EXPERIMENTAL
Choline
NEUROLOGY
47,
173-180
Acetyltransferase in the Developing ALVIN
Drparhmt
Receizled
(1975)
and Acetylcholinesterase Rat Spinal Cord M.
BURT
r
of Anatomy, Vanderbilt, Unizwsity NashzJle, Terumsee 37232 Septewber
14, 1974;
rrvision
receizled
School
Nozpember
of Mediciw,
24, 1974
Microchemical procedures were employed for the quantitative assay of choline acetyltransferase (ChAc; EC 2.3.1.6) and acetylcholinesterase (AChE; EC 3.1.1.7) activity in the lumbar spinal cord of the rat during late fetal and early postnatal development. ChAc activity remained low from the 13th to the 17th day of gestation, increased five-fold from the 17th day of gestation through the end of the second postnatal week, and changed little thereafter. AChE activity increased 19-fold from the 13th day of gestation through the end of the second postanatal week. The ontogenetic patterns for the two enzymes were identical after the 16th day of gestation. When these changes in enzyme activity were correlated with morphological and functional differentiation, the relationships were similar to those previously described for the chick. INTRODUCTION
Increased cholineacetyltransferase ( ChAc) activity has been correlated with the development of functional synaptic connections in the embryonic chick spinal cord (4). Based on the marked differences in the ontogenetic patterns of ChAc and acetylecholinesterase (AChE), it was further suggested that ChAc activity was a more reliable index of fuctional neuronal differentiation than was AChE activity. Experimental studies (6) have provided further support for the association of ChAc activity with synaptic differentiation. Recent studies on the development of the rat hippocampus have led to similar conclusions, namely that ChAc activity increases rapidly in parallel with synaptogenesis whereas AChE activity develops independently and is associated with the appearance of nerve fibers 1 Excellent technical assistance was provided by Mr. Charles Fouser. vestigation was supported by a grant from the Dysautonomia Foundation, Research Grants No. NS-07441 f rom the National Institute of Neurological and Stroke, National Institutes of Health. 173 Copyright All rights
1975 by Academic Press. Inc. o6 reproduction in any form reserved.
This inInc., and Diseases
174
ALVIN
M.
BURT
believed to be cholinergic in nature (15, 18). In addition to the association with synatptogenesis, a major component of the ChAc activity in the chick spinal cord appears to be associated with the biochemical and functional differentiation of the motor neuron per se (5). This suggestion was based on correlations between the ChAc activity in the spinal cord and both the appearance and subsequent increase in spontaneous motor activity (neurogenie in origin, 11, 13), and the appearance and subsequent increase in ChAc activity in motor nerve terminals in the differentiating axial and limb musculature ( 10). In the developing spinal cord of the rat, the temporal sequenceof motor neuron growth and differentiation and the development of functional synaptic connections differs from that of the chick (19). The onset of spontaneous motor neuron firing, resulting in spontaneous motor activity in the rat is not separated temporally from the appearance of synaptic connections and the onset of reflex responsesto exteroceptive stimuli. In the chick, however, development is characterized by an extensive “prereflexogenic” period, a period characterized by the presence of spontaneous motor activity and the absenceof functional reflex connections ( 11-13). If, therefore, the relationship between the enzymes ChAc and AChE and these processesof neurogenesisobserved for the chick (4-6) are valid for the rat, one would expect the ontogenetic patterns for the two enzymes to be similar during rat development. In order to test this hypothesis, the development of ChAc and AChE activities was studied in the lumbar spinal cord of the rat. METHODS
AND
MATERIALS
Pregnant albino rats, obtained from the Holtzman Company, Madison, Wis., were used throughout this study. The pregnant rats (13-20 days of gestation) were anesthetized with ether and the uterus was exposed by a midline incision in the abdomen. The individual fetuses were removed through an incision in the anti-mesometrial wall of the uterus. Newborn rats and rats of one through 11 days of postnatal development were killed by decapitation. Prejxmztion of Tissue Samples. The lumbar enlargement (representing spinal segments T1x-L4, inclusive) was dissected free from surrounding tissue. The enlargement from each fetal, newborn, or young rat was removed to a chilled glass microhomogenizer and ground in distilled water. Aliquots of the homogenate were transferred to small culture tubes (6 x 50 mm), immediately frozen, and lyophilized at -40 C. Following the lyophilization, sampletubes were sealedand stored at -75 C until assayed. There was no detectable loss of activity for either enzyme, ChAc or AChE, after freezing and lyophilization or after storage of the lyophilysate at -75 C for up to 4 months.
