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Neocortical VIP neurons are increased in the hemisphere containing focal cerebrocortical microdysgenesis in New Zealand Black mice
232
Brain Research, 532 (1990) 232-236 Elsevier
BRES 16012
Neocortical VIP neurons are increased in the hemisphere containing focal cerebrocortical microdysgenesis in New Zealand Black mice Gordon F. Sherman, Jennifer S. Stone, Glenn D. Rosen and Albert M. Galaburda The Dyslexia Research Laboratory and Charles A. Dana Research Institute, Beth Israel Hospital, Department of Neurology, Harvard Medical School, and Beth Israel Hospital, Boston, MA 02215 (U.S.A.) (Accepted 15 May 1990) Key words: Vasoactive intestinal polypeptide (VIP); Autoimmune mouse; New Zealand Black mouse; Microdysgenesis; Ectopic neuron; Cerebral cortex
Twenty to forty percent of New Zealand Black mice, a strain that develops severe autoimmune disease and learning deficits, exhibit focal unilateral collections of ectopic neurons and glia in layer I of the neocortex with underlying laminar dysplasia. This type of anomaly traditionally has been considered to represent disordered neuronal migration. In an attempt to further characterize these abnormalities, we compared counts of immunohistochemically-stained VIP-neurons in cortical regions containing ectopias and in adjacent cortex to homologous regions of the opposite hemisphere. There was an overall increase in the number of these neurons in the hemisphere containing the ectopias, which resulted from an increase in the number of VIP neurons both in the column of cortex within and underlying the ectopias and in the medially adjoining columns. We concluded that the presence of ectopias in the cerebral cortex not only represent abnormal migration, but also an increase in the number of at least one subset of neurons.
INTRODUCTION Twenty to forty percent of New Z e a l a n d Black (NZB) mice show ectopic collections of neurons and glia in layer I and underlying dysplasia of the cerebral cortex (together termed focal cerebrocortical microdysgenesis)24' 25. The underlying laminar dysplasia can be demonstrated best with the use of antibodies to neurofilament protein26. Usually one area of abnormality is seen in each brain, which is most often located in the somatosensory cortex. N e u r o n s located within the ectopia have stained positively for vasoactive intestinal polypeptide (VIP), somatostatin, neuropeptide Y (NPY), and ~,-aminobutyric acid ( G A B A ; paper in preparation). A b n o r m a l i t i e s of similar morphology occur in a variety of developmental brain disorders in humans 1'11'19 and we have seen these anomalies in brains of individuals with developmental dyslexia 9'1°. The ectopias are often associated with more severe forms of cortical malformation (e.g., micropolygyria, pachygyria), and have been attributed with the latter to reflect disorders of n e u r o n a l migration 1'5'17'18"23. It is not known whether ectopias represent a primary disorder of n e u r o n a l migration or are secondary to an event such as injury occurring during that period. In either case, it is also not known whether the ectopias represent simply abnormally positioned n e u r o n s
that would normally be located in the subjacent cortical laminae, or whether they represent additional neurons that are also misplaced. The present study addressed this question as it pertains to VIP n e u r o n s located within the cerebral cortex. VIP is a neuropeptide contained within bipolar and multipolar interneurons and stains distinctly in layers II and III of the rodent neocortex 12. By counting the n u m b e r of these neurons in the affected and unaffected hemisphere of the brains of N Z B mice containing focal cerebrocortical microdysgenesis, we could address the question of whether these abnormalities represent abnormal placement of neurons, a b n o r m a l n u m b e r s , or both, and thus could better u n d e r s t a n d the mechanism underlying this form of cerebral malformation.
