1400
m
120-
+GARFISH
TURTLE
100-
-g 80-
p FROG
r
0
u)
I
60-
c21 0 0
I
80
I
120
I
180
I
200
I
240
I
280
I
320
ChAc activity (mmolcs/kg dry wt./hr,25"C)
.2 40c
2
I
40
20-
0 0-
Rs Rn
FIG. 1. Distribution of ChAc activity in retinal layers of pigeon (left) and rat (right). The pattern of distribution of ChAc activity is illustrated for the 2 animals in which the inner nuclear layer was divided into 6 sublayers. ACtivity is expressed as mmol/kg dry wt per h at 25°C. Vertical lines denote +s.E.M. Samples were dissected from 8 eyes from 4 rats and 6 eyes from 3 pigeons. Since no significant differences were found in ChAc activities between brown or albino rats, the data were pooled. Abbreviations are Rs (outer and inner photoreceptor segments), Rn (photoreceptor nuclei), OP (outer plexiform layer), oIN, mIN, iIN (outer, middle and inner thirds respectively of inner nuclear layer), IP (inner plexiform layer), GC (ganglion cell layer), F (fibre layer), and OpN (optic nerve). The space representing the photoreceptors in the figure is proportionately smaller than that occupied in a retinal cross section.
in the number of amacrine cell synapses in the inner plexiform layer. Visual information in vertebrate retinas is transmitted from the photoreceptors through the bipolar cells to the ganglion cells for transfer to other brain centers. This information may be modulated in the inner plexiform layer by lateral interactions between amacrine cells (DOWING,1970). These interactions allow some processing of visual information, particularly the detection of movement, to occur at the retinal level. There is a variety of synaptic arrangements between bipolar, amacrine, and ganglion cells in the
\\\\
PIGEON
inner plexiform layer (DOWING, 1970; DUBIN,1974). A bipolar cell may form a synapse on both an amae rine and a ganglion cell, thereby providing direct input to the ganglion cell. Or a bipolar cell may contact only amacrine cells, which then make synaptic contact with the ganglion cell. In the latter case, amacrine cells, rather than bipolars, provide the direct input to the ganglion cells. The incidence of these two types of synaptic arrangements varies from one species to another. Those species exhibiting a high degree of involvement of amacrine cells in the input to the ganglion cells also have a high density of amacrine synapses in the inner plexiform layer (DUBIN,1970). While the density of amacrine synapses varies significantly from one species to another, the density of bi1970, 1974). The ratio polar synapses does not (DUBIN, of the incidence of amacrine synapses to bipolar synapses for each species (Table 2) affords a convenient way to indicate the variation in both the density of amacrine synapses in the inner plexiform layer as well as the involvement of amacrine cells in gang-
% 60-
r+ RABBIT
4-
FIG.3. Comparison of ChAc activity in inner nuclear and inner plexiform layers from poikilotherms. The total bar represents activity in inner plexiform layer +s.E.M. The striped portion of the bar represents activity in the inner nuclear layer, either the innermost third (goldfish, turtle, and frog) or the entire layer (garfish) +s.E.M. Data are from Table 1.
MOUSE
I-
40-
W 0
30-
z
MONKEY
Ra Rn OPdNmlNirJ
@CAT
I
I
I
I
I
I
I
I
I
0
20
40
60
80
100
120
140
I60
ChAc aclivily(rnrnoles/kg dry *l./hr,25'Cl
FIG.2. Comparison of ChAc activity in inner nuclear and inner plexiform layers from homeotherms. The total bar represents activity in inner plexiform layer _+S.E.M. The striped portion of the bar represents activity in the innermost sixth (pigeon, rat; data from Fig. 1) and innermost third (rabbit, mouse, monkey, and cat; data from Table 1) bf the inner nuclear layer +s.E.M.
IP GC F OpN
FIG.4. Choline acetyltransferase activity in retinal layers from 10 vertebrates expressed relative to the activity in the inner plexiform layer (= 100%). The average of these percentage values is represented for each retinal layer +s.E.M. Abbreviations are defined in legend with Fig. 1. Percentages were calculated from absolute values in Table 1 and Fig. 1. Cell types in the diagram representing retinal neural connections are (from left to right) photoreceptor, horizontal, bipolar, amacrine, and ganglion cells.