CHAC
AND
ACHE
IN
RAT
SPINAL
CORD
175
Choline Acetyltra.nsfemse Assay. The enzymatic activity of choline acetyltransferase was measured by a sensitive radiochemical assay procedure in which [ I-‘“Clacetyl-CoA and choline were used as substrates and the radioactivity of the reaction product, [ 1-14C] acetylcholine, was measured ( 16,20). Optimal assay conditions were determined for a preparation of newborn rat brain. The modified incubation medium contained in final concentration : 0.1 nr phosphate buffer, pH 7.3, 0.29 nr NaCl, 15 m&f MgSO,, 0.05% b ovine serum albumin, 0.2 mM physostigmine sulfate, 10 m&f choline and 0.2 mM [ l-14C]acetyI-CoA (2.35 uc/pmole), The radiochemically labeled acetyl-CoA was obtained from New England Nuclear, Boston, Mass. the unlabeled acteyl-CoA from Sigma Chemical Company, St. Louis, MO. In order to insure an optimal rate of reaction throughout the incubation period, sample concentrations were selected so that only from 1 to 5% of the substrate, [ 1-‘“Clacetyl-CoA, was consumed. The incubation was initiated by transferring the entire rack of tubes with the full complement of incubation components from an ice-water bath (&2 C) to a 37 C water bath and the reaction was terminated by returning the tubes to the ice-water bath. With the use of relatively thin-walled (0.5 mm) disposable glass tubes, the chillin g of the incubation medium (100 ~1) was sufficiently rapid to stop the enzymatic reaction. There was no detectable synthesis of additional acetylcholine in the most active samples alter transfer to the ice-water bath for periods of up to 2 hr. After incubation for 30 min at 37 C, the chilled reaction mixture was transferred to an anion exchange column (0.6 cm diameter X 5 cm high ; Bio-Rad AG l-X2 resin, chloride form) and the [ l-14C] acetylcholine formed was washed through the resin into a scintillation vial with 2 ml distilled water. All assays with appropriate blanks, were run in triplicate and enzymatic activity was calculated as pmoles of acetylcholine synthesized/gram tissue protein/hr at 37 C. Acrtylcholinesterase Assay. The enzymatic activity of AChE was measured with a modification of the spectrophotometric procedure of Ellman et al. (9) in which acetylthiocholine (AThCh) is used as the substrate. The incubation medium contained in final concentration : 0.1 31 phosphate buffer, pH 8.0, 0.5 mM AThCh iodide, 0.3 mM DTNB (5 : 5-dithiobis-2-nitrobenzoic acid), 0.01% Triton X-100, and from 1.0 to 20 pg protein of tissue sample in a final volume of 300 ~1. In addition to these reactants, the blank contained 3 x 1O-5 nf physostigmine sulfate. Thus AChE activity described herein is defined as that enzymatic hydrolysis of AThCh which is sensitive to physostigmine sulfate at a concentration of 3 X 1O-5 hr. All tissue samples were assayed in triplicate and enzymatic activity was expressed as pmoles AThCh split/g protein/min at 25 C. Protein Assay. Protein determinations for each sample were made in triplicate with the Folin phenol reagent (14), employing human serum albumin as a standard.
176
ALVIN
M.
BURT
TABLE CHOLINE
ACETYLTRANSFERASE AND IN THE LUMBAR ENLARGEMENT
Age
ChAc
1 ACETYLCHOLINESTERASE OF THE RAT SPINAL
activitya
ACTIVITY CORD
AChE
activity*
Days, gestation 13 14 17 18 19 20 Newborn
9.8 8.9 10.6 12.7 14.3 15.8
f f f f f f
1.3 1.9 1.1 0.5 0.7 1.7
(6)~ (6) (4) (5) (6) (6)
15.9 f
2.0 (9)
21.1 17.2 21.3 27.8 35.7 34.5 46.8 49.9 52.4 47.6 53.0 58.4 52.0
1.2 1.2 0.3 3.2 4.6 2.0 0.3 3.1 2.4 1.5 1.9 1.8 3.6
15.7 f
1.9 (5)d
20.4 f 21.3 f
1.4 (6)d 1.9 (5)d
6.0 10.4 17.6 23.7 28.2 33.3
f f f f f f
31.3 f
1.9 1.2 1.5 2.2 2.5 3.3
(6) (6) (4) (5) (6) (6)
43.1
f
4.8 (5)d
81.5 f 79.3 f
6.6 (6)d 12.4 (5)d
5.1 (9)
Days, postnatal 1 12 3 -4 ~6 8 10 12 14 16 20 24 41 (adult)
f f f f f f f f f f f f f
(4) (4) (4) (4) (3) (3) (4) (4) (3) (3) (4) (3) (4)
62.9 49.2 48.9 73.7 77.7 82.1 98.1 114.8 90.3 94.4 101.1 113.9 98.2
a ChAc activity: rmoles acetylcholine synthesized/g, * AChE: pmoles acetylthiocholine split/g protein/min c Average f standard deviation (number of samples done series of 17, 19, and 20-day gestation embryos both enzymes (see text for details).