MATERIALS AND METHODS lmmunohistochemistry NZB mice (approximately 8 weeks old) were anesthetized with ether and perfused transcardially with buffered saline for 1 min and PBS-buffered 2% paraformaldehyde/0.1% glutaraldehyde (pH 7.6) for 20 min. The brains were removed, immersed in the same fixative for 1 h at room temperature, and stored in buffered 10% sucrose overnight at 4 °C. The following day, they were transferred to buffered 30% sucrose at 4 °C for 24 h or until they sank. The brains were frozen using dry ice and sectioned at 30 /~m on sliding microtome. Every 10th section was stained with thionin and the
Correspondence: G.F. Sherman, Department of Neurology, Beth Israel Hospital, 330 Brookline Avenue, Boston, MA 02215, U.S.A. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
233 remaining sections were stored in 0.1 M sodium phosphate buffer (PBS; pH 7.4) at 4 °C until initiation of processing for immunohistochemistry. Ten mice (9 males and 1 female) were selected for immunohistochemical processing based on the presence of a large ectopic collection of neurons in layer I of the somatosensory cortex. Six brains contained ectopias in the right hemisphere, 3 were in the left, and 1 brain was not coded for side. A series of every 5th section was immunohistochemically processed for vasoactive intestinal polypeptide (Incs~ar Corporation, Stillwater, MN) using the biotin-avidin system. Free-floating sections were rinsed in phosphate-buffered saline (pH 7.4) for 5 min and transferred to a buffered 0.6% hydrogen peroxide solution for 20 min in order to block staining of endogenous peroxidases. Each ensuing step was followed by rinsing with agitation 2 times for 5 min in PBS. The sections were placed into a 1/2000 solution of primary antibody (Incstar) overnight at 4 °C. The vehicle (diluent) for all antibody incubations was 5% goat serum in PBS. The next day, sections were transferred into a biotinylated anti-rabbit immunoglobulin solution (Vectastain) diluted 1/60 for 2 h at room temperature. Then, the sections were placed into ABC complex (Vectastain) for 2 h at room temperature. The tissue was rinsed in PBS and 0.05 mM Tris buffer (pH 7.8) and developed using 0.05% diaminobenzidine and 0.005% hydrogen peroxide diluted in Tris. After rinsing with Tris, sections were mounted on chromealum-coated slides, dehydrated, counterstained with Methyl Green, and coverslipped with Permount. VIP neuron
measurements
In each of the 10 brains, a section containing the ectopia at its greatest extent was selected for quantitative analysis (Fig. 1).
Sections from 4 brains were judged to pass through the rostrocaudal center of the ectopia on the basis of the appearance of underlying laminae II through IV, which at the center of ectopias usually display dysplastic changes. Sections from the remaining brains passed close to the ectopias' centers, judging from the size and shape of each abnormality. The mediolateral extent of the ectopias ranged from 360 to 720 #m and the median size was approximately 500/~m. VIP neurons were identified by their darkly staining brown color and their neuronal profiles (Figs. 1 and 2). The neurons were counted at 400 x (oil) magnification from layer I to layer VI using a square reticule (240 by 240 pm) to insure the radial alignment of the counts. VIP neurons were counted in the ectopia and subjacent cortex within a quadrilateral measuring 480 pm in width (2 adjacent reticules), except in 1 case in which the ectopia measured 360 btm at its greatest diameter and only 1.5 reticules wide were counted. In order to include the whole depth of the cortex in the counts, 5 radially aligned reticules (1.2 mm) were counted within each column. Therefore, in most cases, the counting area that contained the ectopia measured 480 by 1200 pm. The exact size, architecture, and location of the counting fields were recorded so that the control (contralateral) hemispheres could be counted in an identical manner. In addition, we counted 2 consecutively adjoining quadrilaterals (480 by 1200pm) lateral (L1-2) and medial (M1-2) to that containing the ectopia and a quadrilateral in the center of the ectopia, and the same number of fields in the homologous areas of the contralateral hemisphere (Fig. 3). A consistent bias in cell size or section thickness between hemispheres can affect neuronal counts. In order to determine whether correction factors were needed to adjust for these parameters, we sampled lengths of neurons at their longest axis and measured the thickness of the sections in each hemisphere. The
Fig. 1. A low-power photomicrograph of VIP-stained cortical neurons within and surrounding an ectopia (arrows) in layer I of the cerebral cortex (left is medial and right is lateral). Bar - 175 jtm.
234 section. Paired t-tests were used to compare the n u m b e r and size of
VIP neurons and section thickness between the hemispheres.
RESULTS
Fig. 2. A high-power photomicrograph of a VIP-positive neuron in the cerebral cortex. Bar = 35/~m. lengths of a r a n d o m sample of VIP neurons (10 in each quadrilateral
used for neuronal counts) were measured at 400 x. Section thickness was determined as the distance between the perfectly focused cellular elements at the extreme superior and inferior surfaces of the
Ect
\
~~Medi a l ~<~M 1 M2
,
\~ "-~
/ Lateral Fig. 3. A n example of the quadrilateral fields used to count VIP neurons within and surrounding the ectnpias. E l - 2 are contained within the borders o l the ectopia, M ] - 2 arc medially adjacent to the cctopia and L I - 2 are laterally adiaccnt. The homologous fields of the opposite hemisphere are counted for comparisons. Some ectopias extended outside the E1-2 columns and in 1 case was
smaller than 2 columns. However, in each case the appropriate columns were counted as controls in the opposite hemisphere.
