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Microtubular disarray in cortical dendrites and neurobehavioral failure. II. Computer reconstruction of perturbed microtubular arrays
Developmental Brain Research, 5 (1982) 299-309 Elsevier Biomedical Press
299
Microtubular Disarray in Cortical Dendrites and Neurobehavioral Failure. II. Computer Reconstruction of Perturbed Microtubular Arrays N. BODICK, J. K. STEVENS, S. SASAKI and D. P. PURPURA*
Department of Neuroscwnce and the Rose F. Kennedy Center for Mental Retardation and Human Development, Albert Einstein College of Medicine, Bronx, N Y 10461 (U.S.A.) and (J.K.S. and S.S.) Playfair Neuroscience Unit, University of Toronto, Toronto Western Hospital, 399 Bathurst Street, Toronto, Ont. MST 2S8 (Canada)
(Accepted March 26th, 1982) Key words: neurobehavioral failure - - mental retardation - - dendritic vancosities - - microtubules - cytoskeleton - - computer reconstruction
A previous report a details morphological alterations in dendritic structure of corUcal neurons m severe neurobehavioral retardation of unknown etiology. Using computer graphic techniques, the present study describes perturbations m the 3dimensional character of the microtubular array, which correspond to degenerative change in dendritic geometry. In large proximal processes, two types of array have been reconstructed. Segmented microtubules may form a continuous helical swirl which underlies a bulge in the dendriUc cylinder. Alternatively, small groups of microtubules, while maintaining orderly internal orgamzatlon, may be disoriented with respect to the long axis of the process. In varicose regions of the dendrite the rmcrotubular array is discontinuous. Microtubules course side by side through constricted regions, only to splay out and terminate within expanded regions. These pathological alterations in the mlcrotubular array contrast sharply with the cortical dendritic microtubular array reconstructed from the normal adult mouse. Perturbation in those parameters which determine packing of microtubules within the dendritic process is also documented. In the pathological condition, microtubules lose the abihty to exclude one another from close approach. The role of cross-linking molecules m maintaining the integrity of the microtubular array, and the role of microtubules in maintaining the geometry of the dendrite, are considered. INTRODUCTION T h e preceding r e p o r t 9 describes changes in cortical n e u r o n s in 5 b i o p s i e d cases o f severe neurod e v e l o p m e n t a l r e t a r d a t i o n . These alterations include f o r m a t i o n o f varicosities along distal dendritic processes, loss o f cylindrical s y m m e t r y in p r o x i m a l processes, p r e d o m i n a c e o f long thin spines, a n d perturbation of normal microtubule deployment within these dendrites. The present study a t t e m p t s to establish a relation between changes in the microt u b u l a r a r r a y a n d changes in the external m o r p h o l ogy o f i n d i v i d u a l cortical cells. The basis for this s t u d y derives f r o m two n o t i o n s : (1) m i c r o t u b u l e s f o r m a structurally i n t e g r a t e d a r r a y within the dendrite; a n d (2) the external f o r m o f the dendrite is d e p e n d e n t on the integrity o f t h a t array. The validity o f the first s t a t e m e n t is reinforced b y recent studies o f K i m et al. 7 a n d Shelanski et a1.13,14. Cross-linking
functions, b o t h between m i c r o t u b u l e s a n d between m i c r o t u b u l e s a n d neurofilaments, have been identified b y b i o c h e m i c a l a n d m i c r o s c o p i c methods. T h e applicability o f the second statement is largely untested. W h i l e drugs which interfere with the p o l y m e r i z a t i o n o f t u b u l i n affect cell shape in vitro15, le, the mechanistic role o f m i c r o t u b u l e s in the genesis a n d m a i n t e n a n c e o f n e u r o n a l shape remains to be elucidated. Recently d e v e l o p e d c o m p u t e r graphic techniques 17,1s have been used to describe the structural p a r a m e t e r s o f the n o r m a l dendritic m i c r o t u b u l a r a r r a y 12. It is against this b a c k d r o p that the microt u b u l a r d i s o r g a n i z a t i o n in n e u r o b e h a v i o r a l failure is d o c u m e n t e d . The present study correlates local, p a t h o l o g i c a l l y derived p e r t u r b a t i o n s in the integrity o f the m i c r o t u b u l a r a r r a y with local changes in dendritic geometry.
* Present address for correspondence: Dr. D. P. Purpura, Stanford Umversity School of Medicine, Office of the Dean, Stanford, CA 94305, U.S.A. 0165-3806/0000-0000/$03.00 © 1982 Elsevier Biomedical Press
300 MATERIALS AND METHODS Cortical biopsies from infants between 4 and 21 months in age were obtained as described previously 9, and normal mouse neocortex was prepared by similar procedures. With methods described elsewhere1,17 series of 100 or fewer consecutive thin (silver gold) sections were collected on Formvarcoated ultra-rigid slot grids (Synaptec, Toronto). Sections were post-stained in 2 % uranyl acetate and lead citrate and photographed at 5000 × in the Jelco 100 cx or the Philips 200. Micrographs were copied at high resolution and in sequence onto 35 mm film. The film was then mounted in a high speed film transport which interfaces with a video-computer graphic device previously described 17, and ultrastructural features of individual dendrites were traced mto computer memory. Critical to reconstruction at high resolution ~s the process of microalignment. For various reasons (sublimation of Epon in the electron beam, differential compression during sectioning, lens aberration) sequential electron micrographs cannot be perfectly superimposed. Microalignment compen-
sates for this distortion by creating, ['or each feature to be reconstructed (e.g. for each microtubule), a separate alignment set. After a ~tructure has been aligned in this local, high resolution fashion it ts traced into computer memory l-or many structures, mtcroahgnment is essentml to the tracing process. Without it, continuity ts lost. Graphic display of individual dendrites was accomphshed with computer methods already descnbed17,1s. RESU LTS
Computer display of dendritic process'es Because few descriptions of the neuronal microtubular array have been published'-', 3.12, it is important to characterize the normal structure of cortical dendrites in this regard. The mouse was selected for this purpose because it is readily accessible, and because it is likely that much of the character of the dendritic cytoskeleton is conserved across species 12. A computer-generated plot of a cortical dendrite from the 24-day-old mouse is shown in Fig. 1. Because only a small portion of the cell has been
Fig. 1. Cortical dendrmc process of the 24-day-old mouse. Various spine types (thin, mushroom-shaped, stubby) extend from a long cyhndrlcal process. Within the dendrite, the microtubules course beside one another, occasionally exchanging nearest neighbors. Those regions inside the process which are devoid of microtubules are largely occupied by mitochondria and by smooth endoplasmic reticulum. Asterisks denote synapses. Bar I ,,m
301 reconstructed, cell type cannot be determined unambiguously. However, given the size and orientation of the process, it is likely to be part of the apical arborization of a pyramidal cell. The external features of the process are also consistent with those of a Golgi-impregnated pyramidal cell. In particular, the various types of spines (thin, mushroomshaped and stubby) project from an essentially smooth cylindrical shaft. Both axodendritic and axospinal-dendritic synapses are noted. Multiple synapses often occur on a single spine. Important to the present study is the arrangement of microtubules within the dendritic shaft. They form a largely parallel, interdigitated array, which tends to occupy a central core of the dendritic cylinder. The reconstructed microtubules are discrete, relatively short segments with an average length calculated 3 to be 8.5/~m. The array is interrupted by the membranous inclusions of the cytosol:mitochondria and smooth endoplasmic reticulum. In fact, individual tubules very often terminate near these membranous structures as well as near the plasma membrane. Although the microtubular array is largely parallel, it is not entirely orderly. Nearest neighbors can change along the length of any given microtubule and, in that sense, the array has a minimally woven quality. The normal dendrite is filled with microtubules and membranous components. It is rare to find an expanse of cytosol without either. As noted above, the microtubules tend to occupy a central core of the dendritic cylinder. Outside of this core, in the region under the plasma membrane, large saccules of smooth endoplasmic reticulum and mitochondria predominate. Although the number of reconstructed tubules (55) is not large, a range of microtubule lengths is observed. The largest microtubule extends beyond the length of the reconstruction (10 #m), while the shortest is 0.4/zm in length. Microtubular arrays within cortical dendrites in neurobehavioral failure Seven dendritic segments from cases 1, 2 and 3 have been reconstructed to reveal changes m the cytoskeleton which accompany changes in neuronal form. Proximal and distal segments of the dendrite
