EXPERIMENTAL NEUROLOGY 109, 35-56 (1990)

Boundaries during Normal and Abnormal Brain Development: In Vivo and in Vitro Studies of Glia and Glycoconjugates D. A. STEINDLER, *'1 T. F. O'BRIEN,*'~ E. LAYWELL,* K. HARRINGTON,* A. FAISSNER,$ AND M. SCHACHNER$'§

Departments of *Anatomy and Neurobiology and tPathology, University of Tennessee, Memphis, Tennessee 38163; SDepartment of Neurobiology, University of Heidelberg, Heidelberg, Germany; and §Swiss Federal Institute of Technology, Zurich, Switzerland

biology. The idea of specific cellular interactions mediated by unique macromolecules has stimulated our thinking as to how functional cytoarchitectonics and specific neuronal connections arise during embryonic and postnatal development of the central nervous system (CNS) (13, 35, 43, 63). In a classic review of developmental neurobiology published in Scientific American in 1979 (13), Cowan elegantly proposed numerous genetic, cellular, and molecular mechanisms involved in the construction of the nervous system. This review included discussions on (i) the work by Rakic and Sidman (65, 69) on glial-neuronal interactions during cortical histogenesis; (ii) functional sorting of neurons based on selective adhesiveness that " . . . seems to be a general property of all living cells and is probably due to the appearance on their surfaces of specific classes of large molecules that serve both to 'recognize' cells of the same kind and to bind the cells t o g e t h e r . . . " (13); (iii) the use of the "whisker-barrel" system of rodents, ingenuously explored by Woolsey, Van der Loos, and others (36, 82, 85, 89, 90) as a model for studying functional localization and pattern formation in the mammalian CNS; and (iv) specific genetic mutations affecting nervous system development that can provide insights into normal CNS morphogenetic mechanisms (4, 5). The present paper touches upon some of these recurring themes of genetic, cellular, and molecular mechanisms at work during development of the mouse CNS. Findings on particular cells and putative "recognition" molecules which may be involved in the development of functional cytoarchitectonic units, e.g., the "barrel system," are discussed. Further, genetic and experimentally induced perturbations of developmental processes are exploited to distinguish intrinsic from extrinsic factors involved in CNS pattern formation. Our focus is on the existence of boundaries that appear during development of the CNS and may play an important role in segregating different neuronal assemblies and their projections. Glia and glycoconjugates have been proposed to play important roles during development of functional patterns in the CNS (78). Classic studies by Rakic (65, 66) have shown that radial glial cells provide a blueprint for neuronal migration and that postmitotic neurons use these cells as guideposts while migrating great distances

T h i s p a p e r f o c u s e s on t r a n s i e n t b o u n d a r i e s of glia and g l y c o c o n j u g a t e s d u r i n g d e v e l o p m e n t of the m o u s e central n e r v o u s s y s t e m (CNS). L e c t i n - b o u n d g l y c o c o n j u gates, glial fibrillary acidic p r o t e i n , and the J 1 / t e n a s c i n glycoprotein are distributed coextensively within boundaries around developing substructural arrangem e n t s (e.g., d e v e l o p i n g n u c l e i , and at a finer l e v e l , som a t o s e n s o r y c o r t i c a l " b a r r e l s " r e l a t e d to i n d i v i d u a l facial v i b r i s s a e ) t h r o u g h o u t the C N S d u r i n g p a t t e r n f o r m a t i o n e v e n t s . E l e c t r o n m i c r o s c o p y has s h o w n that the J 1 / t e n a s c i n g l y c o p r o t e i n , for e x a m p l e , is p r e s e n t in i m m a t u r e a s t r o c y t e s , on glial and n e u r o n a l p l a s m a m e m b r a n e s , and w i t h i n the p e r i c e l l u l a r s p a c e that could b e e x t r a c e l l u l a r m a t r i x (ECM). T h e findings pres e n t e d on the e x p r e s s i o n o f this w e l l - c h a r a c t e r i z e d ECM m o l e c u l e s u g g e s t that p r e v i o u s l y d e s c r i b e d glial and g l y c o c o n j u g a t e b o u n d a r i e s r e p o r t e d b y our group a r e in part c o m p o s e d of specific r e c o g n i t i o n m o l e c u l e s . The J1/tenascin glycoprotein, a chondroitin sulfatec o n t a i n i n g a n t i g e n t e r m e d t h e 4 7 3 p r o t e o g l y c a n , and the a d h e s i o n m o l e c u l e on glia a r e e x p r e s s e d w i t h i n disc r e t e b o u n d a r y r e g i o n s and a s s o c i a t e d a x o n a l pathw a y s . T h e r e , t h e y m a y s c u l p t u r e fine a s p e c t s of funct i o n a l c y t o a r c h i t e c t o n i c a r r a n g e m e n t s and help g u i d e a x o n s to specific t a r g e t s . T h e e x p r e s s i o n and d e v e l o p m e n t a l r e g u l a t i o n o f g l y c o p r o t e i n s such as J 1 / t e n a s c i n m a y thus be i n t e g r a l e v e n t s d u r i n g p a t t e r n f o r m a t i o n and s y n a p t o g e n e s i s in the C N S . T h e p r e s e n c e o f abnorm a l glial a r r a n g e m e n t s and g l y c o c o n j u g a t e b o u n d a r i e s in t h e c o r t i c e s of the g e n e t i c m u t a n t m o u s e r e e l e r , and findings on p l a s t i c i t y o f b o u n d a r i e s f o l l o w i n g v a r i o u s p e r t u r b a t i o n s , s u g g e s t that b o u n d a r y e x p r e s s i o n is cont r o l l e d b y both g e n e t i c and e p i g e n e t i c factors. S o m e fut u r e d i r e c t i o n s for s t u d y i n g d e v e l o p m e n t a l b o u n d a r i e s , i n c l u d i n g u s e of c u l t u r e d e x p l a n t s for in v i t r o "bioass a y s , " are also d i s c u s s e d . © 1990 AcademicPress,Inc.

INTRODUCTION The problem of assembling neurons into functional circuits has been a major focus of developmental neuro1 To whom reprint requests should be addressed at Department of Anatomy and Neurobiology, College of Medicine, University of Tennessee, Memphis, The Health Science Center, 875 Monroe Ave., Memphis TN 38163. 35

0014-4886/90 $3.00 Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

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from their site of genesis to their final position. Roles for glia during neuron migration and neurite growth and sorting have since been demonstrated in invertebrate systems by Goodman and his collaborators (34) and in mammalian systems using both in vitro and in vivo approaches developed by Hatten, Silver, and others ( 17, 31, 70-72). Adhesion and extracellular matrix (ECM) molecules comprise a growing class of characterized proteins which might underly such distinct cell-cell interactions during CNS histogenesis, leading to specific patterns of developing functional organization (57, 68, 81, 84). These molecules are developmentally regulated and exhibit site specificity during neuronal migration and differentiation in different areas of the nervous system. Besides the well-documented neural cell adhesion molecule (NCAM) and neuron-glia cell adhesion molecule L1 (7, 30, 46, 58, 60, 87), the list of adhesion and extracellular matrix molecules putatively associated with the development of neurocytoarchitecture is continuously growing. Several adhesion and ECM molecules have been associated with neurite growth. These include L1 and laminin (for review, see (42, 45)), and unique cell surface and ECM molecules such as CAT 301 (33) and the limbic associated membrane protein (LAMP (91)) have been implicated in the generation of system specificity during neurogenesis (16). In addition to neuronal sorting and axonal pathfinding, adhesion or ECM molecules also appear to play a role in other aspects of neuronal differentiation, including dendritic growth. For example, Prochiantz and others (2) have described biochemically distinct subsets of glia that affect dendritic growth and orientation, and glia from different areas of the CNS may express distinct glycoconjugates during development that affect neuronal and neuritic patterning (6). The existence of glycoconjugate-rich boundaries around developing CNS substructure has now been described in the developing somatosensory cerebral cortex (10), in neostriatum (53, 76), and in numerous subcortical structures including the thalamus and the brain stem (54, 75). These boundaries appear prior to the final segregation of neurons into functional patterns (e.g., in the vibrissae-related, somatosensory cortical "barrel field" of rodents (10)), as well as during a postnatal period in which circuitries are undergoing stabilization (75). The transience of these boundaries that cordon off functionally distinct neuron groups and their apparent relationship with immature glial elements (11, 54, 75) suggest that they play a role in the shaping of functional patterns during development of CNS cytoarchitecture and connectivity. Using radiolabeled fucose incorporation into glycoconjugates during development, we observed specific timetables for the appearance and disappearance of the glycan moieties within boundaries that may designate molecular interactions that are unique to the developing or "plastic" brain (75). Our most recent studies have determined that the extracellular matrix mole-

cule J1/tenascin is a major boundary constituent in the barrel field and other CNS boundaries (77, 78). The present paper summarizes some of this work on glial and glycoconjugate boundaries during development of the mouse CNS, focusing on four areas: (i) the similarity of lectin cytochemistry and immunocytochemistry for glial fibrillary acidic protein (GFAP), J1/tenascin and a chondroitin sulfate proteoglycan (20), and the novel adhesion molecule on glia, a 50-kDa glycoprotein referred to as AMOG (1) that carries a unique carbohydrate epitope, L3 (40), for revealing boundaries and pathways; (ii) malleability of boundaries following deafferentation of the whisker-barrel system, and the consequence of such lesions for boundary pattern expression; (iii) abnormal and normal boundaries in the developing forebrain of the reeler mouse, an autosomal recessive mutation that results in abnormal cytoarchitectonic patterns but appropriate connectivity in the cortices (4, 5, 73); and (iv) future areas of study of boundaries, looking at their expression and functions in vitro, as well as relating boundary expression to neural cell lineage. Even though data are presented using a variety of labeling methods at different times during development and in different regions of the normal or perturbated (genetically or experimentally induced) CNS, a consistent theme of glial and glycoconjugate boundary patterns and their possible functions during development prevails throughout the paper. MATERIAL AND METHODS Detailed descriptions and protocols for studying glial and glycoconjugate boundaries in normal and reeler mutant mice during development have appeared elsewhere (10, 11, 53, 54, 75-77). However, we present a brief description of the methodologies used in the present study. Experimental animals and tissue handling. Mice from the ICR strain (Harland/Sprague-Dawley) or the B6C3Fe-a/a-rl strain (reeler mice, rl/rl mice showing the abnormal phenotype, and +/rl and + / + animals appearing normal; from our breeding colony originally obtained from The Jackson Laboratory, Bar Harbor, ME) were used in this study. The material presented here is compiled from over 100 mice ranging in age from Embryonic Day 17 (E17; day of appearance of vaginal plug is E0) to adult (1-2 months). Many of the examples photodocumented here are from studies of early postnatal mice (P0 = birth, P1 = the first 24 h after birth, P2 = the second 24 h after birth, and so on) since boundaries are most pronounced in the early postnatal period. All animals were deeply anesthetized with intraperitoneal injections of Avertin and/or by hypothermia prior to whisker lesions or perfusion. Postnatal and adult mice were perfused through the left ventricle with mixed aldehydes (e.g., 4% paraformaldehyde, 0.1% glutaraldehyde in Tris-buffered saline or 2-4% paraformaldehyde/0.5-

