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Trans-synaptic regulation of growth and development of adrenergic neurones in a mouse sympathetic ganglion
229
BRAIN RESEARCH
TRANS-SYNAPTIC REGULATION OF GROWTH AND DEVELOPMENT OF ADRENERGIC NEURONES IN A MOUSE SYMPATHETIC GANGLION
I. B. BLACK, I. A. H E N D R Y AND L. L. IVERSEN
Department of Pharmacology, University of Cambridge, Cambridge (Great Britain) (Accepted May 27th, 1971)
INTRODUCTION
The study of developmental events within the central nervous system has involved anatomical1°,z4, ultrastructural2,5, 6, electrophysiological9, and biochemicaPS, 21 approaches. The complexity of organization of the central nervous system, however, has led to interpretative difficulties, and studies in the periphery x4 have provided simpler models of neural ontogeny. In the present study the maturation of mouse superior cervical ganglion in vivo has been examined utilizing a combination of biochemical and morphological methods. Choline acetyltransferase (ChAc), the enzyme catalyzing the conversion of acetyl-CoA and choline to the neurotransmitter acetylcholine, was used as a marker for the development of presynaptic cholinergic fibres. The enzyme is highly localized to these presynaptic terminals16. Maturation of postsynaptic neurones was followed by measuring the activity of tyrosine hydroxylase (T-OH), the rate-limiting enzyme in the biosynthesis of noradrenaline20, the postganglionic neurotransmitter. Visualization of ganglion synapses with the electron microscope allowed estimation of the development of synaptic connections. Correlation of these biochemical and morphological parameters suggested that presynaptic nerve terminals might regulate the development of the postsynaptic neurone. The results of surgical transection of the preganglionic nerve trunk in neonatal animals were consistent with this hypothesis. A preliminary report of these findings has appeared 3. METHODS
Experimental animals
Litters of Swiss albino mice were used, and mothers were offered OXO Diet 41B and water ad libitum. All litters were standardized, by reassigning offspring to mothers, so each had 10 pups within a day of birth. Groups of 6 animals, randomly chosen, were used in studies of enzyme development. The mice were killed by exposure Brain Research, 34 (1971) 229-240
230
I.B. BLACKet al.
to ether vapour and superior cervical sympathetic ganglion pairs were removed under a dissection microscope. Left and right ganglia from each pup were homogenized in 50/tl of distilled water and 10/~1 aliquots were taken for the assay of ChAc, T-OH and for the estimation of total protein. E n z y m e assays
ChAc activity was assayed using minor modifications of the method of Fonnum 11. The assay was initiated by the addition of 10 #1 of ganglion homogenate to 10 #1 of a solution containing, in final concentration, [3H]acetyl-CoA 0.1 mM; choline bromide 10 mM; physostigmine sulphate 1 mM; in sodium phosphate buffer, 50 mM, with a final pH of 7.40. For maximal activity the following ingredients were also present: sodium ethylene diamine tetraacetic acid (EDTA), 10 mM; sodium cyanide, 10 mM; sodium chloride, 300 mM;and albumin 0.I ~. [aH]Acetyl-CoA (247 #Ci//zmole) was synthesized before the enzyme assay using [3H]acetic anhydride and CoA. To 30 mg o f C o A (Boehringer) dissolved in 1 ml 1 M potassium bicarbonate was added 3.5 ml of an ether solution of [all]acetic anhydride (500 mCi/mole, Radiochemical Centre, Amersham; total 25 mCi), in 0.5 ml aliquots. The solution was acidified to pH 1.0 with 4 N hydrochloric acid, and was extracted 8 times with 1.5 ml of acid-saturated ether to remove excess [3H]acetate. The aqueous solution was assayed for acetyl-CoA by the citrate synthase method 25 to determine the concentration and specific activity. The ChAc reaction was terminated after 60 min at 37°C, by the addition of 3.5 ml of iced 0.01 M phosphate buffer, pH 7.4, containing acetylcholine bromide 0.25 mg/7.0 ml as carrier. The tritiated acetylcholine formed was extracted by liquid cation exchange chromatography into 0.5 ml of butyl ethyl ketone containing tetraphenylboron, 15 mg/ml. After low speed centrifugation an aliquot of the organic mixture in ethoxy ethanol and 0 . 4 ~ butyl PBD (CIBA) in toluene was counted by liquid scintillation spectroscopy. With this procedure the assay was linear for 60 min and for 1-50 #g of adult ganglion tissue. Ganglion T-OH was assayed by methods previously described 17. A 10 #1 aliquot of tissue homogenate was added to 15/~1 of a solution containing, in final concentration, [3H]tyrosine 0.004 m M (spec. act. = 11.5 mCi/#mole); 6,7-dimethyl tetrahydropterine 1.25 mM; 2-mercaptoethanol 0.3125 mM; NSD 1055 0.15 mM; and potassium phosphate buffer 400 m M at a final pH of 6.0. The reaction at 37°C was terminated after 20 min by the addition of 0.25 ml 0.4 N perchloric acid containing L-dihydroxyphenylalanine (L-DOPA) and dopamine 2 #g/ml. This mixture was brought to pH 8.6 with 0.1 M Tris containing sodium EDTA 0.05 M and N a O H 0.075 M and passed over an alumina column (dimensions 5.0 cm × 0.25 cm) previously equilibrated with 0.1 M sodium phosphate buffer, pH 8.6. After two successive washes of 5 and 15 ml with 0.005 M Tris buffer pH 8.6, the tritiated L-DOPA was eluted with 1.5 ml of 0.5 M acetic acid. The eluate was counted in a liquid scintillation counter after the addition of 12 ml of a 2:1 (v/v) mixture of 0.6 ~ butyl PBD (CIBA) in toluene and Triton X-100.
Brain Research, 34 (1971) 229-240
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Fig. 1. Electron micrograph of mouse superior cervical ganglion stained with EPTA. Two synaptic complexes are seen with presynaptic projections, intrasynaptic line and postsynaptic membrane. Horizonta! bar equals 0.5 ~m. Total protein was estimated in samples of ganglion homogenates by the procedure of Lowry2L Electron microscopy Ganglion synaptic junctions were visualized for electron microscopy using ethanolic phosphotungstic acid (EPTA) as described by Bloom and AghajanianL Ganglia were removed from mice and fixed by immersion in 0.16 M sodium phosphate buffer, pH 7.4, containing 5 ~ glutaraldehyde for ! h. The ganglia were then washed with phosphate buffer dehydrated in ethanol and stained with 1 ~o PTA in ethanol before embedding in Epon. Sections averaging 75 nm in thickness were prepared with an LKB Ultratome III in a plane parallel to the long axis of the ganglia. The density of synaptic junctions was estimated by counting the number observed in systematic searches of l0 grid squares (45/~m × 45 #m). Synapses were scored only if the presynaptic specialization, intercleft substance and postsynaptic membrane were visualized (Fig. 1). Synapses were identified and counted by 3 different observers. Appropriate corrections for synapse diameter were made according to the method of Abercrombie 1. Synapse density measurements were converted to total numbers per ganglion by estimating total ganglion volume from sections of the same ganglia. Brain Research, 34 (1971) 229-240
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I.B. BLACKet al.