exhibit profound alteration m their microtubular arrays 9. However, the effects in each region are dissimilar enough to merit separate description. Perturbed microtubular arrays in proximal processes In proximal regions, alterations in dendritic form include thinning of the shaft, formation of irregular bumps on the cylindrical surface, loss of mushroomshaped and stubby spines and predominance of long, thin, often tortuous spines. Two basic types of perturbed array are found to underlie changes in proximal dendritic form. An example of the first type of proximal microtubular disarray has been reconstructed from the biopsy tissue of case 2 (Figs. 2 and 3). The microtubular array has lost many of the qualities described for the normal cortical cell. (A characteristic micrograph is shown in ref. 9, Fig. 5B.) Microtubules are no longer parallel to one another, or to the long axis of the cell. Focally, microtubules spiral along the length of
Fig. 2. A cutaway view of a proximal cortical dendrite reconstructed from case 2 (ref. 9). A slight swelling in the p r o c e s s occurs in the region where the rnicrotubules form a hehcai swirl. Inside the dendrite, mitochondna occupy s o m e o f the interstices in the microtubular array. However, relatively large regions of cytoplasm are devoid of microtubules or membranous c o m p o n e n t s . Bar = 1 ~ m .
302
Fig. 3. Mlcrotubular disarray m the same dendrite shown m Fig. 2. Spines are reduced in number relatwe to the corncal process of the normal mouse M~crotubules form a continuous helical swirl as they course through the process. Many short fragments are observed. The mlcrotubules frequently display radical curvature. One short fragment occupies a spine neck (lower left). Asterisks represent synapses Bar 1 jim
the process. The continuity of the microtubular array is loosely maintained as all of the microtubules curve in unison, completing a helical turn. Short fragments of microtubules, with seemingly random orientation, are inte~ spersed among the longer tubules which form the helical swirl. On close examination, individual microtubules both inside and outside of the helical swirl occasionally bend sharply, and do so in a manner independent of the direction taken by neighboring microtubules. In a most striking example, a single microtubule turns, leaving its neighbors, to enter the neck of a dendritic spine This is noteworthy in that microtubules are not part of the normal ultrastructure of the spine. The 65 reconstructed microtubules have an average calculated 3 length of 3.5 #m with a range extending from 0.15 t~m for the short fragments to
greater than 6.5 /,m for microtubules extending beyond one border of the reconstruction. The short fragments, seemingly disconnected from the rest of the microtubular array, can demonstrate radical curvature, at times doubling back upon themselves reside a diameter of 0.3/~m. As is the case in mouse dendrites, mierotubules often terminate near vesicles of smooth endoplasmlc reticulum and near mitochondrm. However~ ~t ~s more common, especially for the shorter microtubules, for terminations to occur m the cytoplasm. An ultrastructure for mlcrotubule endings has been previously suggested 3. The present preparation shows some evidence of diffuse and filled terminations but, as noted previously ~, tile classification is often difficult if not ambiguous. in the process of forming the hehcal swirl, microtubules have seemingly left behind relatively large expanses of cytoplasm (Fig. 2). These volumes approaching a cubic/tin, are bare of both microtubules and membranous structures, and ~eemmgly have no counterpart m the normal corttcal dendrite described above. In this case, the helical swIrl underlies a shght swelling in the profile of the process, while those parts of the array which maintain the semblance of parallel deployment underhe a region of the process with a more cylindrical profile (Figs 2 and 3) A second type of microtubular disarray is evident in the proximal process displayed in Fig. 4 (case 1). (A characteristic micrograph is found in rel: 9, Fig. 4B.) Microtubules maintain parallel deployment with respect to their immediate neighbors. However, groups of mlcrotubules are disoriented, both with respect to other groups and with respect to the long axis of the process. Reconstruction reveals occasional microtubules which turn rather sharply to run from one group to a second group with a grossly different axis. However, it is also clear that many microtubules begin and end with a single group. While this disarray may correspond to some asymmetry m the process, this is not the normal cytoskeletal structure which underlies a curving process. In curving dendrites, reconstructed from normal tissuO 2, individual microtubutes change orientation, with respect to one another, continually and gradually, wJthout the abrupt transitions m axial ahgnment observed in the pathological case.
303
Fig. 4. Cutaway view of a large cortical dendrite reconstructed from case 1. Few spines are in evidence.Within the process, small groups of microtubules are disoriented with respect to each other and with respect to the long axis of the process. Individual microtubules occasionallyturn sharply to run from one group to another. Most often, however, microtubules begin and terminate w~thln a group. Asterisks represent synapses. Bar = 1 pm. Perturbed microtubular arrays in distal processes Varicose processes (Figs. 5 and 6, cases 2 and 3 respectively) have shapes characteristic of more distal portions of the dendritic arborization defined in Golgi preparations. In general, expanded regions of the process are connected by thin necks which range from 0.5 to 10 /~m in length. The number of dendritic spines is reduced, and those spines which do occur are most often of the long, thin type. Within the field of view in Fig. 5, no spines project from the neuronal surface. Synapses persist along the length of the process, both on varicose and constricted segments. The 3-dimensional ultrastructure described below is typical for all varicose dendritic segments reconstructed from cases 1, 2 and 3. (Micrographs characteristic of these processes are shown in ref. 9, Fig. 6.) Membranous structures, both mitochondria and smooth endoplasmic reticulum, extend through varicose and constricted regions of the process. Again, individual microtubules often terminate near these structures. The microtubular array itself displays a periodicity synchronous with the occurrence of varicosities along the length of the dendrite. Within constricted neck regions microtubules maintain the semblance of parallel array. In these regions a small number of mlcrotubules course beside one another, maintaining their long axis parallel to the axis of the
process. Upon entering the varicosity, the microtubules most often splay away from one another and terminate within the varicosity. These terminations may be near vesicular smooth endoplasmic reticulum, near mitochondria, or in free cytoplasm. Most notably, the microtubular terminations within the varicose regions create distinct discontinuities in the microtubular array. These cytoskeletal elements no longer form a roughly parallel lattice which extends through the entire length of the process. One small varicosity (Fig. 5) does not exhibit microtubular discontinuity. Nonetheless the microtubules do splay away from one another within its volume. Rotation of the process displayed in Fig. 5 shows that the microtubules form long spirals as they course through the varicose section of the process. The most apt comparison to these pathologically altered varicose processes is perhaps to those spontaneously occurring varicosities in normal neurons. The A2 amacrine cell of the cat retina has been reconstructed using similar methods (Stevens, manuscript in preparation). In this case, small smooth varicosities occur at regular (approximately 10 #m) intervals along the length of the process. While the external appearance of the A2 amacrine dendrite is similar to that of a pathologically altered process, the underlying microtubular arrays are dissimilar. Importantly, in the amacrine cell microtubules form a continuous system through the entire length of the
304
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Fig. 5. A a n d B: a varicose dendrite reconstructed f r o m case 2 m two rotated wews. N o spines are evident. Mlcrotubules course t h r o u g h the thin segments o f the process a n d splay a w a y f r o m one a n o t h e r on entering t h e varicosity. T h e varicosities represent regions o f discontinuity in the m i c r o t u b u l a r array. M i t o c h o n d r i a a n d s m o o t h e n d o p l a s m i c retlculum are f o u n d in both constricted a n d varicose regions. Asterisks denote synapses. Bar =: 1 tern. Fig. 6. A varicose process reconstructed f r o m case 3. O n e s p i n e is visible. In general, rracrotubules occupy the constricted regions of the process while the varicosities represent areas o f discontinuity in the m i c r o t u b u t a r array. Asterisks represent synapses. Bar = 1
~m.
