Dopamine-β-hydroxylase-like immunoreactivity in the fetal cerebral cortex of the rat: Noradrenergic ascending pathways and terminal fields

073&5748/t?4 $0X00fO.00

In!. J. Devi. Neuroscrcncr. Vol. 2. No. 5, pp. 491-503, 1984. Printed in Great Britain

Per.qmon Press Ltd. @ il)XJ lSDN

DOPAMINE-P-HYDROXYLASE-LIKE IMMUNOREACTIVITY FETAL CEREBRAL CORTEX OF THE RAT: NORADRENERG~C ASCENDING PATHWAYS AND TERMINAL FIELDS

IN THE

CATHERINEVERNEY,* BRIGI-ITEBERGER.* MICHEL BAuLAc,*t KAREN B. HELLES and CHANTAL ALVAREZ* ‘Unite INSERM 134. Laboratoirede NeuropathologieCharles Foix, HBpital Salpetriere, 75651 Paris cedex itaboratoire d’Anatomie, CHU Pitii Salpetriere. 75634 Paris cedex 13, France: and ~Department of Physiology. PKI, University of Bergen, Norway (Accepted

13;

1984)

29 March

Abstract-A topographical analysis of the noradrenergic innervation in the fetal rat cerebral cortex was carried out from embryonic day 15 (E15) until birth using antibodies raised against dopamine-& hydroxylase (DBH). During late gestation DBH-like immunoreactjve axons were coursing through the basal forebrain along three pathways: (1) a medial component reached the medial cortex and then ran caudally along the anlage of the cingulum bundle; (2) a lateral component reached the frontal pole and curved ventro-dorsally in the primordium of the external capsule; (3) a few fibers were observed along the ventral amygdaloid bundle toward the amygdaloid complex and the surrounding cortex. No DBH positive fibers were observed in the main body of the internal capsule. The first noradrenergic axons were seen at El7 in the frontal pole, the lateral frontal cortex, and in the medial frontal cortex which also receives a dopaminergic input. The innervation then extended caudally. but the dorsal part of the cortex was reached after a a-day delay when compared to the medial and lateral parts. The arrival of noradrenergic axons did not parallel the gradient of cortical neurogenesis; however. all cortical areas were innervated at birth. DBH positive fibers reached a given cortical region simultaneously through the marginal and intermediate zones and then invaded the cortical plate. Key words: Dopamine-B-hydroxylase, Cerebral cortex.

Immunocytochemistry.

Noradrenaline.

Prenatal

development,

In the last decade, several morphological studies5.‘4.‘5.‘“,30,~2,34h ave dealt with the ontogeny of the catecholaminergic input to the cerebral cortex, mainly with regard to its possible functional role in neuronal development. Indeed, catecholaminergic axons penetrate the frontal cortical anlage on embryonic day 16 (El@ and, thereafter, invade rostro-caudally the different cortical areas along two main plexuses located above and below the cortical plate and parallel to the pial surface.“” As known in the adult rat the catecholaminergic input includes the noradrenergic and dopaminergic systems that have different topographical distribution in the cerebral cortex. ‘~2~‘3*‘7*‘x Therefore, it seemed necessary to analyze the respective participation of these two systems in the early catecholaminergic innervation. The classical technique available, essentially fluorescence histochemistry but also radioautography, did not allow a selective morphological visualization during prenatal development. The most accurate data, using fluorescence histochemistry, showed the first noradrenergic axons at E17, l4 but the authors observed only the cortical areas that did not receive any dopaminergic input. These fibers were visualized in the marginal and intermediate zones of the frontal and lateral cortex and their rostro-caudal progression was confirmed. More recently, immunocytochemistry of the synthetic enzymes has provided a new technical option. Indeed, we have shown that during embryonic life, tyrosine-hydroxylase-the first enzyme in the catecholaminergic synthetic pathway+ould be used as a selective marker of the ingrowing prenatal dopaminergic axons.42 Dopamine+ h ydroxylase (DBH) could be employed as a specific marker of noradrenergic neurons as demonstrated in the adult rat brain35 and especially the cerebral cortex.23+25 Nevertheless, this enzyme is present in noradrenergic, as well as adrenergic neurons3(j but no adrenaline-containing elements have yet been described in this part of the brain.” Therefore, the purpose of this study is to use DBH immunocytochemist~ in the developing forebrain as a specific marker of noradrenergic axons and to determine: firstly, the main pathways _. Correspondence to: C. Verney, 75651 Paris cedex 13. France.

