Metabolic requirements for differentiation of embryonic sympathetic ganglia cultured in the absence of exogenous nerve growth factor.

The embryonic mouse superior cervical ganglion (SCG) in culture was employed to define the role of ongoing metabolic processes in morphological and biochemical development. The 14 gestational day SCG does not require added nerve growth factor (NGF) for differentiation in vitro. Consequently, its use allows study of intraganglionic regulation of neuronal growth in the absence of complicating, exogenous growth factors. Ganglia were cultured without added NGF, in medium containing various metabolic inhibitors; neurite elaboration and development of tyrosine hydroxylase (T-OH) activity, a biochemical marker of adrenergic maturation, were evaluated. Neurite elaboration proceeded normally with inhibition of RNA synthesis by actinomycin D, or of protein synthesis by cycloheximide or puromycin. In contrast, inhibition of RNA or protein synthesis prevented normal development of T-OH activity. However, neurites and T-OH developed normally in the presence of DNA synthesis inhibition by cytosine arabinoside, which markedly reduced the nonneuronal cell population. These observations suggest that neurite elaboration and the ontogenetic increase in T-OH activity are regulated differently in ganglia cultured in the absence of exogenous NGF. Moreover, the initial outgrowth of neurites and increase in T-OH activity may occur independent of peripherally migrating support cells in this embryonic ganglion.     

BIOCHEMICAL AND MORPHOLOGICAL EFFECTS OF TESTOSTERONE TREATMENT ON DEVELOPING SYMPATHETIC NEURONS

Abstract— Neonatal rats were treated with testosterone propionate (TP) or isoproterenol (ISO) to determine whether (1) increases in salivary gland mass are associated with alteration of developing sympathetic neurons and (2) whether effects on neuron growth are secondary to altered target mass itself or to increases in salivary growth factors. TP treatment is known to result in salivary tubule hypertrophy and elevated nerve growth factor (NGF) content whereas IS0 treatment results in acinar hypertrophy and no known alteration in NGF. TP treatment increased submaxillary gland weight as well as tyrosine hydroxylase (T-OH) activity, adrenergic neuron numbers and total protein in the innervating superior cervical ganglion (SCG). Unilateral sialectomy prevented the increase in T-OH activity in the SCG, suggesting that the salivary glands were necessary for this effect. T-OH activity and total protein were elevated in the distant sixth lumbar sympathetic ganglion after TP treatment, suggesting that sympathetic development as a whole was affected and that humoral factors may be involved. Salivary gland weight was also elevated following ISO administration, but T-OH activity in the SCG was not affected. These observations suggest that TP treatment increases T-OH activity and sympathetic neuron numbers by alteration of specific salivary humoral growth factor(s).     

Stage-dependent stimulation of neurite outgrowth exerted by nerve growth factor and chick heart in cultured embryonic ganglia

The ability of embryonic chick heart to elicit neuritic outgrowth in different ganglia was tested to examine (1) whether stimulative activity is possessed by the heart only at specific stages and (2) whether the ability of the ganglionic neurons to respond is limited to certain periods of development. As an assay, ganglia were explanted into thin collagen gels with ventricular tissue placed at a distance of about 1 mm. Neuritic outgrowth was measured after 2 days. Control ganglia and ganglia cultured with added nerve growth factor (NGF) were also scored. Four types of tested ganglia, including the ciliary ganglion, showed a peak in neuritic outgrowth when cultured with heart of embryonic Day 18, at about which age the heart becomes sympathetically innervated in ovo. No age-related size differences that could account for this temporal pattern were found among the heart explants when measuring their protein content. A peak in neuronal susceptibility to heart tissue was evident in the 6-day ciliary ganglion and in the 8-day paravertebral, Remak, and spinal ganglia, roughly coinciding with the onset of fibre outgrowth in ovo. Neurite extension is concluded to have been triggered by a factor spread from the heart explants and being distinct from the mouse type of NGF since anti-NGF did not at any stage block the events and since added NGF at all stages failed to evoke neurite formation in the ciliary ganglia. A testable hypothesis is that this factor regulates the growth of sympathetic and possibly parasympathetic and sensory fibres in the developing chick heart.     

