EXPERIMENTAL
NEUROLOGY
53, 703-713 (1976)
Choline Acetyltransferase, Choline Kinase, and Acetylcholinesterase Activities during the Development of the Chick Ciliary Ganglion ALVIN
M. BURT 1 AND C. H. NARAYANAN
Department of Anatomy, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, and Department of Anatomy, Louisiana State University School of Medicine, New Orleans, Louisiana 70112 Received
July
12,1976
The specific activity of choline acetyltransferase, choline kinase, and acetylcholinesterase was measured in ciliary ganglia of chick embryos from 9 days of incubation to hatching. The specific activity of choline acetyltransferase increased eightfold during this period and the activity per cell increased from 29.5 fmol/hr/cell at 9 days to 2428 at hatching. The specific activity of both choline kinase and acetylcholinesterase was highest at 9 days of incubation although the total activity per ganglion increased nearly sixand eightfold, respectively, during the developmental period studied. The specific activity of choline acetyltransferase in the choroid cells was significantly higher than that in the ciliary cells after 16 days of incubation, with values of 767 and 589 pmol/g dry weight/hr, respectively, at 20 days of incubation. When correlated with the morphological and physiological development, the results of this study indicate that relatively low levels of acetylcholine synthetic capacity (29.5 fmol/hr) are sufficient for a functional cholinergic synapse. The major increase in choline acetyltransferase activity parallels synaptic maturation and the establishment of functional connections by the postganglionic neurons with the peripheral field.
INTRODUCTION A series of experimental and normal studies has related the development of choline acetyltransferase ( ChAc ; acetyl-CoA :choline O-acetyltrans1 This investigation was supported by a grant from the Dysautonomia Foundation, Inc., and by Research Grant NS-07441 from the National Institute of Neurological and Communicative Disorders and Stroke, National Institutes of Health. A preliminary report has been presented (11). Send correspondence to Dr. Burt at the Vanderbilt University address. 703 ItIC.
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ferase, EC 2.3.1.6) activity in the chick spinal cord to synaptogenesis (3, 5, 9). These data, however, do not indicate the particular phase of synaptogenesis concerned but only that the increased activity of ChAc was dependent on synapse formation and that, when synapse formation was experimentally reduced, there was a corresponding reduction in ChAc activity, a reduction that was not reflected in the levels of acetylcholinesterase (AChE ; acetylcholine acetylhydrolase, EC 3.1.1.7) activity and in the activity of enzymes of intermediary carbohydrate metabolism (9, 10). Because both the preganglionic and postganglionic elements are cholinergic (24), the chick ciliary ganglion should provide a model system in which to study cholinergic synaptogenesis. Although some adrenergic nerve endings are present, they do not make direct synaptic contact with either cholinergic element (12). In addition, the developmental morphology and physiology have been well documented (19, 20, 22) and precise correlations can be made with the developmental neurochemistry. The preganglionic fibers of the ciliary ganglion arise in the accessory oculomotor nucleus and terminate in a one-on-one fashion with the neurons of the ganglion (22). Two morphologically distinct populations of neurons, the choroid and ciliary cells, are recognizable as early as 10 days of incubation and give rise to the postganglionic choroid and ciliary nerves, respectively (19). Synaptic transmission begins at 5 days and involves 100% of the neurons as early as 8 days of incubation, although morphological evidence for presynaptic and postsynaptic thickenings is still scant (19). At this stage, the transmission in both populations of neurons is chemical and acetylcholine is the apparent neurotransmitter. The morphological maturation of the synapse is much slower, and by 14 days of incubation synaptic vesicles are still sparse (19). At 15 days of incubation, direct electrical coupling is first noted in the ciliary cell population and, by 1 to 2 days after hatching, 80% of the ciliary cells show direct electrical transmitting synapses in addition to the chemical synapse. Synapses on the choroid cells, on the other hand, remain entirely chemical throughout development (19). In the present study, the activity of ChAc, AChE, and choline kinase (CK ; ATP : cholinephosphotransferase, EC 2.7.1.32) was studied in individual ciliary ganglia of chick embryos from 9 to 21 days of incubation. In addition, the choroid and the ciliary neurons, along with their corresponding presynaptic terminals, were assayed separately for ChAc activity. Our data continue to support the direct association of ChAc with synaptogenesis and further suggest that very low levels of enzyme activity are sufficient for a physiologically functional synapse. The major increase in ChAc activity is related to the morphological and physiological maturation of the synapse and to the establishment of functional contact between the postganglionic neuron and its peripheral field.
chAc,
CK,
AND
AchE
METHODS
IN
AND
CILIARY
705
GANGLIA
MATERIALS
Preparation of Samples. Fertile white Leghorn eggs were incubated at 37.8”C in a forced-air incubator for 9 to 21 days. Individual ciliary ganglia were dissected free from surrounding tissue, frozen rapidly in isopentane chilled to approximately -155°C with liquid Na, lyophilized at -40°C and stored at -76°C until assayed. Individual lyophilized ganglia were freed from adhering tissues, trimmed of nerve roots, weighed on a Cahn electrobalance, and homogenized in microglass tissue grinders with 0.05% Triton X-100 at a final concentration of from 0.2 to 0.5 pg tissue dry weight/$. Individual ganglia ranged, in dry weight, from 9 to 10 pg at 9 days of incubation to 50 to 55 g at hatching (Table 1). Homogenate aliquots were assayed immediately for AChE, ChAc, and CK activities. Other ganglia were rapidly frozen and cut at 10 p in a cryostat. The frozen sections were lyophilized at -40°C and stored at -76°C until assayed. Discrete cell groups which contained primarily choroid or ciliary cells were dissected free from capsular tissue and weighed on a quartzfiber microbalance (2, 4). Individual samples, from 80 to 300 ng dry weight, were transferred directly to 10 ~1 chilled ChAc incubation medium in 2.5 X SO-mm glass tubes and the entire tube was immediately frozen. When 15 to 20 tubes had accumulated, the samples were incubated at 37°C. Preparation of [ 1J4C] acetyl-CoA. Labeled acetyl-CoA was prepared from [ 1J4C] acetic anhydride (Amersham/Searle, 116 mCi/mmol) and CoA (P-L Biochemicals) according to the procedure of Hebb et al. (18). TABLE .4nalyses
Days incubated
9 II 13 1.5 17 I9 21
AChE”
Ganglion dry weight (/a) 9.8 19.3 23.9 30.9 42.6 45.8 51.0
a Micromoles a Micromoles c Micromoles J All values
f f f f f f f
1
of Embryonic Chick Ciliary Ganglia Stages of Development
1.8 (9)J 3.8 (8) 5.0 (8) 4.8 (10) 7.5 (8) 8.1 (9) 8.1 (6)
171.3 154.6 147.3 121.4 121.1 109.2 130.0
zk f f f f f f
49.3 30.8 27.7 43.1 28.4 36.6 29.8
at Successive
ChAch
(5) (6) (6) (7) (6) (7) (6)
18.1 23.4 30.5 59.8 69.3 113.5 142.9
xk f f f i f f
2.9 6.6 7.6 14.4 22.6 38.4 27.5
CK”
(4) (5) (5) (6) (6) (7) (6)
40.7 39.3 27.5 27.8 24.3 21.2 22.1
f 4.8 f 7.2 f 12.2 f 9.0 zk 3.7 f 3.0 f 4.3
(4) (3) (2) (5) (4) (6) (6)
of acetylthiocholine hydrolyzed per gram dry weight per minute at 22’C. of acetylcholine synthesized per gram dry weight per hour at 37’C. of choline phosphorylated per gram dry weight per hour at 37%. f standard deviation (n).