Laboratoire

de Neuropathologie

DN2:5-F 491

Charles

Foix. Hopital

Salpetriere,

47 Blvd de I’Hopital,

192

PI (Il.

(‘. Verncy

of noradrenergic axons reaching the cerebral wall and compare them to those described in the adult rat: and secondly, the timing and pattern of the first noradrenergic innervation in the different cortical areas and especiaily the medial frontal cortex also receiving a dopaminergic input. Although it is well established that noradrenergic axons extend rostro-caudally, it remains unclear in which developmental time course the medial, dorsal, and lateral parts of the cerebra1 wall are innervated. Thirdly, the ingrowth of the noradrenergic arborizations through the different cortical layers and their morphological aspects will be studied. This work brings ~omplement~~ry knowledge on the cortical development of the monoainine innervation since the early topographical pattern and time course of the dopaminergic”’ and serotoninergic systems’“..‘3 have recently been described.

EXPERIMENTAL

PROCEDURES

Three to six Sprague-Dawley rat embryos ofeach age: E15, E16, E17, ElX. E19. E20, PO were used; the day following the mating night was designed as E 1, the day of birth as PO. Pregnant and early newborn rats were anesthetized with ketamine (100 mg/kg i.p.). Pups and embryos removed from the uterus were perfused through the heart for 15 min with ice cold 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2-7.4). The brains were removed and postfixed in the same fixative for a further 105 min and thereafter rinsed for 48 h in the same phosphate buffer solution to which 5% sucrose was added. The brains of the younger fetuses (EtS, Elfi) were directly immersed for 2 h in the fixative. Coronal. sagittal and horizontal semi-serial 10 pm cryostat sections were alternatively processed for DBH immunocytochemistry using the peroxidase-antiperoxidase method (PAP) and classical thionine staining. The immunocytochemical procedure consisted of the sequential incubation of tissue sections in PBS buffer with DBH antiserum 111200 in the presence of 0.3% triton for f8 h at 4°C; sheep anti-rabbit lgG l/IO for 30 min at 20°C; PAP,complex (Nordic) l/SO for 30 min at 20°C. PAP was visualized by reacting the tissue with 0.05% 3.3’-diaminobenzidine (DAB) (Sigma) to which O.OOS% hydrogen peroxide was added. Some sections were slightly counterstained with toluidine blue (0.15%). dehydrated and coverslipped with Depex. The preparation and specificity of the antiserum to rat adrenal DBH has been described in detail elsewhere.’ Briefly, antisera were raised against rat chromaffin granule protein by injections of the dialyzed Na-deoxycholate soluble antigen into the rabbit. The specificity of the antisera was examined by crossed immunoelectrophoresis yielding a single precipitation line. The anti-DBH did not crossreact with antirat PNMT (phenylethanol-amine-N-methyltransferase) (Helle, unpublished observation) and only one antibody specificity was detected against rat adrenal medulla homogenate, rat plasma or particulate or supernatant fractions of rat liver. The tissues were observed either in light- or dark-field microscopy. Drawings were performed with the use of an XY plotter adapted on a Leitz ortoplan microscope.

RESULTS We will describe first the main DBH immunoreactive pathways reaching the anlage cerebral cortex and, secondly, the topographical distribution of the first noradrenergic penetrating the different cortical layers and areas of the telencephalic wall. Forebrain

noradrenergic

As already

yathwuys reaching

the cerebral

of the axons

wall

described? the noradrenergic axons leave locus coeruleus to follow the dorsal noradrenergic bundle and mix with the medial forebrain bundle (MFB). From El7 on, the main DBH immunoreactive bundles leaving the ventral MFB towards the cerebral wall were present, but they contained only a few fibers. At E20, the density had increased and the following description of the noradrenergic pathways was primarily done at this stage. Indeed, later on, the development of the terminal arborizations may mask the noradrenergic trajectories. Lateral and rostra1 to the anterior hypothalamus, DBH positive axons fanned out through the basal forebrain (Fig. la,b). Dorsally, this bundle encountered the lateral ventricle (Fig. lb). The ventricular area is very large during late gestation as the ventricle extends ventrally and rostrally within the olfactory bulb and is surrounded by a thick periventricular zone. A few positive axons