Developmental changes of nerve growth factor levels in sympathetic ganglia and their target organs

The predominant source of nerve growth factor (NGF) used by mature sympathetic neurons originates in their target organs (Heumann, R., Korsching, S., Scott, J., and Thoenen, H. (1984), EMBO J. 3, 3183-3189; Korsching, S., and Thoenen, H. (1985), J. Neurosci. 5, 1058-1061). We have determined the NGF content of two sympathetically innervated mouse organs, submandibular gland and heart ventricle, and of sympathetic ganglia from mouse and rat between embryonic Day 12 (E12) and adulthood. NGF levels were measured by a two-site enzyme immunassay (Korsching, S., and Thoenen, H. (1983), Proc. Natl. Acad. Sci. USA 80, 3513-3516). In heart ventricle and submandibular gland, NGF first became detectable around the time of initial innervation by sympathetic neurons (E12 and E13, respectively) and increased respectively 14- and 7-fold in the following 2 days, to reach adult levels already at E14 for heart ventricle (1.4 +/- 0.2 ng NGF/g wet wt). NGF in the superior cervical ganglion (SCG) was first detected at the same time as in its target organ, the submandibular gland. NGF content in the SCG then increased 6-fold during the next 2 days and continued to increase until the end of the third postnatal week, when adult levels were reached. Although the levels of NGF in the adult mouse submandibular gland are sexually dimorphic and six orders of magnitude higher than those in other sympathetic target organs, no sex difference in the NGF content was found in either developing submandibular gland or SCG until the end of the third postnatal week. Moreover, the steep NGF increase observed in the male submandibular gland after postnatal Day 18 (250-fold within the following 3 days and up to the 55,000-fold in the next 7 days) was not reflected in a corresponding increase in the NGF content of the male SCG. These data indicate that, in accordance with earlier findings (see Levi-Montalcini, R., and Angeletti, P. U. (1968), Physiol. Rev. 48, 534-569), SCG neurons do not have access to the large amounts of NGF synthesized during and after adolescence in the mouse submandibular gland. Our results support the concept that initial fiber outgrowth of sympathetic neurons is neither dependent on NGF nor mediated by it. The time course of NGF levels in the SCG is consistent with the concept that sympathetic neurons are provided with NGF by means of retrograde axonal transport from the innervated organs already early in development.     

Studies on the action of nerve growth factor: I. Characterization of a simplified in vitro culture system for dorsal root and sympathetic ganglia

Neurite outgrowth from dorsal root (DRG) and sympathetic ganglia has been studied utilizing a simplified in vitro culture system for intact ganglia. Attachment of ganglia to tissue culture plates was achieved after a brief incubation of ganglia on the plates in the presence of 100% fetal calf serum or 5% ovalbumin in F12 medium. Neurite outgrowth from dorsal root and sympathetic ganglia was dependent on the continued presence of nerve growth factor (NGF) and on the NGF concentration. The NGF induced neurite outgrowth from DRG cultured in serum-free medium was delayed approximately 24 hr compared to the outgrowth in serum-containing medium.     

Stimulative effect of nerve growth factor on α-aminoisobutyric acid uptake and Na,K-ATPase activity in superior cervical sympathetic ganglia excised from adult rats