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Following synthesis, the [ 1J4C]acetyl-CoA was assayed enzymatically with citrate synthase (13) and the specific radiochemical activity of the substrate was calculated. The [ 1-14C] acetyl-CoA prepared for these studies had a specific radiochemical activity of 2.33 mCi/mmol. With this substrate, blank values for the ChAc assay were routinely 35 to 45 cpm over background, or from 0.07 to 0.08% of the total counts in the incubation medium. Choline Acetyltransferase Assay. The enzymatic activity of ChAc was measured by a sensitive radiochemical assay procedure in which [ l-14C]acetyl-CoA and choline were used as substrates and the radioactivity of the reaction product, [ 1J4C] acetylcholine, was measured (16, 18). The incubation medium contained, in final concentrations: 0.068 M phosphate buffer, pH 7.4; 0.3 M NaCl; 0.5 mM EDTA (ethylenediaminetetraacetic acid) ; 0.14 mM physostigmine sulfate; 12 ITlM choline chloride; 0.05% bovine serum albumin ; and 0.45 mM [ 1-14C]acetyl-CoA (2.33 mCi/ mmol). Homogenate preparations of whole ganglia were incubated from 1 to 2 hr at 37°C; the 80 to 300-ng samples of choroid and ciliary cells were incubated in a final volume of 10 d for 3 hr at 37°C. After incubation, the tubes were transferred to an ice bath and the [ 1-14C] acetylcholine formed was extracted from the medium by the addition of 150 ~1 organic cationic exchange medium (30 mg sodium tetraphenylboron/ml ally1 cyanide) modified from Fonnum (16). Homogenate assays were made in triplicate and the enzyme activity was expressed as micromoles acetylcholine synthesized/g dry weight/hr at 37°C. Acetylcholinesterase Assay. The enzymatic activity of AChE was measured with a modification of the spectrophotometric procedure of Ellman et al. (15) in which acetylthiocholine (AThCh) is used as the substrate, The incubation medium contained in final concentration : 0.1 M phosphate buffer, pH 8.0; 0.5 mM AThCh iodide; 0.3 mM DTNB (5,5’-dithiobis-2nitrobenzoic acid) ; 0.01% Triton X-100 ; and from 1.O to 3.0 pg dry weight of tissue sample in a final volume of 300 ~1. In addition to these reactants, the blank contained 3 x 1G-5 M physostigmine sulfate. Thus AChE activity described herein is defined as that enzymatic hydrolysis of AThCh which is sensitive to physostigmine at a concentration of 3 x 1O-5 M. All ganglia were assayed in triplicate and enzymatic activity was expressed as micromoles of AThCh hydrolyzed per gram dry weight per minute at 22°C. Cholke Kinase Assa.y. The activity of CK was measured by a sensitive radiochemical assay procedure which permits the measurement of low levels of CK activity in the presence of other ATP (adenosine triphosphate) -metabolizing enzymes (8). In nervous tissue preparations ATPases (adenosine triphosphatases) and adenylate kinase activities are so high, relative to CK activity, that inhibitory levels of ADP (adenosine diphosphate) and AMP (adenosine 5’-monophosphate) accumulate rapidly. To
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CK,
AND
AchE
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GANGLIA
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overcome this problem, an enzymatic ATP-regenerating system consisting of phospho (en01)pyruvate and pyruvate kinase and a Na-K-ATPase inhibitor, ouabain, were included in the incubation medium. The medium contained, in final concentrations, 0.1 M Z-amino-2-methyl-1,3-propanediol buffer, pH 9.0 ; 10 mM MgClz ; 10 mM ATP ; 10 mM [methyZ-14C]choline ( 1 mCi/mmol) ; 1 .O mM ouabain ; 20 mM phospho (enol)pyruvate ; and 81 units/ml pyruvate kinase (8). After incubation, the unphosphorylated [metlzyV*C]choline was extracted from the incubation medium with an organic cationic exchanger (30 mg sodium tetraphenylboron/ml ally1 cyanide), and an aliquot of the remaining aqueous phase containing the [methyl-l*C] phosphorylcholine was counted in a liquid scintillation counter (8). Assays were made in triplicate, in a final volume of 25 ~1, and enzyme activity was expressed as micromoles of choline phosphorylated per gram dry weight per hour at 37’ C. RESULTS Enzyme data from the individual ciliary ganglia are summarized in Table 1. The specific activity of both AChE and CK decreased throughout the period studied, although the total activity per ganglion continued to increase. The specific activity of ChAc, on the other hand, increased nearly eight-fold throughout the same period (Table 1). Utilizing the cell-count data of Landmesser and Pilar (20), the enzyme activities were recalculated as activity per cell, with one cell representing the ganglionic neuron and its preganglion terminal(s). These data are plotted as a function of developmental age in Fig. 1. In femtomoles per cell per unit time, CK and AChE activity increased nearly six- and eightfold, respectively, during this period of development. The ChAc activity increased more than SO-fold during the same period; from 29.5 to 2428 fmol ACh synthesized/cell/hr at 37°C. Data from the separate assay of choroid and ciliary cells, with their corresponding preganglionic terminals, are summarized in Table 2. The rate of increase in specific activity from 12 to 20 days of incubation for both cell populations is similar to the rate of increase in specific activity for the entire ganglion during the comparable period (Table 1). However, the actual values for specific activity are five to six times greater than those obtained for whole ganglia. A major decrease in the specific activity for both cell populations was noted in ganglia at 1 day after hatching. A decrease of similar magnitude in the specific activity of ChAc at hatching has been noted in previous studies on the chick spinal cord (9, 21). DISCUSSION The development of ChAc and AChE activity in the chick ciliary ganglion has been described in two recent publications (23, 25). Our
708
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sooo2000.
1000.
500.
E ‘Z .% 200. 3 *
100: :
E 2 E ,o
50.
