THE JOURNAL OF COMPARATIVE NEUROLOGY 314:415-428 (1991)

Serotoninergic Innervation of the Ferret Cerebral Cortex. 11. Postnatal Development THOMAS VOIGT AND ANA DOLABELA DE LIMA Max-Planck-Institut fur Entwicklungsbiologie, 7400 Tubingen, Germany

ABSTRACT We have investigated the serotoninergic innervation of the ferret cortex from the day of birth to adulthood with immunohistochemical techniques. Due to the premature birth of ferrets, this period spans the entire generation of cells located within the upper cortical layers and their subsequent migration to their final positions. Already at birth, serotoninergic fibers innervate the developing cortex. This innervation is most dense within the marginal zone, the subplate region, and the lower portion of the cortical plate. As long as cell migration continues, serotoninergic fibers enter the expanding portions of the cortex. Only the region just below the marginal zone where newly arriving cells are added to the cortical plate is not innervated by the ingrowing fibers. When the bulk of cell migration ceases, during the third postnatal week, this gap disappears and the fibers gradually form a continuous innervation from the pia to the ventricle. As the cortex matures, the serotoninergic fibers become successively confined to the upper layers, to generate the adult pattern. In the adult ferret cortex, the highest innervation density is found within layers 1, 2, and 3, with a much sparser innervation within the lower layers (Voigt and de Lima, J. Comp. Neurol. 314:403-414,1991). The dense innervation in the deep cortical layers is only transient, virtually disappearing toward adulthood. These results suggest that serotoninergic axons innervate cortical layers as soon as newly arriving cells reach their final positions within the cortex. This early innervation lends support to the idea that serotonin may play a role during development of the cerebral cortex. Key words: 5-HT, modulatory systems, visual cortex, immunohistochemistly, monoaminergic afferents

In higher vertebrates, serotonin is one of several transmitters involved in the modulation of cerebral cortex function (for review see Foote and Morrison, '87). Serotoninergic neurons of the dorsal and median raphe nuclei extensively innervate the entire cerebral cortex. In mammals, serotonin forms such a dense fiber plexus that it is possible that every cortical neuron might be innervated (Lidov et al., '80; Morrison et al., '82; Morrison and Foote, '86; Papadopoulos et al., '87; Mulligan and Tork, '88; Hornung et al., '90;Voigt and de Lima, '91). In contrast to the topographically organized thalamic afferents with their distinct, small receptive fields, modulatory projections like the serotoninergic one innervate large brain areas (Foote and Morrison, '87). Serotoninergic axons have been found to traverse long distances and form en passant synapses with both projection neurons and intrinsic neurons (Papadopoulos et al., '87; de Lima et al., '88; Seguela et al., '891, suggesting that the system has a global modulatory function. Several lines of evidence suggest that serotonin may play a role in developmental processes in the brain (Lauder and Krebs, '78; Chubakov et al., '86; D'Amato et al., '87; Rhoades et al., '90).In mammals, serotoninergic cells in the raphe nuclei are among the very first neurons to enter the postmitotic phase and begin their differentiation (Olson O 1991 WILEY-LISS. INC.

and Seiger, '72; Seiger and Olson, '73; Lauder and Bloom, '74; Wallace and Lauder, '83).Major subcortical target areas within the brainstem, the thalamus, and the tectum are innervated by serotoninergic fibers at about the time they start their differentiation (Lauder and Krebs, '78; Lauder et al., '82). This has led to the assumption that serotonin might have a role as a differentiation signal during early histogenesis in these subcortical structures (Lauder and Krebs, '78; Lauder et al., '82). In contrast to the early innervation of subcortical areas, it has been found that the rat cerebral cortex is innervated relatively late by the serotoninergic system. Based on the time of innervation, it has been concluded that serotonin is probably not involved in the developmental processes of early cortex formation (Molliver, '82). However, recent studies in the rat somatosensory cortex show that serotonin innervation is already very dense at birth and its role during the formation of the somatosensory maps has been discussed (D'Amato et al., '87;Rhoades et al., '90).Additionally, in slice cultures of newborn rat cortex, serotonin Accepted August 28,1991. Address reprint requests to Dr. Thomas Voigt, Max-Planck-Institut fur Entwicklungsbiologie, Spemannstr. 35/I, D-7400 Tubingen, Germany.

