Neurochemical aspects of the ontogenesis of cholinergic neurons in the rat brain

Brain Research, 118 (1976) 429-440

429

© Elsevier/North-HollandBiomedicalPress, Amsterdam- Printed in The Netherlands

NEUROCHEMICAL ASPECTS OF THE ONTOGENESIS ERGIC NEURONS IN THE RAT BRAIN

OF CHOLIN-

JOSEPH T. COYLE and HENRY I. YAMAMURA* Departments of Pharmacology and Experimental Therapeutics and Psychiatry and the Behavioral Sciences, The Johns Hopkins University School of Medicine, Baltimore, Md. 21205 (U.S.A.)

(Accepted April 13th, 1976)

SUMMARY Ontogenic development of central cholinergic neurons in rat brain was examined by measuring the activity of choline acetyltransferase (CAT), concentration of acetylcholine (ACh) after focused microwave irradiation, the activity of the high affinity uptake process for choline and the apparent muscarinic receptor as quantified by specific binding of [3H]3-quinuclidinyl benzilate (QNB). For whole brain, the specific activity of CAT increases from 1 to 8 ~ of adult between 15 days gestation and 7 days postpartum and then increases linearly to 83 ~ by 4 weeks postpartum. The concentration of ACh is 22 ~ of adult at 15 days gestation, rises to 29 ~ by birth and attains adult levels by 4 weeks postpartum. The developmental rise in specific binding of [3H]QNB is intermediate between CAT and ACh with 10 ~ of adult concentration of receptor at birth and a linear increase to 90 ~ by 4 weeks postpartum. The development of the uptake of [3H]choline parallels that of CAT. In all regions of the neonatal rat brain, the relative level ( ~ adult) of ACh is higher than [ZH]QNB binding, which is higher than CAT. The neonatal medulla-pons has higher levels of [ZH]QNB binding and activity of CAT ( ~ adult) and develops more rapidly than the parietal cortex and corpus striatum; the hypothalamus and midbrain-thalamus exhibit intermediate rates of development.

INTRODUCTION Cholinergic neurons innervate all major regions of the brain and thus exert an influence on a wide variety of brain functions including neuroendocrine regulation in the hypothalamus16, modulation of movement in the caudate-putamen2a and thought * Present address: Department of Pharmacology,Universityof Arizona Schoolof Medicine,Tucson, Ariz. 85721, U.S.A.

430 processing in the cerebral cortex 2°. Although there have been several studies on certain neurochemical aspects of the development of the cholinergic neurons in specific regions of mammalian brain (for review, see refs. 14 and 22), there has yet to be a detailed regional ontogenetic study of cholinergic neurochemistry. Recent advances in the biochemical characterization of these neurons would allow for more detailed examination of their ontogenesis. These advances include the development of a sensitive assay technique to measure acetylcholineIs, the characterization of a high affinity uptake mechanism for choline which is specific for cholinergic processes19,39, and the development of methods to biochemically measure the cholinergic receptors 40. In the present study, we have examined the ontogenetic development in whole rat brain and in major regions of the brain of 3 presynaptic parameters for the cholinergic neurons, choline acetyltransferase (CAT), endogenous acetylcholine (ACh) and the high affinity uptake process for choline and the postsynaptic muscarinic cholinergic receptor. An assessment of the ontogenetic development of these pre- and postsynaptic parameters for the cholinergic neurons in brain can provide better insight into the neurochemical basis for cholinergic function in the immature brain. In addition, such information can be compared with results from studies using similar approaches for the development of the central noradrenergic v, dopaminergic9 and GABAnergic1° neurons and generate data of physiologic and pharmacologic significance concerning the changing relationships among these neurotransmitter systems in the developing nervous system. METHODS