20 I 9
II 13 Developmental
15 I7 Age (days)
2
I9 &h)
FIG. 1. Enzyme activity of the ciliary ganglion expressed as femtomoles per cell per unit time plotted as a function of developmental age. Values were calculated from the enzyme data of Table 1 and the cell-count data of Landmesser and Pilar (20). One cell represents the ganglionic neuron and its associated preganglionic terminal(s) AChE, log femtomoles acetylthiocholine hydrolyzed per cell per minute at 22°C; ChAc, log femtomoles of acetylcholine synthesized per cell per hour at 37°C; CK, log femtomoles of choline phosphorylated per cell per hour at 37°C.
general findings are similar ; however, there are several points of difference that warrant discussion. Although the work of Sorimachi and Kataoka (25) emphasized the posthatching development, the prehatching pattern TABLE Choline
Acetyltransferase Activity Embryonic Chick
2 in Choroid and Ciliary Ciliary Ganglia
Days
of incubation
Choroid
1 day
12 14 16 20 posthatching
173 245 382 767 441
f f f f f
cells 26 18 46 44 24
(5)S (4) (4) (4) (3)
Cells
from
Ciliary 169 329 328 589 326
f f f f f
cells 18 38 31 51 13
(5)” (3) (5) (4)b (7)b
a Choline acetyltransferase activity: micromoles of acetylcholine synthesized per gram dry weight per hour at 37°C f standard deviation (n). * Values for ciliary cells were significantly less than for choroid cells. When the standard t test was used, at 20 days of incubation, P < 0.05, and at 1 day posthatching, P < 0.001.
ChAc, CK, AND AchE IN CILIARY GANGLIA
709
for ChAc activity was similar to ours, in that the specific activity of ChAc increased markedly with most of the increase occurring after 13 to 14 days of incubation. Pilar and his co-workers, (17, 23) described a gradual increase in specific activity after 7 days of incubation with a very pronounced increase occurring after day 10. Different levels of ChAc activity were reported in these studies. At hatching, Sorimachi and Kataoka (25) reported a total activity of 90 nmol/hr/pair of ganglia, whereas our activity was only 14.6 nmol/hr/pair of ganglia (calculated from the data of Table 1). Our values for the specific activity of ChAc (Table 1) are at least four orders of magnitude greater than the 6.37 x IO-10 mol/g/hr of Pilar et al. (23). The values in Table 1 are similar in magnitude to those reported for chick spinal cord (3, 5, 9) and rat spinal cord (5, 6). Buckley et al. (1) found a similar level of activity (24.8 wol/g wet weight/hr) in cat ciliary ganglion and Dolezalova et al. (14) reported 73.77 pmol/g dry weight/hr in sympathetic ganglia from 7-day-old chicks. These are of the same order of magnitude as our data. All synapses in the ciliary ganglion are functional at the earliest age of this study (20), hence the marked increase in activity observed (Table 1) cannot be associated with the initial stage of synaptogenesis. When cellcount data (20) are utilized, the enzyme data of Table 1 can be expressed as activity per cell, where one cell is defined as the postganglionic neuron and its associatedpreganglionic terminal(s) (Fig. 1) . These data indicate that a synthetic capacity of less than 29.5 fmol/hr is sufficient for a functional cholinergic synapse. In the adult, 60% of the ChAc activity is preganglionic and the remainder is postganglionic (17). In this study, it is not possible to estimate the percentage of the activity associated with the preganglionic terminal. Experiments to differentiate between the preganglionic and postganglionic components are in progress. The ganglionic neurons begin to establish functional contact with the peripheral field at 9 to 10 days of incubation, and the morphology of the ganglionic synapsematures rapidly after 15 days (17, 20). Increased ChAc activity in the ganglion is probably associated with both events. In the developing spinal cord, a major component of ChAc activity parallels the establishment of functional neuromuscular junctions in the periphery (5, 9). A sharp decreasein the latency of the evoked responseis one characteristic of synaptic maturation in both the choroid and the ciliary cell populations (19). When these data are expressed as the speed with which the potential traverses the ganglion (the reciprocal of the latency of the evoked response) a marked parallel with ChAc activity is noted (Fig. 2). The reduced latency is due in large measure to an increase in conduction velocity of both preganglionic and postganglionic fibers although these increases alone are not sufficient to account for the entire reduction in
710
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II
AND
13
Developmental
NARAYANAN
15 Age
17
19
2
(days)
FIG. 2. Chick ciliary ganglion development. Choline acetyltransferase activity from Fig. 1 compared with the reciprocal of the latency of the evoked response of the ciliary cells, log (l/milliseconds), as calculated from the data of Landmesser and Pilar (19).
latency time (19). In our comparison, the reciprocal of the latency of the evoked responseserves as an index of synaptic maturation. If other parameters of synaptic maturation such as increased number of synaptic vesicles, shortened duration of the chemical postsynaptic potential, and increased resistance to fatigue with repetitive stimulation (19) were sufficiently quantitated, similar parallels with increased ChAc activity could be made. Both the choroid and the ciliary cell populations (Table 2) have rates of increase in ChAc activity which parallel the rate for the whole ganglion (Table l), although the actual values for the specific activity are much greater. The specific enzymatic activity of the ganglion is diluted by the capsular and connective tissues which are devoid of ChAc activity and by the bundles of intraganglionic nerve fibers which have relatively low ChAc activity (Burt, unpublished observations). The sharp drop in specific activity at hatching is of similar magnitude to that reported by Burt and Narayanan (9) and by Marchisio and Consolo (21) for the chick spinal cord at hatching. There was no significant difference in ChAc activity for the choroid and ciliary cell populations at 12, 14, and 16 days of incubation (Table 2). However, at 20 days of incubation and at 1 day after
chAc,
CK,
AND
AchE
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CILIARY
GANGLIA
711
hatching, the choroid cells had significantly higher levels of ChAc activity than the ciliary cells. With the standard t test, P values of less than 0.05 and 0.001 were obtained for the two ages, respectively. This difference may reflect the fact that the choroid cells are much smaller than the ciliary cells and a sample of ciliary cells, per unit weight, would contain fewer synaptic units (i.e., postganglionic neurons and their associated preganglionic terminals). An alternative explanation is that the advent of direct electrical coupling in the ciliary cells, in addition to the chemical synapse, reduces the demand for acetylcholine synthetic capacity. There was little change in the specific activity of AChE during the period studied (Table 1). Our data are similar in both developmental pattern and in level of activity to those of Sorimachi and Kataoka (25). Pilar et al. (23) described a tenfold increase in the specific activity of AChE which began at stage 32 (7.5 days) and reached adult values at stage 38 (12 days). We found the specific activity of AChE to be highest in the youngest ganglion of this study, 9 days of incubation (Table 1). Total activity (nanomoles per minute per pair of ganglia, calculated from Table 1) did increase nearly fourfold from 9 to 21 days of incubation, an increase that is also reflected in the activity per cell of Fig. 1. The significant point, apparent in all three studies, is that the ontogenetic pattern of AChE activity is quite different from that of ChAc activity. This was also noted in the developing chick spinal cord (3, 5, 9). In both the chick spinal cord and the ciliary ganglion, AChE reached adult levels of activity much earlier in development than did the ChAc. The activity of CK follows a developmental pattern which is nearly identical to that of AChE (Table 1, Fig. 1). In the developing rat spinal cord, the specific activity of choline kinase reached a peak 4 days before birth and subsequently decreased to about 50% of that maximum by birth and remained relatively constant through 46 days of age (7). This transient peak preceded the rapid increase in ChAc activity by 6 to 7 days. In the rat spinal cord, however, the pattern of AChE activity (6) did not parallel that of CK (7). Choline kinase is the initial enzyme in the Kennedy pathway for lecithin synthesis and, if the enzyme activity has a morphological parallel in the developing rat spinal cord, it would be the rapid synthesis and growth of neuronal membranes of which lecithin is a principal constituent (7). A parallel between either CK or AChE activity and a morphological or physiological parameter of ciliary ganglion development is not apparent from these studies. REFERENCES G., S. CONSOLO, E. GIACOBINI, and F. SJOQVIST. 1967.Cholineacetylase h innervatedand denervatedsympatheticganglia and ganglion cells of the
1. BUCKLEY,
cat. Acta Physiol. Scud
71: 348-356.
