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Developmental Changes Revealed by Immunohistochemical Markers in Human Cerebral Cortex The developing human cerebral cortex is distinguished by a particularly wide subplate. a transient zone in which crucial cell-cell interactions occur. To further understand the role of the subplate in human brain development, w e have studied the immunohistochemical expression of certain neuronal (GAP-43, MAP-2, parvalbumin) and astroglial (vimentin, GFAP) markers in the developing visual cortex from gestational ages of 14 weeks to 9 months post-term. At 14-22 weeks, immunoreactivity to GAP-43, a protein involved in axonal outgrowth, was most prominent in the subplate and marginal zone neuropil and in the fibers of the radiations running near the ventricular zone; at 22-42 weeks, GAP-43 immunoreactive fibers were observed in the maturing cortical plate. Immunoreactivity for the microtubule-associated protein MAP-2 was present in the differentiating cortical plate at 14 weeks, but at 22-42 weeks was most prominent in the somata and dendrites of differentiated neurons, particularly the Cajal-Retzius neurons of the marginal zone, in neurons of the subplate and in those forming cortical layer 5. Parvalbumin immunoreactivity did not appear until 26 weeks, when stained neurons were in a sparse band of cells in layer 6 and upper subplate. Vimentin and GFAP did not -stain differentiated neuronal cells. Vimentin immunoreactivity appeared early in neuroepithelial and radial glial cells, decreasing after 35 weeks, with a concomitant increase in GFAP immunoreactivity in radial glial and maturing astrocytic cells. Our results show that despite the greater complexity of the developing human neocortex, molecular markers are expressed in spatial and temporal patterns similar to those observed in non-human primates, carnivores and rodents. These protein markers should prove useful in developmental staging, and in providing a framework in which to examine congenital disorders of cerebral development The mammalian neocortex is a multilaminar structure that develops in an inside-first, outside-last pattern (Caviness et al, 1981; Sidman and Rakic, 1982). Neurons of the innermost cortical layer 6 are born earlier than those of the more superficial layers 2 and 3 (see reviews by McConnell, 1991; Allendoerfer and Shatz, 1994). In addition to the neurons constituting the permanent adult cortical layers 1-6, a transient population of neurons is present in the subplate layer of the developing cerebral wall (Allendoerfer and Shatz, 1994). Studies in experimental mammals (carnivores and primates) have shown that subplate neurons are among the first generated post-mitotic neurons of the telencephalon (Kostovic and Rakic, 1980, 1990; Luskin and Shatz, 1985a,b; Chun and Shatz, 1989a; Kostovic, 1990; McConnell, 1991; Allendoerfer and Shatz, 1994). These subplate neurons differentiate morphologically and function physiologically during fetal stages, prior to the maturation of the definitive neurons of the mature cortex (Friauf etal,\ 990; Friauf and Shatz, 1991). During normal postnatal development the subplate neurons largely disappear (Valverde and Facal-Valverde, 1987; Chun and Shatz, 1989b), although some persist as Lawrence S. Honig1-2-3, Kathrin Herrmann1-4 and CarlaJ. Shatz15 Departments of 'Neurobiology and 2Neurology, Stanford University Medical Center, Stanford, CA 94305, USA Current addresses: 'Department of Neurology (F2-318), University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd, Dallas, TX 75235-9036, ''Laboratory of Neurophysiology, NIMH/NIH Animal Center, POB 608, Poolesville, MD 20837 and 5Howard Hughes Medical Institute & Department of Molecular & Cell Biology (LSA #3200), University of California, Berkeley, CA 94720-3200, USA interstitial neurons of the adult subcortical white matter (Valverde and Facal-Valverde, 1988; Chun and Shatz, 1989b; Kostovic and Rakic, 1990; Meyer etal, 1992). The subplate is a region through which growing axons as well as migrating neurons travel. It is thought to function as an early scaffold involved in the formation of connections between thalamus and cortex (Shatz et al, 1988; McConnell et al., 1989; Ghosh and Shatz, 1992a; Allendoerfer and Shatz, 1994). The subplate layer is more pronounced in width as one ascends the mammalian phylogenetic tree (reviewed in Allendoerfer and Shatz, 1994). For example, it is larger in cats than in rodents (Uylings and van Eden, 1990; Allendoerfer and Shatz, 1994), and in monkeys than in cats (Kostovic and Rakic, 1980,1990), and it is particularly prominent in the developing human brain (Kostovic and Rakic, 1980, 1990). Clinically, this region is susceptible to injury in utero or perinatally. For example, it is affected in both of the two major neonatal brain conditions, periventricular leukomalacia and germinal matrix hemorrhage, which are responsible for significant congenital developmental delay and subsequent mental retardation (Volpe, 1989, 1990). We have undertaken a developmental study of human visual cortex to examine the spatio-temporal pattern of immunostaining for selected markers of neuronal and glial development. To follow neuronal development, antibodies to GAP-43, MAP-2 and parvalbumin were used. GAP-43 is a protein linked to axon outgrowth during both development and regeneration (Skene, 1989; Biffo et al., 1990). With the exception of the earliest developmental stages, at which time it is also present at