(Accepted April 13th, 1977). SUMMARY. The biochemical properties and morphological localization of the cholinergic system in the chicken retina were studied ...
Brain Research, 138 (1977)469-485
469
© Elsevier/North-HollandBiomedicalPress
BIOCHEMICAL CHARACTERIZATION AND CELLULAR LOCALIZATION OF THE CHOLINERGIC SYSTEM IN THE CHICKEN RETINA
ROBERT W. BAUGHMAN and CHARLES R. BADER Department o f Neurobiology, Harvard Medical School, Boston, Mass. 02115 (U.S.A.)
(Accepted April 13th, 1977)
SUMMARY The biochemical properties and morphological localization of the cholinergic system in the chicken retina were studied with the following results. (1) The uptake of [3H]choline saturated with increasing choline concentration, and could be accounted for by the presence of two saturable processes, a 'high affinity' system with a Km of 1.1/~M and Vmax of 8.0 pmole/min/mg protein, and a 'low affinity' system with a Km of 214/~M and Vmax of 578 pmole/min/mg protein. (2) Following an incubation with 5 × 10-7 M Jail]choline for 10 min, the intracellular composition of labeled products consisted of 44 ~ acetylcholine, 46 ~o choline and 10 ~ phosphorylcholine. (3) Incubation in the presence of 5 × 10-5 M hemicholinium-3 or in the absence of sodium blocked synthesis of acetylcholine and reduced the total uptake of [3HIcholine by an amount which could be accounted for by blockage of the high affinity system. (4) Labeled acetylcholine synthesized from [aH]choline could be released by increasing the extracellular potassium concentration, and this release was calcium dependent. (5) By the use of freeze drying and autoradiography with a dry emulsion film, the [3H]choline taken up was found to be concentrated ill 6 ~ of the cell bodies in the inner nuclear layer, in 19 ~ of the cell bodies in the ganglion cell layer and in two bands in the inner plexiform layer. (6) When autoradiography was repeated following an incubation carried out in the presence of hemicholinium-3, no localization of aH uptake was observed. (7) Following an incubation with 5/~M [aH]choline, the total uptake of choline and the contributions of the high and low affinity uptake systems were estimated from an autoradiograph by grain counting, and the values obtained closely matched those found with the biochemical methods.
470 INTRODUCTION Considerable evidence suggests that acetylcholine (ACh) may be a neurotransmitter in the vertebrate retina2,14,1G,21,25,2s,30,40. The localization of the cholinergic system has been the object of numerous studies. Investigations based on assays of sections cut horizontally through the retina indicate that in several species choline acetyltransferase and acetylcholinesterase are concentrated in and close to the inner plexiform layer14,33. This agrees with histochemical studies of cholinesterase staining which showed localization of reaction products within and on either side of the inner plexiform layerZg, 35. Similarly, binding of l'~al labeled a-bungarotoxin was strongest in the inner plexiform layer, although some binding also was present in the outer plexiform layer40, 44. The cholinergic cells, however, have been neither visualized nor identified. The present study was undertaken to try to localize directly cholinergic cells in the chicken retina by visualizing autoradiographically the cells that take up [3H]choline by a high affinity transport mechanism. A similar approach has been used previously to localize cells that take up amino acids and catecholamines m the vertebrate central nervous system and retina 10,11,12,18. Since both choline and ACh are water soluble molecules that cannot be fixed with standard techniques, a procedure involving freeze drying and autoradiography with dry emulsion film was used 4,27,42. As a prerequisite to this study, the uptake systems for choline were characterized biochemically. In addition, the synthesis of [3H]ACh from pH]choline and the release of [aH]ACh were investigated. MATERIALS AND METHODS Five-week-old, light adapted, White Leghorn chicks were decapitated and one eye was quickly removed and transferred into a 37 °C salt solution (solution A, Table I) bubbled with a CO2-air mixture (pH 7.6). The eye was hemisected at the equator, and the anterior half and the vitreous were removed. A strip of retina, approximately 2 m m × 8 mm, free from pigment epithelium, was dissected from the inferior half of the retina. The long axis of the retinal strip was perpendicular to the pecten axis. The strip was further sectioned into 4 pieces of about 1 mg wet weight (100/~g protein) each. The pieces then were preincubated in 1 ml of choline free medium with gentle shaking for 10 rain at 37 °C in a Lucite chamber gassed with a water saturated COz-air mixture adjusted to give a pH of 7.6. The medium consisted of either choline-free MEM (Gibco) prepared without glutamate or glutamine in view of the deleterious effect of glutamate on some retinal neurons aI, or a salt solution (see Table I). In some experiments, hemicholinium-3 (Aldrich) was added to the incubation medium. Following the preincubation, 1 ml of medium containing [3H]choline (Amersham Searle; 10.1 Ci/mmole) and unlabeled choline chloride in the amounts used for the various experiments was substituted. After incubation, tissues were briefly rinsed twice with choline-free medium, and were either freeze-dried for autoradiography or homogenized for biochemistry.
