Quantitative evidence for selective dendritic growth in normal human aging but not in senile dementia

Brain Research, 2 1 4 ( 1 9 8 1 ) 2 3 - 4 1 © Elsevier/North-Holland Biomedical Press

23

QUANTITATIVE EVIDENCE FOR SELECTIVE D E N D R I T I C G R O W T H IN N O R M A L H U M A N A G I N G BUT NOT IN SENILE D E M E N T I A

STEPHEN

J. B U E L L

and PAUL D. COLEMAN

Departments of Neurology and Anatomy, School of Medicine and Dentistry, University of Rochester Rochester, N. Y. 14642 (U.S.A.) ( A c c e p t e d O c t o b e r 3 0 t h , 1980)

Key words: d e n d r i t e s - - a g i n g - - c e r e b r a l c o r t e x - - p l a s t i c i t y - - q u a n t i f i c a t i o n - - h u m a n - - senile

dementia -- growth

SUMMARY

Parahippocampal gyrus was sampled from human brains at autopsy to form three groups: adult (n = 5, mean age 51.2 years), normal aged (n = 5, mean age 79.6), and senile dementia (SD)(n -- 5, mean age 76.0). Classification as normal aged or senile demented was based on both behavioral and neuropathological criteria. Tissue was processed for Golgi-Cox, cresyl violet, hematoxylin and eosin and Bodian silver stains. Both atrophied and normal dendritic trees were seen in all cases. Dendrites of layer II pyramidal neurons were quantified with a computer-microscope system. Quantitative data showed that normal aged individuals had longer and more branched dendrites than either adult or SD individuals. There was a slight tendency for SD individuals to have shorter, less-branched dendrites than adults. Differences among groups were greater in apical than in basal portions of the dendritic tree. These differences were largely accounted for by the lengthening and branching (apical dendrites) or lengthening only (basal dendrites) of terminal dendritic segments. These data suggest a model in which aging cortex contains both regressing, dying neurons and surviving, growing neurons. In normal aging it is the latter group that predominates. This is the first demonstration of plasticity in the adult human brain.

INTRODUCTION

The aged human shows many clinical signs which indicate that the brain is subject to age-associated degenerative processes. For example, rates and amount of motor activity decrease 1, EEG patterns are disrupted zz, and performance on tests of mnemonic functions deteriorate 13. Consistent with these findings is a large body of histological evidence of degeneration. Neuronal loss or decreased packing density

24 have been reported in the aged human cerebral 5 and cerebellar r4 cortices and locus coeruleus a7. Often, dendritic and axonal processes of surviving neurons are found in abnormal configurations known as senile plaques as. There are some reports that dendritic trees undergo progressive regression 26-zs, sometimes to such an extent as to leave a single stump projecting from the soma. In senile dementia similar changes have been described but the severity is reportedly greater than in normal aging3,26-2s,3L There are, on the other hand, a small number of isolated studies which suggest that dendritic growth may occur in the aging brain. In some cases, this growth appears abnormal. The earliest of such reports is described abnormal-appearing dendritic neoformations in the hippocampus of an aged dog. Andrew 2 described occasional granular cells which were polydendritic rather than monodendritic in the dentate gyrus in a variety of species in aged individuals. Mervis 20 has also reported new dendritic growths on the apical shafts of regressed neurons in aged dog cortex. Scheibel and Tomiyasu 25 observed abnormal dendritic neoformations in presenile dementia. Dense clusters of spined, branched dendritic material were seen sprouting from the soma and dendrites of regressed neurons in the cortex. A stereological electron microscopic study 16 of rat olfactory bulb at a series of ages showed maintenance of the inferred total length of mitral cell dendrites in the neuropil even in the face of significant mitral cell loss between 24 and 27 months. This maintenance was explained by a calculated 50 % increase in dendritic length per mitral cell. The nature of this study makes it impossible to be certain whether this dendritic growth was normal. In preliminary reports 7--9 we have presented data which suggest that there is dendritic growth in the aging human brain and that this growth is largely normal. In addressing questions regarding the status of the morphological substrate for cereblal functioning, the dendritic tree is of special interest since it represents about 95 % of the receptive surface of cortical neurons ~4. With the loss of many neurons in aging and the massive accumulation of lipofuscin and neurofibrillary material in many remaining neurons, it is important to have an appreciation of the quantitative and qualitative nature of any alterations in dendritic morphology which might accompany these degenerative changes. The purpose of the present study was to gain such an appreciation by applying computerized methodology to analyze quantitatively dendritic parameters of single Golgi-stained neurons of normal adult and normal aged human cases as well as of cases of senile dementia. METHODS