f f f f f f f f f f f f f
6.3 7.7 4.0 10.7 6.8 3.4 11.0 9.1 8.8 7.7 4.7 4.6 9.1
(4) (4) (4) (4) (3) (3) (4) (4) (3) (31 (4) (3) (4)
protein/hr at 37 C. at 25 C. assayed in triplicate). exhibited very high
values
for
RESULTS Data from AChE and ChAc assays are summarized in Table 1 and the ontogenetic patterns are compared in Fig. 1. The activity of ChAc remained low and relatively unchanged from the 13th through the 17th day of gestation; however, from the 17th day of gestation through the 12th to 14th day of postnatal development, activity increased nearly five-fold (Table 1). The subsequent period of development showed little significant change in level of ChAc activity. AChE activity, on the other hand, increased rapidly from the 13th day of gestation through the 12th day of postnatal development, a 19-fold increase in activity ; the subsequent period of development
CrrAc
AND
ACHE
DEVELOPMENTAL
IN
RAT
SPINAL
CORD
AGE
FIG. 1. A comparison of AChE activity (O-O) and ChAc activity ( l - - - -0 ) during development in the lumbar enlargement of the rat spinal cord. AChE activity in pmoles acetylthiocholine split/g protein/min at 25 C; ChAc activity in pmoles acetylcholine synthesized/g protein/hr at 37 C.
showed little significant change in level of AChE activity (Table 1). When the ontogenetic patterns are compared, both enzymes are strikingly similar after the 16th day of gestation (Fig. 1). The only suggestion of a difference in pattern is between the 13th and 17th days of gestation: ChAc activity showed little change whereas AChE activity increased nearly three-fold.
178
ALVIN
M.
BURT
One group of fetal rats showed levels of activity which were markedly higher than others of similar developmental age (Table 1). All of the pregnant female rats for this series were obtained from Holtzman in a single shipment. Although the ontogenetic patterns for both enzymes were similar, these values were much larger (ChAc values about 140% and AChE values nearly 260% those from comparable embryos). There was no apparent difference in the degree of morphological development between the embryos having these high values and the others of comparable developmental age. DISCUSSION The levels of activity for both ChAc and AChE in the rat spinal cord increase markedly with development (Table 1) . Increases of similar magni.tude have been described for the embryonic chick spinal cord (4). During rat spinal cord development, the patterns for the two enzymes are nearly identical (Fig. 1) ; however, in the chick, the ontogenetic patterns of AChE and ChAc are dissimliar (4). Thus, there are marked species differences in the development of these two enzymes. However, the functional and behavioral development of the rat spinal cord also differs markedly from that observed in the chick. Spontaneous motor activity in the chick develops prior to sensory input or connections with higher neural centers (1 l-13). This spontaneous activity reaches a maximum by day 13 and is subsecluently replaced by a “goal-directed” prehatching activity. The electrophysiological studies of Corner and Bot (8) indicate an increase in complexity of the reflex response during the latter stages of development, suggesting the differentiation of multisynaptic reflex arcs. Thus in the chick, the functional development is characterized by an extensive “prereflexogenie” period, a period that is biochemically characterized by a plateau in the level of ChAc activity. In contrast, this “prereflexogenic” period is not present during fetal rat development (19). At 16 days’ gestation, there is the first indication of both spontaneous motor activity and locally evoked reflex responses. By 18 days, gestation, the frequency of the spontaneous activity bursts increases rapidly and the local responses to exteroceptive stimuli increase in both degree of complexity and degree of integration (19). During this period of development, the activity of both ChAc and AChE is increasing and this simultaneous increase in activity continues through the first 2 weeks of postnatal development (Fig. 1). The rat pup undergoes marked maturation in locomotor development during the early postnatal period (19 and Narayanan, personal communication). These investigators would rate the motor coordination as mature by 8 days of development and by 13 to 14 days of postnatal development the patterns of locomotor activity would be considered mature. In the rat, there is not the temporal lag between the onset of spontaneous motor nueron firing
and the subsequent establishment of reflex arcs and “goal-directed” behavior. Similarly, there is no temporal lag in the increase in ChAc activity during development (Fig. 1). \\‘hen the differences in morphological and functional development are considered, hypotheses previously developed from studies on the chick (-M) appear equally valid for the rat ; that is, AChE activity increases in parallel with morphological growth and arborization of the neuron while Cl1Ac activity parallels both synatpogenesis and the functional differentiation of the motor neuron per SC. Studies on the development of the hippocampus in the rat have led to similar conclusions (15, 18). Although these two enzymes of acetylcholine metabolism develop in parallel in the rat, there is no basis for postulating a common mechanism for the