Brahz Research, 127 (1977) 159-163 © Elsevier/North-Holland Biomedical Press
159
Choline acetyltransferase in developing rat brain and spinal cord
VIJENDRA K. SINGH and EDITH G. McGEER
Kinsmen Laboratory qf Neurological Research, Department of Psychiatry, University o]'British Columbia, Vancouver, B.C. V6T 1 t4/5 (Canada) (Accepted February 3rd, 1977)
Developmental studies of the cholinergic innervation in the nervous system have been concerned with changes in the levels of acetylcholine (ACh), choline acetyltransferase (CAT) and acetylcholine esterase (AChE)l,2,1°-14. The enzyme CAT is responsible for the synthesis of the neurotransmitter ACh. Because it is more specifically localized than ACHE, the former enzyme may serve as a very reliable biochemical marker of the onset of functional maturation of cholinergic neurons in brain. In addition to a neurotransmitter function, the chemical acetylcholine may have some role in the growth and maturation of neurons and their structures 11. This communication reports the postnatal development of CAT in rat brain and spinal cord, and describes the differential sensitivity of this enzyme to NaC1 and Triton X-100. Neonatal Wistar rats (3-30 days old) and adult rats (weighing about 500 g) were obtained from the vivarium of this University. Animals were sacrified by cervical dislocation, the brain or specific brain areas and spinal cord (thoracic segment) dissected out, weighed and immediately homogenized. Usually a 5 - 1 0 ~ (w/v) homogenate was prepared in 0.32 M sucrose buffered with l0 m M potassium phosphate, pH 7.4 using a Potter-Elvehjem homogenizer. The enzyme activity was measured radiochemically according to our procedure as described elsewhere 17. The results are given as the mean values of 3 separate experiments (3-5 rats in each) of duplicate determinations. In each group, the S.D. was about 5-10 ~. The data summarized in Fig. 1 show the pattern of development of CAT activity in spinal cord, whole brain, caudate-putamen and cerebral cortex during the maturation of rat nervous system. The spinal cord displayed the highest CAT activity of all other areas during maturation. The enzyme activity peaked at about 25 days after birth. The CAT activity of spinal cord from the older rats was found to be significantly lower than that from younger adults (25 days old). The enzyme activity of brain appeared to develop in a linear fashion. On the other hand, the CAT activity of caudate-putamen and cerebral cortex initially showed very low values, especially when compared to spinal cord, and started to mature gradually at 10-15 days after birth. Between 15-25 days after birth, there was a 3-4 fold increase in the CAT activity in the
160 12
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17 21 Age in days
25
30
Adult
Fig. 1. The developmental profile of CAT activity in rat brain, caudate-putamen, cerebral cortex and spinal cord. The enzyme activity in whole brain (O Q), caudate-putamen (O O), cerebral cortex A A) and spinal cord (A . . . . A).
caudate-putamen as well as in the cerebral cortex, and thereafter no further increase in the enzyme activity with increasing age was seen in these nervous tissues. The addition of NaC1 to the reaction mixture activated the CAT activity of tissue homogenates in vitro (Fig. 2). The enzyme activity of spinal cord or brain from baby rats (3 days old) was stimulated by 0.3 M NaCI to 165 °/o and 125 ~ respectively. At the same concentration, NaC1 enhanced the CAT activity to about 195 ~ and 230 ~ respectively for the adult rat spinal cord and brain. Similar results were found when the supernatant fraction, prepared by centrifuging the isotonic sucrose-tissue homogenates at 27,000 × g for 30 min, was used as the enzyme source. Various concentrations of Triton X-100 ( 0 . 0 5 ~ - 0 . 4 ~ ) added into the CAT assay mixture affected the enzyme activity differentially with the animal age (Fig 3). The enzyme activity in the homogenates of brain or spinal cord of baby rats (3 days old) was unaffected by this detergent while the activity of brain and spinal cord from adult rats was activated to about 230 ~ and 135 ~ respectively. In general, Triton X100 greatly activated the CAT from adult rat brain. The postnatal development profiles found for CAT in rat spinal cord, brain, cerebral cortex and caudate nucleus compare closely with those previously reported 2, lo,1~,13. In such prior work, however, no comparison was made between brain and spinal cord. The enzyme has been shown to exist in soluble as well as particulate formsa,7,8; the membrane binding is dependent on the ionic strength and pH of the homogenizing
161 250
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Fig. 2. The effect of NaCI on CAT activity of homogenates of spinal cord and brain from baby and adult rats. The enzyme activity of adult rat brain (O O) or spinal cord (O . . . . O) and that of whole brain ( & Ak) or spinal cord (A . . . . A) from 3-day-old rats.