Selection of material Tissues were collected at autopsy from 50 cases aged from 17 to 96 years of age. From these, 5 cases were selected to form the adult group (mean age 51.2 years, range "!.A. 55), 5 for the normal aged group (mean age 79.6 years, range 68-92), and 5 for the senile dementia (SD) group (mean age 76.0 years, range 70-81). Selection was based on clarity of the clinical history to insure: (1) that the adult and aged cases were neurologically normal, and (2) that the SD cases represented accurate diagnoses of

25 senile dementia and were free of other severe neurological abnormalities. Retrospective clinical evaluations were based on information obtained from hospital charts and/or interviews with the patients' physicians, nulses, and/or pathologists. Any evidence of severe stroke, seizure disorder, alcoholism or physical trauma to the head was cause for rejection of a case from the study. Inclusion in the aged group required that there be clear evidence that the patient was alert, well oriented, and capable of functioning relatively independently shortly before death, i.e. that there be no evidence of senile dementia. Inclusion in the SD group required that there be clear evidence of severe, progressive dementia consistent with the diagnosis of senile dementia as described in The World Health Organization's International Classification of diseases. (The authors wish to thank John Romano, M.D., Distinguished University Professor of Psychiatry for his aid in reviewing cases with us.) It was also required that there be abundant senile plaques and neurofibrillary degeneration in a section of contralateral parahippocampal cortex prepared by the Bodian method 4. Other goals of the selection process were to maximize the quality of the Golgi preparations and to minimize postmortem delay before fixation. Causes of death were cardiovascular/pulmonary (11 cases) or neoplastic disease without cerebral involvement (4 cases). We were not able to detect any relationship between cause of death or nature and duration of the agonal period and dendritic parameters. A summary of clinical information for each case appears elsewhere 9.

Tissue preparation Within 2.25-21.50 h after death blocks of tissue approximately 2 cm long were taken from the parahippocampal gyri (PHG) at a level just caudal to the uncus. Tissue from the right gyrus was prepared by the Van der Loos modification 35 of the Golgi-Cox method of controlled impregnation. When impregnation was complete terminal segments ended without trailing off as a series of dots. Tissues were dehydrated, embedded in Cedukol, and sectioned coronally at alternate thicknesses of 200 and 40/zm. The 200/am sections wele processed through solutions of ammonium hydroxide and photographic fixel (Tetanal), dehydrated in ethanol and cover-slipped with Canada Balsam. The slides were then coded by a technician not involved in the data collection procedures. The 40 /~m sections were stained with cresyl violet, dehydrated, cleared, and coverslipped with Permount. Tissues from the left gyri were fixed in formalin, embedded in paraffin and sectioned at 10 #m. These sections were used for routine hematoxylin and eosin, cresyl violet and Bodian preparations for pathological survey and for demonstrations of lipofuscin, senile plaques and neurofibrillary degeneration. There was no evidence of infarct, tumor, cyst, or physical trauma in the P H G of any of the cases selected. Data collection and analysis A cursory observation of sections showed that there was an extremely wide range of sizes of dendritic trees within each of the cases in each of the 3 groups. It was important, therefore, to insure that the selection of cells for analysis was random. The