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stimulates glial proliferation, neuron differentiation, and synaptogenesis (Chubakov et al., '86). Thus serotoninergic systems may also be involved in the regulation of various processes during cortical development. However, at present it is not known at what time the serotoninergic system starts to become active, nor what its precise function during development might be. The whole cortical histogenesis of a small laboratory animal like the rat is confined to a brief period of not more than 5 to 6 days, during the later stages of a 3-week gestation period (Berry et al., '64). As the entire brain is generated in such a short time period, many processes that last for several days in animals with larger brains happen within several hours in this small animal. In ferrets (Mustela putorzus furo), a small carnivore with a relative large gyrated brain, the duration of cortical neurogenesis is similar to that in cat, spanning a period of more than 4 weeks (Jackson and Hickey, '85; Jackson et al., '89). This prolonged phase of histogenesis allows a resolution of events on a much finer time scale than is possible in the rat brain. In addition, ferrets are born very immature. With a gestation period 3 weeks shorter than the cat, a considerable amount of cortical development occurs postnatally in the ferret (Jackson and Hickey, '85; Luskin and Shatz, '85a; Jackson et al., '89). In this study we used immunohistochemical techniques to describe the time course of the innervation of serotoninergic fibers in the ferret cerebral cortex from the day of birth to adulthood. We found that the entire cerebral cortex is already densely innervated at a time when the cortical histogenesis is at its peak. Newly formed layers were innervated just after migrating cells reached their final position. The obtained findings support the idea that serotonin might be functionally involved in early processes of cortical development.

MATERIALS AND METHODS A total of 17 normal pigmented ferrets (M.putorius furo) ranging from newborn to adult were used. Pregnant animals were obtained from Marshall Research Animals (North Rose, NY)and from our own colony. Prior to perfusion, animals with ages ranging from postnatal day zero (PO) to P28 were deeply anesthetized with ether. Older animals were anesthetized with a ketamine-xylazine mixture. They were then transcardially perfused with 0.9% saline followed by 4% paraformadehyde in phosphate buffer (PB, pH = 7.4). After the perfusion, brains were immersed in the same fixative for an additional 2 hours, impregnated in 10%and 30% sucrose, and then cut coronally on a freezing microtome (50 pm sections). Free aldehydes were removed by washing the sections 3 x 10 minutes in cold 50 mM ammonium chloride dissolved in phosphate-buffered saline (PBS, pH = 7.4). Every sixth section was stained free-floating for immunohistochemistry; adjacent sections were counterstained for Nissl. A polyclonal rabbit antiserotonin antibody (Incstar) was used as primary antibody at a concentration of 1:2,000. The primary antibody was visualized with the peroxidaseantiperoxidase technique (PAP-method) (SternbergerMeyer) or with a rhodamine-conjugated goat anti-rabbit antibody (Dianova).Antibodies were dissolved to the appropriate dilution in PBS, which contained 2% bovine serum albumin (BSA), 10% goat serum, and 5% sucrose. To

increase the penetration, 0.5% Triton X-100 was added with the primary antibodies. Incubation times for the primary antibodies were 12 hours at 4°C and for all consecutive incubation steps, 2 hours at room temperature. Between every incubation step, the sections were washed thoroughly three times for 10 minutes in PBS. The peroxidase complex was visualized by incubating the tissue in 0.07% diaminobenzidine (DAB) containing 0.003% hydrogen peroxide in phosphate buffer (pH = 7.4, for 7-10 min). Sections were placed on gelatinized slides and air-dried. The reaction product was intensified by serial immersions in 0.005% osmium tetroxide (6-7 m i d , 0.5% thiocarbohydrazide (10 min), and 0.005% osmium tetroxide (2 min). Sections were then dehydrated in a graded series of alcohols and mounted in Permount (Fisher). When rhodamine was used as secondary antibody a Nissl-like counterstaining with m-phenylene-diamine was performed (Quinn and Weber, '88). Within most cortical regions, subplate cells were found to be stained between P7 and about P28. The staining was quite weak but noticeable. Control experiments showed that these cells were nonspecifically stained by various secondary antibodies. The following antibodies were tested, whereas the primary antibodies were omitted: goat antirabbit RITC (Dianova),goat anti-rabbit PAP (Sternberger), rabbit anti-mouse PAP (Sternberger), goat anti-rat PAP (Sternberger), and goat anti-rabbit peroxidase (Dianova). All these antibodies labeled subplate cells unspecifically with similar intensity and frequency. The staining was found in sections processed for the PAP-method as well as in sections processed with rhodamine-labeled secondary antibodies. Fibers were never labeled in these control sections in which the primary antibody was omitted. In concordance with the convention used by Linden et al. ('Sl), the day of birth was considered to be PO.

