hoc. Null. Acad. Sci. USA

Vol. 74, No. 12, pp. 5524-5528, December 1977
Biochemistry

Muscarinic acetylcholine receptors of the developing retina

(synapse formation/receptor localization/3-quinuclidinyl benzilate)

HIROYUKI SUGIYAMA, MATHEW P. DANIELS, AND MARSHALL NIRENBERG

I.&oratory of Biochemical Genetics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda. hlarvland 20014

Contributed by Marshall Nirenberg, October 7, 1977

AB!?TRACT   Six- and U-day chicken embryo retinas contain
IO and 320 fmol per mg of protein of specific binding sites for
q3H]quinuclidinyl benzilate, a ligand of muscarinic acetyl-
choline receptors. Most of the receptors of U-day embryo retina
were found, b
bands within t autoradiography, to be localized in two sharp
  K P
       e inner syna tic layer of the retina. In the adult,
the receptors were found a most exclusively in three bands in
the inner synaptic layer of the retina. A possible mechanism for
generating sets of stratified or columnar neurons and relating
one set to another is proposed.

The vertebrate retina provides a model system for synapse
formation because synaptic circuits may be assembled with
relatively few types of cells and because cultured neurons dis-
sociated from retina form synapses in profusion in vitro (1,2).
Biochemical (3-7), histological (8, 9), and electrophysiological
(10-13) evidence strongly suggests that acetylcholine (ACh)
functions as a neurotransmitter in the retina. Developmental
and histological studies of chicken retina acetylcholinesterase
(EC 3.1.1.7 AChE) (8), ACh (14), choline acetyltransferase (EC
2.3.1.6) (2), and nicotinic ACh receptors (15, 16) have been
reported.

In this report, the properties of muscarinic ACh receptors of
chicken retina, the number of receptors, and their distribution
within the retina during embryonic development are de-
scribed.

MATERIALS AND METHODS

Homogenate Preparations. Neural retinas of White Leg-
horn chicken embryos were homogenized in 50 mM sodium
phosphate buffer, pH 7.4 (buffer A). In some experiments,
homogenates were diluted several times with buffer A and
centrifuged at 17,300 X g for 20 min at 3". The pellet was
suspended in buffer A (membrane fraction). All experiments
were performed with freshly prepared homogenates or mem-
branes.

Binding Assay. (3*)-Q uinuclidinyl benzilate (QNB), a gift
from Hoffman-La Roche, Inc., was labeled by catalytic 3H
exchange and purified as described by Yamamura and Snyder
(17); the specific activity was 8.4 Ci/mmol. 3(~t)-[3-~H] QNB
used in some experiments was obtained from Amersham/Searle
(13 Ci/mmol).

13H]QNB b' d' g
     m m was measured by a modification of the
method of Yamamura and Snyder (17). Homogenates were
combined with [3H]QNB in buffer A and incubated for various
periods. Each lOO- to 150-~1 portion of the reaction mixture
(usually containing IOO-ZOO pg of protein) then was diluted into
5 ml of ice-cold buffer A, immediately filtered, and washed
three times, each with 5 ml of buffer A. Binding kinetics were

The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
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measured at 25" by using Whatman glass fiber GF/C filters,
The concentration of (&)-[3H]QNB in the reaction mixture was
0.5-1.0 nM. When the effects of competing ligands were tested,
homogenates were incubated with desired concentrations of
ligands for 5-10 min at 25" and then mixed with [3H]QNB
solution containing the same concentrations of the ligands.
Equilibrium studies were performed at 4" with Millipore
HAWP filters or, in some cases, GF/C filters (results were es-
sentially the same). For the determination of nonspecific
binding, homogenates were incubated with 0.4-10 PM atropinr
sulfate for 10 min in ice and then mixed with 13H]QNB solution
containing the same concentration of atropine. The number of
13H]QNB binding sites was determined by Scatchard analysis
in some experiments but more often was determined at one
saturating concentration of (i)-[3H]QNB (6-10 nM).