low levels in neuronal somata, the protein is present at high levels in axons and neuronal growth cones. MAP-2 (microtubuleassociated protein 2) is a microtubule- associated protein found exclusively in neuronal dendrites and somata (Caceres et al, 1984a,b; Sims et al., 1988; Chun and Shatz, 1989a; Crandall, 1989; Schoenfeld et al, 1989). MAP-2 immunoreactivity can be detected in the cortical and subplate neurons of developing mammals and non-human primates. Parvalbumin is a calciumbinding protein associated with certain subpopulations of GABA-ergic cells in the developing mammalian cerebral cortex, including subplate neurons (Antonini and Shatz, 1990; Hendrickson et al, 1991; Van Brederode et al, 1991). In addition, to confirm the specificity of our immunocytochemistry and correlate with the neuronal markers, we examined the staining patterns for two astroglial markers, GFAP and vimentin. Vimentin is an intermediate filament protein of the cytoskeleton known to be present early in development in primitive neuroepithelial cells and radial glia (Sasaki et al, 1988; Bignami and Dahl, 1989; Stagaard and M0llgard, 1989; Sarnat, 1992). GFAP, a different intermediate filament protein, is well recognized as a marker for astroglial cells including mature astrocytes of the central nervous system (Antanitus et al, 1976; Cerebral Cortex Nov/Dec 1996;6:794-806; 1047-32Il/96/$4.00 Levitt and Rakic, 1980; Stagaard and Mellgard, 1989; Sarnat, 1992). The immunostaining patterns of these five antibodies at different ages can provide information about the progression of developmental changes in the neurons and glia of the subplate and cortical plate. We studied this progression in human cerebral cortex at developmental stages ranging from 14 gestational weeks to early postnatal life. The patterns of distribution of these antigen markers observed here in the human cortex are highly similar to those observed in the developing cerebral cortex of other mammals, implying similarities in the underlying developmental events of neuronal maturation and formation of connections. vimentin (clone Vim 3B4; Boehringer-Mannheim; 1:40). These antibodies all cross-reacted with their human counterpart protein antigens, consistent with the strong amino acid sequence conservation exhibited by these proteins. Antibody directed against GFAP was a rabbit anti-human polyclonal antibody (1:250; Dr L. Eng, Stanford University). Following incubation with primary antibody, sections were washed with PBS, incubated with appropriate anti-mouse or anti-rabbit biotinylated secondary antibody, washed and stained using the Vectastain ABC-elite kit (Vector) avidin-biotin linked peroxidase method. Diaminobenzidine (Sigma) was used as chromogen. All slides were dehydrated and mounted using Permount (Fisher Scientific; Pittsburgh, PA). Control sections, using normal mouse serum (1:500) instead of 'primary antibody' or omitting either primary antibody or biotinylated secondary antibody, did not show reaction product, except for some occasional stain in blood cells persisting despite peroxide pre-treatment. Sections near those used for immunohistochemistry were stained histologically by the Nissl method using cresyl violet. Materials and Methods Tissue Hunan fetal brains were obtained at autopsy, or following therapeutic abortions, and immersion-fixed in buffered 10% formalin for 14-21 days at 4°C. Use of autopsy specimens received approval by the Department of Pathology, Stanford University School of Medicine. The Stanford Institutional Review Board for Human Subjects in Medical Research gave Human Subjects Approval. Gestational age (GA) is defined using the clinical convention, as total weeks (including postnatal survival time) from the last menstrual period. When this information was not known, specimens were dated by use of the prior physical measurements obtained upon ultrasound in utero, or those made at post-mortem autopsy. By the convention used here, GA is 2 weeks greater than the actual conceptional (post-ovulatory, or fertilization) embryonic age; normal term is GA 40 weeks (40 weeks). Any brains showing anatomical anomalies, parenchymal hemorrhages or macroscopic autolytic changes were excluded. The average post-mortem interval was 18 h (range 1-36 h). With the exclusion of several brains showing microscopic post-mortem change on hcmatoxylin & eosin staining, there were no effects of post-mortem interval noted. Twenty brains were examined in this study, including several post-term (>40 weeks GA) brains; GA ranged from 14 to 79 weeks GA. The nomenclature of the developing cortical layers is a modification (Allendoerfer and Shatz, 1994) of that of the Boulder Committee (1970). After brain fixation, blocks (-5 mm thick slabs) of left or right occipital cortex were manually cut in the coronal plane. The calcarine fissure was included in these slabs for those stages at which the fissure was evident (after -27 weeks). The tissue blocks were infiltrated for 1-3 days at 4°C in 25% sucrose in 0.1 M phosphate buffer, pH 7.4, and then freeze-embedded in Tissue-Tek® O.C.T. Compound (Mites; Elkhart, IN) on a block of dry ice before storage at -80°C or sectioning. Cryostat sections were cut at 12 urn thickness and mounted onto slides previously made adhesive using a subbing solution of 0.1% chrome alum-1.5% gelatin-30% ethanoL Slides were then stained as below, or in some cases stored desiccated at -20°C prior to staining. Immunohistochemistry Sections were usually pretreated