regulation of their development. In fact, cell fusion studies (2, 17) and in vitro studies of selected embryonic nerve cell populations (1) support the concept of different mechanisms for the regulation of ,4ChF_ and ChAc activity. In order to further test the validity of hypotheses developed from estensive studies on the chick embryo (4-6), the surgical manipulation of the fetal rat will be required. When the necessary i,l ztfero surgical techniques are perfected, these esperiments will be performed. The very high values for AChE and ChL4c in 16 of the 105 embryos studied (Table 1) is puzzling, and we have no explanation for these observations. All of the pregnant rats used in this study were obtained from the same source and were presumed to be of the same strain. In addition, these embryos were the same size and apparent stage of morphological development as others of comparable age. A two-fold difference in level of glucosed-phosphate dehydrogenase activity in the spinal cord of embryonic chicks of different breeds has been reported (7) and, as in this study, the ontogenetic patterns were similar for the two breeds. However. Bennett et al. (3) described a difference of only 6% to 10% in the cholinesterase activity of rat brains from different strains. In the present study a marked and real difference was observed for 16 fetal rats. These data have been included because although we have assumed that the lower values are the “true” values, the study would not be complete if the higher values were omitted and because it illustrates the problem which biological variation and/or strain differences may introduce in a study of this type. REFERENCES R., and G. TEITELMAN. 1974. Aggregates formed by mixtures of embryonic neural cells: Activity of enzymes of the cholinergic system. Dcvrlop. Bid. 39: 317-321. 2. AMANO, T., B. HAMPRECHT, and W. KEMPER. 1974. High activity of choline acetyltransferase induced in neuroblastoma X glia hybrid cells. Exp. Cell Res. 85 : 399-40s. 1. ADLER,
180
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BURT
3. BENNETT, E. L., M. R. ROSENZWEIG, D. KRECH, N. D. KARLSSON, and A. OHLANDER. 1958. Individual, strain and age differences in cholinesterase activity of the rat brain. J. Ncr~rocZrc~rz. 3: 144-152. 4. BURT, A. M. 1968. Acetylcholinesterase and choline acetyltransferase activity in the developing chick spinal cord. J. Exp. 2001. 169: 107-112. 5. BURT, A. M. 1973. Choline acetyltransferase and neuronal maturation, pp. 245-252. In “Neurobiological Aspects of Maturation and Aging, Progress in Brain Research,” Vol. 40. D. H. Ford [Ed.]. Elsevier, Amsterdam. 6. 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. Exp. Nezcrol. 29: 201-210. 7. BURT, A. M., and C. H. NARAYANAN. 1972. Development of glucose-&phosphate, malate and glutamate dehydrogenase activities in the ventral half of the chick spinal cord in the absence of extrinsic neuronal connections. Exp. Ncurol. 34: 342-353. 8. CORNER, M. A., and A. P. C. BOT. 1967. Developmental patterns in the central nervous system of birds. III. Somatic motility during the embryonic period and its relation to behavior after hatching. pp. 214-236. In “Developmental Neurology, Progress in Brain Research,” Vol. 26. C. G. Bernhard and J. P. Schade [Eds.]. Elsevier, Amsterdam. 9. ELLMAN, G. L., K. D. COURTNEY, V. ANDRES, JR., and R. M. FEATHERSTONE. 1961. A new and rapid calorimetric determination of acetylcholinesterase activity. Biochcm. Pharmacol. 7 : 88-95. 10. GIXOBINI, G. 1972. Embryonic and postnatal development of choline acetyltransferase activity in muscles and sciatic nerve of the chick. J. Nez~roc/zcna. 19: 1401-1403. 11. HAMBURGER, V., M. BALABAN, R. OPPENHEIM, and E. WENGER. 1965. Periodic motility of normal and spinal chick embryos between 8 and 17 days of incubation. J. Exp. 2001. 159 : 1-14. 12. HAMBURGER, V., and R. OPPENKEIM. 1967. Prehatching motility and hatching behavior in the chick. J. Exp. 2001. 166 : 171-204. 13. HAMBURGER, V., E. WENGER, and R. OPPENHEIM. 1966. Motility in the chick embryo in the absence of sensory input. J. Exp. Zool. 162: 133-160. 14. LOWRY, 0. H., N. J. ROSEBROUGH, A. L. FARR, and R. J. RANDALL. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chcm. 193: 265-275. 15. MATTHEWS, D. A., J. V. NALDER, G. S. LYNCH, and C. W. COTMAN. 1974. Development of cholinergic innervation in the hippocampal formation of the rat. I. Histochemical demonstration of acetylcholinesterase activity. Develop. Biol. 36 : 130-141. 16. MCCAMAN, R. E., and J. M. HUNT. 1965. Microdetermination of choline acetylase in nervous tissue. J. Neurochem. 12: 253-259. 17. MCMORRIS, F. A., and F. H. RUDDLE. 1974. Expression of neuronal phenotypes in neuroblastoma cell hybrids. Develop. Biol. 39: 226-246. 18. NADLER, J. V., D. A. MATTHEWS, C. W. COTMAN, and G. S. LYNCH. 1974. Development of cholinergic innervation in the hippocampal formation of the rat. II. Quantitative changes in choline acetyltransferase and acetylcholinesterase activities. Dcvclop. Biol. 36: 142-154. 19. NARAYANAN, C. H., M. W. Fox, and V. HAMBURGER. 1971. Prenatal development of spontaneous and evoked activity in the rat (Ratfu Norvcgicus albinus). Behavior 40 : 100-134. 20. SCHRIER, B. K., and L. SHUSTER. 1967. A simplified radiochemical assay for choline acetyltransferase. I. Neurochem. 14 : 977-986.