26 population of cells from which our cell sample was selected consisted of fully impregnated layer 11 pyramidal neurons in the region of the parahippocampal gyrus lying between the occipitotemporal sulcus and the apex of the gyrus. Beginning at the sulcus and moving toward the apex, as each such neuron was encountered a judgement was made as to whether it was suitable for analysis based on a set of criteria discussed below. If it was suitable, a die was cast to randomize the selection of cells. If an odd number was cast the cell was included in the study and a rough hand drawing of the dendritic tree was made to aid in the tracking process. Three cells were selected fiom each of 5 slides from each case for a total of 15 cells per case, 75 cells per group and 225 cells for the study. The criteria for selection of cells for analysis required that the dendritic processes be completely impregnated and not obscured by other elements in the tissue section. In addition, it was required that the soma lie near the center of thickness of the section. This criterion was adopted in favor of the alternate one of requiring that no segments of the tree be cut in the sectioning process because the latter method might introduce inadvertent bias toward smaller cells. In our sample, fewer than 6 ~ of dendritic ends resulted from sectioning. The number and length of cut ends did not differ significantly among the three groups (P > 0.30, non-parametric analysis of variance). Therefore, cut and uncut ends were treated as a single group. Data were collected using a computerized image processing system TM which tracks dendrites in three dimensions and stores 1000-3000 points to represent a tracked dendritic tree in 3 dimensions. All tracking was done under a special Leitz Ks F1 Oel 100 × / 1.32 long working distance oil objective in order to accommodate the 200 /zm thick sections. Analysis of the data by the computer included measurements of length and numbers of dendritic segments with the trees ordered in somatofugal (lst order begins at cell body and branches into second order, etc.) and somatopetal (terminal segments, next-to-terminal etc.) directions. In addition, we employed a three-dimensional modification of Sholl's concentric circle method 29. The computer constructed concentric spheres centered at the cell body and spaced 10 # m apart. The number of intersections of dendrites with successive spheres and the number of branch and end points between successive spheres were recorded. For all analyses, data from the apical trees were kept separate from those for the basal trees. Sizes of layer II pyramidal cell bodies were measured in the Golgi material using a Zeiss MOP III X, Y tablet and microcomputer. These measures were made to assess the possibility that any group differences in dendritic trees might be related to differential loss of neurons as a function of size. Golgi-stained material was used for this analysis so we could be certain that measures of cell body size would be made on the same population of cells used to obtain dendritic measures. Statistical tests of significance consisted of the Kruskall-Wailis non-parametric analysis of variance, the Mann-Whitney U-test and the sign test 30. Sizes of cell bodies were evaluated by means of a parametric analysis of variance.

27 RESULTS Qualitative

Routine neuropathological survey of the left parahippocampal gyri stained with hematoxylin and eosin (H + E) did not show any severe atherosclerosis, tumors, spongiform changes, or other pathological processes unrelated to SD. It was not possible to distinguish groups qualitatively on the basis of the H + E slides. In cresyl violet-stained paraffin sections lipofuscin accumulations distinguished the older cases from the younger cases. Differential loss of cells as a function of size was not readily apparent. Bodian preparations showed abundant senile plaques and neurofibrillary tangles in the SD group. There was wide variation in the appearance of the Golgi preparations of the right parahippocampal gyri of the 15 cases in terms of the relative densities of neurons which impregnated, impregnation of non-neuronal elements, formation of nonspecific precipitate and clarity of background. Within every section from each of the 15 cases there was a great deal of variation in the appearance of even closely adjacent impregnated neurons. Neurons ranged from those with rich dendritic arborizations extending over large distances to those with apparently shrunken, atrophied, or regressed arborizations, sometimes consisting of only a single segment. Examples of these two extremes could be found side by side. Examination of sections with Normarski optics showed that this variability was not an artifact of incomplete impregnation. Perhaps in part because of this large degree of variability there were no detectable qualitative differences among the adult, aged and senile dementia Golgiimpregnated tissues. Neither was there any qualitative trend in the appearance of cases with different postmortem delays before fixation. While there was a tendency for TABLE I Summary of quantitative data of dendrites for the 3 groups of subjects