Preparation of tissues. Sprague-Dawley rats, sperm positive on a specific date, were obtained from Hormone Assay Laboratories, (Chicago) and were housed in separate cages after 16 days of gestation. For all assays except for the determination of the levels of acetylcholine, the rats were killed by decapitation; and the brain was dissected at 2 °C into component regions by the method of Glowinski and Iversen 17. For the measurement of the levels of acetylcholine in postnatal rats, the animals were killed by microwave irradiation focused on the skull (1300 W; 2450 MHz) in an oven adapted by Medical Engineering Consultants (Lexington, Mass.); optimal exposure varied for each age from 2.0 to 3.4 sec. Choline acetyltransferase. The activity of choline acetyltransferase was measured in 5-50/~1 portions of tissue homogenized in 20 vol. (wt./vol.) of 0.05 M Tris • HC1, pH 7.4, containing 0.2 ~ (vol./vol.) Triton X-100 according to the method of Bull and Oderfeld-Nowak3. Endogenous acetylcholine. Concentration of acetylcholine in formic acidacetone (15:85) extracts of brain tissue was measured by the enzymatic radiometric assay of Goldberg and McCaman Is. Muscarinic cholinergic receptor. For the muscarinic cholinergic receptor binding assay, tissue was homogenized in 10 vol. of 0.32 M sucrose with a Brinkman Polytron (model PT-10; setting 6) for 1 rain 41. Portions of the homogenate (50-100/zl) were incubated at 25 °C in 2 ml of 0.05 M sodium-potassium phosphate buffer, pH 7.4,

431 containing 1 nM [3H]3-quinuclidinyl benzilate (QNB; 5.1 Ci/mmole; New England Nuclear Corp.) for 60 rain. The amount of [ZH]QNB bound was measured by isolating membranes on glass filters (GF/B; Whatman), which were counted for radioactivity by scintillation spectrometry. [ZH]QNB binds to non-specific sites as well as to the muscarinic-cholinergic receptor; to assess the amount of non-specific binding, duplicate samples were assayed in the presence of oxotremorine (100/~M), a potent muscarinic receptor agonist that blocks the binding of [ZH]QNB to muscarinic receptor but not to the non-specific sites. The difference between the binding of [3H]QNB in the presence and in the absence of oxotremorine represents the amount of [3H]QNB bound to the cholinergic receptor. Since the concentration of [3H]QNB used in the assay is saturating, the absolute amount of [3H]QNB specifically bound reflects the number of available muscarinic receptors in a preparation4k For newborn and adult brains, inhibition curves for the muscarinic receptor agonist, oxotremorine, and the muscarinic receptor antagonist, atropine, on the binding of [3H]QNB were determined. Protein. Protein was determined by the method of Lowry et al. 27 with bovine serum albumin as the standard. Uptake of [3H]choline. Fresh whole brain or striatum was homogenized with 4-6 strokes in 10 vol. of 0.32 M sucrose with a Potter-Elvehjem glass homogenizer fitted with a Teflon pestle and subjected to subcellular fractionation according to the method of Whittaker 38 to obtain the P2 fraction. The P2 fraction was gently resuspended in 10 vol. of 0.32 M sucrose (original homogenizing volume) with 3 strokes of the homogenizer. Portions (100/A) were preincubated for 4 rain at 37 °C in 0.9 ml of Krebs-phosphate buffer or sodium-free Krebs-phosphate buffer (made iso-osmolar with sucrose) and then incubated for 4 min after the addition of [ZH]choline (4.3 Ci/ mmole; New England Nuclear Corp.) at a final concentration of 50 nmolar 34. Uptake was terminated by the addition of 3 ml of ice-cold buffer and centrifugation at 2 °C at 20,000 × g for 10 rain. The pellet was solubilized in 1 ml of Protosol (New England Nuclear Corp.) and radioactivity was measured by liquid scintillation spectrometry. The difference between the amount of [ZH]choline taken up in the presence of sodium and the amount accumulated in the absence of sodium reflects the specific uptake occurring into cholinergic terminals. All ages were run on the same day under the same conditions to ensure validity of comparisons. RESULTS

Activity of choline acetyltransferase in developing brain. Choline acetyltransferase activity is present in whole rat brain at 15 days of gestation when the brain weighs 60-fold less than that of the adult (Fig. 1). The specific activity of the enzyme is quite low, being 1.4 ~ of the adult activity, and increases 2.5-fold during the last week in utero. Although the specific activity increases only an additional 2-fold during the first week postpartum, there is an inflection at 7 days after birth with a linear 10-fold increase in specific activity during the subsequent 3 weeks. There is marked regional heterogeneity in the distribution of the activity of choline acetyltransferase in the adult brain; the striatum has a 3-fold higher specific activity than the next highest

432

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Conceptual Age (Days) Fig. 1. Development of cholinergic neurons in whole rat brain. Activity of choline acetyltransferase, acetylcholine content, specific binding of [3H]QNB, and sodium-dependent uptake of [3H]choline were measured as described in Methods. Values are expressed in terms of mg wet weight of tissue. Each point is the mean from at least 5 separate brain preparations derived from at least 2 litters with S.E.M. indicated by the bars. Results are expressed in terms of conceptual age with birth occurring at 22 days. TABLE I Regional activity of choline acetyltransferase in fetal rat brain Results are means ± S.E.M. of 6 preparations derived from 2 litters and are expressed in terms of mgwet weight. Region