Serial sections of the ganglia, cut parallel to their long axes, were mounted on glass slides, stained with toluidine blue and measured with a light microscope using a micrometer eye piece to determine the major and minor ganglion diameters. Ganglia were assumed to conform most closely in geometry to prolate spheroids and ganglion volume was calculated on the basis of the formula V = 4/3 :rab 2, where V = volume, a = major radius and b = minor radius. Estimation of synapse numbers by this method involves the assumption that synaptic centers are randomly distributed and oriented in the ganglia. Such an assumption is basic to morphometric investigation 26. It has also been assumed that there is no age-related alteration in synapse geometry. This has been confirmed, to some extent, by noting no significant change in observed synapse diameter with age (mean synapse diameter was 0.23/~m). Errors introduced by assuming that ganglia are prolate spheroids may alter the absolute, but not the relative, values. Surgical procedure Ganglia were decentralized in mice anaesthetized with ether by unilateral transection of the preganglionic trunk in the neck under a dissecting microscope. The surgical wound was closed with celloidin and mice were returned to their cages. This procedure resulted in a variable mortality rate, but those which survived surgery thrived and grew normally for the duration of the experiment. RESULTS
Groups of mice were randomly selected from litters of different ages and ganglion pairs were assayed for enzyme activities and total protein. ChAc activity increased 30-40-fold during the course of development. From low levels on day l, enzyme activity rose rapidly during the first 2 weeks of life reaching a hyperbolic plateau by approximately 3 weeks (Fig. 2). This increase in enzyme activity may reflect either ongoing invasion of the ganglion by presynaptic nerve endings and/or transport of the enzyme to nerve endings already present in the ganglion. The developmental curve for T-OH activity differed significantly from that of ChAc. T-OH activity increased 6-8-fold from birth to adulthood. The rise in activity, however, occurred in two distinct phases. From birth to day 3, T-OH activity nearly doubled, but from day 3 to 7 a highly reproducible plateau phase occurred with no change in T-OH activity. The major increase took place during the second week of development when enzyme activity underwent nearly a 3-fold rise with little subsequent elevation to the 38th day of life (Fig. 2). These 3 phases of development, the initial increase in activity, the plateau phase and the phase of major increase have been observed in all experiments. During development total ganglion protein increased only 3-fold, thus the rises in enzyme specific activities were highly significant (Fig. 2). This relatively modest increase in total protein has been observed previously during the development of ganglia 8,14. Brain Research, 34 (1971) 229-240
DEVELOPMENT OF SYMPATHETIC GANGLION NEURONES
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Fig. 2. Developmental increases of transmitter enzyme activities and total protein in mouse superior cervical ganglia. Groups of 6 mice were taken from litters of varying ages and ganglion pairs from each animal were assayed for enzyme activities and total protein as indicated under Methods. Choline acetyltransferase activity is expressed as mean (nmoles product per ganglion pair) per h -4- S.E.M. (vertical bars). Tyrosine hydroxylase activity is expressed as (10 -11 moles product per ganglion pair) per h. Total protein is expressed as mean/~g per ganglion pair 4- S.E.
Developmentalformation of synapticjunctions To appreciate the functional significance o f these biochemical correlates o f maturation, we attempted to define their temporal relation to the d e v e l o p m e n t o f Brain Research, 34 (1971) 229-240
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inter-neural connections. Synaptic j u n c t i o n s (Fig. 1) were identified and counted, as described in M e t h o d s in ganglia o f mice aged 1-60 days. D u r i n g this p e r i o d total synapses per ganglion increased f r o m a p p r o x i m a t e l y 8,000 on d a y 1 to 3,000,000 by d a y 60 (Fig. 3). Synapse density increased 10-fold while total ganglion v o l u m e rose over a 50-fold range. The n u m b e r o f synapses r e m a i n e d relatively c o n s t a n t d u r i n g the first 2 days after birth, b u t rose d r a m a t i c a l l y between days 5 and 11 to an a s y m p t o t i c p l a t e a u (Fig. 3). F r o m d a y 11 to 60 the further increase in the n u m b e r o f synapses just reached statistical significance. C o m p a r i s o n o f synapse d e v e l o p m e n t with the d e v e l o p m e n t a l p a t t e r n o f C h A c
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DEVELOPMENTOF SYMPATHETICGANGLIONNEURONES
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activity revealed some similarities (Fig. 4). Both functions display roughly hyperbolic curves with relatively modest increases during the first 3-4 days of life, and rapid rises early in the second week of development. On the basis of the data presented, however, more precise temporal relationships were not evident. The development of T-OH activity contrasts interestingly with synapse formation (Fig. 5). The early rise in T-OH activity precedes the increase in synapses. Enzyme activity remains at plateau levels during the initial phase of synaptic rise, days 5-7. Immediately following the steep increase in synapse formation, however, T-OH activity increases markedly to adult levels. These observations suggested that development of T-OH activity in postsynaptic neurones might be dependent on contact with presynaptic nerve endings.
Effect of decentralization of the superior cervical ganglion To determine whether the presynaptic cholinergic nerve terminals regulate the development of T-OH activity in the postsynaptic neurone, ganglia were decentralized in neonatal mice. The preganglionic trunk was transected unilaterally in mice aged 5-6 days. The contralateral normal ganglion served as control. Rats were killed 13 days postoperatively, ipsilateral ptosis and reduced ganglion ChAc activity indicating success of the procedure (Fig. 6). As expected ChAc activity was reduced to approximately 1 0 ~ of control values. T-OH activity failed to increased above normal 7-day plateau levels, remaining at approximately 30 ~ of the activity of contralateral unoperated ganglia (Fig. 5). These findings indicate that the increase in T-OH activity occurring during the second week of development (Fig. 2) is dependent on innervation of the postsynaptic neurone. In additional studies sham operations were performed in which ganglia were
Brain Research, 34 (1971) 229-240
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Fig. 6. Effect of preganglionic decentralization on ganglion tyrosine hydroxylase activity. Five mice aged 7 days were subjected to unilateral ganglion decentralization and killed 13 days thereafter, as described in Methods. Choline acetyl transferase and tyrosine hydroxylase activities were assayed in individual decentralized and contralateral control ganglia. Results are expressed as mean (nmoles product per single ganglion) per h ± S.E.M. (vertical bars) for choline acetyl transferase and as (10-11 moles product per ganglion) per h for tyrosine hydroxylase. * and ** differ from experimental group at P < 0.001.