13H]QNB Autoradiography. Neural retinas were dissected
in cold Dulbecco's phosphate-buffered saline with Ca*+ and
Mg2+ (PBS). Pieces of retina from 1Sday embryos or adult
chickens were incubated for 90 min in 5 ml of PBS containing
4 or 2 nM (*)j3H]QNB (13 Ci/mmol), respectively, and then
washed eight times, each with 5 ml of PBS. In control experi-
ments, pieces of retina were preincubated in 5 ml of PBS con-
taining 0.4 PM atropine sulfate for 10 min, followed by incu-
bation in 5 ml of [3H]QNB solution in PBS containing 0.4 pLM
atropine sulfate for 90 min. The tissue then was washed twice
with 5 ml of PBS containing 0.4 PM atropine sulfate and six
times with 5 ml of PBS. Samples were kept in an ice bath at each
step. Both experimental and control retinas were washed for
25 min (all washes). Retinas then were sandwiched between two
pieces of mouse liver and frozen quickly in liquid Freon cooled
in liquid nitrogen. Frozen pieces were sectioned (I2 pm thick)
and thaw-mounted onto glass slides coated with Kodak NTB-9
nuclear emulsion. To minimize diffusion of [3H]QNB, mounted
sections were immediately dried under a stream of nitrogen gas.
Slides were stored in the dark at 4" with a desiccant. Autora-
diographs were developed, fixed, and then immersed in 2.5c/;
glutaraldehyde in 0.1 M phosphate buffer, pH 7.0, for 1 hr at
room temperature. Some slides were stained with 0.02% toln-
idine blue for 5 min at room temperature.

Retina Cell Cultures. Cells were prepared from 8-day em-
bryos and cultured in rotating petri dishes as described (I) with
minor, modifications: 1.5 X lo7 cells in 3 ml of medium (95';
Eagle's basal medium with Earle's salts and 5% fetal boviw
serum) were cultured in a bacterial petri dish (35 mm; FakclJt
no. 1008) placed on a rotary shaker with an excursion of 2.6 cm
(75-80 rpm) in a 37" incubator in a humidified atmosphere of
5% CO2/95% air. Half of the medium was replaced each
day.

Abbreviations: ACh, acetylcholine; AChE, acetylcholinesterase: QNe.
Squinuclidinyl benzilate; PBS, phosphate-buffered saline with (:a"+
and Mg*+.

5524


Biochemistry: Sugiyama et al.

          MINUTES      nM&!j(+) QNB
FIG. 1. QNB binding to receptors in homogenates of 13-day
chicken embryo retina. (A) Kinetics of QNB binding to and release
from sites on membrane preparations at 25'. The ordinate represents
specific binding of L"H]QNB per 0.273 mg of protein (0.15 and 3.0 ml
reaction mixtures for on and off reactions, respectively). The initial
concentration of (k)-("H]QNB was 2.0 nM. For the release kinetics
experiment, the reaction mixture was incubated for 3 min and then
diluted with 19 volumes of buffer A containing 0.4 rM atropine.
Fifty-seven min after the first dilution, the reaction mixture was again
diluted 20-fold with buffer A with 0.4 PM atropine. No release of$NB
was observed during further incubation (not shown). (B) QNB con-
centration curve and Scatchard plot (Inset). Specific binding (0) is
the difference between total binding (0) and nonspecific binding (0)
in the presence of 0.4 PM atropine. Each point represents the mean
of triplicate values. B and F correspond to concentrations (nM) of
specifically bound QNB and free (-)-QNB, respectively. The line
without points represents the concentration of active isomer (-)-QNB
added, The concentration of protein in the reaction mixture was 2.86
mg/ml. Tubes were incubated at 4' for 100 min.

RESULTS

Receptor Properties. The rates of 13H]QNB binding to and
release from receptors in homogenates of 13-day chicken em-
bryo retina are shown in Fig. 1A. [3H]QNB bound rapidly to
retina membranes; in the presence of 2 nM (+z)-[~H]QNB, half
maximal binding was achieved in 2 min and maximal binding,
in approximately 15 min. Some, but not all, of the reactions are
reversible. The addition of 0.38 PM atropine and dilution of
reaction mixtures 2O-fold resulted in the dissociation of ap-
proximately 50% of the [3H]QNB-receptor complex. QNB
association and dissociation reactions both exhibited biphiasic
kinetics with fast and slow association reactions and dissociation
reactions. The kinetics will be discussed elsewhere; however,
the rate constants (k) for fast and slow QNB association reactions
were estimated, by assuming bimolecular irreversible reactions
as a first approximation, to be 2.7 X IOR M-' min-' and 1.4 X
l@ M-l min-`, respectively. Both fast and slow QNB-receptor
dissociation reactions were first-order reactions with rate con-
stants of 1.2 min-' and 0.041 min-I, respectively.