Effects of nerve growth factor (NGF) on the uptake of non-metabolizable -aminoisobutyric acid (AIB) and on Na,K-ATPase activity in superior cervical sympathetic ganglia (SCG) excised from adult rats were examined during aerobic incubation in vitro. Active uptake of labelled AIB into isolated SCG during 1 to 5 hours incubation at 37°C was significantly accelerated by the addition of NGF to the incubation medium in a dose-dependent manner. Although theK _m value of the AIB uptake by the SCG did not change with the addition of NGF,V _max was nearly doubled. The NGF-evoked increase in AIB uptake was antagonized by the further addition of its specific antiserum in a dose-dependent fashion, and was largely suppressed in a mdium containing ouabain. In SCG, axotomized one week prior to the examination, from which most of the neurons had disappeared and reactive proliferation of satellite glial components was in progress, the NGF-induced acceleration of AIB uptake was completely absent. The ganglionic Na,K-ATPase activity was greatly stimulated in the presence of NGF, and the effect was completely eliminated in the axotomized SCG. These results strongly suggest that the NGF-induced acceleration of active AIB uptake by the isolated SCG occurs not in glial cells but exclusively in the neuronal components with the apparent coupling of an Na ion extrusion process.Dedicated to Professor Yasuzo Tsukada.     

A COMPARISON OF THE NEURAL REGULATION OF TYROSINE HYDROXYLASE ACTIVITY IN SYMPATHETIc GANGLIA OF ADULT MICE AND RATS

Surgical decentralization of the superior cervical ganglion (SCG) in rats and mice led to a fall in ganglionic tyrosine hydroxylase (T-OH) activity, and a loss of more than 90 per cent of the preganglionic neurone marker, choline acetyl transferase. T-OH activity was reduced by more than 50 per cent in mice SCG ten days after surgery, but fell by only 25 per cent in rat SCG after 21 days. The surgical procedure did not cause obvious histo-logical damage or loss of SCG cells in either species. Both T-OH and choline acetyl transferase activities in rat and mouse SCG recovered to normal three months after surgery. Reserpine treatment was more effective in rats in causing increased ganglionic T-OH activity than in mice. Neither decentralization nor reserpine treatment caused any changes in DOPA-decarboxylase or monoamine oxidase activities in rat SCG. These results demonstrate that T-OH activity in SCG is subject to trans-synaptic regulation in both rats and mice; this regulation does not apply to DOPA-decarboxylase or monoamine oxidase. Differences in basal sympathetic tone may explain the different results obtained in mice and rats.     

Nerve growth factor-independent development of embryonic mouse sympathetic neurons in dissociated cell culture

Although ganglia from neonatal mouse sympathetic ganglia require nerve growth factor (NGF) for survival in culture, explanted sympathetic ganglia from early embryonic stages do not require added NGF for survival and growth. To determine whether the change in growth factor requirement is due to changes in the neurons themselves, to variations in neuronal populations, or to changes in nonneuronal cells, we examined the response to growth factors by dissociated sympathetic neurons at various stages of development. Results indicate that neurons from the 14-day gestational (E14) superior cervical ganglion (SCG) do not require NGF for initial survival and neurite extension, but do require the conditioned medium neurite extension factor, CMF. By 2 to 3 days thereafter, whether in vivo or in culture, most neurons have developed a requirement for NGF for survival in culture. During the same period, there is a concomitant increase in responsiveness to NGF alone as a trophic agent. Changes in response to NGF are not due to changes in NGF content of ganglia, to interactions in culture with nonneuronal cells, or to age-related differences in NGF requirements for maximum survival. The changes in growth factor requirements may be related to mechanisms regulating specificity of nerve-target connections.     

Responses of Mature and Aged Sympathetic Neurons to Laminin and NGF: An in Vitro Study

Whilst the potent effects of NGF and laminin on developing neurons are well documented, relatively little is known about the effects of, or altered availability of or altered responsiveness to, these substances on the growth of adult neurons. We have therefore examined this question using explant cultures of sympathetic neurons from the superior cervical ganglion (SCG) of mature and aged rats. Explants were grown on substrata containing different doses of laminin, either with or without added NGF in culture medium containing FCS. Individually, laminin and NGF had relatively small effects on neurite outgrowth and length, which tended to be reduced in old neurons. In contrast, laminin in the presence of exogenous NGF exerted a powerful effect on nerve growth which was substantially greater than the sum of the effects of the individual factors. This synergy was evident in all experimental groups and was greatest in old explants at high doses of laminin, where growth was comparable to that of mature neurons. The dose-response curve of old neurons to laminin in the presence of added NGF indicated reduced responsiveness. These results suggest that variations in the availability of laminin and/or exogenous NGF, together with altered patterns of neuronal responsiveness, may contribute to impaired neuronal plasticity in old age.     

