Anat. Embryol. 148, 279--301 (1975) 9 by Springer-Verlag 1975

Loss of Dendritic Spines in Aging Cerebral Cortex* M a r t i n L. F c l d m a n a n d C. D o w d Department of Anatomy, Boston University School of Medicine, Boston, Massachusetts, USA Received April 9, 1975

,S'ummafy. Previous work has shown that lbhe dendritic spines of pyramidal neurons of the cerebral cortex are sensitive to a wide variety of environmental and surgical manipulations. The present study shows that the normal aging process also affects these spines. The spines were studied with the light microscope in Golgi preparations from rats ranging in age from 3 to 29.5 months. Visible spines were counted on either 25 or 50 ft, segments of the basal dendrites, apical dendrites, oblique branches, and terminal tufts of layer V pyramidal cells in area 17. A progressive loss of spines occurred at each of these loci. The smallest observed spine loss (24%) occurred on the dendrites of the terminal tuft, and the largest (40%) on the oblique branches. Age-related spine loss appears to affect all animals, and for animals of any one age the overall loss is similar. However, the cell-to-cell variability within an individual animal is pronounced, some cells with high spine densities being present at every age examined. As a general rule, there is a positive relationship between visible spine density along the apical dendrite as it traverses layer IV and the thickness of the dendrite. Wit h advancing age, the relatively thick dendrites decrease in number so that the thinner dendrites make up an increasingly larger proportion of the total apical dendrite population. Questions that remain for the future include the genesis of the spine loss, its relation to other aging changes, and its functional significance for the neuron. Key words: CNS - - Cerebral cortex - - Aging - - Spines.

Introduction D e n d r i t i c spines are small finger-like protrusions, often t e r m i n a t i n g in bulbous expansions, which e x t e n d from t h e d e n d r i t e s of m a n y t y p e s of neurons. One class of n e u r o n whose d e n d r i t e s are p a r t i c u l a r l y rich in spines is t h e p y r a m i d a l cell of t h e m a m m a l i a n cerebral cortex. All of the m a j o r d e n d r i t e s of this cell t y p e - - t h e basal dendrites, t h e apical d e n d r i t e , t h e oblique branches of t h e apieM dendrite, a n d t h e t e r m i n a l t u f t of t h e apical d e n d r i t e - - b e a r spines. P y r a m i d a l cells are p a r t i c u l a r l y p r o m i n e n t in l a y e r V of t h e cortex, h a v e w i d e l y r a m i f y i n g dendrites, a n d h a v e axons which c o l l e c t i v e l y comprise t h e chief c o n d u c t i o n p a t h w a y s for n e r v e impulses l e a v i n g t h e c o r t e x (Lorente de Nd, 1949; R a m d n y Ca,jal, 1911; Shell, 1956). P y r a m i d a l d e n d r i t i c spines can be visualized in t h e light microscope in Golgis t a i n e d p r e p a r a t i o n s . A l t h o u g h e x a m i n a t i o n of such p r e p a r a t i o n s , a n d of correl a t e d electron micrographs, allows classification of spines according to shape (Jones a n d Powell, 1969; P e t e r s a n d K a i s e r m a n - A b r a m o f , 1970), no differential functions h a v e been d e t e r m i n e d for t h e various spine types. I n t h e electron micro* Supported by United States Public tIealth Service Program Project Grant HDO-5796-03 and Research Gran~ NB-07016.