The relationship between 13H]QNB concentration and
binding to receptors in retina is shown in Fig. 1B. The binding
of the pharmacologically active isomer, (-)-[3H]QNB, to retina
receptors is a saturable process. In the presence of 0.4 &I at-
ropine, relatively little nonspecific (3H]QNB binding was found
with homogenates prepared from l&day chicken embryo
retina; however, nonspecific (3H]QNB binding was markedly
increased when homogenates are prepared from >15-day
chicken embryo retina or posthatched retina. The dissociation
constant (Ko) determined by Scatchard analysis (Fig. 1B and
inset) was 0.12 nM (-)-13H]QNB. However, we consistently
observed higher apparent dissociation constants (4.3 nM) with
homogenates from retina of chickens 2 weeks after hatching
4 of adult chickens. The calculated concentration of specific
QNI% binding sites in 13-day chicken embryo retina is 32.5

hoc. Natl. Acad. Sci. USA 74 (1977)   5525

FIG. 2. (A) Inhibition of pH]QNB binding by various com-
pounds. (B) Hill plot. Thirteen-day embryo retinas were used. Whole
homogenates and membrane fractions were used and no significant
difference was noted. The final concentration of (A)-["H]QNB was
0.5 nM (1.0 nM in some experiments). Protein concentrations in re-
action mixtures were 0.5-2.6 mg/ml. Receptor concentrations were
0.1-1.0 nM. Initial rates of binding (usually 0 to 34 min) were fitted
to a model of bimolecular irreversible reaction mechanism, and the
bimolecular association rate constant was calculated. The apparent
rate constant in the presence of protecting drugs was expressed as the
percentage of the control value in A. Hill plots were obtained by as-
suming that the percentage decrease of ["H]QNB binding rate rep-
resents the percentage of the receptor sites occupied by unlabeled
ligands which corresponds to B/B,,,. When ACh was tested the ho-
mogenate was preincubated with 3 JIM eserine for 30 min at 25" before
addition of ACh. Symbols: O , scopolamine; 0, atropine sulfate; +,
oxotremorine; o , AChCl; A, carbamylcholine chloride; t, pilocarpine;
*, muscarine.

fmol/mg of protein and each retina contained 818 fmol per
retina (4.9 X 10" sites per retina) of specific QNB binding sites.
The apparent Hill coefficient is 1.0 (plot not shown), which
suggests that QNB binds to independent, noninteracting re-
ceptors. Although only one population of QNB binding sites was
detected by Scatchard analysis, kinetics of the QNB binding
to and release from receptors show that !3H]QNB-receptor
complexes are heterogeneous.

The effects of different concentrations of unlabeled ligands
known to activate or inhibit muscarinic LiCh receptors on the
initial rate of [3H]QNB binding to receptors in homogenates
prepared from I3 day chick embryo retina are shown in Fig.
2A. [3H]QNB binding was markedly decreased in the presence
of antagonists of muscarinic ACh receptors such as scopolamine
or atropine or receptor activators such as oxotremorine, ACh,
carbamylcholine, pilocarpine, or muscarine at expected
physiological concentrations. [3H]QNB binding was not af-
fected by prior incubation of homogenates with 10 nM w
bungarotoxin for 3 hr at 25" (not shown). Thus, thespecificity
of QNB binding sites for ligands closely resembles that of
muscarinic ACh receptors.

As shown in Fig. 2B, the apparent Hill coefficients of acti-
vators of the muscarinic ACh receptor such as oxytremorine,
ACh, and carbamylcholine were 0.6 to 0.8, whereas those of
receptor antagonists were approximately 1. These results agree
well with those of Birdsall et al. (18). The apparent Hill coef-
ficients of pilocarpine and muscarine were approximately I.
Pilocarpine has been shown to be both an activator and an an-


5526  Biochemistry. Sugiyama et al.

Table 1.  Apparent dissociation constants and Hill coefficients
   of ligands lirr muscarinic acetylcholine receptors
         of 13 dav chick emhrvo retina

Ligands

ApI) Kl,.*
nM        h

Atropine
Scopolamine
Activators
Oxotremorine
Acetylcholine
Carhamylcholine
Muscarine
I'ilocarpine
I,ocal anesthetics
Dibucaine