NORMAL AND ABNORMAL BRAIN BOUNDARIES 2% glutaraldehyde in Tris-buffered saline or cacodylate buffer for electron microscopy). Embryonic mice were immersion fixed in mixed aldehydes. Animals used in boundary perturbation studies, e.g., using sensory deprivation (whisker lesions), had their middle row (C row), and sometimes their C and D rows, of large vibrissae removed on P 2 - P 4 using electrocauterization as previously described (12). Parasagittal or flattened tangential vibratome sections were processed for light and electron microscopic cytochemistry or immunocytochemistry using methods briefly described below and detailed in previous papers (76, 77).

Lectin cytochemistry. Sections to be processed for lectin cytochemistry were washed in Tris-buffered saline (10 m M Tris, 0.9% NaC1, 1 m M CaC12, 1 m M MnC12, pH 7.3) containing 1% bovine serum albumin. These sections were then incubated for 3-24 h in horseradish peroxidase-conjugated, or biotinylated, peanut agglutinin (PNA) or Tetragonolobus purpureas agglutinin (TPA) (Sigma or Vector Laboratories). These lectins show a pronounced binding to fl-1, 3-N-acetylgalactosamine and a-L-fucosyl residues of glycoconjugates, respectively. Control sections were incubated in labeled lectins in the presence of the appropriate hapten sugar that can completely block all labeling (10, 74). The sections were further processed for peroxidase histochemistry. Some sections were counterstained with cresyl violet, and all sections were examined using bright-field microscopy. Immunocytochemistry. Sections processed for GFAP immunocytochemistry were incubated in a monoclonal antibody to the 51-kDA protein (Immunon). The glial protein was isolated from human astrocytomas and the IgG antibody, which is non-species-specific, was raised in mice. The sections were further processed for peroxidase histochemistry using an ABC kit (Vector Laboratories), and positive and negative controls were performed in accordance with the manufacturer's suggestions (e.g., substitution of nonimmune serum for the primary antibody; see 11). Sections were also processed for J1/tenascin, the 473 proteoglycan (20), and AMOG (1) immunocytochemistry. The monoclonal antibodies to J1/tenascin (22) recognize 200/220-kDa polypeptides described in detail by Faissner, Schachner, and Kruse (22, 39) in previous reports. A protocol for light and electron microscopic immunocytochemistry of J1/tenascin distribution in the developing mouse CNS appeared in a recent report (77). Antibodies to the 473 antigen and AMOG have been described in earlier reports (1, 20). Roller tube explant cultures. Some very preliminary data are presented using organotypic slice cultures for studies of boundaries and boundary molecules. We have followed the protocols for roller tube cultures developed in the laboratories of Drs. B. Gahwiler of the Brain Research Institute, University of Zurich, Rick Robertson, University of California, Irvine, and Jens Zimmer, Uni-

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versity of Aarhus (23). Vibratome slices, 200-400 ~m thick, from fresh brains of P 0 - P 7 mice were collected in Gey's balanced salt solution containing glucose. The slices were cultured on coverslips containing a chick plasma clot and in medium consisting of Basal medium without glutamine, horse serum, and Hank's or Earle's balanced salt solution. The cultures were rotated for 2 days to 2 weeks using a Bellco Roller Drum, in a humid incubator (Forma) maintained at 36°C. Explants were fixed and processed for immunocytochemistry as whole mounts or, depending on their thickness, sectioned, or embedded and sectioned, and further processed as described above. RESULTS

Lectin Cytochemistry and Immunocytochemistry of Glial Antigens Reveal Transient Boundaries Lectin cytochemistry, using a variety of labeled lectins with different sugar-binding specificities, reveals distinct glycoconjugate distributions throughout the developing CNS. The a-L-fucosyl binding lectin T P A nicely delineates boundaries around many different CNS substructural arrangements, including nuclei in the thalamus, midbrain, and brain stem (Fig. 1A). Immunocytochemistry of the J1/tenascin glycoprotein is almost identical to T P A in its labeling of boundaries during the first postnatal week (Fig. 1B). J1/tenascin boundaries are visible throughout the neuraxis in late embryonic and early postnatal periods, and they define patterns unique to every structure (e.g., see Fig. 1C that shows boundaries around subdivisions of the facial nucleus and more dense J1/tenascin labeling of the pons in a P0 mouse). Figures 1A and 1B compare T P A and J1/tenascin boundaries around the thalamic ventrobasal complex and lateral geniculate nucleus in parasagittal sections through a P2 brain. It previously has been determined that fucose can be readily incorporated into the J1/tenascin glycoprotein (39), and in fact T P A and other lectins may bind to J1/tenascin (14); hence, it should perhaps not be so surprising that fucose binding lectins and J1/tenascin immunocytochemistry reveal similar boundary patterns. Striking similarities in the disclosure of tissue boundaries by lectin binding, J1/tenascin, the 473 proteoglycan (not shown), AMOG, and GFAP immunocytochemistry are further illustrated in Figs. 1G-1J where these markers delineate boundaries around the inferior olivary complex. It is noteworthy that boundaries such as those shown in Figs. 1C and 1G1J, around brain stem structures, have been detected quite early during embryonic development. For example, J1/tenascin has already been described in a boundary around the vestibular nuclear complex in the embryonic mouse brain (78), and some of the earliest glycoconjugate boundaries may be associated with cranial nerve, reticular, and other nuclei of the brain stem (e.g., around

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STEINDLER ET AL.

•

~

i~¸

FIG. 1. Boundaries and axon pathways revealed by lectin cytochemistry versus immunocytochemistry. (A) Lectin cytochemistry, using T P A - H R P , of the Postnatal Day 2 (P2) brain; parasagittal section with rostral to the left and dorsal up. Boundaries are visible around the ventral tier nuclei of the thalamus (curved arrow) and dorsal lateral geniculate nucleus (asterisk). A pale area can be seen in the rectum (open arrow) that can be compared with B. Arrowhead points to a cerebellar peduncle. Scale = 500 #m. (B) J1/tenascin immunocytochemistry on P2 reveals similar boundaries around the thalamic ventral tier and lateral geniculate (curved arrow and asterisk, respectively), but increased staining throughout the neuropil by J1/tenascin versus lectin is obvious by more staining in nonboundary sites of these structures and the tectum (open arrow points to the inferior colliculus). Arrowhead points to a boundary around a cerebellar peduncle. Scale = 500 #m. (C) At birth (P0), J1/tenascin is present in boundaries around and within the facial nerve nucleus (open arrow) and the inferior olivary complex (curved arrow), and dense labeling is present withln'the pons (long arrow). Parasagittal section with rostral to the left and dorsal up. Scale = 500 ttm. (D) On P2, J1/tenascin in pathways such as the pyramidal tract is most distinct, with the molecule most heavily distributed in a tube-like fashion, small arrows). This pathway can be seen in close proximity to the pons (long arrow) that again exhibits heavy labeling. This section was from a case that was fixed with lower glutaraldehyde that can produce lighter labeling as seen in surrounding structures. Parasagittal section. Scale = 500 #m. (E) Closer inspection of labeling of a pathway. In this case the internal capsule on P2 reveals J1/tenascin labeling of distinct axon bundles that course in different directions (e.g., open arrow). Scale = 50 ~m. (F) Lectin binding reveals white matter labeling in the adult mouse brain. In this case, P N A - H R P labeling in the adult labels highways such as the subcortical white matter (short, filled arrow), the internal capsule (long, filled arrow), and the white matter of the cerebellum (open arrow). Parasagittal section with rostral to the left and dorsal up. Scale = 1 ram. (G-J) Boundaries around the inferior olive and its subnuclei on P0 with J1/tenascin (G), P2 with J1/tenascin (H), P6 with AMOG (I), and GFAP+ astrocytes on P6 (J). GFAP+ astrocytes occupy boundary regions between various divisions of the inferior olivary complex, see Ref. (75) for details. Scales = 100 #m.