Choline acetyltransferase in vertebrate retina TABLE2. INCIDENCEOF
AMACRINE:BIPOLAR SYNAPTIC ELE-
MENTS IN INNER PLEXIFORM LAYER
monkey cat rat rabbit pigeon frog
Dubin (1970)
Sosula & Glow (1970)
1.9-2.6 2.6-3.0 3.3
8
5.1-5.3 10.8
8.8-109
lion Cell input (DUBIN,1970, 1974). Among the homeotherms, monkey and cat have low ratios of amacrine synapses to bipolar synapses; rabbit has an intermediate ratio; pigeon has a high ratio. This arrangement of species resembles the order of relative activities of ChAc. A possible inconsistency in this correlation is the rank for rat, which had higher ChAc activity in inner plexiform layer, but a lower ratio of amacrine to bipolar synapses, than did rabbit. Although SOSULA & GLOW(1970) reported a higher ratio than that found by DIJBIN (1970), results from electrophysiological studies are more consistent with a relatively low incidence of amacrine cell synapses (BROWN& ROJAS,1965). Further study will be needed to resolve this issue. Since the density of bipolar synapses is relatively constant from species to species, while the ChAc activity varied over a 35-fold range, it seems unlikely that the bipolar cells are cholinergic. The variation of ChAc activity in inner plexiform layer between homeotherms (35-fold) was much greater than the variation in amacrine to bipolar synapses ratio (4- to 5-fold). This lack of quantitative correspondence affords a clue that the differences in ChAc activity reflect more than simply the differences in total number of amacrine cells or concentration of amacrine cell synapses. This idea is supported by the enzyme activity found in retinas from the poikilotherms. Since the amacrine to bipolar synapse ratio in carp is 6 7 (WITKOVSKY & DOWLING, 1969), the ratio in goldfish, a close relative of the carp, may well be less than that in frog (Table 2), while the ChAc activity found in goldfish inner plexiform layer was 45 times greater than that in frog. Since different types of amacrine cells have been recognized histologically (C..uArd, 1972; KOLB& FAMIGLIETTI, 1974), it seems possible that only one of these may prove to be cholinergic. Thus, the apparent quantitative inconsktencies between the amacrine to bipolar synapse ratios and choline acetyltransferase activities could be explained by cholinergic amacrine cells contributing in large percentage to the total amacrine number in goldfish retina but to a much smaller percentage in frog retina. This suggestion is based on the assumption that the amount of enzyme per cholinergic amacrine cell is about the same in all species. It is well known that axons of cholinergic cells contain significant amounts of ChAc (HEBB, 1963; FON-
525
N m et a/., 1973), some or all of which is in transit from the cell bodies to the terminals (HEBB & WAITES, 1956; PARTLOW et al., 1972). Therefore, in view of the low ChAc activity in the fiber layer and optic nerve (Table l),ganglion cells are probably not tholinergic. Since samples of ganglion cell layer contain some neuropil in addition to ganglion cell bodies, the ChAc activity in this layer probably reflects the terminals of cholinergic neurons in this neuropil. It has been demonstrated in pigeon that centrifugal fibers from the isthmo-optic nucleus terminate around amacrine cell bodies near the inner plexiform layer (DOWLING & COWAN, 1966). Cholinesterase staining studies suggest these fibers may be cholinergic (NICHOLS & KOELLE, 1968). The extent to which these terminals contribute ChAc activity to samples of inner nuclear and inner plexifom layers is not known, but the number of synapses in pigeon from the centrifugal fibers is very small with respect to the number of amacrine synapses (MATURANA & FRENK, 1965; YAZULLA, 1974). Centrifugal fibers like those in pigeon have not been demonstrated in retinas from the other species used in this study. The low ChAc activity in the photoreceptor and outer plexiform layers from all species tested suggests the photoreceptor and horizontal cells are not cholinergic. The enzyme activity found in photoreceptors was estimated to be no more than 0.1-050/, of the total retinal ChAc. The activity is comparable to that found in other sensory systems. The activity in photoreceptors of frog, for instance, was not significantly different from that in spinal sensory root (PARTLOW et al., 1972). The enzyme activity in the inner plexiform layer of most animals, however, was very high compared to most other nervous systems regions, including caudate nucleus and spinal motor root (HEBB & SILVER, 1956) and interpeduncular nucleus (KATAOKA et aI., 19'73). The values reported here correlate well with those from other studies. The ChAc activity in mouse whole retina, calculated by adding the proportionate contribution of each layer (Table la), was estimated to be 12.2mmol/kg dry wt per h. After allowing for a difference in temperature of incubation ( x 3), this value is indistinguishable from the 37.8 & 0.8 mmolfig dry wt per h, found in homogenate of mouse whole retina (Ross et al., 1975). The value for ChAc activity in turtle photoreceptor layer (Table lb) agrees with that found by Lam for isolated turtle photoreceptors without endings (LAM,1972). The relative distribution of ChAc in retinal layers of pigeon, rat, and frog, the differences in enzyme activity between these species, and the absolute activities are similar to those found by GRAHAM(1974). The highest enzyme activity was found by Graham to be in the inner plexiform layer and the highest activity within the inner nuclear layer to be in the inner-most half. ChAc activity in the photoreceptor layer of mudpuppy was about one-third of the value for inner plexiform layer, in contrast to the extremely
