Journal of Neuroscience Research 1 :31 5-33 1 ( 1 975)

NEURONALCONTROLOF NEUROTRANSMITTERS BIOSYNTHESIS DURING DEVELOPMENT Ezio Giacobini Laboratory of Neuropsychopharmacology, Department of Biobehavioral Sciences, University of Connecticut, Storrs

Studies on neuronal control mechanisms of neurotransmitters biosynthesis during the development of peripheral and centralautonomic synapses are reviewed. Particular emphasis is placed on investigations of developing peripheral sympathetic ganglia and brain in chick embryo and chick. Studies on the development of autonomic neurons and synapses under different pharmacological conditions are reported. Principally the effect of a) the administration of drugs and precursors such as L-dopa, 3H-dopa, 6-OH dopa; b) the prenatal administration of reserpine; c) the blockade of cholinergic receptors; d) the nerve growth factor (NGF) is analyzed. Results of developmental studies on chick ciliary ganglia are summarized. The review particulary underlines the importance of combining the use of sensitive microchemical methods to pharmacological tools in exploring the development of regulatory mechanisms at the cellular level. INTRODUCTION

The initial differentiation an'd maturation of the dopamine (DA) and noradrenaline (NA) containing systems take place during the early prenatal period of ontogeny; this applies to peripheral ganglia (Enemar et a]., 1965) as well as to the CNS (Olsson and Seiger, 1972). In sympathetic ganglia the immature monoamine neuron is able to synthesize and store catecholamines (CA) at a very early stage of development. In fact, these processes begin long before the development of the terminal effector areas of these neurons and thus begins apparently independently of the establishment of the peripheral field of innervation (Giacobini, 1971; Olsson and Seiger, 1972). Due t o the extremely low amounts and concentrations of monoamines and acetylcholine present in the early prenatal monoaminergic neurons and ganglion synapses, respectively, biochemical assays of the endogenous level of transmitters are difficult to perform. Therefore, either fluorescence histochemistry, uptake of labeled NAS and dopa, or histochemical staining of acetylcholinesterase (AChE) is used in prenatal studies. In contrast, numerous biochemical studies have been done on the postnatal development of

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monoamine neurons, including levels of amines and precursors, metabolites, and activity of biosynthetic enzymes in the brain. The ganglion synapse, which represents the fundamental constituent of the sympathetic circuit, also develops during the prenatal period. However, the relationship between the development of these two components, the ganglion synapse and the postganglionic neuron, is still largely a matter of speculation. Several studies have focused on variations of enzyme activities related to the metabolism of neurotransmitters during (a) prenatal development in sympathetic ganglia of the chick, (b) postnatal development in the cervical superior ganglion of the newborn mouse and rat, and (c) postnatal development in sympathetic neurons of rat brain. Such studies were made possible through the recent development of sensitive micromethods for assaying enzyme activity in microgram samples of nervous tissue (Giacobini, 1970). The micromethods applied in our own studies are represented by sensitive isotopic microassays which have been originally described and applied in our laboratory or developed by other authors: ChAc (Buckley et al., 1967; Fonnum, 1969); AChE and cholinesterase (Koslow and Giacobini, 1969); coenzyme A and acetylcoenzyme A (Allred and Guy, 1969); TH (Waymire et al., 1971); DDC (Giacobini and Nor& 1971; Filogamo et al., 1971); DBH (Molinoff et al., 1969, 1971); PNMT (Molinoff et al., 1969, 1971); MA0 (Consolo et al., 1968; Giacobini et al., 1970); and COMT (Giacobini and Kerpel-Fronius, 1969). STUDIES O N R A T A N D MOUSE SYMPATHETIC GANGLIA A N D BRAIN