NORMAL AND ABNORMAL BRAIN BOUNDARIES

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substructural arrangements of the Vth, VIIth, and VII- tributed within boundary or nonboundary sites, we were Ith cranial nerve nuclei, and inferior olivary complex, fortunate to subcellularly localize it in the Golgi apparasee Figs. 1, 2, 4, and 6). The exact time course of appear- tus of immature astrocytes (77). These findings were in ance of J1/tenascin versus the 473 proteoglycan versus agreement with in vitro studies (39) suggesting that asAMOG in these early boundaries has not yet been estab- trocytes are the major source of J1/tenascin expression lished, but they may be uniquely developmentally regu- in the young brain. Figure 2 shows light and electron milated. Crossin and her colleagues (14), for example, have croscopic immunocytochemistry of J1/tenascin and the established that a proteoglycan ligand for their cytotac- 473 proteoglycan in the P1-P2 trigeminal interpolaris tin (cytotactin is most likely the same as J1/tenascin) is nucleus. Light microscopy reveals that these molecules expressed slightly later than cytotactin in the barrel field are concentrated in boundaries between rows of whisker boundaries. It is possible that different adhesion and representations (Figs. 2A and 2B). We attempted to take ECM molecules and their putative ligands may thus sections from such a boundary for EM immunocytoshow unique times of appearance and disappearance in chemistry of J1/tenascin (Fig. 2C). It is our impression boundaries throughout the developing neuraxis. that more J1/tenascin is expressed in the pericellular All of the aforementioned markers delineate bound- space (possibly associated with an extracellular matrix) aries for a limited time in development, and by the end "and on the membranes of neuronal and glial processes of the second postnatal week boundaries are for the most in neuropil areas containing few neuronal cell bodies. part no longer apparent (10-12, 53, 54, 75, 76). However, However, we did not quantify these data nor did we atlectin cytochemistry of labeled PNA delineates white tempt to associate the labeling with particular cellular matter pathways in the more mature (i.e., adult) CNS processes. However, J1/tenascin and the 473 proteogly(76). This is clear in Fig. 1F where there is PNA binding can appear to be expressed in boundaries between brain to undefined constituents of the subcortical white mat- stem trigeminal whisker row representations that may ter of the cerebral cortex, as well as in the internal cap- become neuron-sparse areas in the adult. This conforms sule, cerebral peduncle, anterior commissure, and cere- to our previous findings of greater amounts of J1/tenasbellar white matter. The glycoconjugates recognized in cin in barrel boundaries during development that are these fiber pathways may be associated with oligoden- destined to become the neuron-sparse interbarrel septae drocytes or perinodal astrocyte processes (23, 48, 59), in the adult. and these labeling patterns become most clear at a time when boundaries around nuclei and barrels disappear. Boundaries around N o r m a l and Perturbated Barrels The J1/tenascin glycoprotein labels such fiber pathways during postnatal development (Figs. 1D, 1E, 2, and 6). We have previously characterized the times of appearThe labeling associated with fibers in the internal cap- ance and disappearance of lectin-bound glycoconjusule is quite distinctive, with pathways of different tra- gates, GFAP, and J1/tenascin in the barrel field of the jectories contributing to a sort of meshwork (Fig. 1E). normal mouse, reeler mouse, and the whisker-lesioned We do not know if this J1/tenascin expression in path- normal mouse (10-12, 54). As described above, all of ways is exclusively associated with white matter astro- these markers are present in developing brain tissue but cytes, or also possibly with oligodendrocytes or common they are concentrated in boundaries during the first progenitor cells (64). Nonetheless, the expression of J1/ postnatal week. By the end of the second postnatal week, tenascin in fiber systems might relate to pathfinding and glial and glycoconjugate markers no longer reveal sorting of different projection systems, for it is present boundary patterns. Figure 3 shows the similarity bein growing axon systems early during development. Fig- tween lectin binding and immunocytochemistry for ure 1D documents J1/tenascin expression in the neona- GFAP, J1/tenascin, and the 473 proteoglycan for cortal pyramidal tract where the molecule has been seen to doning offthe P6 barrel field (Figs. 3A and 3C-3E). Even distribute in a tube-like fashion around presumptive cor- though lectin-positive and immunoreactive elements are ticospinal axons en route to their spinal targets. present throughout the developing barrel field, greater Immunocytochemistry of the J1/tenascin glycopro- amounts of these labels are concentrated in boundaries tein is distinctly the best marker of transient CNS between individual barrels. It is therefore conceivable boundaries. A previous light and electron microscopic that we should interpret the incipient barrel pattern study (77, 78) demonstrated t h a t this macromolecule is seen with these markers as a "hollowing out" of prospecpresent on surfaces of glia and neurites and also appears tive barrel hollows with a persistence of certain glia and to be present in the ECM in agreement with other data, glycoconjugates in surrounding boundaries. These soindicating matrix associations (18, 78, 79). J1/tenascin called glial and glycoconjugate boundaries are complewas expressed in larger amounts in boundaries of devel- mentary to the appearance of afferent innervation of the oping barrels (also see below), and even though it could barrel field during the first postnatal week. In fact, one not be directly established in either the light or electron marker we have studied actually produces a reverse immicroscopic study that the glycoprotein was associated age in the same time frame. Immunocytochemistry of with a certain type of cell or process differentially dis- the synaptic vesicle protein p38 (synaptophysin (51))

FIG. 2. Light (A, B) and electron microscopic (EM) (C) immunocytochemistry of J1/tenascin and the 473 proteoglycan in vibrissae row boundaries of the brain stem trigeminal interpolaris nucleus. (A) On P2, J1/tenascin is concentrated in boundaries (e.g., straight arrows) between whisker row representations (e.g., stars) in the interpolaris nucleus. A J1/tenascin boundary is also present between this nucleus of the trigeminus and the caudalis nucleus (open arrow). The curved arrow points to labeling associated with an axonal pathway, in this case a cerebellar peduncle (curved arrow). The micrograph in C was taken from a region corresponding to the boundary delineated by the straight arrows. Parasagittal section with rostral to the left and dorsal up. Scale 400 ttm. (B) On P1.5, the 473 proteoglycan is also distributed in 40

NORMAL AND ABNORMAL BRAIN BOUNDARIES (Fig. 3B), as well as of o t h e r m a r k e r s such as G A P - 4 3 (19), shows a developing f u n c t i o n a l l y segregated innerv a t i o n of individual b a r r e l hollows also during the first p o s t n a t a l week. T h i s labeling in developing barrel hollows is p r e s u m e d to be associated with t h a l a m o c o r t i c a l projections f r o m t h e barreloid field of t h e v e n t r o b a s a l complex. Cooper a n d Steindler (12) h a v e s h o w n t h a t electroc a u t e r y lesions of the w h i s k e r p a d b e t w e e n P1 a n d P 4 results in altered glycoconjugate a n d G F A P - p o s i t i v e glial cell distributions in b o u n d a r i e s associated with the c o r r e s p o n d i n g d e a f f e r e n t e d barrels in mice e x a m i n e d on P 6 - P 7 . Figure 3G shows such a case where a lesion t h a t involved whiskers of the c row on P2 resulted in a loss of J 1 / t e n a s c i n b o u n d a r i e s in this row, in addition to the altered t o p o g r a p h y of t h e a a n d b rows in c o m p a r i s o n to the c row of t h e c o n t r a l a t e r a l s o m a t o s e n s o r y cortex w h e n looked at on P7. I f in addition to a c row lesion, the entire n e o n a t a l w h i s k e r p a d is immobilized using a n adhesive, the resulting glycoconjugate b o u n d a r y p a t t e r n in the c o n t r a l a t e r a l barrel field on P6 is quite different. Figure 3F shows a case where a loss of c row b o u n d a r i e s can be discerned as previously discussed, b u t in addition, b o u n d a r i e s b e c o m e inconspicuous t h r o u g h o u t the barrel field. T h i s image is similar to t h a t seen in our earliestdetected barrel b o u n d a r y p a t t e r n seen on P2 using J 1 / t e n a s c i n i m m u n o c y t o c h e m i s t r y (Fig. 3 H a n d inset). Figure 3H a n d its inset show the p r e s e n c e of a barrel fieldlike p a t t e r n on two adjacent sections t h r o u g h the P2 som a t o s e n s o r y cortex using J 1 / t e n a s c i n i m m u n o c y t o chemistry. E v e n t h o u g h a " p r o t o b a r r e l field" can only be i m p u t e d because of the lack of clear-cut b a r r e l delineations t h r o u g h o u t five discernible rows, individual barrels c a n be recognized, a n d in addition to an overall pale staining p a t t e r n t h a t often characterizes the developing barrel field in glycoconjugate-labeled material, these put a t i v e early barrel fields have a n overall t o p o g r a p h y (and g e o m e t r y ) t h a t r e s e m b l e s t h a t of the m o r e m a t u r e barrel field (e.g., p r e s e n c e of p o s t e r o m e d i a l a n d a n t e r o l a t e r a l fields; a tissue crack t h a t is associated with a group of blood vessels often p o s i t i o n e d above the a n t e r o l a t e r a l field, see the curved arrows in Figs. 3 H a n d 5A; a n d dist i n c t curvilinear a r r a n g e m e n t s of the d a n d e rows, e.g., see open arrow in t h e inset in Fig. 3H).