526
C. D. Ross and D. B. MCDOUGAL, JR.
HEBBC. 0. & WAITESG. M. H. (1956) J . Physiol. Lond. 132, 667-671. HEBBC. 0. & SILVERA . (1956) J . Physiol. Lond. 134, 71&728. KATAOKA K., NAKAMURA Y. & HASSLERR. (1973) Brain Res. 62, 264-267. KOLBH. & FAMIGLIETTI E. V. (1974) Science, N.Y. 1% 4749. LAMD. M. K. (1972) Proc. natn. Acad. Sci. U.S.A. 69, 1987-1991. LINDEMAN V. F. (1947) Am. J . Physiol. 148, 4 M 4 . LOWRY0. H., ROSEBROUGH N. J., FARR A. L. & RANDALL Acknowledgements-Appreciation is expressed t o Drs. A. R. J. (1951) J . biol. Chem. 193, 265-275. COHENand 0.LOWRY for their encouragement and helpful LOWRY 0. H., ROBERTS N. R. & LEWISC. (1956) J . biol. suggestions. Chem. 220, 879-892. LOWRY 0. H. & PASSONNEAU J. V. (1972) A Flexible SysREFERENCES tem for Enzymatic Analysis. Academic Press, New York. AMES A. & POLLEND. A. (1969) J . Neurophysiol. 32, MARSHALLJ. & VOADEN M. (1975) Vision Res. 15,459-460. 424-442. MATSCHINSKY F. M., PASSONNEAU J. V. & LOWRY 0. H. ANFINSEN C. B. (1944) J . biol. Chem. 152, 267-278. (1968) J. Histochem. Cytochem. 16, 29-39. BROWNJ. E. & ROJASJ. A. (1965) J . Neurophysiol. 28, MATSCHINSKY F. M. & MCDOUGAL D. B. (1968) in Pro1073-1090. gress in Clinco-Chemical Methods, p p . 71-86. Karger, CAJALS. RAMONY (1972) The Structure of the Retina. New York. Thomas, Springfield. MATURANA H. R. & FRENK S. (1965) Science, N.Y. 150, DOMINGJ . E. (1970) Invest. Ophthal. 9, 655680. 359-361. DOWLING J. E. & COWANW. M. (1966) Z . Zellforsch mikMCCAMANR. E. & HUNTJ. M. (1965) J . Neurochem. 12, rosk. Anat. 71, 14-28. 253-259. DUBINM. W. (1970) J . comp. Neurol. 140, 470-506. NACHMANSOHN D. (1959) Chemical and Molecular Basis of DUBINM. W. (1974) in The Eye, Vol. 6. Comparative PhysiNerve Activity. Academic Press, New York. ology (DAVSONH. & GRAHAML. T., JR., eds.) pp. NICHOLSC. W. & KOELLEG. B. (1968) J . comp. Neurol. 227-256. Academic Press, New York. 133, 1-16. EHINGER B. (1972). Brain Res. 46, 297-311. EHINGER B. & FALCK B. (1969) Z . Zellforsch mikrosk. Anat. NICHOLSC. W., HEWTTJ. & LATIESA. M. (1972) J . Histochem. Cytochem. 20, 130-136. 100, 364-375. L. M., Ross C. D., MOTWANI R. & MCD~UGAL EHINGERB. & FALCK B. (1971) Brain Res. 33, 157-172. PARTLOW D. B., JR. (1972) J . gen. PhysioI. 60, 388-405. ESILAR. (1963) Acta Ophthalmologica Supp. 77, 9-113. M. (1971) J . Histochem. REALEE., LVCIANO L. & SPITZNAS FELDBERG W. (1945) Physiol. Rev. 25, 596-642. Cytochem. 19, 85-96. FELDBERG W. & MA" T. (1946) J . Physiol. Land. 104, Ross C. D. & MCDOUGALD. B., JR. (1974) Fedn Proc. 41 1425. Fedn Am. SOCSexp. Biol. 33, 477. FONNUM F. (1969) Biochem. J. 113, 291-298. D. B., JR. (1975) FONNUM F., FRIZELL M. & SJ~STRAND J. (1973) J . Neuro- Ross D., COHENA. I. & MCDOUGAL Invest. Ophthal. 14, 756761. chem. 21, 1109-1120. GLOVERV . A. S. & POTTERL. T. (1972) J . Neurochem. SILVERA. (1967) Int. Rev. Neurobiol. 10, 57-109. SOSULAL. & GLOWP. H. (1970) J . comp. Neurol, 140, 18, 571-580. 435478. GRAHAM L. T., JR. (1972) Brain Res. 36, 476-479. VON BREDOW J., BAYE. & ADAMSN. (1971) Expl Neurol. GRAHAM L. T., JR. (1974) in The Eye, Vol. 6. Comparative 33, 45-52. H. & GRAHAM L. T., JR., eds.) pp. Physiology (DAVSON WHITE H. L. & WU J. C. (1973) Biochem. J . 12, 841-846. 283-342. Academic Press, New York. WITKOVSKY P. & DOWING J. E. (1969) Z . Zellforsch. mikHEBBC. 0. (1957) Physiol. Rev. 37, 196-220. rosk. Anat. 100, 6Ck82. HEBBC. 0. (1963) in Cholinesterase and Anticholinesterase S. (1974) J. comp. Neurol. 153, 309-324. Agents, Handbook of Experimental Pharmacology YAZULLA (KOELLE G. B., ed.) Vol. XV pp. 5688. Springer, Berlin.
low activity found in photoreceptors in the three other species studied by both Graham and us and in the seven additional species in this study. Graham found no obvious correlation between ChAc activities in the different animals and retinal structure or function. The data from our study, however, strongly suggest that the high ChAc activity in the innermost part of the inner nuclear layer and in the inner plexiform layer of the retina is associated with cholinergic amacrine cells.