exposed unilaterally without transection of the preganglionic trunk. This procedure did not alter the normal pattern of development of ChAc or T-OH activities. DISCUSSION The superior cervical ganglion receives presynaptic innervation from axons arising in the intermediolateral column of the lateral horn of thoracic spinal segments 1-3 (ref. 23). ChAc is, presumably, synthesized within these spinal neurone cell bodies and transported peripherally as in other cholinergic neurone systems 13. Since no natural activator or inhibitor (except possibly acetylcholine itself 18) of ChAc has been identified 12 it is likely that the increased ganglionic enzyme activity observed during maturation represents an actual increase in the number of enzyme molecules. Thus the increase in ChAc activity in the ganglion during development may represent a summation of the rates of enzyme synthesis and degradation in the spinal cell body, the rate of transport of ChAc along the axon, of ingrowth of cholinergic nerve terminals, and of degradation of enzyme in the nerve endings. The marked increase in ChAc activity, and most probably in ChAc molecules, closely approximates adult levels by the second to third week of life. At this time the capacity for neurotransmitter synthesis is thus present. These results also suggest that acetylcholine synthesis does not suddenly appear, in an all-or-none fashion, but increases progressively over the first few postnatal weeks. The similarities in the patterns of ChAc activity and synapse development suggest that the increased activity of this enzyme reflects functional neuronal maturaBrain Research, 34 (1971) 229-240
DEVELOPMENT OF SYMPATHETIC GANGLION NEURONES
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tion. Such a formulation is consistent with that of Burt 7 who has noted association of ChAc activity with the development of motor activity in embryonic chick spinal cord. In addition since ChAc activity appears shortly after acetylcholine storage vesicles are present 7, and since enzyme is associated with cholinergic nerve terminals in the ganglion15, ChAc activity may reflect functional synaptic development as well as neurotransmitter synthesis. The developmental pattern of ganglionic T-OH activity occurs in a context different from that of ChAc. T-OH is largely restricted to postsynaptic cell bodies4 and thus the activity measured directly reflects development of the noradrenergic neurone, and is dependent on enzyme turnover within that neurone. T-OH activity rises in two separate and well-defined phases. The initial 2-fold rise occurs from birth to day 3. After a 4-day plateau period, the major 3-fold increase in T-OH takes place. These two phases of increase may reflect different events in the ganglion. The initial rise occurs prior to the period of synapse formation, and before ChAc activity has risen to any significant degree. Hence, it is unlikely that this initial phase is regulated by the presynaptic neurone. This elevation of T-OH may occur in perikarya which later respond to synaptic influences. However, it is possible that the early elevation of T-OH activity reflects a separate enzyme pool whose turnover is regulated by other factors. The ganglionic chromaffin cells27 may represent such a separate enzyme pool, and could be responsible for the early increase in enzyme activity. Consideration of events transpiring during the early plateau phase of T-OH activity further suggests independence of the two phases of enzyme development. Levi-Montalcini and Booker 19have demonstrated that from days 3 to 9 the neuroblasts of mouse superior cervical ganglion undergo intense mitotic activity. This period corresponds almost precisely to the T-OH early plateau phase. Furthermore, the second rise in T-OH activity is virtually synchronous with cessation of mitosis in the postsynaptic cells. These observations suggest that prior to the plateau phase, the blast progenitors of postganglionic neurones are in an undifferentiated state, most probably incapable of synthesizing T-OH. The relatively synchronous mitotic activity of the postsynaptic primordia may indicate that biochemical maturation of these cells occurs simultaneously and not in separate stages. The virtually synchronous increase of ganglionic synapses and T-OH activity suggested that maturation of the noradrenergic neurones depends on synapse formation. Abolition of the major increase in T-OH activity by transection of the preganglionic nerve trunk supported this view. Thus the presynaptic cholinergic nerve terminals regulate development of the postsynaptic cells. Although acetylcholine itself may constitute the stimulus evoking T-OH development postsynaptically, further study of this question is clearly needed. It should be stressed that while presynaptic terminals may be necessary for the development of ganglionic neurones, they may not be sufficient, and other unidentified mechanisms may also participate. These studies further suggest that, in some sense, the synapses demonstrated by electron microscopy are functional. That is, surgical destruction of the synaptic junctions prevents normal T-OH development. However, the observations presented
Brain Research, 34 (1971) 229-240
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I.a. BLACKet al.
do not indicate whether the demonstration of synapses morphologically can be correlated with the onset of cholinergic synaptic transmission. Synapse numbers remain at a basal level for at least the first 5 days of life. Preliminary investigations also indicate that a similar number of synapses exist in the superior cervical ganglion in the foetus near full term. After day 5, however, there is an abrupt and marked increase in synapses. Thus, once again, there is a striking degree of synchrony in a developmental parameter, suggesting an almost simultaneous staging of many cell functions within the ganglion. The development of synapses in the mouse sympathetic ganglion exhibits interesting similarities to that previously described in various regions of brain 2. In both cases an early plateau phase in synapse density is replaced by a sudden steep increase in synapse numbers to near the adult values within a few days. Such a pattern may thus be typical of synapse formation during development in the peripheral as well as the central nervous system. SUMMARY
The development of transmitter enzymes and the formation of synapticconnections have been studied in mouse superior cervical ganglion in vivo. Ganglion choline acetyl transferase (ChAc) activity increased by 40-fold during development and described a hyperbolic curve with time. Tyrosine hydroxylase (T-OH) activity rose in two separate and distinct phases during maturation. Estimates of total synapses per ganglion were obtained electron microscopically after staining with ethanolic phosphotungstic acid. Ganglion synapses increased 500-fold during development. From plateau levels during the first few days of life, synapse numbers increased markedly between days 5 and 11 to reach an asymptotic plateau. The developmental curve for ChAc was virtually congruent with that of synapse numbers, suggesting that increased enzyme activity reflected the maturation of preganglionic cholinergic terminals. On the other hand, the major increase in T-OH activity occurred synchronously with the marked increase in synapses, suggesting that the formation of synaptic contacts might be necessary for T-OH activity development. To examine this possibility ganglia were unilaterally decentralized in neonatal mice. Surgical transection of the preganglionic nerve trunk prevented normal development of T-OH activity in the postsynaptic adrenergic neurones. These observations indicate that presynaptic nerve terminals regulate the biochemical maturation of the postsynaptic adrenergic neurones. ACKNOWLEDGEMENTS
We are grateful to Dr. F. Bloom for advice and assistance in the use of the ethanolic phosphotungstic acid method for staining ganglion synapses. I.B.B. is the recipient of a William O. Moseley Travelling Fellowship from Harvard Medical School. I.A.H. is the recipient of a Fellowship from the Postgraduate Medical Foundation of the University of Sydney, Australia. These studies Brain Research, 34 (1971) 229-240
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were supported by grants to L.L.I. from the Medical Research Council and the Mental Health Research Fund. We thank Miss Margaret Hickman for excellent technical assistance.