Abnormal and Normal Boundaries in the Reeler Mouse Brain T h e reeler m o u s e offers a unique setting in which to s t u d y b o u n d a r i e s in relation to the d e v e l o p m e n t of functional cytoarchitecture. In the adult reeler mouse, neuronal position a n d o r i e n t a t i o n are a b n o r m a l in all of the

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cortical s t r u c t u r e s (4, 5). It has b e e n suggested t h a t cortical l a m i n a e are i n v e r t e d in the cerebral cortex, a n d t h e r e is a m i x i n g of cortical n e u r o n s in the cerebellum t h a t n o r m a l l y occupy distinct layers. Dendritic p a t t e r n s are also a b e r r a n t , yet a x o n a l projections for the m o s t p a r t seek out a n d t e r m i n a t e on t h e i r a p p r o p r i a t e t a r g e t n e u r o n s (4, 73). T h e reeler m o u s e offers a p o t e n t i a l l y valuable model for studying b o u n d a r i e s because we have d e m o n s t r a t e d altered glial a n d glycoconjugate dispositions in the i m m a t u r e s o m a t o s e n s o r y cortex (54) t h a t predict a n a b n o r m a l c y t o a r c h i t e c t o n i c barrel field seen in t h e adult (86), yet the w h i s k e r - r e l a t e d barreloids of t h e t h a l a m i c v e n t r o b a s a l c o m p l e x a p p e a r to h a v e a completely n o r m a l glycoconjugate b o u n d a r y p a t t e r n . T h i s is d o c u m e n t e d in Figure 4 where lectin c y t o c h e m i s t r y of a flattened t a n g e n t i a l section t h r o u g h the P6 reeler foreb r a i n reveals t h r e e t y p e s of glycoconjugate b o u n d a r y p l a n s all within one section. T h e s o m a t o s e n s o r y cortex c o n t a i n s glycoconjugate b o u n d a r i e s a r o u n d grossly miss h a p e n barrels t h a t can only be recognized b y the presence of occasional glycoconjugate-poor hollows. T h e t h a l a m i c barreloid complex, however, possesses completely n o r m a l barreloid b o u n d a r i e s t h a t can be seen to i n t e r v e n e b e t w e e n N i s s l - s t a i n e d barreloid n e u r o n s (inset). In this N i s s l - c o u n t e r s t a i n e d P N A c y t o c h e m i c a l p r e p a r a t i o n , one gets the i m p r e s s i o n t h a t the t h a l a m i c n e u r o n s of individual barreloids are often e m b e d d e d in the glycoconjugate boundaries, a n d it is intriguing to consider t h e ways in which the cytoarchitectonically " n o r m a l " barreloids m u s t s o m e h o w i n t e r a c t with the m a l f o r m e d cortical barrels. In b e t w e e n these related b o u n d a r y p a t t e r n s involved in r e p r e s e n t i n g the facial vibrissae, glycoconjugate b o u n d a r i e s can be seen a r o u n d a t h i r d b o u n d a r y p l a n related to two different c o m p a r t m e n t s of the c a u d a t e - p u t a m e n (neostriatum). As we h a v e already described in the n o r m a l mouse n e o s t r i a t u m (76), glycoconjugate b o u n d a r i e s also cordon off t h e neurochemically distinct p a t c h a n d m a t r i x c o m p a r t m e n t s of the n e o s t r i a t a l mosaic in the early p o s t n a t a l reeler mouse. B u t to date we have not b e e n able to establish w h e t h e r this mosaic p a t t e r n is n o r m a l in the reeler striaturn since individual p a t c h e s c a n n o t yet be recognized f r o m a n i m a l to animal. In reeler, however, it is clear t h a t glycoconjugate b o u n d a r i e s do s u r r o u n d p a t c h e s b o t h t e m p o r a l l y a n d spatially in a m a n n e r similar to t h a t seen in the n o r m a l mouse.

Boundaries around Perturbated Barrels in the Reeler Mouse As already discussed, the reeler gene(s) c o n t r i b u t e s to a b n o r m a l c y t o a r c h i t e c t o n i c a r r a n g e m e n t s in the corti-

whisker row (e.g., stars) boundaries in the interpolaris nucleus, as well as in a boundary (arrow) between the interpolaris and caudalis nuclei. Scale = 400 ttm. (C) EM of J1/tenascin in a P2 whisker row boundary in the interpolaris nucleus reveals immunolabeled elements around cellular processes. Within the cellular layer (asterisk is within neuronal nucleus), labeling (e.g., curved arrows) is not as prevalent as that seen near or within a putative boundary (area to the right of the dashed line). Scale = 500 nm.

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S T E I N D L E R E T AL.

NORMAL AND ABNORMAL BRAIN BOUNDARIES ces, y e t a p u t a t i v e l a y e r IV b a r r e l field is d i s c e r n i b l e i n t h e a d u l t u s i n g cell s t a i n i n g m e t h o d s (86). O ' B r i e n et al. (54) d e t e r m i n e d t h a t d e v e l o p m e n t a l b o u n d a r i e s , s e e n with lectin cytochemistry, predict the altered organizat i o n of t h e r e e l e r b a r r e l field w h e r e a t o p o g r a p h i c a l l y app r o p r i a t e b a r r e l field is p r e s e n t y e t i n d i v i d u a l b a r r e l s are g r o s s l y m i s s h a p e n . I n s e c t i o n s p r o c e s s e d for l e c t i n c y t o c h e m i s t r y or i m m u n o c y t o c h e m i s t r y of G F A P or J 1 / t e n ascin, b o u n d a r i e s a r o u n d developing barrels in the reeler somatosensory cortex reveal presumptive a b e r r a n t barrel a r r a n g e m e n t s . O n e is h a r d p u t t o e v e n a t t e m p t row a s s i g n m e n t s i n t h e b o u n d a r y - d e l i n e a t e d b a r r e l field (e.g., Figs. 5A a n d 5B); h o w e v e r , l e c t i n c y t o c h e m i s t r y a n d J 1 / t e n a s c i n i m m u n o c y t o c h e m i s t r y do s o m e t i m e s p r o d u c e i m a g e s t h a t i n v i t e s u c h a s s i g n m e n t s . F i g u r e s 5A a n d 5B are j u s t s u c h cases: l e c t i n b i n d i n g u s i n g l a b e l e d PNA and J1/tenascin immunoreactivity resulted in b o u n d a r y - d e l i n e a t e d P 6 r e e l e r b a r r e l fields i n w h i c h t h e p r e s e n c e of b o t h a n t e r o l a t e r a l a n d p o s t e r o m e d i a l b a r r e l s c o n t r i b u t e s to a field w h e r e t h e e x i s t e n c e of rows a - e c a n be imputed. E v e n t h o u g h barrel hollows appear miss h a p e n a n d s m a l l e r i n t h e s e p r e p a r a t i o n s , b o u n d a r i e s do delineate clear-cut barrel-like arrangements. H o w e v e r , w h i s k e r l e s i o n s i n reeler, as d e s c r i b e d a b o v e in the normal mouse, produce changes in the contralateral b a r r e l field i n r e e l e r t h a t d e f i n i t e l y do allow a recogn i t i o n of b a r r e l rows. T h i s is p h o t o d o c u m e n t e d i n Fig. 5D w h e r e a c row l e s i o n o n P 2 l e a d s t o a c l e a r - c u t loss of J1/tenascin boundaries in corresponding barrels within a m i d field p o s i t i o n i n t h e c o n t r a l a t e r a l P 7 reeler cortex. I n s o m e cases, t h i s " d e a f f e r e n t e d " c row also s h r i n k s i n a d d i t i o n to e x h i b i t i n g loss of b o u n d a r i e s b e t w e e n i n d i vidual whisker representations. A n a c c o m p a n y i n g inc r e a s e i n size o f t h e d row a n d its b o u n d a r i e s c a n also b e o b s e r v e d (Fig. 5D). T h i s c a n also b e s e e n i n t h e n o r m a l mouse following the same whisker lesions a n d using J 1 / t e n a s c i n or 473 p r o t e o g l y c a n i m m u n o c y t o c h e m i s t r y

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(Fig. 5C), a n d h a s b e e n d e s c r i b e d b y W o o l s e y , V a n der Loos, a n d t h e i r c o l l a b o r a t o r s (36, 82, 90). I t is o u r i m p r e s s i o n t h a t t h e w h i s k e r - b a r r e l s y s t e m , a n d likewise b o u n d a r i e s a r o u n d e l e m e n t s of t h i s s y s t e m , are i n a p r o cess of f o r m a t i o n a n d r e f o r m a t i o n u n d e r n o r m a l a n d les i o n e d c o n d i t i o n s . D e p e n d i n g o n t h e e x t e n t of t h e per i p h e r a l l e s i o n , a n d also o n t h e e x t e n t of r e g r o w t h of follicles p r i o r t o s a c r i f i c i n g t h e a n i m a l s , t h e i m a g e s of barrels a n d barrel b o u n d a r i e s can be visualized in different configurations in both n o r m a l a n d reeler phen o t y p e s . F o r e x a m p l e , w i t h i n t h e s h r u n k e n c row i n t h e 473 p r o t e o g l y c a n - d e l i n e a t e d b a r r e l field of a c row-les i o n e d n o r m a l a n i m a l (Fig. 5C), s o m e b o u n d a r i e s c a n b e seen. T h e w h i s k e r p a d of t h i s a n i m a l w a s p a r a f f i n - s e c tioned and stained with hematoxylin and eosin (data not s h o w n ) , a n d a c o u p l e of v i b r i s s a e w i t h i n t h i s row h a d a p p a r e n t l y r e g e n e r a t e d or p e r h a p s e v a d e d t h e e l e c t r o c a u t e r i z a t i o n . T h u s , t h e a p p e a r a n c e of b o u n d a r i e s a r o u n d b a r r e l s i n t h e d e v e l o p i n g n o r m a l or r e e l e r b a r r e l field is d e p e n d e n t o n t h e p r e s e n c e or s t a t e of a c t i v i t y of t h e w h i s k e r s (see a b o v e r e s u l t s r e l a t e d t o i m m o b i l i z a t i o n of w h i s k e r s ) . T h e f i n d i n g s of a l t e r e d b o u n d a r y e x p r e s s i o n i n t h e r e e l e r b a r r e l field f o l l o w i n g p e r i p h e r a l l e s i o n s s u g g e s t t h a t e v e n t h o u g h t h e reeler s h o w s s t r i k i n g a b n o r m a l i t i e s of n e u r o n a l a n d glial (54) p a t t e r n i n g i n t h e c e r e b r a l cortex, g l y c o c o n j u g a t e b o u n d a r y p a t t e r n s a p p e a r m a l l e a b l e i n a m a n n e r t h a t is p o s s i b l y n o t so d i f f e r e n t f r o m t h a t seen in the normal mouse.