Means (per cell) £ S.E.M. n = 75 for total dendritic length (TDL) and number of segments; n is variable for average segment length (ASL) depending on number of segments per group. See figure legends for statistical evaluations. All segments

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27.4il.5 32.6£1.0 26.4~-1.8

298.9+40.0 404.7+52.3 253.8±16.1

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683.6-E101.826.7+4.0 777.1±47.2 25.8±0.8 691.0±87.8 27.1±3.0

25.5±0.9 30.1:~1.4 25.6±1.9

468.8±64.0 15.7:~2.2 568.8+96.3 15.2i0.5 486.7+60.3 15.8+1.6

9.1+1.0 9.9+0.8 8.4±0.3

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30 impregnation to be less dense in the superficial layers of the cortex than in the deeper layers, this tendency was not related to increasing age, decreased level of intellectual function, or duration of postmortem delay before fixation. The same held true for irregular somata and kinked dendritic processes.

Quantitative In general, the quantitative data show that layer II pyramids in the parahippocampal gyrus of aged individuals had longer and more branched dendrites than either adult or SD individuals. There was a slight tendency for SD individuals to have shorter, less branched dendrites than adults. Differ ences among groups were greater in apical than in basal portions of the dendritic tree. These differences were largely accounted for by the terminal portions of the dendritic tree. These comparisons are shown in Table I. AGED VS ADULT Concentric spheres analysis Apical tree. The number of intersections of dendrites of the average apical tree with each successively larger concentric sphere (Fig. 1A) was consistently greater in the aged than in the adult cases (P < 0.001, sign test). The absolute magnitudes of the differences were greatest in the distances intermediate from the soma (50-150 #m) and least at the proximal (10-40 ktm) and distal (160-300/zm) extremes of the dendritic tree. There were no statistically significant differences between the adult and aged groups in the number of branch or end points between successive concentric spheres (Fig. 1B and 1C). Basal tree. In the basilar system, the profile of number of dendritic intersections with successive concentric spheres was higher for the aged than for the adult cases (P < 0.001, sign test, Fig. 2A) but the magnitude of the differences was substantially smaller than in the apical trees. As in the apical trees there were no statistically significant differences in the number of branch (Fig. 2B) or end points (Fig. 2C) occurring in the space between successive concentric spheres. Figs. 1 (top) and 2 (bottom). A: numbers of intersections of dendrites with successive concentric spheres spaced 10 p m apart. B: numbers of branch points between successive concentric spheres. C: numbers of endpoints between successive concentric spheres. Points represent averages for all 75 cells in each group. Sign test for significance of differences between group profiles.

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31

Length and number analyses: Somatofugal analysis Apical tree. Total dendritic length was significantly greater in the*aged cases than in the adult cases as a consequence of more segments per tree and greater average length of segments. Analysis of dendritic trees ordered in a somatofugal direction was based on the dendritic trees of the 'average' cell for each group. This average was obtained for each point by summing values for a given parameter across the entire group and dividing by the total number of cells in the group, i.e. 75. Thus, low mean values seen at the higher orders reflect the small number of cells whose branching extended to those orders. The profile of total dendritic length as a function of order was significantly higher for the aged than the adult group (P < 0.025, sign test, Fig. 3A). The differences were accentuated in the fourth through the seventh orders. At these orders, the adult vs aged number of segments (Fig. 3B) and average segment length (Fig. 3C) were also significantly different (P < 0.01, non-parametric ANOVA, Mann-Whitney U-test). Basal tree. In the basal tree, plots of total dendritic length (Fig. 4A) and average segment length (Fig. 4C) as a function of order were significantly higher in the aged than adult groups (P < 0.02 and 0.01, respectively, sign test). In contrast to the apical tree the number of segments as a function of order was not significantly different (Fig. 4B).