Black (1973) and Black et al. (1971, a, b, c; 1972 a, b) in a series of papers reported a significant correlation between the development of ganglion synapses, determined by their number and by cholineacetylase (ChAc) activity, and the maturation of ganglion adrenergic neurons, expressed as tyrosine hydroxylase (TH) activity. According to these authors, this correlation strongly suggests that presynaptic nerve terminals might be involved in regulating the development of the postsynaptic neurone, particularly in establishing functional levels of enzymes related to neurotransmitter synthesis. Another group of authors (Thoenen et al., 1972a) has reported findings in the rat which t o some extent contradict the observations on mice. They showed that ChAc activity as a measure of the formation of ganglion synapses does not seem to be of primary importance for the modulation of enzymes which are specific for the adrenergic neuron, such as dopamine-/3-hydroxylase (DBH). These experiments in the rat have recently been confirmed by the same authors (Thoenen, 1972; Thoenen et al., 1971,1972b); they emphasize the importance of factors other than presynaptic terminals, such as the nerve growth factor (NGF) necessary for normal development of the synthetic apparatus in adrenergic neurons in ganglia. Thoenen et aL(l972c) also reported on an increased activity of ChAc in sympathetic ganglia after prolonged administration of NGF. In the brain, Karki et al. (1962) did a pioneer study of the newborn rat, whose functional development is relatively poor compared, for example, to the chick. They showed that the brain levels of NA, S H T , and monoamine oxidase (MAO) reach those of the adult rat only after several weeks postnatally, when functional activity has developed. In contrast, they found that the levels of monoamines and MA0 in the newborn guinea pig, whose functional activity is well developed, are almost as high as in the adult. According to these

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findings, the low amine levels of the newborn rat appear to result from a low capacity to store and synthesize NA and 5-HT. Consequently, these biochemical processes might be important with respect t o the profound functional changes occurring in the CNS during early life. Developmental data for TH activity distribution (McGeer et aI., 1967, 1971) correlate with known neonatal changes in endogenous CA levels. In a more recent study, Coyle and Axelrod (1972b) examined the development of DBH in the rat brain. They found that the appearance of DBH precedes the presence of NA by several days and that a gradual increase in enyzme activity occurs with maturation. The enzyme first appears in the caudal parts of the brain where the cell bodies are localized; with maturation, there is a progressive increase in DBH activity and a shift of distribution to the rostra1 parts of the brain where nerve terminals are localized. The increase in DBH activity correlates with the development of the specific uptake mechanism for NA (Coyle and Axelrod, 1971) and with the increase in TH activity (Breese and Traylor, 1972; Coyle and Axelrod, 1972a). The levels of dopamine and noradrenaline were determined in the brains of fetal and newborn rats (Coyle and Henry, 1973). Both amines were present at 15 days of gestation and increased 15-fold in content during the last week of gestation. The regional distribution of these neurotransmitters correlated with the distribution of their biosynthetic enzymes. STUDIES O N CHICK AND CHICK EMBRYO SYMPATHETIC GANGLIA A N D BRAIN

An advantage of studying chick autonomic ganglia is that their development can be followed step by step from prenatal, before hatching (b.h.), stages, starting from a very early degree of maturation (day 4-5 of incubation), to postnatal (a.h.) stages and through adulthood. In chicks, the presence of the post ganglionic transmitter NA can be detected histochemically as early as at 3-4 days of incubation (Enemar et al., 1965). However, no quantitative data are found in the literature with regard to the levels of transmitters in sympathetic ganglia throughout development. Marchisio and Consolo (1968) demonstrated in our laboratory that in the ganglion synapse, the developmental trend of the enzyme related to acetylcholine synthesis (ChAc) shows a peak in its specific activity 48 hr after the period of maximal proliferation of the neuroblasts (that is, after 8 days of incubation). This peak clearly correlates with the period of morphological maturation of ganglion synapses at day 8-9 of incubation, according to the electronmicroscopic (EM) data of Wechsler and Schmekel, (1967). AChE shows a peak of activity at day 12 of incubation, in a developmental phase characterized by both synaptic development and proliferation of the neuroblasts (Giacobini et al., 1970). The activities of three enzymes related t o the adrenergic neurons have recently been studied in our laboratory; MAO, dopadecarboxylase (DDC), and DBH (Giacobini et al., 1970; Filogamo et al., 1971; Dolezalova et al., 1974). All three enzymes show an interesting developmental aspect - that is, a first phase of steady increase occurs from early development to hatching, with a sudden and significant decline on the day 4 a.h., followed by a second phase of steady increase until 30 days a.h. (Figs. 1 and 2).

