Altered postnatal development of the visual cortex in trisomy 19 mice

Brain Research Bulletin, Vol. 34, No. 6, pp. 563-570, 1994 Copyright 6 1994 Elsevier Science Ltd Printed in the USA. All rights reserved 0361-9230/94 $6.00 + .OO

0361-9230(94)EOO66-9

Altered Postnatal Development of the Visual Cortex in Trisomy 19 Mice DIETRICH E. LORKE’ AND HARRO ALBRECHT Anatomisches Institut, Abteilungfir Neuroanatomie, Universitiitskrankenhaus Eppendor$ Martinistr. 52, D 20251 Hamburg, Germany Received 23 November 1993; Accepted 21 March 1994 LORKE, D. E. AND H. ALBRBCHT. Alteredpostnatal development of the visual cortex in Trisomy 19 mice. BRAIN RES BULL 34(6) 563-570, 1994.-The development of the visual cortex was studied in 30 Trisomy 19 (Ts19) mice aged 1-16 days postpartum and their euploid littermates. Morphogenesis of the Ts19 visual cortex, though delayed in development, followed the regular sequence observed in control littermates. Early morphogenetic events, such as obliteration of the ventricular lumen, disappearance of the ventricular zone, formation of a visible apical dendrite, as well as disappearance of both migrating neurons and the columnar organization of bipolar preneurons were delayed by 1 day; maturation steps occurring later such as appearance and disappearance of perikaryal basophilia were delayed by 2 days. Myelination of the white matter was similarly retarded by 2 days. The fronto-occipital length of the cerebral hemispheres and the thickness of the visual cortex were decreased by about 20%, consistent with a hypoplasia of the Ts19 neocortex. Unlike in the cerebral cortex of human Ts21, morphometric analysis of the visual cortex of Ts19 mice did not give any indication of a selective deficit in a particular neuron population; the increased cell

density and the reduced nuclear volume observed during early postnatal development are attributable to a maturational delay. The relevance of these results with respect to the mechanisms underlying neuropathological alterations in human Ts21 is discussed. Aneuploidy

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Development

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and the cerebellum, though delayed by 2 days, proceeds in the orderly sequence observed in control littermates (26-28,30,31). The cerebellum of Ts19 mice is markedly hypoplastic (28,30). Cerebral ganglioside concentration is decreased and the developmental expression of the individual ganglioside fractions is retarded by 2-3 days, but not disrupted (29). However, differential reductions in neurotransmitter synthesizing enzymes have been observed in Ts 19 cerebral hemispheres and the diencephaion/brain stem (40). In addition, alterations in specific behaviors have been reported (8). The purpose of the present report was to determine morphological and morphometric development of the Ts 19 cerebral cortex, comparing its histological changes with those observed in DS individuals. In previous detailed studies of the visual cortex of DS individuals, structural abnormalities, decreased cell density, and enlarged nuclear volume have been observed (12,38,49). Therefore, this area has been chosen for the analysis of neocortical development in Ts19.

MENTAL retardation is an invariable consequence of human Trisomy 21 (Ts21), Down syndrome (DS) (34). In addition, DS individuals suffer from numerous other functional deficits of the CNS, such as frequent seizure disorders and abnormalities in neuromuscular tone and visual acuity (13). Morphological alterations associated with these clinical symptoms are reduction in cell number and neuron density in the cerebral cortex accompanied by a selective deficit of small granule cells (12,38,49). Little is known about the mechanisms whereby trisomy leads to these neuropathological changes. An experimental system allowing detailed investigations of the consequences of mammalian aneuploidy is provided by a mouse model originally described by Gropp (17): primary trisomy for any of the 19 murine autosomes can be generated by appropriate breeding of animals carrying Robertsonian (Rb) translocation chromosomes. A general feature of murine trisomy is reduced viability with only Ts19 being compatible with limited postnatal survival (8,16,18,27). Murine Ts19, therefore, permits systematic studies on the effect of an extra chromosome upon neurogenetic processes that mainly occur postnatally, such as the morphogenesis of the cerebral cortex. Apart from a decreased viability (survival beyond the second postnatal week is extremely rare), Ts19 mice among the progeny of NMRI-outbred mice have a reduced body weight and delayed development without gross malformations (4,7,27). Studies of CNS development have failed to detect structural abnormalities; morphogenesis of the retina, the optic nerve, the locus coeruleus,