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Fig. 1. E20: drawings of three horizontal levels of the ventral forebrain, showing schematic representation of DBH-like immunoreactive axons. (a) The most ventral level: DBH positive axons fanned out towards the hemispheric pole. (b) More dorsally: DBH positive axons in the medial forebrain bundle (mfb) are divided in a medial and lateral component by the ventricular zone (vz). Some fibers from the lateral component reach the rhinal fissure (rf) and the amygdaloid area (aa). (c) At the level of the anterior commissure (ac): the lateral positive axons cross the ventral striatum; the medial positive bundle is observed along the primordium of the vertical limb of the diagonal band as well as rostrally to it. lot, lateral olfactory tract; ob, olfactory bulb; oln, anterior olfactory nucleus; ot, optic tract; db, horizontal limb of the diagonal band: aa, amygdaloid area; v, ventricle.

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were observed within this periventricular zone (Fig. 2). However, most of the fibers coursed either medially or laterally to the ventricular area. Consecutively. two main components of DBH positive axons could bc described (Figs 1b and 2). The lateral pathways. Among this contingent, a small subset left the MFB just rostrally to the optic tract in a lateral direction (Fig. 1b). Most of these axons coursed towards the ventral rhinal fissure and reached the lateral rim of the cerebral hemisphere. Then they curved rostrally into the marginal zone of the ventro-lateral cortical anlage along the lateral olfactory tract. A few other axons ran later-o-caudally towards the primordium of the amygdaloid complex and its surrounding cortical anlage. The main lateral contingent fanned out rostrally towards the anterolateral part and the pole of the cerebral hemisphere. Positive axons ran mainly ventrally to the caudal limb of the anterior commissure, but some passed dorsally. across the primordium of the ventral pallidal and striatal areas (Fig. lc). The more ventral contingent spread within the anlage of the anterior olfactory nucleus (pars lateralis and pars posterior). Therefrom, positive axons entered ventro-dorsally the telencephalic pole. first restricted to the marginal and intermediate zones (Figs 3a and 4a). The bulk of the dorsal contingent curved dorso-caudally in the deep fronto-lateral cortex which represents the primordium of the external capsule.

0a

0

b

oln Fig. 3. EX: drawings of two sagittal significant levels, lateral and medial. (a) Lateral level: DBH posittve axons course mainly under the anterior commissure (ac) and reach the marginal and intermediate zones of the lateral frontal cortex. Numerous fibers enter the deep cortex along the rostra1 limb of the external capsule (ec). No positive axons arc observed in the internal capsule (ic). (b) Medial level: DBH positive axons course dorso-medially along the vertical limb of the diagonal band and rostrally to it. Upon reachmg the primordium of the corpus callosum (cc), two bundlca arc formed. one running above the corpus callosum which enters the cingulum bundle, the other coursing under the corpus callosum entering the septal anlage(s). Notice positive axons in the hippocampal formations. v. ventricle; h. hippocampal formations: st. striatum anlage; oln. anterior olfactory nucleus: lot, lateral olfactory tract.

The medial pathways. The large DBH immunoreactive component of the MFB which travelled medially to the ventricular area was widely overlapping the horizontal limb of the diagonal band (Fig. lb). A rostra1 contingent reached the ventro-medial frontal pole through the primordium of the anterior olfactory nucleus (pars medialis) and curved dorsally towards the marginal and intermediate zones of the medial frontal cortex (Fig. 3b).

Development

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Fig. 2. Photomicrograph at the level of Fig. lb. E20, DBH positive axons visualized by PAP method and observed with dark field illumination. The noradrenergic axons of the medial forebrain bundle (MFB) give rise to a lateral and medial positive bundle separated by the ventricular zone (VZ). However, some fibers are seen within this ventricular zone. IF, interhemisphere fissure. x 100.