305 process. Each varicosity corresponds to an underlying mitochondrion positioned beside the continuous microtubule array. In this instance, the normally occurring mature dendritic varicosity appears to have a distinctly different 3-dimensional ultrastructure.
Histograms have also been generated for large cortical cells of the spiral array types from cases 1 and 2 (ref. 9). Specifically, those areas in which the array is relatively well conserved (microtubules in parallel array) have been analyzed. In areas where microtubules follow more tortuous paths, it is difficult to determine distance of closest approach. In each of the pathological cases (Fig. 7), the average distance between microtubular pairs is slightly reduced relative to mouse cortex. Important for present purposes, is the distance of closest approach between microtubules. For case 1, this value is 0.025 /am, for case 2 it is 0.03/am. Each of these values is considerably smaller than values obtained for normal cells.
Relationship between adjacent microtubules A graphics program 12 has been devised to calculate the perpendicular distance (center to center) between nearest neighboring microtubules. Histograms which are assembled from large ( > 350) numbers of these measurements, display inter-microtubular distances as a function of their frequency in the microtubular array. Inter-microtubular histograms (Fig. 7) for the mouse cortex were calculated from the same preparation used for the graphic reconstruction of cortical dendrites and are similar to those generated from the retinal ganglion cell of the cat 12. The average distance between microtubules is 0.10/am and the distance of closest approach in this population is 0.05/am.
DISCUSSION
Pathological changes in the microtubular array Golgi and EM analyses of cortical biopsies obtained from 5 children suffering severe neurobehavioral retardation have revealed striking changes in the dendritic systems of cortical neurons a. These
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Fig. 7. Inter-microtubule distance histograms. The perpendicular distance between nealest neighbor pairs is plotted as a function of its frequency for the mouse cortical process (A), case 1 (B) and case 2 (C). Each plot is generated from more than 350 measurements. In the pathological cases, measurements were taken in areas of the dendrite in which the parallel character of the array is maintained. The abscissa is calibrated in microns.
306 changes include formation of varicoslties m distal processes, irregular and thin proximal processes, and the predominance of long thin spines throughout the arborization. The present study describes the alterations m the 3-dimensional structure of the m~crotubular array, which accompany these changes in dendritic geometry. For purposes of comparison, the mlcrotubular array within a cortical dendrite of the normal mouse has been reconstructed (Fig. 1 I. The array is made up of relatively short, segmented mlcrotubules which course beside one another, occasionally exchanging neighbors, along the length of the procesb. In forming this array, microtubules strictly exclude each other from close approach (Figs. 7 and 8). This structure is similar to that determined for the dendrite of the retinal ganglion cell~K In the pathological condition, microtubules of the proximal dendritic process turn and twist with respect to one another and with respect to the long ax~s of the process. Two basic patterns have been reconstructed (Figs. 3 and 4). The array may comprise a continuous helical swM involving all microtubules in the locale, or the array may be made up of small groups of tubules which, while maintaining parallel internal orgamzation, are disoriented with respect to each other and with respect to the long axis of the process, in forming these bizarre arrays, microtubules do not consistently exclude one another from close approach. Inappropriately placed microtubules (for example in dendritic spines) and short fragmentary microtubules are also observed In the distal region of the arborlzation, where pathological change includes the formation of varicose processes, the continuity of the microtubular array is lost. In the normal dendrite, microtubules interdigltate along the entire length of the process In the degenerative varicosity (Figs. 5 and 6), expanses of cytosol within the expanded regmn separate clusters of microtubules contained within the intervening constricted segments. It may be reasonable to think that microtubule length is affected in the pathological process. Average length in the large cortical dendrite of the mouse (8.5 #m) is greater than that of the large pathologically altered process (3.5/~m). Breakage of mlcrotubules as a result of stresses imposed by the twisted array might be anticipated. However. it
remains difficult to compare this parameter across species. In fact, variations m average length have been described across cell typesK across species :3,~ and may be anticipated m different regions of mdlwdual neurons 5. A more constant parameter may be the distance measured perpendicular to the long axis, between nearest neighboring dendritic mlcrotubules. The average inter-microtubular distance, and distance of closest approach for cat retinal ganglion dendrltes 1", for mouse cortical ceil dendrites (Fig. 71, and for in vitro preparations of tubuhn-MAP2 polymers 7, are very similar It is suggested that this packing parameter for dendritic microtubules may be conserved across many neuronal types. However, in the pathological condition, microtubules have lost the abihty to exclude one another from close approach. These changes m microtubular packing w~thin the process are documented m inter-microtubular histograms (Fig. 7J. Possible mechan&ms in the perturhatton oj the mtcrotubular array attd alteratton in dendritic geontetr~ In Golgl preparations, it ~s apparent that individual cells, in the pathological condition, vary m the extent of varicosity tbrmation Cells appear in various stages of degeneration in each of the biopsies. This suggests that varmoslty formatmn may not be directly dependent on conditions external to the cell. It is unlikely that a diffusable substance or osmotic condition directly triggers varicosity formatlon in all cells simultaneously Rather, varicosity formation and the underlying microtubular disarray would seem a more subtle response of the cell's metabolism to external conditions or to an independently expressed anomaly in the cell's development. With the above considerations in mind, recent suggestions about the role of MAP2 in the cytoskeleton are noteworthy. In double fluorescence labeling of neuroblastoma 6, anti-MAP2 and antitubulin show similar distribution~ m neurites. We have previously suggested 12 that MAP2 or a similar molecule is at least in part responsible for maintaining the mlcrotubular array within the dendrite. MAP2 would do so by binding to tubulin and creating a cyhndrical volume around each microtubule which excludes other similarly bound microtubules from close approach In ~vddltlon, attractzve
307
Fig. 8. Cortical dendrites of normal mouse (A) and tissue samples of case 2 (B). In addition to the disorientation of microtubules in the dendritic pathology, reduction in the distance of close approach is evident. In the pathological condition, microtuhules (oriented perpendicular to the plane of section) are seen in close approximation (0.03 pm center to center), while in the normal cell, minimal distance is larger (0.05 #m). Bar ----0.5/~rn.