Length and number analyses: Somatopetal analysis Apical tree. Analysis in a somatopetal direction allows one to look at terminal segments as a separate population. In our sample, the terminal and next-to-terminal segments constituted over 90 ~ of the segments. Therefore the remaining segments were grouped together. Although most of the differences found in this fbrm of analysis did not reach significance at the P < 0.05 level, thete was clearly a trend for the group differences identified by the concentric spheres and somatofugal analyses to be consequent to effects in the terminal dendritic segments. Fig. 5A suggests that the greater complexity of apical dendritic trees in aged cases shown in Fig. 1A by the concentric spheres analysis and in Fig. 3 by the somatofugal analysis, is due largely to changes in the terminal segments. Next-toFigs. 3 (top) and 4 (bottom). Dendritic lengths (A), segment numbers (B) and average segment lengths (C) as functions of dendritic order for trees ordered in a somatofugal direction. Points represent means for the 75 cells in each group. Low values for the higher orders reflect the small number of cells which had segments of those orders. Sign test for significant (P < 0.05) differences between group profiles.

Fig. 3 Apical Fig. 4 Basal

Adult vs Aged Aged vs SD Adult vs SD Adult vs Aged Aged vs SD Adult vs SD

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34 terminal segments contribute to the differences among groups to a lesser extent. Fig. 5B and 5C shows that in apical dendrites these differences in total length of the terminal segments are due to differences in both the number and average length of segments. Basal tree. The pattern seen in the basilar system is different from that seen in the apical system. Fig. 5D shows that the differences between these two groups seen in basal dendrites (Figs. 2 and 4) are confined to the terminal segments. The differences are due entirely to changes in average length of terminal dendritic segments with no differences apparent among groups in the number of terminal dendritic segments. Next-to-terminal dendritic values are very similar among groups. Regression analysis on age Because of the wide range of ages within both the adult and normal elderly group, it was possible to pool these 10 cases to look at aging as a continuum. Using regression analysis and the best fit regression line determined by the least squares method we were able to calculate that the length of terminal segments increases significantly (r ---- -}-0.57; P < 0.05) with advancing age (from 44 to 92 years of age) at a rate of 0.21/~m per terminal segment per year, or 1.99/~m per cell per year since there were an average of 9.48 terminal segments per cell. When a number of other parameters were examined in this manner there were trends toward positive correlations with advancing age, but the slopes of the best fit lines did not reach statistical significance. SD VS N O R M A L A D U L T A N D AGED Concentric spheres analysis Apical tree. The profile for the number of dendritic intersections with successive concentric spheres was significantly lower for the SD group than for the normal aged or the adult groups (P < 0.001, P < 0.03, respectively, sign test, Fig. IA). The profile for branch point numbers was also significantly lower in the SD than in the aged group (P < 0.001, sign test, Fig. 1B) but the magnitude of this difference was small. All other comparisons of SD branch and endpoint numbers as a function of distance from the cell body failed to reach significance at the 5 °/o level. Basal tree. The profile for the number of intersections with concentric spheres (Fig. 2A) was significantly lower (P < 0.001, sign test) for SD basal dendrites than for basal dendrites of the comparably aged normal cases. The magnitude of this difference was very small in comparison to that seen in the apical trees. Number of intersections

Fig. 5. Dendritic lengths (A), segment numbers (B) and average segment lengths (C) as functions of dendritic order for trees ordered in a somatopetal direction. Means for 75 cellsin each group 4- S.E.M. Aged group differed significantlyfrom SD in average length of terminal segments of the apical trees (£ < 0.01, non-parametric analysis of variance and Mann-Whitney U-tests). No other differenceswere significant beyond the P < 0.05 level.

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36 was similar in the SD and younger adult cases. Numbers of branch points (Fig. 2B) and dendritic ends (Fig: 2C) per interval were similar among the three groups.