REFERENCES 1 ABERCROMBIE,M., Estimation of nuclear population from microtome sections, Anat. Rec., 94 (1946) 239-247. 2 AGHAJANIAN,G., AND BLOOM,F. E., The formation of synaptic junctions in developing rat brain: a quantitative electron microscopic study, Brain Research, 6 (1967) 716-727. 3 BLACK,I. B., BLOOM,F. E., HENDRY,L, ANDIVERSEN,L. L., Growth and development of a sympathetic ganglion: Maturation of transmitter enzymes and synapse formation in the mouse superior cervical ganglion, J. Physiol. (Lond.), 215 (1971) 24P. 4 BLACK,I. B., HENDRY,I., AND IVERSEN,L. L., Differences in the regulation of tyrosine hydroxylase and DOPA decarboxylase in sympathetic ganglia and adrenal, Nature (Lond.), 231 (1971) 27-29. 5 BLOOM,F. E., AND AGHAJANIAN, G. K., Fine structural and cytochemical analysis of the staining of synaptic junctions with phosphotungstic acid, J. Ultrastruct. Res., 22 (1968) 361-375. 6 BUNGE, R. P., AND BUNGE, i . B., Ultrastructural characteristics of synapses forming in cultured spinal cord, Anat. Rec., 151 (1965) 329. 7 BURT, A. M., Acetylcholinesterase and choline acetyltransferase activity in the developing chick spinal cord, J. exp. Zool., 169 (1968) 107-112. 8 COHEN, S., Purification of a nerve-growth promoting protein from the mouse salivary gland and its neuro-cytotoxic antiserum, Proc. nat. Acad. Sci. (Wash.), 46 (1960) 302-311. 9 DEZA, L., AND EIDELBERG, E., Development of cortical electrical activity in the rat, Exp. Neurol., 17 (1967) 425--438. 10 EAYRS, J. T., AND GOODHEAD,B., Postnatal development of the cerebral cortex in the rat, J. Anat. (Land.), 93 (1959) 385~,02. 11 FONNUM,F., Radiochemical micro-assays for the determination of choline acetyltransferase and acetylcholinesterase activities, Biochem. J., 115 (1969) 465472. 12 FONNUM, F., Studies of choline acetyltransferase with particular reference to its subcellular localization, Norwegian Defence Research Establishment, Report No. 58 (1970). 13 FRIZELL, M., HASSELGREN,P. O., AND SJ()STRAND,J., Axoplasmic transport of acetylcholinesterase and choline acetyltransferase in the vagus and hypoglossal nerve of the rabbit, Exp. Brain Res., 10 (1970) 524-531. 14 GIACOBINI, G., MARCHISIO, P. C., GIACOBINI,E., AND KOSLOW, S. H., Developmental changes of cholinesterases and monoamine oxidase in chick embyro spinal and sympathetic ganglia, J. Neurochem., 17 (1970) 1177-1186. 15 HEBB, C. O., Choline acetylase in the developing nervous system of the rabbit and guinea-pig, J. Physiol. (Lond.), 133 (1956) 566-570. 16 HEBB, C. O., AND WAITES, G. M. H., Choline acetylase in antero-and retrograde degeneration of a cholinergic nerve, J. Physiol. (Lond.), 132 (1956) 667-671. 17 HENDRY, l., AND IVERSEN,L. L., Effect of nerve growth factor and its antiserum on tyrosine hydroxylase activity in mouse superior cervical ganglion, Brain Research, 29 (1971) 159-162. 18 KAITA, A. A., AND GOLDBERG,A. i . , Control of acetylcholine synthesis. The inhibition of choline acetyltransferase by acetylcholine, J. Neurochem., 16 (1969) 1185-1191. 19 LEvI-MONTALCINI,R., AND BOOKER,B., Excessive growth of the sympathetic ganglia evolved by a protein isolated from mouse salivary glands, Proc. nat. Acad. Sci. (Wash.), 46 (1960) 373-384. 20 LEVITT, M., SPECTOR,S., S.IOERDSMA,A., AND UDENFRIEND,S., Elucidation of the rate-limiting step in norepinephrine biosynthesis in the perfused guinea pig heart, J. Pharmacol. exp. Ther., 148 (1965) 1-8. 21 LOGNADO,J. R., AND HARDY, i . , Brain esterases during development, Nature (Lond.), 214 (1967) 1207-1210. 22 LOWRY, O. H., ROSEBROUGH,N. J., FARR, A. L., AND RANDALL,R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 23 MONNIER,M., Functions of the Nervous System, Vol. I, General Physiology, Autonomic Functions, Elsevier, Amsterdam, 1968, pp. 91-129. Brain Research, 34 (1971) 229-240
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24 PETERS, V. B., AND FLEXNER, L. B., Biochemical and physiological differentiation during morphogenesis. VIII. Quantitative morphologic studies on the developing cerebral cortex of the fetal guinea pig, Amer. J. Anat., 86 (1950) 133-157. 25 TUBBS, P. K., AND GARLAND, P. B., The assay of CoA and acyl derivatives of CoA, Methods Enzymol., 13 (1969) 535-551. 26 WEIBEL, E. R., Stereological principles for morphometry in electron microscopic cytology, Int. Rev. Cytol., 26 (1969) 235-302. 27 WILLIAMS, T. H., AND PALAY, S. L., Ultrastructure of the small neurons in the superior cervical ganglion, Brain Research, 15 (1969) 17-34.