Choline
NEUROLOGY
47,
173-180
Acetyltransferase in the Developing ALVIN
Drparhmt
Receizled
(1975)
and Acetylcholinesterase Rat Spinal Cord M.
BURT
r
of Anatomy, Vanderbilt, Unizwsity NashzJle, Terumsee 37232 Septewber
14, 1974;
rrvision
receizled
School
Nozpember
of Mediciw,
24, 1974
Microchemical procedures were employed for the quantitative assay of choline acetyltransferase (ChAc; EC 2.3.1.6) and acetylcholinesterase (AChE; EC 3.1.1.7) activity in the lumbar spinal cord of the rat during late fetal and early postnatal development. ChAc activity remained low from the 13th to the 17th day of gestation, increased five-fold from the 17th day of gestation through the end of the second postnatal week, and changed little thereafter. AChE activity increased 19-fold from the 13th day of gestation through the end of the second postanatal week. The ontogenetic patterns for the two enzymes were identical after the 16th day of gestation. When these changes in enzyme activity were correlated with morphological and functional differentiation, the relationships were similar to those previously described for the chick. INTRODUCTION
Increased cholineacetyltransferase ( ChAc) activity has been correlated with the development of functional synaptic connections in the embryonic chick spinal cord (4). Based on the marked differences in the ontogenetic patterns of ChAc and acetylecholinesterase (AChE), it was further suggested that ChAc activity was a more reliable index of fuctional neuronal differentiation than was AChE activity. Experimental studies (6) have provided further support for the association of ChAc activity with synaptic differentiation. Recent studies on the development of the rat hippocampus have led to similar conclusions, namely that ChAc activity increases rapidly in parallel with synaptogenesis whereas AChE activity develops independently and is associated with the appearance of nerve fibers 1 Excellent technical assistance was provided by Mr. Charles Fouser. vestigation was supported by a grant from the Dysautonomia Foundation, Research Grants No. NS-07441 f rom the National Institute of Neurological and Stroke, National Institutes of Health. 173 Copyright All rights
1975 by Academic Press. Inc. o6 reproduction in any form reserved.
This inInc., and Diseases
174
ALVIN
M.
BURT
believed to be cholinergic in nature (15, 18). In addition to the association with synatptogenesis, a major component of the ChAc activity in the chick spinal cord appears to be associated with the biochemical and functional differentiation of the motor neuron per se (5). This suggestion was based on correlations between the ChAc activity in the spinal cord and both the appearance and subsequent increase in spontaneous motor activity (neurogenie in origin, 11, 13), and the appearance and subsequent increase in ChAc activity in motor nerve terminals in the differentiating axial and limb musculature ( 10). In the developing spinal cord of the rat, the temporal sequenceof motor neuron growth and differentiation and the development of functional synaptic connections differs from that of the chick (19). The onset of spontaneous motor neuron firing, resulting in spontaneous motor activity in the rat is not separated temporally from the appearance of synaptic connections and the onset of reflex responsesto exteroceptive stimuli. In the chick, however, development is characterized by an extensive “prereflexogenic” period, a period characterized by the presence of spontaneous motor activity and the absenceof functional reflex connections ( 11-13). If, therefore, the relationship between the enzymes ChAc and AChE and these processesof neurogenesisobserved for the chick (4-6) are valid for the rat, one would expect the ontogenetic patterns for the two enzymes to be similar during rat development. In order to test this hypothesis, the development of ChAc and AChE activities was studied in the lumbar spinal cord of the rat. METHODS
AND
MATERIALS
Pregnant albino rats, obtained from the Holtzman Company, Madison, Wis., were used throughout this study. The pregnant rats (13-20 days of gestation) were anesthetized with ether and the uterus was exposed by a midline incision in the abdomen. The individual fetuses were removed through an incision in the anti-mesometrial wall of the uterus. Newborn rats and rats of one through 11 days of postnatal development were killed by decapitation. Prejxmztion of Tissue Samples. The lumbar enlargement (representing spinal segments T1x-L4, inclusive) was dissected free from surrounding tissue. The enlargement from each fetal, newborn, or young rat was removed to a chilled glass microhomogenizer and ground in distilled water. Aliquots of the homogenate were transferred to small culture tubes (6 x 50 mm), immediately frozen, and lyophilized at -40 C. Following the lyophilization, sampletubes were sealedand stored at -75 C until assayed. There was no detectable loss of activity for either enzyme, ChAc or AChE, after freezing and lyophilization or after storage of the lyophilysate at -75 C for up to 4 months.