THE DISTRIBUTION OF CHOLINE ACETYLTRANSFERASE ACTIVITY IN VERTEBRATE RETINA' C. D. Ross2 and D. B. MCDOUGAL, JR. Department of Pharmacology, Washington University School of Medicine, St. Louis, M O 631 10, U.S.A. (Received 7 May 1975. Accepted 4 August 1975) Abstract-Choline acetyltransferase (ChAc) activity was determined in retinal layers from 10 vertebrates. In all animals, the highest activity was in the inner plexiform layer, intermediate activity in the inner nuclear and ganglion cell layers, and very low activity in the photoreceptor and outer plexiform layers and optic nerve. The pattern of distribution of enzyme activity within the inner nuclear layer corresponds quantitatively to the distribution of amacrine cells within that layer. A species difference of almost 90-fold was found between the lowest and highest values for ChAc activity in inner plexiform layer. The variation in enzyme activity found among homeotherms in inner nuclear and inner plexiform layers is related to the number ofamacrine cell synapses in the inner plexiform layer. But the differences in enzyme activity are generally greater than those which have been found in numbers of amacrine cell synapses between species. The data suggest that cholinergic neurons in retina are to be found predominantly among the amacrine cell types and that not all amacrine cells will bc found to be cholinergic.
IT HAS been suggested for a number of years that @LA, 1963; NICHOLS & KOELLE,1968; REALErt aE., acetylcholine might be involved in neural transmis- 1971; NICHOLSet al., 1972). Following complete inhision in the retina. Application of ACh evokes electri- bition of AChE by diisopropyl Auorophosphate, syncal responses from retinal cells, which can be blocked thesis of new cnzyme was observed first in amacrine by ACh antagonists (AMES & POLLEN, 1969; VON BRE- cells (NICHOLS & KOELLE,1968). DOW eta]., 1971). The retinas of most animals contain One possible interpretation of results from AChE significant amounts of ACh (FELDBERG histochemical studies is that some of the cells, most & MA", 1946; LINDEMAN,1947), choline acetyltransferase likely amacrine, which form synapses in the inner (ChAc, acetyl-CoA: choline 0-acetyltransferase, EC plexiform layer are cholinergic. However, conclusions 2.3.1.6; HEBB& SILVER, 1956), and acetylcholinester- that cells are cholinergic based only on the fact that ase (AChE, EC 3.1.1.7; ANFINSEN,1944; NACHMAN- they contain AChE must be tentative, since non-choSOHN, 1959). A review has appeared recently dealing linergic neurons may also contain this enzyme (HEBB, et al., 1972). Identificawith some comparative aspects of neurotransmitters, 1957; SILVER,1967; PARTLOW tion of the cholinergic neurons in retina as amacrine including ACh, in the retina (GRAHAM,1974). Most attempts to identify cholinergic cells in the cells is further obscured by evidence that other putaretina have involved the localization of cholinesterase tive neurotransmitters, such as dopamine (EHINGER 1972; activity by histochemical staining procedures. The & FALCK, 1969, 1971) and GABA (GRAHAM, & VOADEN,1975) have product of the histochemical reaction is consistently EHINGER,1972; MARSHALL associated with amacrine cells and ganglion cells also been attributed to amacrine cells. Since specific cell types and synaptic regions are and with their processes in the inner plexiform layer concentrated in strata or layers within the retina, the problem of localization of the enzymes of neurotransPreliminary reports of this study were presented to the mitter metabolism can be approached using the tech58th Annual Meeting of the Federation of American Socieniques of quantitative histochemistry ( L o m y et al., ties for Experimental Biology (Ross & M c D o u ~ u ,1974) 1956; MATSCHMSKY & MCDOUGAL, 1968). Of the two and to the 1974 Meeting of the Association for Research enzymes involved in the metabolism of ACh, ChAc in Vision and Ophthalmology. Supported by Grants NS06800, EY-00258, and NS-05221 of the National Institutes and AChE, ChAc is considered the more reliable indicator of the presence of cholinergic mechanisms of Health. 1945). * Present address: Department of Anatomy, Washington (HEBB,1957, 1963; SILVER,1967; FELDBERG, The purpose of this study was to determine the University School of Medicine, 660 So. Euclid Ave., St. activity of ChAc in layers of retina from various verteLouis, MO 63110, U.S.A. Abbreviations used: ChAc, choline acetyltransferase; Na brate species in an effort to determine which cell type(s) in the retina might be cholinergic. TPB, sodium tetraphenylboron. 52 1