RESULTS In this study, we have processed and analyzed coronal sections from all anterioriposterior levels. In the cortex histogenesis does not begin simultaneously over the entire surface, but spreads in rostrocaudal and laterodorsal direction over the hemispheres from a focus on the lateral wall (Smart and Smart, '82; McSherry, '84; McSherry and Smart, '86). Thus at a given time, rostra1 and lateral portions of the cortex are more developed than caudal and dorsal parts. All figures (with the exception of Fig. 2) are taken from the dorsal half of the occipital cortex, where the visual areas are located (McConnell and LeVay, '84; McConnell and LeVay, '86; LeVay et al., '87; Law et al., '88). This part of the cortex was chosen because many studies on cell migration and early cortical development have been performed on the visual cortex of various species. In addition, this region is the latest developing part of the cortex, allowing one to observe more stages of development after birth (Smart and Smart, '82; McSherry, '84; McSherry and Smart, '86).

First postnatal day At the day of birth, serotoninergic fibers can already be found throughout the entire cerebral cortex. Most fibers are concentrated in the marginal zone directly below the pial surface, in the subplate region, and in the lower part of the cortical plate (Fig. 1).The intermediate zone as well as the

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Fig. 1. Serotonin innervation of a ferret cortex at the day of birth (PO). This frontal section was taken from the occipital portion of the cerebral cortex (future visual cortex). Immunofluorescent serotoninergic fibers are more numerous within the upper portion of the marginal zone, within the subplate region and the lower part of the cortical plate.

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The upper part of the cortical plate contains noticeably less serotoninergic fibers. Often fibers are found that crossed the cortical plate in vertical directions (arrow). MZ = marginal zone, CP = cortical plate, SP = subplate, IZ = intermediate zone, VZ = ventricular zone. Scale bar = 50 +m.

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Fig. 2. Comparison of a serotonin-stained section with an adjacent Nissl section at PO. These sections are taken from a more anterior level of the cortex than that of Figure 1.A. Darkfield photograph of the serotonin innervation. Here nearly the entire cortical plate is innervated by serotoninergic fibers but a zone of lower fiber density just below the marginal zone is clearly visible. B. Adjacent Nissl section. The

area just below the marginal zone contains a band of high cell density (white arrows). C. Superposition of both stainings from A and B clearly shows that the zone of low fiber density in A and the band of high cell density in B coincide. MZ = marginal zone, CP = cortical plate, SP = subplate, IZ = intermediate zone, VZ = ventricular zone. Scale bar = 100 pm.

ventricular zone contains fewer processes. Within the cortical plate, the innervation follows the rostrocaudal gradient described for the histogenesis in the ferret cortex (McSherry, '84; McSherry and Smart, '86). At caudal positions, where histogenesis is less advanced, the entire upper half of the cortical plate is nearly free of fibers, with processes only occasionally crossing from the subplate to the marginal zone (Fig. 1). At more lateral and more anterior positions where the histogenesis is more advanced, the innervation reaches farther up toward the marginal zone. The innervation pattern found in more anterior levels of the cortex at PO is comparable to that at more caudal positions some days later (compare Fig. 2A with Figs. 4 and 5 ) . But even within most developed areas, a small region directly below the marginal zone has a markedly lower fiber density. Closer inspection of Nissl sections shows a band of somewhat higher cell density directly below the marginal zone. The density difference in comparison to lower layers is not very dramatic but clearly noticeable (Fig. 2B, arrows). A superposition of serotonin-stained sections with adjacent

Nissl sections, or direct counterstaining of fluorescent sections, shows that the fiber-free zone coincides exactly at all cortical locations with this band of increased cell density (Fig. 2C). In more caudal regions, where the zone of lower fiber density is broader, the region of higher cell density was also broader. Within the marginal zone, the fibers tend to run directly below the pial surface, dividing this zone into two parts. The upper half contains the bulk of the innervation, whereas the lower half often contains substantially fewer processes (see Fig. 6). The majority of fibers are of fine caliber, with irregularly spaced ovoid varicosities. They are of the same type as most of the fibers in the adult ferret brain (Voigt and de Lima, '911, although the axons generally seem to be thicker and straighter in the neonate than in the adult brains. In addition to these fine fibers, many growing axons are observed (Fig. 3). These axons are much thicker and usually grew straight (see Fig. 3B for comparison of both fiber types). Directly behind the growth cones, the processes have one to several thick ovoid varicosities.