J1/Tenascin Boundaries in the Embryonic Brain and Possible Lineage Relationships T h e r e l a t i o n s h i p b e t w e e n glial a n d g l y c o c o n j u g a t e b o u n d a r i e s a n d n e u r o n a l p r o g r a m s for f u n c t i o n a l aggreg a t i o n is n o t y e t k n o w n . F o r e x a m p l e , it w o u l d b e i n t e r e s t i n g if l i n e a g e - r e l a t e d c l u s t e r s of n e u r o n s were s u r -

FIG. 3. The developing and lesioned barrel field in flattened tangential sections, labeled with boundary and complementary markers. A, B, and F are oriented with anterior to the right and medial up; C-H are oriented with anterior to the left. (A) PNA-HRP on P6 labels boundaries around developing barrels of the posteromedial (rows a-e) and anterolateral (open arrow) subfields. Stars are in some representative hollows. Scale = 200 #m. (B) Immunocytochemistry of the putative synaptic vesicle marker p38 (synaptophysin) on P6 is a complementary marker in that it labels the neuropil of developing barrel hollows (e.g., star). Scale = 200 tLm. (C) J1/tenascin, on P6, is more concentrated in barrel boundaries then in hollows similar to that seen with lectin binding. This is shown at higher magnification in D. Scale in C = 200 ttm and in D = 100 ~m. (E) GFAP immunocytochemistry on P6 reveals labeled radial glia and astrocytes that are more densely distributed within barrel boundaries (e.g., between arrowheads) compared to hollows. Scale = 100 #m. (F) Following an electrocautery lesion of the c row of whiskers, and immobilization of the remaining whiskers on P2, lectin-bound glycoconjugates (using PNA-HRP) in the P6 contralateral barrel field are reduced in boundaries between the deafferented barrels of the c row (curved arrow, although some intact boundaries are apparent, large stars) as well as throughout all five rows of the larger posteromedial and smaller anterolateral (open arrow) barrels. Scale = 350 #m. (G) J1/tenascinimmunopositive boundaries are also lost between deafferented barrels of the P6 c row (curved arrow) following a lesion of just the c row of whiskers on P2. The a and b rows also appear affected by the lesion that may have involved whiskers of these rows as well, and some boundaries associated with these rows appear to be more dense (e.g., open arrows). Curved arrow points out the unaffected smaller barrels of the anterolateral subfield. Scale = 200 t~m. (H and inset) On P2, J1/tenascin in the normal developing barrel field labels a subtle boundary pattern where posteromedial and anterolateral (open arrows) subfields are barely visible. The presumed barrel fields are outlined by arrows in these adjacent sections (H and the inset are adjacent sections, satisfying one criterion for "barrels" in that they are visible one more than one section, see Refs. (89, 86), with some barrel hollows visible as a result of near complete enclosure by boundaries (e.g., stars). Asterisks mark some barely visible barrel hollows. The curved arrow points to some blood vessels that are often associated with a tissue crack in these flattened preparations (see legend to Fig. 5A). The open arrow in the inset points to a curvilinear boundary pattern possibly associated with a developing a row of barrels. There is often intense J1/tenascin labeling that seems to be associated with vascular elements in these putative boundary sites (also see Fig. 6C), and boundary-blood vessel relationships need further study. Scale = 50 t~m in H and 100 ttm in the inset.

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rounded by a boundary early in development. This type of information is currently under study in collaboration with Karl Herrup of the Shriver Center using chimeras and other lineage models; however, some preliminary observations in the early postnatal reeler cerebellum suggest that such early lineage-boundary relationships may exist. Figures 6A and 6B show J1/tenascin boundaries in the neonatal reeler cerebellum that surround groups of displaced cortical neurons. Clusters of displaced Purkinje cells referred to as the central cerebellar mass by Mariani and his collaborators (47) appear to be surrounded by boundaries that segregate these clusters from each other and the underlying cerebellar nuclei. The clusters of Purkinje cells line up with corresponding cerebellar nuclei (i.e., dentate, anterior and posterior interpositus, fastigius nuclei), and a J1/tenascin (Fig. 6A) and also a 473 proteoglycan (Fig. 6B) boundary surround each one of these clusters of displaced Purkinje neurons in the P2 and P6 reeler cerebellum. In addition, within a cluster, more boundaries are visible that appear to relate to subsets of Purkinje cells (Fig. 6B). Herrup and Bower (32) have related Purkinje cell lineage to a variegated mosaic of the tactile map in the adult cerebellar cortex, and it would be most intriguing if the boundaries we see in the displaced (in reeler) and migratory (in the normal) Purkinje cell clusters could be related to such a boundary-defined functional map. We have not yet performed a systematic study of when the" earliest cortical or subcortical boundaries appear with J1/tenascin or any of the other probes. However, we do have data from many antibody screenings on boundaries present in the embryonic brain that suggest that boundaries recapitulate the ontogenetic timetables already assembled on generations of different neuron groups in the brain using other methodologies (e.g., tritiated thymidine birthday labeling). We predict, on the basis of findings already presented on J1/tenascin boundaries in the embryonic brain stem, that boundaries are visible at least when neurons and their processes begin to functionally segregate, and more information is needed on potential pre-map boundary patterns composed of this or other recognition molecules. Nonetheless, J1/tenascin does reveal an interesting distribution in the developing cortices. In the late embryonic cerebral cortex (Fig. 6C), the molecule is densely

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distributed in a subplate boundary as well as beneath the subplate in the ventricular, subventricular, and intermediate zones. In the developing cortical plate, J1/tenascin immunoreactivity is present just above the subplate in a band that exhibits periodicities of labeling (Fig. 6C). The periodicities of dense J1/tenascin immunostaining appear to be related to ensheathment of radial vessels in these regions of the cortical plate. Their overall appearance is modular and may relate to an embryonic cordoning off of presumptive cortical "columns."

J1/Tenascin Expression and Boundaries Persist in Organotypic Cultures The determination of functions of glial and glycoconjugate boundaries requires in vitro bioassays where molecules or antibodies can be plated or introduced to evaluate the resulting cellular responses. We have begun to use organotypic cultures as a way of evaluating such interactions and responses. Figures 7 and 8 present some findings from pilot studies using the roller tube explant method, perfected by Gahwiler and his collaborators (24), for determining boundary persistence in culture, and subsequent bioassays will utilize such in vitro boundary preparations that have the advantage of retaining a reasonable amount of their spatial (and functional) organization as seen in vivo. Explants of neonatal cortex survive in roller tube cultures, and they can even mature to some extent and reveal laminar arrangements (Figs. 7A-7E). For example, an explant taken from the P2 somatosensory cortex and cultured for 1 week can show laminar arrangements of GFAP-positive astrocytes not so unlike those seen in sections from the in vivo P9 cortex (compare Figs. 7A and 7B). Most prominent is a dense band of GFAP-immunoreactive astrocytes within the molecular layer in both preparations that are destined to become GFAP-positive astrocytes of layer I in the adult. Such layering characteristics are also visible in J1/tenascin-immunostained preparations. Figures 7D and 7E show J1/tenascin staining in the P2 cortex prior to culturing and after 5 days in vitro. In the P2 cortex, J1/tenascin is expressed within laminae that do not easily correspond with developing layers or other subdivisions of the cortical plate and subplate, but may relate to boundaries between such developing substruc-

F I G . 4. Glycoconjugate boundaries around normal and abnormal structures in the P6 reeler brain. (A) Flattened tangential section (with rostral to the right and dorsal up) through the reeler forebrain processed for P N A - H R P . In this single section, boundaries are visible around three different structural elements: abnormal somatosensory cortical barrels (filled arrow), patches of the neostriatal mosaic (e.g., curved arrows), and apparently normal boundaries around barreloids of the thalamic ventrobasal complex (open arrow, star is within a representative barreloid hollow). Arrowhead points to the anterior commissure. Scale = 250 ttm. (B) Higher magnification of the thalamic barreloid complex from this section, following counterstaining with cresyl violet, shows thalamic neurons along and often embedded within barreloid boundaries, some of which are marked by arrowheads. In a black and white photomicrograph of a counterstained preparation such as this, it is difficult to resolve the glycoconjugate barreloid boundaries that appeared golden brown and distinct from purple cells seen under the microscope. A representative cell-sparse barreloid hollow (e.g., star) can be seen between two barreloid boundaries. Scale = 30 #m. (C) Glycoconjugate boundaries also reveal apparently "normal" boundaries within the reeler trigeminal interpolaris complex. In this case, immunocytochemistry of the 473 proteoglycan shows boundaries between these brain stem whisker row representations (stars), and J1/tenascin and lectin binding similarly reveals these apparently normal patterns in the reeler brain stem and thalamus. Scale = 200 ttm.