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A. M. 1966. Modification of the Lowry quartz fiber “fishpole” ultramicrobalance. dlicrorhcln. J. 11 : 18-25. 3. BURT, A. M. 1968. Acetylcholinesterase and choline acetyltransferase activity in the developing chick spinal cord. J. Eafi. Zoo/. 169: 107-112. 4. BURT, A. M. 1969. Quantitative histochemistry : The weighing and manipulation of nanogram tissue samples, pp. 183-190. III. “Autoradiography of Diffusible Substances.” I,. J. Roth and W. E. Stumpf [Eds.]. Academic Press, New York. 5. BURT, A. M. 1973. Choline acetyltransferase and neuronal maturation, pp. 245252. Zrr “Neurobiological Aspects of Maturation and Aging, Progress in Brain Research,” Vol. 40. D. H. Ford [Ed.]. Elsevier, Amsterdam. 6. BURT, A. M. 1975. Choline acetyltransferase and acetylcholinesterase in the developing rat spinal cord. E-VP. ~Vc~rrol. 47 : 173-180. 7. BURT, A. M. 1977. Choline kinase activity in the developing rat spinal cord: Differential development of hemicholinium-3 sensitive and insensitive activity. Submitted for publication. 8. BURT, A. M., and S. A. BRODY. 1975. The measurement of choline kinase activity in rat brain: The problem of alternate pathways of ATP metabolism. Amd. Biochew. 65 : 215-224. 9. BURT, A. M., and C. H. NARAYANAN. 1970. Effect of extrinsic neuronal connections on development of acetylcholinesterase and choline acetyltransferase activity in the ventral half of the chick spinal cord. Esfi. Netwol. 29: 201-210. 10. BURT, A. M., and C. H. NARAYANAN. 1972. Development of glucose-6-phosphate, malate and glutamate dehydrogenase activities in the ventral half of the chick spinal cord in the absence of extrinsic neuronal connections. Exp. Neural. 34: 342-353. 11. BURT, A. M., and C. H. N~RA~AKAN. 1975. The developmental patterns of choline acetyltransferase, choline kinase and acetylcholinesterase activity in the chick ciliary ganglion. Neurosci. i2bstr. 1 : 386. 12. CANTINO, D., and E. MUGNAINI. 1974. Adrenergic imlervation of the parasympathetic ciliary ganglion in the chick. Science 185: 279-281. 13. CHASE, J. F. A. 1967. pH-Dependence of carnitine acetyltransferase activity. Biochem. J. 104: 503-509. 14. DOLEZALOVA, H., E. GIACOBINI, G. GIACOBIXI, A. ROSSI, and G. TOSCHI. 1974. Developmental variations of choline acetyltransferase, dopamine+-hydroxylase and monoamineoxidase in chicken embryo and chicken sympathetic ganglia. Bra& Rex. 73: 309-320. 15. ELLMAN, G. L., Ii. D. COURTNEY, V. ANDRES, JR., and R. M. FEATHERSTONE. 1961. A new and rapid calorimetric dete&&tion of acetylcholinesterase activity. Biochcvr. Pharmacol. 7 : 88-95. 16. FONNUM, F. 1969. Radiochemical micro assays for the determination of choline acetyltransferase and acetylcholinesterase activities. Biochem. J. 115 : 465-472. 17. GIACOBINI, E. 1975. Neuronal control of neurotransmitter biosynthesis during development. J. Neurosci. Res. 1: 315-331. 18. HEBB, C., S. P. MANN, and J. MEAD. 1975. Measurement and activation of choline acetyltransferase. Biochenz. Pimmacol. 24 : 1007-1011. 19. LANDMESSER, L., and G. PILAR. 1972. The onset and development of transmission in the chick ciliary ganglion. J. Ph>wioZ. 222: 691-713. 20. LANDMESSER, L., and G. PILAR. 1974. Synaptic transmission and cell death during normal ganglionic development. J. Physiol. 241 : 737-749. 21. MARCHISIO, P. C., and G. GIACOBINI. 1969. Choline acetyltransferase activity in the central nervous system of the developing chick. Brain Res. IS: 301-304. 2.