Brain Research, 34 (1971) 229-240
BRAIN RESEARCH
TRANS-SYNAPTIC REGULATION OF GROWTH AND DEVELOPMENT OF ADRENERGIC NEURONES IN A MOUSE SYMPATHETIC GANGLION
I. B. BLACK, I. A. H E N D R Y AND L. L. IVERSEN
Department of Pharmacology, University of Cambridge, Cambridge (Great Britain) (Accepted May 27th, 1971)
INTRODUCTION
The study of developmental events within the central nervous system has involved anatomical1°,z4, ultrastructural2,5, 6, electrophysiological9, and biochemicaPS, 21 approaches. The complexity of organization of the central nervous system, however, has led to interpretative difficulties, and studies in the periphery x4 have provided simpler models of neural ontogeny. In the present study the maturation of mouse superior cervical ganglion in vivo has been examined utilizing a combination of biochemical and morphological methods. Choline acetyltransferase (ChAc), the enzyme catalyzing the conversion of acetyl-CoA and choline to the neurotransmitter acetylcholine, was used as a marker for the development of presynaptic cholinergic fibres. The enzyme is highly localized to these presynaptic terminals16. Maturation of postsynaptic neurones was followed by measuring the activity of tyrosine hydroxylase (T-OH), the rate-limiting enzyme in the biosynthesis of noradrenaline20, the postganglionic neurotransmitter. Visualization of ganglion synapses with the electron microscope allowed estimation of the development of synaptic connections. Correlation of these biochemical and morphological parameters suggested that presynaptic nerve terminals might regulate the development of the postsynaptic neurone. The results of surgical transection of the preganglionic nerve trunk in neonatal animals were consistent with this hypothesis. A preliminary report of these findings has appeared 3. METHODS
Experimental animals
Litters of Swiss albino mice were used, and mothers were offered OXO Diet 41B and water ad libitum. All litters were standardized, by reassigning offspring to mothers, so each had 10 pups within a day of birth. Groups of 6 animals, randomly chosen, were used in studies of enzyme development. The mice were killed by exposure Brain Research, 34 (1971) 229-240
230
I.B. BLACKet al.
to ether vapour and superior cervical sympathetic ganglion pairs were removed under a dissection microscope. Left and right ganglia from each pup were homogenized in 50/tl of distilled water and 10/~1 aliquots were taken for the assay of ChAc, T-OH and for the estimation of total protein. E n z y m e assays
ChAc activity was assayed using minor modifications of the method of Fonnum 11. The assay was initiated by the addition of 10 #1 of ganglion homogenate to 10 #1 of a solution containing, in final concentration, [3H]acetyl-CoA 0.1 mM; choline bromide 10 mM; physostigmine sulphate 1 mM; in sodium phosphate buffer, 50 mM, with a final pH of 7.40. For maximal activity the following ingredients were also present: sodium ethylene diamine tetraacetic acid (EDTA), 10 mM; sodium cyanide, 10 mM; sodium chloride, 300 mM;and albumin 0.I ~. [aH]Acetyl-CoA (247 #Ci//zmole) was synthesized before the enzyme assay using [3H]acetic anhydride and CoA. To 30 mg o f C o A (Boehringer) dissolved in 1 ml 1 M potassium bicarbonate was added 3.5 ml of an ether solution of [all]acetic anhydride (500 mCi/mole, Radiochemical Centre, Amersham; total 25 mCi), in 0.5 ml aliquots. The solution was acidified to pH 1.0 with 4 N hydrochloric acid, and was extracted 8 times with 1.5 ml of acid-saturated ether to remove excess [3H]acetate. The aqueous solution was assayed for acetyl-CoA by the citrate synthase method 25 to determine the concentration and specific activity. The ChAc reaction was terminated after 60 min at 37°C, by the addition of 3.5 ml of iced 0.01 M phosphate buffer, pH 7.4, containing acetylcholine bromide 0.25 mg/7.0 ml as carrier. The tritiated acetylcholine formed was extracted by liquid cation exchange chromatography into 0.5 ml of butyl ethyl ketone containing tetraphenylboron, 15 mg/ml. After low speed centrifugation an aliquot of the organic mixture in ethoxy ethanol and 0 . 4 ~ butyl PBD (CIBA) in toluene was counted by liquid scintillation spectroscopy. With this procedure the assay was linear for 60 min and for 1-50 #g of adult ganglion tissue. Ganglion T-OH was assayed by methods previously described 17. A 10 #1 aliquot of tissue homogenate was added to 15/~1 of a solution containing, in final concentration, [3H]tyrosine 0.004 m M (spec. act. = 11.5 mCi/#mole); 6,7-dimethyl tetrahydropterine 1.25 mM; 2-mercaptoethanol 0.3125 mM; NSD 1055 0.15 mM; and potassium phosphate buffer 400 m M at a final pH of 6.0. The reaction at 37°C was terminated after 20 min by the addition of 0.25 ml 0.4 N perchloric acid containing L-dihydroxyphenylalanine (L-DOPA) and dopamine 2 #g/ml. This mixture was brought to pH 8.6 with 0.1 M Tris containing sodium EDTA 0.05 M and N a O H 0.075 M and passed over an alumina column (dimensions 5.0 cm × 0.25 cm) previously equilibrated with 0.1 M sodium phosphate buffer, pH 8.6. After two successive washes of 5 and 15 ml with 0.005 M Tris buffer pH 8.6, the tritiated L-DOPA was eluted with 1.5 ml of 0.5 M acetic acid. The eluate was counted in a liquid scintillation counter after the addition of 12 ml of a 2:1 (v/v) mixture of 0.6 ~ butyl PBD (CIBA) in toluene and Triton X-100.
Brain Research, 34 (1971) 229-240
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Fig. 1. Electron micrograph of mouse superior cervical ganglion stained with EPTA. Two synaptic complexes are seen with presynaptic projections, intrasynaptic line and postsynaptic membrane. Horizonta! bar equals 0.5 ~m. Total protein was estimated in samples of ganglion homogenates by the procedure of Lowry2L Electron microscopy Ganglion synaptic junctions were visualized for electron microscopy using ethanolic phosphotungstic acid (EPTA) as described by Bloom and AghajanianL Ganglia were removed from mice and fixed by immersion in 0.16 M sodium phosphate buffer, pH 7.4, containing 5 ~ glutaraldehyde for ! h. The ganglia were then washed with phosphate buffer dehydrated in ethanol and stained with 1 ~o PTA in ethanol before embedding in Epon. Sections averaging 75 nm in thickness were prepared with an LKB Ultratome III in a plane parallel to the long axis of the ganglia. The density of synaptic junctions was estimated by counting the number observed in systematic searches of l0 grid squares (45/~m × 45 #m). Synapses were scored only if the presynaptic specialization, intercleft substance and postsynaptic membrane were visualized (Fig. 1). Synapses were identified and counted by 3 different observers. Appropriate corrections for synapse diameter were made according to the method of Abercrombie 1. Synapse density measurements were converted to total numbers per ganglion by estimating total ganglion volume from sections of the same ganglia. Brain Research, 34 (1971) 229-240
232
I.B. BLACKet al.