1.8
1.2
6.1
140.6 140.6 149.6 149.6 156.9 1.0
1.0 1.0 1.0 1.0 1.0 --
24.0 24.0 24.0 24.0 -1.2
-----10.0
----10.0
256
__ __ __ __ --
10.0
5.0 5.0 5.0 5.0 10.0
Glucose
--
0.01" 0.01 0.01 0.01 --
**
0.005* 0.005 0.005 0.005 **
Neostigmine Choline bromide chloride
** A m i x t u r e o f J a i l ] c h o l i n e a n d u n l a b e l e d c h o l i n e a p p r o p r i a t e t o r e a c h t h e d e s i r e d c o n c e n t r a t i o n a n d s p e c i f i c a c t i v i t y w a s a d d e d p r i o r t o e a c h u p t a k e incubation.
* Neostigmine bromide and choline chloride were included only for the ACh release experiments.
2.5
1.2 1.2 20.0 20.0 1.2
S042-
--
1.8 1.8 --1.8
HCOa
F
2.5 25.0 2.5 25.0 2.5
HAP04-
157.1 134.6 130.6 108.6 149.4
CI-
A B C D E
M g 2+
Tris base Sucrose
Ca 2+
Na +
Solution
K+
are given in mmole/liter.
I
T h e s o l u t i o n s w e r e b u b b l e d w i t h a C O ~ - a i r m i x t u r e t o r e a c h a p H o f 7.6. A l l c o n c e n t r a t i o n s
Solutions
TABLE
4~
472 Biochemistry After rinsing, each piece of retina was transferred into a 1 ml microhomogenizer (Kontes) to which were added 100 #1 of a solution containing 0.5)/o SDS, 0.1 N formic acid, and 10-4 M neostigmine bromide (Sigma). Tissue homogenates were centrifuged at 1720 × g for 5 min. Aliquots of the supernatant were taken for scintillation counting and protein determinationZL The pellet, which included lipid bound choline, such as phosphatidylcholine, contained less than 2~o of the total counts. When the distribution of labeled compounds in the supernatant was studied, 50 #1 of a solution containing ACh (20 mg/ml) and choline (40 mg/ml) in 2 N formic acid was added to 50 #1 supernatant. The mixture was centrifuged for 5 rain at 1720 × g and 20/~1 of the supernatant were spotted on a 1 mm Avicel cellulose thin layer plate (Analtech). The plate was developed with butanol-acetic acid-water (12:3:5). Iodine vapor was used to reveal the ACh and choline spots. The phosphoryl choline position was determined by the Rf (0.21-0.27) of a phosphoryl choline standard. The regions corresponding to phosphoryl choline, choline and ACh were scraped off the plate and the cellulose was soaked in 0.01 N HCI for 30 rain after which scintillation fluid (PCS (Amersham Searle)-xylene 2:1) was added. The samples were counted in a Packard scintillation counter with an efficiency of 3 5 ~ . Recovery of the radioactivity spotted was between 90 and 95 ~o. For kinetic analysis of choline uptake each piece of retina was incubated for 10 min in 1 ml of medium (solution E, Table I) containing 2/~Ci of [3H]choline (30 #Ci were used at 300 ~ M choline), and an appropriate amount of cold choline to reach the desired choline concentration. At the end of the incubation, the tissue was treated as described above. The total counts measured in the supernatant, normalized to 100 #g protein, were corrected for the counts from [3H]choline present in the extracellular space of the tissue. From the studies of choline uptake as a function of time, the extracellular space was estimated to be 37 ~o of the total volume of the retina, a value similar to that obtained in other retinas 1,13. At 2 #Ci/ml [3H] choline, thecorrection factor was 630 counts/rain/100/~g protein. In order to determine the values of the uptake kinetic parameters (Kin and Vmax), and to estimate the error in these parameters, a non-linear least squares curve fitting program was used. This program fitted the individual data with an equation corresponding to the sum of two MichaelisMenten equationsL The program, kindly provided by W. W. Cleland, was