0.12
0.44
0.29+
0.69
0.17

130
1,100
1,700
8,700
1,100

30,000

1.0
1.0


1.0
1.1

0.7
0.8
0.6
1.1
1.0

1.0

Tetracaine         " 1,000       1.1

* Values at 2.5" except otherwise specified. The KI, values for QNH
were c,btained by determining the binding of I:`H]QNR at equilih-
rium. The Ki,,,,, values for the other antagonists and activators
represent the concentrations that result in 50'~~ inhihition of' the
initial rate of I"HjQNH binding: values were not corrected l'or h <
1. The K,,,,, values L'or local anesthetics are estimated from experi-
ments where the retina homogenate with or without difl'erent cc,n-
cenrrations of a local anesthetic were incubated for 60 min at 25'
in the absence of I:`H]QNB, then 0.50 nM (+)-I:`H]QNR was added
and the reaction mixtures were incubated fur an additional 5
min.
+ 0.29 and 4.4 nM (-I-QNH are the dissociation constant values de-
lermined from rate constants for slow and fast association and
dissociation reactions, respectively.

tagonist of the muscarinic ACh receptor; although muscarine
is an activator of the muscarinic ACh receptor in other organ-
isms, the apparent Hill coefficient with chicken embryo retina
receptors resembles that of a receptor antagonist. The apparent
Hill coefficients of <l observed with oxytremorine, ACh, and
carbamylcholine can be interpreted in various ways such as
negative cooperativit),, heterogeneity of binding sites, or de-
sensitization of the ACh receptor. The demonstration by W.
Klein in this laboratory that heterogeneity of muscarinic ACh
receptors can be detected by kinetic studies was confirmed.

Muscarinic ACh Receptors during Embryonic Develop-
ment. The concentration and number of muscarinic ACh re-
ceptors in chicken embryo retina are shown in Fig. 3 A and B,
respectively, as a function of developmental age. Values re-
ported for nicotinic ACh receptors (16) also are shown for
comparison. Muscarinic ACh receptors were detected in 5.5-
day embryo retina, but the concentration was relatively low (10
fmol/mg of protein). Between the 6th and 14th days of em-
bryonic development, the concentration of specific QNB
binding sites increased 30.fold. In the 5.5- to g-day chicken
embryo retina, specific QNB binding sites accumulated expo-
nentially with a doubling time of approximately 26 hr; in 9- to
I+day embryo retina, the doubling time was approximately
60 hr. The maximal concentration of QNB binding sites (320
fmoljmg of protein) was attained in the retina of the l3- to
l4-day embryo. No further change was detected during later
embryonic development; however, receptor concentrations
were lower in retina 2 weeks after hatching and in adult retina.
Although the concentration of receptors decreased after
hatching, the number of receptors per retina increased slightly
after hatching (Fig. 3B). The adult chick retina contained 1200

0  5  10  1520   I4aDUT5  IO  1520   14 ADULT
             DAYS

FI(;. 3.  The accumulation of`QNH binding sites in chicken em-
hry retina as a I'unction of development age. The number of specific
sites per mg protein (A) or per retina (H) is shown. For comparison,
tr-hungarotoxin (tr-HT) binding to nicotinic ACh receptors (16) i>
shown (broken lines). Circles, intact retina in WIW; triangles, cultured
cells dissociated from X-day chicken embryo retina. Filled circles are
values obtained by Scatchard analysis. Open symbols represent the
specific hinding ohtained at G-10 nM (h)-(:`H]QNB. Tuhes were in-
cuhated at 4O for 60 min. In some cases, the retina dissection was not
complete; the amount of protein per retina then was estimated lrr)m
the puhlished values (19). Each point represents the mean of at least
three determinations.

fmol of specificQNn binding sites per retina(S.2 X 10" sites per
retina).

These results show that genes for muscarinic ACh receptors
are expressed early in the development of the retina and suggest
that some neurons synthesize muscarinic ACh receptors but not
neuroblasts as reported for nicotinic.ACh receptors (16). The
number of muscarinic and nicotinic ACh receptors increase
more than 30-fold and the receptors accumulate at similar rates
between the sixth and ninth days in embryos, The maximal
concentration of muscarinic ACh receptors is attained in the
retina of the 13-day embryo, whereas nicotinic ACh receptors
continue to increase until hatching. The concentration of spe-
cific QNB binding sites in retina of the 6- to 13-day embryo is
2- to S-fold higher than the concentration of tr-bungarotoxin
binding sites; however, this ratio is reversed in the adult retina.
Thus, the ratio of muscarinic to nicotinic ACh receptor changes
markedly during retina development.