A histochemical study of the distribution of choline acetyltransferase and acetylcholinesterase activity in sensory ganglia and nerve roots of the bullfrog

Synopsis Histochemical techniques were employed for the localization of choline acetyltransferase (ChAc; EC 2.3.1.6.), acetylcholinesterase (AChE; EC 3.1.1.7) and cholinesterase (ChE; EC 3.1.1.8) activities in dorsal and ventral roots and dorsal root ganglia of the bullfrog. AChE activity was present in most of the neuronal elements of dorsal root ganglia, in some nerve fibres in the dorsal roots, and in all nerve fibres in ventral roots. ChE activity in dorsal root ganglia and in the dorsal roots was confined to non-neuronal elements. No ChE activity was demonstrable in the ventral roots. ChAc activity was localized in many neurons of the dorsal root ganglia and in some nerve fibres of the dorsal roots; however, none of the ventral root fibres were visibly reactive. Some supportive cells of the dorsal roots and ganglia contained small amounts of ChAc activity. Except for the ventral roots, the histochemical distribution of AChE and ChAc activity was similar. The results of solubility studies indicated that under the histochemical conditions, approximately 50% of the ChAc remained bound to the dorsal roots and ganglia, whereas more than 90% of the ChAc in the ventral roots was soluble. This would account for the lack of reactivity in ventral root fibres. Differences in ChAc solubility are discussed in relation to the interpretation of histochemical data and in relation to the concept of multiple forms of ChAc. The results of this study indicate that at least one-third of the neurons of the dorsal root ganglia contain significant levels of the enzymes involved in both the synthesis and hydrolysis of acetylcholine.     

SEPARATION OF APPARENT MULTIPLE FORMS OF HUMAN BRAIN CHOLINE ACETYLTRANSFERASE BY ISOELECTRIC FOCUSING

Abstract— Choline acetyltransferase (acetyl-CoA: choline O -acetyl transferase; EC 2.3.1.6; ChAc) purified from human brain (basal ganglia) and sciatic nerve were separated into apparent multiple enzyme forms by the method of isoelectric focusing (pH gradient 3-10) on acrylamide gel. A preparative separation of enzyme forms of human brain was accomplished by the column method, by using a sucrose gradient. When each separated form was re-electrofocused, only a portion of the ChAc activity was observed in its original pH region while more than one-half of the recovered activity for each fraction appeared at pH 7.8-8. Gel filtration and kinetic studies of separated forms indicated that the more acidic forms might be aggregates, while more basic forms might be configurational isomers. Human ChAc of sciatic nerve did not exhibit acidic forms on electrofocusing, but otherwise yielded an electrofocusing profile similar to that of human brain. ChAc of rabbit brain and sciatic nerve each exhibited only a single form at pH 7.1 ± 0.2. Although ChAc differs among species, the enzyme of brain and sciatic nerve of the same species cannot be clearly distinguished by electrofocusing.     