run in a modified form on a DEC PDP-10 computer. For release experiments a piece of retina was incubated for 20 min with 5 × 10-6 M [3H]choline, rinsed for 10 min with flesh medium (solution A, Table I) and transferred into another vial for the remainder of the experiment. In this vial, the piece of retina was rinsed for 5 min periods at 37 °C and pH 7.6 with 140 #1 of the solution to be tested (solutions A-D, Table 1). At the end of each 5 min period, 120 ~1 of the rinse solution were removed for analysis of [all]choline and [3H]ACh content, and an equal amount of flesh solution was added to the vial. During the experiment, care was taken to avoid mechanically damaging the piece of retina or exposing it to air.
473
A utoradiography The method for localizing the uptake of [aH]choline makes use of procedures described previously 4,27,42 with some modifications that were necessary to prevent diffusion of choline and ACh. Following incubation and rinsing, each piece of retina was frozen by quickly transferring it on a 2 mm × 5 mm piece of lens paper with a minimal amount of adhering medium to a propane slurry cooled with liquid nitrogen. After the excess paper was trimmed away, the tissue was maneuvered into a small aluminum dish which was transferred to a two stage thermoelectric cooling device (Cambion cat. no. 801-1004-01) in a bell jar containing 1-2 g of P205 desiccant. The cold stage was precooled to - - 7 0 °C and the transfer was made with liquid propane in the aluminum dish to prevent thawing. After residual propane was pumped off, the bell jar was evacuated to 10-4 torr and the cold stage was maintained at --70 °C for 36 h, at which time the temperature was increased to ambient gradually over 3-4 h. The system was then returned to atmospheric pressure by introduction of dry air via a CaClz drying tube, and the aluminum dish containing the tissue was quickly transferred to another glass vacuum system. There the tissue was fixed for 1 h in OsO4 vapor at 25 °C. The fixed tissue was embedded directly in fresh, degassed epoxy resin (13 g nonenylsuccinic anhydride, 10 g Epon 812, 1.5 ~ DMP-30) for 24 h and cured for 36 h at 60 °C. Sections 1.5/~m thick were cut dry with a glass knife on a Porter-Blum microtome and flattened individually on a glass slide in a small amount of xylene. The sections were coated with a film of nuclear track emulsion (Kodak NTB-2) that had been dried for one day at 30 ~ humidity. The film was prepared by dipping loops of nickel-chromium wire into molten emulsion. The dried emulsion was gently pressed against the sections through Teflon tape. Any moisture on the sections or emulsion at any point of the procedure, including the exposure, caused a loss of localization. After a few days exposure in a desiccator at 20 °C the emulsion was developed for 2 min in Kodak D-19, briefly dipped in a hardening stop bath, fixed for 2 min in Kodak Rapidfix, and washed for 15 min in distilled water. One or two breaths on the exposed emulsion immediately before developing helped to prevent the emulsion from separating from the slide. Some sections were counterstained by dipping the slides sequentially in 1 ~ sodium borate buffer for 3 min, 0.05 ~ Toluidine blue 0 in 1 ~ sodium borate buffer for 3 min, 1 ~ sodium borate briefly, and pH 5.2 m M sodium maleate buffer twice for 5 min to clear the stain from the emulsion. After dehydration in a graded series of ethanol solutions, the sections were cleared with xylene and mounted with Permount. Bright- and dark-field light micrographs were taken with Zeiss 10 × and 40 × Planapochromat objectives.