CHAC
AND
ACHE
IN
RAT
SPINAL
CORD
175
Choline Acetyltra.nsfemse Assay. The enzymatic activity of choline acetyltransferase was measured by a sensitive radiochemical assay procedure in which [ I-‘“Clacetyl-CoA and choline were used as substrates and the radioactivity of the reaction product, [ 1-14C] acetylcholine, was measured ( 16,20). Optimal assay conditions were determined for a preparation of newborn rat brain. The modified incubation medium contained in final concentration : 0.1 nr phosphate buffer, pH 7.3, 0.29 nr NaCl, 15 m&f MgSO,, 0.05% b ovine serum albumin, 0.2 mM physostigmine sulfate, 10 m&f choline and 0.2 mM [ l-14C]acetyI-CoA (2.35 uc/pmole), The radiochemically labeled acetyl-CoA was obtained from New England Nuclear, Boston, Mass. the unlabeled acteyl-CoA from Sigma Chemical Company, St. Louis, MO. In order to insure an optimal rate of reaction throughout the incubation period, sample concentrations were selected so that only from 1 to 5% of the substrate, [ 1-‘“Clacetyl-CoA, was consumed. The incubation was initiated by transferring the entire rack of tubes with the full complement of incubation components from an ice-water bath (&2 C) to a 37 C water bath and the reaction was terminated by returning the tubes to the ice-water bath. With the use of relatively thin-walled (0.5 mm) disposable glass tubes, the chillin g of the incubation medium (100 ~1) was sufficiently rapid to stop the enzymatic reaction. There was no detectable synthesis of additional acetylcholine in the most active samples alter transfer to the ice-water bath for periods of up to 2 hr. After incubation for 30 min at 37 C, the chilled reaction mixture was transferred to an anion exchange column (0.6 cm diameter X 5 cm high ; Bio-Rad AG l-X2 resin, chloride form) and the [ l-14C] acetylcholine formed was washed through the resin into a scintillation vial with 2 ml distilled water. All assays with appropriate blanks, were run in triplicate and enzymatic activity was calculated as pmoles of acetylcholine synthesized/gram tissue protein/hr at 37 C. Acrtylcholinesterase Assay. The enzymatic activity of AChE was measured with a modification of the spectrophotometric procedure of Ellman et al. (9) in which acetylthiocholine (AThCh) is used as the substrate. The incubation medium contained in final concentration : 0.1 31 phosphate buffer, pH 8.0, 0.5 mM AThCh iodide, 0.3 mM DTNB (5 : 5-dithiobis-2-nitrobenzoic acid), 0.01% Triton X-100, and from 1.0 to 20 pg protein of tissue sample in a final volume of 300 ~1. In addition to these reactants, the blank contained 3 x 1O-5 nf physostigmine sulfate. Thus AChE activity described herein is defined as that enzymatic hydrolysis of AThCh which is sensitive to physostigmine sulfate at a concentration of 3 X 1O-5 hr. All tissue samples were assayed in triplicate and enzymatic activity was expressed as pmoles AThCh split/g protein/min at 25 C. Protein Assay. Protein determinations for each sample were made in triplicate with the Folin phenol reagent (14), employing human serum albumin as a standard.
176
ALVIN
M.
BURT
TABLE CHOLINE
ACETYLTRANSFERASE AND IN THE LUMBAR ENLARGEMENT
Age
ChAc
1 ACETYLCHOLINESTERASE OF THE RAT SPINAL
activitya
ACTIVITY CORD
AChE
activity*
Days, gestation 13 14 17 18 19 20 Newborn
9.8 8.9 10.6 12.7 14.3 15.8
f f f f f f
1.3 1.9 1.1 0.5 0.7 1.7
(6)~ (6) (4) (5) (6) (6)
15.9 f
2.0 (9)
21.1 17.2 21.3 27.8 35.7 34.5 46.8 49.9 52.4 47.6 53.0 58.4 52.0
1.2 1.2 0.3 3.2 4.6 2.0 0.3 3.1 2.4 1.5 1.9 1.8 3.6
15.7 f
1.9 (5)d
20.4 f 21.3 f
1.4 (6)d 1.9 (5)d
6.0 10.4 17.6 23.7 28.2 33.3
f f f f f f
31.3 f
1.9 1.2 1.5 2.2 2.5 3.3
(6) (6) (4) (5) (6) (6)
43.1
f
4.8 (5)d
81.5 f 79.3 f
6.6 (6)d 12.4 (5)d
5.1 (9)
Days, postnatal 1 12 3 -4 ~6 8 10 12 14 16 20 24 41 (adult)
f f f f f f f f f f f f f
(4) (4) (4) (4) (3) (3) (4) (4) (3) (3) (4) (3) (4)
62.9 49.2 48.9 73.7 77.7 82.1 98.1 114.8 90.3 94.4 101.1 113.9 98.2
a ChAc activity: rmoles acetylcholine synthesized/g, * AChE: pmoles acetylthiocholine split/g protein/min c Average f standard deviation (number of samples done series of 17, 19, and 20-day gestation embryos both enzymes (see text for details).