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chx,
CK,
AND
AchE
IN
CILIARY
GANGLIA
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R., G. PILAR, and J. N. WEAKLY. 1971. Characterization of two ganglion cell populations in avian ciliary ganglia. Bruin Res. 25: 317-334. 23. PILAR, G., V. CHIAPPINELLI, H. UCHIMURA, and E. GIACOBINI. 1974. Changes of acetylcholinesterase ( AChE) and cholineacetyltransferase (ChAc) correlated with the formation of cholinergic synapses in the chick embryo. Physiologist 17 : 307. 24. PILAR, G., D. J. JENDEN, and B. CAMPBELL. 1973. Distribution of acetylcholine in the normal and denervated pigeon ciliary ganglion. Brain Rcs. 49: 245-256. 25. SORIMACHI, M., and K. KATAOKA. 1974. Developmental change of choline acetyltransferase and acetylcholinesterase in the ciliary and the superior cervical ganglion of the chick. Brain Res. 70: 123-130. 22. MARWITT,
NEUROLOGY
53, 703-713 (1976)
Choline Acetyltransferase, Choline Kinase, and Acetylcholinesterase Activities during the Development of the Chick Ciliary Ganglion ALVIN
M. BURT 1 AND C. H. NARAYANAN
Department of Anatomy, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, and Department of Anatomy, Louisiana State University School of Medicine, New Orleans, Louisiana 70112 Received
July
12,1976
The specific activity of choline acetyltransferase, choline kinase, and acetylcholinesterase was measured in ciliary ganglia of chick embryos from 9 days of incubation to hatching. The specific activity of choline acetyltransferase increased eightfold during this period and the activity per cell increased from 29.5 fmol/hr/cell at 9 days to 2428 at hatching. The specific activity of both choline kinase and acetylcholinesterase was highest at 9 days of incubation although the total activity per ganglion increased nearly sixand eightfold, respectively, during the developmental period studied. The specific activity of choline acetyltransferase in the choroid cells was significantly higher than that in the ciliary cells after 16 days of incubation, with values of 767 and 589 pmol/g dry weight/hr, respectively, at 20 days of incubation. When correlated with the morphological and physiological development, the results of this study indicate that relatively low levels of acetylcholine synthetic capacity (29.5 fmol/hr) are sufficient for a functional cholinergic synapse. The major increase in choline acetyltransferase activity parallels synaptic maturation and the establishment of functional connections by the postganglionic neurons with the peripheral field.
INTRODUCTION A series of experimental and normal studies has related the development of choline acetyltransferase ( ChAc ; acetyl-CoA :choline O-acetyltrans1 This investigation was supported by a grant from the Dysautonomia Foundation, Inc., and by Research Grant NS-07441 from the National Institute of Neurological and Communicative Disorders and Stroke, National Institutes of Health. A preliminary report has been presented (11). Send correspondence to Dr. Burt at the Vanderbilt University address. 703 ItIC.
reserved.
704
BURT
AND
NARAYANAN
ferase, EC 2.3.1.6) activity in the chick spinal cord to synaptogenesis (3, 5, 9). These data, however, do not indicate the particular phase of synaptogenesis concerned but only that the increased activity of ChAc was dependent on synapse formation and that, when synapse formation was experimentally reduced, there was a corresponding reduction in ChAc activity, a reduction that was not reflected in the levels of acetylcholinesterase (AChE ; acetylcholine acetylhydrolase, EC 3.1.1.7) activity and in the activity of enzymes of intermediary carbohydrate metabolism (9, 10). Because both the preganglionic and postganglionic elements are cholinergic (24), the chick ciliary ganglion should provide a model system in which to study cholinergic synaptogenesis. Although some adrenergic nerve endings are present, they do not make direct synaptic contact with either cholinergic element (12). In addition, the developmental morphology and physiology have been well documented (19, 20, 22) and precise correlations can be made with the developmental neurochemistry. The preganglionic fibers of the ciliary ganglion arise in the accessory oculomotor nucleus and terminate in a one-on-one fashion with the neurons of the ganglion (22). Two morphologically distinct populations of neurons, the choroid and ciliary cells, are recognizable as early as 10 days of incubation and give rise to the postganglionic choroid and ciliary nerves, respectively (19). Synaptic transmission begins at 5 days and involves 100% of the neurons as early as 8 days of incubation, although morphological evidence for presynaptic and postsynaptic thickenings is still scant (19). At this stage, the transmission in both populations of neurons is chemical and acetylcholine is the apparent neurotransmitter. The morphological maturation of the synapse is much slower, and by 14 days of incubation synaptic vesicles are still sparse (19). At 15 days of incubation, direct electrical coupling is first noted in the ciliary cell population and, by 1 to 2 days after hatching, 80% of the ciliary cells show direct electrical transmitting synapses in addition to the chemical synapse. Synapses on the choroid cells, on the other hand, remain entirely chemical throughout development (19). In the present study, the activity of ChAc, AChE, and choline kinase (CK ; ATP : cholinephosphotransferase, EC 2.7.1.32) was studied in individual ciliary ganglia of chick embryos from 9 to 21 days of incubation. In addition, the choroid and the ciliary neurons, along with their corresponding presynaptic terminals, were assayed separately for ChAc activity. Our data continue to support the direct association of ChAc with synaptogenesis and further suggest that very low levels of enzyme activity are sufficient for a physiologically functional synapse. The major increase in ChAc activity is related to the morphological and physiological maturation of the synapse and to the establishment of functional contact between the postganglionic neuron and its peripheral field.
chAc,
CK,
AND
AchE
METHODS
IN
AND
CILIARY
705
GANGLIA
MATERIALS
Preparation of Samples. Fertile white Leghorn eggs were incubated at 37.8”C in a forced-air incubator for 9 to 21 days. Individual ciliary ganglia were dissected free from surrounding tissue, frozen rapidly in isopentane chilled to approximately -155°C with liquid Na, lyophilized at -40°C and stored at -76°C until assayed. Individual lyophilized ganglia were freed from adhering tissues, trimmed of nerve roots, weighed on a Cahn electrobalance, and homogenized in microglass tissue grinders with 0.05% Triton X-100 at a final concentration of from 0.2 to 0.5 pg tissue dry weight/$. Individual ganglia ranged, in dry weight, from 9 to 10 pg at 9 days of incubation to 50 to 55 g at hatching (Table 1). Homogenate aliquots were assayed immediately for AChE, ChAc, and CK activities. Other ganglia were rapidly frozen and cut at 10 p in a cryostat. The frozen sections were lyophilized at -40°C and stored at -76°C until assayed. Discrete cell groups which contained primarily choroid or ciliary cells were dissected free from capsular tissue and weighed on a quartzfiber microbalance (2, 4). Individual samples, from 80 to 300 ng dry weight, were transferred directly to 10 ~1 chilled ChAc incubation medium in 2.5 X SO-mm glass tubes and the entire tube was immediately frozen. When 15 to 20 tubes had accumulated, the samples were incubated at 37°C. Preparation of [ 1J4C] acetyl-CoA. Labeled acetyl-CoA was prepared from [ 1J4C] acetic anhydride (Amersham/Searle, 116 mCi/mmol) and CoA (P-L Biochemicals) according to the procedure of Hebb et al. (18). TABLE .4nalyses
Days incubated
9 II 13 1.5 17 I9 21
AChE”
Ganglion dry weight (/a) 9.8 19.3 23.9 30.9 42.6 45.8 51.0
a Micromoles a Micromoles c Micromoles J All values
f f f f f f f
1
of Embryonic Chick Ciliary Ganglia Stages of Development
1.8 (9)J 3.8 (8) 5.0 (8) 4.8 (10) 7.5 (8) 8.1 (9) 8.1 (6)
171.3 154.6 147.3 121.4 121.1 109.2 130.0
zk f f f f f f
49.3 30.8 27.7 43.1 28.4 36.6 29.8
at Successive
ChAch
(5) (6) (6) (7) (6) (7) (6)
18.1 23.4 30.5 59.8 69.3 113.5 142.9
xk f f f i f f
2.9 6.6 7.6 14.4 22.6 38.4 27.5
CK”
(4) (5) (5) (6) (6) (7) (6)
40.7 39.3 27.5 27.8 24.3 21.2 22.1
f 4.8 f 7.2 f 12.2 f 9.0 zk 3.7 f 3.0 f 4.3
(4) (3) (2) (5) (4) (6) (6)
of acetylthiocholine hydrolyzed per gram dry weight per minute at 22’C. of acetylcholine synthesized per gram dry weight per hour at 37’C. of choline phosphorylated per gram dry weight per hour at 37%. f standard deviation (n).