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35. L. Feldman and C. Dowd

scope normal pyramidal dendritic spines, regardless of morphology, appear to be postsynaptie structures. In fact, the large majority of the axon terminals which form synapses with the pyramidal cell surface do so on the spines, rather than on the inter-spinous portion of the dendritic trunk or on the perikaryon (Colonnier, 1968; Colonnier and t~ossignol, 1969). It is this fact which makes the study of dendritic spines significant. During the decade following Ramdn y Cajal's discovery of the dendritic spines, it became apparent that these structures were sensitive to a wide spectrum of conditions, including disease states, the administration of certain chemical substances, non-physiologicM stimulation, and manipulation of factors such as food intake and sleep. Frequently, spine changes were accompanied by the appearance of nodules, or beads, in the parent dendrites. Examples of these early findings are to be found in the papers of Demoor (1898), Monti (1895), Querton (1898), and Soukhanoff (1898a, b). Within the past 10 years, there has been a resurgence of interest in these types of changes. The modern work has demonstrated that alterations in the numerical density of spines along pyramidal cell dendrites can be produced by interruption of the afferent axons synapsing on the spines (Ben Hamida, Ruiz de Pereda, and Hirseh, 1970; Globus and Scheibel, 1967a, b; Gruner, Hirsch, and Sotelo, 1974; Rutlege, Duncan, and Cant, 1972; Valverde and Estdban, 1968), by manipulation. of sensory stimuli or stimulus processing at the level of the receptor (Cant and Rutlege, 1973; Fifkovs 1970; Globus and Scheibel, 1967b; Parnavelas, Globus, and Kaups, 1973; Valverde, 1967, 1971b; Valverde and Estgban, 1968), by Xirradiation (Schad6 and Caveness, 1968), by intracortieal injection of alumina cream (Westrum, White, and Ward, 1964), and by variation of what is assumed to be the "complexity" of the cage environment (Globus et al., 1973; Greenough and Volkmar, 1973). In addition, it has been determined that the rate of development of certain spines may be influenced by multidimensional sensory stimulation (Schapiro and Vukovich, 1970). Finally, alterations in dendritic spine number and form are observed in certain types of human clinical material (Marin-Padilla, 1972, 1974, Purpura, 1974). Taken together, the data of both the classical and modern studies establish unequivocally the sensitivity of the pyramidal dendritic spines to experimentallyinduced changes in the external and internal environments. The purpose of the present study was to determine whether the spines are also affected by a naturallyoccurring variable, the passage of time. To this end, spine counts were undertaken in Golgi preparations from adult rats of progressively advanced ages. Although a considerable body of information exists concerning changes in the brain that accompany advancing age (Feldman and Peters, 1974; Gaitz, 1972; Wright, 1974), the past lack of exploitation of the Golgi method in the study of the aging brain has deprived us of systematic data concerning aging changes in the morphology of dendrites and their spines. Methods

The animals used in this study were male albino Sprague-D~wley rats, purchased from Charles River Breeding Laboratories, Wilmington, Massachusetts. All animals were reared