T h e r e was no difference in section thickness between the hemispheres containing ectopias and their opposite mates (t = 1.50, df = 9), nor was there any hemispheric difference in the lengths of VIP-positive neurons (ts < 1). Thus, no correction factors were used for section thickness of neuronal size, since, although the counts m a y not reflect absolute numbers, they provide a d e q u a t e relative comparisons b e t w e e n the hemispheres that do or do not contain neuronal e c t o p i a s - t h e focus of this study. Comparisons were m a d e between the counts of VIPpositive neurons in the hemispheres containing a molecular layer ectopia and the unaffected contralateral hemispheres. There was an increase in the total n u m b e r of VIP neurons in the m e a s u r e d areas of the hemispheres with ectopias as o p p o s e d to those without ectopias ($s = 123.6 vs. 102.3, respectively; t = 3.327, df = 9, P < 0.01). This difference was accounted for by m o r e VIP neurons in the quadrilaterals containing ectopias than in those in the homologous areas of the opposite hemispheres ($s = 47.7 vs. 34.6, respectively; t = 2.68, df = 9, P < 0.025), as well as by m o r e V I P neurons in the quadrilaterals medial to the ectopias than in their mates in the opposite hemispheres ($s = 41.6 vs. 34.5, respectively; t = 2.44, df = 9, P < 0.05). The n u m b e r of V I P neurons counted in the center of the ectopia also differed from the contralateral h o m o l o g u e s ($s = 25.5 vs. 17.8, respectively; t = 2.22; P < 0.053). V I P counts in the quadrilaterals lateral to those containing ectopias, however, did not differ from those in their contralateral homologues ($s = 34.3 vs. 32.2, respectively; t < 1, df = 9). N o quadrilateral in the affected hemisphere had fewer VIP neurons than its h o m o l o g u e in the unaffected hemispheres. Within-hemisphere comparisons showed that the quadrilateral in the center of the ectopia had m o r e V I P neurons than the m e a n of the m e a s u r e d regions lateral to the ectopia ($s = 25.5 vs. 17.15, respectively; t = 3.01; P < 0.015), but was not different from the mean of the medial regions ($s = 25.5 vs. 20.8, respectively; t = 1.70). There were no differences among the m e a s u r e d regions in the hemispheres without ectopias (ts < 1). DISCUSSION
VIP-like neurons were increased in cortical regions containing molecular layer ectopias as well as in medially adjoining cortex. This occurred in the face of actual cell-free areas that often characterize the dysplastic
235 cortex underlying the ectopias. No cortex neighboring the area of ectopia actually had fewer VIP-stained neurons than the corresponding areas in the opposite hemisphere. This would suggest that there is an absolute increase in VIP-stained neurons in the affected hemisphere, rather than a relative increase in the area of ectopia caused by neurons that have migrated laterally from adjacent cortex to p o p u l a t e the ectopic region. Lateral migration from m o r e distant areas within the affected hemisphere cannot be excluded, but seems unlikely. Thus, we may conclude that in this form of cortical malformation VIP-stained neurons are increased, as well as abnormally migrated. Yet, alternative explanations must be considered ~z. A difference in the n u m b e r of positively-stained VIP neurons between two homologous structures could reflect either a difference in the n u m b e r of cells containing VIP or a difference in the concentration and activity of VIP in the neurons such that some neurons fail to stain although they contain varying concentrations of VIP. H o w e v e r , H a j 6 s and Zilles 12 showed that colchicine t r e a t m e n t , which blocks axoplasmic transport and presumably V I P activity, did not alter the staining of VIP, indicating that there is a slow turnover of VIP in the cortex. Thus, the increase of VIP-positive neurons in the hemisphere with an ectopia cannot be explained by selective changes in neurochemical activity. It is also possible that the presence of ectopias heralds an increase in numbers of all neurons, signifying a disorder in the regulation of neuronal numbers in addition to abnormal neuronal migration. H o w e v e r , this possibility cannot be addressed here, since total neuronal counts were not m a d e and some neuronal subtypes may be decreased while the V I P neurons are increased. In any REFERENCES 1 Barth, P.G., Disorders of neuronal migration, Canad. J. Neurol. Sci., 14 (1987) 1-16. 