with 3% hydrogen peroxide in Dulbecco's sodium phosphate-buffered saline (PBS), pH 7.4, to attenuate endogenous peroxidase staining associated with blood cells and vessels (Nilaver and Kozlowski, 1989), then washed in PBS. For the antibodies used in this study, this pretreatment did not affect the antibody-specific staining. Prior to treatment with primary antibody, slides were preincubated for 1-3 h in blocking solution consisting of 2% horse serum (Vector, Burlingame, CA), 1% bovine serum albumin (Sigma Chemical Co., St Louis, MO) and 0.1% Triton X-100 (Sigma) in PBS. This step was followed by incubation with primary antibody, diluted in blocking solution, for 12-18 h at 4°C. Antibodies (with their dilutions) included mouse monoclonal antibodies generated against: rat GAP-43 (clone 9-1E12; 1:10 000), originally provided by Drs D. J. Schreyer and J. H. P. Skene (Schreyer and Skene, 1991) and now available from Boehringer-Mannheim (Indianapolis, IN); rat MAP-2 (clone HM-2; Sigma; 1:2000): carp parvalbumin (clone PA-135; Sigma; 1:1000); and bovine Results During development, the human cerebral hemisphere, like that of all mammals, can be subdivided into several embryonic zones. Initially, the neuroepithelium is a single layer, which develops into a trilayered structure consisting of a ventricular zone, a preplate and a marginal zone. The first appearance of the cortical plate occurs at -10 weeks gestational age (Sidman and Rakic, 1982). Thus by 14 weeks, the earliest stage studied here, four zones are present (Fig. IB). From ventricle to outer brain surface these are: the ventricular zone (VZ), which contains progenitor cells undergoing cell division; the intermediate zone (IZ), which can be further subdivided into the radiations (RA) and the subplate (SP); the cell-dense cortical plate (CP), which contains the accumulating postmigratory neurons of the adult cerebral cortex; and the marginal zone (MZ) (Boulder Committee, 1970; Allendoerfer and Shatz, 1994). In humans at -40 weeks GA, the generative ventricular zone has disappeared, leaving a differentiated ependymal layer, whereas the intermediate zone has become the definitive white matter. In experimental mammals, including nonhuman primates, it is known that the subplate and marginal zones contain the first postmitotic neurons of the cerebral cortex. However, by early maturity many of these have been eliminated by cell death. The subplate becomes the mature white matter, which has few neurons, and the marginal zone becomes the molecular layer, which contains very few neurons and is known as cortical layer 1. Distribution of GAP-43 Immunoreactivity GAP-43 immunoreactivity is present in the cerebral cortex throughout the stages examined in this study. At the earliest stage examined (14 weeks), GAP-43 is present through nearly the full width of the cerebral wall (Fig. L4), excepting the ventricular zone (VZ) (compare Fig. L4 and B). The most intense regions of GAP-43 immunoreactivity at the 14 week stage are the marginal zone and intermediate zone. In the intermediate zone, staining is present in dense fiber fascicles running generally parallel to the ventricular zone (Figs \A and 2/4). However, the forming cortical plate at 14 weeks, which spans -30% of the width of the cerebral wall, does show some modest immunoreactivity. This is evident in cell somata and neuropil, with some fine fibers running radially within the cerebral wall, most likely axonal processes of differentiating cortical cells. Thus, at the 14 week stage, with the exception of the minor cell body immunoreactivity found in the cortical plate, anti-GAP-43 antibody is predominantly a fiber stain. At stages 20-25 weeks (Figs ID and 2B), fiber staining in the Cerebral Cortex Nov/Dec 1996, V 6 N 6 795 14w sp IZ *6w 1 2/3 sp 1mm Figure 1 . Visual cortex at gestational ages 14-36 weeks. This figure of nine panels consists of three rows showing sections of brains from different fetal ages and three columns with different stains. The ages are 14 weeks in the top row (4, B, C), 22 weeks in the middle row (0, E. F) and 36 weeks in the bottom row (6, H. I). Immunohistochemical stains are for GAP-43 (4. D. G) and for MAP-2 [C. F. I), and comparable sections are shown with cresyl violet (CV) staining (fl. f, tf). Brain regions are labeled in the central column panels: mz. marginal zone (future cortical layer 1); cp. cortical plate (future cortical layers 2-6); iz, intermediate zone (containing the subplate, sp, a transient zone containing subplate neurons, and the radiations, ra), destined to become the definitive white matter; vz, ventricular zone (early proliferative zone and future ependyma). All panels are at the same magnification; the scale bar shown in G is 1 mm. 796 Immunohistochemtstry in Developing Cortex • Honig et at. W^^ '^ vz vz B Figure 2. GAP-43 immunoreactivity in the intermediate zone. Dense fiber staining is evident in the optic radiations and developing white matter. Sections through the inner half of the cerebral wall of 14 week Ifl) and 22 week (8) brains, and the central portion of a gyrus of a 36 week brain (C) are shown. At 14 weeks ifl) there is a relative absence of staining in the ventricular zone (vz), but marked staining in the optic radiations (ra) present in the intermediate zone, which are shown at higher magnification in the inset (*). At 22 weeks (fl) the intermediate zone is wider and the layered arrangement of the radiations can be appreciated; the staining pattern is similar, with