Serial sections of the ganglia, cut parallel to their long axes, were mounted on glass slides, stained with toluidine blue and measured with a light microscope using a micrometer eye piece to determine the major and minor ganglion diameters. Ganglia were assumed to conform most closely in geometry to prolate spheroids and ganglion volume was calculated on the basis of the formula V = 4/3 :rab 2, where V = volume, a = major radius and b = minor radius. Estimation of synapse numbers by this method involves the assumption that synaptic centers are randomly distributed and oriented in the ganglia. Such an assumption is basic to morphometric investigation 26. It has also been assumed that there is no age-related alteration in synapse geometry. This has been confirmed, to some extent, by noting no significant change in observed synapse diameter with age (mean synapse diameter was 0.23/~m). Errors introduced by assuming that ganglia are prolate spheroids may alter the absolute, but not the relative, values. Surgical procedure Ganglia were decentralized in mice anaesthetized with ether by unilateral transection of the preganglionic trunk in the neck under a dissecting microscope. The surgical wound was closed with celloidin and mice were returned to their cages. This procedure resulted in a variable mortality rate, but those which survived surgery thrived and grew normally for the duration of the experiment. RESULTS
Groups of mice were randomly selected from litters of different ages and ganglion pairs were assayed for enzyme activities and total protein. ChAc activity increased 30-40-fold during the course of development. From low levels on day l, enzyme activity rose rapidly during the first 2 weeks of life reaching a hyperbolic plateau by approximately 3 weeks (Fig. 2). This increase in enzyme activity may reflect either ongoing invasion of the ganglion by presynaptic nerve endings and/or transport of the enzyme to nerve endings already present in the ganglion. The developmental curve for T-OH activity differed significantly from that of ChAc. T-OH activity increased 6-8-fold from birth to adulthood. The rise in activity, however, occurred in two distinct phases. From birth to day 3, T-OH activity nearly doubled, but from day 3 to 7 a highly reproducible plateau phase occurred with no change in T-OH activity. The major increase took place during the second week of development when enzyme activity underwent nearly a 3-fold rise with little subsequent elevation to the 38th day of life (Fig. 2). These 3 phases of development, the initial increase in activity, the plateau phase and the phase of major increase have been observed in all experiments. During development total ganglion protein increased only 3-fold, thus the rises in enzyme specific activities were highly significant (Fig. 2). This relatively modest increase in total protein has been observed previously during the development of ganglia 8,14. Brain Research, 34 (1971) 229-240
DEVELOPMENT OF SYMPATHETIC GANGLION NEURONES
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Fig. 2. Developmental increases of transmitter enzyme activities and total protein in mouse superior cervical ganglia. Groups of 6 mice were taken from litters of varying ages and ganglion pairs from each animal were assayed for enzyme activities and total protein as indicated under Methods. Choline acetyltransferase activity is expressed as mean (nmoles product per ganglion pair) per h -4- S.E.M. (vertical bars). Tyrosine hydroxylase activity is expressed as (10 -11 moles product per ganglion pair) per h. Total protein is expressed as mean/~g per ganglion pair 4- S.E.
Developmentalformation of synapticjunctions To appreciate the functional significance o f these biochemical correlates o f maturation, we attempted to define their temporal relation to the d e v e l o p m e n t o f Brain Research, 34 (1971) 229-240
234
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Fig. 3. Development of ganglion synapses. Total synapses per ganglion were estimated in mice of different ages as indicated under Methods. Each point represents the estimated total synapses in a superior cervical ganglion from a single mouse. Vertical bars represent S.E. of determinations of synapse numbers in the 10 grid squares sampled for each ganglion.
inter-neural connections. Synaptic j u n c t i o n s (Fig. 1) were identified and counted, as described in M e t h o d s in ganglia o f mice aged 1-60 days. D u r i n g this p e r i o d total synapses per ganglion increased f r o m a p p r o x i m a t e l y 8,000 on d a y 1 to 3,000,000 by d a y 60 (Fig. 3). Synapse density increased 10-fold while total ganglion v o l u m e rose over a 50-fold range. The n u m b e r o f synapses r e m a i n e d relatively c o n s t a n t d u r i n g the first 2 days after birth, b u t rose d r a m a t i c a l l y between days 5 and 11 to an a s y m p t o t i c p l a t e a u (Fig. 3). F r o m d a y 11 to 60 the further increase in the n u m b e r o f synapses just reached statistical significance. C o m p a r i s o n o f synapse d e v e l o p m e n t with the d e v e l o p m e n t a l p a t t e r n o f C h A c
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Fig. 4. Comparison of developmental increases in choline acetyltransferase activity and total ganglion synapses. Results are expressed as in Figs. 2 and 3. Solid curve represents total ganglion synapses and dashed curve represents choline acetyltransferase activity. Brain Research, 34 (1971) 229-240
DEVELOPMENTOF SYMPATHETICGANGLIONNEURONES
235
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activity revealed some similarities (Fig. 4). Both functions display roughly hyperbolic curves with relatively modest increases during the first 3-4 days of life, and rapid rises early in the second week of development. On the basis of the data presented, however, more precise temporal relationships were not evident. The development of T-OH activity contrasts interestingly with synapse formation (Fig. 5). The early rise in T-OH activity precedes the increase in synapses. Enzyme activity remains at plateau levels during the initial phase of synaptic rise, days 5-7. Immediately following the steep increase in synapse formation, however, T-OH activity increases markedly to adult levels. These observations suggested that development of T-OH activity in postsynaptic neurones might be dependent on contact with presynaptic nerve endings.
Effect of decentralization of the superior cervical ganglion To determine whether the presynaptic cholinergic nerve terminals regulate the development of T-OH activity in the postsynaptic neurone, ganglia were decentralized in neonatal mice. The preganglionic trunk was transected unilaterally in mice aged 5-6 days. The contralateral normal ganglion served as control. Rats were killed 13 days postoperatively, ipsilateral ptosis and reduced ganglion ChAc activity indicating success of the procedure (Fig. 6). As expected ChAc activity was reduced to approximately 1 0 ~ of control values. T-OH activity failed to increased above normal 7-day plateau levels, remaining at approximately 30 ~ of the activity of contralateral unoperated ganglia (Fig. 5). These findings indicate that the increase in T-OH activity occurring during the second week of development (Fig. 2) is dependent on innervation of the postsynaptic neurone. In additional studies sham operations were performed in which ganglia were
Brain Research, 34 (1971) 229-240
236
I.a. BLACKet al.