f f f f f f f f f f f f f
6.3 7.7 4.0 10.7 6.8 3.4 11.0 9.1 8.8 7.7 4.7 4.6 9.1
(4) (4) (4) (4) (3) (3) (4) (4) (3) (31 (4) (3) (4)
protein/hr at 37 C. at 25 C. assayed in triplicate). exhibited very high
values
for
RESULTS Data from AChE and ChAc assays are summarized in Table 1 and the ontogenetic patterns are compared in Fig. 1. The activity of ChAc remained low and relatively unchanged from the 13th through the 17th day of gestation; however, from the 17th day of gestation through the 12th to 14th day of postnatal development, activity increased nearly five-fold (Table 1). The subsequent period of development showed little significant change in level of ChAc activity. AChE activity, on the other hand, increased rapidly from the 13th day of gestation through the 12th day of postnatal development, a 19-fold increase in activity ; the subsequent period of development
CrrAc
AND
ACHE
DEVELOPMENTAL
IN
RAT
SPINAL
CORD
AGE
FIG. 1. A comparison of AChE activity (O-O) and ChAc activity ( l - - - -0 ) during development in the lumbar enlargement of the rat spinal cord. AChE activity in pmoles acetylthiocholine split/g protein/min at 25 C; ChAc activity in pmoles acetylcholine synthesized/g protein/hr at 37 C.
showed little significant change in level of AChE activity (Table 1). When the ontogenetic patterns are compared, both enzymes are strikingly similar after the 16th day of gestation (Fig. 1). The only suggestion of a difference in pattern is between the 13th and 17th days of gestation: ChAc activity showed little change whereas AChE activity increased nearly three-fold.
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One group of fetal rats showed levels of activity which were markedly higher than others of similar developmental age (Table 1). All of the pregnant female rats for this series were obtained from Holtzman in a single shipment. Although the ontogenetic patterns for both enzymes were similar, these values were much larger (ChAc values about 140% and AChE values nearly 260% those from comparable embryos). There was no apparent difference in the degree of morphological development between the embryos having these high values and the others of comparable developmental age. DISCUSSION The levels of activity for both ChAc and AChE in the rat spinal cord increase markedly with development (Table 1) . Increases of similar magni.tude have been described for the embryonic chick spinal cord (4). During rat spinal cord development, the patterns for the two enzymes are nearly identical (Fig. 1) ; however, in the chick, the ontogenetic patterns of AChE and ChAc are dissimliar (4). Thus, there are marked species differences in the development of these two enzymes. However, the functional and behavioral development of the rat spinal cord also differs markedly from that observed in the chick. Spontaneous motor activity in the chick develops prior to sensory input or connections with higher neural centers (1 l-13). This spontaneous activity reaches a maximum by day 13 and is subsecluently replaced by a “goal-directed” prehatching activity. The electrophysiological studies of Corner and Bot (8) indicate an increase in complexity of the reflex response during the latter stages of development, suggesting the differentiation of multisynaptic reflex arcs. Thus in the chick, the functional development is characterized by an extensive “prereflexogenie” period, a period that is biochemically characterized by a plateau in the level of ChAc activity. In contrast, this “prereflexogenic” period is not present during fetal rat development (19). At 16 days’ gestation, there is the first indication of both spontaneous motor activity and locally evoked reflex responses. By 18 days, gestation, the frequency of the spontaneous activity bursts increases rapidly and the local responses to exteroceptive stimuli increase in both degree of complexity and degree of integration (19). During this period of development, the activity of both ChAc and AChE is increasing and this simultaneous increase in activity continues through the first 2 weeks of postnatal development (Fig. 1). The rat pup undergoes marked maturation in locomotor development during the early postnatal period (19 and Narayanan, personal communication). These investigators would rate the motor coordination as mature by 8 days of development and by 13 to 14 days of postnatal development the patterns of locomotor activity would be considered mature. In the rat, there is not the temporal lag between the onset of spontaneous motor nueron firing