more intense staining in the radiations (ra) than in the subplate (sp) or ventricular zone. At 36 weeks (C) layers 4-6 and the developing white matter (wm) are labeled; dense fiber staining is evident in the white matter. Scale bars are 100 jim. intermediate zone increases in prominence. A dense layer of axon fascicles coursing superficial to the ventricular zone becomes distinct from the overlying subplate; this represents the radiations portion of the intermediate zone (Fig. IE). At 20-30 weeks (Figs ID and 2B) the GAP-43 immunoreactive fascicles of the radiations continue to be prominent. These fascicles presumably contain corticofugal fibers as well as the corticopetal (thalamocortical) fibers of the optic radiations emanating from the lateral geniculate nucleus. At 36 weeks and later, following deep sulcation of the developing cortex with gyrus formation, the GAP-43 immunostained fibers are present in the center of the gyri, anatomically representing the subcortical white matter (Figs lGand2QAt early stages, GAP-43 staining is very low in the cortical plate (Fig. 34, B); no immunoreactivity is visible in cell bodies at 19 weeks. The staining is markedly less than that in the intermediate zone, where there is robust fiber staining (Fig. 5A). However, between 21 and 40 weeks, increasing amounts of GAP-43 immunoreactivity appear in the cortical plate present in fine radial fiber bundles (Fig. 3C, E, G). At 22 weeks these fibers are evident only in the deep regions of the cortical plate (Fig. ID); subsequently, at 26-27 weeks (Fig. 3C-F), immunostaining extends throughout the entire plate, reaching the outermost cortical cellular layers 2/3 (Fig. 3E, F). By 36 weeks (Figs \G and 3G, H) the cortical plate staining is non-uniform, with a laminar accumulation of immunoreactivity. The most intense staining is present in three bands: a top band located in the marginal zone, a middle band corresponding to layer 4 and a bottom band corresponding to upper layer 6/lower layer 5 (Figs Distribution ofMAP-2 Immunoreactivity At 14 weeks gestation, anti-MAP-2 monoclonal antibody stains neuronal cell bodies and processes in the forming cortical plate (Figs \C and 44, B, D). The ventricular zone is essentially unstained (Fig. 4A), while the marginal zone (Fig. 4A, B) and subplate (Fig. 4A, B, D) contain immunostained cell bodies at low density. This distribution of MAP-2 immunoreactivity (Fig. 1O is almost complementary to that of GAP-43 (Fig. L4) staining: at 14 weeks, the densest MAP-2 immunoreactivity is located in a closely packed lower part of the marginal zone and within the neurons of the cortical plate (Figs \C and 4A, B), whereas GAP-43 staining is most intense in the marginal and intermediate zones and lowest in the cortical plate (Fig. 1/4). At 22 weeks, intense MAP-2 staining is detectable in the sparsely distributed neurons of layer 1, known as the Cajal-Retzius neurons (Figs 4B, C, E and 5A), and in maturing cortical layers 5 and 6 (Figs AC and 5E). By 36 weeks, at which time the cortical layers are clearly evident (Caviness et al., 1981; Sidman and Rakic, 1982; Kostovic and Rakic, 1984; Kostovic et al, 1988, 1989), staining is present not only in layer 1 neurons and the large pyramidal cells of layers 5 and 6, but also in the maturing neurons of layers 2-4 (Fig. 5C, D, G, H). This immunostaining is prominent in cell somata and dendrites, especially the apical and parabasal dendrites of the layer 5 pyramidal cells (Fig. 5D, E). Subplate neurons show MAP-2 immunoreactivity throughout Cerebral Cortex Nov/Dcc 1996, V 6 N 6 797 36w Figure 3. GAP-43 immunoreactivity in the developing cortical plate. Photographs of GAP-43-stained sections (left: A C, E, G) are paired with corresponding cresyl violet (CV)-stained sections (right: B. D.F.H). These latter are labeled with abbreviations as in Figure 1, and with the gestational ages: 19 weeks (4, B), 26 weeks (C. 0), 27 weeks (£, F) and 36 weeks (G, H). GAP-43-immunoreactive fibers running perpendicular to the pial surface increase in prominence during development, as described in the text. Magnification is the same in all panels; the scale bar shown in E is 500 urn. the period under study. In the 14 weeks subplate, neuronal cell body and process staining is most evident in the upper subplate, immediately below the cortical plate (Figs \C and 44, Q. At 22-27 weeks MAP-2 somatic staining is most prominent in the subplate cells, as well as the pyramidal neurons of layer 5 (Figs IF and 5A, B, E, F). The population of immunostained bipolar or multipolar subplate neurons appears denser in the upper, compared with the lower, portions of the subplate. By 36 weeks, subplate somata and processes are intensely immunoreactive for MAP-2 (Figs 1/ and 5C, G, H), and can be seen scattered throughout the subplate and developing white matter (Fig. 5G, H). Parvalbumin Immunoreactivity At the earliest ages studied here (14-25 weeks), no parvalbumin 798 Immunohistochcmistry in Developing Cortex • Honigetal. immunostaining could be detected in the cerebral wall. At subsequent stages, from -26 weeks onwards, increasing numbers of neurons staining for parvalbumin are present. These neurons consist of a sparse population located in the upper subplate or deepest part of cortical layer 6 (Fig. 6/1, C, E, F). Comparison with adjacent MAP-2 immunostained (not shown) or Nissl-stained sections (Fig. 6B, D) reveals that these Immunoreactive cells are only a small fraction of the total neuronal population in