Grain counting Individual silver grains were counted over a section after autoradiography. The background grain density (grain/sq./~m) was determined by averaging the grain density over three different regions of emulsion that were not in contact with the labeled section. Grain counting over the section and evaluation o f the various surfaces was performed as follows. Using a camera lucida, patches and bands of high grain density were outlined on drawing paper, and the grains inside and outside these
474 delimited regions were counted. The surfaces of the patches and bands and total area of the retina were determined by cutting and weighing the corresponding surfaces of drawing paper. RESULTS
Biochemistry In the chicken retina the uptake of choline increased linearly with time up to 12 min, when the rate of uptake began to diminish. When the concentration of choline was varied, the rate of uptake of choline tended to saturate at higher con-
i
Q.
E X
'7
E x
2
0
E
O b ~
1
o
~
J.
6
a
V/C (10-61 x m i n - l x rngprot )-1 Fig. 1. Eadie-Hofstee plot of the rate of choline uptake as a function of choline concentration. The mean ~ate of uptake ,V, was plotted as a function of V/C, where C is the choline concentration. The rate of uptake in pieces of retina was determined after a 10 min incubation in solution E (Table l) containing choline at concentrations varying between I and 300/~M. The rates of uptake were corrected for [3H]chotine present in the extracellular space (see Methods). Each point is the mean of 2-5 measurements, and a total of 40 measurements were made. The standard deviations were between 10 and 25 ~ of the mean values of V. The curved line drawn through the points was computed for the sum of two Michaelis-Menten equations, using the kinetic parameters given in the text. The straight lines labeled H and L represent the individual contributions of the high and low affinity uptake systems respectively.
475 centrations, and the presence of both a high and low affinity uptake mechanism for choline was suggested by an Eadie-Hofstee plot of these data (Fig. 1). Estimates of the kinetic parameters of the two uptake mechanisms were obtained by a non-linear least squares treatment (see Methods). For the high affinity mechanism, the calculated Kra was 1.1 i 1 . 0 # M (mean ± S.D.) and the Vmax was 8.0 ± 3.8 pmole/min/mg protein. For the low affinity uptake, the Km was 214 ± 58 #M, and the Vraax was 578 ± 94 pmole/min/mg protein. The curved line drawn through the points in Fig. 1 represents the combined contribution of the two uptake mechanisms computed with these parameters. The straight lines labeled H and L represent the contribution at the high and low affinity transport systems acting individually. In an attempt to distinguish the two uptake systems biochemically, the choline uptake experiments were repeated in the absence of sodium by substituting sucrose (solution F, Table I), or in the presence of 5 × 10-5 M hemicholinium-3 (HC-3). HC-3 reduced the total uptake of choline by 78 ~ and 0 Na by 6 7 ~ (Table II). From kinetic data, if the high affinity uptake system solely was blocked, a reduction of the total uptake by 65 ~ would be expected at the choline concentration used in this experiment. After a 10 min incubation at 5 × 10-7 M choline only three labeled compounds were found, ACh, choline and phosphorylcholine. At this time, less than 2 ~ of the label taken up in the tissue was present as lipid bound choline. The presence of HC-3 or the absence of Na, in addition to reducing the uptake of choline, caused a marked decrease in ACh synthesis (Table II). To provide evidence of a synaptic role for the ACh in the retina, the release of ACh by depolarization with K and the dependence of this release on external Ca were examined. A piece of retina incubated for 20 min with [all]choline was rinsed repeatedly with small volumes of normal salt solution (see Methods) until the amount of radioactivity released per rinse was approximately constant. The tissue was then rinsed with a high potassium solution and, as shown in Fig. 2, the release of [aH]ACh
TABLE I! [3HjCholine metabolism and effect of hemieholinium-3 and 0 Na on choline uptake and metabolism
Pieces of retina were incubated for 10 rain with 5 × 10-7 M [all]choline in solution E with or without 5 × 10-5 M hemicholinium-3 or in solution F (Table III). The total uptake of [all]choline in each experiment is expressed as the mean percentage of the uptake under control conditions. The contribution of ACh, choline and phosphorylcholine is expressed as the mean percentage of the total uptake in each experimental condition. The standard deviation of the mean values was 10--15%
Control Hemicholinium-3 0 Sodium
Total [SH] eholine uptake*
[3H]ACh**
[3H]Choline**
[zH]Phosphorylcholine**
100 22 33
44 9 6
46 68 76
10 23 18
* ~ control. ** ~ total choline uptake in each experimentalcondition.