Cells dissociated from &day chicken embryo retina were
cultured for various times in rotating petri dishes. At various
times, homogenates were assayed for specific binding of
J3H]QNB (Fig. 3A). The concentration of QNB binding sites
increased from 50 fmol of specific QNB binding sites per m$
of protein in retina of the g-day embryo to 225 fmol/mg (11.
protein after 4 days of culture. Thus, the accumulation ()t
muscarinic ACh receptors in cultured retina cells resemblt'tl
that in the intact retina.

Receptor Distribution in Retina. The distribution of
13H]QNB binding sites in intact I3-day chicken embryo retina
and in adult retina is shown in Fig. 4. In 13-day embryo retina.
most of the silver grains were localized in two narrow bands
within the inner synaptic layer of the retina (also termed "innrr
plexiform layer") (Fig. 4 A and B). In adult retina (Fig. 4 C and


Biochemistry: Sugiyama et af.

FI(;. 4.  Autoradiography of sections of chicken retina incubated
with ["HIQNB in the absence of atropine. (A) Phase-contrast view
and (H) dark-field view of stained section of IS-day embryo retina
exposed for SO days. (C) Phae-contrast view and (D) dark-field view
,$stained section of adult chicken.retina exposed for 173 days. (Bars
represent 100 urn.) Lines at the left of each photograph represent the
h,,undaries of layers: R, photoreceptor layer; 0, outer synaptic layer;
IN, inner nuclear layer; IS, inner synaptic layer: G, ganglion cell layer:
A. ganglion axon layer.

D), two or three bands of silver grains could be seen within the
inner synaptic layer of the retina.

Histograms relating the density of silver grains on autora-
dingraphs that had been exposed for shorter times with grain
location over the retina are shown in Fig. 5. Two sharplv de-
fined bands of silver grains of equal density can be seen within
the inner synaptic layer of 13-day embryo retina. Fewer silver
grains were associated with the lower portion of the inner nu-
clear layer (cell bodies of amacrinr and bipolar neurons and
M&r cells) and with ganglion neuron soma and axons but were
not associated with other regions of the retina. The average
number of silver grains over the entire retina incubated in the
absence or presence of 0.1 &I a&opine (nonspecific [SHJQNB
binding) was 3.83 and 0.87 grain per 100 prn2, respectively.

FI(;. 5.  Histograms showing the grain distribution in [:`H]QNB
autoradiographs of sections of I:<-day chicken (A) or adult chicken
10) retina. Sections of retina of both ages, treated f'or both total
binding and nonspecific binding, were subjected to autoradiography
ior 65 days. (irains were counted at X600 magnification by using a
(`amera lucida. Specific binding was obtained by subtracting non-
hprcific from total binding, Number of grains counted were: 54~1 and
220 (`or total and nonspecific binding, respectivelv, for l:I-day embryo
retina; 15% and 847 for total and nonspecific binding, respectively.
lor adult retina. Abbreviations are ah in Fig. -1.

Proc. Natl. Acad. Sci. USA 74 (1977)   5527

-5

FIG. 6.  Schematic representation of the relative distributions and
concentrations of muscarinic (MI and nicotinic (N) ACh receptors
(16) and AChE activity (8) in the inner synaptic layer of chick retina.
M and N are from IS-day embryo or adult retinas; AChE is from
12-day emhryo and newly hatched chicken retinas. Open, stippled.
hatched. and filled areas represent relative ACh receptor concen-
trations or AChE activity in increasing order. Numbers refer to peak
positions. The top and the hottom of the figure (0 and 1OOYn) corre-
spond to the inner nuclear and ganglion neuron boundaries of inner
synaptic layer. respectively. Cajal's layers for chicken retina are from
plates 1 and 5 of ref. `LO.

Thus, specific QNB binding accounted for 77% of total QNB
binding. In the adult retina (Fig. 5B), three bands of specific
QNB binding sites were localized in the inner synaptic layer
of the retina. Few, if any, specific binding sites for QNB were
detected elsewhere in the retina; thus, muscarinic ACh recep-
tors are localized to a greater extent in the adult retina than in
the I3-day embryo retina. In other sections, the first band of
specific QNB binding sites near the inner nuclear layer over-
lapped the first and second fractions of the inner synaptic layer
and the demarcation between the second and third bands was
less distinct than that shown. The average number of silver
grains over the entire retina in the absence or presence of 0.4
KM atropine was 1.39 and 0.96 grain per 100 pm', respectively.
Specific ["HIQNB binding was 31% of total QNB binding, in
accord with ligand binding results, Adult retina was incubated
with 2 rather than 4 nM ["H)QNB to decrease nonspecific
("HjQNB binding; however, the grain density was somewhat
lower than expected.