and the subsequent establishment of reflex arcs and “goal-directed” behavior. Similarly, there is no temporal lag in the increase in ChAc activity during development (Fig. 1). \\‘hen the differences in morphological and functional development are considered, hypotheses previously developed from studies on the chick (-M) appear equally valid for the rat ; that is, AChE activity increases in parallel with morphological growth and arborization of the neuron while Cl1Ac activity parallels both synatpogenesis and the functional differentiation of the motor neuron per SC. Studies on the development of the hippocampus in the rat have led to similar conclusions (15, 18). Although these two enzymes of acetylcholine metabolism develop in parallel in the rat, there is no basis for postulating a common mechanism for the regulation of their development. In fact, cell fusion studies (2, 17) and in vitro studies of selected embryonic nerve cell populations (1) support the concept of different mechanisms for the regulation of ,4ChF_ and ChAc activity. In order to further test the validity of hypotheses developed from estensive studies on the chick embryo (4-6), the surgical manipulation of the fetal rat will be required. When the necessary i,l ztfero surgical techniques are perfected, these esperiments will be performed. The very high values for AChE and ChL4c in 16 of the 105 embryos studied (Table 1) is puzzling, and we have no explanation for these observations. All of the pregnant rats used in this study were obtained from the same source and were presumed to be of the same strain. In addition, these embryos were the same size and apparent stage of morphological development as others of comparable age. A two-fold difference in level of glucosed-phosphate dehydrogenase activity in the spinal cord of embryonic chicks of different breeds has been reported (7) and, as in this study, the ontogenetic patterns were similar for the two breeds. However. Bennett et al. (3) described a difference of only 6% to 10% in the cholinesterase activity of rat brains from different strains. In the present study a marked and real difference was observed for 16 fetal rats. These data have been included because although we have assumed that the lower values are the “true” values, the study would not be complete if the higher values were omitted and because it illustrates the problem which biological variation and/or strain differences may introduce in a study of this type. REFERENCES R., and G. TEITELMAN. 1974. Aggregates formed by mixtures of embryonic neural cells: Activity of enzymes of the cholinergic system. Dcvrlop. Bid. 39: 317-321. 2. AMANO, T., B. HAMPRECHT, and W. KEMPER. 1974. High activity of choline acetyltransferase induced in neuroblastoma X glia hybrid cells. Exp. Cell Res. 85 : 399-40s. 1. ADLER,
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3. BENNETT, E. L., M. R. ROSENZWEIG, D. KRECH, N. D. KARLSSON, and A. OHLANDER. 1958. Individual, strain and age differences in cholinesterase activity of the rat brain. J. Ncr~rocZrc~rz. 3: 144-152. 4. BURT, A. M. 1968. Acetylcholinesterase and choline acetyltransferase activity in the developing chick spinal cord. J. Exp. 2001. 169: 107-112. 5. BURT, A. M. 1973. Choline acetyltransferase and neuronal maturation, pp. 245-252. In “Neurobiological Aspects of Maturation and Aging, Progress in Brain Research,” Vol. 40. D. H. Ford [Ed.]. Elsevier, Amsterdam. 6. 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. Exp. Nezcrol. 29: 201-210. 7. BURT, A. M., and C. H. NARAYANAN. 1972. Development of glucose-&phosphate, malate and glutamate dehydrogenase activities in the ventral half of the chick spinal cord in the absence of extrinsic neuronal connections. Exp. Ncurol. 34: 342-353. 8. CORNER, M. A., and A. P. C. BOT. 1967. Developmental patterns in the central nervous system of birds. III. Somatic motility during the embryonic period and its relation to behavior after hatching. pp. 214-236. In “Developmental Neurology, Progress in Brain Research,” Vol. 26. C. G. Bernhard and J. P. Schade [Eds.]. Elsevier, Amsterdam. 9. ELLMAN, G. L., K. D. COURTNEY, V. ANDRES, JR., and R. M. FEATHERSTONE. 1961. A new and rapid calorimetric determination of acetylcholinesterase activity. Biochcm. Pharmacol. 7 : 88-95. 10. GIXOBINI, G. 1972. Embryonic and postnatal development of choline acetyltransferase activity in muscles and sciatic nerve of the chick. J. Nez~roc/zcna. 19: 1401-1403. 11. HAMBURGER, V., M. BALABAN, R. OPPENHEIM, and E. WENGER. 1965. Periodic motility of normal and spinal chick embryos between 8 and 17 days of incubation. J. Exp. 2001. 159 : 1-14. 12. HAMBURGER, V., and R. OPPENKEIM. 1967. Prehatching motility and hatching behavior in the chick. J. Exp. 2001. 166 : 171-204. 13. HAMBURGER, V., E. WENGER, and R. OPPENHEIM. 1966. Motility in the chick embryo in the absence of sensory input. J. Exp. Zool. 162: 133-160. 14. LOWRY, 0. H., N. J. ROSEBROUGH, A. L. FARR, and R. J. RANDALL. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chcm. 193: 265-275. 15. MATTHEWS, D. A., J. V. NALDER, G. S. LYNCH, and C. W. COTMAN. 1974. Development of cholinergic innervation in the hippocampal formation of the rat. I. Histochemical demonstration of acetylcholinesterase activity. Develop. Biol. 36 : 130-141. 16. MCCAMAN, R. E., and J. M. HUNT. 1965. Microdetermination of choline acetylase in nervous tissue. J. Neurochem. 12: 253-259. 17. MCMORRIS, F. A., and F. H. RUDDLE. 1974. Expression of neuronal phenotypes in neuroblastoma cell hybrids. Develop. Biol. 39: 226-246. 18. NADLER, J. V., D. A. MATTHEWS, C. W. COTMAN, and G. S. LYNCH. 1974. Development of cholinergic innervation in the hippocampal formation of the rat. II. Quantitative changes in choline acetyltransferase and acetylcholinesterase activities. Dcvclop. Biol. 36: 142-154. 19. NARAYANAN, C. H., M. W. Fox, and V. HAMBURGER. 1971. Prenatal development of spontaneous and evoked activity in the rat (Ratfu Norvcgicus albinus). Behavior 40 : 100-134. 20. SCHRIER, B. K., and L. SHUSTER. 1967. A simplified radiochemical assay for choline acetyltransferase. I. Neurochem. 14 : 977-986.