’ To whom requests

Trisomy

METHOD

Animals

Ts19 mice were obtained according to the breeding scheme described by Gropp (17): Laboratory strain Han:NMRI females with a normal set of 40 acrocentric chromosomes were mated with males doubly heterozygous for metacentric Rb translocation

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chromosomes Rb(9.19)1638 and Rb(819)lCt (generous gift of PD Dr. Heinz Winking, Medizinische Universitl zu Ltibeck). Each of these metacentric chromosomes contains one chromosome 19. The birth date of the pups, denoted as postnatal day 0 (PO), was determined by two inspections of the pregnant females per day. All litters were reduced to three to four animals at birth to provide adequate nutrition and maternal care for the Tsl9 pups. Tsl9 mice were distinguished from the controls by their markedly reduced body weight and retarded development. On each day between PO and P16, at least one Ts19 mouse and its chromosomally balanced control littermate were prepared for examination. From PO to PS, two to three Ts 19 mice were collected each day for morphological studies, making the total number of animals collected 60.

contours were traced with the Videoplan cursor within fields of 300 cm’, corresponding to 9500 pm2 in the original section, to determine nuclear diameters (magnification: x 1800). Two to seven such nonoverlapping fields were evaluated in the middle of each zone, so that at least iO0 nuclei were measured per zone. Cell density was determined by dividing the total number of nuclei counted by the volume evaluated. The crude nuclear counts were then corrected for double counting according to Konigsmark’s formula (21). Wilcoxon’s signed rank test for matched pairs was used to determine statistical significance. The periods from PO to P8 and from P9 to PI6 were evaluated separately to distinguish between the phase of rapid cortical expansion and the subsequent phase of more gradual growth.

Histology

Chromosome

The animals were weighed and anesthesized deeply with an intraperitoneal injection of pentobarbital (100 mg/kg body weight). For chromosome analysis, the abdomen was opened and the spleen was removed in animals aged l-9 days; after PlO, the femur was collected. After incision of the jugular veins, a fixative consisting of 6% glutaraldehyde in 0.05 M phosphate buffer was perfused through the left ventricle of the heart for 15 min (pH 7.3; 760 mosM; pressure: 100 mmHg). Following perfusion, the whole animals were left in the same fixative for 12 h (4°C) before the skull was opened. The brain was then removed carefully and the fronto-occipital and the medio-lateral distances were measured using vernier callipers. The fronto-~cipital length was determined as the distance between the most anterior and the most posterior points of the right hemisphere (excluding the olfactory bulb) on a line parallel to the longitudinal fissure. To calculate the medio-lateral width, a line pe~ndicul~ to the longitudinal fissure extending from the midsagittal line to the most lateral point was used. Afterwards, the occipital pole of the telencephalon was dissected free of the rest of the neocortex at the posterior edge of the corpus callosum, a level corresponding to sections 340-350 in Sidman et al. (44). This slab of the visual cortex was kept in fresh fixative for another 24 h, then rinsed in a mixture of 0.1 M phosphate buffer and 0.1 M saccharose, postfixed with buffered osmium tetroxide (1% for 2 h), dehydrated in an ascending series of ethanols, cleared in propylene oxide, and embedded in Epon 812. Semithin sections (1 pm) of the occipital pole of the telencephalon were cut in the coronal plane, stained with toluidine bluelpyronine red, and used for quantitative analysis. Care was taken to section the brain as perpendicular to the cortical surface as possible. For morphometric analysis, semithin sections were projected by a Zeiss light microscope and a Panasonic camera (Osaka) into a MOP-Videoplan (Kontron, Mtinchen) semiautomatic image analyzer. One Ts19 mouse and its control littermate (aged PO-P16) were selected for study. To estimate the inte~ndi~dual variations, three Ts19 pups and their corresponding littermates were examined on P2, P4, and P5. The region evaluated was located above the dentate gyms, corresponding to area 17/l 8a and b (11). To determine the thickness of the visual cortex, the distance from the pial surface to the border between layer VIb and the white matter was traced manually with the cursor on each section (magnification: x450-900). At younger ages (PO-P3), only layer I, cortical plate, and the subplate were measured; the intermediate zone was not included in the measu~ments. For the measurement of nuclear diameters and cell densities, the visual cortex was subdivided into three zones: layers II-IV, which at earlier stages could not be delimited from each other with sufficient precision, layer V, and layer VIa,b. All nuclear