522
JR. C. D. Ross and D. B. MCDOUGAL,
METHODS Protein was determined by the Lowry method ( L O W Y Anulytical methods et al., 1951). Tissue handling. Histologically defined samples, ranging Experimental materials in dry weight from 0.03 to 2 pg, were dissected from frozenEyes from mice, turtles, goldfish, and pigeons were prodried sections cut tangentially to the eyeballs of several, poikilothermic and homeothermic vertebrates (LOWRY ' vided by Dr. Adolph Cohen, Department of Ophthalmology. Frozen-dried sections from monkey and rabbit a/., 1956) and weighed on a quartz-fiber balance (LOWRY retinas were provided by Dr. Oliver Lowry. Rats were ob& PASSONNEAU, 1972). tained from National Laboratories, St. Louis, Mo., frogs Et1zyrne and protein assays. The assay of ChAc was based on the radiometric method developed by MCCAMAN from J. R. Schettle Biologicals, Oshkosh, Wisconsin, and & HUNT(1965), using the sodium tetraphenylboron (Na garfish from E. Saeugling, Gutenberg, Iowa. Cats were obTPB) procedure of FONNUM(1969) for extraction of tained from a local supplier. [1-'4C]acety1-CoA (46-54 mCi/mmol) and [I-l4C]ace[1-14Clacetylcholine. Samples were incubated in oil tylcholine (2-5 mCi/mmol) were obtained from New Enget ul., 1968; LOWRY & PASSONNEAU, wells (MATSCHINSKY 1972) for 45-60 min at 25°C in 2 pl of a reagent containing land Nuclear. 75 p~-[l-~~c]acetyl-CoA, 6 mM-choline bromide, 250 m ~ - N a C l , 0.1% Triton x-100, 0.08 mM-neostigmine RESULTS methylsulphate, 0.1% bovine serum albumin, 110 mMThe patterns of distribution of ChAc activity sodium phosphate buffer, pH 7.6. After incubation, the aqueous droplet was removed and added to 5 pl 10mM- among retinal layers were similar for all vertebrates sodium phosphate pH 7.8 in a 400 pl plastic centrifuge tube, studied (Table la and b, Fig. 1). Almost all the enwhich was placed in an ice bath. Forty-five p1 of Na TPB zyme activity was confined to the inner retinal layers, (15 mg/ml) in 3-heptanone was added, the contents mixed with the highest activity being in the inner plexiform well,and layers separated by centrifugation (Beckman Micro- layer. The activity in the outer and inner segments fuge model 152). The organic phase was removed and and nuclei of the photoreceptors was very low comadded to another tube containing 1M)pl Na TPB pared to the value for inner plexiform layer. The ratio (0.5 mg/ml) in 10 mM-sodium phosphate buffer pH 7% of enzyme activity between inner plexiform and After mixing and centrifugation, a 38 p1 aliquot of the or- photoreceptor layers was at least loo0 in mouse, rat, ganic layer was removed and added to a scintillation vial cat, pigeon, garfish, and goldfish, about 500 in turtle, containing 0.4% 2,5-diphenyloxazole (PPO) and 001% p50 in frog. The activities rabbit, monkey, and about bis-[2-(4-methyl-5-phenyloxazoyl)] benzene (dimethyl of the in the outer plexiform layer, the outer third POPOP) in toluene with 0.1 ml hyaminebhydroxide added to each 8 ml volume. The blank, including background, for inner nuclear layer, and the optic nerve were also the ChAc assay was about 50 c.p.m. at 91% counting effi- very low relative to the inner plexiform layer. ChAc activities showed a graded increase within ciency, equivalent to 0.55pmol or 055 mmol/kg dry wt per h for a 1 pg sample incubated 1 h. The reaction rate the inner nuclear layer, with the lowest value being was linear for at least 1 h. Sample sizes and incubation in the outer third and highest value in the inner third times were adjusted with activity so that AcCoA concen- next to the inner plexiform layer. Because the retinas tration was not limiting. were cut tangentially, invaginations of inner plexiform The assay of ChAc activity in homogenates was similar layer into inner nuclear layer were easy to see in the except that the volumes used during incubation, extraction, and washing were increased, the incubation was done in frozen-dried sections and were dissected away from plastic tubes, and the incubation medium was not covered the inner nuclear samples. It is estimated that not more than 10% of the enzyme activity ascribed to with oil (Ross et al., 1975). No enzyme activity was detected when choline was omit- the inner third of inner nuclear was due to inner plexted from the incubation medium. The product formed dur- iform layer contamination. ing incubation with both substrates was demonstrated to Although the relative distribution of ChAc activity be ACh by the addition of AChE (electric eel), which re- between retinal layers was similar, a significant differduced the