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Fig. 3. Growing serotoninergic fibers at PO. A, B. Migrating axons of horizontal orientation within the upper portion of the cortical plate. Note the size difference between an ingrowing axon and a mature axon (arrow in B). C . Axon migrating vertically through the upper portion of the cortical plate toward the marginal zone. The border between the cortical plate and the marginal zone can be seen in the upper part of the

picture. D. Growing axons at the outer side of the marginal zone. E. Growth cone within the lower portion of the cortical plate. F. Growth cone within the cortical plate of a P14 animal leads a much finer fiber than most seen at PO or P7. MZ = marginal zone, CP = cortical plate. Scale bar = 10 pm.

With increasing distance from the growing tip, the axons become thinner until they are indistinguishable from the surrounding axons. These growing fibers show a remarkably high immunoreactivity within and shortly behind the growth cones allowing their easy detection at low power magnification (see Figs. 4, 5, 7). Axonal growth can be found in all parts of the cerebral cortex. The angle of growth

varies from parallel to the pial surface to vertical, toward the pia (Figs. 3A,B). The zone of low fiber density below the marginal zone is often crossed by ingrowing processes that come from the subplate region (Fig. 3C). When these fibers reach the marginal zone, they join the main fiber stream running parallel to the pial surface (Fig. 1, arrow). Within the marginal zone itself, they grow mostly within the outer

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Fig. 4. Serotonin innervation in a ferret cortex at P7. This frontal section is taken from the rostra1 portion of the occipital cortex at a dorsomedial position. The cortical plate increases dramatically through the first postnatal week. The serotoninergic innervation follows closely the front of newly added cells and only the uppermost portion where the

migration cells are just arriving has a lower fiber content. Many ingrowing axons (arrow) show that in this cortical zone new axons are continuously being added. MZ = marginal zone, CP = cortical plate. Scale bar = 100 pm.

portion of this layer (Fig. 3D). Growth cones are occasionally found heading downward to the ventricle.

of the cells destined for layer 4 are still on their way, whereas cells of the upper cortical layers have not yet been born (Jackson and Hickey, '85; Jackson et al., '89). Thus during the first 2 postnatal weeks, the thickness of the cortical wall shows an enormous increase in size (compare cortical plate in Figs. 1 and 4, and in Fig. 6A,B). The

First and second postnatal week ('1-'14) In the ferret, only neurons of the lower layers 5 and 6 have finished their cell migration by the day of birth. Most

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Fig. 5. Higher magnification of the upper part of the cerebral cortex at (P7). The picture shows more clearly the zone of low fiber density at the top of the cortical plate. As the cortex expands, growing axons innervate this zone continuously (arrow). MZ = marginal zone, CP = cortical plate. Scale bar = 50 pm.

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I Fig. 6. Drawings of a cortex at P1 (A) and at P7 (B) show the tremendous increase of brain thickness within 1 week. At P1, the suhplate region has the highest content of fibers, but this zone has moved by P7 into the lower cortical plate. At both ages, the innervation

gap between marginal zone and cortical plate is clearly visible. The insets show the positions from where the drawings were made. MZ = marginal zone, CP = cortical plate, SP = subplate, IZ = intermediate zone, VZ = ventricular zone. Scale bar = 100 p,m.

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Fig. 7. Upper portion of a cerebral cortex at 6 weeks of age (P42) shows the continuous innervation of layer 1and layers 213 as was found from the time cell migration ended until adulthood. At P42, growth cones can be observed only very rarely (straight arrow). A serotonin-

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ergic fiber of the smooth long type can be seen within the marginal zone (curved arrow). MZ = marginal zone, CP = cortical plate. Scale bar = 50 pm.

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innervation of the serotoninergic fibers closely follows this expansion. As long as cell migration continues, both the area of low fiber density just below the marginal zone (Figs. 4 , 5 ) and the corresponding band of higher cell density seen in the Nissl stained sections are visible (Figs. 2B,C). A large number of growing axons are found at the lower border and within this area of low fiber density (Figs. 4, 5, arrows). During the first postnatal weeks, the overall number of fibers is somewhat lower than that found in the newborn animals. Between PO and P14 many growth cones can still be found within the cortex.