308 forces between the p e r i p h e r a l regions o f these cylindrical v o l u m e s c o u l d gel the m i c r o t u b u l e s into a coherent, s t r u c t u r a l l y d e t e r m i n e d array. T h e studies o f K i m et al. 7 can be interpreted as s u p p o r t i v e o f this structure. E l e c t r o n m i c r o g r a p h s o f pelleted fractions o f purified t u b u l i n reveal r e d u c e d inter-microt u b u l a r distances in the absence o f M A P 2 . A d d i t i o n o f M A P 2 to the p o l y m e r i z a t i o n mixture induces larger, a n d m o r e regular spacings between microtubules. I n t e r - m i c r o t u b u l a r h i s t o g r a m s o f cortical b i o p s y tissue show t h a t in the dendritic p a t h o l o g y , microtubules lose the ability to exclude one a n o t h e r from close a p p r o a c h . This i n a p p r o p r i a t e p a c k i n g o f mtc r o t u b u l e s within the a r r a y m a y reflect m a l f u n c t i o n of, or r e d u c t i o n in M A P 2 . W i t h the loss o f M A P 2 function, it ~s p o s t u l a t e d , the integrity a n d the o r g a n i z a t i o n o f the m i c r o t u b u l a r a r r a y is impaired. O t h e r features o f the r e c o n s t r u c t e d array, m i c r o tubules in spurious p o s i t i o n s and i n a p p r o p r i a t e c u r v a t u r e o f m i c r o t u b u l e s also suggest the b r e a k d o w n o f those m e c h a n i s m s responsible for maintaining regular a l i g n m e n t a m o n g n e i g h b o r i n g m~-
m a t t e r o f speculation. Changes m the a r r a y m a y induce changes in shape, or changes in the a r r a y m a y be s e c o n d a r y to o t h e r p a t h o l o g i c a l m e c h a nisms. W h i l e the present study c a n n o t differentiate between these alternatives, recent w o r k on the gangliosidoses 8,1°.m19 can be b r o u g h t to bear. In these disorders, a p p a r e n t l y evolving from the fmlure o f r e g u l a t i o n o f m e m b r a n e mass, the cellular g e o m e t r y is d i s t o r t e d by the p r o l i f e r a t i o n o f m e m b r a n o u s inclusions in the cytosol a n d b,~ the f o r m a t i o n o) meganeurites near the a x o n hillock. W i t h i n these processes, m i c r o t u b u l a r structure is intact. In sp~tc o f the p r o d i g i o u s l o a d o f m e m b r a n o u s inclusions. m i c r o t u b u l e s course in parallel array. A t least in this dmorder, ~t is a p p a r e n t t h a t the a r r a y is r o b u s t ; that changes in cellular g e o m e t r y do not induce alterattons in the c h a r a c t e r o f the m i c r o t u b u l a r array. The possibility r e m a i n s t h a t flaws intrinsic to the cytoskeleton m a y induce p a t h o l o g i c a l change in dendritic g e o m e t r y
crotubules. The r e l a t i o n s h i p between a l t e r a t i o n in the mtcrot u b u l a r a r r a y a n d a l t e r a t i o n in cell shape r e m a i n s a
This w o r k was s u p p o r t e d m p a r t b y N I H G r a n t HD-01799 a n d M R C M A 7345.
REFERENCES
9 Purpura, D. P, Bodlck, N., Suzuki, K., Rapm, l. and Wurzelmann, S., Mlcrotubular disarray m cortical dendrites and neurobehavioral fadure. 1 Golgl and electron mtcroscopic studies, Develop. Brain Res., 5 (1982) 287-297 10 Purpura, D. P., Pappas, G D and Baker, H J., Fine structure of meganeurites and secondary growth processes in fehne GMt gangliosidosis, Brain Res., 143 (1978) 1-12. 11 Purpura, D. P. and Suzuki, K , DlStOmon of neuronal geometry and formation of aberrant synapses in neuronal storage disease, Brain Res., 116 (1976) 1-21. 12 Sasakl, S., Stevens, J. K. and Bodlckj N , Serial reconstruction of microtubular arrays within dendrites of the cat retinal ganghon cell: the cytoskeleton of a vertebrate dendrite, Brain Res., in press. 13 Shelartski, M. L. and Liem, R. K. H., Neurofdaments, J Neurochem., 33 (1979) 5-13. 14 Shelanskl, M. J., Letterier, J. F. and Liem, t L K . H., Evidence for interactions between neurofilaments and microtubules, cytoskeletons and the architecture of the nervous system, Neurosct. Res. Progr. Bull., 19 (1981) 32-43. 15 Solomon, F , Neuroblastoma cells recapitulate their detailed neur,te morphologies alter reversible microtubular d)sassembly, Cell, 21 (1981)) 333-338 16 Solomon, F and Magendantz, M , Cytochalasm separales rmcrotubule disassembly from I~,~s of asymmetrw moT-
1 Bodlck, N., The self assembly o/the embryomc optic net re mto a retinotopically ordered array, Ph.D. thesis, Columbia University, 1980. 2 Bray, P. and Bunge, M. P , Serml analysis of mlcrotubules in cultured rat sensory axons, J. NeuroeytoL, 10 (1981) 589-605. 3 Chalfie, M. and Thomson, J. N., Organization of neuronal microtubules in the nematode Coenorhabditis elegam, J. Cell Biol., 82 (1979) 278-289. 4 Ellias, S. A. and Stevens, J. K., The dendritic varicosity a mechamsm for electrically isolating the dendrites of cat retinal amacrine cells? Brain Res., 196 (1980) 365-372. 5 Gonzes, I. and Sweadner, K. J., Multiple tubuhn forms are expressed by a single neurone, Nature (Lond.), 294 (1981) 477-480. 6 Isant, J. G. and Mclntosh, R., Microtubule-assoclated proteins. A monoctonal antibody to MAP brads to differentiated neurons, Proc nat Acad Sci. U.S A.. 77 (1980) 4741-4745. 7 Kit'n, H., Binder, L. I. and Rosenbaum, J. L., The periodic assocmtlon of MAP2 with. brain microtubules m wtro, J Cell Biol, 80 (1979) 266-276. 8 Purpura, D. P. and Baker, H. J, Neunte induction m mature cortical neurons m feline GMt-ganghoslde storage diseases, Nature (Lond.), 266 (1977) 553-554
ACKNOWLEDGEMENTS
309 phology, J. Cell. Biol., 89 (1981) 157-161. 17 Stevens, J. K., Reconstructing neuronal microc~rcuitry, Bull. Microscop. Soc. Canada, 9 (1980) 4-11. 18 Stevens, J. K., Davis, T. L., Friedman, N. and Sterling, P., A systematic approach to reconstructing microcircuitry by electron microscopy of serial sections, Brain
Res. Rev., 2 (1980) 265-293. 19 Walldey, S. U., Wurzelmann, S. and Purpura, D. P., Ultrastructure of neurites and meganeurites of cortical pyramidal neurons in feline gangliosidosls as revealed by the combined Golgi-EM technique, Brain Res., 211 (1981) 393-398.