476 10 ~'q
25 mM
K
• •
20 mM
Mg Ca
0 mM
-8 0
E:
E 6' Q. v
:
0 4 "0 0
v
2
c~
0
20
40
60
80
t(min)
Fig. 2. Potassium induced release of [SH]ACh. After incubation of a piece of retina (220 #g protein) with 5 × 10-6 M [3H]choline, rinses were collected every 5 min and their ACh and choline content determined by thin layer chromatography. The [SH]ACh counts/min released during each 5 min rinse are plotted, normalized to 1 mg protein. The periods during which high potassium (solutions B or D, Table 1) or low Ca and high Mg (solutions C or D) were used are indicated by the arrows. Similar results were obtained in two other experiments with different retinas.
increased 10-fold with respect to the preceding rinse in normal solution. Under these conditions the [3H]choline efflux increased 2.7-fold (not shown). The levels returned to control values when the normal salt solution was restored. A low Ca, high Mg solution was introduced and, following a few rinses, a repeat of the high potassium rinse produced no increase in ACh or choline release. The normal salt solution was restored and another high potassium rinse was carried out. This time the release of ACh increased 26-fold and that of choline 2.6-fold.
Autoradiography The results presented so far indicate that a functional cholinergic system is present in the chicken retina. In order to localize the cells that constitute this system, incubation with [ZH]choline was followed by freeze-drying and autoradiography with a dry emulsion film. The morphology of the retina is satisfactorily preserved by this procedure. In Fig. 3A, a bright-field micrograph of a freeze-dried, Toluidine blue counterstained section, the different layers of the vertebrate retina can be recognized
477 with the photoreceptors at the top. Fig. 3B, a dark-field micrograph of a similar section at the same magnification, shows the distribution of label following incubation with [SH]choline. Silver grains, which appear white in dark-field, are present in patches in the inner nuclear and ganglion cell layers and in two well-defined bands in the inner plexiform layer. No localization is observed in the photoreceptor layer, outer plexiform layer or optic nerve fibers. The following controls suggest that the observed localization was not artifactual. Repetition of the procedure with no [SH]choline in the incubation medium produced a grain density no greater than emulsion background over the sections. Incubation with [SH]choline followed by fixation with aqueous 2 700 formaldehyde-4 ~ glutaraldehyde yielded only a few diffuse grains over the sections. This is consistent with most of the label being present in ACh, choline and phosphorylcholine. Additional confirmation of the diffusability of the labeled compounds was provided by an experiment in which freeze-dried sections were dipped in liquid emulsion. With this procedure, even when the Epon was prepared with silicone oil aT, no localization was observed and instead grains were distributed diffusely over the sections. In order to better characterize the distribution of the label seen in Fig. 3B, high power light micrographs of this section were made following counterstaining with Toluidine blue. A montage of such micrographs extending from the outer plexiform layer to the optic nerve fibers is shown in Fig. 4. On the left the focus is on the grains and on the right the focus is on the section. It can be seen that label in the inner nuclear and ganglion cell-layers is present over cell bodies. In the entire field shown in Fig. 3B, label was present in 6 700of the cells in the inner nuclear layer and in 19 of the cells in the ganglion cell layer. In the inner nuclear layer, half of the labeled cells coincided with smaller cells roughly in the middle of the layer, possibly bipolar cells, while the other half coincided with larger cells near the inner margin of the layer, possibly amacrine cells. The question remains, however, as to whether the label seen to be localized in cell bodies and bands is associated with the high affinity choline transport and ACh synthesis characterized biochemically. Two approaches were used to try to answer this question. The first was to repeat the autoradiographic procedure after a choline uptake experiment done in the presence of HC-3. This experimental condition, as described above, blocks both the high affinity uptake of choline and the synthesis of ACh. Grains associated with these two processes therefore should disappear from the autoradiogram. The result of such an experiment (Fig. 3C) was that the patches and bands of label were indeed no longer seen. A second approach was to use grain counting to compare quantitatively the results for total choline uptake and for high affinity choline uptake obtained with autoradiography with those obtained in the biochemical studies. Individual grains were counted on part of the sample section shown in Fig. 3B. The surface counted was 65,250 sq./~m (approximately 180/~m × 360/~m, where 180 ~m was the thickness of the retina). The total number of grains counted in the different labeled regions, after correction for background grains, are given in column B of Table III. From the total number of grains over the section, the uptake of choline was calculated to be 23 pmole/
.=
4~ C~
479
Fig. 3. Morphology and distribution of [all]choline uptake in chicken retina after freeze-drying. All micrographs are at the same magnification. The calibration bar equals 50/~m. Abbreviations: PC, photoreceptor cell layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GC, ganglion cell layer; ON, optic nerve fibers. A: bright-field illumination of a Toluidine blue stained section. B: dark-field illumination of an autoradiograph obtained following incubation ~ith 5 × 10-6 M [all]choline for 15 min. C: dark-field illumination of an autoradiograph obtained following similar Jail]choline incubation including hemicholinium-3. Sections from hemicholinium-3 incubated retinas were autoradiographed and processed together on the same slides with sections from normal [SHlcholine incubated retinas which showed localization as seen in Fig. 3B.