Third postnatal week to adulthood At the end of the second postnatal week, the dense zone of cells found directly below the marginal zone in Nisslstained sections disappears in more rostral positions. One week later this zone has also gone from the occipital cortex. The disappearance of this zone is accompanied by the development of a continuous innervation between layer I and layer 11. This type of innervation persists throughout the further development until adulthood (Figs. 7,s).At P7 the intermediate zone still contains a high number of serotoninergic fibers, but this innervation shifts with the maturation of the cortex toward the upper layers. At 6 weeks of age, the serotoninergic innervation closely resembles the adult pattern, although the fiber density across the cortex is more even than in the adult. In addition, most fibers are thicker than in adult and have a very straight morphology, so that they can be followed over long distances within a section. In the adult cortex, layers 1and 213 have a much higher innervation density compared to the 6-week-old animal, whereas the lower layers contain few fibers (Fig. 8) (Voigt and de Lima, '91).This change is due to the addition of fine, highly branched processes in the upper layers. Growth cones are still found between P14 and P21. In addition to the thick growing fibers frequently observed at earlier ages, now finer axons have growth cones (Fig. 3F). The number of growing axons declines continuously and at 6 weeks of age, growth cones are observed very rarely (Fig. 7, straight arrow). At birth, only fine fibers with ovoid irregular varicosities are present. Most of the serotoninergic axons seen during development and in the adult ferret are of this fiber type (Voigt and de Lima, '91). The two other fiber types found in adult animals seem to develop later. Smooth long fibers with rare varicosities are first observed around P21 (Fig. 7, curved arrow),whereas the first thick varicose fibers appear around P29 (not illustrated).

DISCUSSION Development of serotoninergic inputs to the cortex In the present work, we show that serotoninergic fibers innervate the developing cerebral cortex at a time when the upper layer neurons are still migrating. Comparison of different brain areas shows that all parts of the cortex have roughly the same pattern of innervation. At the day of birth (PO),the marginal zone is heavily innervated throughout the entire cortex, as is the subplate region and the lower portion of the cortical plate. However, directly below the marginal zone there is a region of low fiber density. Its extension varies according to the known lateromedial and rostrocaudal gradients of development (McSherry, '84;

McSherry and Smart, '86). In the occipital cortex, the zone of low fiber density is largest (Fig. 11, whereas in more rostral locations or more lateral, it is restricted to just below the marginal zone (Fig. 2A). In the occipital cortex, this zone is particularly broad at the day of birth, as is the band of higher cell density. Its thickness indicates that many cells arrive simultaneously at the top of the cortical plate, mirroring the fact that the occipital cortex undergoes a tremendous expansion during the first postnatal weeks. Thus at the time of birth, the cell proliferation and the number of newly arriving cells are apparently much higher in the occipital cortex than in more rostral regions (Figs. 1, 2). A comparison of different cortical locations and different ages reveals that there is a link between low fiber density and high cell density in this region just below the marginal zone (Fig. 2). It is known from 3H-thymidine labelling studies that the cortex is generated in an inside-out fashion by the addition of newly arrived cells at the top of the cortical plate (Angevine and Sidman, '61; Berry et al., '64; Rakic, '74; Jackson and Hickey, '85; Luskin and Shatz, '85a; Jackson et al., '89). The zone where the cells are added can be seen in Nissl-stained sections as a band of higher cell density immediately below the marginal zone. This has been demonstrated in the cat by comparing 3H-thymidine labelled and Nissl-stained sections (Shatz and Luskin, '86). The fact that serotoninergic axons can be found directly below this zone indicates that the innervation occurs as soon as the migrating cells have reached their final position. This interpretation fits well with the fact that this zone becomes narrower as the proliferation rate slows after the first postnatal week (compare Fig. 1and Figs. 4 , 5 ) and that it disappears entirely as soon as the cell migration has ceased (Fig. 7).