299
Microtubular Disarray in Cortical Dendrites and Neurobehavioral Failure. II. Computer Reconstruction of Perturbed Microtubular Arrays N. BODICK, J. K. STEVENS, S. SASAKI and D. P. PURPURA*
Department of Neuroscwnce and the Rose F. Kennedy Center for Mental Retardation and Human Development, Albert Einstein College of Medicine, Bronx, N Y 10461 (U.S.A.) and (J.K.S. and S.S.) Playfair Neuroscience Unit, University of Toronto, Toronto Western Hospital, 399 Bathurst Street, Toronto, Ont. MST 2S8 (Canada)
(Accepted March 26th, 1982) Key words: neurobehavioral failure - - mental retardation - - dendritic vancosities - - microtubules - cytoskeleton - - computer reconstruction
A previous report a details morphological alterations in dendritic structure of corUcal neurons m severe neurobehavioral retardation of unknown etiology. Using computer graphic techniques, the present study describes perturbations m the 3dimensional character of the microtubular array, which correspond to degenerative change in dendritic geometry. In large proximal processes, two types of array have been reconstructed. Segmented microtubules may form a continuous helical swirl which underlies a bulge in the dendriUc cylinder. Alternatively, small groups of microtubules, while maintaining orderly internal orgamzatlon, may be disoriented with respect to the long axis of the process. In varicose regions of the dendrite the rmcrotubular array is discontinuous. Microtubules course side by side through constricted regions, only to splay out and terminate within expanded regions. These pathological alterations in the mlcrotubular array contrast sharply with the cortical dendritic microtubular array reconstructed from the normal adult mouse. Perturbation in those parameters which determine packing of microtubules within the dendritic process is also documented. In the pathological condition, microtubules lose the abihty to exclude one another from close approach. The role of cross-linking molecules m maintaining the integrity of the microtubular array, and the role of microtubules in maintaining the geometry of the dendrite, are considered. INTRODUCTION T h e preceding r e p o r t 9 describes changes in cortical n e u r o n s in 5 b i o p s i e d cases o f severe neurod e v e l o p m e n t a l r e t a r d a t i o n . These alterations include f o r m a t i o n o f varicosities along distal dendritic processes, loss o f cylindrical s y m m e t r y in p r o x i m a l processes, p r e d o m i n a c e o f long thin spines, a n d perturbation of normal microtubule deployment within these dendrites. The present study a t t e m p t s to establish a relation between changes in the microt u b u l a r a r r a y a n d changes in the external m o r p h o l ogy o f i n d i v i d u a l cortical cells. The basis for this s t u d y derives f r o m two n o t i o n s : (1) m i c r o t u b u l e s f o r m a structurally i n t e g r a t e d a r r a y within the dendrite; a n d (2) the external f o r m o f the dendrite is d e p e n d e n t on the integrity o f t h a t array. The validity o f the first s t a t e m e n t is reinforced b y recent studies o f K i m et al. 7 a n d Shelanski et a1.13,14. Cross-linking
functions, b o t h between m i c r o t u b u l e s a n d between m i c r o t u b u l e s a n d neurofilaments, have been identified b y b i o c h e m i c a l a n d m i c r o s c o p i c methods. T h e applicability o f the second statement is largely untested. W h i l e drugs which interfere with the p o l y m e r i z a t i o n o f t u b u l i n affect cell shape in vitro15, le, the mechanistic role o f m i c r o t u b u l e s in the genesis a n d m a i n t e n a n c e o f n e u r o n a l shape remains to be elucidated. Recently d e v e l o p e d c o m p u t e r graphic techniques 17,1s have been used to describe the structural p a r a m e t e r s o f the n o r m a l dendritic m i c r o t u b u l a r a r r a y 12. It is against this b a c k d r o p that the microt u b u l a r d i s o r g a n i z a t i o n in n e u r o b e h a v i o r a l failure is d o c u m e n t e d . The present study correlates local, p a t h o l o g i c a l l y derived p e r t u r b a t i o n s in the integrity o f the m i c r o t u b u l a r a r r a y with local changes in dendritic geometry.
* Present address for correspondence: Dr. D. P. Purpura, Stanford Umversity School of Medicine, Office of the Dean, Stanford, CA 94305, U.S.A. 0165-3806/0000-0000/$03.00 © 1982 Elsevier Biomedical Press
300 MATERIALS AND METHODS Cortical biopsies from infants between 4 and 21 months in age were obtained as described previously 9, and normal mouse neocortex was prepared by similar procedures. With methods described elsewhere1,17 series of 100 or fewer consecutive thin (silver gold) sections were collected on Formvarcoated ultra-rigid slot grids (Synaptec, Toronto). Sections were post-stained in 2 % uranyl acetate and lead citrate and photographed at 5000 × in the Jelco 100 cx or the Philips 200. Micrographs were copied at high resolution and in sequence onto 35 mm film. The film was then mounted in a high speed film transport which interfaces with a video-computer graphic device previously described 17, and ultrastructural features of individual dendrites were traced mto computer memory. Critical to reconstruction at high resolution ~s the process of microalignment. For various reasons (sublimation of Epon in the electron beam, differential compression during sectioning, lens aberration) sequential electron micrographs cannot be perfectly superimposed. Microalignment compen-
sates for this distortion by creating, ['or each feature to be reconstructed (e.g. for each microtubule), a separate alignment set. After a ~tructure has been aligned in this local, high resolution fashion it ts traced into computer memory l-or many structures, mtcroahgnment is essentml to the tracing process. Without it, continuity ts lost. Graphic display of individual dendrites was accomphshed with computer methods already descnbed17,1s. RESU LTS
Computer display of dendritic process'es Because few descriptions of the neuronal microtubular array have been published'-', 3.12, it is important to characterize the normal structure of cortical dendrites in this regard. The mouse was selected for this purpose because it is readily accessible, and because it is likely that much of the character of the dendritic cytoskeleton is conserved across species 12. A computer-generated plot of a cortical dendrite from the 24-day-old mouse is shown in Fig. 1. Because only a small portion of the cell has been
Fig. 1. Cortical dendrmc process of the 24-day-old mouse. Various spine types (thin, mushroom-shaped, stubby) extend from a long cyhndrlcal process. Within the dendrite, the microtubules course beside one another, occasionally exchanging nearest neighbors. Those regions inside the process which are devoid of microtubules are largely occupied by mitochondria and by smooth endoplasmic reticulum. Asterisks denote synapses. Bar I ,,m
301 reconstructed, cell type cannot be determined unambiguously. However, given the size and orientation of the process, it is likely to be part of the apical arborization of a pyramidal cell. The external features of the process are also consistent with those of a Golgi-impregnated pyramidal cell. In particular, the various types of spines (thin, mushroomshaped and stubby) project from an essentially smooth cylindrical shaft. Both axodendritic and axospinal-dendritic synapses are noted. Multiple synapses often occur on a single spine. Important to the present study is the arrangement of microtubules within the dendritic shaft. They form a largely parallel, interdigitated array, which tends to occupy a central core of the dendritic cylinder. The reconstructed microtubules are discrete, relatively short segments with an average length calculated 3 to be 8.5/~m. The array is interrupted by the membranous inclusions of the cytosol:mitochondria and smooth endoplasmic reticulum. In fact, individual tubules very often terminate near these membranous structures as well as near the plasma membrane. Although the microtubular array is largely parallel, it is not entirely orderly. Nearest neighbors can change along the length of any given microtubule and, in that sense, the array has a minimally woven quality. The normal dendrite is filled with microtubules and membranous components. It is rare to find an expanse of cytosol without either. As noted above, the microtubules tend to occupy a central core of the dendritic cylinder. Outside of this core, in the region under the plasma membrane, large saccules of smooth endoplasmic reticulum and mitochondria predominate. Although the number of reconstructed tubules (55) is not large, a range of microtubule lengths is observed. The largest microtubule extends beyond the length of the reconstruction (10 #m), while the shortest is 0.4/zm in length. Microtubular arrays within cortical dendrites in neurobehavioral failure Seven dendritic segments from cases 1, 2 and 3 have been reconstructed to reveal changes m the cytoskeleton which accompany changes in neuronal form. Proximal and distal segments of the dendrite