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Fig. 4. E20: photomicrographs taken at the same sagittal levels as Fig. 3a and b (see boxes). PAP method, dark field. x 65. (a) Lateral: see numerous positive axons along the external capsule (white arrows) separated from the marginal positive fibers by the cortical plate (CP). (b) Medial: notice positive axons in the cingulum bundle (CB) and the DBH immunoreactive axons entering the cortical plate (CP), especially at a frontal level. LOT, lateral olfactory tract; OB, olfactorv bulb

Development

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Fig. 6. E17: photomicrographs of coronal sections corresponding to Fig. 5b and c (see boxes). PAP method, dark field. (a) Anterior frontal cortex: DBH-like immunoreactive fibers are located in the marginal (M) and intermediate (IZ) zones of the medial, dorsal and lateral areas. x 130. (b) Posterior frontal cortex: positive axons are observed in its medial and lateral parts. x80. IF, interhemispheric fissure; cp, cortical plate; V, ventricle.

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Fig. 7. E19: medial frontal cortex (PAP method, dark field). DBH-like immunoreactive axons are entering the cortical plate (cp) (white arrows). They appear to arise either from the outer plexus in the marginal zone (M) or from the inner plexus in the intermediate zone (IZ). x 210. IF, interhemispheric fissure. Fig. 8. Morphological changes of DBH-like immunoreactive axons from El7 to PO (PAP method, bright field). Pial surface on the left. x 1040. (a) E17: marginal zone, short positive segments. (b) E17: intermediate zone, long and coarse positive axons. (c) E20: marginal zone, sequence of small dots suggesting varicosities. (d) PO: layer VI, closely spaced varicosities linked by intervaricose segments (aspect similar to that observed in the adult).

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A caudal contingent curved dorsally along the vertical limb of the diagonal band of Broca (Figs lc and 3b). Several days after the arrival of the first DBH immunoreactive axons, the corpus cahosum starts to cross the midline”” (between El9 and E20). Therefore, as it enlarged, it divided this caudal positive contingent into two components (Fig. 3b): one looping dorsally above the corpus callosum in the deep medial intermediate zone, the primordium of the cingulum bundle (Fig. 4b); the other coursing under the corpus callosum innervating the septal area and more caudally the fimbria and the hippocampal formations (Fig. 3b). Spreading of the noradrenergic

innervation in the cortical anlage

In the Sprague-Dawley strain, the formation of the cortical plate which initiates the development of the main cortical layers starts on days 15-16 of embryonic life, dividing the cerebral wall into four layers: marginal zone, cortical plate, intermediate zone, and periventricular zone. In the following results, the white matter anlage and the future layer Vib (see review in ref. 21), both displaying early DBH positive axons, were considered as components of the intermediate zone. At El5E16, no DBH-like immunoreactive fibers were detected in the developing cortex. At E17, the first DBH-positive fibers were observed in the anterior half of the hemisphere (Fig. 5). In the frontal pole (anterior half of the frontal cortex), DBH-like immunorea~tive fibers were seen in the medial, dorsal and lateraf cortex (Figs 5a, b and 6a). In the posterior part of the frontal cortex and at the levels of the anterior par&al and cingulate cortex, DBH reactive axons were only present in the medial and lateral cortex, the dorsal cortex being devoid of them (Figs 5c, d and 6b). No positive axons were observed caudally in the posterior part of the cingulate and parietal cortex and in the occipital cortex. DBH-like immunoreactive axons were seen both in the marginal and intermediate zones, just above and below the cortical plate (Fig. 5). In these two layers, they ran more or less parallel to the pial surface as seen on sagittal and coronal sections (Figs 4-6). In the other layers, sparse positive fibers could be seen, crossing the periventricular zone or entering the cortical plate of the medial frontal cortex. From El7 to birth, three main features characterized the development of DBH-like immunoreactivity. A slow rostro-caudal extension to the different cortical areas took place simultaneously in the marginal an&dintermediate zones. This sagittal progression occurred first along the medial and lateral parts of the hemisphere, whereas the dorsal part was invaded with a 2-day delay. Therefore, at E20, the retrosplenial, temporal and entorhinal cortex displayed positive axons. At the same age, the dorsal fibers just reached the rostra1 parietal cortex. They were observed in the dorsal occipital cortex only at birth. lmmunoreactive axons invade gradually other cortical layers, also following a rostro-caudal gradient. Thus from El8 on, once the positive axons had invaded the marginal zone, they penetrated the cortical plate (Fig. 7). Several morphological aspects suggested that positive axons arose from both superficial and deep plexuses to invade simultaneously the cortical plate. Nevertheless, as the density of positive axons is greater in the intermediate zone, the respective participation of these two bundles in the intracortical innervation appeared to be more important for the deeper plexus than the superficial one. ~orp~~ological aspect of developing noradrenergic axons. The overall morphological aspect of cortical DBH immunoreactive fibers was not identical at the different ages observed. At E17, positive axons appeared as short positive segments in the marginal zone (Fig. 8a), whereas in the intermediate zone they were longer and coarse (Fig. 8b). At E20, they became much thinner, being reduced to small positive dots (Fig. 8~). This aspect was observed in ali layers and was compatible with the presence of varicosities. Finally, at birth, the overall aspect of noradrenergi~ axons was that of closely spaced varicosities linked by very thin intervaricose segments, as observed in the adult (Fig. 8d).