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The prenatal phase corresponds to the growth and maturation of the adrenergic neuron. M A 0 and DDC activity both remain at a steady level between days 5 and 8 b.h.; this activity then progressively increases until day 16 b.h. (MAO) (Fig. 1) and until day 2 a.h. (DDC). This increase is about 3-fold for MA0 and 2-fold for DDC, and occurs immediately after the peak in ChAc activity, indicating a temporal relation with the biochemical and morphological maturation of the presynaptic structures. This relation is less clear for DBH activity (Fig. 2), which has already begun t o increase at day 5-6 b.h., during a preceding stage of development. The sudden decrease a.h. (Figs. 1, 2) might be related to a particular stage of the functional relationship between cell bodies in ganglia and peripheral terminals, resulting from the sudden initiation of visceral functions in the newborn animals. For comparison with sympathetic ganglia, we studied the activity of AChE, ChAc, MAO, and DDC in chicken spinal ganglia at corresponding stages of development, revealing much lower levels of activity and a totally different pattern throughout the whole embryonic life (Marchisio and Consolo, 1968; Giacobini et al., 1970; Filogamo et al., 1971). Some quantitative aspects of enzymatic variations in developing chick sympathetic ganglia deserve particular attention. ChAc shows a 27-fold increase between days 1-7 a.h.; ChAc activity then declines until day 15 a.h. and remains practically unchanged in the adult animal Fig. 3). The pattern of DBH activity shows a peak (Fig. 2) at day 8 of incubation, during the period of intensive proliferation of neuroblasts and morphological as well as biochemical maturation of ganglion synapses (see ChAc activity). A subsequent increase of DBH activity until day 2 a.h. (Fig. 2) occurs in the absence of an increase in total ganglion proteins, indicating a specific functional maturation related, in this case, to the synthesis of NA (Dolezalova et al., 1974). The most significant postnatal variation in enzyme activity is the sudden and pronounced decline of both specific and total (per ganglion) DBH activity between days 2 and 4 (Fig. 2), in the absence of any corresponding decrease in total proteins of the ganglion. This event appears during an advanced phase of the development of both ganglion synapses and ganglion cells, and might depend on a significant change in the functional state of the ganglion neurons. The functional relation between the cell bodies and peripheral terminals probably undergoes a period of transition as a result of the sudden establishment of visceral activity (cardiovascular, intestinal, and so on) and the new circuit of sympathetic innervation. At the cellular level, the most plausible explanation for this sudden decline could be a massive migration of DBH from the cell body to the terminals, carried by the fast transport of granules to which DBH is bound. This phenomenon is similar to the variation of DBH and TH activity found in the rat brain by Coyle and Axelrod (1972a, b). The “day 4 fall” phenomenon of DBH activity in sympathetic ganglia described above occurs simultaneously with a similar decrease in DDC and MA0 activity. ChAc activity, however, which is related to the preganglionic component, not only does not show a similar decrease at day 4 a.h. but increases significantly (Fig. 3) at this stage ( Dolezalova et al., 1974). The induction of an intense functional activity in the ganglion cells during the early postnatal period is indicated by these enzyme variations and is further strongly suggested by the explosive 27-fold increase in ChAc activity in ganglion synapses which starts between day 1-2 a.h., immediately prior to the decline of DBH, DDC, and MA0 activity (Fig. 3).