exhibit profound alteration m their microtubular arrays 9. However, the effects in each region are dissimilar enough to merit separate description. Perturbed microtubular arrays in proximal processes In proximal regions, alterations in dendritic form include thinning of the shaft, formation of irregular bumps on the cylindrical surface, loss of mushroomshaped and stubby spines and predominance of long, thin, often tortuous spines. Two basic types of perturbed array are found to underlie changes in proximal dendritic form. An example of the first type of proximal microtubular disarray has been reconstructed from the biopsy tissue of case 2 (Figs. 2 and 3). The microtubular array has lost many of the qualities described for the normal cortical cell. (A characteristic micrograph is shown in ref. 9, Fig. 5B.) Microtubules are no longer parallel to one another, or to the long axis of the cell. Focally, microtubules spiral along the length of
Fig. 2. A cutaway view of a proximal cortical dendrite reconstructed from case 2 (ref. 9). A slight swelling in the p r o c e s s occurs in the region where the rnicrotubules form a hehcai swirl. Inside the dendrite, mitochondna occupy s o m e o f the interstices in the microtubular array. However, relatively large regions of cytoplasm are devoid of microtubules or membranous c o m p o n e n t s . Bar = 1 ~ m .
302
Fig. 3. Mlcrotubular disarray m the same dendrite shown m Fig. 2. Spines are reduced in number relatwe to the corncal process of the normal mouse M~crotubules form a continuous helical swirl as they course through the process. Many short fragments are observed. The mlcrotubules frequently display radical curvature. One short fragment occupies a spine neck (lower left). Asterisks represent synapses Bar 1 jim
the process. The continuity of the microtubular array is loosely maintained as all of the microtubules curve in unison, completing a helical turn. Short fragments of microtubules, with seemingly random orientation, are inte~ spersed among the longer tubules which form the helical swirl. On close examination, individual microtubules both inside and outside of the helical swirl occasionally bend sharply, and do so in a manner independent of the direction taken by neighboring microtubules. In a most striking example, a single microtubule turns, leaving its neighbors, to enter the neck of a dendritic spine This is noteworthy in that microtubules are not part of the normal ultrastructure of the spine. The 65 reconstructed microtubules have an average calculated 3 length of 3.5 #m with a range extending from 0.15 t~m for the short fragments to
greater than 6.5 /,m for microtubules extending beyond one border of the reconstruction. The short fragments, seemingly disconnected from the rest of the microtubular array, can demonstrate radical curvature, at times doubling back upon themselves reside a diameter of 0.3/~m. As is the case in mouse dendrites, mierotubules often terminate near vesicles of smooth endoplasmlc reticulum and near mitochondrm. However~ ~t ~s more common, especially for the shorter microtubules, for terminations to occur m the cytoplasm. An ultrastructure for mlcrotubule endings has been previously suggested 3. The present preparation shows some evidence of diffuse and filled terminations but, as noted previously ~, tile classification is often difficult if not ambiguous. in the process of forming the hehcal swirl, microtubules have seemingly left behind relatively large expanses of cytoplasm (Fig. 2). These volumes approaching a cubic/tin, are bare of both microtubules and membranous structures, and ~eemmgly have no counterpart m the normal corttcal dendrite described above. In this case, the helical swIrl underlies a shght swelling in the profile of the process, while those parts of the array which maintain the semblance of parallel deployment underhe a region of the process with a more cylindrical profile (Figs 2 and 3) A second type of microtubular disarray is evident in the proximal process displayed in Fig. 4 (case 1). (A characteristic micrograph is found in rel: 9, Fig. 4B.) Microtubules maintain parallel deployment with respect to their immediate neighbors. However, groups of mlcrotubules are disoriented, both with respect to other groups and with respect to the long axis of the process. Reconstruction reveals occasional microtubules which turn rather sharply to run from one group to a second group with a grossly different axis. However, it is also clear that many microtubules begin and end with a single group. While this disarray may correspond to some asymmetry m the process, this is not the normal cytoskeletal structure which underlies a curving process. In curving dendrites, reconstructed from normal tissuO 2, individual microtubutes change orientation, with respect to one another, continually and gradually, wJthout the abrupt transitions m axial ahgnment observed in the pathological case.
303
Fig. 4. Cutaway view of a large cortical dendrite reconstructed from case 1. Few spines are in evidence.Within the process, small groups of microtubules are disoriented with respect to each other and with respect to the long axis of the process. Individual microtubules occasionallyturn sharply to run from one group to another. Most often, however, microtubules begin and terminate w~thln a group. Asterisks represent synapses. Bar = 1 pm. Perturbed microtubular arrays in distal processes Varicose processes (Figs. 5 and 6, cases 2 and 3 respectively) have shapes characteristic of more distal portions of the dendritic arborization defined in Golgi preparations. In general, expanded regions of the process are connected by thin necks which range from 0.5 to 10 /~m in length. The number of dendritic spines is reduced, and those spines which do occur are most often of the long, thin type. Within the field of view in Fig. 5, no spines project from the neuronal surface. Synapses persist along the length of the process, both on varicose and constricted segments. The 3-dimensional ultrastructure described below is typical for all varicose dendritic segments reconstructed from cases 1, 2 and 3. (Micrographs characteristic of these processes are shown in ref. 9, Fig. 6.) Membranous structures, both mitochondria and smooth endoplasmic reticulum, extend through varicose and constricted regions of the process. Again, individual microtubules often terminate near these structures. The microtubular array itself displays a periodicity synchronous with the occurrence of varicosities along the length of the dendrite. Within constricted neck regions microtubules maintain the semblance of parallel array. In these regions a small number of mlcrotubules course beside one another, maintaining their long axis parallel to the axis of the
process. Upon entering the varicosity, the microtubules most often splay away from one another and terminate within the varicosity. These terminations may be near vesicular smooth endoplasmic reticulum, near mitochondria, or in free cytoplasm. Most notably, the microtubular terminations within the varicose regions create distinct discontinuities in the microtubular array. These cytoskeletal elements no longer form a roughly parallel lattice which extends through the entire length of the process. One small varicosity (Fig. 5) does not exhibit microtubular discontinuity. Nonetheless the microtubules do splay away from one another within its volume. Rotation of the process displayed in Fig. 5 shows that the microtubules form long spirals as they course through the varicose section of the process. The most apt comparison to these pathologically altered varicose processes is perhaps to those spontaneously occurring varicosities in normal neurons. The A2 amacrine cell of the cat retina has been reconstructed using similar methods (Stevens, manuscript in preparation). In this case, small smooth varicosities occur at regular (approximately 10 #m) intervals along the length of the process. While the external appearance of the A2 amacrine dendrite is similar to that of a pathologically altered process, the underlying microtubular arrays are dissimilar. Importantly, in the amacrine cell microtubules form a continuous system through the entire length of the
304
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5B
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Fig. 5. A a n d B: a varicose dendrite reconstructed f r o m case 2 m two rotated wews. N o spines are evident. Mlcrotubules course t h r o u g h the thin segments o f the process a n d splay a w a y f r o m one a n o t h e r on entering t h e varicosity. T h e varicosities represent regions o f discontinuity in the m i c r o t u b u l a r array. M i t o c h o n d r i a a n d s m o o t h e n d o p l a s m i c retlculum are f o u n d in both constricted a n d varicose regions. Asterisks denote synapses. Bar =: 1 tern. Fig. 6. A varicose process reconstructed f r o m case 3. O n e s p i n e is visible. In general, rracrotubules occupy the constricted regions of the process while the varicosities represent areas o f discontinuity in the m i c r o t u b u t a r array. Asterisks represent synapses. Bar = 1
~m.