2 Cavines, Jr., V.S., Patterns of cell and fiber distribution in the neocortex of the reeler mutant mouse, J. Comp. Neurol., 170 (1976) 435-448. 3 Caviness, Jr., V.S., Neocortical histogenesis and reeler mice: a developmental study based upon [3IJ]thymidine autoradiography, Dev. Brain Res., 4 (1982) 293-302. 4 Caviness, Jr., V.S. and Frost, D.O., Thalamocortical projections in the reeler mutant mouse, J. Comp. Neurol., 219 (1983) 182-202. 5 Caviness, Jr., V.S. and Yorke, Jr., C.H., Interhemispheric neocortical connections of the corpus callosum in the reeler mutant mouse: a study based on anterograde and retrograde methods, J. Comp. Neurol., 170 (1976) 449-460. 6 Caviness, Jr., V.S., Misson, J.-P. and Gadisseux, J.-F., Abnormal neuronal patterns and disorders of neocortical development. In A.M. Galaburda (Ed.), From Reading to Neurons. M.I.T. Press/Bradford Books, Cambridge, MA, 1989, pp. 405-442. 7 Dvor~ik, K. and Feit, J., Migration of neuroblasts through partial necrosis of the cerebral cortex in newborn rats - - Contribution to the problems of morphological development and developmental period of cerebral microgyria, Acta Neuropathol., 38
case the present findings suggest a change in the p r o p o r t i o n of neuronal types associated with the presence of focal microdysgenesis and hence an alteration in the functional architecture. Studies on induced focal cerebrocortical microdysgenesis provide useful clues about mechanisms by which ectopias are p r o d u c e d and V I P neurons increased. Freezing lesions to the rat or mouse brain on the first postnatal day p r o d u c e dysplastic layering and ectopic neuronal nests in the molecular layer similar to, albeit m o r e severe than, the spontaneous changes seen in the N Z B mouse 7"8"13"22. This leads us to speculate that the microdysgenesis of the a u t o i m m u n e mice also occurs after early d a m a g e to the cortex. In addition, the p r o p o s e d d a m a g e may cause the release of trophic factors that in turn permit the survival of neurons that would ordinarily d i s a p p e a r during cortical ontogenesis. It is likely that the functional p r o p e r t i e s of the cortex exhibiting microdysgenesis differ from the n o r m a l case. Unlike the situation in the R e e l e r mouse, in which abnormally migrated neurons in the cortex m a k e appropriate interhemispheric and thalamic connections 2 - 4 , 6 , cerebrocortical microdysgenesis in the N Z B mouse is accompanied by a b n o r m a l n u m b e r s of neurons and anomalous patterns of connections as shown on neurofilament staining. This raises the possibility that the resulting neuronal assemblies have altered functional capacities and m a y help to explain the learning difficulties r e p o r t e d in N Z B mice 14-16'2°'21'27'28.
Acknowledgements. This research supported, in part, by NIH Grant HD-20806, and grants from the Carl W. Herzog Foundation, and the Research Division of the Orton Dyslexia Society.
(1977) 203-212. 8 Dvor~ik, K., Feit, J. and Jurfinkov~i, Z., Experimentally induced focal microgyria and status verrucosus deformis in rats - Pathogenesis and interrelation histological and autoradiographical study, Acta Neuropathol., 44 (1978) 121-129. 9 Galaburda, A.M. and Kemper, T.L., Cytoarchitectonic abnormalities in developmental dyslexia; a case study, Ann. Neurol., 6 (1979) 94-100. 10 Galaburda, A.M., Sherman, G.F., Rosen, G.D., Aboitiz, E and Geschwind, N., Developmental dyslexia: four consecutive cases with cortical anomalies, Ann. Neurol., 18 (1985) 222-233. 11 Grcevid, N. and Robert, F., Verrucose dysplasia of the cerebral cortex, J. Neurosurg. Exp. Neurol., 20 (1961) 399-411. 12 Haj6s, F. and Zilles, K., Quantitative immunohistochemical analysis of VIP-neurons in the rat cortex, Histochemistry, 90 (1988) 139-144. 13 Humphreys, P., Rosen, G.D., Sherman, G.F. and Galaburda, A.M., Freezing lesions of the newborn rat brain: a model for cerebrocortical microdygenesis, Soc. Neurosci. Abstr., 15 (1989) 1120. 14 Lal, H. and Forster, M.J., Cognitive disorders related to immune dysfunction: novel animal models for drug development, Drug Dev. Res., 7 (1986) 195-208. 15 Lal, H., Forster, M.J. and Nandy, K., Immunologic factors related to cognitive/behavioral dysfunctions in aging. In J.