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If an intense functional activity between day 2-4 a.h. is responsible for the depletion of DBH from the ganglion depots and the possible accumulation of DBH in the terminals, then a mechanism of transsynaptic induction similar to that described for a comparable situation by Thoenen and Axelrod (Axelrod, 1972) could explain the rapid rise in the ganglionic level of DBH between day 4 and 7 a.h. (Fig. 2). From our own studies (Dolezalova et al., 1974), a temporal relationship between the variations in “presynaptic” and “postsynaptic” enzymes in sympathetic ganglia of chick is evident throughout their development. Our attention, therefore, should be focused on two critical periods of development: (a) around the day 8 b.h., when ChAc and DBH activity increase simultaneously, indicating a relationship between the two components of the synapse in the ganglia; and around the day 16 b.h., when the second peak of ChAc activity closely precedes the second pronounced increase of DBH activity; and (b) the first few postnatal days when a 27-fold increase in ChAc activity and the “4th day fall” phenomenon of DBH activity occur simultaneously. Our results indicate that during embryonal development the biochemical and functional

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maturation of the synapse in sympathetic ganglia, expressed as ChAc activity, might be strictly correlated with the development of both the synthesizing (DBH) and the inactivating (MAO) intracellular machinery of the adrenergic transmitter in the ganglionic neuron. After hatching, the sudden and intense functional activity of this synapse is correlated with a pronounced migration of DBH and other intrasomal enzymes (DDC and MAO) toward the terminals. Waymire et al. (1974) studied the development of TH, MAO, and DDC in several regions of the chick brain. The highest levels of both NA and TH were found in the diencephalonmidbrain just prior to hatch. For all areas of the brain examined, the most rapid rate of TH increase occurred between day 16 and day 18 b.h. Vernadakis (1974) reported on the in vitro uptake and storage of 3H-noradrenaline in the cerebral hemispheres and cerebellum of chicks during development. She found that in the cerebral hemispheres, mechanisms for the cellular uptake of NA develop before mechanisms for intraneuronal storage of NA. THE EFFECT OF PREGANGLIONIC DECENTRALIZATION ON THE DEVELOPMENT OF SYNAPTIC GANGLIA

Black et al. (1971 c) demonstrated that surgical transection of the preganglionic nerve trunk prevents the normal development of TH activity in the postsynaptic adrenergic neurons in mice. Surgical decentralization of the superior cervical ganglion (SCG) in rats and mice led to a 50% fall in TH (Hendry et al., 1973). Neither decentralization nor reserpine treatment caused any changes in DDC or MA0 activities in rat SCG. Administration of NGF to young mice failed to eliminate the differences in ganglion size, cell numbers, and TH activity between normally innervated and decentralized ganglia (Black et al, 1972b). Likewise, NGF was unable to reverse the effect of the ganglion blocking drug pempidine (mimicking decentralization). These observations indicate that the presynaptic nerve terminals play a role in the regulation of the biochemical maturation of the postsynaptic neurons, constituting, perhaps, the most important factor. STUDIES ON DEVELOPING CHICKEN HEART

In the chicken heart, dopa and DA are first detected on day 4 and 6 of incubation, respectively (Ignarro and Shideman, 1968). Cardiac NA is first detected on day 3 of incubation; TH, DDC, DBH, and phenylethanolamine-N-methyl-transferase (PNMT) activity can be measured on days 1 , 2 , 4 , and 6 of incubation, respectively (Ignarro and Shideman, 1968), showing an early and sequential appearance of the enzymes responsible for the synthesis of the various precursors. Ignarro and Shideman (1968) pointed out the fact that although endogenous NA and adrenaline are initially detected on day 3 of incubation, they are presumably not synthesized by the embryo until days 4 and 6, respectively, suggesting that the early embryo obtains these catecholamines from extraembryonic sources. According to these authors, the presence of catecholamines in the yolk of fertilized eggs indicates that these amines pass from the yolk to the embryo at an early stage of development. In view of the proposed regulatory and end-product inhibition exerted by the tissue

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levels of catecholamines on tissue levels of biosynthetic enzymes, specifically TH (Dairman et al., 1972), it should be important to examine the relation between the level of neurotransmitters and the level of their respective synthesizing enzymes at each stage of development . BIOSYNTHETIC REGULATORY MECHANISMS I N DEVELOPING AUTONOMIC NEURONS UNDER DIFFERENT PHARMACOLOGICAL CONDITIONS