305 process. Each varicosity corresponds to an underlying mitochondrion positioned beside the continuous microtubule array. In this instance, the normally occurring mature dendritic varicosity appears to have a distinctly different 3-dimensional ultrastructure.
Histograms have also been generated for large cortical cells of the spiral array types from cases 1 and 2 (ref. 9). Specifically, those areas in which the array is relatively well conserved (microtubules in parallel array) have been analyzed. In areas where microtubules follow more tortuous paths, it is difficult to determine distance of closest approach. In each of the pathological cases (Fig. 7), the average distance between microtubular pairs is slightly reduced relative to mouse cortex. Important for present purposes, is the distance of closest approach between microtubules. For case 1, this value is 0.025 /am, for case 2 it is 0.03/am. Each of these values is considerably smaller than values obtained for normal cells.
Relationship between adjacent microtubules A graphics program 12 has been devised to calculate the perpendicular distance (center to center) between nearest neighboring microtubules. Histograms which are assembled from large ( > 350) numbers of these measurements, display inter-microtubular distances as a function of their frequency in the microtubular array. Inter-microtubular histograms (Fig. 7) for the mouse cortex were calculated from the same preparation used for the graphic reconstruction of cortical dendrites and are similar to those generated from the retinal ganglion cell of the cat 12. The average distance between microtubules is 0.10/am and the distance of closest approach in this population is 0.05/am.
DISCUSSION
Pathological changes in the microtubular array Golgi and EM analyses of cortical biopsies obtained from 5 children suffering severe neurobehavioral retardation have revealed striking changes in the dendritic systems of cortical neurons a. These
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Fig. 7. Inter-microtubule distance histograms. The perpendicular distance between nealest neighbor pairs is plotted as a function of its frequency for the mouse cortical process (A), case 1 (B) and case 2 (C). Each plot is generated from more than 350 measurements. In the pathological cases, measurements were taken in areas of the dendrite in which the parallel character of the array is maintained. The abscissa is calibrated in microns.
306 changes include formation of varicoslties m distal processes, irregular and thin proximal processes, and the predominance of long thin spines throughout the arborization. The present study describes the alterations m the 3-dimensional structure of the m~crotubular array, which accompany these changes in dendritic geometry. For purposes of comparison, the mlcrotubular array within a cortical dendrite of the normal mouse has been reconstructed (Fig. 1 I. The array is made up of relatively short, segmented mlcrotubules which course beside one another, occasionally exchanging neighbors, along the length of the procesb. In forming this array, microtubules strictly exclude each other from close approach (Figs. 7 and 8). This structure is similar to that determined for the dendrite of the retinal ganglion cell~K In the pathological condition, microtubules of the proximal dendritic process turn and twist with respect to one another and with respect to the long ax~s of the process. Two basic patterns have been reconstructed (Figs. 3 and 4). The array may comprise a continuous helical swM involving all microtubules in the locale, or the array may be made up of small groups of tubules which, while maintaining parallel internal orgamzation, are disoriented with respect to each other and with respect to the long axis of the process, in forming these bizarre arrays, microtubules do not consistently exclude one another from close approach. Inappropriately placed microtubules (for example in dendritic spines) and short fragmentary microtubules are also observed In the distal region of the arborlzation, where pathological change includes the formation of varicose processes, the continuity of the microtubular array is lost. In the normal dendrite, microtubules interdigltate along the entire length of the process In the degenerative varicosity (Figs. 5 and 6), expanses of cytosol within the expanded regmn separate clusters of microtubules contained within the intervening constricted segments. It may be reasonable to think that microtubule length is affected in the pathological process. Average length in the large cortical dendrite of the mouse (8.5 #m) is greater than that of the large pathologically altered process (3.5/~m). Breakage of mlcrotubules as a result of stresses imposed by the twisted array might be anticipated. However. it
remains difficult to compare this parameter across species. In fact, variations m average length have been described across cell typesK across species :3,~ and may be anticipated m different regions of mdlwdual neurons 5. A more constant parameter may be the distance measured perpendicular to the long axis, between nearest neighboring dendritic mlcrotubules. The average inter-microtubular distance, and distance of closest approach for cat retinal ganglion dendrltes 1", for mouse cortical ceil dendrites (Fig. 71, and for in vitro preparations of tubuhn-MAP2 polymers 7, are very similar It is suggested that this packing parameter for dendritic microtubules may be conserved across many neuronal types. However, in the pathological condition, microtubules have lost the abihty to exclude one another from close approach. These changes m microtubular packing w~thin the process are documented m inter-microtubular histograms (Fig. 7J. Possible mechan&ms in the perturhatton oj the mtcrotubular array attd alteratton in dendritic geontetr~ In Golgl preparations, it ~s apparent that individual cells, in the pathological condition, vary m the extent of varicosity tbrmation Cells appear in various stages of degeneration in each of the biopsies. This suggests that varmoslty formatmn may not be directly dependent on conditions external to the cell. It is unlikely that a diffusable substance or osmotic condition directly triggers varicosity formatlon in all cells simultaneously Rather, varicosity formation and the underlying microtubular disarray would seem a more subtle response of the cell's metabolism to external conditions or to an independently expressed anomaly in the cell's development. With the above considerations in mind, recent suggestions about the role of MAP2 in the cytoskeleton are noteworthy. In double fluorescence labeling of neuroblastoma 6, anti-MAP2 and antitubulin show similar distribution~ m neurites. We have previously suggested 12 that MAP2 or a similar molecule is at least in part responsible for maintaining the mlcrotubular array within the dendrite. MAP2 would do so by binding to tubulin and creating a cyhndrical volume around each microtubule which excludes other similarly bound microtubules from close approach In ~vddltlon, attractzve
307
Fig. 8. Cortical dendrites of normal mouse (A) and tissue samples of case 2 (B). In addition to the disorientation of microtubules in the dendritic pathology, reduction in the distance of close approach is evident. In the pathological condition, microtuhules (oriented perpendicular to the plane of section) are seen in close approximation (0.03 pm center to center), while in the normal cell, minimal distance is larger (0.05 #m). Bar ----0.5/~rn.