Length and number analyses: Somatofilgal analysis Apical tree. The profiles of total dendritic length (Fig. 3A), number of segments (Fig. 3B) and the average length of segments (Fig. 3C) at each order, were significantly (P < 0.05, sign test) lower for SD than for the normal aged or adult groups. These differences were at times quite large (e.g. 45 ~ in the comparison of total length of fourth order dendrites of SD and aged cases). Basal tree. In the basilar system the profiles for dendritic length per cell (Fig. 4A) and average segment length per cell (Fig. 4C) were significantly lower (P < 0.05, sign test) in the SD group than in the aged, but SD did not differ from the adult. Differences were accentuated in the middle range of dendritic orders (orders 2 and 3). Number of segments per cell (Fig. 4B) was not different among groups.

Length and number analyses: Somatopetal analysis Apical tree. Analysis in a somatopetal direction showed that the decreased complexity of dendritic trees in the SD cases relative to the normal aged shown by concentric spheres analysis and by the somatofugal length and number analyses (Figs. 1A and 3) are due largely to differences in terminal segments (Fig. 5A). These differences were present in lengths, numbers and average segment length (Fig. 5A-C) of terminal segments. Differences were generally smaller or not apparent in next-toterminal and remaining segments. Basal tree. Group differences demonstrated by concentric spheres and somatofugal analyses were more sharply confined to terminal segments than in the apical tree. Values for total length (Fig. 5D) and average length (Fig. 5F) of terminal segments were lower for SD than for the aged group while the SD and adult groups were very similar. In contrast to the apical trees, no differences were apparent in the number of terminal segments (Fig. 5E) in the 3 groups, suggesting that the apical but not the basal dendrites altered their numbers of branches. In spite of substantial differences among the adult, aged and SD groups in the magnitudes of a number of dendritic parameters, the shapes of curves and histograms derived from all forms of analysis were strikingly similar for the 3 groups. Maximum and minimum values for the three groups occurred at identical or nearly identical points on the abscissas of Figs. 1, 2, 3 and 4.

Effect of postmortem delay Postmortem delay before fixation is an unavoidable variable in studies utilizing autopsy-derived tissues. In our samples, the mean postmortem delay did not differ significantly among the 3 groups (P > 0.30, non-parametric analysis of variance). In addition, it was found that there was no significant relationship between postmortem delay (from 2.25 to 21.50 h) and total dendritic length per cell in either the apical or basilar dendritic systems (r = --0.26, apical; --0.21, basal, P > 0.20, linear regression). Thus, it is unlikley that this factor contributed to the differences obtained among the groups in this study.

37 Cell body size

Measures of Golgi-stained cell body sizes gave an average size of 204 sq./~m for adult and 182 sq.#m for the aged groups. These differences were not statistically significant (P > 0.10, ANOVA). DISCUSSION Dendritic trees were significantly larger in the normal aged than in the adult human. In contrast, in aged humans who became demented with advancing age, dendritic trees were smaller than those of both the normal aged and the adult groups. Differences between groups were greatest in the terminal segments of apical trees at distances intermediate from the soma rather than at the proximal or distal extremes of the dendritic tree. The differences were found in both the number and length of terminal segments in the apical portion of the tree and in length but not number of terminal segments in the basal dendritic tree. Adult vs normal aged cases