17 days gestation (pmoles/mg/h)

20 days gestation

Telencephalon Mesencephalon-diencephalon Rhombencephalon

60 ± 2 114 ± 9 511 ± 23

156 ± 5 166 ± 6 677 ± 63

region, the medulla-pons, which is 20-fold higher t h a n the lowest region, the cerebellum. I n utero, the r h o m b e n c e p h a l o n , which includes the cerebellar anlage, has a several-fold higher activity t h a n the more cephalad m e s e n c e p h a l o n a n d telencephalon (Table I). The medulla-ports m a i n t a i n s the highest specific activity at birth, being 3-10-

433 TABLE II Development of choline acetyltransferase activity in cerebellum Results are means ± S.E.M. of 6 preparations derived from 2 litters and are expressed in terms of mg wet weight. Age

nmole/mg/h

Birth 7 days 14 days 28 days Adult

0.39 -4- 0.04 0.66 4- 0.04 0.51 4- 0.01 0.74 4- 0.12 0.61 4- 0.04

fold higher than all other brain regions. All brain regions except the cerebellum exhibit a similar inflection in the developmental rise in choline acetyltransferase activity at 7 days after birth (Figs. 2 and 3). Whereas the medulla-pons achieves adult activity by 4 weeks after birth, the other regions have attained only 70-80 ~ of adult level by this date. Although in terms of absolute activity, the cerebellum has the lowest activity of all brain regions, the specific activity of choline acetyltransferase remains relatively constant during development as previously observed in chick brain (Table I1) ~9. Concentration o f acetylcholine in developing brain. The concentration of acetylcholine in the fetal brain is relatively high, being 22 ~ of the adult level at 15 days of gestation and increasing to 38 ~ of adult by 2 days before birth (Fig. 1). There is a decrement in the level at birth to 27 ~ of adult concentration. It should be emphasized that the levels of the neurotransmitter in fetal brain were determined after decapitation whereas all postnatal measurements were done in brains of animals instantaneously killed by focused microwave irradiation; thus, the difference between the fetal and postnatal values may be due in part to the change in the method of sacrifice. The whole brain concentration of acetylcholine exhibits a rather linear increase between birth and 4 weeks after birth, when adult levels are achieved. TABLE llI Regional distribution of acetyleholine The levels of endogenous acetylcholine were measured in various regions of the brains of rats that were killed by focused microwave irradiation. Each value is the mean 4- S.E.M. of at least 6 preparations and expressed in terms of mg wet weight of tissue. ND -- not determined. Region

A dult

Newborn pmole/mg

Medulla-pons Midbrain-thalamus Hypothalamus Corpus striatum Parietal cortex Cerebellum

27.0 2:3.3 40.0 4- 2.9 46.5 4- 3.7 65.4 4- 5.7 20.6 T 2.2 9.4 4- 1.5

23.4 ± 4.4 10.8 4- 0.3 15.3 ~ 3.8 9.3 ± 1.8 ND

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Age (Days P0st-Partum) Fig. 2. Development of cholinergic neurons in rat striatum. Activity of choline acetyltransferase, acetylcholine, specificbinding of [3H]QNB, and sodium-dependent uptake of [3H]cholinewere measured as described in Methods. Values are expressed in terms of mg wet weight of tissue. Each point is the mean from at least 5 preparations derived from at least 2 litters with S.E.M. indicated by the bars. There is a marked regional heterogeneity in the distribution of endogenous acetylcholine that does not exhibit a close proportionality with the regional activity of choline acetyltransferase (Table III). The striatum has the highest content and the parietal cortex and cerebellum the lowest, with midbrain, hypothalamus and medullapans intermediate. At birth the concentration of acetylcholine in the medulla-pans is not significantly different from that of the adult; in contrast, the combined midbrainhypothalarnus is 25 ~ and the parietal cortex is 50 ~ of adult levels. In the striatum, the concentration of acetylcholine remains constant until I week after birth when there occurs a linear increase to adult levels by 4 weeks after birth (Fig. 2). Uptake of [3H]choline by P2 fractions of developing rat brain. The uptake of [3H]choline was measured in resuspended Pz fractions prepared from brains of rats from birth through to adulthood. The washed Pz fraction was utilized to reduce significant contamination by endogenous choline which varies in concentration during development z6. Sodium-dependent uptake is first detectable in Pz fractions prepared from whole brain at 1 week after birth; the activity increases dramatically during the next 7 days, attaining 50 ~ of adult levels by 2 weeks postpartum (Fig. 1). It increases another 30 ~ in the subsequent 2 weeks. The striatum exhibits a similar developmental pattern although the rapid rise in sodium-dependent uptake is delayed until 2 weeks after birth (Fig. 2). The developmental increase in sodium insensitive uptake closely