Chromosome spreads were obtained from minced spleen (PI P9) or femoral bone marrow (PlO-P16) of all mice examined. The tissues were incubated in a culture medium containing 0.2 fig/ml Colcemid (37”C, 90 min), treated with hypotonic 0.075 M KC1 and fixed in methanol-acetic acid (3:l). After centrifugation, the pellet was resuspended in acetic acid (60%) and drops of the suspended cells were dripped on alcohol-swab~d slides, air dried, and stained with Giemsa before evaluation at a magnification of about Xl000 [for details see (27)]. Chromosome spreads from Ts19 mice contained two metacentric chromosomes and a total of 41 chromosome arms, while those of control littermates had only 1 Rb metacent~c and 40 arms.

Analysis

RESULTS Morphogenesis

At birth (PO), the visual cortex of Ts19 mice displayed an immature, but laminated appearance composed of five layers (Fig. la). The ventricular zone consisted of two to three rows of proliferating cells with scarce cytoplasm and oval nuclei oriented ~~ndicul~ to the surface of the lateral ventricle, which at this stage of development still had a visible lumen. Numerous mitotic figures were observed close to the ventricular cavity. The adjacent subventricular zone lacked radial cell arrangement and contained smaller, more darkly stained, irregularly oval or rounded nuclei. Subven~cular cells were relatively densely packed and some of them were undergoing cell division, Next to them, in the much broader intermediate zone, cells were morphologically indistinct from subventricular cells, but were packed less densely and were occasionally oriented ho~zontally. Next to the intermediate zone lay neurons of the subplate and the adjacent preneurons of the cortical plate. This zone could be divided into three regions. The subplate and the inner part of the cortical plate contained rather differentiated preneurons with relatively large pale, round to oval nuclei, and a wide intemuclear space. Between these cells, migrating neuroblasts with dark elongated, spindle-shaped nuclei oriented radially were found. Often, these migrating cells had a leading process that stained intensely. In the middle of the cortical plate, the preneurons were characterized by lightly stained polygonal nuclei and sparse cytoplasm. Two types of cells were found in the densely packed outer part of the cortical plate: the most common cell type with medium sized oval bipolar nuclei was tightly packed together in vertical columns. Only occasionally was the second cell type found; it was distinguished by its smaller, darkly stained, elongated nucleus reminiscent of that of the migrating neurons observed previously. Underneath the pial surface lay the cell-scarce molecular layer (layer I).

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By P2, the ventricular zone had disappeared in Ts19 mice. Fibre number in the intermediate zone had increased. In the inner part of the cortical plate, only occasional neurons had a basophilic apical dendrite, and numerous presumably migrating neurons with dark, spindle-shaped nuclei oriented vertically were found in the middle and upper parts of the cortical plate. With P2 control mice, the cortical plate was considerably thicker. Particularly in its inner part, the internuclear space had increased further. Migrating cells were found mainly in the upper part of the cortical plate. Almost all neurons exhibited a noticeable cytoplasmic cone at their apical pole, indicating the outgrowth of the primary dendrite. On P4, the differentiation of the visual cortex of Ts19 mice was similar to that of P2 control mice: the majority of the cortical plate neurons had an apical cytoplasmic cone. Numerous migrating cells with darkly stained elongated nuclei were still found, predominantly in the outer part of the cortical plate (Fig. 3a). Here, the bipolar oval nuclei were still arranged in vertical columns. Control mice of the same age had virtually no dark elongated nuclei of migrating neurons (Fig. 3b). In the outer part of the cortical plate, cell size had increased, and the nuclei had become larger and roundish in shape with markedly paler nucle-

FIG. 1. Semithin sections of the visual cortex of a newborn (a) Ts19 mouse and (b) its control littermate. Ventricular (v), subventricular (s) and intermediate (i) zones, cortical plate (c) and molecular layer (m) can be distinguished. Whereas a lumen of the lateral ventricle (asterisk) is still visible in the Ts19 mouse, it is already obliterated in the control animal. Note the difference in thickness of the cortical plate. X 180. Insert: Magnified view of the inner part of the visual cortex: In the Ts19 mouse, numerous mitotic figures are still present (arrowheads). Cells in the intermediate layer (arrow) lack a preferential orientation, whereas they are already arranged parallel to the cortical surface in the control mouse. X250.