DISCUSSION Technical considerations

In two previous works, using catecholamine fluorescence histochemistry, the arrival of the catecholaminergic axons in the cortical anlage was investigated. Schlumpf et al.““observed the first fibers as early as E16. whereas Levitt & Moore.14 who studied neocortical areas receiving no

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Fig. 5. El7: seml-scrtal coronal sections ot the anteriot- hall 01 the hcmlsphere where the first DBH immunoreactivc axons are ohserved in three mn~n xcu\ (note that the corpuscallosum is not yet developed), (a.b) In the frontal cortex in its medial. dorsal and lateral part. (c.d) In the medial and lateral cortex more caudally (frontal. anterior parietal and clngulate cortex). At these Icvcls. no positive axons are visualized in the dorsal part of the hemisphere. Within the cerebral wall. fiber> xc pl-esent in the marginal and intermediate zones: the cortical plate (cp) and the periventrwulal- Lone show only sparse positive axons. ac. anterior commissure; lot. Iatcral olfactory tract: \. wptal nnlage: v. wntriclc.

dopaminergic input. lateral, frontal, dorsal and parietal cortex. detected the noradrenergic axons at E17. Our data confirm the arrival of the earliest noradrenergic cortical fibers in the frontal cortex at E 17. The catecholaminergic axons detected by Schlumpf ct (11.at E I6 could be dopaminergic as we observed their arrival in the developing cerebral cortex at this age.‘” In the hippocampus. we noticed the first DBH positive axons only from El8 on. at the same stage when Loy & Moore”’ visualized the endogenous norepinephrine with fluorescence histochemistry. These results indicate that, as suggested by the detection of DBH activity before and concomitant with the increase in endogenous norepinephrine content in the embryonic brain.“ DBH immunocytochemistry may be at least as sensitive as the formaldehyde-induced fluorescence method for the labelling of noradrenergic axons. However, this depends on the antibody used. In fact, the developmental time course of DBH positive innervation reported in this study is at difference with our previous results obtained with another batch of DBH antiserum. ” In this former study. DBH-containing axons were first visualized in the marginal zone at E 19 and in the intermcdiatc Lone at E21. These different results were obtained with two equally sensitive and purified homologous polyclonal antibodies.‘.’ One of the possible reasons for this discrepancy could he the use of antibodies raised against DBH extracted from adrenal medulla of young Sprague-Dawley rats’ in the present study, whereas the former antibody was raised against adrenal DBH obtained from adults of the same strain.” Pathways

ferent

ofdeveloping

pathways

reaching

corticipetal

rtoradrener~ic~ih~~.s.