m i n / m g protein.* This value c o m p a r e s well with the m e a n value o f 20 p m o l e / m i n / m g p r o t e i n o b t a i n e d for the same choline c o n c e n t r a t i o n with liquid scintillation counting in the b i o c h e m i c a l studies. The close agreement between the two techniques should be interpreted with some c a u t i o n because the efficiency o f the a u t o r a d i o g r a p h y might differ f r o m the value used a n d the thickness o f the section is k n o w n only a p p r o x i m a t e l y . A n a t t e m p t was then m a d e to d e t e r m i n e whether the increased density o f grains over the cell bodies a n d in the b a n d s could be a c c o u n t e d for by the label t a k e n up via the high affinity mechanism. F r o m the total n u m b e r o f grains associated with low affinity a n d high affinity u p t a k e (columns C a n d D, Table III), the c o n t r i b u t i o n o f the high I
* The uptake with autoradiography is equal to the total number of grains (7195) divided by the amount of protein in the sample (9.78 × 10-6 mg protein, assuming that the protein represents 10700of the tissue mass and that the tissue has a density of 1), the duration of the incubation with labeled choline (15 min), the duration of the exposure (18 h), the specific activity of the [all]choline (10,1 Ci/mmole), the disint./min/Ci (2.2 × 1012)and by the efficiency of the production of silver grains for each isotopic decay. From literature values for the efficiency of grain production in NTB-2 emulsion over methyl methacrylate sections, the efficiency for the 1.5/~m section used jn the present case was estimated to be 0,09 grain/decay82,
480
Fig. 4. Montage at high magnification made from the section shown in Fig. 3B following counterstaining with Toluidine blue. On the right focus is on the section and on the left focus is on both the grains and the section. The calibration bar equals 10 t~m. Abbreviations as in Fig. 3.
481 TABLE III
Grain counting analysis of autoradiography following [all]choline uptake The various regions were outlined, their surfaces measured (column A), individual grains in each region were counted with a camera lucida and background grains (2.3 × 10-a grain/sq./~m) were subtracted to give the total grain number (column B) (see Methods). Grains in regions with low grain density were assumed to be associated with the low affinity uptake. It was further assumed that the low affinity contribution was evenly distributed across all regions, and the grains associated with this system (column C) were subtracted from the total grain number (column B) to yield the grains associated with the high affinity uptake (column D). Grain densities (columns E and F) were obtained by dividing respectively the grain number (column B) and the high affinity grain (column D) by the surface (column A).