The same pattern of cell layers was observed in the visual cortex of newborn control littermates as well (Fig. lb). However,

in euploid mice, the lumen of the ventricle below the visual cortex was already obliterated on PO. The ventricular zone consisted of only a few polygonal cells that lacked an orientation perpendicular to the ventricular surface. Only rare mitotic figures were observed adjacent to the subventricular zone consisting of several rows of cells. The intermediate zone contained numerous fibres surrounding spindle-shaped nuclei, which were predominantly oriented parallel to the ventricular surface. The cortical plate was considerably thicker than that of Ts19 littermates, and the preneurons of its inner aspect were not packed as densely as those in Ts19 mice. On Pl, no ventricular lumen was visible any longer beneath the visual cortex of Ts19 mice (Fig. 2a). The ventricular zone consisted of only one cell row with an occasional mitotic figure. The cells of the intermediate zone had begun to assume a parallel arrangement. In control littermates (Fig. 2b), the ventricular zone had disappeared completely so that the visual cortex and the hippocampal formation were separated from each other by only a thin subventricular zone. In contrast to trisomic mice, an apical dendrite appearing as a basophilic process directed to the cortical surface had already become visible in some neurons of the inner part of the cortical plate.

FIG. 2. Internal part of the visual cortex on Pl. A ventricular

layer and occasional periventricular mitotic figures (arrow) are still found in the (a) Ts19 mouse, while they have completely disappeared in the (b) control littermate. Basophilic dendrites (arrowhead), which are recognizable at the apical cone of several control neurons, are not yet observed in Ts19. x280.

LORKE AND ALBRECHT

The cortical dimensions of Ts 19 mice were markedly reduced as compared to control littermates. The fronto-occipital length (Fig. 5a) and the medio-lateral width of the cerebral hemispheres increased with age in both trisomic and euploid mice; both measurements were reduced significantly in Tsl9 mice (p I 0.001). The thickness of the visual cortex, which increased rapidly until PlO in both genotypes and increased more gradually thereafter (Fig. 5b), was reduced by 15-20% in Ts19 mice between PO and P16 (p 5 0.001). In both Tsl9 and control visual cortices, the nuclear diameter increased between PO and PlO in layers II-IV (Fig. 5c) and until P6 in layers V and VIa,b. Nuclear diameter was smaller in all layers of the Ts19 cortex (p 5 0.05) during the growth period (PO-P@, but did not differ thereafter. Numerical cell packing density in layers II-IV changed with deveIopment in both Ts19 and control mice (Fig. 6a). While packing density decreased from 160 to 20 between PO and P16 in Tsl9 mice, it decreased from 130 to 20 during the same period in control mice and was greater in the Ts19 visual cortex both during the period of rapid decline (PO-P& p 5 0.01) and thereafter (Pg-Pl6; p =YZ 0.01). The decrease in cell density during development was less dramatic in layer V (Fig. 6b); here, the cells were also packed more densely in Tsl9 mice both from PO to P8 (p ‘= 0.001) and from P9 to P16 (p c 0.01). In both genotypes, cell density decreased with age in layer VIa,b (Fig. 6~). It was higher in Ts19 mice between PO and P8 (p 5 O.Ol), but no different thereafter. DISCUSSION

FIG. 3. Outer layers of the visual cortex of a Lt-day-old (a) Tsl9 mouse and fb) its control littermate. Dark spindle-shaped migrating neurons (arrowheads) are still observed in the Ts19 mouse, and the bipolar nuclei of superficial conical neurons are arranged in vertical columns (arrows). In the conml mouse, neurons have a larger, paler nucleoplasm and already are more widely dispersed; migrating neurons are no longer observed. X280.

oplasm. The typical columnar organization was no longer visible. A comparable morphology of the visual cortex was not seen in Ts19 mice until P5. With P6 Ts19 mice, basophilic staining of the cortical neurons was still confined to the apical pole. In contrast, the entire circumference of the nuclei was intensely basophilic in cortical neurons of control littermates. This accumulation of basophilic material in the p&nuclear and basal cytoplasm was observed at only P8 in Ts19 mice. It persisted in the visual cortex of both genotypes for approximately 1 week. The basophilia started to decrease in control littermates at P14 (Fig. 4a and b), but was still visible in cortical neurons of Tsl9 mice at P16. At the end of the period examined, the laminar ~angement of the visual cortex of both Ts19 and control mice was comparable (Fig. 4a and b): beneath the cell-scarce molecular layer (layer I), neurons in layers II to IV were densely packed, and large pyramidal cells lay in layer V. The polymorphous neurons of layer Via and b lay close to the white matter. Thus, layer patterns of the visual cortex were not severely disrupted in Ts19. Myelinated axons were first observed in the white matter of control mice at P13, but appeared in Ts19 mice only at P15. Therefore, histogenesis of the visual cortex and its underlying white matter was delayed by I-2 days in Ts19 mice.