236
16 17
18 19 20 21
22
Traber and W.H. Gispen (Eds.), Senile Dementia of Alzheimer Type. Springer-Verlag, Berlin, Heidelberg, 1985 pp. 343-354. Lal, H., Bennett, M., Bennett, D., Forster, M.J. and Nandy, K., Learning deficits occur in young mice following transfer of immunity from senescent mice, Life Sci., 39 (1986) 507-512. Levine, D.N., Fisher, M.A. and Caviness, V.S., Porencephaly with microgyria: a pathologic study, Acta Neuropathol., 29 (1974) 99-113. McBride, M.C. and Kemper, T.L., Pathogenesis of four-layered microgyric cortex in man, Acta Neuropathol., 57 (1982) 93-98. Morel, E and Wildi, E., Dysg6n6sie nodulaire diss6min6e de l'6corce frontale, Rev. Neurol., 87 (1952) 251-270. Nandy, K., Significance of brain-reactive antibodies in serum of aged mice, J. Gerontol., 30 (1975) 412-416. Nandy, K., Lal, H., Bennet, M. and Bennett, D., Correlation between a learning disorder and elevated brain-reactive antibodies in aged C57BL/6 and young NZB mice, Life Sci., 33 (1983) 1499-1503. Rosen, G.D., Humphreys, P., Sherman, G.F. and Galaburda, A.M., Connectional anomalies associated with freezing lesions to the neocortex of the newborn rat, Soc. Neurosci. Abstr., 15
(1989) 1120. 23 Sarnat, H.B., Disturbances of late neuronal migration in the perinatal period, Am. J. Dis. Child., 141 (1987) 969-980. 24 Sherman, G.F., Galaburda, A.M. and Geschwind, N., Cortical anomalies in brains of New Zealand mice: a neuropathologic model of dyslexia?, Proc. Natl. Acad. Sci. U.S.A., 82 (1985) 8072-8074. 25 Sherman, G.F., Galaburda, A.M., Behan, P.O. and Rosen, G.D., Neuroanatomical anomalies in autoimmune mice, Acta Neuropathol., 74 (1987) 239-242. 26 Sherman, G.F., Stone, J.S., Press, D.M., Rosen, G.D. and Galaburda, A.M., Abnormal architecture and connections disclosed by neurofilament staining in the cerebral cortex of autoimmune mice, Brain Research, (1990) in press. 27 Spencer, D.G. and Lal, H., Specific behavioral impairments in association tasks with an autoimmune mouse., Soc. Neurosci. Abstr., 9 (1983) 96. 28 Spencer, D.G., Humphries, K., Mathis, D. and Lal, H., Behavioral impairments related to cognitive dysfunction in the autoimmune New Zealand Black mouse, Behav. Neurosci., 100 (1986) 353-358.
Brain Research, 532 (1990) 232-236 Elsevier
BRES 16012
Neocortical VIP neurons are increased in the hemisphere containing focal cerebrocortical microdysgenesis in New Zealand Black mice Gordon F. Sherman, Jennifer S. Stone, Glenn D. Rosen and Albert M. Galaburda The Dyslexia Research Laboratory and Charles A. Dana Research Institute, Beth Israel Hospital, Department of Neurology, Harvard Medical School, and Beth Israel Hospital, Boston, MA 02215 (U.S.A.) (Accepted 15 May 1990) Key words: Vasoactive intestinal polypeptide (VIP); Autoimmune mouse; New Zealand Black mouse; Microdysgenesis; Ectopic neuron; Cerebral cortex
Twenty to forty percent of New Zealand Black mice, a strain that develops severe autoimmune disease and learning deficits, exhibit focal unilateral collections of ectopic neurons and glia in layer I of the neocortex with underlying laminar dysplasia. This type of anomaly traditionally has been considered to represent disordered neuronal migration. In an attempt to further characterize these abnormalities, we compared counts of immunohistochemically-stained VIP-neurons in cortical regions containing ectopias and in adjacent cortex to homologous regions of the opposite hemisphere. There was an overall increase in the number of these neurons in the hemisphere containing the ectopias, which resulted from an increase in the number of VIP neurons both in the column of cortex within and underlying the ectopias and in the medially adjoining columns. We concluded that the presence of ectopias in the cerebral cortex not only represent abnormal migration, but also an increase in the number of at least one subset of neurons.