Na TPB-heptanone extractable radioactivity to ence was found in the absolute activities in inner nuG blank value. The possibility of synthesis of ACh by carni- lear and inner plexiform layers. The highest activity tine acetyltransferase, however, cannot be excluded (WHITE for inner plexiform layer among the homeotherms & WU, 1973). This enzyme could be significant in samples (Fig. 2) was found in pigeon, intermediate values were with low synthetic activity. found in rat, rabbit, and mouse, and the lowest activiNo significant differences were found among Km,AeCaA) values for ChAc in homogenates of frozen-dried tissues ties were in monkey and cat. The ChAc activity in of four species. Individual figures were 1 5 p ~(mouse pigeon inner plexiform layer was 35 times that in cat. retina), 12 p~ (goldfish retina), 14 p~ (turtle retina), and An even greater range in ChAc activity was found 17 p~ (rat cerebral cortex). These values are comparable among the poikilotherms (Fig. 3). Activity in goldfish values obtained for ChAc: 10 PM (bovine inner plexiform layer was the highest of all the anito other K,,,(A~C,,A) striate nuclei, GLOVER & POTTER,1972), 10-25 p~ (rabbit mals tested, almost 45 times the value for frog and & HUNT, 1965), and 8 p~ (cat brain, D. brain, MCCAMAN twice the value for pigeon. GODFREY, personal communication). The ChAc activity in homogenates of mouse retina inDISCUSSION creased between 25" and 38°C by almost 3-fold. No detectThe consistency in the pattern of distribution of able loss in enzyme activity occurred during the freeze-drying process. ChAc in the retinas of 10 species from 9 orders and
Choline acetyltransferase in vertebrate retina
TABLE la. ChAc ACTIVITY IN RETINAL
Outer and inner photoreceptor segments Photoreceptor nuclei Outer plexiform layer Inner nuclear layer (outer third)
1
LAYERS FROM FOUR HOMEOTHERMS
Cat
Monkey
0 (5)
0'06' (12)0'01
0 (2)
0.013 I 0.01 (10) 0.12 k 0.01 (8)
*
0 (1)
0.267
& 0.17
0 (6)
(middle third)
523
0438 i 0.3
}
Mouse
O.O43(&0Ql
Rabbit
0.121 i 0.09 0.012
0 (5)
*
(8)
0.01
(4) 0.39 (2)
0.256 f 0.08 (12) 6.93 1.0
(inner third) Inner plexiform layer Ganglion cell layer Fiber layer Optic nerve
(4) 3.61 t 0.52
(3) 9.53 i0.98
1.16
6.87 k 0.89 (5) 1.08 (2) -
0.23 (5) -
TABLElb. ChAc ACTIVITYIN
RETINAL LAYERS FROM POIKILOTHERMS
Frog Outer and inner photoreceptor segments Photoreceptor nuclei Outer plexiform layer Inner nuclear layer (outer third) (middle third)
(inner third)
(7) 40.4 k 1.4 (67) 23.9' +'13 (15) 0.36 k 0.5 (14)
Turtle
0.055
+ 0.02
1.90 k 0.44 (4) 5.78 f 037 (10) 21.6 f 4.7
0225 f 006 (7) 1.71 f 0.4 (6) 5.60 k 1.2
Garfish
Goldfish
0.01 i 0.033 .
13.2 & 3.9 (7) 53.8 k 4.5 (6) 125.0 k 12.0
Inner plexiform layer Ganglion cell layer Fiber layer Optic nerve
-+
Values are expressed in mmol/kg dry wt per h at 25°C S.E.M. Numbers in parentheses indicate number of samples assayed. Samples were dissected from frozen-dried sections from 2 eyes from 1 cat, 1 eye from 1 monkey, 12 eyes from 10 mice, 1 eye from 1 rabbit, 4 eyes from 2 frogs, 5 eyes from 3 turtles, 2 eyes from 2 garfish, and 3 eyes from 2 goldfish. Since no significant differences were found in ChAc activities between black Cg7BL(6J)and albino mice, the data for pigmented and nonpigmented animals were pooled. The photoreceptor layer was divided into segments (inner and outer) and nuclei in all species except frog and mouse. The inner nuclear layer was divided into sublayers in all species except garfish. ChAc activity in undivided inner nuclear layer of garfish (24% of inner plexiform layer) compares well with undivided inner nuclear layers from rat (21%) and from turtle (26%), and with the average of inner nuclear layer subdivisions in goldfish (20%).
6 classes of vertebrates is quite remarkable (Fig. 4), especially when it is considered that the absolute range of activities in inner plexiform layer is nearly 90-fold. This suggests that the distribution of a single cell type and its processes may be responsible for the major portion of the pattern seen. TWOlines of evidence from this study suggest that the cholinergic neurons in retina are predominantly
among the amacrine cell types. First, the pattern of increase in ChAc activity within the inner nuclear layer (Table 1, Fig. 1) follows the distribution of amacrine cell bodies found histologically (CAJAL, 1972). Amacrine cell terminals are distributed throughout the inner plexiform layer, and a few even extend into the ganglion cell layer. Second, the variation in ChAc activity among species resembles the variation
C. D. ROSSand D. B. MCDOUGAL,JR.
524
0
.= .