These results show that muscarinic ACh receptors are lo-
calized in the inner synaptic layer of the retina and suggest that
the receptors are present in some amacrine and ganglion neu-
rons but not in other cell types in the retina

DISCUSSION

The distribution of muscarinic ACh receptors within the inner
synaptic layer of chicken embryo and adult retina is compared
with previously repotted distributions of nicotinic ACh re-
ceptors (16) and AChE activity (8) in Fig. 6. Muscarinic and
nicotinic ACh receptors and AChE activity are localized in
bands within the inner plexiform layer of chick retina. In the
embryo, two bands, each with high concentrations of muscar-
inic ACh receptors and high AChE activity, can be seen; nico-
tinic ACh receptors are distributed diffusely in two broad bands
throughout most of the inner synaptic layer. After hatching,
the inner synaptic layer of the retina contains three muscarinic
ACh receptor bands, four nicotinic ACh receptor bands (161,
and four bands with high AChE activity (8). Nicotinic ACh
receptors are present in the outer synaptic layer (16). but not
muscarinic receptors. Most, but not all, of the ACh receptor
bands are associated with AChE bands. The bands appear in


5528  Biochemistry: Sugiyama et al.

an ordered sequence during development, with respect to
temporal and positional relationships. The maximal concen-
trations of muscarinic and nicotinic ACh receptors are attained
on the 13th and 21st days of embryonic development. Thus,
most of the synapses mediated by muscarinic ACh receptors
probably are formed at an earlier developmental stage in retina
than those mediated by nicotinic ACh receptors. Vogel et al.
(21) have shown that nicotinic ACh receptors are localized at
sites of synapses in chicken retina. The localized bands of
muscarinic ACh receptors within the inner synaptic layer and
the apparent absence of the receptors from cell bodies and axons
of ganglion neurons in adult retina raise the possibility that
muscarinic receptors also may be localized at certain synapses.
Further work is needed to resolve this question.

Bipolar neurons and photoreceptors of retina form double
or triple synapses (ribbon synapses) wherein one cell transmits
information across one synapse simultaneously to two or three
neurons. ACh probably is the transmitter at some double sy-
napses of bipolar neurons because localized nicotinic ACh re-
ceptors have been found on the processes of one or both post-
synaptic cells (21). Because three species of ACh receptor-
muscarinic excitatory, muscarinic inhibitory, and nicotinic-are
widely, distributed in the nervous system, ACh released at one
synapse may excite and/or inhibit the recipient neurons, de-
pending on the species of ACh receptor that are present.

The inner synaptic layer is composed predominantly of
neurites of amacrine, bipolar and ganglion neurons, and syn-
aptic connections with processes of ganglion, amacrine, or bi-
polar neurons. Five layers can be distinguished by phase-con-
trast microscopy but not by transmission electron microscopy
within the inner synaptic layer. However, Dubin (22) has shown
that three classes of synapses that can be identified by ultra-
structural features are stratified in different ways in the inner
synaptic layer of pigeon retina; stratification was not detected
in the retina of other organisms examined.

Eleven layers can be distinguished within the inner synaptic
layer of chick retina on the basis of ACh receptor concentrations
and AChE activity. Three additional layers rich in catechol-
amines have been identified in the inner synaptic layer of
chicken retina (5), and four or five glutamic acid decarboxylase
bands have been detected in rat retina (23). These results show
that neurites of one type sort out from those of other types. A
neuron that forms synaptic connections with two or more
neurons is, in effect, a polyvalent crosslinking agent. Thus,
neighboring neurons that form synapses with two or more cells
of the same type, at the same stage of development, become
linked to one another and sort out from other sets of neurons,
Since a single neuron may both send and receive information

across synapses and may form multiple kinds of synapses, such
neurons may link sets of neurons that form different types of
synapses. The extent of sorting out and the relationship of one
class of neurons to another may be determined by the number
and kinds of synapses formed by each class of neurons (both pre-
and postsynaptic connections), the sequence of synapse for-
mation, and the initial spatial relationships of the neurons.

We thank Linda Lee for excellent assistance.

1.


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12.


13.
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17.


18.


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20.

21.


22.
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