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FIG. 5. Reeler barrel field boundaries compared with the normal and following lesions. Flattened tangential sections with anterior to the left. ~A) Lectin binding (using P N A - H R P ) again reveals boundaries around abnormal barrel patterns in the P6 reeler somatosensory cortex, with some hint of subfield (e.g., smaller barrels in an anterolateral region, open arrow) and row-like organization (e.g., stars are within hollows of two representative barrels within a presumed posteromedial subfield). The curved arrow points out a crack that is often observed in these flattened preparations, in the vicinity of blood vessels above the anterolateral subfield. Scale = 500 ttm. (B) J1/tenascin immunolabeling in the P6 reeler barrel field also reveals row- and barrel-like patterns (stars are within representative hollows), but again it is difficult to reliably assign rows a-e. Scale = 500 t~m. (C) Following lesions of the c row of whiskers on P2 in a normal mouse, the 473 proteoglycan (as well as J1/tenascin, see Fig. 3) reveals boundary changes in the contralateral barrel field. This section, from a P7 animal, shows a shrunken c row (curved arrow) that can follow such peripheral lesions, with a couple of barrels visible (e.g., asterisk), and an accompanying increase in size of the d row (stars in some hollows). The open arrow points to the anterolateral field. The a and b rows of barrels were lost from this section during the sectioning process but are present in adjacent sections. Scale = 500 ttm. (D) Following a similar lesion of the c row in a P2 reeler, J1/tenascin labeling in the P7 contralateral somatosensory cortex also reveals a shrunken row presumed to represent the deafferented c row (white arrow) and a corresponding enlarged d row (stars in some enlarged hollows) above. The open arrow points to barrels of the presumed anterolateral field. Scale = 500 #m. tures. In the cultured neocortex, J1/tenascin-positive l a m i n a e p e r s i s t t o a d e g r e e (e.g., s e e t h e l i g h t z o n e p r e s ent in Figs. 7C-7E), but staining is reduced in a manner t h a t is n o t s o d i f f e r e n t t h a n t h a t s e e n d u r i n g m a t u r a t i o n

in vivo ( c o m p a r e F i g . 7 D , a P 2 e x p l a n t c u l t u r e d f o r 5 d a y s , w i t h F i g . 7 E f r o m t h e n o n c u l t u r e d in vivo P 7 c o r tex). Both preparations reveal a persistent, dense J1/ tenascin expression within the molecular layer (most

NORMAL AND ABNORMAL BRAIN BOUNDARIES

47

FIG. 6. Other boundaries in the reeler cerebellum and in the normal embryonic cerebral cortex. (A) P2 reeler cerebellum in a flattened tangential section reveals J1/tenascin boundaries around ectopic Purkinje cell clusters (curved arrows) of the central cerebellar mass. A larger boundary (arrowhead) has a more radial orientation. A labeled pathway (long arrow) displays a unique course in the vicinity of the cochlear nucleus. In this section, rostral is to the left and dorsal is up, and the cerebellum appears distended as a result of flattening. Scale - 250 #m. (B) Higher magnification of reeler cerebellar boundaries, looking at the 473 proteoglycan on P6, reveals a continuous boundary around a large cluster of Purkinje cells of the central cerebellar mass (arrowheads), with discontinuous and sometimes tortuous boundaries associated with other smaller clusters (e.g., open arrows). Scale = 250 #m. (C) J1/tenascin immunolabelingof the E17.5 somatosensory cortex in the normal mouse is dense within a subplate boundary (open arrow) and the cortical surface (above the marginal zone, mz, of the molecular layer). There is a more homogeneous labeling of the deep intermediate zone (iz), but J1/tenascin labelingjust beneath the subplate boundary and within the deep cortical plate shows intermittent densities and hollows (arrows). Scale = 100 ~m. likely associated with the persistently "reactive" p i a l glial a s t r o c y t e s of t h i s layer), b u t s o m e d i f f e r e n c e s are p r e s e n t w i t h i n o t h e r l a y e r s of t h e e x p l a n t . T h e s e differe n c e s i n e x p r e s s i o n of J 1 / t e n a s c i n i n e x p l a n t s v e r s u s in vivo c o u l d be r e l a t e d to t h e c o n s e q u e n c e s of d e a f f e r e n t a t i o n (see D i s c u s s i o n ) . A s u c c e s s f u l b i o a s s a y of b o u n d a r y f u n c t i o n s r e q u i r e s t h e p e r s i s t e n c e of c h a r a c t e r i s t i c b o u n d a r i e s c o m p r i s i n g recognizable cellular e l e m e n t s in addition to the novel e x p r e s s i o n s of b o u n d a r y m o l e c u l e s as j u s t d i s c u s s e d . Neocortical explants contain GFAP-immunoreactive

b o u n d a r i e s as s e e n in vivo t h a t c a n be u s e d as f u t u r e t e m p l a t e s for a s s a y i n g n e u r o n r e s p o n s e s i n c o c u l t u r e s . E x p l a n t s f r o m P 2 n e o c o r t e x c u l t u r e d for 5 d a y s r e v e a l G F A P - p o s i t i v e glial cells t h a t r e t a i n t h e i r b o u n d a r y top o g r a p h y (Fig. 8). T h e s u b c o r t i c a l w h i t e m a t t e r is a s o r t of b o u n d a r y b e t w e e n t h e n e o c o r t e x a n d t h e s u b c o r t i c a l s t r u c t u r e s (e.g., t h e n e o s t r i a t u m a n d h i p p o c a m p u s ) , a n d t h i s b o u n d a r y , i n a d d i t i o n to n e u r o n s of t h e s e s t r u c t u r e s , p e r s i s t s i n e x p l a n t c u l t u r e s (Figs. 8A a n d 8B). T h e s u b c o r t i c a l w h i t e m a t t e r b o u n d a r y t h a t p e r s i s t s i n slice cult u r e s r e s e m b l e s s u c h a b o u n d a r y p r e s e n t in vivo (Fig. 8B,

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FIG. 8. GFAP immunolabelingin an explant boundary versus that seen in vivo. (A) An explant that was slice cultured from a P2 forebrain, tangentially sectioned, 5 days in vitro. GFAP-positive astrocytes of a subcortical white matter "boundary" (arrows) separate the emerging subiculum and hippocampus from neocortical structures. Scale = 500 #m. (B) Higher magnification of this boundary shows GFAP-positive elements (between arrows) that are concentrated at the interface between the different telencephalic structures. Inset shows the same boundary from an in vivo P7 brain also processed for GFAP. I n vivo, this boundary often appears as two separate plates of GFAP+ astrocytes. Scale in B and inset = 100 um. (C) Higher magnification through the cultured boundary reveals immunolabeledradial elements within bundles (e.g., open arrow), as well as astrocytes possibly derived from white matter (closed arrows). Scale = 50 #m. i n s e t ) a n d m a y b e m a d e u p of p r e d o m i n a n t l y f i b r o u s ast r o c y t e s r e l a t e d t o T y p e II a s t r o c y t e s d e s c r i b e d b y Raft, Miller, a n d their colleagues in the optic nerve a n d corpus c a l l o s u m (48). Close i n s p e c t i o n of t h e G F A P - p o s i t i v e ele m e n t s w i t h i n s u c h a c u l t u r e d b o u n d a r y (Fig. 8C) rev e a l s r a d i a l - l i k e e l e m e n t s as well as a s t r o c y t e s i n p e r h a p s d i f f e r e n t s t a t e s of d i f f e r e n t i a t i o n or d e d i f f e r e n t i a t i o n t h a t c a n b e r e l a t e d to a s t r o c y t e s a n d p r o g e n i t o r cells s e e n i n vivo. I t is i m p o r t a n t to d e t e r m i n e w h e t h e r t h e s e w h i t e m a t t e r astrocytes are d e t e r r e n t s to a x o n a l growth i n lesion a n d r e g e n e r a t i o n models, a n d t h e p r e l i m i n a r y results f r o m slice c u l t u r e s suggest t h a t we c a n n o w t e s t t h i s b y t h e i n t r o d u c t i o n of l a b e l e d n e u r o n s i n t h e s e cultures.

DISCUSSION T h e findings p r e s e n t e d here contribute the following pieces of i n f o r m a t i o n o n b o u n d a r y e x p r e s s i o n : (i) Boundaries around developing CNS substructure (nuclei, layers, or d i s c r e t e f u n c t i o n a l u n i t s s u c h as w h i s k e r r e l a t e d b a r r e l s ) a p p e a r to b e g r o s s l y s i m i l a r u s i n g l e c t i n cytochemistry and ECM molecule immunocytochemistry, b u t t h e r e m a y b e d i f f e r e n c e s i n t h e t e m p o r a l e x p r e s s i o n of t h e v a r i o u s m a r k e r s : (ii) B o u n d a r i e s m a y a p p e a r in conjunction with previously established ontogenetic t i m e t a b l e s of n e u r o n a l o r i g i n , s o r t i n g , a n d n e u r i t e e l a b o r a t i o n , a n d t h e p r e s e n c e of e a r l i e s t b o u n d a r i e s does n o t

FIG. 7. GFAP and J1/tenascin immunolabelingin vivo and in cultured explants of neocortex. Parasagittal sections with dorsal up, and the scale bars = 50 #m in A-E. (A) In the P9 cortex in vivo, GFAP staining is most prominent in astrocytes of the molecular layer (m) and glial limitans (pial-glial cells), with only a few stained astrocytes (e.g., arrow) in other layers. (B) When a slice of neocortex taken at P2 is cultured for 7 days, GFAP labeling again is most prominent within astrocytes of the molecular layer, with scattered GFAP-positive astrocytes present in other layers. Neurons from these explants appear darker, and some of them may be pyknotic. Some of the laminar features appear similar to those seen in the P9 neocortex. (C) J1/tenascin staining in the P2 cortex shows layering (e.g., most apparent in the lower magnification inset), with labeled radial elements in the molecular layer (curved arrow) and alternating light (e.g., straight arrow) and dark staining laminae. (D) Explants cultured from the P2 neocortex for 5 days show more subtle laminar variations in J1/tenascin immunostaining{most apparent in the low magnification inset), with a darker molecular layer and persistence of a lightly stained layer beneath the molecular layer (arrow), but possibly more dense labeling present in all layers in the explant compared with the in vivo cortex (E). (E) J1/tenascin labeling of the explant shown in D resembles that seen in the in vivo neocortex on P7. J1/tenascin labeling on P7 is still somewhat laminar, with a light layer (arrow) in a position similar to that present in the explant.