_ 200 o c
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Fig. 3. The influence of Triton X-100 on CAT activity of homogenates of whole brain and spinal cord from baby and adult rats. The enzyme activity of whole brain (& Ak) or spinal cord (A . . . . A) from 3-day-old rats and that of adult rat brain ( O 0 ) or spinal cord (O . . . . O.)
162 m e d i u m ~. It is generally believed that the soluble f o r m is derived f r o m the cholinergic cell soma, dendrites and axons whereas the particulate form is o b t a i n e d from the s y n a p t o s o m e s L The latter can be further activated by treatments with organic solvent 7 or detergents 4. As r e p o r t e d here the activity o f enzyme C A T was influenced by NaC1 or T r i t o n X-100 differently with age. The salt activation a p p e a r s to be a general feature o f this enzyme and results in an increase in kinetic p a r a m e t e r s for b o t h substrates 9,15. Different forms o f squid-head ganglia C A T have been shown to be s t i m u l a t e d by salts to varying degrees 16. It is not yet clear whether the differential magnitudes o f salt activation o f C A T from a d u l t as c o m p a r e d with b a b y rats reveal a change in enzymic form or subcellular distribution with age. The detergent increases the solubility o f the enzyme from the cellular particulate fractions a n d thereby stimulates the enzyme activity in tissue homogenates. The differential response o f T r i t o n as described in Fig. 3 m a y therefore reflect a differential cellular localization o f this enzyme in nervous tissues from b a b y a n d a d u l t rats. The enzyme in b a b y rats m a y be more soluble a n d more o f cell s o m a origin whereas the enzyme in a d u l t rats may be more particulate a n d m o r e s y n a p t o s o m a l in origin. The close parallelism between p o s t n a t a l d e v e l o p m e n t a l patterns for C A T a n d for the cholinergic type o f synapses in the rat n e o s t r i a t u m 6 lends some s u p o r t to this possibility. W e are grateful to the M e d i c a l Research Council o f C a n a d a for the financial s u p p o r t o f this work.
1 Bull, G., Hebb, C. and Ratkovic, D., Choline acetyltransferase activity of human brain tissue during development and at maturity, J. Neurochem., 17 (1970) 1505-1516. 2 Burt, A. M., Choline acetyltransferase and acetylcholinesterase in the developing rat spinal cord, Exp. NeuroL, 47 (1975) 173-180. 3 De Robertis, E., De lraldi, P. A., Arnaiz, G. R. and Salganicoff, L., cholinergic and non-cholinergic nerve endings in rat brain. Isolation and subcellular distribution of acetylcholine and acetylcholinesterase, J. Neurochem., 9 (1962) 23-35. 4 Fonnum, F., A radiochemical method for the estimation of choline acetyltransferase, Biochem. J., 100 (1966) 479-484. 5 Fonnum, F. and Malthe-Sorenssen, D., Molecular properties of choline acetyltransferase and their importance for the compartmentation of acetylcholine synthesis, Progr. Brain Res., 36 (1972) 13-27. 6 Hattori, T. and McGeer, P. L., Synaptogenesis in the corpus striatum of infant rat, Exp. Neurol., 38 (1973) 70-79. 7 Hebb, C. O. and Smallman, B. N., lntracellular distribution of choline acetylase, J. Physiol. (Lond.), 134 (1956) 385-392. 8 Hebb, C. O. and Whittaker, V. P., lntracellular distributions ofacetylcholine and choline acetylase, J. Physiol. (Lond.), 142 (1958) 187-196. 9 Kuczenski, R., Segal, D. S. and Mandell, A. J., Regional and subcellular distribution and kinetic properties of rat brain choline acetyltransferase - - some functional considerations, J. Neurochem., 24 (1975) 39-45. 10 Ladinsky, H., Consolo, S., Peri, G. and Garratini, S., Acetylcholine, choline and choline acetyltransferase activity in the developing brain of normal and hypothyroid rats, J. Neurochem., 19 (1972) 1947-1952. 1 ! Marchisio, P. C. and Consolo, S., Developmental changes of choline acetyltransferase (ChAc) activity in chick embryo spinal cord and sympathetic ganglia, J. Neurochem., 15 (1968) 759-764.