Comparison with other species Thymidine labeling studies reveal that the generation of individual layers during cortical histogenesis is nearly identical in the cat and the ferret, a related carnivore (Jackson and Hickey, '85; Luskin and Shatz, '85a; Jackson et al., '89).In both animals, cortical neurons are born over a period of 33-35 days. In ferrets, the genesis of cortical neurons begins on embryonic day 20 (E20) of the 41 2 1 day gestational period and continues postnatally until 2 weeks after birth (P14) (Jackson et al., '89). In cats, the cortical histogenesis starts at E24 and lasts until E57 of a 65 +- 2 days gestational period (Luskin and Shatz, '85a). Additionally, the time from conception to eye opening (an event that occurs postnatally in both species) differs in the cat and ferret by only 1 to 3 days (Greiner and Weidman, '81; Linden et al., '81). Thus it is likely that at a given day after conception, the maturation of the brain is very similar in both species. However, ferrets are born about 24 days earlier than cats. Consequently, most of the upper cortical layers that are generated in cats during the last weeks of gestation are produced in ferrets during early postnatal life, allowing easy access to the early stages of cortical development (Jackson and Hickey, '85; Luskin and Shatz, '85a; Jackson et al., '89). This makes ferrets an ideal model for developmental investigations of the cerebral cortex (Jackson and Hickey, '85). Investigations of the development of the cortical serotoninergic innervation in cats and monkeys have been performed at stages when cortical histogenesis is finished and the adult pattern of cortical layering has already been

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Fig. 8. Drawings show the serotonin-immunoreactive fibers in the cortex of a 6 weeks old animal (A) and of an adult ferret (B).Here it is clearly shown that the shift of innervation density during development is mainly achieved by addition of fine, highly branched processes in the upper layers and by reduction of fiber density in the lower layers. The insets show the positions from where the drawings were made. WM = white matter. Scale bar = 100 Fm.

established (Foote and Morrison, '84; Gu et al., '90). If one wants to compare the early postnatal stages of cat and ferret, one has to take into account the premature birth date of ferrets. The earliest age in which the serotoninergic

innervation has been investigated in cat is 2 weeks postnatally (Gu et al., '90). Based on the thymidine studies (Jackson and Hickey, '85; Luskin and Shatz, '85a; Jackson et al., '89), the maturity of a 2-week-old cat cortex corre-

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Fig. 9. Schematic illustration shows the shift of innervation over the cortex during development. See text for further details. MZ = marginal zone. CP = cortical plate, SP = subplate, IZ = intermediate zone, VZ = ventricular zone.

sponds roughly to the developmental stage of a 5-week-old ferret cortex. At this stage, the serotoninergic innervation of area 17 is very similar in both species. The innervation is less dense than in the adult and the fibers are scattered over all cortical layers. In the cat and in the ferret, the fiber density increases within the upper cortical layers as the cortex matures, whereas the lower layers remain less densely innervated (Gu et al., '90). In monkeys, data is only available for the postnatal development of the serotoninergic innervation in area 17 (Foote and Morrison, '84). The adult innervation of monkey area 17 differs from ferret and cat in that layer 4 and not layer 1is most densely innervated (Morrison et al., '82; Kosofsky et al., '84). In macaque monkeys the infragranular layers and layer 4 contain a dense innervation at the day of birth, the supragranular layers are only sparsely innervated. As in carnivores, the serotoninergic innervation pattern of primates at these later stages of development is mainly modified by an increase of fiber density (Foote and Morrison, '84). Data about earlier stages of cortical development, when neuronal histogenesis is not yet complete, is only available for the rat (Lidov and Molliver, '82; Wallace and Lauder, '83).These data indicate that the serotoninergic fibers seem to innervate the cortex at considerably later stages than in ferret. At E l 7 when the histogenesis of the rat cortex is at its peak, serotoninergic fibers reach the frontal neocortical pole (Lidov and Molliver, '82; Molliver, '82; Wallace and Lauder, '83).When the last cortical neurons are generated at E21 (Berry et al., '64), the serotoninergic axons have just reached but do not innervate most areas of the telencephalon. Four days later at P3, only the marginal zone and the subplate region of the developing rat cortex contain seroto-