A quantitative study of the developmental expression of nerve growth factor (NGF) receptor in rats

The developmental expression of nerve growth factor (NGF) receptor was quantitated in either homogenates or plasma membrane-enriched preparations from whole rat embryos or from isolated tissues. The assay involved crosslinking 125I-NGF to receptors followed by immunoprecipitation with a monoclonal antibody to rat NGF receptor. In some cases, the pellet was resuspended and subjected to a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) autoradiographic analysis. The NGF receptor was found in whole embryo homogenates as early as embryonic Day 10 (E10) (earliest age examined). The NGF receptor content in whole embryos per milligram protein increased about 3-fold from E11 to E18 and decreased slightly at E20. SDS-PAGE autoradiography showed that the molecular weights of 125I-NGF-bound receptors did not vary with age. The NGF receptor content in sciatic nerve homogenates decreased 23-fold from newborn to adulthood. The change of NGF receptor level in hindleg muscle had a profile similar to that seen in sciatic nerve. The NGF receptor content in superior cervical ganglion (SCG) or dorsal root ganglion (DRG) homogenate preparations was expressed in two ways. On a per milligram protein basis, in SCG, the receptor density was decreased slightly from E20 to adulthood; in DRG, it was relatively constant from E15 through postnatal Day 0 (PND-0) and then dropped 6.7-fold in adults. On a per ganglion basis, in SCG, it increased 4.4-fold from E20 to adult; in DRG, it increased 9-fold from E15 to PND-0 and then stayed constant through adulthood. In brain membrane preparations, the NGF receptor level decreased 11-fold from E15 to adulthood. In spinal cord membrane preparations, it decreased 7-fold from E18 to adulthood. Levels of receptor in cord were always greater than in brain. These data suggest that alterations in the NGF receptor density may have a role in changes in tissue responsiveness to NGF during development.     

Characterization of a neuronal growth factor from mouse heart-cell-conditioned medium

A fraction of medium conditioned by embryonic mouse heart cells in culture promotes the growth of sympathetic and parasympathetic neurons in vitro. The factor stimulates neurite outgrowth, elevates specific activities of tyrosine hydroxylase and choline acetyltransferase in sympathetic ganglion explants, and enhances survival of dissociated sympathetic neurons in culture. The growth-promoting activity, which has a profound effect on survival of mouse sympathetic and parasympathetic neurons but little effect on mouse sensory neuron survival, is sensitive to trypsin and elevated temperature, suggesting association with a polypeptide or protein. Unlike nerve growth factor (NGF), the conditioned medium fraction is insensitive to anti-NGF antiserum, and fosters growth of mouse parasympathetic neurons. Consequently, the conditioned medium appears to contain a new nerve growth-promoting factor.     

The cranial sensory ganglia in culture: Differences in the response of placode-derived and neural crest-derived neurons to nerve growth factor

Explants of cranial sensory ganglia and dorsal root ganglia from embryonic chicks of 4 to 16 days incubation (E4 to E16) were grown for 24 hr in collagen gels with and without nerve growth factor (NGF) in the culture medium. NGF elicited marked neurite outgrowth from neural crest-derived explants, i.e., dorsal root ganglia, the dorsomedial part of the trigeminal ganglion, and the jugular ganglion. This response was first observed in ganglia taken from E6 embryos, reached a maximum between E8 and E11, and gradually declined through E16. Explants in which the neurons were of placodal origin varied in their response to NGF. There was negligible neurite outgrowth from explants of the ventrolateral part of the trigeminal ganglion and the vestibular ganglion grown in the presence of NGF. The geniculate, petrosal, and nodose ganglia exhibited an early moderate response to NGF. This was first evident in ganglia taken from E5 embryos, reached a maximum by E6, and declined through later ages, becoming negligible by E13. Dissociated neuron-enriched cultures of vestibular, petrosal, jugular, and dorsal root ganglia were established from embryos taken at E6 and E9. At both ages NGF elicited neurite outgrowth from a substantial proportion of neural crest-derived neurons (jugular and dorsal root ganglia) but did not promote the growth of placode-derived neurons (vestibular and petrosal ganglia). Our findings demonstrate a marked difference in the response of neural crest and placode-derived sensory neurones to NGF. The data from dissociated neuron-enriched cultures suggest that NGF promotes survival and growth of sensory ganglionic neurons of neural crest origin but not of placodal origin. The data from explant cultures suggest that NGF promotes neurite outgrowth from placodal neurons of the geniculate, petrosal, and nodose ganglia early in their ontogeny. However, we argue that this fibre outgrowth emanates not from the placodal neurons but from neural crest-derived cells which normally give rise only to satellite cells of these ganglia.     