the developing

cerebral

wall

On the whole. our description of the difis in concordance with the observations

Development of the noradrenergic cortical innervation

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of Seiger & Olson32 in the embryo and Ungerstedt38 and Lindvall & BjGrklund’7*‘8 in the adult rat. However, some points are noteworthy: (1) The cingulum bundle appears to innervate the rostrocaudal extension of the medial cortex but not the lateral cortex, as also demonstrated by Morrisson et a1.26in adult lesioned rats, using the same immunocytochemical technique. (2) The lateral tioradrenergic inne~ation arises in the frontal lobe, along the rostra1 limb of the external capsule, as (3) The third catecholaminergic pathway towards described by other authors in the adult. 1’~26,27,33 the cerebral cortex described along the internal capsule by some authors is probably not noradrenergic. Indeed, catecholamine fluorescence histochemistry alone,17 or after loading with 6showed positive axons presumed to be noradrenergic, in the internal capsule hydroxydopa, 10*29,37 at the level of the caudo-putamen. However, the specificity of 6-hydroxydopa loading for visualizing noradrenergic axons is uncertain. Moreover, selective DBH immunocytochemistry in the adult3” or in the fetal rat (this study) failed to reveal any positive axons in this part of the internai capsule. On the other hand, as we saw tyrosine-hydroxylase immunoreactive fibers (personal data) in the same area at the same prenatal stage, the catecholaminergic fibers described by previous authors in the rostra1 internal capsule are likely to be dopaminergic. In fact, we observed a small fascicle of DBH-positive axons crossing the internal capsule, but through its most ventral and caudal part, coursing towards the amygdaloid complex and the periamygdaloid cortical anlage. These fibers corresponded to the ventral amygdaloid bundle-ansa peduncularis, as described in the adult rat.“.‘7 Cortical ingrowth of noradrenergic axons and neurogenesis. The use of DBH immunoreactivity as specific marker of noradrenergic innervation allowed us to follow its arrival and development even in cortical areas receiving a dopaminergic input such as the medial and frontal and rhinal cortices. Thus we have visualized a simultaneous ingrowth of noradrenergic fibers both in the medial and lateral frontal cortex at E17, the dorsal cortex becoming innervated with a 2-day delay. Therefore, the medial and lateral bundles achieve their rostrocaudal extension when the dorsal axons begin their progression. This means that the ingrowth of the noradrenergic innervation in the developing cerebral cortex proceeds from the frontal pole in three sagittal bands: medial, lateral and dorsal (Fig. 5). These data confirm the observations made by ~orrisson et al. 24-26Using different corticai lesions in the adult rat, these authors demonstrated a tangentially and sagittally organized pattern of noradrenergic innervation from the frontal pole and throughout the telencephalon, mainly arising from two bundles, medial and lateral. Moreover, we show that this type of distributon is already present in the fetal brain from the incoming of the noradrenergic fibers. The early arrival of the noradrenergic projections in the fetal cerebral cortex and their location in the layers (marginal and intermediate zones), where the first synaptic contactsI occur and the dendritic maturation22 starts, have raised the question of a possible trophic role on the cortical neurogenesis. This hypothesis has been recently largely debated. Studies of the cortical neurons after destruction of the noradrenergic input furnished contradictory results about its possible influence on various parameters of neuronal growth (see ref. 3 for review). In any event, our data showed that the spreading of the noradrenergic terminal fields in the rat cortex does not follow the latero-medial gradient of cortical development, contrarily to previous statements.‘” It has been reported that the lateral areas of the cerebral cortex develop l-2 days before the medial areas regarding cell density, cell migration and gross neuropil development. x.28As the noradrenergic neurons grow simultaneously in the medial and lateral cortex, they do not seem to act as a signal at least for these processes. The noradrenergic system is not the only one to arrive in the cerebral wall during late gestation. Indeed, the serotoninergic innervation displayed at El7 a very similar distribution through the different cortical layers.‘“,43 Moreover, the dopaminergic axons are already present from E16, but restricted to the intermediate zone. Thereafter, the three aminergic systems have a different time course of intracortical development. The dopaminergic system is well developed at birth in the prefrontal cortex”‘*42 but far less so in the cingular” and entorhina14’ cortex. The noradrenergic system is present in all cortical areas at birth whereas the serotoninergic arborizations develop mainly during postnatal life. ” These different schedules of development could underfine some complex interactions with neurogenesis. Evolution of the morphological aspect of incoming DBH immunoreactive fibers. The cortical DBH-like immunoreactive fibers showed different morphological aspects according to the fetal

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stage but not the topography. Indeed, very thick with apparently a large amount of synthetic enzyme at E17, they became thinner and reduced to dots at E20, suggesting the f~~rmation of varicosities which were clearly visualized at birth. The presence of such large noradrenergic fibers in the embryonic cortex has also been observed in the developing cerebeIlum.J”The cortical dopaminergic fibers also look thicker in the early embryonic stage. Perhaps this aspect could be compared to the pile up phenomenon described in catecholaminergic fibers after interruption of the axonal flow.‘” It could retlect the unbalance between the well-developed synthetic capacity of the locus coeruteus neurons’ and the small terminal fields during early ~Ievel~~pment as compared to the adult, REFERENCES Berger B.. Tassin J. P.. Blanc G.. Moyne M. A. & Thierry A. M. 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