435 Midbrain

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Age (Days Post-Partum) Fig. 3. Regional development in the activity of choline acetyltransferase in rat brain. Each point is the mean of at least 6 preparations derived from 2 litters with S.E.M. indicated by the bars. Values are expressed in terms of mg wet weight of tissue.

parallels the increase in protein content of the P2 fraction for both whole brain and striatum. Muscarinic cholinergic receptor in developing brain. The apparent muscarinic cholinergic receptor in developing rat brain was quantified by measuring the specific binding of [3H]QNB to membrane fragments in sucrose homogenates. At 15 days of gestation, the brain exhibits low but detectable levels of specific binding of [3H]QNB which is at 1 ~ of that of the adult. The apparent muscarinic receptor content increases in a relatively linear fashion during the last week of gestation and first month postpartum, when the concentration of specific binding sites for [~H]QNB reaches adult levels (Fig. 1). The concentrations of atropine and oxotremorine that inhibit the specific binding of [3H]QNB by 50 ~ are not significantly different for neonatal and adult brain preparations; this suggests that the same receptor is being measured throughout development. In the adult brain, the cortex and striatum possess the highest concentration of muscarinic receptor being 4-5-fold higher than midbrain, hypothalamus or medullapons. At birth, the striatum possesses the highest concentration of receptor as is the case in adulthood; the medulla-pons has the next highest concentration although the parietal cortex surpasses it by 2 weeks after birth (Fig. 4). The more caudal regions of the brain achieve adult concentrations of receptor much more rapidly than the rostral ones. Thus, at 2 weeks after birth, the medulla-pons and hypothalamus have nearly

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Age ( Doys Post-PQrtum) Fig. 4. Regional development of the specificbinding of [ZH]QNBin rat brain. Each point is the mean of at least 5 preparations derived from 2 litters with S.E.M. indicated by the bars. Values are expressed in terms of mg wet weight of tissue. 80 ~ concentration of the muscarinic receptor whereas the parietal cortex and striatum are less than 40 ~ of adult levels. DISCUSSION The developmental increase in the specific activity of choline acetyltransferase in whole brain is similar to that previously reported for chick 4, mouse 39, and rat ~6 with pronounced sigmoid characteristics to the curve. In the rat, the activity remains relatively low in whole brain until 1 week after birth and then exhibits a 10-fold increase during the subsequent 3 weeks. This delay in the developmental rise until 1 week postpartum is mirrored in all regions examined except the cerebellum, which has a relatively constant specific activity throughout the postnatal period. In spite of this delay, the medulla-pons and the hypothalamus develop more rapidly, achieving adult levels of activity in advance of the parietal cortex and striatum. The more rapid maturation of choline acetyltransferase activity in the caudal regions of the brain probably reflects the fact that cell division ceases and synaptogenesis commences in these regions much in advance of the subcortical and cortical areas1, 7. The phase of differentiation occurring after cessation of cell division 2 and the later formation of cholinergic synaptic terminals 6,80 are both associated with marked increases in the activity of choline acetyltransferase.