The presence of at least two regulatory mechanisms operating at the enzymatic level and controlling the synthesis of the sympathetic neurotransmitter NA seems now to be a well established notion. (Giacobini,l972). The first mechanism is a modification of the activity of TH in response to short periods of increased nerve activity produced in various ways (Weiner et al., 1972; Dairman et al., 1972). It has been suggested that tissue levels of catecholamines are regulative through end-product inhibition (Dairman et al., 1972). A second type of regulatory mechanism is the induction or modifications of tissue levels of TH after administration of various drugs leading indirectly to increased sympathetic nerve activity (Axelrod, 1972). In the latter case, the increased synthesis of the enzyme could represent an attempt to compensate for the depleted store of neurotransmitters. This second mechanism can be elicited transsynaptically in sympathetic ganglia. In the ganglia, besides an enhanced rate of preganglionic input, other factors have been suggested such as (a) optimal levels of corticosteroids (Hanbauer et al., 1973) and (b) an increase of cAMP/cGMP ratio (Costa et al., 1974). Very little is known about the effect of various factors or pharmacological agents on transmitter-synthesizing enzymes in developing sympathetic ganglia. However, two aspects are suggestive: first, the total, selective, and permanent destruction of sympathetic ganglia of newborn mice and rats after injection of 3 doses, 50 pg/g body weight of 6-hydroxydopamine (6-OHDA) (Angeletti and Levi-Montalcini, 1970). Second, a premature rise in ganglionic TH activity in mice after administration of NGF (Hendry and Iversen, 197 I), and selective induction of NGF of TH and DBH in rat superior cervical ganglia (Thoenen et al., 1971). Thoenen et al. ( 1 9 7 2 ~ )described an increase of ChAc activity in sympathetic ganglia of newborn rats after prolonged administration of NGF, and Breese and Traylor (1972) found that the intracisternal administration of 6-OHDA to 7-day-old rats reduced the concentration of NA, DA, and TH by 72 hr. Effect of the Administration of L-dopa to Chick and Rat Embryos

The administration of a tritiated precursor of monoamines into the amniotic fluid immediately surrounding the embryo can be used as an efficient means of labeling the tissue catecholamines in the fetus (Ignarro and Shideman, 1968). A study on the production of 3H-catecholamines following the peripheral administration of H-dopa during pre- and postnatal development has been performed in the rat brain by Kellogg and Lundborg (1972). They found that peripheral L-aminoacid-decarboxylase activity is low at birth and develops with age. Their results also indicate a gradual, postnatal development of mechanisms involved in the neuronal storage of NA. The administration of large amounts of L-dopa to rats over a period of 2-7 days was

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found to lead to a progressive decrease in the activity of TH in adrenals, heart, mesenteric arteries and so on but not in the CNS (Dairman et al., 1972). This effect of L-dopa has now been demonstrated in several other species and in other tissues, and it appears that the L-dopa mediated decrease in tissue levels of TH activity represents an actual reduction in enzyme protein (Dairman et al., 1972). The experimental data of Dairman and his co-workers indicate that the increased formation of catecholamines after the administration of L-dopa can reduce the tissue level of TH independently of changes in sympathetic neuronal activity. Under the same conditions, DDC activity decreases only in the liver and not in the neural tissue. Effect of 6-OHDA on Sympathetic Neuroblasts

Angeletti and Levi-Montalcini (1 970) injected newborn mice daily with 6-OHDA (50 pg/g body weight). The result was a complete and irreversible degeneration of sym-

pathetic neuroblasts, indicating that in sympathetic cells undergoing differentiation, the uptake of biogenic amines and 6-OHDA may occur not only at the site of the synapse, as in the adult, but also along growing fibers and cell bodies. The mechanism by which 6-OHDA, once having entered the cell body, produces such dramatic damage has not been explained. It is known that the administration of 6-OHDA in large doses to the adult rat decreases the synthesis of DBH but not of TH (Brimijoin and Molinoff, 1971; Brimijoin, 1972). Moreover, 6-OHDA prevents the increase in DBH and TH activity which normally occurs in ganglia after treatment with reserpine (Brimijoin and Molinoff, 1971; Thoenen, 1971). Effect of Prenatal Administration of Reserpine