this region. The number of parvalbumin immunoreactive cells increases during development such that a prominent band is visible at stages 36 weeks (Fig. 6A) and later. However, this broad band has rather low cell density, and consists of cells separated at some lateral distance from each other, located in layer 6 and the upper subplate. The neurons are often spindle shaped at 26-30 weeks. At later stages, the labeled mz. Figure 4. MAP-2 immunoreactivity in the developing cerebral wall. Sections from 14 week (4, B. D) and 22 week (C, E] brains are stained with antibody to MAP-2. For the 14 week brain the entire cerebral wall is shown 14), along with higher magnification views of the cortical (6) and subplate zones [D, magnified view of box \nA). Prominent staining of subplate neurons can be seen (0, arrowheads). The Cajal-fietzius neurons of the marginal layer also stain densely (£, short arrows). These neurons are somewhat obscured at 14 weeks by extensive neuropil staining [B). but they are obvious at 22 weeks (C and E. which is a magnified view of box in C). Staining is also observed in the forming cortical plate. Abbreviations are as in Figure 1. All scale bars are 100 |xm. neurons appear larger, more polygonal and with more processes (Fig. 6E, F). At term and during the postnatal period, these cells are more numerous, with greater abundance of more ramified processes, than present during previous stages (e.g. 26-36 weeks). In the postnatal 79 weeks brain shown in Figure 6C, D, F a greater number of parvalbumin immunoreactive cells are evident than at 36 weeks; while at 79 weeks these cells are still mostly located in layer 6, scattered cells are seen throughout other layers of the cortex, particularly in layers 3, 5 and the white matter. The ramified cell processes stain prominently (Fig. 6F). Vimentin and GFAP Immunoreactivity In agreement with previous studies (Stagaard and Mollgard, 1989; Wilkinson et al, 1990; Sarnat, 1992), vimentin immunoreactivity at the ages studied here is evident in cells that appear morphologically to be neuroepithelial cells and radial glia. At 14 weeks there is some immunoreactivity for vimentin distributed throughout the entire width of the cerebral wall (Fig 7A). The most dense staining is in the cells of the ventricular zone. There is moderately dense staining in the intermediate zone, in both cell bodies and 'radially' oriented cell processes (Fig. 7A). The cortical plate contains only light staining, present in radially oriented fibers. A thin band of moderate staining is present at the boundary between the marginal zone and the cortical plate. At later ages (19-36 weeks) vimentin immunoreactivity persists in cells of the neuroepithelium, and in cell bodies and processes in the intermediate zone that morphologically fit criteria for radial glia (Rakic, 1978; Schmechel and Rakic, 1979; Voigt, 1989). Radial glia have their cell bodies situated in the ventricular and subventricular zones, and long fibers extending from ventricular surface to marginal zone (Fig. IB, C). At 36 weeks the most intense staining for vimentin persists in the periventricular region adjacent to the ventricular zone and in the forming subventricular zone (Fig. 7O- Subsequently, particularly during postnatal development, the vimentin immunoreactivity evident in the brain diminishes. The remaining vimentin staining Cerebral Cortex Nov/Dcc 1996, V 6 N 6 799 - stt~^r*H+»* 90> f>,Sk^%: m wm Figure 5. MAP-2 immunoreactivity in the developing cortical plate. Anti-MAP-2 immunostaining is seen in low magnification views of the cortical plate of 22 week ifi), 27 week (B) and 36 week (C) brains. Higher magnification panels show selected regions from sections of the same brains: 22 weeks layer 5 (£), 27 weeks subplate If). 36 weeks cortical plate layers 5-6 (D), and 36 weeks upper subplate (6) and subplate and forming white matter (H). At 22 weeks staining is most prominent in the Cajal-Retzius cells of the marginal zone IA. arrowheads), the pyramidal cells of layer 5-6 and subplate cells (A, £). While staining of subplate cells and pyramidal layer 5 cells remains strong at subsequent stages (5, C. D. F. G. H), prominent staining is also seen in differentiating neurons of layers 4,3 and 2 (5, C). Abbreviations are as in Figure 1. All scale bars are 100 \un. observed at 79 weeks includes intense staining of the ependyma itself, as well as some cell bodies and tortuous fibers located in the periventricular region (Fig. ID). Staining in blood vessels and capillaries is also pronounced. GFAP immunostaining by our methods is very light at the earlier developmental stages studied here (brains of 14-26 weeks of age); there is some staining of cells in the ventricular and subventricular rones (Fig. &4). Immunostaining increases in the subventricular zone during the period 26-36 weeks, and the cells and processes that are stained are morphologically characteristic of radial glia. However, during this period, GFAP staining by our methods appears less intense and in fewer cells than that observed with antibody to vimentin. By 36 weeks, differentiated ramified astrocytes are evident throughout tfee intermediate zone and cortical plate (Fig. 8B). Postnatally, there is prominent staining of process-bearing astrocytes as well as persistent ventricular zone immunoreactivity (Fig. 8QDiscussion We have studied the development of human cerebral cortex during the second and third trimesters of fetal development, from gestational ages 14-40 