437 The level of acetylcholine in whole brain is remarkably high at early stages of development in contrast to the activity of its biosynthetic enzyme. When expressed in terms of per cent of adult levels, the concentration of acetylcholine at 17 days of gestation is 38 ~ of adult whereas the specific activity of choline acetyltransferase is only 3 ~o of adult. Similar disparities between acetylcholine and choline acetyltransferase in immature brain have been previously observed in chick4 and rat brain 2~. As in the whole brain, disparities between the levels of acetylcholine and activity of choline acetyltransferase are observed in all brain regions examined at birth with levels of acetylcholine at 25-85 ~ of adult when the activity of choline acetyltransferase is less than 10~ of adult in the regions. Several factors may contribute to the high levels of acetylcholine present in the fetal and neonatal rat brain. The low activity of acetylcholinesterase in the immature brain may allow for a much greater accumulation of acetylcholine than occurs in the mature brain 13,28. In addition, the turnover of acetylcholine in adult brain, which reflects the firing rate of the neurons, is quite high with a half-life estimated to be only a few minutes 21,z2. Since the firing rate of immature neurons is known to be low31, the relatively slow turnover of the neurotransmitter in the immature brain may result in proportionately higher stores of acetylcholine in the neuron. Finally, there is considerable evidence from developmental studies that acetylcholine may serve a function other than that of a neurotransmitter in early embryogenesis~4; thus, the acetylcholine may not be localized exclusively in cholinergic neurons at early stages of development. Choline can be transported by a low affinity and a high affinity process into slices of brain and partially purified synaptosomal fractions~9,3L The latter transport process is markedly dependent on sodium; and most of the choline taken up by this process is converted to acetylcholine34,4°. Furthermore, lesion studies indicate that the sodium-dependent high affinity uptake process is specific for cholinergic neurons 25. Since both low and high affinity uptake processes are temperature-dependent, the development of the transport process for choline specific for cholinergic neurons was quantified by measuring that portion of choline transport that was sodium-dependent. In washed P2 fractions prepared from both whole brain and the striatum, the sodiumdependent high affinity uptake process for choline is quite low until 7 days after birth, after which a marked increase in the activity of the uptake process occurs. Our results are in general agreement with those of Sorimachi et al. 37 who examined the development of temperature-dependent uptake of choline into synaptosomes. Similar studies done in developing rat brain with the norepinephrine and dopamine transport mechanism suggest that there is a close correlation between the development of uptake of these neurotransmitters into P2 fractions and the elaboration of terminals as documented by histofluorescent studiesS, 9. In the case of choline uptake, however, this relationship between uptake and terminal density is more tenuous since recent studies by Simon and Kuhar 3~ have shown that the high affinity uptake process for choline is markedly affected by the antecedent activity of the neurons. In spite of this caveat, it is noteworthy that after the initial delay in appearance of the choline uptake process, the development of the transport mechanism for choline into partially purified

438 synaptosomal fractions coincides closely with the developmental rise in the activity of choline acetyltransferase. Pharmacologic, electrophysiologic and autoradiographic studies strongly suggest that the specific binding of [3H]QNB provides a reliable assessment of muscarinic receptor density in tissue36,41. The apparent muscarinic receptor is detectable at low levels at 15 days of gestation in the rat brain. It exhibits a rather linear increase in density during subsequent maturation of the brain. If the developmental increases are plotted in terms of per cent of adult levels, the development of the apparent muscarinic receptor in whole brain parallels but precedes that of choline acetyltransferase. In all regions examined, the receptor achieves adult concentration well before that of the presynaptic marker of choline acetyltransferase. In addition, the caudal regions of the brain exhibit more rapid maturational increases than the rostral regions. Extensive investigations in vivo and in vitro of the neuromuscular junction indicate that the nicotinic cholinergic receptor appears on the muscle membrane prior to the development of cholinergic innervation 12,15. If the activity of choline acetyltransferase is a reliable index of presynaptic cholinergic differentiation in brain, the development of the postsynaptic muscarinic receptor appears to antedate that of its cholinergic innervation as is the case for the neuromuscular junctions. It must be emphasized that the muscarinic receptor represents only one of several types of cholinergic receptors as characterized by electrophysiologic and pharmacologic studies 24. The development of a nicotinic cholinergic receptor, that can be measured by the specific binding of the snake venom a-bungarotoxin, has been examined in the cortex of the postnatal rat 33. In contrast to the muscarinic receptor, the nicotinic receptor exhibits only a 2-fold change in concentration between birth and adulthood; however the density of this receptor in adult neocortical tissue is 30fold lower than that of the muscarinic receptor as measured by the specific binding of the muscarinic antagonists, [3H]QNB41 and [ZH]N-2'-chloroethyl-N-[2',3'-~H2]-propyl 2-aminoethylbenzilateL The neurochemical aspects of the development of the central cholinergic neurons exhibit some significant parallels and differences when compared to the differentiation of the central catecholaminergic and GABAnergic neurons. Unlike the catecholaminergic neurons 7,9 but similar to the GABAnergic system~°, there appears to be a marked disparity between the levels of GABA and acetylcholine in comparison to the activity of their biosynthetic enzymes at early stages of development with disproportionately high concentrations of the neurotransmitters. However, similar to the catecholaminergic neurons 8,9 and unlike the GABAnergic neurons lo, there is a close correlation between the developmental increase in the activity of the biosynthetic enzymes for their respective neurotransmitters and the increase in the high affinity uptake processes in partially purified synaptosomal fractions37. Using the activity of the biosynthetic enzymes for the neurotransmitters as the discriminator for neuronal differentiation, we observe that in whole brain and in the striatum there is a sequential maturation of the 3 neurotransmitter systems with the catecholaminergic neurons first, GABAnergic second and cholinergic last. Since the catecholaminergic and GABAnergic inputs are predominantly inhibitory, it is noteworthy that their development precedes that of the