Reserpine induces an increase of DBH and TH activity in adult rat sympathetic ganglia within 24 hr after its administration (Molinoff et al., 1972; Axelrod, 1972). The administration of dopa and MA0 inhibitors halts this increase, and both procedures increase the level of catecholamines in sympathetic nerves (Axelrod, 1972). The latter effect probably results from a depressed formation of biosynthetic enzymes. The administration of reserpine and 6-OHDA elicits an increase in adrenal TH and DBH activity in rats 7-9 days old (Angeletti et al., 1972). Alpha-methyl-p-tyrosine (a-MT), a specific inhibitor of catecholamine synthesis (Spector et al., 1965), decreases catecholamine levels in adult and developing sympathetic structures and increases DBH (Loizou, 1971;Molinoff et al., 1972). However, desmethylmipramine (DMI), which blocks the re-uptake of CA, does not seem to alter either the ganglionic content of NA or DBH activity (Bhatnagar and Moore, 1971). Sparber and Shideman (1968, 1969) demonstrated that when reserpine is injected into the yolk sac of a fertilized egg prior to incubation, in a dose that has little or no effect upon hatching, it significantly decreases the concentration of catecholamines in the brain of chick embryos 14-18 days old. The administration of this drug, however, has profound effects upon the fluorometric estimation of catecholamines in the brain of both embryonic and newly hatched chicks (Sparber and Shideman, 1969), which can lead to an erroneous interpretation of the quantitative effect. The effect of prenatal reserpine administration upon uptake of tritiated NA by the newly hatched chick brain suggests

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an interference of catecholamine uptake and/or binding (Sparber and Shideman, 1970). Catecholamine levels in the whole brain of chicks hatched from drug-injected eggs are significantly higher than in controls (Sparber and Shideman, 1970). a finding which the authors interpret as supporting the hypothesis that ontogenetic alteration of systems exhibiting end-product inhibition can be long lasting or perhaps can induce a permanent change. It has been shown by Mandell and Morgan (1969) that reserpine (5 mg/kg) administered twice daily for 3 days to 2-week-old chickens increases ChAc activity in all areas of the brain. This study indicates that ChAc could be involved in regulatory mechanisms of neurotransmission in the same way as TH. In the adult rat, Oesch and Thoenen (1973) and Oesch (1974) reported an induction of ChAc in the preganglionic cholinergic neuron following increased activity of the peripheral sympathetic after treatment with reserpine. Evidence for a critical period for postnatal elevation of brain TH activity, resulting from reserpine administration during embryonic development to chick embryos has been presented by Lydiard and Sparber (1974). Waymire et al. (1974) after injecting reserpine (3-10 pg) in the yolk sak at day 1 of incubation found that NA levels were still totally depleted in brain by 18 days b.h. but found no change in TH activity. These results are in contrast to those of Lydiard and Sparber (1974) reported above. Attempts to elevate TH in developing chick brains with treatment of CAMPanalogs or phosphodiesterase inhibitors failed (Waymire et al., 1974). Effect of the Blockade of Cholinergic Receptors on the Development of Biosynthetic Enzymes

Results from our own and other laboratories indicate a correlation between the biochemical maturation of the synapse in sympathetic ganglia and the development of both synthesizing and inactivating enzymes in the adrenergic neuron. If the observed increase in DBH, DDC, and MA0 activity is triggered, or at least influenced, by the release of acetylcholine during the early embryonal period (that is, around day 8 of incubation), then it should be possible to prevent this elevation by blocking nicotinic and/or muscarinic receptors on the postsynaptic membrane. Using specific nicotinic blockers, Mueller et al. (1970) abolished the increase in TH activity induced by reserpine in adult rat superior cervical ganglia. Gisiger (1971) has shown that the neuronal stimulation by endogenous or exogenous acetylcholine increases RNA synthesis in sympathetic ganglia of adult rats and that the signal inducing the observed metabolic changes originates from the postsynaptic membrane. Hendry (1973) and Black (1973) showed that the ganglion-blocking drug pempidine when administered to neonatal mice mimicked the effects of surgical decentralization; TH activity of the superior cervical ganglia treated with this drug failed t o develop normally. It was also found (Hendry, 1973) that, as in surgically decentralized animals, NGF was unable to reverse the effects of pempidine. This would suggest that the most important regulation mechanism for the ganglion development is the influence of transsynaptic activity, most probably mediated by the depolarizing action of acetylcholine (Hendry, 1973). These results are compatible with the hypothesis that variation in enzyme synthesis induced by neuronal activity might be associated with the interaction between the newly formed transmitter (ACh) in the developing ganglion synapse and receptors and that the