706
BURT
AND
NARAYANAN
Following synthesis, the [ 1J4C]acetyl-CoA was assayed enzymatically with citrate synthase (13) and the specific radiochemical activity of the substrate was calculated. The [ 1-14C] acetyl-CoA prepared for these studies had a specific radiochemical activity of 2.33 mCi/mmol. With this substrate, blank values for the ChAc assay were routinely 35 to 45 cpm over background, or from 0.07 to 0.08% of the total counts in the incubation medium. Choline Acetyltransferase Assay. The enzymatic activity of ChAc was measured by a sensitive radiochemical assay procedure in which [ l-14C]acetyl-CoA and choline were used as substrates and the radioactivity of the reaction product, [ 1J4C] acetylcholine, was measured (16, 18). The incubation medium contained, in final concentrations: 0.068 M phosphate buffer, pH 7.4; 0.3 M NaCl; 0.5 mM EDTA (ethylenediaminetetraacetic acid) ; 0.14 mM physostigmine sulfate; 12 ITlM choline chloride; 0.05% bovine serum albumin ; and 0.45 mM [ 1-14C]acetyl-CoA (2.33 mCi/ mmol). Homogenate preparations of whole ganglia were incubated from 1 to 2 hr at 37°C; the 80 to 300-ng samples of choroid and ciliary cells were incubated in a final volume of 10 d for 3 hr at 37°C. After incubation, the tubes were transferred to an ice bath and the [ 1-14C] acetylcholine formed was extracted from the medium by the addition of 150 ~1 organic cationic exchange medium (30 mg sodium tetraphenylboron/ml ally1 cyanide) modified from Fonnum (16). Homogenate assays were made in triplicate and the enzyme activity was expressed as micromoles acetylcholine synthesized/g dry weight/hr at 37°C. Acetylcholinesterase Assay. The enzymatic activity of AChE was measured with a modification of the spectrophotometric procedure of Ellman et al. (15) in which acetylthiocholine (AThCh) is used as the substrate, The incubation medium contained in final concentration : 0.1 M phosphate buffer, pH 8.0; 0.5 mM AThCh iodide; 0.3 mM DTNB (5,5’-dithiobis-2nitrobenzoic acid) ; 0.01% Triton X-100 ; and from 1.O to 3.0 pg dry weight of tissue sample in a final volume of 300 ~1. In addition to these reactants, the blank contained 3 x 1G-5 M physostigmine sulfate. Thus AChE activity described herein is defined as that enzymatic hydrolysis of AThCh which is sensitive to physostigmine at a concentration of 3 x 1O-5 M. All ganglia were assayed in triplicate and enzymatic activity was expressed as micromoles of AThCh hydrolyzed per gram dry weight per minute at 22°C. Cholke Kinase Assa.y. The activity of CK was measured by a sensitive radiochemical assay procedure which permits the measurement of low levels of CK activity in the presence of other ATP (adenosine triphosphate) -metabolizing enzymes (8). In nervous tissue preparations ATPases (adenosine triphosphatases) and adenylate kinase activities are so high, relative to CK activity, that inhibitory levels of ADP (adenosine diphosphate) and AMP (adenosine 5’-monophosphate) accumulate rapidly. To
chAc,
CK,
AND
AchE
IN
CILIARY
GANGLIA
707
overcome this problem, an enzymatic ATP-regenerating system consisting of phospho (en01)pyruvate and pyruvate kinase and a Na-K-ATPase inhibitor, ouabain, were included in the incubation medium. The medium contained, in final concentrations, 0.1 M Z-amino-2-methyl-1,3-propanediol buffer, pH 9.0 ; 10 mM MgClz ; 10 mM ATP ; 10 mM [methyZ-14C]choline ( 1 mCi/mmol) ; 1 .O mM ouabain ; 20 mM phospho (enol)pyruvate ; and 81 units/ml pyruvate kinase (8). After incubation, the unphosphorylated [metlzyV*C]choline was extracted from the incubation medium with an organic cationic exchanger (30 mg sodium tetraphenylboron/ml ally1 cyanide), and an aliquot of the remaining aqueous phase containing the [methyl-l*C] phosphorylcholine was counted in a liquid scintillation counter (8). Assays were made in triplicate, in a final volume of 25 ~1, and enzyme activity was expressed as micromoles of choline phosphorylated per gram dry weight per hour at 37’ C. RESULTS Enzyme data from the individual ciliary ganglia are summarized in Table 1. The specific activity of both AChE and CK decreased throughout the period studied, although the total activity per ganglion continued to increase. The specific activity of ChAc, on the other hand, increased nearly eight-fold throughout the same period (Table 1). Utilizing the cell-count data of Landmesser and Pilar (20), the enzyme activities were recalculated as activity per cell, with one cell representing the ganglionic neuron and its preganglion terminal(s). These data are plotted as a function of developmental age in Fig. 1. In femtomoles per cell per unit time, CK and AChE activity increased nearly six- and eightfold, respectively, during this period of development. The ChAc activity increased more than SO-fold during the same period; from 29.5 to 2428 fmol ACh synthesized/cell/hr at 37°C. Data from the separate assay of choroid and ciliary cells, with their corresponding preganglionic terminals, are summarized in Table 2. The rate of increase in specific activity from 12 to 20 days of incubation for both cell populations is similar to the rate of increase in specific activity for the entire ganglion during the comparable period (Table 1). However, the actual values for specific activity are five to six times greater than those obtained for whole ganglia. A major decrease in the specific activity for both cell populations was noted in ganglia at 1 day after hatching. A decrease of similar magnitude in the specific activity of ChAc at hatching has been noted in previous studies on the chick spinal cord (9, 21). DISCUSSION The development of ChAc and AChE activity in the chick ciliary ganglion has been described in two recent publications (23, 25). Our
708
BURT
AND
NARAYANAN
sooo2000.
1000.
500.
E ‘Z .% 200. 3 *
100: :
E 2 E ,o
50.