439 predominantly excitatory cholinergic neuronal system as has been suggested by electrophysiologic studies 24,al. ACKNOWLEDGEMENTS

The authors wish to thank Robert Zaczek for his excellent technical assistance and Nancy Hiatt and Vickie Rhodes for their secretarial assistance. This research was supported by USPHS Grants DA 00266 (J.T.C.) and MH 26967 and MH 27257 (H.I.Y.). REFERENCES 1 Altman, J., Autoradiographic and histologic studies of postnatal neurogenesis, J. comp. Neurol., 136 (1969) 269-294. 2 Amano, T., Richelson, E. and Nirenberg, M. W., Neurotransmitter synthesis by neuroblastoma clones, Proc. nat. Acad. Sci. (Wash.), 69 (1972) 258-263. 3 Bull, G. and Oderfeld-Nowak, B., Standardization of a radiochemical assay of choline acetyltransferase and a study of the activation of the enzyme in rabbit brain, J. Neurochem., 18 (1971) 935-947. 4 Burdick, C. J. and Strittmatter, C. F., Appearance of biochemical components related to acetylcholine metabolism during the embryonic development of chick brain, Arch. Biochem., 109 (1965) 293-301. 5 Burgen, A. S. V., Hiley, C. R. and Young, J. M., The properties of muscarinic receptors in mammalian cerebral cortex, Brit. J. PharmacoL, 51 (1974) 279-285. 6 Burt, A. M., Choline acetyltransferase and acetylcholinesterase in the developing rat spinal cord, Exp. NeuroL, 47 (1975) 173-180. 7 Coyle, J. T., Biochemical aspects of the catecholaminergic neurons in the brain of the fetal and neonatal rat. In K. Fuxe, L. Olson and Y. Zotterman (Eds.), Dynamics of Degeneration and Growth in Neurons, Pergamon Press, New York, 1974, pp. 425-434. 8 Coyle, J. T. and Axelrod, J., Development of the uptake and storage of L[aH]norepinephrine in rat brain, J. Neurochem., 18 (1971) 2061-2075. 9 Coyle, J. T. and Campachiaro, P,, Ontogenesis of dopaminergic-cholinergic interactions in rat striatum: a neurochemical study, J. Neurochem., (1976) in press. 10 Coyle, J. T. and Enna, S. J., Ontogenesis of GABAnergic neurons in the rat brain, Brain Research, 111 (1976) 119-132. 11 Das, G. D. and Altman, J., Postnatal neurogenesis in the caudate nucleus and nucleus accumbens septi in the rat, Brain Research, 21 (1970) 122-127. 12 Diamond, J. and Miledi, R., A study of fetal and newborn rat muscle fibers, J. Physiol. (Lond.), 162 (1962) 393-408. 13 Elkes, J. and Todrick, A., On the development of the cholinesterases in the rat brain. In H. Waelsch (Ed.), Biochemistry of the Developing Nervous System, Academic Press, New York, 1955, pp. 304-314. 14 Filogamo, G. and Marchisio, P. C., Acetylcholine system and neural development, Neurosci. Res., 4 (1971) 29-64. 15 Fishbach, G. D. and Cohen, S. A., The distribution of acetylcholine sensitivity over uninnervated and innervated muscle fibers grown in cell cultures, Develop. BioL, 31 (1973) 147 162. 16 Ganong, W. F., The role of catecholamines and acetylcholine in the regulation of endocrine function, Life Sci., 15 (1975) 1401-1414. 17 Glowinski, J. and Iversen, L. L., Regional studies of catecholamines in rat brain. I. The disposition of [ZH]norepinephrine, [3H]-dopamine and [3HI-DOPA in various regions of the brain, J. Neurochem., 13 (1966) 655-669. 18 Goldberg, A. M. and McCaman, R. E., The determination of picomole amounts of acetylcholine in mammalian brain, J. Neurochem., 20 (1973) 1 8. 19 Haga, T. and Noda, H., Choline uptake systems of rat brain synaptosomes, Biochim. biophys. Acta (Amst.), 291 (1973) 564-572.

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