In this study we found that the laminar arrangement of the visual cortex is not disturbed in Tsl9. The sequence of neocortical histogenesis is the same as in control littermates, but is delayed in Tsl9 mice. It is difficult to evaluate mo~hogenetic events, such as cell division, migration, and differentiation, in the visual cortex because neocortical histogenesis is distinguished from the development of most other brain regions by the formation of two additional structures: the subventricular zone and the cortical plate. Layers of the visual cortex are not composed of largely homogeneous neuron populations existing at the same stage of development; rather, at the same time most layers contain migrating, resting, and differentiating preneurons. Neurons of the visual cortex are generated in the ventricular zone, a germinal matrix that disappears in control mice after PO and in Ts 19 mice after P 1. The secondary proliferative zone, the subventricular zone, is characteristic of mammalian neocortical histogenesis and forms around El4 in mice (47). It contains both locally proliferating cells and young neurons sojourning there before migrating to the cortical plate (2). The subventricular zone gradually disappears during development in the occipital region, but a vestige persists rostrally (46). Although some of the proliferating cells of the subventricular zone may generate neurons (42) the majority gives rise to astrocytes and oligodendrocytes (1,35,42,46). During development, me subventricular zone is progressively incorporated into the overlying intermediate zone (37), which is later transformed into the white matter by the ingrowth of corticopetal and corticofugal fibres. Because the cells in both layers are morphologically indistinct and only cell densities differ between them (36,37), it is not possible to assess the exact date of disappearance of the subventricular zone by morphological methods. Neocortical neurons migrate in two waves: ~stmitotic neurons of the first wave migrate from the ventricular zone towards the surface where they meet with ingrowing corticopetal fibres to form the preplate (22), or primordial plexiform layer (32).

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FIG. 4. On P14, the layering pattern of the visual cortex is similar in (a) Ts19 and (b) control mice. While the cytoplasm of Ts19 cortical neurons is still markedly basophilic, dark cytoplasmic staining has already decreased in the control littemate. In addition. cortical thickness is considerably reduced in Ts19. x 150.

These earliest generated neurons are destined to become cells of the superficial molecular layer (layer I) and the deep subplate (layer VIb) (5,32,37,48,) and to differentiate quickly. Generation and migration of these first wave neurons have been completed in the mouse by birth and, therefore, have not been analyzed in this study. Given the morphology of the visual cortex that we observe in Ts19 mice, it is, however, unlikely that the formation of these layers is disrupted. From El4 on, a second wave of later generated neurons migrates within the preplate to split it into layer I and the deep subplate (layer VIb) (5,37,47,48). These younger neurons do not initiate differentiation immediately; rather, they enter a considerable period of stasis and form a zone of densely packed cell columns, the cortical plate (37). Subsequently, the cortical plate increases progressively in thickness due to the addition of new postmitotic cells which migrate through both the intermediate zone and the inner part of the cortical plate in such a manner that progressively younger cells form progressively superficial layers (3,6,20,37,43). Thus, generation of the cortical plate follows an inside-out gradient (3,20,37,43). Migrating cells are distinguished morphologically by a dark spindle-shaped nucleus and an intensely stained leading process (6,9,15,43). They are found in the intermediate zone and in all parts of the cortical plate in

both newborn Ts19 mice and control littermates. After P4, the number of migrating cells decreases markedly in control mice, but this decline is delayed until P5 in Ts19 mice. Occasionally, darkly stained spindle-shaped nuclei are visible for a few more days, perhaps as a result of the considerable variation in the duration of neuronal migration (20,43) and the continuous migration of morphologically indistinguishable glial cells until at least PlO (2,10,20,36). The marked decline in the number of dark spindle-shaped nuclei in the outer part of the cortical plate of control mice on P4 indicates that migration has been completed for the majority of the neurons. This decline is observed in Ts19 mice on P5, suggesting that Ts 19 neocortical neurons arrive 1 day later at their destination. Thus, delay in neuronal migration accompanies other delays in Ts19 mice. The duration of neuronal migration cannot be assessed by these morphological studies, because data on birth dates of the cells occupying the Ts19 cortical plate are not available. After finishing their migration to the surface of the cortical plate, the preneurons assume a bipolar shape, arrange themselves in vertical columns and remain unchanged for a considerable time before their differentiation begins (37). With the onset of differentiation, the columnar organization is lost, the nuclei become pale and round, and the cytoplasm expands in volume