163 12 McCaman, R. E. and Aprison, M. H., The synthetic and catabolic enzyme systems for acetylcholine and serotonin in several discrete areas of the developing rabbit brain, Progr. Brain Res., 9 (1964) 220-233. 13 McGeer, E. G., Fibiger, H. C. and Wickson, V., Differential development of caudate enzymes in neonatal rat, Brain Research, 32 (1971) 433-440. 14 McGeer, E. G., Parkinson, J. and McGeer, P. L., Neonatal enzymic development in the interpeduncular nucleus and surrounding ventral tegmentum, Exp. Neurol., 53 (1976) 109-114. 15 Morris, D., Maneckjee, A. and Hebb, C., The kinetic properties of human placental choline acetyltransferase, Biochem. J., 125 (1971) 857-863. 16 Prempeh, A. B. A., Prince, A. K. and Hide, E. G. J., The reaction of acetyl-coenzyme A with choline acetyltransferase, Biochem. J., 129 (1972) 991-994. 17 Singh, V. K. and McGeer, P. L., Studies on choline acetyltransferase isolated from human brain, Neurochem. Res., (1977) In press.
159
Choline acetyltransferase in developing rat brain and spinal cord
VIJENDRA K. SINGH and EDITH G. McGEER
Kinsmen Laboratory qf Neurological Research, Department of Psychiatry, University o]'British Columbia, Vancouver, B.C. V6T 1 t4/5 (Canada) (Accepted February 3rd, 1977)
Developmental studies of the cholinergic innervation in the nervous system have been concerned with changes in the levels of acetylcholine (ACh), choline acetyltransferase (CAT) and acetylcholine esterase (AChE)l,2,1°-14. The enzyme CAT is responsible for the synthesis of the neurotransmitter ACh. Because it is more specifically localized than ACHE, the former enzyme may serve as a very reliable biochemical marker of the onset of functional maturation of cholinergic neurons in brain. In addition to a neurotransmitter function, the chemical acetylcholine may have some role in the growth and maturation of neurons and their structures 11. This communication reports the postnatal development of CAT in rat brain and spinal cord, and describes the differential sensitivity of this enzyme to NaC1 and Triton X-100. Neonatal Wistar rats (3-30 days old) and adult rats (weighing about 500 g) were obtained from the vivarium of this University. Animals were sacrified by cervical dislocation, the brain or specific brain areas and spinal cord (thoracic segment) dissected out, weighed and immediately homogenized. Usually a 5 - 1 0 ~ (w/v) homogenate was prepared in 0.32 M sucrose buffered with l0 m M potassium phosphate, pH 7.4 using a Potter-Elvehjem homogenizer. The enzyme activity was measured radiochemically according to our procedure as described elsewhere 17. The results are given as the mean values of 3 separate experiments (3-5 rats in each) of duplicate determinations. In each group, the S.D. was about 5-10 ~. The data summarized in Fig. 1 show the pattern of development of CAT activity in spinal cord, whole brain, caudate-putamen and cerebral cortex during the maturation of rat nervous system. The spinal cord displayed the highest CAT activity of all other areas during maturation. The enzyme activity peaked at about 25 days after birth. The CAT activity of spinal cord from the older rats was found to be significantly lower than that from younger adults (25 days old). The enzyme activity of brain appeared to develop in a linear fashion. On the other hand, the CAT activity of caudate-putamen and cerebral cortex initially showed very low values, especially when compared to spinal cord, and started to mature gradually at 10-15 days after birth. Between 15-25 days after birth, there was a 3-4 fold increase in the CAT activity in the
160 12
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/
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,
/
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3
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17 21 Age in days
25
30
Adult
Fig. 1. The developmental profile of CAT activity in rat brain, caudate-putamen, cerebral cortex and spinal cord. The enzyme activity in whole brain (O Q), caudate-putamen (O O), cerebral cortex A A) and spinal cord (A . . . . A).