Differential regulation of survival and growth in adult sympathetic neurons: an in vitro study of neurotrophin responsiveness

The survival and growth of embryonic and postnatal sympathetic neurons is dependent on both NGF and NT3. While it has been established that adult sensory neurons survive independently of neurotrophins, the case is less clear for adult sympathetic neurons, where the studies of survival responses to neurotrophins have relied upon using long-term cultures of embryonic neurons. We have previously established a method to culture purified young (7 day) and adult (12 week) sympathetic neurons isolated from adult rat superior cervical ganglia (SCG) in order to examine their survival and growth responses to neurotrophins. We now show that by 12 weeks after birth virtually all neurons (90%) survive for 24 h in the absence of neurotrophins. Neuron survival is unaffected by treatment with anti-NGF antibodies (anti-NGF) or with the tyrosine kinase inhibitor, K252a, confirming the lack of dependence on extrinsic neurotrophins. Duration of neuron survival in culture increases significantly between E19 and day 7 and week 12 posnatally, and is similarly unaffected by the presence of anti-NGF or K252a. Saturating concentrations of NGF and NT3 are equipotent in promoting neurite extension and branching. However, we find that NGF is more potent than NT3 in promoting neurite growth, irrespective of postnatal age. The growth-promoting effects of NGF and NT3 are almost entirely blocked by K252a, demonstrating that these effects are mediated via activation of Trk receptors, which therefore appear to remain crucial to plasticity of adult neurons. Our results indicate that maturing neurons acquire protection against cell death, induced in the absence of neurotrophin, while retaining their growth responsiveness to these factors.     

Retrograde Transport of Endogenous Nerve Growth Factor in Superior Cervical Ganglion of Adult Rats

The concentration of naturally synthesized nerve growth factor (NGF) was measured in various tissues of adult rats, using a highly sensitive two-site enzyme immunoassay. The highest concentration was found in the superior cervical sympathetic ganglion (SCG). Transection of the postganglionic external carotid nerve (ECN) reduced the ganglionic level of NGF more than did section of the internal carotid nerve (ICN). When both the preganglionic nerve and the ECN were cut, the ganglionic NGF level decreased even more. On the other hand, when the preganglionic nerve and the ICN were both sectioned, leaving the ECN intact, endogenous NGF content in the SCG was significantly enhanced 3-9 h after operation. Bilateral extirpation of submaxillary gland produced a rapid decrease in ganglionic NGF 3-6 h after operation, and even unilateral removal of one salivary gland caused a decrease in both ganglia, which was however much greater in the ipsi- than in the contralateral ganglion. Removal of the eyeballs caused a much smaller reduction in ganglionic NGF than did removal of the glands. These results suggest that the endogenous NGF that accumulates in the SCG is mostly synthesized in the submaxillary gland rather than in the iris, and that it is transported to the SCG, mostly via the ipsilateral ECN.     

Morphological and biochemical studies on the development of cholinergic properties in cultured sympathetic neurons. II. Dependence on postnatal age