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signal for modifying enzyme synthesis comes directly from the postsynaptic membrane via RNA synthesis. Effect of N G F (Nerve Growth Factor) on Developing Sympathetic Neurons and Adrenal Medulla

Angeletti et al. (1972) studied the effect of NGF and NGF antiserum (NGF-AS) on the sympathetic ganglia and adrenal medulla in newborn rats. They found an increase in TH and DBH activity in both locations after NGF and a reduction in activity of these enzymes in the ganglia after the antibody NGF-AS. In the adrenal medulla, however, both procedures caused increased TH and DBH activity. This paradoxical effect was explained as being the resslt of a stress-induced increase in the activity of splanchnic nerves, leading to a neurally mediated induction of enzymes. In our observations, however, the “physiological” stress condition of hatching (Dolezalova et al., 1974) produces a dramatic fall in the enzymes of a developing monoaminergic neuron. Stockel et al., 1974 showed that the effect of NGF on TH synthesis is quantitatively distinct from the effect on tubulin synthesis. DEVELOPMENTAL STUD1ES O N CI L I ARY GANGLIA

The onset and development of synaptic transmission in the chick ciliary ganglion has been described by Landmesser and Pilar (1972, 1974a, b). Synaptic transmission begins at day 5 b.h. in both ciliary and choroid cells and reaches 100%at day 8 b.h. in spite of the fact that morphologically (EM) only a few synapses can be distinguished at this stage. Sorimachi and Kataoka (1974) reported significant developmental changes of ChAc and AChE activity in the ciliary and superior cervical ganglion of the chick. Their study emphasizes mostly the postnatal period rather than the establishment of synaptic connections. Suszkiw et al. (1 973) and Giacobini et al. (1974) showed that in the adult ciliary ganglion, 60% of ChAc activity is localized in the preganglionic and 40% in the postganglionic elements. Practically all of the iris ChAc activity occurs in the ciliary nerve terminals. AChE activity is localized 25% preganglionically and 7 5% postganglionically. In the iris neuromuscular junction 15% AChE activity is localized to the terminals and 85%to the muscle. Both enzyme’s activities were measured (Pilar et al., 1974) during the period of synapse formation (day 5-7 b.h.), cell death (day 8-12 b.h.), and synapse maturation (after day 7 b.h.) (Landmesser and Pilar, 1974a, b). In ganglia, a 10-fold increase in AChE activity starts at day 7 b.h. and adult values are reached at day 10 b.h. The rapid increase in the iris between day 8 and day 12 b.h. coincides with the formation of synapses in the muscle. ChAc in ganglia is present at day 5 b.h. but increases progressively only after day 7 b.h. From day 10 b.h. a very pronounced increase is observed. In the iris, ChAc activity is rather low from day 5 to day 9 b.h., then a gradual 10-fold increase occurs. From these results it is inferred (Pilar et al., 1974) that (a) in the iris muscle the establishment of the nerve terminals may play a role ih regulating the appearance and development of AChE activity postsynaptically, (b) that ciliary ganglion cells are biochemically differentiated, as far as ACh metabolism is concerned, from the onset of synaptic transmission, and (c) the formation of muscular junctions in the iris may influence

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the initial increase in ChAc activity in the ganglion cells. Are Developmental Increases in Enzyme Activity Due to a True Accumulation of Specific Enzyme Protein?