caudate-putamen as well as in the cerebral cortex, and thereafter no further increase in the enzyme activity with increasing age was seen in these nervous tissues. The addition of NaC1 to the reaction mixture activated the CAT activity of tissue homogenates in vitro (Fig. 2). The enzyme activity of spinal cord or brain from baby rats (3 days old) was stimulated by 0.3 M NaCI to 165 °/o and 125 ~ respectively. At the same concentration, NaC1 enhanced the CAT activity to about 195 ~ and 230 ~ respectively for the adult rat spinal cord and brain. Similar results were found when the supernatant fraction, prepared by centrifuging the isotonic sucrose-tissue homogenates at 27,000 × g for 30 min, was used as the enzyme source. Various concentrations of Triton X-100 ( 0 . 0 5 ~ - 0 . 4 ~ ) added into the CAT assay mixture affected the enzyme activity differentially with the animal age (Fig 3). The enzyme activity in the homogenates of brain or spinal cord of baby rats (3 days old) was unaffected by this detergent while the activity of brain and spinal cord from adult rats was activated to about 230 ~ and 135 ~ respectively. In general, Triton X100 greatly activated the CAT from adult rat brain. The postnatal development profiles found for CAT in rat spinal cord, brain, cerebral cortex and caudate nucleus compare closely with those previously reported 2, lo,1~,13. In such prior work, however, no comparison was made between brain and spinal cord. The enzyme has been shown to exist in soluble as well as particulate formsa,7,8; the membrane binding is dependent on the ionic strength and pH of the homogenizing
161 250
"~ 200
~
c
o
/
/ /
,< ~. < (3
150
.----- --'-~
~
-
/ /
IooUF ~
!
!
0.1
I
0.2 NaCI
0.3
(M)
Fig. 2. The effect of NaCI on CAT activity of homogenates of spinal cord and brain from baby and adult rats. The enzyme activity of adult rat brain (O O) or spinal cord (O . . . . O) and that of whole brain ( & Ak) or spinal cord (A . . . . A) from 3-day-old rats.
_ 200 o c
8
/
o,,~ o
~.0~
,.0
/
100
0
o'.1
Triton X-IO0
o'.4
Fig. 3. The influence of Triton X-100 on CAT activity of homogenates of whole brain and spinal cord from baby and adult rats. The enzyme activity of whole brain (& Ak) or spinal cord (A . . . . A) from 3-day-old rats and that of adult rat brain ( O 0 ) or spinal cord (O . . . . O.)
162 m e d i u m ~. It is generally believed that the soluble f o r m is derived f r o m the cholinergic cell soma, dendrites and axons whereas the particulate form is o b t a i n e d from the s y n a p t o s o m e s L The latter can be further activated by treatments with organic solvent 7 or detergents 4. As r e p o r t e d here the activity o f enzyme C A T was influenced by NaC1 or T r i t o n X-100 differently with age. The salt activation a p p e a r s to be a general feature o f this enzyme and results in an increase in kinetic p a r a m e t e r s for b o t h substrates 9,15. Different forms o f squid-head ganglia C A T have been shown to be s t i m u l a t e d by salts to varying degrees 16. It is not yet clear whether the differential magnitudes o f salt activation o f C A T from a d u l t as c o m p a r e d with b a b y rats reveal a change in enzymic form or subcellular distribution with age. The detergent increases the solubility o f the enzyme from the cellular particulate fractions a n d thereby stimulates the enzyme activity in tissue homogenates. The differential response o f T r i t o n as described in Fig. 3 m a y therefore reflect a differential cellular localization o f this enzyme in nervous tissues from b a b y a n d a d u l t rats. The enzyme in b a b y rats m a y be more soluble a n d more o f cell s o m a origin whereas the enzyme in a d u l t rats may be more particulate a n d m o r e s y n a p t o s o m a l in origin. The close parallelism between p o s t n a t a l d e v e l o p m e n t a l patterns for C A T a n d for the cholinergic type o f synapses in the rat n e o s t r i a t u m 6 lends some s u p o r t to this possibility. W e are grateful to the M e d i c a l Research Council o f C a n a d a for the financial s u p p o r t o f this work.