weeks and postnatally, by examining the distribution of certain neuronal and astroglial protein markers (summarized in part in Table 1), whose distribution and developmental regulation during development has previously been investigated in lower animals. Compared with lower mammals of similar stages, human cerebral cortex 800 Immunohtstochcmistry in Developing Cortex • Honig et a]. has a much more prominent subplate and a slower, more elaborate pace of development of connections and cortical layers. However, the spatio-temporally patterned sequence of immunostaining for the neuronal and astroglial proteins studied here, while prolonged in time, shows broad similarities in pattern to that of other mammals. In particular, GAP-43 staining was present mostly in axonal fiber tracts, and in locations filled with growth cones and with ingrowing axons (Table 1). MAP-2 stained a number of neuronal cell bodies and dendrites in the developing brain, particularly those of subplate, marginal zone and large cortical projection neurons. Parvalbumin was present in a much more restricted set of upper subplate and lower cortical plate neurons. The astroglial marker proteins vimentin and GFAP were seen in primitive radial glia cell bodies near the ventricular zone and their fibers. Astrocytes in the subplate and cortical plate were evident in later stages, using antibody to GFAP. These observations indicate that the patterns of cortical development as assessed by these markers are highly similar in humans and lower mammals. GAP-43 is a Marker for Axonal Development GAP-43 immunoreactivity was present in the developing human cortex predominantly in the thalamic radiations, and in the neuropil of the marginal zone and subplate. These zones are known from previous anatomical studies in experimental mammals to contain many growing axons (Marin-Padilla, 1971; Molliver etal,l973; Kostovic and Rakic, 1980, 1990; Ghosh and 1 ' • •-.'. — B ' * • ' ' . • ' • . ' • • ' ' sp-: - • • • " L _ v b :• >:. i > : . . ' ^ «: 4 Figure 6. Parvalbumin immunoreactivity in the developing brain. Sections from late fetal and postnatal brains of gestational ages 36 weeks (A, B, E] and 79 weeks (C, D, F) respectively are shown. Low magnification views show parvalbumin immunostained sections (4, C) adjacent to comparable Nissl-stained sections (ft D), labeled with abbreviations as in Figure 1. Parvalbumin-immunoreactive neurons are mostly confined to the subplate and lower cortical plate at 36 weeks {A, B. E), although more numerous than when first observed at 26-27 weeks (not shown). At 79 weeks (C, D, F) parvalbumin-immunoreactive cells are distributed throughout other cortical layers as well, although there remains a higher density of these cells in the deeper layers of cortex and white matter (C, F). The higher magnification pictures show the morphology of the stained subplate neurons at 36 weeks (£) and 79 weeks (f). All scale bars are 100 \m. Shatz, 1992a). This pattern of dominant fiber staining is consistent with the fact that GAP-43 is synthesized in neurons extending processes and axonally transported to growth cones (Skene, 1989). Since the protein does not accumulate in cell bodies but is rapidly transported, there are relatively complementary, or mutually exclusive, patterns of mRNA expression and protein distribution for GAP-43 (Biffo et aL, 1990). The presence of GAP-43 protein or mRNA in the developing brain has been demonstrated biochemically in a number of systems, including cat visual cortex (Mclntosh et aL, 1990) and a 20-22 week human fetal brain cDNA library (Neve et aL, 1987). The anatomic distribution of GAP-43 protein in developing brain has also been established previously using immunohistochemistry in embryonic rodents, including mice (Biffo et aL, 1990) and rats (Dani et aL, 1991). Rodents do not have a very well developed subplate. However, the prominent concentration of GAP-43 in deep fiber tracts seen in our study of developing human brains is consistent with similar patterns seen in the developing mouse and rat (Biffo et aL, 1990; Dani et al., 1991). It is also consistent with a recent report in which GAP-43 was studied in human brain development in several embryos at earlier gestational stages (6-10 weeks GA) than those in this report (Milosevic et aL, 1995). These authors noted no GAP-43 immunoreactivity at 6-8 weeks, but the development of some fiber staining below the forming cortical plate at 10 weeks GA (Milosevic et aL, 1995). The striking intensity of staining in the neuropil of the marginal zone in humans resembles that seen in small mammals. At the earliest age studied, 14 weeks, GAP-43 immunoreactivity was observed in the cortical plate. The presence of immunostaining in the cortical plate at this stage, by analogy with experimental mammals (De Carlos and O'Leary, 1992; Ghosh and Shatz, 1994), likely reflects GAP-43 production by early resident cortical neurons of layers 5 and 6 that are beginning to extend axons towards their targets. At subsequent stages, staining in the cortical plate was minimal. This relative absence of staining in the cortical plate during the major period of neuronal migration has also been noted in rat (Dani et aL, 1991). The gradual pattern of intrusion of GAP-43 immunoCerebral Cortex Nov/Dcc 1996, V 6 N 6 801 I <*•*#*" r- < 14w Figure 7. Changes in the pattern of vimentin immunoreactivity from gestational age 14 weeks to 79 weeks. The entire cerebral wall at 14 weeks (4) shows immunoreactivity for vimentin, including intense immunoreactivity at the ventricular surface (v), and in fibers coursing perpendicular to the ventricular surface (arrowheads). Subsequent stages shown include 19 weeks (S). 