INTRODUCTION Twenty to forty percent of New Z e a l a n d Black (NZB) mice show ectopic collections of neurons and glia in layer I and underlying dysplasia of the cerebral cortex (together termed focal cerebrocortical microdysgenesis)24' 25. The underlying laminar dysplasia can be demonstrated best with the use of antibodies to neurofilament protein26. Usually one area of abnormality is seen in each brain, which is most often located in the somatosensory cortex. N e u r o n s located within the ectopia have stained positively for vasoactive intestinal polypeptide (VIP), somatostatin, neuropeptide Y (NPY), and ~,-aminobutyric acid ( G A B A ; paper in preparation). A b n o r m a l i t i e s of similar morphology occur in a variety of developmental brain disorders in humans 1'11'19 and we have seen these anomalies in brains of individuals with developmental dyslexia 9'1°. The ectopias are often associated with more severe forms of cortical malformation (e.g., micropolygyria, pachygyria), and have been attributed with the latter to reflect disorders of n e u r o n a l migration 1'5'17'18"23. It is not known whether ectopias represent a primary disorder of n e u r o n a l migration or are secondary to an event such as injury occurring during that period. In either case, it is also not known whether the ectopias represent simply abnormally positioned n e u r o n s
that would normally be located in the subjacent cortical laminae, or whether they represent additional neurons that are also misplaced. The present study addressed this question as it pertains to VIP n e u r o n s located within the cerebral cortex. VIP is a neuropeptide contained within bipolar and multipolar interneurons and stains distinctly in layers II and III of the rodent neocortex 12. By counting the n u m b e r of these neurons in the affected and unaffected hemisphere of the brains of N Z B mice containing focal cerebrocortical microdysgenesis, we could address the question of whether these abnormalities represent abnormal placement of neurons, a b n o r m a l n u m b e r s , or both, and thus could better u n d e r s t a n d the mechanism underlying this form of cerebral malformation.
MATERIALS AND METHODS lmmunohistochemistry NZB mice (approximately 8 weeks old) were anesthetized with ether and perfused transcardially with buffered saline for 1 min and PBS-buffered 2% paraformaldehyde/0.1% glutaraldehyde (pH 7.6) for 20 min. The brains were removed, immersed in the same fixative for 1 h at room temperature, and stored in buffered 10% sucrose overnight at 4 °C. The following day, they were transferred to buffered 30% sucrose at 4 °C for 24 h or until they sank. The brains were frozen using dry ice and sectioned at 30 /~m on sliding microtome. Every 10th section was stained with thionin and the
Correspondence: G.F. Sherman, Department of Neurology, Beth Israel Hospital, 330 Brookline Avenue, Boston, MA 02215, U.S.A. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
233 remaining sections were stored in 0.1 M sodium phosphate buffer (PBS; pH 7.4) at 4 °C until initiation of processing for immunohistochemistry. Ten mice (9 males and 1 female) were selected for immunohistochemical processing based on the presence of a large ectopic collection of neurons in layer I of the somatosensory cortex. Six brains contained ectopias in the right hemisphere, 3 were in the left, and 1 brain was not coded for side. A series of every 5th section was immunohistochemically processed for vasoactive intestinal polypeptide (Incs~ar Corporation, Stillwater, MN) using the biotin-avidin system. Free-floating sections were rinsed in phosphate-buffered saline (pH 7.4) for 5 min and transferred to a buffered 0.6% hydrogen peroxide solution for 20 min in order to block staining of endogenous peroxidases. Each ensuing step was followed by rinsing with agitation 2 times for 5 min in PBS. The sections were placed into a 1/2000 solution of primary antibody (Incstar) overnight at 4 °C. The vehicle (diluent) for all antibody incubations was 5% goat serum in PBS. The next day, sections were transferred into a biotinylated anti-rabbit immunoglobulin solution (Vectastain) diluted 1/60 for 2 h at room temperature. Then, the sections were placed into ABC complex (Vectastain) for 2 h at room temperature. The tissue was rinsed in PBS and 0.05 mM Tris buffer (pH 7.8) and developed using 0.05% diaminobenzidine and 0.005% hydrogen peroxide diluted in Tris. After rinsing with Tris, sections were mounted on chromealum-coated slides, dehydrated, counterstained with Methyl Green, and coverslipped with Permount. VIP neuron
measurements
In each of the 10 brains, a section containing the ectopia at its greatest extent was selected for quantitative analysis (Fig. 1).