1 Bull, G., Hebb, C. and Ratkovic, D., Choline acetyltransferase activity of human brain tissue during development and at maturity, J. Neurochem., 17 (1970) 1505-1516. 2 Burt, A. M., Choline acetyltransferase and acetylcholinesterase in the developing rat spinal cord, Exp. NeuroL, 47 (1975) 173-180. 3 De Robertis, E., De lraldi, P. A., Arnaiz, G. R. and Salganicoff, L., cholinergic and non-cholinergic nerve endings in rat brain. Isolation and subcellular distribution of acetylcholine and acetylcholinesterase, J. Neurochem., 9 (1962) 23-35. 4 Fonnum, F., A radiochemical method for the estimation of choline acetyltransferase, Biochem. J., 100 (1966) 479-484. 5 Fonnum, F. and Malthe-Sorenssen, D., Molecular properties of choline acetyltransferase and their importance for the compartmentation of acetylcholine synthesis, Progr. Brain Res., 36 (1972) 13-27. 6 Hattori, T. and McGeer, P. L., Synaptogenesis in the corpus striatum of infant rat, Exp. Neurol., 38 (1973) 70-79. 7 Hebb, C. O. and Smallman, B. N., lntracellular distribution of choline acetylase, J. Physiol. (Lond.), 134 (1956) 385-392. 8 Hebb, C. O. and Whittaker, V. P., lntracellular distributions ofacetylcholine and choline acetylase, J. Physiol. (Lond.), 142 (1958) 187-196. 9 Kuczenski, R., Segal, D. S. and Mandell, A. J., Regional and subcellular distribution and kinetic properties of rat brain choline acetyltransferase - - some functional considerations, J. Neurochem., 24 (1975) 39-45. 10 Ladinsky, H., Consolo, S., Peri, G. and Garratini, S., Acetylcholine, choline and choline acetyltransferase activity in the developing brain of normal and hypothyroid rats, J. Neurochem., 19 (1972) 1947-1952. 1 ! Marchisio, P. C. and Consolo, S., Developmental changes of choline acetyltransferase (ChAc) activity in chick embryo spinal cord and sympathetic ganglia, J. Neurochem., 15 (1968) 759-764.
163 12 McCaman, R. E. and Aprison, M. H., The synthetic and catabolic enzyme systems for acetylcholine and serotonin in several discrete areas of the developing rabbit brain, Progr. Brain Res., 9 (1964) 220-233. 13 McGeer, E. G., Fibiger, H. C. and Wickson, V., Differential development of caudate enzymes in neonatal rat, Brain Research, 32 (1971) 433-440. 14 McGeer, E. G., Parkinson, J. and McGeer, P. L., Neonatal enzymic development in the interpeduncular nucleus and surrounding ventral tegmentum, Exp. Neurol., 53 (1976) 109-114. 15 Morris, D., Maneckjee, A. and Hebb, C., The kinetic properties of human placental choline acetyltransferase, Biochem. J., 125 (1971) 857-863. 16 Prempeh, A. B. A., Prince, A. K. and Hide, E. G. J., The reaction of acetyl-coenzyme A with choline acetyltransferase, Biochem. J., 129 (1972) 991-994. 17 Singh, V. K. and McGeer, P. L., Studies on choline acetyltransferase isolated from human brain, Neurochem. Res., (1977) In press.