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usefulness of ganghosides as biochemicat markers for brain maturation (39). Thus, the delayed decline in GQIb in the Ts19 telencephalon (29) represents a good indicator that neuronal migration and initial fibre outgrowth are delayed in the Tsl9 mouse. GM1 is a major component of myelin. Disparity in its increase in Ts19 and control mice (29) corresponds well with the delay in myelinat~on observed in this study. Although the mo~hology of the Ts19 visual cortex is not abnormal, morphometric parameters are altered. Measurements of both the fronto-occipital and the medio-lateral extensions of the Ts19 forebrain are reduced significantly and the reduction exceeds that expected from developmental retardation. The diminished cerebral size we observed corresponds well with the 2545% reduction in dry weight observed previously in the telencephalon of Tsl9 mice during pre- (40) and postnatal (29) development. In addition, the thickness of the visual cortex remains reduced in Ts19 animals, even at later postnatal stages when its growth is minimal. All these measurements indicate that a considerable hypoplasia develops in the Ts19 telencephalon. Because cell densities decrease with postnatal age in all Iayers of the visual cortex, their elevation in postnatal Ts19 mice is most likely attributable to the retarded development. Prenatally, celt density appears to be reduced in the Ts19 cerebral cortex (40),

NUCLEAR DIAMETER

FIG. 5. Quantitative analysis of the Ts.19 and control telencephalon. (a) Fronto-occipital length of the cerebral hemispheres and (b) thickness of the visual cortex are reduced significantly in Ts19 (y s 0.001). The (c) nuclear diameter in Iayers II-IV of the visual cortex is only decreased between PO and P8 (p 5 0.01). Values represent single measurements and the lines connect the medians.

layerr V -

100

(20,37,43). During this increase In perikaryal size, free ribosomes and rough endoplasmic reticulum first accumulate in the outgrowing primary dendrite which, thus, becomes intensely basophihc (IO, 15,19). subsequently, further differen~at~on of the neurons is refTec&edat the hght microscopical level by an intense perinuclear basophilia (IO). Only towards the end of neuronal maturation, when formation of dendrites and axons is essentially completed, does the amount of perikaryal ribosomes decrease, resulting in a decline in basophilic staining (10). The morphogenesis of the visual cortex in these mice corresponds well to that reported previously in rats (10,19) and other mice (33). Delay is observed at each step of maturation in Tst9 mice, whether the early developmental steps such as formation of a basophilic apical dendrite and disappearance of the columnar organization of bipolar preneurons in the superficial cortical plate (1 day lag) or morphogenetic events occurring later such as the appearance and disappearance of perikaryal basophilia and the onset of mye~inat~on (2 day lag). These results indicate a delay similar to that observed previously in developmental profiles of cerebral ganglioside concentrations in Ts19 and control mice (29) and further confirm the

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FIG. 6. Numerical celi densities in (a) layers II-IV, (b) Iayer V and (cc) layer VIa,b of the T’s19 and control visual cortex. Depicted are single measurements, the lines connect the medians.

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which may represent the basis for developmental alterations observed postnatally. The decrease in the specific activity of choline acetyltransferase (ChAT) described previously in the telencephalon of fetal Ts19 mice (40) is not paralleled by specific histological changes of the Ts19 cerebral cortex. This may not be surprising because over 70% of the telenceph~ic ChAT is synthesized by neurons located in the septum and the basal forebrain complex (23). Delayed development and hypoplasia occur in other parts of the Ts19 CNS, such as the retina (31). the optic nerve (27), the cerebellum (28,30), and the locus coeruleus (26). Although development is delayed in all these brain regions by l-2 days, the degree of hypoplasia varies, the cerebellum being the most markedly affected (2830) and the retina and the locus coeruleus the least (26,31). Morphometric alterations characterize the brain of individuals with human trisomy 21, or DS. Brain weight is reduced markedly (49) and the fronto-occipital diameter diminished (49). Cortical thickness of areas 10 and 28 of DS individuals older than 11 years is significantly smaller (49). However, cortical thickness in other areas, including area 17, is not signific~tly different (12,49). The reduction in numerical density of neurons observed in several cortical areas (3849) is associated with a selective depletion of small granule cells, particularly in area 17 (38). Moreover, in DS individuals, nuclear volume is enlarged by 50% in the visual cortex (12). When seeking to define the mechanisms underlying these changes, it is important to distinguish whether these alterations are the nonspecific result of excess genetic material disrupting overall histogenesis or whether these changes are the consequence of dosage effects of specific genes. To an-