20 I 9
II 13 Developmental
15 I7 Age (days)
2
I9 &h)
FIG. 1. Enzyme activity of the ciliary ganglion expressed as femtomoles per cell per unit time plotted as a function of developmental age. Values were calculated from the enzyme data of Table 1 and the cell-count data of Landmesser and Pilar (20). One cell represents the ganglionic neuron and its associated preganglionic terminal(s) AChE, log femtomoles acetylthiocholine hydrolyzed per cell per minute at 22°C; ChAc, log femtomoles of acetylcholine synthesized per cell per hour at 37°C; CK, log femtomoles of choline phosphorylated per cell per hour at 37°C.
general findings are similar ; however, there are several points of difference that warrant discussion. Although the work of Sorimachi and Kataoka (25) emphasized the posthatching development, the prehatching pattern TABLE Choline
Acetyltransferase Activity Embryonic Chick
2 in Choroid and Ciliary Ciliary Ganglia
Days
of incubation
Choroid
1 day
12 14 16 20 posthatching
173 245 382 767 441
f f f f f
cells 26 18 46 44 24
(5)S (4) (4) (4) (3)
Cells
from
Ciliary 169 329 328 589 326
f f f f f
cells 18 38 31 51 13
(5)” (3) (5) (4)b (7)b
a Choline acetyltransferase activity: micromoles of acetylcholine synthesized per gram dry weight per hour at 37°C f standard deviation (n). * Values for ciliary cells were significantly less than for choroid cells. When the standard t test was used, at 20 days of incubation, P < 0.05, and at 1 day posthatching, P < 0.001.
ChAc, CK, AND AchE IN CILIARY GANGLIA
709
for ChAc activity was similar to ours, in that the specific activity of ChAc increased markedly with most of the increase occurring after 13 to 14 days of incubation. Pilar and his co-workers, (17, 23) described a gradual increase in specific activity after 7 days of incubation with a very pronounced increase occurring after day 10. Different levels of ChAc activity were reported in these studies. At hatching, Sorimachi and Kataoka (25) reported a total activity of 90 nmol/hr/pair of ganglia, whereas our activity was only 14.6 nmol/hr/pair of ganglia (calculated from the data of Table 1). Our values for the specific activity of ChAc (Table 1) are at least four orders of magnitude greater than the 6.37 x IO-10 mol/g/hr of Pilar et al. (23). The values in Table 1 are similar in magnitude to those reported for chick spinal cord (3, 5, 9) and rat spinal cord (5, 6). Buckley et al. (1) found a similar level of activity (24.8 wol/g wet weight/hr) in cat ciliary ganglion and Dolezalova et al. (14) reported 73.77 pmol/g dry weight/hr in sympathetic ganglia from 7-day-old chicks. These are of the same order of magnitude as our data. All synapses in the ciliary ganglion are functional at the earliest age of this study (20), hence the marked increase in activity observed (Table 1) cannot be associated with the initial stage of synaptogenesis. When cellcount data (20) are utilized, the enzyme data of Table 1 can be expressed as activity per cell, where one cell is defined as the postganglionic neuron and its associatedpreganglionic terminal(s) (Fig. 1) . These data indicate that a synthetic capacity of less than 29.5 fmol/hr is sufficient for a functional cholinergic synapse. In the adult, 60% of the ChAc activity is preganglionic and the remainder is postganglionic (17). In this study, it is not possible to estimate the percentage of the activity associated with the preganglionic terminal. Experiments to differentiate between the preganglionic and postganglionic components are in progress. The ganglionic neurons begin to establish functional contact with the peripheral field at 9 to 10 days of incubation, and the morphology of the ganglionic synapsematures rapidly after 15 days (17, 20). Increased ChAc activity in the ganglion is probably associated with both events. In the developing spinal cord, a major component of ChAc activity parallels the establishment of functional neuromuscular junctions in the periphery (5, 9). A sharp decreasein the latency of the evoked responseis one characteristic of synaptic maturation in both the choroid and the ciliary cell populations (19). When these data are expressed as the speed with which the potential traverses the ganglion (the reciprocal of the latency of the evoked response) a marked parallel with ChAc activity is noted (Fig. 2). The reduced latency is due in large measure to an increase in conduction velocity of both preganglionic and postganglionic fibers although these increases alone are not sufficient to account for the entire reduction in
710
BURT
9
II
AND
13
Developmental
NARAYANAN
15 Age
17
19
2
(days)
FIG. 2. Chick ciliary ganglion development. Choline acetyltransferase activity from Fig. 1 compared with the reciprocal of the latency of the evoked response of the ciliary cells, log (l/milliseconds), as calculated from the data of Landmesser and Pilar (19).
latency time (19). In our comparison, the reciprocal of the latency of the evoked responseserves as an index of synaptic maturation. If other parameters of synaptic maturation such as increased number of synaptic vesicles, shortened duration of the chemical postsynaptic potential, and increased resistance to fatigue with repetitive stimulation (19) were sufficiently quantitated, similar parallels with increased ChAc activity could be made. Both the choroid and the ciliary cell populations (Table 2) have rates of increase in ChAc activity which parallel the rate for the whole ganglion (Table l), although the actual values for the specific activity are much greater. The specific enzymatic activity of the ganglion is diluted by the capsular and connective tissues which are devoid of ChAc activity and by the bundles of intraganglionic nerve fibers which have relatively low ChAc activity (Burt, unpublished observations). The sharp drop in specific activity at hatching is of similar magnitude to that reported by Burt and Narayanan (9) and by Marchisio and Consolo (21) for the chick spinal cord at hatching. There was no significant difference in ChAc activity for the choroid and ciliary cell populations at 12, 14, and 16 days of incubation (Table 2). However, at 20 days of incubation and at 1 day after
chAc,
CK,
AND
AchE
IN
CILIARY
GANGLIA
711
hatching, the choroid cells had significantly higher levels of ChAc activity than the ciliary cells. With the standard t test, P values of less than 0.05 and 0.001 were obtained for the two ages, respectively. This difference may reflect the fact that the choroid cells are much smaller than the ciliary cells and a sample of ciliary cells, per unit weight, would contain fewer synaptic units (i.e., postganglionic neurons and their associated preganglionic terminals). An alternative explanation is that the advent of direct electrical coupling in the ciliary cells, in addition to the chemical synapse, reduces the demand for acetylcholine synthetic capacity. There was little change in the specific activity of AChE during the period studied (Table 1). Our data are similar in both developmental pattern and in level of activity to those of Sorimachi and Kataoka (25). Pilar et al. (23) described a tenfold increase in the specific activity of AChE which began at stage 32 (7.5 days) and reached adult values at stage 38 (12 days). We found the specific activity of AChE to be highest in the youngest ganglion of this study, 9 days of incubation (Table 1). Total activity (nanomoles per minute per pair of ganglia, calculated from Table 1) did increase nearly fourfold from 9 to 21 days of incubation, an increase that is also reflected in the activity per cell of Fig. 1. The significant point, apparent in all three studies, is that the ontogenetic pattern of AChE activity is quite different from that of ChAc activity. This was also noted in the developing chick spinal cord (3, 5, 9). In both the chick spinal cord and the ciliary ganglion, AChE reached adult levels of activity much earlier in development than did the ChAc. The activity of CK follows a developmental pattern which is nearly identical to that of AChE (Table 1, Fig. 1). In the developing rat spinal cord, the specific activity of choline kinase reached a peak 4 days before birth and subsequently decreased to about 50% of that maximum by birth and remained relatively constant through 46 days of age (7). This transient peak preceded the rapid increase in ChAc activity by 6 to 7 days. In the rat spinal cord, however, the pattern of AChE activity (6) did not parallel that of CK (7). Choline kinase is the initial enzyme in the Kennedy pathway for lecithin synthesis and, if the enzyme activity has a morphological parallel in the developing rat spinal cord, it would be the rapid synthesis and growth of neuronal membranes of which lecithin is a principal constituent (7). A parallel between either CK or AChE activity and a morphological or physiological parameter of ciliary ganglion development is not apparent from these studies. REFERENCES G., S. CONSOLO, E. GIACOBINI, and F. SJOQVIST. 1967.Cholineacetylase h innervatedand denervatedsympatheticganglia and ganglion cells of the
1. BUCKLEY,
cat. Acta Physiol. Scud
71: 348-356.