(A) Surface (sq.pm)
(B) Total grain number
(C) Grains associated with low affinity uptake
Regions with low density of grains 58556
4127
4127
0
0.07
0
Heavily labeled cell bodies
1970
1402
139
1263
0.71
0.64
Heavily labeled bands
4717
1666
330
1336
0.35
0.28
65243
7195
4596
2599
--
--
Total
(D) Grains associated with high affinity uptake
(E) (F) Mean density Mean density of total of high grains affiity grains (g/sq.pm) (g/sq.pm)
affinity u p t a k e to the total choline u p t a k e was calculated to be 36%*. F r o m the kinetic p a r a m e t e r s derived in the biochemical u p t a k e studies, the c o n t r i b u t i o n o f the high affinity u p t a k e at the choline c o n c e n t r a t i o n used should be 33 %. This suggests t h a t the label t a k e n u p via the high affinity m e c h a n i s m might a c c o u n t for the increased density o f grains over the heavily labeled cell bodies a n d bands. DISCUSSION The aim o f this study was to p r o v i d e a d d i t i o n a l b i o c h e m i c a l c h a r a c t e r i z a t i o n o f the cholinergic system in the chicken retina ~md to localize the n e u r o n s t h a t m a k e u p this system. The biochemical a p p r o a c h d e m o n s t r a t e d t h a t b o t h high a n d low affinity u p t a k e systems for choline are present. T h e values o f Km calculated for the u p t a k e agree well with those f o u n d for o t h e r preparationsg,es, 41. A s r e p o r t e d for o t h e r systems,
* This calculation assumes that the cholinergic cells possess the low as well as the high affinity uptake system for choline. If instead it is assumed that all grains over the cell bodies and bands are derived from high affinity uptake, then the contribution of the high affinity uptake to total choline uptake would be 43 %
482 the high affinity uptake of choline appeared to be Na dependentlS,~s, 4',3 and was blocked when HC-3 was present during the incubationlS, 43. The small additional reduction in uptake seen with HC-3 might be accounted for by a partial block of the low affinity choline uptake systemt~, 43. As might be anticipated from the high level of choline acetyltransferase present 16, [aH]ACh was synthesized from [3H]cboline at a high rate. For example, after 10 rain in an incubation medium with 5 x 10 v M choline, 4 4 ~ of the tritium label found in the tissue was present in ACh. The marked reduction in ACh synthesis in the presence of HC-3, or in the absence of Na, is consistent with previous observations that high affinity uptake of choline is required for ACh synthesis~5,2s,36, 43. In the present results 7 0 ~ of the choline taken up by the high affinity mechanism was converted to ACh. This study provides the additional observation that [3H]ACh synthesized from [3H]choline could be released by increasing the extracellular potassium concentration. The release of ACh required the presence of Ca in the medium and was specific in the sense that Jail]choline release did not increase to the same extent, a result that is consistent with the recent report of a light induced, Ca dependent release of ACh in the rabbit retina 26. The small increase in choline release that does occur in the presence of high potassium might be due to an incomplete blockage of the acetylcholinesterase by neostigmine. Localization of the neurons labeled after incubation with [3H]choline was achieved autoradiographically. The label was concentrated in cell bodies in the inner nuclear and ganglion cell layers and in two bands in the inner plexiform layer. The association of the labeled cells with the cholinergic system characterized biochemically is suggested by the following three points. When HC-3, which blocks high affinity uptake and ACh synthesis, was included in the incubation, no localization of label was observed. Secondly, at the choline concentration used for the autoradiography, both grain counting and the kinetic parameters determined biochemically gave the same value for the contribution of the high affinity uptake to the total uptake. Finally, from grain counting and biochemical results, approximately 69% of the highly localized grains seen autoradiographically are associated with ACh. The present results show that a large proportion of the label taken up with high affinity is present in cell somas. This raises the question of whether high affinity uptake sites are present on somas as well as terminals. In the chicken, it has been reported38, 39 that for the ciliary neurons which innervate the iris, high affinity uptake of choline is not found in the cell bodies in the ciliary ganglion but appears to be ~estricted to the terminals in the iris. In the retina the distance from terminals to cell bodies is normally only a few hundred microns. It can be calculated that even if all of the high affinity choline uptake occurred at synaptic terminals, diffusion of choline or ACh from these sites could produce a significant concentration of label in cell bodies during the time used for incubation and rinsing*. Thus, the fact that a large fraction of the label is present in cell bodies does not necessarily imply that high affinity uptake sites are present on the cell bodies. Since the autoradiographic results following [3H]choline uptake appear to localize the cholinergic system it is of interest to compare these results with other morphological studies of this system in the chicken retina. With cholinesterase staining