swer this question, the comparison of alterations observed in the brain of human Ts21 with both those found in murine Ts16, involving a chromosome with some genetic homology to human chromosome 21, and those found in murine Ts 19 is of particular interest. Abnormal features shared by all these trisomies are most probably the result of general effects, whereas alterations that are only found in human Ts21 and murine Ts16, but not in murine Ts19, are most likely attributable to specific gene dosage effects. Delayed neocortical development and a decrease in weight, in fronto-occipital diameter and in cortical thickness occur in the brains of both human Ts21 (41,49) and murine Ts16 (45). Their occurrence in tbe Ts19 telencephalon, thus, most likely represents the nonspecific results of gene imbalance and is similar to the comparable alterations observed after postnatal undemourishment (14,25). Both Ts19 and postnatal undernourishment (25) result in increased cortical cell density, attributable to a maturational delay, and have no effect on nuclear volume. In contrast, cell density is lower and nuclear volume elevated in the DS cerebral cortex, due in large part to a selective loss of small granule cells. The present study, therefore, indicates that these mo~home~cal changes are the result of dosage effects caused by the overexpression of specific genes located on human chromosome 21, due to their triplication in DS. ACKNOWLEDGEMENTS The authors are grateful for the valuable technical assistance given by E. Bohm, S. Feldbaus, I. Schade, and K. Siebert. We also wish to thank PD Dr. H. Winking for kindly providing the Rb(9.19)163W

Rb(8.19)lCt translocation males.

REFERENCES 1. Altman, J. ~oliferation and migration of undifferentiat~ precursor cells in the rat during postnatal gliogenesis. Exp. Neurol. l&263278; 1966. 2. Altman, J.; Bayer, S. A. Horizontal compartmentation in the germinal matrices and intermediate zone of the embryonic rat cerebral cortex. Exp. Neurol. 107:36-47; 1990. 3. Angevine, J. B.; Sidman, R. L. Autoradiographic study of cell migration during histogenesis of cerebral cortex in the mouse. Nature 192:766-768; 1961. 4. Bacchus, C.; Sterz, H.; Buselmaier, W.; Sahai, S.; Winking, H. Genesis and systematization of cardiovascular anomalies and analysis of skeletal malformations in murine trisomy 16 and 19. Two animal models for human trisomies. Hum. Genet. 77: 12-22; 1987. Bayer, S. A.; Altman, J. Development of layer I and the subplate in the rat neocortex. Exp. Neurol. 107:48-62; 1990. Berry, M.; Rogers, A. W. The migration of neuroblasts in the developing cerebral cortex. 3. Anat. 99:691-709, 1965. Bersu, E. T. Morphologic development of the fetal trisomy 19 mouse. Teratology 29:117-129; 1984. Buselmaier, W.; Bacchus, C.; Grohe, G.; Winking, H. Behavioural and developmental abnormalities in mouse trisomy 19: An animal model of mental retardation induced by chromosome imbalance. Teratology 37:167-174; 1988. 9. Butler, A. B.; Caley, D. W. An ultras~cmr~ and ra~~utographic study of the migrating neuroblast in hamster neocortex. Brain Res. 44:83-97; 1972. 10. Caley, W. D.; Maxwell, D. S. An electron microscopic study of neurons during postnatal development of the rat cerebral cortex. J. Comp. Neurol. 133:17-44; 1968. 11. Caviness, V. S. Architectonic map of neocortex of the normal mouse. J. Comp. Neural. 164:247-264; 1975. 12. Colon, E. J. The structure of the cerebral cortex in Down’s syndrome-A quantitative analysis. Neurop&liatrie 3:362-376; 1972. 13. Coyle, J. T. ; Oster-Granite, M. L.; Reeves, R.; Hohmann, C.; Co&, P.; Gearhart, J. Down syndrome and trisomy 16 mouse: Impact of

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