nin positive axons, whereas the cortical plate itself is free of fibers. It is not until 3 weeks postnatally that a significant number of serotoninergic axons can be found in lateral cortex (Lidov and Molliver, '82; Molliver, '82). Whereas the first fibers in the rat were found above and below the cortical plate, the plate itself seems not to be innervated until much later. This is not the case in the ferret where serotoninergic fibers enter the individual cortical layers as soon as they have been formed at the top of the cortical plate, and long before cell proliferation and cell migration has ceased. However, more recent data describe a hyperinnervation of serotoninergic fibers in the rat somatosensory cortex starting around birth (Rhoades et al., '90). Our study does not include stages of cortical development earlier than the generation of layer 4 (at PO). For this reason, it is not known at what age the first serotoninergic fibers enter the ferret cortex. But at PO the innervation of the marginal zone and the subplate region was so dense that the first serotoninergic fibers must have arrived there well before birth. Golgi studies as well as birth-dating studies with radioactive thymidine showed that cells in the marginal zone and the subplate are generated before the rest of the cortical plate (Marin-Padilla, '71, '83; Luskin and Shatz, '85a,b; Jackson et al., '89). At this early stage, these cells form an undifferentiated pseudostratified neuroepithelium that is later split apart by the ingrowing neurons of the cortical plate (Marin-Padilla, '71, '78, '83, '88). It is possible that the serotoninergic fibers enter the primordial cortex at this early stage, forming a continuously innervated layer that is subsequently separated by the ingrowing neurons of the cortical plate. In this way these ingrowing neurons could generate the innervation

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The role of serotonin during the cortex development In the rat somatosensory cortex, serotoninergic s o n s form a transient hyperinnervation within the lower part of the cortical plate that precedes the formation of the somatosensory maps by thalamic fierents (D'Amato et al., '87; Rhoades et al., '90). This finding has led to the proposal that serotonin may exert a trophic influence on the development of thalamocortical pathways or on their cortical target cells (D'Amato et al., '87). In slice cultures of the rat visual cortex, exogenous application of serotonin or serotoninergic agonists can alter glial proliferation, neuronal differentiation, and synaptogenesis (Chubakov et al., '86). Some of these effects induced by serotonin seem to be secondary to the stimulation of glial cells and their subsequent proliferation. Indeed, treatment of primary astroglial cultures with serotonin induces morphological changes in glial cells and causes them to produce substances that regulate the growth and development of serotoninergic neurons in culture (Whitaker-Azmitia and Azmitia, '89; Azmitia et al., '90; Whitaker-Azmitia et al., '90). Our data and those of others (Lauder et al., '82; Lidov and Molliver, '82; Martin et al., '87) indicate that serotonin first appears within the marginal zone and within the subplate region, both structures that are known to contain transient neuronal elements likely to be involved in the organization of the cortical layers (Valverde and FacalValverde, '87, '88; Marin-Padilla, '88; Shatz et al., '88; McConnell et al., '89; Ghosh et al., '90; Kostovic and Rakic, '90). The innervation of the marginal zone is of particular interest, since the leading processes of migrating cells reach this layer when they arrive at the top of the cortical plate. Here they could come into direct contact with serotoninergic axons once they have finished their migration. Although the marginal zone, which in the adult becomes layer 1,is heavily innervated from birth to adulthood (Figs. 6, 8, 9), this is not true for all of the other layers of the cortex. In the adult ferret, the lower cortical layers contain substantially fewer fibers than the upper layers (Voigt and de Lima, '911.From this innervation pattern, one can conclude that the supragranular layers are under a more profound influence of serotonin than the infragranular layers. The situation is different during development. While the cortex is forming, each layer receives a similarly dense innervation by serotonin (Fig. 9). This happens once the cells have arrived at the top of the cortical plate. Thus processes such as the induction of growth factor release, possibly necessary for synapse formation and differentiation, could take place successively in every layer as it is formed. At the moment it is not certain whether serotonin fulfills the same biological functions during development and adult life. If this is the case, processes that occur during early cortical development in all layers would be confined in the adult animal to the upper cortical layers. It is possible that one function of serotonin is its involvement in plastic remodeling of synapses and dendrites. The regional variations in innervation density found in the adult cortex could then be an indication of differences in plastic capability between cortical areas.

ACKNOWLEDGMENTS We thank Christiane Schmidt for skillful technical assistance, Friedrich Bonhoeffer, Christian Miiller, and Eckhard Friauf, for helpful comments on an earlier version of the manuscript, and Daniel St. Johnston for critically reading and improving the English.

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Serotoninergic innervation of the ferret cerebral cortex. II. Postnatal development.

We have investigated the serotoninergic innervation of the ferret cortex from the day of birth to adulthood with immunohistochemical techniques. Due t...
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