3 160-
h\q
f 1400
m
120-
+GARFISH
TURTLE
100-
-g 80-
p FROG
r
0
u)
I
60-
c21 0 0
I
80
I
120
I
180
I
200
I
240
I
280
I
320
ChAc activity (mmolcs/kg dry wt./hr,25"C)
.2 40c
2
I
40
20-
0 0-
Rs Rn
FIG. 1. Distribution of ChAc activity in retinal layers of pigeon (left) and rat (right). The pattern of distribution of ChAc activity is illustrated for the 2 animals in which the inner nuclear layer was divided into 6 sublayers. ACtivity is expressed as mmol/kg dry wt per h at 25°C. Vertical lines denote +s.E.M. Samples were dissected from 8 eyes from 4 rats and 6 eyes from 3 pigeons. Since no significant differences were found in ChAc activities between brown or albino rats, the data were pooled. Abbreviations are Rs (outer and inner photoreceptor segments), Rn (photoreceptor nuclei), OP (outer plexiform layer), oIN, mIN, iIN (outer, middle and inner thirds respectively of inner nuclear layer), IP (inner plexiform layer), GC (ganglion cell layer), F (fibre layer), and OpN (optic nerve). The space representing the photoreceptors in the figure is proportionately smaller than that occupied in a retinal cross section.
in the number of amacrine cell synapses in the inner plexiform layer. Visual information in vertebrate retinas is transmitted from the photoreceptors through the bipolar cells to the ganglion cells for transfer to other brain centers. This information may be modulated in the inner plexiform layer by lateral interactions between amacrine cells (DOWING,1970). These interactions allow some processing of visual information, particularly the detection of movement, to occur at the retinal level. There is a variety of synaptic arrangements between bipolar, amacrine, and ganglion cells in the
\\\\
PIGEON
inner plexiform layer (DOWING, 1970; DUBIN,1974). A bipolar cell may form a synapse on both an amae rine and a ganglion cell, thereby providing direct input to the ganglion cell. Or a bipolar cell may contact only amacrine cells, which then make synaptic contact with the ganglion cell. In the latter case, amacrine cells, rather than bipolars, provide the direct input to the ganglion cells. The incidence of these two types of synaptic arrangements varies from one species to another. Those species exhibiting a high degree of involvement of amacrine cells in the input to the ganglion cells also have a high density of amacrine synapses in the inner plexiform layer (DUBIN,1970). While the density of amacrine synapses varies significantly from one species to another, the density of bi1970, 1974). The ratio polar synapses does not (DUBIN, of the incidence of amacrine synapses to bipolar synapses for each species (Table 2) affords a convenient way to indicate the variation in both the density of amacrine synapses in the inner plexiform layer as well as the involvement of amacrine cells in gang-
% 60-
r+ RABBIT
4-
FIG.3. Comparison of ChAc activity in inner nuclear and inner plexiform layers from poikilotherms. The total bar represents activity in inner plexiform layer +s.E.M. The striped portion of the bar represents activity in the inner nuclear layer, either the innermost third (goldfish, turtle, and frog) or the entire layer (garfish) +s.E.M. Data are from Table 1.
MOUSE
I-
40-
W 0
30-
z
MONKEY
Ra Rn OPdNmlNirJ
@CAT
I
I
I
I
I
I
I
I
I
0
20
40
60
80
100
120
140
I60
ChAc aclivily(rnrnoles/kg dry *l./hr,25'Cl
FIG.2. Comparison of ChAc activity in inner nuclear and inner plexiform layers from homeotherms. The total bar represents activity in inner plexiform layer _+S.E.M. The striped portion of the bar represents activity in the innermost sixth (pigeon, rat; data from Fig. 1) and innermost third (rabbit, mouse, monkey, and cat; data from Table 1) bf the inner nuclear layer +s.E.M.
IP GC F OpN
FIG.4. Choline acetyltransferase activity in retinal layers from 10 vertebrates expressed relative to the activity in the inner plexiform layer (= 100%). The average of these percentage values is represented for each retinal layer +s.E.M. Abbreviations are defined in legend with Fig. 1. Percentages were calculated from absolute values in Table 1 and Fig. 1. Cell types in the diagram representing retinal neural connections are (from left to right) photoreceptor, horizontal, bipolar, amacrine, and ganglion cells.