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Fig. 6. Effect of preganglionic decentralization on ganglion tyrosine hydroxylase activity. Five mice aged 7 days were subjected to unilateral ganglion decentralization and killed 13 days thereafter, as described in Methods. Choline acetyl transferase and tyrosine hydroxylase activities were assayed in individual decentralized and contralateral control ganglia. Results are expressed as mean (nmoles product per single ganglion) per h ± S.E.M. (vertical bars) for choline acetyl transferase and as (10-11 moles product per ganglion) per h for tyrosine hydroxylase. * and ** differ from experimental group at P < 0.001.
exposed unilaterally without transection of the preganglionic trunk. This procedure did not alter the normal pattern of development of ChAc or T-OH activities. DISCUSSION The superior cervical ganglion receives presynaptic innervation from axons arising in the intermediolateral column of the lateral horn of thoracic spinal segments 1-3 (ref. 23). ChAc is, presumably, synthesized within these spinal neurone cell bodies and transported peripherally as in other cholinergic neurone systems 13. Since no natural activator or inhibitor (except possibly acetylcholine itself 18) of ChAc has been identified 12 it is likely that the increased ganglionic enzyme activity observed during maturation represents an actual increase in the number of enzyme molecules. Thus the increase in ChAc activity in the ganglion during development may represent a summation of the rates of enzyme synthesis and degradation in the spinal cell body, the rate of transport of ChAc along the axon, of ingrowth of cholinergic nerve terminals, and of degradation of enzyme in the nerve endings. The marked increase in ChAc activity, and most probably in ChAc molecules, closely approximates adult levels by the second to third week of life. At this time the capacity for neurotransmitter synthesis is thus present. These results also suggest that acetylcholine synthesis does not suddenly appear, in an all-or-none fashion, but increases progressively over the first few postnatal weeks. The similarities in the patterns of ChAc activity and synapse development suggest that the increased activity of this enzyme reflects functional neuronal maturaBrain Research, 34 (1971) 229-240
DEVELOPMENT OF SYMPATHETIC GANGLION NEURONES
237
tion. Such a formulation is consistent with that of Burt 7 who has noted association of ChAc activity with the development of motor activity in embryonic chick spinal cord. In addition since ChAc activity appears shortly after acetylcholine storage vesicles are present 7, and since enzyme is associated with cholinergic nerve terminals in the ganglion15, ChAc activity may reflect functional synaptic development as well as neurotransmitter synthesis. The developmental pattern of ganglionic T-OH activity occurs in a context different from that of ChAc. T-OH is largely restricted to postsynaptic cell bodies4 and thus the activity measured directly reflects development of the noradrenergic neurone, and is dependent on enzyme turnover within that neurone. T-OH activity rises in two separate and well-defined phases. The initial 2-fold rise occurs from birth to day 3. After a 4-day plateau period, the major 3-fold increase in T-OH takes place. These two phases of increase may reflect different events in the ganglion. The initial rise occurs prior to the period of synapse formation, and before ChAc activity has risen to any significant degree. Hence, it is unlikely that this initial phase is regulated by the presynaptic neurone. This elevation of T-OH may occur in perikarya which later respond to synaptic influences. However, it is possible that the early elevation of T-OH activity reflects a separate enzyme pool whose turnover is regulated by other factors. The ganglionic chromaffin cells27 may represent such a separate enzyme pool, and could be responsible for the early increase in enzyme activity. Consideration of events transpiring during the early plateau phase of T-OH activity further suggests independence of the two phases of enzyme development. Levi-Montalcini and Booker 19have demonstrated that from days 3 to 9 the neuroblasts of mouse superior cervical ganglion undergo intense mitotic activity. This period corresponds almost precisely to the T-OH early plateau phase. Furthermore, the second rise in T-OH activity is virtually synchronous with cessation of mitosis in the postsynaptic cells. These observations suggest that prior to the plateau phase, the blast progenitors of postganglionic neurones are in an undifferentiated state, most probably incapable of synthesizing T-OH. The relatively synchronous mitotic activity of the postsynaptic primordia may indicate that biochemical maturation of these cells occurs simultaneously and not in separate stages. The virtually synchronous increase of ganglionic synapses and T-OH activity suggested that maturation of the noradrenergic neurones depends on synapse formation. Abolition of the major increase in T-OH activity by transection of the preganglionic nerve trunk supported this view. Thus the presynaptic cholinergic nerve terminals regulate development of the postsynaptic cells. Although acetylcholine itself may constitute the stimulus evoking T-OH development postsynaptically, further study of this question is clearly needed. It should be stressed that while presynaptic terminals may be necessary for the development of ganglionic neurones, they may not be sufficient, and other unidentified mechanisms may also participate. These studies further suggest that, in some sense, the synapses demonstrated by electron microscopy are functional. That is, surgical destruction of the synaptic junctions prevents normal T-OH development. However, the observations presented
Brain Research, 34 (1971) 229-240
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I.a. BLACKet al.
do not indicate whether the demonstration of synapses morphologically can be correlated with the onset of cholinergic synaptic transmission. Synapse numbers remain at a basal level for at least the first 5 days of life. Preliminary investigations also indicate that a similar number of synapses exist in the superior cervical ganglion in the foetus near full term. After day 5, however, there is an abrupt and marked increase in synapses. Thus, once again, there is a striking degree of synchrony in a developmental parameter, suggesting an almost simultaneous staging of many cell functions within the ganglion. The development of synapses in the mouse sympathetic ganglion exhibits interesting similarities to that previously described in various regions of brain 2. In both cases an early plateau phase in synapse density is replaced by a sudden steep increase in synapse numbers to near the adult values within a few days. Such a pattern may thus be typical of synapse formation during development in the peripheral as well as the central nervous system. SUMMARY
The development of transmitter enzymes and the formation of synapticconnections have been studied in mouse superior cervical ganglion in vivo. Ganglion choline acetyl transferase (ChAc) activity increased by 40-fold during development and described a hyperbolic curve with time. Tyrosine hydroxylase (T-OH) activity rose in two separate and distinct phases during maturation. Estimates of total synapses per ganglion were obtained electron microscopically after staining with ethanolic phosphotungstic acid. Ganglion synapses increased 500-fold during development. From plateau levels during the first few days of life, synapse numbers increased markedly between days 5 and 11 to reach an asymptotic plateau. The developmental curve for ChAc was virtually congruent with that of synapse numbers, suggesting that increased enzyme activity reflected the maturation of preganglionic cholinergic terminals. On the other hand, the major increase in T-OH activity occurred synchronously with the marked increase in synapses, suggesting that the formation of synaptic contacts might be necessary for T-OH activity development. To examine this possibility ganglia were unilaterally decentralized in neonatal mice. Surgical transection of the preganglionic nerve trunk prevented normal development of T-OH activity in the postsynaptic adrenergic neurones. These observations indicate that presynaptic nerve terminals regulate the biochemical maturation of the postsynaptic adrenergic neurones. ACKNOWLEDGEMENTS
We are grateful to Dr. F. Bloom for advice and assistance in the use of the ethanolic phosphotungstic acid method for staining ganglion synapses. I.B.B. is the recipient of a William O. Moseley Travelling Fellowship from Harvard Medical School. I.A.H. is the recipient of a Fellowship from the Postgraduate Medical Foundation of the University of Sydney, Australia. These studies Brain Research, 34 (1971) 229-240
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were supported by grants to L.L.I. from the Medical Research Council and the Mental Health Research Fund. We thank Miss Margaret Hickman for excellent technical assistance.