The question remains open whether the increase in enzyme activity seen by different authors in the course of development might be due to a true increase of the specific enzyme protein or a simple activation of enzyme molecules which already preexisted. The fact that cycloheximide blocks the postnatal increase in enzyme activity (Black et a]., 1972b) is indirect evidence suggesting that an increase in protein synthesis is responsible for a normal development of the enzyme activity. Black et al. (1974) studied recently the developmental increase in TH activity in the SCG from mice and rats using a specific antibody to TH. Immunotitration studies (Black et al., 1974) demonstrated that the ontogenetic rise in ganglion TH activity is entirely dependent on an increase in the number of enzyme molecules and not on an

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Vig. 4. Factors involved in the regulation of biosynthetic processes during development of the autonomic neuron. TE = development of preganglionic transmitter enzymes. NTR = neurotransmitterreceptor (R) interaction. NGF = nerve growth factor. TR = establishment of neurotransmission at the effector (E) site. RAF = retrograde axoplasmic flow.

activation of preexistent enzyme. Furthermore, these studies suggested that the neonatal and adult TH molecules are immunochemically similar and that they have equivalent thermostabilities. These observations suggest that there are no important structural differences between neonatal and adult TH and that maturation does not involve the appearance of different species of TH molecules. Black et al. also demonstrated that the postnatal accumulation of specific TH protein could be prevented by ganglion decentralization or pharmacologic ganglionic blockade. It appears, therefore, that the release of the presynaptic transmitter and the following postsynaptic interaction with the receptor constitute specific signals which regulate the development of a specific postsynaptic protein (Fig. 4).

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CONCLUSIONS

The present lack of knowledge about the most fundamental mechanisms responsible for the development of interneuronal connections in both the peripheral and central nervous system still precludes the evaluation of the consequences of both external and internal factors which may disturb such development and the evaluation of their impact on subsequent function in the mature animal. Recent studies in various laboratories including our own have shown that enzymes specifically related to biosynthesis or inactivation of neurotransmitters can profitably be used as markers of development for a specific type of neuron or synapse. Moreover, their activity can be related to dynamic conditions of the neuron during growth. This view is supported by the present knowledge that enzymatic regulatory mechanisms act on the adult neuron, including transsynaptic induction of enzymes in the cell bodies and terminals (Axelrod, 1972; Giacobini, 1972). The final goal of the studies reported here is the possibility of elucidating whether (a) the formation of synaptic contacts in ganglia during development triggers or modulates the synthesis of biosynthetic enzymes in the autonomic neuron, (b) the formation of the peripheral field of innervation, or establishment of functional activity, influences or regulates the functional levels of these specific enzymes. It is conceivable that an interplay of several factors, including nerve growth factors and hormonal factors, might be responsible for the final adjustment of the functional levels of specific enzymes and that no one single factor alone is able to significantly alter the development of the biochemical apparatus of the neuron (Fig. 4). The emphasis of our own approach lies in considering the developing neuron as being continuously subjected to influences arising both peripherally and centrally, and as a cell which is able to interact at different stages of development with both the periphery and centers and to continuously readjust itself, making use of an intrinsic plasticity. This concept of neuronal plasticity has been the subject of discussion in some of our previous publications (Giacobini, 1970) and is supported by our present knowledge of regulatory mechanisms acting on the adult neuron (Axelrod, 1972 ; Giacobini, 1972). By using different microcliemical methods combined to pharmacological tools which have already proved to be valuable in exploring the adult autonomic neuron and by comparing normal ganglia with ganglia deprived of their synaptic connections at early stages of postnatal development, a new method of analyzing the development of regulatory mechanisms at the cellular level is provided.

ACKNOW L E DGMENTS

The work from our own laboratory reported in this paper was supported by grant no. GB-41475 from the National Science Foundation, PHS grant no. NS-11496-01 from NINDS, and from grants 35-048, 35-071, and 35-099 from the University of Connecticut Research Foundation. The support of the Swedish Medical Research Council during the years 1965-1971 is acknowledged.

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Neuronal control of neurotransmitters biosynthesis during development.

Journal of Neuroscience Research 1 :31 5-33 1 ( 1 975) NEURONALCONTROLOF NEUROTRANSMITTERS BIOSYNTHESIS DURING DEVELOPMENT Ezio Giacobini Laboratory...
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