This is the first demonstration of dendritic plasticity in the adult human brain. There have been reports of dendritic growth in rodent brain past the period normally defined as the developmental phase (i.e. beyond 30 or 40 days post-partum)ll,16AT, 23, 24,34. Further evidence for growth of dendritic trees in the mature, non-primate mammalian nervous system is found in demonstrations that dendrites can grow in response to prolonged electrical ~3 and environmental 34 stimulation. It has also been reported that growth of dendrites can occur in non-primate mammals subsequent to experimental lesions22, 31 well after the period of normal neuronal maturation has passed. As in our study, several of these reportsll,17,2z, 34 demonstrated that dendritic growth was confined largely to terminal dendritic segments. In addition, in a report on aging in rat olfactory bulb t6, data were presented showing a spurt of dendritic growth coincident with a period of rapid cell loss (24-27 months of age). It is tempting to speculate that this spurt of growth occurs as a compensatory response to neuronal loss. In the present study, quantitative estimates of total neuronal numbers were not attempted. Thus, it is not known whether the dendritic growth we observed was a continuation of a preprogrammed life-long process of sustained growth, a compensatory response to partial loss of cortical neuronsS,6, tr, a response to environmental stinlulation 34, or some combination of these. The differences between the adult and normal aged cases indicate that there is either a significant net growth of dendrites by both branching and segment elongation with advancing age, or that there is selective fallout of smaller neurons. However, measurements of cell body size showed that the latter was not the case. In fact, cell bodies were actually larger in the adult than in the aged group. Thus, it is difficult to accept selective loss of smaller neurons with advancing age as an explanation of our results. Rather, it seems likely that the data represent net continued growth of dendrites from adulthood through normal old age. It is known that in a number of conditions there is widespread abnormal growth

38 of dendrites into formations which, simply by their appearance, seem unlikely to enter into normal synaptic relationships in the surrounding neuropi121, 25. In material used in the present study, such neoformations were not observed. The close similarities in the shapes of profiles of data from the adult and aged groups which were generated by the spheres and centrifugal analyses are important in this context. They indicate that the growth process which produced the differences from the adult to the aged group was similar to the normal growth process of development (which gave rise to the adult profiles) in that it left undisturbed the normal overall pattern of branching and segment elongation. Our study does not, however, provide evidence as to whether the growth of dendrites late in life involves the generation of functionally operational material.

SD vs a&dt and normal aged cases In our SD group, in which there was a failure to maintain a normal level of intellectual and cognitive functioning late in life, there was an apparent failure to maintain the pattern of continued net dendritic growth seen in the normal aged group. In fact, in certain dendritic parameters, dendritic trees appeared to be smaller in SD than in the adult group. This failure to maintain net dendritic growth would allow for the full effects of neuronal loss to be incurred. It is possible that the data from the SD cases do not represent only a failure of growth. Such growth may have occurred only to be followed by regression to and beyond the adult level. In the study by Hinds and McNelly 16 a period of dendritic growth occurred from 24 to 27 months of age and was followed by a collapse back to adult (3 months) levels by 30 months of age. If such a process occurs in normal human aging, and if dementia represents an acceleration of the aging process, then data from the SD cases in this study may therefore reflect this period of collapse rather than an actual failure to continue to grow. This possibility is an object of further study in our laboratory. Previous reports of regression in aged brain While the results of this study are consistent with one study le of aging in rodent brain, they are at sharp variance with a number of reports of aging in human, dog and rodent brain. Scheibel et al. 26-2s conducted a series of qualitative studies of Golgi preparations of human cerebral cortex from aged and senile dementia cases. The consistent observation reported for all areas of cortex examined was of a progressive regression of dendritic trees sometimes to such an extent as to leave only the soma and a single dendritic stump in some affected neurons. Very similar descriptions of dendritic regression have been reported in the central nervous system of the aged mouse 19 and dog 20. In agreement with these studies we saw numerous neurons with dendritic trees which fit the previous descriptions of regression, often adjacent to normal-appearing cells. However, we saw many such cells in every section from every case regardless of age. We were unable to distinguish one group from another on the basis of the number of cells involved or the severity of the regression. It is possible that the dendritic regression seen in Golgi preparations is a