308 forces between the p e r i p h e r a l regions o f these cylindrical v o l u m e s c o u l d gel the m i c r o t u b u l e s into a coherent, s t r u c t u r a l l y d e t e r m i n e d array. T h e studies o f K i m et al. 7 can be interpreted as s u p p o r t i v e o f this structure. E l e c t r o n m i c r o g r a p h s o f pelleted fractions o f purified t u b u l i n reveal r e d u c e d inter-microt u b u l a r distances in the absence o f M A P 2 . A d d i t i o n o f M A P 2 to the p o l y m e r i z a t i o n mixture induces larger, a n d m o r e regular spacings between microtubules. I n t e r - m i c r o t u b u l a r h i s t o g r a m s o f cortical b i o p s y tissue show t h a t in the dendritic p a t h o l o g y , microtubules lose the ability to exclude one a n o t h e r from close a p p r o a c h . This i n a p p r o p r i a t e p a c k i n g o f mtc r o t u b u l e s within the a r r a y m a y reflect m a l f u n c t i o n of, or r e d u c t i o n in M A P 2 . W i t h the loss o f M A P 2 function, it ~s p o s t u l a t e d , the integrity a n d the o r g a n i z a t i o n o f the m i c r o t u b u l a r a r r a y is impaired. O t h e r features o f the r e c o n s t r u c t e d array, m i c r o tubules in spurious p o s i t i o n s and i n a p p r o p r i a t e c u r v a t u r e o f m i c r o t u b u l e s also suggest the b r e a k d o w n o f those m e c h a n i s m s responsible for maintaining regular a l i g n m e n t a m o n g n e i g h b o r i n g m~-
m a t t e r o f speculation. Changes m the a r r a y m a y induce changes in shape, or changes in the a r r a y m a y be s e c o n d a r y to o t h e r p a t h o l o g i c a l m e c h a nisms. W h i l e the present study c a n n o t differentiate between these alternatives, recent w o r k on the gangliosidoses 8,1°.m19 can be b r o u g h t to bear. In these disorders, a p p a r e n t l y evolving from the fmlure o f r e g u l a t i o n o f m e m b r a n e mass, the cellular g e o m e t r y is d i s t o r t e d by the p r o l i f e r a t i o n o f m e m b r a n o u s inclusions in the cytosol a n d b,~ the f o r m a t i o n o) meganeurites near the a x o n hillock. W i t h i n these processes, m i c r o t u b u l a r structure is intact. In sp~tc o f the p r o d i g i o u s l o a d o f m e m b r a n o u s inclusions. m i c r o t u b u l e s course in parallel array. A t least in this dmorder, ~t is a p p a r e n t t h a t the a r r a y is r o b u s t ; that changes in cellular g e o m e t r y do not induce alterattons in the c h a r a c t e r o f the m i c r o t u b u l a r array. The possibility r e m a i n s t h a t flaws intrinsic to the cytoskeleton m a y induce p a t h o l o g i c a l change in dendritic g e o m e t r y
crotubules. The r e l a t i o n s h i p between a l t e r a t i o n in the mtcrot u b u l a r a r r a y a n d a l t e r a t i o n in cell shape r e m a i n s a
This w o r k was s u p p o r t e d m p a r t b y N I H G r a n t HD-01799 a n d M R C M A 7345.
REFERENCES
9 Purpura, D. P, Bodlck, N., Suzuki, K., Rapm, l. and Wurzelmann, S., Mlcrotubular disarray m cortical dendrites and neurobehavioral fadure. 1 Golgl and electron mtcroscopic studies, Develop. Brain Res., 5 (1982) 287-297 10 Purpura, D. P., Pappas, G D and Baker, H J., Fine structure of meganeurites and secondary growth processes in fehne GMt gangliosidosis, Brain Res., 143 (1978) 1-12. 11 Purpura, D. P. and Suzuki, K , DlStOmon of neuronal geometry and formation of aberrant synapses in neuronal storage disease, Brain Res., 116 (1976) 1-21. 12 Sasakl, S., Stevens, J. K. and Bodlckj N , Serial reconstruction of microtubular arrays within dendrites of the cat retinal ganghon cell: the cytoskeleton of a vertebrate dendrite, Brain Res., in press. 13 Shelartski, M. L. and Liem, R. K. H., Neurofdaments, J Neurochem., 33 (1979) 5-13. 14 Shelanskl, M. J., Letterier, J. F. and Liem, t L K . H., Evidence for interactions between neurofilaments and microtubules, cytoskeletons and the architecture of the nervous system, Neurosct. Res. Progr. Bull., 19 (1981) 32-43. 15 Solomon, F , Neuroblastoma cells recapitulate their detailed neur,te morphologies alter reversible microtubular d)sassembly, Cell, 21 (1981)) 333-338 16 Solomon, F and Magendantz, M , Cytochalasm separales rmcrotubule disassembly from I~,~s of asymmetrw moT-
1 Bodlck, N., The self assembly o/the embryomc optic net re mto a retinotopically ordered array, Ph.D. thesis, Columbia University, 1980. 2 Bray, P. and Bunge, M. P , Serml analysis of mlcrotubules in cultured rat sensory axons, J. NeuroeytoL, 10 (1981) 589-605. 3 Chalfie, M. and Thomson, J. N., Organization of neuronal microtubules in the nematode Coenorhabditis elegam, J. Cell Biol., 82 (1979) 278-289. 4 Ellias, S. A. and Stevens, J. K., The dendritic varicosity a mechamsm for electrically isolating the dendrites of cat retinal amacrine cells? Brain Res., 196 (1980) 365-372. 5 Gonzes, I. and Sweadner, K. J., Multiple tubuhn forms are expressed by a single neurone, Nature (Lond.), 294 (1981) 477-480. 6 Isant, J. G. and Mclntosh, R., Microtubule-assoclated proteins. A monoctonal antibody to MAP brads to differentiated neurons, Proc nat Acad Sci. U.S A.. 77 (1980) 4741-4745. 7 Kit'n, H., Binder, L. I. and Rosenbaum, J. L., The periodic assocmtlon of MAP2 with. brain microtubules m wtro, J Cell Biol, 80 (1979) 266-276. 8 Purpura, D. P. and Baker, H. J, Neunte induction m mature cortical neurons m feline GMt-ganghoslde storage diseases, Nature (Lond.), 266 (1977) 553-554
ACKNOWLEDGEMENTS
309 phology, J. Cell. Biol., 89 (1981) 157-161. 17 Stevens, J. K., Reconstructing neuronal microc~rcuitry, Bull. Microscop. Soc. Canada, 9 (1980) 4-11. 18 Stevens, J. K., Davis, T. L., Friedman, N. and Sterling, P., A systematic approach to reconstructing microcircuitry by electron microscopy of serial sections, Brain
Res. Rev., 2 (1980) 265-293. 19 Walldey, S. U., Wurzelmann, S. and Purpura, D. P., Ultrastructure of neurites and meganeurites of cortical pyramidal neurons in feline gangliosidosls as revealed by the combined Golgi-EM technique, Brain Res., 211 (1981) 393-398.