Choline acetyltransferase in vertebrate retina TABLE2. INCIDENCEOF
AMACRINE:BIPOLAR SYNAPTIC ELE-
MENTS IN INNER PLEXIFORM LAYER
monkey cat rat rabbit pigeon frog
Dubin (1970)
Sosula & Glow (1970)
1.9-2.6 2.6-3.0 3.3
8
5.1-5.3 10.8
8.8-109
lion Cell input (DUBIN,1970, 1974). Among the homeotherms, monkey and cat have low ratios of amacrine synapses to bipolar synapses; rabbit has an intermediate ratio; pigeon has a high ratio. This arrangement of species resembles the order of relative activities of ChAc. A possible inconsistency in this correlation is the rank for rat, which had higher ChAc activity in inner plexiform layer, but a lower ratio of amacrine to bipolar synapses, than did rabbit. Although SOSULA & GLOW(1970) reported a higher ratio than that found by DIJBIN (1970), results from electrophysiological studies are more consistent with a relatively low incidence of amacrine cell synapses (BROWN& ROJAS,1965). Further study will be needed to resolve this issue. Since the density of bipolar synapses is relatively constant from species to species, while the ChAc activity varied over a 35-fold range, it seems unlikely that the bipolar cells are cholinergic. The variation of ChAc activity in inner plexiform layer between homeotherms (35-fold) was much greater than the variation in amacrine to bipolar synapses ratio (4- to 5-fold). This lack of quantitative correspondence affords a clue that the differences in ChAc activity reflect more than simply the differences in total number of amacrine cells or concentration of amacrine cell synapses. This idea is supported by the enzyme activity found in retinas from the poikilotherms. Since the amacrine to bipolar synapse ratio in carp is 6 7 (WITKOVSKY & DOWLING, 1969), the ratio in goldfish, a close relative of the carp, may well be less than that in frog (Table 2), while the ChAc activity found in goldfish inner plexiform layer was 45 times greater than that in frog. Since different types of amacrine cells have been recognized histologically (C..uArd, 1972; KOLB& FAMIGLIETTI, 1974), it seems possible that only one of these may prove to be cholinergic. Thus, the apparent quantitative inconsktencies between the amacrine to bipolar synapse ratios and choline acetyltransferase activities could be explained by cholinergic amacrine cells contributing in large percentage to the total amacrine number in goldfish retina but to a much smaller percentage in frog retina. This suggestion is based on the assumption that the amount of enzyme per cholinergic amacrine cell is about the same in all species. It is well known that axons of cholinergic cells contain significant amounts of ChAc (HEBB, 1963; FON-
525
N m et a/., 1973), some or all of which is in transit from the cell bodies to the terminals (HEBB & WAITES, 1956; PARTLOW et al., 1972). Therefore, in view of the low ChAc activity in the fiber layer and optic nerve (Table l),ganglion cells are probably not tholinergic. Since samples of ganglion cell layer contain some neuropil in addition to ganglion cell bodies, the ChAc activity in this layer probably reflects the terminals of cholinergic neurons in this neuropil. It has been demonstrated in pigeon that centrifugal fibers from the isthmo-optic nucleus terminate around amacrine cell bodies near the inner plexiform layer (DOWLING & COWAN, 1966). Cholinesterase staining studies suggest these fibers may be cholinergic (NICHOLS & KOELLE, 1968). The extent to which these terminals contribute ChAc activity to samples of inner nuclear and inner plexifom layers is not known, but the number of synapses in pigeon from the centrifugal fibers is very small with respect to the number of amacrine synapses (MATURANA & FRENK, 1965; YAZULLA, 1974). Centrifugal fibers like those in pigeon have not been demonstrated in retinas from the other species used in this study. The low ChAc activity in the photoreceptor and outer plexiform layers from all species tested suggests the photoreceptor and horizontal cells are not cholinergic. The enzyme activity found in photoreceptors was estimated to be no more than 0.1-050/, of the total retinal ChAc. The activity is comparable to that found in other sensory systems. The activity in photoreceptors of frog, for instance, was not significantly different from that in spinal sensory root (PARTLOW et al., 1972). The enzyme activity in the inner plexiform layer of most animals, however, was very high compared to most other nervous systems regions, including caudate nucleus and spinal motor root (HEBB & SILVER, 1956) and interpeduncular nucleus (KATAOKA et aI., 19'73). The values reported here correlate well with those from other studies. The ChAc activity in mouse whole retina, calculated by adding the proportionate contribution of each layer (Table la), was estimated to be 12.2mmol/kg dry wt per h. After allowing for a difference in temperature of incubation ( x 3), this value is indistinguishable from the 37.8 & 0.8 mmolfig dry wt per h, found in homogenate of mouse whole retina (Ross et al., 1975). The value for ChAc activity in turtle photoreceptor layer (Table lb) agrees with that found by Lam for isolated turtle photoreceptors without endings (LAM,1972). The relative distribution of ChAc in retinal layers of pigeon, rat, and frog, the differences in enzyme activity between these species, and the absolute activities are similar to those found by GRAHAM(1974). The highest enzyme activity was found by Graham to be in the inner plexiform layer and the highest activity within the inner nuclear layer to be in the inner-most half. ChAc activity in the photoreceptor layer of mudpuppy was about one-third of the value for inner plexiform layer, in contrast to the extremely
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C. D. Ross and D. B. MCDOUGAL, JR.
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low activity found in photoreceptors in the three other species studied by both Graham and us and in the seven additional species in this study. Graham found no obvious correlation between ChAc activities in the different animals and retinal structure or function. The data from our study, however, strongly suggest that the high ChAc activity in the innermost part of the inner nuclear layer and in the inner plexiform layer of the retina is associated with cholinergic amacrine cells.