39 preliminary stage in the age-related process of neuronal fallout which has been demonstrated in Nissl preparations 5. Our data suggest that, at a given point in time, not all neurons are at such a stage. Rather, a number of neurons must be in a growth phase. In normal aging, the sum of the regressive and progressive events results in a net average increase in dendritic length per cell at least into the tenth decade of life. However, in view of the great variability in size and extent of dendritic trees relative to the magnitude of the net growth we have demonstrated, it is not surprising that the growth has gone undetected in qualitative studies. In senile dementia, the magnitude of any growth which may occur is insufficient to overcome the effects of regression. Our data are also at variance with two well controlled quantitative studies of dendritic parameters in aged rat cortex15,36 in which regression was reported. A single difference in methodology may be the source of the discrepancy. In both of these previous studies, cells with dendrites cut in sectioning were eliminated from the sample. The sections used in both studies were 125/,m thick. In contrast, we required only that the soma be near the center of a 2 0 0 / , m thick section. If a cut end was encountered in the tracking process, it was labeled as such, but was not used as a selection criterion. (In the present study, fewer than 6 ~ of the terminal segments were cut in sectioning and the number of cut ends per cell did not differ significantly amopg the 3 study groups - - P > 0.30, non-parametric analysis of variance). In our sample of apical trees we found a significant correlation between total dendritic length and the number of ends per cell cut in sectioning (r = +0.67; P < 0.01). The best fit line predicts that in 200/zm thick sections a sample consisting only of apical trees with less than 226/tm total dendritic length will have no dendrites cut. This is smaller than the smallest average apical tree tracked in any of our cases. Thus, in this study, elimination of trees which were cut in sectioning would most certainly have biased our sample toward smaller cells. If both regressive and progressive changes occur in normal aging, such constraint on selection of cells for analysis would cause overestimation of regressive changes and underestimation of progressive ones. (Note that this analysis applies only to sections parallel to the apical dendrite and only to the apical tree of layer II pyramids. However, the same general conclusions would apply to other cell types, although the values of the parameters would differ.) While the 200 ~m section thickness used in this study plays a role in increasing the validity of comparison among groups (relative validity), it does not totally eliminate the effect of cut dendritic ends on absolute validity. The probability that a dendrite will be cut in sectioning decreases as its point of exit from the section deviates from the two points defined by a line from the cell body perpendicular to the plane of section. As this line deviates from the perpendicular, the decrease in probability of cutting a dendrite is proportional to the inverse o f the cosine of the angle of deviation. Thus, inclusion of cells with cut ends does not impose any simple numerical limit (e.g. half the section thickness) on the data. Our data showed that the basal and apical portions of the dendritic tree respond differently to aging. Terminal dendritic segments in the apical tree grow and branch, whereas terminal segments in the basal tree show a net growth (similar in magnitude to that seen in the apical tree) but not net branching. This difference between two regions

40 of the dendritic tree may be a reflection of: (1) intrinsic differences in the organization of these two regions of the dendritic trees of the pyramidal cells studied, or (2) differences in the afferent input to these two regions of the dendritic tree, with these afferent inputs differentially affected by aging. Thus, the neurological deterioration associated with normal advancing age is not reflected in reduced dendritic extent of single neurons in the parahippocampai gyrus. On the contrary, in the face of widespread neuronal loss, lipofuscin accumulation and neurofibrillary degeneration, neurons continue to elaborate dendritic material into the 10th decade of life. That this elaboration is sufficient to compensate totally for neuronal loss seems doubtful, but must be left to further study, In senile dementia, where there is a devastating degree of neurological deterioration, we found no evidence for such continued dendritic growth. Neither did we find a profound regression of trees which might be expected in view of the decimation of cognitive capacities in this condition. This suggests one of two possibilities. (1) The dendritic morphology of the system we studied may not be directly affected in senile dementia. The lesions causing the behavioral deterioration may be purely biochemical (e.g. neurotransmitter dysfunction) or physiological, or, if morphological, they may be confined to other cell types or brain regions. (2) Alternately, there may be a threshold effect in which the dendritic growth in normal aging is sufficient to compensate partially for the effects of neuronal loss, lipofuscin accumulation and neurofibrillary degeneration. Any margin of safety which remains may decrease as these degenerative changes accumulate. In SD, the result of a failure to maintain net dendritic growth demonstrated by the data in this report could allow the full force of the deleterious effects of these degenerative processes to fall on the organism. Clearly we do not have sufficient data to distinguish between these two classes of hypotheses. Further studies are underway in our laboratory as part of an effort to address these issues.

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