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necessarily precede premaps of neurons seen with other markers (e.g., lineage markers (62), axonal tracing of blueprint-conveying afferents (19)). (iii) Boundaries can be sculptured by deafferentation and their expression may be activity dependent: (iv) There may be boundary mutants, with some or all boundary constituents being altered as a result of single gene mutations. (v) Boundary constituents persist for given amounts of time in vitro as well as in vivo, and changes in their disposition may reflect their physical/molecular dependence on inputs or activity. The lectin or antibody delineated boundaries described here all appear at times during development when either neurons, their dendrites, or afferents (19) are functionally sorting. Thus, the boundaries could relate to one or all of these processes. We believe that because glial and glycoconjugate boundaries are most prominent when neurons are elaborating their dendrites, and afferents are positioning themselves for synaptogenesis, that boundaries are most likely barriers (52, 70, 72) between functionally different zones. Molecules like J1/tenascin may be inhibitory to neurite growth and thus stabilize an emerging functional arrangement just before the ultimate stabilizing event occurs--synapse formation with accompanying cessation of neurite exploration, and other more permissive macromolecules that work in concert with repulsive molecules would thus help shape neuritic distributions (patterning). Glial and glycoconjugate boundaries that surround barrels or any other functional unit in the developing CNS are unquestionably under the infuence of both genetic programs and extrinsic factors (e.g., afferent axons). The studies of Tolbert and Oland provide dramatic evidence for the interaction between and dependence on mapconveying afferents, glial intermediaries, and neuronal/ dendritic sorting that occurs during development of the olfactory glomerular unit in Manduca sexta (55, 80). It should be emphasized that in our original reports of glial and glycoconjugate boundaries in the developing barrel field (10, 11), we noted that the boundaries appeared before (i.e., P2-P6) the barrel neurons themselves completed their final sorting within their prospective barrels. Thus, our initial speculations on boundary functions (10, 11) included a proposal t h a t map-conveying afferents could interact with glial and glycoconjugate boundaries (10, 11) to help accomplish this functional sorting of postsynaptic target neurons. In a subsequent study looking at the incorporation of tritiated fucose into glycoconjugates during development (75), we noted that the temporal and spatial organization of radiolabeled fucose-incorporated glycoconjugates, compared with lectin cytochemistry and GFAP immunocytochemistry, followed a boundary plan throughout the neuraxis that could also relate to dendritic growth that is certainly occurring in the same time frame in disparate CNS structures. This suggested the possibility that boundaries could be involved in deterring neurites (espe-

cially growing dendrites) from crossing functionally distinct domains. The studies of Tolbert and Oland go beyond speculation regarding the roles for boundaries in organizing dendritic patterning, with their experimental manipulations in Manduca proving a role for glial boundaries in shaping neuritic growth. In a study of a newly uncovered biological phenomenon such as tissue boundary expression, it is important to have several technological approaches as well as some existing body of knowledge to help determine its functions. In the case of transient brain boundaries, it is useful that we already have three different sources of information that can help us propose models of boundary structure and function. Those three sources are some nature and nurture information, genetic and induced perturbation data, and findings on boundaries in more "primitive" species. There is some information available on what boundaries are made of, what cells might make them, how genetic mutants and certain lesions affect boundary expression, and how boundaries are expressed during morphogenesis in certain invertebrate systems. Boundaries and the Reeler M u t a n t Mouse

A gene mutation that affects boundary expression would be useful for determining the structure and function of boundaries. Several such possible mutations are present in the insect world, especially in Drosophila (for review, see (78)), but it is possible that there are boundary mutants in mammals (29, 88). The numerous neurological mutations in mice hold promise for providing us with a mammalian gene mutation that if it does not primarily affect a putative brain boundary gene, it at least causes malformations that include boundary cells and molecules. The reeler mutation certainly affects boundaries in CNS areas affected most severely by the mutation (e.g., the cortices 5, 4, 27), and it is possible that the gene directly involves the altered expression of boundary molecules (54). In the present study, we have now demonstrated abnormal glycoconjugate (including J1/tenascin and the 473 proteoglycan) boundaries in the developing reeler somatosensory cortical barrel field, and at the same time found a strikingly normal boundary delineation of subcortical whisker-associated boundaries in the thalamic barreloid complex and brain stem trigeminus. These findings suggest that thalamocortical afferents, presumably normal in reeler (54, 73), alone are insufficient to induce a normal development of cortical barrels during development. The reeler mouse has been hypothesized to be a problem in cell-cell communication during development (4, 5, 69). Rakic, Sidman, Caviness, and their collaborators have categorized both normal and abnormal circuitry components in reeler (4, 5, 69), with the cortical structures revealing the most gross aberrancies in cytoarchitecture and connectivity, while subcortical structures seem to be fairly unaffected. Developmental studies have

NORMAL AND ABNORMAL BRAIN BOUNDARIES described abnormal migratory patterns of cortical neurons (4, 5), possibly malaligned and altered distributions of glia (54), and altered neurochemistry (26) including glycoconjugate dispositions (54). Goffinet and his collaborators (27) have shown t h a t a variety of adhesion molecules and associated carbohydrate epitopes are expressed in the embryonic reeler brain, but there may be a delay in the appearance of molecules such as L1 and J1 in the mutant cerebral cortex. In the present report, findings on boundaries in the reeler barrel and barreloid fields, and their malleability following sensory deprivation, indicate that a boundary system is in place in reeler. But reeler is clearly a problem in distributing cortical neurons in appropriate laminar arrangements. How might glycoconjugates such as ECM molecules be involved? Caviness and his collaborators (4, 5) pointed to the possibility that the interaction between migrating cortical neurons and their glial guideposts may be abnormal in reeler. This could be mediated by an inappropriate expression of an adhesion molecule, through alterations of the molecule itself or its time of expression, distribution, or amount. The timetables for expression of adhesion and ECM molecules may also be different (27), and this could alter the separation of young neurons from their radial glial fibers during migration and lamination in the reeler cortices. Nakanishi (50) has described altered ECM patterns in the developing reeler cerebral cortex, and Derer has noted that reeler has a problem with its glial limitans on the surface of the cortex that might result from abnormal astrocytic (radial glial) endfeet that do not constitute a continuous glial limitans (15). It is interesting that destruction of meningeal cells using 6-hydroxydopamine during a critical period in development also results in cerebellar cortical malformations including displaced neurons (83) that are not so different from t h a t seen in the reeler cortices. One could envision ways that abnormal glial and glycoconjugate dispositions during early cortical development could result in abnormal interactions with mesodermal cells of the meninges, and how such abnormal interactions might lead to malformations in the radial organization of the cortices. The reeler mouse offers another insight into the expression of boundaries during development. As briefly alluded to above, reeler is an example of how barrel pattern formation comes about from interactive events between afferents and target neurons. If afferents alone were enough to generate functional neuronal patterns in the cortex, as has been suggested in numerous studies (e.g. (19)), then why don't the apparently normal mapconveying thalamocortical projections give rise to a normal barrel field in reeler? There is a reasonably wellconstructed barrel field in reeler, as shown for the first time in the present study using boundary labeling and whisker lesions, yet there is no question that the barrel field is not normal. The barrel boundaries in reeler actually appear "fuzzy." This "fuzziness" could relate to the inability of glial elements to respond to afferent cues to

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down-regulate J1/tenascin, possibly due to a generally excessive expression of the molecule in reeler. A consequence of such an impaired down-regulation could be a relatively imprecise targeting of axons including thalamic afferents. Abnormalities in cell-cell interactions, and misregulation of recognition molecule (e.g., J1/tenascin) expression in the somatosensory cortex of reeler could thus affect afferent patterning. All of this suggests that functional cell and neurite sorting is a result of interactions between afferents, glia, and target neurons, and alterations of any one of these elements can lead to inappropriate patterns of neuronal organization.

How Lesions Affect Boundaries, or How Boundary Expression Might Be Regulated The reeler mouse then shows us that afferents alone may not be able to induce functional patterning in the developing brain. The lesion studies presented here indicate that boundaries can be influenced by afferents (e.g., deafferentation) in both phenotypes. Our previous studies have suggested that boundaries may serve to help position neurons and their dendritic elaborations. As already proposed by Tolbert and Oland, afferents may interact with glia to parcellate dendritic arborizations within a model system in M. sexta that has many similarities with the barrel field (80). Then how might boundaries interact with, or even be partially created by, afferents during normal development and in lesion models? During early development of the barrel field in normal mice, it appears that molecules like J1/tenascin may be rather homogeneously distributed in the prospective layer IV barrel field, but at some point after birth a "hollowing out" of prospective barrel hollows occurs with this molecule showing less expression in the future hollows and more within boundaries (the future interbarrel septae). This event appears to correspond in time with the arrival of map-conveying afferents (e.g., carrying vibrissae topography) from the thalamic barreloid complex. It is therefore possible that the afferents induce a down-expression of molecules such as J1/tenascin. However, whisker lesions during this critical period also result in a down-expression of boundary molecules like J1/tenascin. We have also observed a down-expression of the molecule within the developing neostriatum following pharmacological lesions of the neonatal nigrostriatal pathway (53). Thus, how can one reconcile a down-expression of a "recognition" (or "boundary") molecule upon arrival of an afferent projection with down-regulation that occurs following loss of afferents? During normal development, the arrival of an appropriate number of axons could result in a down-regulation of boundary molecules within their path, but deviations below (and possibly above, we really need to also look at "hyperinnervation" models as well) this number also result in reduced expression of such molecules. In the developing barrel field, as already alluded to under Re-