REFERENCES 1 ABERCROMBIE,M., Estimation of nuclear population from microtome sections, Anat. Rec., 94 (1946) 239-247. 2 AGHAJANIAN,G., AND BLOOM,F. E., The formation of synaptic junctions in developing rat brain: a quantitative electron microscopic study, Brain Research, 6 (1967) 716-727. 3 BLACK,I. B., BLOOM,F. E., HENDRY,L, ANDIVERSEN,L. L., Growth and development of a sympathetic ganglion: Maturation of transmitter enzymes and synapse formation in the mouse superior cervical ganglion, J. Physiol. (Lond.), 215 (1971) 24P. 4 BLACK,I. B., HENDRY,I., AND IVERSEN,L. L., Differences in the regulation of tyrosine hydroxylase and DOPA decarboxylase in sympathetic ganglia and adrenal, Nature (Lond.), 231 (1971) 27-29. 5 BLOOM,F. E., AND AGHAJANIAN, G. K., Fine structural and cytochemical analysis of the staining of synaptic junctions with phosphotungstic acid, J. Ultrastruct. Res., 22 (1968) 361-375. 6 BUNGE, R. P., AND BUNGE, i . B., Ultrastructural characteristics of synapses forming in cultured spinal cord, Anat. Rec., 151 (1965) 329. 7 BURT, A. M., Acetylcholinesterase and choline acetyltransferase activity in the developing chick spinal cord, J. exp. Zool., 169 (1968) 107-112. 8 COHEN, S., Purification of a nerve-growth promoting protein from the mouse salivary gland and its neuro-cytotoxic antiserum, Proc. nat. Acad. Sci. (Wash.), 46 (1960) 302-311. 9 DEZA, L., AND EIDELBERG, E., Development of cortical electrical activity in the rat, Exp. Neurol., 17 (1967) 425--438. 10 EAYRS, J. T., AND GOODHEAD,B., Postnatal development of the cerebral cortex in the rat, J. Anat. (Land.), 93 (1959) 385~,02. 11 FONNUM,F., Radiochemical micro-assays for the determination of choline acetyltransferase and acetylcholinesterase activities, Biochem. J., 115 (1969) 465472. 12 FONNUM, F., Studies of choline acetyltransferase with particular reference to its subcellular localization, Norwegian Defence Research Establishment, Report No. 58 (1970). 13 FRIZELL, M., HASSELGREN,P. O., AND SJ()STRAND,J., Axoplasmic transport of acetylcholinesterase and choline acetyltransferase in the vagus and hypoglossal nerve of the rabbit, Exp. Brain Res., 10 (1970) 524-531. 14 GIACOBINI, G., MARCHISIO, P. C., GIACOBINI,E., AND KOSLOW, S. H., Developmental changes of cholinesterases and monoamine oxidase in chick embyro spinal and sympathetic ganglia, J. Neurochem., 17 (1970) 1177-1186. 15 HEBB, C. O., Choline acetylase in the developing nervous system of the rabbit and guinea-pig, J. Physiol. (Lond.), 133 (1956) 566-570. 16 HEBB, C. O., AND WAITES, G. M. H., Choline acetylase in antero-and retrograde degeneration of a cholinergic nerve, J. Physiol. (Lond.), 132 (1956) 667-671. 17 HENDRY, l., AND IVERSEN,L. L., Effect of nerve growth factor and its antiserum on tyrosine hydroxylase activity in mouse superior cervical ganglion, Brain Research, 29 (1971) 159-162. 18 KAITA, A. A., AND GOLDBERG,A. i . , Control of acetylcholine synthesis. The inhibition of choline acetyltransferase by acetylcholine, J. Neurochem., 16 (1969) 1185-1191. 19 LEvI-MONTALCINI,R., AND BOOKER,B., Excessive growth of the sympathetic ganglia evolved by a protein isolated from mouse salivary glands, Proc. nat. Acad. Sci. (Wash.), 46 (1960) 373-384. 20 LEVITT, M., SPECTOR,S., S.IOERDSMA,A., AND UDENFRIEND,S., Elucidation of the rate-limiting step in norepinephrine biosynthesis in the perfused guinea pig heart, J. Pharmacol. exp. Ther., 148 (1965) 1-8. 21 LOGNADO,J. R., AND HARDY, i . , Brain esterases during development, Nature (Lond.), 214 (1967) 1207-1210. 22 LOWRY, O. H., ROSEBROUGH,N. J., FARR, A. L., AND RANDALL,R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 23 MONNIER,M., Functions of the Nervous System, Vol. I, General Physiology, Autonomic Functions, Elsevier, Amsterdam, 1968, pp. 91-129. Brain Research, 34 (1971) 229-240
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24 PETERS, V. B., AND FLEXNER, L. B., Biochemical and physiological differentiation during morphogenesis. VIII. Quantitative morphologic studies on the developing cerebral cortex of the fetal guinea pig, Amer. J. Anat., 86 (1950) 133-157. 25 TUBBS, P. K., AND GARLAND, P. B., The assay of CoA and acyl derivatives of CoA, Methods Enzymol., 13 (1969) 535-551. 26 WEIBEL, E. R., Stereological principles for morphometry in electron microscopic cytology, Int. Rev. Cytol., 26 (1969) 235-302. 27 WILLIAMS, T. H., AND PALAY, S. L., Ultrastructure of the small neurons in the superior cervical ganglion, Brain Research, 15 (1969) 17-34.
Brain Research, 34 (1971) 229-240