Superior cervical ganglion (SCG) neurons taken from perinatal rats and dissociated in culture develop cholinergic properties. This report examines this "plasticity" of neurotransmitter function with regard to its dependence on the stage of neuronal development. Explants of SCG from rats ranging in age from 2 d to adult were cultured, and the number of neurons surviving after 6 wk in culture was evaluated. The activities of choline acetyltransferase (ChAc) and DOPA decarboxylase (DDC) were assayed for each age group over time in culture, and the cytochemistry of the synaptic vesicle population was studied after norepinephrine loading and KMnO4 fixation. The specific activity of ChAc in all explants fell during the first 3--4 d in culture (secondary to degeneration of presynaptic terminals), with an increase during the next 30 d in explants from all age groups except in those from the 22-d- old and adult rats. The highest activity found after 1 mo in culture was in explants from 2-d-old rats (62.5 mmol per kg dry wt per h); the lowest was in explants from adults (1.3 nmol per kg dry wt per h). After 1 mo in vitro, there were no significant differences in DDC activity among explants from animals of any age (similar to approximately 220 mmol per kg dry wt per h). Co-culture of the SCG explants with heart muscle increased even further the ChAc activity in explants from 2-d-old rats but not in explants from 16-d-old and 6.5-wk- old animals. The cytochemistry of the synaptic vesicle population in 1- mo-old cultures correlated well with the ChAc activity; when the ChAc activity was high, the proportion of synaptic vesicles with clear centers was 71--88%. In explants from adult animals, only 12% of the vesicles contained clear centers. From these data we conclude that the maturity of the SCG neuron influences the degree to which it is able to adjust its neurotransmitter mechanisms. That the axons of this neuron are interacting with target tissues during the time that neurotransmitter plasticity is retained suggests that interaction with the target may play a role in the determination of transmitter type.     

Embryonic rat adrenal glands in organ culture: Effects of dexamethasone,nerve growth factor and its antibodies on pheochromoblast differentiation

Summary In the present study we sought to determine the developmental potentialities and restrictions of adrenal medullary cells (pheochromoblasts) by investigating their morphological and biochemical response to nerve growth factor (NGF), anti-NGF antibodies and dexamethasone (DEX) after explantation into culture at different embryonic stages. With the exception of explants taken at embryonic day 15 (E 15) cultures of embryonic adrenal glands showed neurite outgrowth, which was not influenced by the addition of NGF, anti-NGF antibodies or DEX to the culture medium during the 4-day-culture period.Pheochromoblasts in E 17+4 explants showed spontaneous ultramorphological and biochemical maturation in terms of an increase in the number of catecholamine storage vesicles (CSVs) per m² of cytoplasmic area, diameters of the cores of CSVs, percentages of electron-lucent cores of CSVs indicative of increased storage of adrenaline, overall catecholamine (CA) content and relative amount of adrenaline. NGF did not significantly affect this maturational process. Anti-NGF antibodies slightly decreased the proportion of adrenaline. The most pronounced maturation was seen in response to DEX and DEX plus NGF, although a maturational state equivalent to the E 21 stage was not achieved. E 21+4 explants showed neither spontaneous nor drug-induced biochemical maturation. Medullary cells in NGF-treated E 21 explants frequently retained the morphological features of pheochromoblasts. Treatment with anti-NGF antibodies significantly reduced the portion of adrenaline as compared to any other treatment. We conclude that under the culture conditions employed (1) a few pheochromoblasts spontaneously express a neuronal phenotype, (2) differentiation of pheochromoblasts towards chromaffin cells is enhanced by glucocorticoids but not by NGF, and (3) anti-NGF antibodies do not impair spontaneous neuritic growth and morphological maturation of pheochromoblasts, but cause a small reduction in the relative amount of adrenaline.     

Developmental changes of choline acetyltransferase (ChAc) activity in chick embryo spinal and sympathetic ganglia

The ChAc activity of spinal and sympathetic ganglia was measured throughout the embryonic life of the chick. In spinal ganglia, the ChAc activity reached a first peak when the maximal proliferation of neuroblasts occurred. Then, the relative ChAc activity decreased. After the 12th day of incubation, the enzyme activity increased again and reached a second peak on the 16th day. In sympathetic ganglia, the general course of the development of ChAc activity was similar to spinal ganglia. However, higher enzymic activity was found. Furthermore, the earlier peak of ChAc activity occurred 48 hr later than the corresponding peak in spinal ganglia. The behaviour of ChAc activity in these two areas of the developing nervous system is interpreted as a function of their histogenesis.