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sults, afferents might grow into a "sea" of relatively elevated J1/tenascin which they then "hollow out." This hypothesis needs further study in other deafferentation/activity-altering models; however, preliminary studies using explant cultures might offer additional insights into the role of afferents in boundary expression. For example, is it true that the more neuronal afferents, the less J1/tenascin expression? We presented data (Fig. 7) that suggested J1/tenascin expression in cultured neocortical explants is in some ways similar to and in other ways distinct from that seen during development in vivo. In addition to a persistence of expression of J1/tenascin, in laminar patterns, explants reveal a possible increased expression of the molecule compared to age-matched in vivo preparations. Neocortical layers that normally exhibit modest amounts of J1/tenascin immunoreactivity seem to express elevated amounts of the molecule in explants cultured for 5 days. Though quantification of this is required, using other methods, it is possible t h a t these findings suggest that a precise number of afferents is required for normal boundary expression. During normal development, partial deafferentation (e.g., whisker lesions leading to loss of some thalamocortical inputs) may cause a down-expression of J1/tenascin in boundaries. Massive deafferentation, as exists in cultured explants, may result in increased expression due to greater numbers of afferents lost in this "drastic" lesion model. Pilot experiments of placing lesions in the mature cortex suggest that J1/tenascin as well as the 473 proteoglycan are reexpressed in much greater amounts in and around the wound (Laywell et al., unpublished observations). It is therefore possible that severity of lesions during development and in the mature brain have different effects on the induction of J1/tenascin expression. It will be very important to resolve these boundary regulatory possibilities in the future. However, findings presented here from the reeler mouse and the deafferentation studies strongly support the notion that boundary molecules, and possibly even the accumulation of glial cells and their processes in boundaries, are regulated by inductive-interactive mechanisms. It appears most likely that the first afferents set up a glial and glycoconjugate "scaffold" by either their physical arrival, or, as implied by the whisker lesion and immobilization data, through activity. The scaffold is then usefifl for the orientation of the following incoming afferents or for the shaping of dendritic targets in accordance with the afferent distributions. Thus the prime activity of the first afferents sets the scaffold for the followers, pre- and postsynaptically. Then, since the boundaries are no longer needed, it follows that more neuronal activity or the final complement of neuronal activity shuts off J1/tenascin expression. CONCLUSIONS: WHAT ARE BOUNDARIES MADE OF AND WHY MIGHT THEY BE EXPRESSED? Boundaries around developing functional units within the brain are made up, at least in part, of developmen-

tally regulated molecules like J1/tenascin. Glial cells are the major cellular element of boundaries. T h a t does not mean that other cellular elements are not present within boundaries. Indeed, processes of neurons and even neurons themselves have been observed in boundaries and they may be degenerating within them, or even attempting evasive maneuvers in order to avoid or escape these sites. What we do know however is that boundaries may contain repulsive molecules such as J1/tenascin (21), and in the adult these sites tend to be neuronal cell bodyfree zones where either dendrites or axons seem to accumulate (e.g., in the barrel field, developmental boundaries become the interbarrel septae in the adult where dendrites of pyramidal cells and other neurites course to and from supra- and infragranular layers). We believe that astrocytes, expressing the bulk of the boundary molecules, are the primary boundary cells (78). Then one could ask if there are distinct classes of boundary versus nonboundary astrocytes and are they lineage-determined or somehow induced to differentiate along different lines. Astrocytes within boundaries appear to express more J1/tenascin than those of nonboundary neuropilar sites, and again our studies in the barrel field indicate that many astrocytes are present within barrel hollows for example (44), but they do not express significant amounts of GFAP, J1/tenascin, and the 473 proteoglycan under normal conditions during the first postnatal week. It is noteworthy t h a t the timetable for GFAP labeling of boundary glia differs from that of glycoconjugate boundaries. GFAP seems to label boundary astrocytes between P2 and P16, yet this marker does not reveal boundaries before or after these times. It is possible that other markers including vimentin would reveal glial boundary patterns earlier, or perhaps later, but we have not yet explored this. Lectin binding or adhesion and ECM molecule (e.g., J1/tenascin, 473 proteoglycan, AMOG) immunocytochemistry does reveal boundary patterns in the late embryonic through P16 mouse brain, and hence the regulation of glycoconjugate expression in boundaries is distinct from that of at least one glial filament protein examined to date. However, lesions during development do reveal many astrocytes within nonboundary regions of barrels (e.g., the hollows) that obviously possess a distinct biochemistry (e.g., they express less J1/tenascin (44)). How did this come about? Do the glia proliferate differently in different (i.e., boundary versus nonboundary) regions, change their distribution during development from more in boundaries to more uniform in the adult, and/or shift the functional makeup of their surfaces? The possible afferent-inductive mechanisms discussed above could lead to a differential distribution of boundary and nonboundary glia, but these cells may also be programmed to be different (37). Through studies of cell lineage using chimeras (28) or retroviral labeling (62), there are now many examples of lineage-determined differences of both neuron and glial phenotypy. There are genes, per-

NORMAL AND ABNORMAL BRAIN BOUNDARIES haps even those related to oncogenes (84) or the homeobox (29, 88) th at could determine the fate of boundary and nonboundary glia. Yet so little information is currently available in support of genetically determined differences in astrocytypy, and in fact the work of Raft, Noble, Miller, and their colleagues on the glia of the optic nerve support the notion of genetically determined proliferative clocks th at are also susceptible to environmental messengers (e.g., certain growth factors) t ha t can alter the differentiation and ultimate phenotype of glia. Geller, on the other hand, has shown t ha t two types of glia can be found in cultures from embryonic hypothalamus, with one type expressing small amounts oftenascin t h a t is associated with neuritic growth and another type t h a t expresses greater amounts of the glycoprotein and is associated with few if any neurites (25). It was also reported th at these cells may be morphologically different as well. It is possible t ha t boundary glia arise from a stem population th at may be lineage-associated within the subventricular layer during early embryogenesis (Silver, personal communication), or they may be induced to be different by factors such as certain molecules or contact with certain cellular processes (e.g., ingrowing axons). Rather than lamenting a mandate for categorizing all types of glia and associated molecules t hat potentially could have unique effects on neurite support and growth, it may be worthwhile to focus on common cellular and molecular characteristics between certain types of glia-e.g., boundary glia, glia in growing axon pathways, and glia in wounds. Schwab, Caroni, Chiquet, Chiquet-Ehrissman, Faissner, Pesheva, Schachner, Erickson, and others (3, 8, 9, 18, 21, 59) have all noted t ha t different molecules may provide inhibitory cues to deter and perhaps incidently guide axonal growth. Joosten and Gribnau (38) have described different types of astrocytes, revealed by vimentin versus GFAP immunoreactivity, as possibly providing inhibitory as well as instructive cues in helping to guide growing axons of the corticospinal tract. Th e "glial scar" may contain some of the same constituents, plus others (e.g., nonneural cells such as fibroblasts and macrophages (67)), t hat along with boundary elements within pathways affect the sprouting of axons across the wound and toward their denervated target. In addition to having to consider astrocytic-derived factors within a wound, oligodendrocytes, or astrocyte (e.g., perinodal astrocyte) molecules within an axonal pathway, we also have to characterize the glia and glycoconjugates associated with the afferent-deprived target neurons. Th e list of trophic/tropic molecules present in such a scenario is also extensive, but we may have enough information at hand to propose why glia and certain glycoconjugates may provide inhibitory cues to neurons and neurites during development and in the lesioned, mature brain. For example, we do know t hat when the glial and glycoconjugate "tissue boundaries" disappear following the stabilization of functional tissue

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patterns (e.g., barrels), some of the same molecules are no longer expressed, but some of them and some mature forms of adhesion and ECM molecules, in association with mature glial processes, may then become apparent in relation to the cordoning off of synapses (41, 74). As part of glial and neuronal membranes and the ECM, a molecular "caulk" around synapses in the mature brain may serve as a boundary to isolate synaptic messages as well as to insulate the synaptic complex from deleterious molecules or metabolites of synaptic transmission (e.g., excitotoxins), or molecules released as a consequence of trauma (e.g., interleukins, prostoglandins, growth factors, ECM, basal lamina, adhesive proteins, and products of phagocytosis). Here is one scenario: During development of the brain, boundaries may be programmed to be expressed at the interface of two distinct zones. The genetically determined cellular patterning and cell-cell interactions t hat occur at this interface contribute to a unique microenvironment where boundary cells and molecules serve as a landmark between the two zones. It is possible that boundaries are programmed to occupy certain positions between different neuronal cohorts early during development (37), but the existence of such protoboundaries related to "protomaps" (66) or other evolutionary forms of structural parcellation (56) needs a great deal of study. There is more compelling evidence t hat boundaries may actively discourage the crossing of neurites from one zone to another. In axonal pathways during development, boundary cells and molecules may serve to cordon off different sets of axons and again limit sprouting from one compartment to another (the functional organization of white matter mosaic compartments is, unfortunately, more difficult to characterize than th a t of gray matter compartments). Following synaptogenesis, gray matter boundaries are no longer visible and white matter continues to exhibit a myriad of glycoconjugates t hat may be associated with mature oligodendrocytes and astrocytes. Close inspection of neurons following synaptic stabilization reveals an incredibly pervasive network of glycoconjugates on their surfaces that surround synapses (74). In the normal mature brain circuitries are for the most part stable and boundary elements do not reveal any obvious patterns. Following lesions, the induction of glial tissue boundaries by the aforementioned lesion-associated molecules is associated with the wound, the axonal pathways, and the synaptic targets. Boundary molecules may deter neurite growth in the wound or the projections between the wounded structure and its targets or they may be counteracted by molecules that support growth. Particular inhibitory molecules expressed by astrocytes or oligodendrocytes (3, 21, 59, 72) may normally prevent undesired axonal sprouting during "standard operating procedures" within the normal adult white matter. Following lesions, glia may be involved in synaptic remodelling (61) within targets as well as in neuropilar areas surrounding a wound, and an

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increased expression of glycoconjugates like J1/tenascin (Laywell et al., unpublished observations) may also protect and secure synaptic arrangements t hat have not been compromised by the lesion. In such a prospectus the battle between inhibition of and inspiration for regeneration is dependent upon the ratios of these various molecules. Yet, it is possible t hat inhibition often prevails in order to preserve t hat which is functionally intact, as well as to deter formation of anomalous synaptic connections. T he mature brain offers other impediments to reestablishing exact circuitry arrangements t hat are not found in the developing brain as well, for example, an established complex network of projection systems t hat span great distances between disparate structures.

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17. ACKNOWLEDGMENTS We thank Dr. Reinhard J a h n for his generous gift of the p38 antibody. The work presented in this report was supported by N I H / NINDS Grant NS20856 and NSF Grant BNS-8911514 to D.A.S. and by DFG (SFB 317) Grant to M.S. and A.F. REFERENCES 1.

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