36 weeks (C) and 79 weeks (D). Staining for vimentin continues to be present at the ventricular surface, including the differentiated ependyma (e) at 79 weeks, and in fibers coursing from ventricular zone to the base of the cortical plate at 19 and 36 weeks. Blood vessels (b) also show immunoreactrvity. As described in the text, the fibers are likely those of radial glia. All scale bars are 200 UJTI. reactive fibers into the differentiating cortical plate that we have been able to elucidate in the developing human has not been demonstrated clearly in lower mammals, although a transient period of increased GAP-43 reactivity in the cortical plate has indeed been shown to occur during the first postnatal week in the rat (Erzurumlu et aL, 1990; Dani et aL, 1991), a time when thalamocortical axons are known to invade the cortical plate (Catalano et aL, 1991; Agmon et aL, 1993)- This accumulation of GAP-43 in the rat is in layers 4 and 6 (Erzurumlu et aL, 1990; Dani et aL, 1991), as we observed in the human neocortex. Thetiming of the appearance of GAP-43 staining in fine perpendicular fibers in the deep cortical plate at 22-28 weeks correlates well with the proposed period of ingrowth of the waiting fibers of the thalamocortical afferents (Kostovic and Rakic, 1990). The appearance at later ages of increased GAP-43 in the more superficial cortical layers, with particularly dense staining in layer 4, is also consistent with the ingrowth of the thalamocortical axonal projection, which ramifies extensively in this layer (Shatz et aL, 1988). Axon pathway tracing studies of experimental animals have revealed that in the cat and monkey, thalamocortical axons do not immediately invade the cortical plate. Rather, they accumulate in the subplate, beneath the forming cortical plate, 802 Immunohistochcmistry in Developing Cortex • Honigetal. for weeks in the cat (Shatz and Luskin, 1986; Ghosh and Shatz, 1992b) or months in the monkey (Rakic, 1983; Kostovic and Rakic, 1990). The extensive GAP-43 immunostaining within the subplate at ages 14-40 weeks likely reflects in part the accumulation of axons waiting in this zone prior to their invasion of the cortical plate. In addition, the intense immunoreactivity observed in the marginal zone is consistent with the very early generation of marginal zone neurons in all species studied (reviewed in Allendoerfer and Shatz, 1994), and the presence there of an extensive synaptic network (Molliver et aL, 1973)- Taken together, these observations on the appearance and timing of GAP-43 immunoreactivity suggest that the presence of GAP-43 protein is correlated with periods and routes of axon growth within the developing human cortex. MAP-2 Staining Marks Subplate and Other Large Neurons The pattern of MAP-2 immunostaining differs markedly from the fiber labeling of GAP-43- Antibody to MAP-2 stains somata and dendrites of the differentiating subplate and cortical neurons. While anti-MAP-2 stains mature neuronal cells throughout the width of the developing human cortex, at early stages (14 weeks) prior to differentiation of the cortical plate the staining of cell • > r Figure 8. Immunoreactivity for the astroglial marker GFAP in the prenatal and postnatal brain. Immunostaining for GFAP is most intense in the periventricular (v) regions, as can be seen in 26 week (4) and 79 week (C) brains. However, from mid-gestation onwards, prominent staining of cellular elements is also observed in the intermediate zone, as seen in panel B (36 weeks) and in the upper portions of panels/1 and C, as well as even in the subpial region postnatally, at 79 weeks (not shown). The morphology of the stained parenchymal cells is that of astrocytes. All magnifications are the same; the scale bar shown in C is 100 urn. Table 1 Summary of immunohistochemical data for neuronal markers Antibody immunoreactivity MAP-2 GAP-43 14 weeks 19-20 weeks 21-26 weeks 27-32 weeks 33-40 weeks MZ CP SP neuropil neuropS + - neuropi neuropB neuropi -L fibers X fibers neuropH:4,6 || fibers II fibers || fibers II fibers II fibers VZ Parvalbumin MZ CP SP CR CR CR CR CR +++ 5,6 3,5,6 3,5,6 2,3,4,5,6 SP SP SP SP SP VZ MZ CP 6 5,6 2,3,4.5.6 SP VZ uSP uSP uSP For cerebral layers MZ (marginal zone). CP (cortical plate). SP (subplate). and VZ (ventricular zone), as defined in the text, the table entries show the type of staining obtained with antibodies directed towards neuronal markers GAP-43, MAP-2. and parvalbumin. The symbols and abbreviations are: -I- (staining present), - (staining absent), 1 (perpendicular to ventricular wall), 11 (parallel to ventricular wall); CR (Cajal-Retzius cells), and |u]SP ([upper) subplate cells). The numbers in the CP columns refer to the cortical layers. somata is most marked in the subplate neurons and the Cajal-Retzius cells of the marginal zone, the two populations of neurons with earliest-known birthdates (Luskin and Shatz, 1985b; Kostovic and Rakic, 1990). Previous studies in the developing cat brain have similarly shown prominent MAP-2 immunoreactivity in the subplate, where it is exclusively associated with subplate neurons (Chun and Shatz, 1989a). As the cortical plate matures, deeper layers such as layers 5 and 6, and then later in turn layers 4 and especially 3 and 2 (each of which have pyramidal cell populations), stain