712
BURT
ANL)
NARAYANAti
A. M. 1966. Modification of the Lowry quartz fiber “fishpole” ultramicrobalance. dlicrorhcln. J. 11 : 18-25. 3. BURT, A. M. 1968. Acetylcholinesterase and choline acetyltransferase activity in the developing chick spinal cord. J. Eafi. Zoo/. 169: 107-112. 4. BURT, A. M. 1969. Quantitative histochemistry : The weighing and manipulation of nanogram tissue samples, pp. 183-190. III. “Autoradiography of Diffusible Substances.” I,. J. Roth and W. E. Stumpf [Eds.]. Academic Press, New York. 5. BURT, A. M. 1973. Choline acetyltransferase and neuronal maturation, pp. 245252. Zrr “Neurobiological Aspects of Maturation and Aging, Progress in Brain Research,” Vol. 40. D. H. Ford [Ed.]. Elsevier, Amsterdam. 6. BURT, A. M. 1975. Choline acetyltransferase and acetylcholinesterase in the developing rat spinal cord. E-VP. ~Vc~rrol. 47 : 173-180. 7. BURT, A. M. 1977. Choline kinase activity in the developing rat spinal cord: Differential development of hemicholinium-3 sensitive and insensitive activity. Submitted for publication. 8. BURT, A. M., and S. A. BRODY. 1975. The measurement of choline kinase activity in rat brain: The problem of alternate pathways of ATP metabolism. Amd. Biochew. 65 : 215-224. 9. BURT, A. M., and C. H. NARAYANAN. 1970. Effect of extrinsic neuronal connections on development of acetylcholinesterase and choline acetyltransferase activity in the ventral half of the chick spinal cord. Esfi. Netwol. 29: 201-210. 10. BURT, A. M., and C. H. NARAYANAN. 1972. Development of glucose-6-phosphate, malate and glutamate dehydrogenase activities in the ventral half of the chick spinal cord in the absence of extrinsic neuronal connections. Exp. Neural. 34: 342-353. 11. BURT, A. M., and C. H. N~RA~AKAN. 1975. The developmental patterns of choline acetyltransferase, choline kinase and acetylcholinesterase activity in the chick ciliary ganglion. Neurosci. i2bstr. 1 : 386. 12. CANTINO, D., and E. MUGNAINI. 1974. Adrenergic imlervation of the parasympathetic ciliary ganglion in the chick. Science 185: 279-281. 13. CHASE, J. F. A. 1967. pH-Dependence of carnitine acetyltransferase activity. Biochem. J. 104: 503-509. 14. DOLEZALOVA, H., E. GIACOBINI, G. GIACOBIXI, A. ROSSI, and G. TOSCHI. 1974. Developmental variations of choline acetyltransferase, dopamine+-hydroxylase and monoamineoxidase in chicken embryo and chicken sympathetic ganglia. Bra& Rex. 73: 309-320. 15. ELLMAN, G. L., Ii. D. COURTNEY, V. ANDRES, JR., and R. M. FEATHERSTONE. 1961. A new and rapid calorimetric dete&&tion of acetylcholinesterase activity. Biochcvr. Pharmacol. 7 : 88-95. 16. FONNUM, F. 1969. Radiochemical micro assays for the determination of choline acetyltransferase and acetylcholinesterase activities. Biochem. J. 115 : 465-472. 17. GIACOBINI, E. 1975. Neuronal control of neurotransmitter biosynthesis during development. J. Neurosci. Res. 1: 315-331. 18. HEBB, C., S. P. MANN, and J. MEAD. 1975. Measurement and activation of choline acetyltransferase. Biochenz. Pimmacol. 24 : 1007-1011. 19. LANDMESSER, L., and G. PILAR. 1972. The onset and development of transmission in the chick ciliary ganglion. J. Ph>wioZ. 222: 691-713. 20. LANDMESSER, L., and G. PILAR. 1974. Synaptic transmission and cell death during normal ganglionic development. J. Physiol. 241 : 737-749. 21. MARCHISIO, P. C., and G. GIACOBINI. 1969. Choline acetyltransferase activity in the central nervous system of the developing chick. Brain Res. IS: 301-304. 2.
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chx,
CK,
AND
AchE
IN
CILIARY
GANGLIA
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R., G. PILAR, and J. N. WEAKLY. 1971. Characterization of two ganglion cell populations in avian ciliary ganglia. Bruin Res. 25: 317-334. 23. PILAR, G., V. CHIAPPINELLI, H. UCHIMURA, and E. GIACOBINI. 1974. Changes of acetylcholinesterase ( AChE) and cholineacetyltransferase (ChAc) correlated with the formation of cholinergic synapses in the chick embryo. Physiologist 17 : 307. 24. PILAR, G., D. J. JENDEN, and B. CAMPBELL. 1973. Distribution of acetylcholine in the normal and denervated pigeon ciliary ganglion. Brain Rcs. 49: 245-256. 25. SORIMACHI, M., and K. KATAOKA. 1974. Developmental change of choline acetyltransferase and acetylcholinesterase in the ciliary and the superior cervical ganglion of the chick. Brain Res. 70: 123-130. 22. MARWITT,