483 two heavy bands are seen in the inner plexiform layer aS, and these coincide with the two bands seen in Fig. 3B. In contrast, binding of a-bungarotoxin was distributed rather uniformly across the entire width of the inner plexiform layer 40. This might indicate that the choline uptake and the es~erase staining do not reveal the full extelat of the cholinergic arborizations. Alternatively, in the retina a-bungarotoxin might bind with high specificity to membranes outside cholinergic synaptic regions. The bands seen with [3H]choline uptake overlap closely with layers 2 and 4 visualized by Cajal in the inner plexiform layer of the chicken with Golgi impregnations 6. Specific subclasses of retinal neurons were seen to send processes predominantly to these two layers 6. In the ganglion cell layer a class of small cells located at the margin of the inner plexiform layer, thought by Cajal to be displaced amacrine cells, projected densely to the fourth layer. The existence of significant numbers of displaced amacrine cells in avian retinas also is consistent with the finding that in the pigeon there are many more cell bodies in the ganglion cell layer than there are axons in the optic nerve 3. In the present study the patches of label seen in the ganglion cell layer most often were over small cell bodies at the margin of the inner plexiform layer. Thus, the labeled cells in the ganglion cell layer may be displaced amacrine cells rather than ganglion cells. This possibility is supported by the observation in the pigeon that following retinal ablation high affinity uptake of choline in the optic rectum did not decrease, but in fact increased 17. A decrease would be expected after denervation a9 if ganglion cells were cholinergic. From their position, the labeled ceils in the inner nuclear layer could be amacrine cells and inner bipolar cells, but in its present form the choline localization does not permit any more precise characterization of these cells. Another possible source of terminals that might take up choline are the efferent fibers described for the chicken and other birdsS, s. These fibers terminate in varicosities that often form nests around cell bodies in the amacrine cell layer. In the 1.5/~m thick sections used in the present study, if these terminals took up Jail]choline, the grains should have been present in halos around label free cell bodies. Instead label appeared to be distributed evenly throughout the cytoplasm of cell bodies, suggesting that the efferent fibers are not cholinergic. Although the anatomical details provided by the present study are informative, it is clear that precise identification of the cholinergic cells requires analysis at the electron microscopic level. With the procedure described here such an investigation may be possible 37. * For this calculation a closed cylindrical model was used. Choline uptake was assumed to occur only at one end of the cylinder. For this system the solution to the diffusion equation is (this solution was provided by Dr. John Hutchinson of the Department of Engineering and Applied Physics, Harvard University) Qr ,! 2L2 oo (_1) n C(x,t)=---L--~l + - Y, ~ e zt2Dr n = 1 n2
Dn2z~2t Dn2zt2r L~ L~ x 1 [e --1]cosnzt(1---~)~
The concentration C was calculated at point x along a cylinder of length L, at time t following an incubation of time r, with a choline influx Q at the end of the cylinder. The coefficientD was taken to be 1.48 × 10-5 cm2/sec~0.
484 A n interesting aspect o f the present results for the localization of cholinergic n e u r o n s in the retina is that only a few cells o f any given type were labeled, that is only a b o u t 6~o in the inner nuclear and 19°/,i in the ganglion cell layer. This lo~' p r o p o r t i o n o f labeling for each cell type is consistent with results from uptake studies with other transmitter candidates such as G A B A 23, glycine '~4, a n d d o p a m i n e 1.9. These observations have been confirmed in the chicken where, for each o f these c o m p o u n d s as well as for choline, a different p a t t e r n o f labeling is seen 34. This suggests that the m o r p h o l o g i c a l subclasses that Cajal f o u n d to project to various levels o f the inner plexiform layer m a y have different t r a n s m i t t e r biochemistry a n d different physiological roles. ACKNOWLEDGEMENTS W e wish to t h a n k Torsten Wiesel for his c o n t i n u a l e n c o u r a g e m e n t a n d s u p p o r t a n d E d w a r d K r a v i t z for his careful reading o f the manuscript. This investigation was s u p p o r t e d by P.H.S. I n t e r n a t i o n a l R e s e a r c h F e l l o w s h i p TW02255 to C.R.B., N . I . H . F e l l o w s h i p EYO2317 to R.W.B. a n d N . I . H . G r a n t EY00606.
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