very darkly. The polymorphic cells of layer 6 and small granular cells of layer 4 stain less intensely. The abundance of MAP-2-positive cells within the subplate, which do not stain with astroglial markers vimentin or GFAP, provides further evidence for their neuronal identity and strengthens the analogy between these neurons in humans and similar neurons described in cats (Shatz et al, 1988) and monkeys (Kostovic and Rakic, 1990). Furthermore, the very prominent early MAP-2 staining of the cell somata and dominant apical dendrites of the pyramidal cells of future layer 5 may provide a useful histochemical landmark for this cortical lamina in humans. Parvalbumin-reactive Neurons Form a Distinctive Population At early human stages, <26 weeks, no cells immunoreactive for parvalbumin were seen in the cerebral cortex. At analogous Cerebral Cortex Nov/Dcc 1996, V 6 N 6 803 early stages in monkeys, parvalbumin immunoreactivity is similarly absent (Hendrickson et al., 1991). Wefindthat from 26 weeks through to term (40 weeks) in humans a layer of initially sparsely distributed, but strongly immunoreactive, cells was seen in layer 6 and the upper part of the subplate. This restriction of parvalbumin-immunoreactive cells in the human third trimester to layer 6 is similar to the limited deep localization of these cells observed in early postnatal rats and gerbils, and in late fetal (E54) and neonatal cats (Stichel et al., 1987; Hogan and Berman, 1994). hi comparison to macaque monkeys, the parvalbumin-positive cells may appear earlier in human development, since in the monkey they have been reported as first appearing at the very end of gestation (Hendrickson et al, 1991), a time-point considerably later than the occurrence of complete formation of the definitive cortical layers. Following the initial appearance of the parvalbuminreactive cells in the deep layers, these cells are subsequently seen increasingly superficially, in layers 2-5, a progressive spread also noted in studies of rodents (Seto-Ohshima et al, 1990), cats (Stichel et al, 1987; Hogan and Berman, 1994) and non-human primates (Hendrickson et al, 1991). Indeed, in the postnatal human specimens, parvalbumin-reactive cells were widely distributed throughout the layers of the cerebral cortex, consistent with the more widespread distribution noted in mature postnatal experimental mammals (Stichel et al, 1987; Hendrickson et al, 1991; Van Brederode etal, 1991; Hogan and Berman, 1994). Parvalbumin-reactive cells are GABA-ergic interneurons (Van Brederode et al, 1990). The developmental appearance and maturation that we observe in humans is in an'inside-out' pattern, like that reported for experimental animals. The timecourse of the marked increase in parvalbuminpositive neurons during human cortical maturation suggests a relationship between their appearance and cortical synaptogenesis, as has previously been suggested for experimental primates (Hendrickson et al., 1991). The period in which the earliest parvalbumin-reactive cells appeared—26 weeks GA—is the same as that in which the earliest synapses within the cortical plate have been observed electron microscopically—25 weeks GA (Molliver et al, 1973). Notes We thank Drs D. J. Schreyer, J. H. P. Skene and L. Eng for antibodies. We acknowledge with thanks the support of NIH grant EY02858 (C.J.S.), the Alzheimer's Association (C.J.S.), Fight for Sight/Prevent Blindness Fellowship (K.H.), Nato Fellowship (K.H.), Dana Fellowship in the Neurosciences (L.S.H.), Walter V. and Idun Berry Fellowship (L.S.H.), and UT President's Research Council Award (L.S.H.). C J.S. is an investigator of the Howard Hughes Medical Institute. Address correspondence to Dr L. S. Honig, Department of Neurology (F2-318), University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd (MC-9036), Dallas, TX 75235-9036, USA. Astroglial Markers Expression in Humans is Like That in Other Mammals Previous studies in lower mammals and in developing humans have shown that vimentin and GFAP are both expressed in the developing brain, but have mostly focused on the ventricular zone. We studied the human brain specimens here for the appearance of these glial markers, with particular attention to the subplate, to confirm the reliability of the immunochemical approach used, to confirm the results of previous studies and to be able to compare the astroglial cell populations with the observed changes in the neuronal markers. Vimentin staining was observed in the cells of the generative neuroepithelium (ventricular zone) and in the cells and fibers of the radial glia. This staining is like that previously reported in rodents (Bignami and Dahl, 1989) and prenatal human brain (Sasaki et al, 1988; Stagaard and M0llg&rd, 1989; Sarnat, 1992). In contrast, using our fixation and staining protocol, we did not observe major GFAP staining until ages >26 weeks. This staining was present in the radial glia, and in maturing astrocytes of the subplate and cortical layers. Our results are consistent with those of previous investigations. Some studies have demonstrated the appearance of GFAP staining in radial glia in the human brain as early as 10 weeks (Antanitus etal, 1976; Stagaard and Mollgard, 1989), like References Agmon A, Yang LT, O'Dowd DK, Jones EG (1993) Organized growth of thalamocortical axons from the deep tier of terminations into layer IV of developing mouse barrel cortex. J Neurosci 13:5365-5382. 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