Sections from 4 brains were judged to pass through the rostrocaudal center of the ectopia on the basis of the appearance of underlying laminae II through IV, which at the center of ectopias usually display dysplastic changes. Sections from the remaining brains passed close to the ectopias' centers, judging from the size and shape of each abnormality. The mediolateral extent of the ectopias ranged from 360 to 720 #m and the median size was approximately 500/~m. VIP neurons were identified by their darkly staining brown color and their neuronal profiles (Figs. 1 and 2). The neurons were counted at 400 x (oil) magnification from layer I to layer VI using a square reticule (240 by 240 pm) to insure the radial alignment of the counts. VIP neurons were counted in the ectopia and subjacent cortex within a quadrilateral measuring 480 pm in width (2 adjacent reticules), except in 1 case in which the ectopia measured 360 btm at its greatest diameter and only 1.5 reticules wide were counted. In order to include the whole depth of the cortex in the counts, 5 radially aligned reticules (1.2 mm) were counted within each column. Therefore, in most cases, the counting area that contained the ectopia measured 480 by 1200 pm. The exact size, architecture, and location of the counting fields were recorded so that the control (contralateral) hemispheres could be counted in an identical manner. In addition, we counted 2 consecutively adjoining quadrilaterals (480 by 1200pm) lateral (L1-2) and medial (M1-2) to that containing the ectopia and a quadrilateral in the center of the ectopia, and the same number of fields in the homologous areas of the contralateral hemisphere (Fig. 3). A consistent bias in cell size or section thickness between hemispheres can affect neuronal counts. In order to determine whether correction factors were needed to adjust for these parameters, we sampled lengths of neurons at their longest axis and measured the thickness of the sections in each hemisphere. The
Fig. 1. A low-power photomicrograph of VIP-stained cortical neurons within and surrounding an ectopia (arrows) in layer I of the cerebral cortex (left is medial and right is lateral). Bar - 175 jtm.
234 section. Paired t-tests were used to compare the n u m b e r and size of
VIP neurons and section thickness between the hemispheres.
RESULTS
Fig. 2. A high-power photomicrograph of a VIP-positive neuron in the cerebral cortex. Bar = 35/~m. lengths of a r a n d o m sample of VIP neurons (10 in each quadrilateral
used for neuronal counts) were measured at 400 x. Section thickness was determined as the distance between the perfectly focused cellular elements at the extreme superior and inferior surfaces of the
Ect
\
~~Medi a l ~<~M 1 M2
,
\~ "-~
/ Lateral Fig. 3. A n example of the quadrilateral fields used to count VIP neurons within and surrounding the ectnpias. E l - 2 are contained within the borders o l the ectopia, M ] - 2 arc medially adjacent to the cctopia and L I - 2 are laterally adiaccnt. The homologous fields of the opposite hemisphere are counted for comparisons. Some ectopias extended outside the E1-2 columns and in 1 case was
smaller than 2 columns. However, in each case the appropriate columns were counted as controls in the opposite hemisphere.
T h e r e was no difference in section thickness between the hemispheres containing ectopias and their opposite mates (t = 1.50, df = 9), nor was there any hemispheric difference in the lengths of VIP-positive neurons (ts < 1). Thus, no correction factors were used for section thickness of neuronal size, since, although the counts m a y not reflect absolute numbers, they provide a d e q u a t e relative comparisons b e t w e e n the hemispheres that do or do not contain neuronal e c t o p i a s - t h e focus of this study. Comparisons were m a d e between the counts of VIPpositive neurons in the hemispheres containing a molecular layer ectopia and the unaffected contralateral hemispheres. There was an increase in the total n u m b e r of VIP neurons in the m e a s u r e d areas of the hemispheres with ectopias as o p p o s e d to those without ectopias ($s = 123.6 vs. 102.3, respectively; t = 3.327, df = 9, P < 0.01). This difference was accounted for by m o r e VIP neurons in the quadrilaterals containing ectopias than in those in the homologous areas of the opposite hemispheres ($s = 47.7 vs. 34.6, respectively; t = 2.68, df = 9, P < 0.025), as well as by m o r e V I P neurons in the quadrilaterals medial to the ectopias than in their mates in the opposite hemispheres ($s = 41.6 vs. 34.5, respectively; t = 2.44, df = 9, P < 0.05). The n u m b e r of V I P neurons counted in the center of the ectopia also differed from the contralateral h o m o l o g u e s ($s = 25.5 vs. 17.8, respectively; t = 2.22; P < 0.053). V I P counts in the quadrilaterals lateral to those containing ectopias, however, did not differ from those in their contralateral homologues ($s = 34.3 vs. 32.2, respectively; t < 1, df = 9). N o quadrilateral in the affected hemisphere had fewer VIP neurons than its h o m o l o g u e in the unaffected hemispheres. Within-hemisphere comparisons showed that the quadrilateral in the center of the ectopia had m o r e V I P neurons than the m e a n of the m e a s u r e d regions lateral to the ectopia ($s = 25.5 vs. 17.15, respectively; t = 3.01; P < 0.015), but was not different from the mean of the medial regions ($s = 25.5 vs. 20.8, respectively; t = 1.70). There were no differences among the m e a s u r e d regions in the hemispheres without ectopias (ts < 1). DISCUSSION
VIP-like neurons were increased in cortical regions containing molecular layer ectopias as well as in medially adjoining cortex. This occurred in the face of actual cell-free areas that often characterize the dysplastic
235 cortex underlying the ectopias. No cortex neighboring the area of ectopia actually had fewer VIP-stained neurons than the corresponding areas in the opposite hemisphere. This would suggest that there is an absolute increase in VIP-stained neurons in the affected hemisphere, rather than a relative increase in the area of ectopia caused by neurons that have migrated laterally from adjacent cortex to p o p u l a t e the ectopic region. Lateral migration from m o r e distant areas within the affected hemisphere cannot be excluded, but seems unlikely. Thus, we may conclude that in this form of cortical malformation VIP-stained neurons are increased, as well as abnormally migrated. Yet, alternative explanations must be considered ~z. A difference in the n u m b e r of positively-stained VIP neurons between two homologous structures could reflect either a difference in the n u m b e r of cells containing VIP or a difference in the concentration and activity of VIP in the neurons such that some neurons fail to stain although they contain varying concentrations of VIP. H o w e v e r , H a j 6 s and Zilles 12 showed that colchicine t r e a t m e n t , which blocks axoplasmic transport and presumably V I P activity, did not alter the staining of VIP, indicating that there is a slow turnover of VIP in the cortex. Thus, the increase of VIP-positive neurons in the hemisphere with an ectopia cannot be explained by selective changes in neurochemical activity. It is also possible that the presence of ectopias heralds an increase in numbers of all neurons, signifying a disorder in the regulation of neuronal numbers in addition to abnormal neuronal migration. H o w e v e r , this possibility cannot be addressed here, since total neuronal counts were not m a d e and some neuronal subtypes may be decreased while the V I P neurons are increased. In any REFERENCES 1 Barth, P.G., Disorders of neuronal migration, Canad. J. Neurol. Sci., 14 (1987) 1-16. 2 Cavines, Jr., V.S., Patterns of cell and fiber distribution in the neocortex of the reeler mutant mouse, J. Comp. Neurol., 170 (1976) 435-448. 3 Caviness, Jr., V.S., Neocortical histogenesis and reeler mice: a developmental study based upon [3IJ]thymidine autoradiography, Dev. Brain Res., 4 (1982) 293-302. 4 Caviness, Jr., V.S. and Frost, D.O., Thalamocortical projections in the reeler mutant mouse, J. Comp. Neurol., 219 (1983) 182-202. 5 Caviness, Jr., V.S. and Yorke, Jr., C.H., Interhemispheric neocortical connections of the corpus callosum in the reeler mutant mouse: a study based on anterograde and retrograde methods, J. Comp. Neurol., 170 (1976) 449-460. 6 Caviness, Jr., V.S., Misson, J.-P. and Gadisseux, J.-F., Abnormal neuronal patterns and disorders of neocortical development. In A.M. Galaburda (Ed.), From Reading to Neurons. M.I.T. Press/Bradford Books, Cambridge, MA, 1989, pp. 405-442. 7 Dvor~ik, K. and Feit, J., Migration of neuroblasts through partial necrosis of the cerebral cortex in newborn rats - - Contribution to the problems of morphological development and developmental period of cerebral microgyria, Acta Neuropathol., 38
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Acknowledgements. This research supported, in part, by NIH Grant HD-20806, and grants from the Carl W. Herzog Foundation, and the Research Division of the Orton Dyslexia Society.
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