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with water and a s t a n d a r d laboratory r a t chow (Purina) available ad libitum. Two or three animals from each of the following ages were prepared: 3, 12, 17, 18, 27, and 29.5 months old. I n addition, one animal at 24 months of age was examined. The animals over 12 months of age were designated by the supplier as retired breeders. Animals exhibiting any grossly detectable pathology (e.g., tumors, paralysis) at the time of perfusion were discarded. All animals used in the study were perfuscd through the aorta with glutarMdehyde-formMdehyde solutions, following procedures previously published (Peters and WMsh, 1972). Tissue blocks including the visual cortex (Montero, Rojas, and Torrealba, 1973) in the central portion of Krieg's area 17 (1946a, b) were dissected out on the day following perfusion. Tissue blocks consisted of roughly coronal slabs. An effort was made to excise the slabs in such a way t h a t their anterior a n d posterior surfaces were perpendicular to the surface of the cortex of area 17. Blocks used for the spine counts were impregnated b y the classical Golgi rapid method using the solutions recommended by Valverde (1965). Generally, single impregnations were employed. The tissue was embedded in low-viscosity nitrocellulose, serially sectioned at approximately 125 ix, and m o u n t e d under cover glasses using gum damar. Such preparations do not exhibit fading or destaining after extensive examination a t high magnification over a period of several years. Spine counts were carried o u t on the dendrites of layer V pyramidal neurons. Visible spines were counted on four segments of the dendritic tree, as shown iu Fig. 1. The first segment, 25 [z in length, was located on a basal dendrite, and began 25 ~ from the origin of the dendrite a t the perikaryon. The second segment, 25 ix in length, was located on an oblique branch of the apical dendrite, a n d began 25 ix from the origin of the oblique dendrite at the apical dendrite. The third segment, 50 a in length, was located along the apical dendrite in layer IV. Since a clear delimitation of the boundaries of layer IV was generally not discernible in the Golgi preparations, 16 ~ thick Nissl-stained sections of r a t area 17 were examined in order to derive criteria for specifying the depth of layer I V from the pial surface. As a result of this analysis, it was determined t h a t a 50 a vertical segment t h a t lies between one-third and one-half of the distance from the pial surface down to the top of the white m a t t e r will fall within layer IV. Since these two points could be clearly determined in the Golgi preparations they were used to specify the position of the t h i r d segment counted. The fourth segment, 50 ~x in length, began a t the major branching point of the apical dendrite into the terminal tuft. This branching point is situated in the vicinity of the layer I / I I border. For each section through area 17, counts were performed on every impregnated layer V pyramidal neuron whose apical dendrite extended up to a t least the point a t which it branched into the terminal tuft. Spines were counted in the light mieroscope using an ocular micrometer and Zeiss oil immersion optics (X 100 long-working-distance Neofluor objective, N.A. 1.1). I t was found t h a t a t any lower magnification the correlation between the spine counts of the two observers (M.L.F. and C.D.) was unsatisfactory. We believe our criteria for inclusion of a structure as a countable spine could be characterized as liberal. All protrusions, whether s t u b b y or peduneuluted, with and without terminal bulbous expansions, were counted as spines if they appeared to be in direct continuity with the dendrite shaft. I n the numerous cases where it was difficult to decide whether a structure should be counted as one spine or two, it was eounted as one unless critical fine focussing revealed a clear separation of images at some point along the structure. No a t t e m p t was made to correct for dendritic obliquity within the section or to compute " t r u e " dendritic lengths. E v e r y effort was made to perform counts only on dendritic segments which appeared to be parallel to the plane of sectioning. Although no counted segments were absolutely straight (see Fig. 2--9), the extent of the small local departures from linearity did n o t appear to change with age. These factors, although influencing the measurem e n t of segment length, m a y be treated as constants which do not affect the relative spine counts across ages. Many dendritic segments, particularly on basal and oblique dendrites, were not counted due to unacceptable obliquity or tortuosity. The nominal segment lengths of 25 ix and 50 ~ were measured with an ocular micrometer. The second type of preparation employed in this study consisted of tangentially oriented 1 ~zsections through layer IV. These sections were derived from ArMdite-embedded osmicated blocks and were stained with toluidine blue a n d pyronin-B. Two animals at 3 months of age and two animals at 29.5 months of age were used in this p a r t of the study.

M. L, Feldman and C. Dowd

282

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Fig. 1. Schematic illustration of the counting loci used in the present study. A layer V pyramidal cell is shown with its perikaryon in layer V, its axon (ax) extending down into the white matter, and its apical dendrite branching into the terminal tuft near the I / I I border. Two basal dendrites are shown extending out from the base of the perikaryon and one oblique dendrite extending diagonally into layer IV. Spine counts were performed on the four dendritic segments indicated by the black rectangles. The basal and oblique dendrite segments were 25 ,u in length, the apical shaft and terminal tuft segments 50 ~ in length. The apical shaft segment was situated midway between two points located one-third and one-hMf of the way down from the piM surface (Pin) to the white matter. See text for further details

Results Q u a l i t a t i v e e x a m i n a t i o n of G o l g i - i m p r e g n a t e d p y r a m i d a l cell d e n d r i t e s in cortices from r a t s of different ages leads to a definite impression of progressive spine loss with a d v a n c i n g age. This impression is gained from observations in a u d i t o r y cortex (area 41) as well as in visual cortex, although our analysis of a u d i t o r y cortex is incomplete. E x a m p l e s of t h e a p p e a r a n c e of young a n d old p y r a m i d a l d e n d r i t e s from visual cortex are shown in Fig. 2 - - 9 . The loss of spines a p p e a r s to affect t h e entire d e n d r i t i c t r e e of t h e p y r a m i d a l neuron. A l t h o u g h c e r t a i n areas of old d e n d r i t e s f r e q u e n t l y seem to be p a r t i c u l a r l y d e n u d e d (see Fig. 9), t h e r e is no a p p a r e n t p a t t e r n to t h e spine loss. F o r example, spine-free regions of d e n d r i t e s do n o t a l t e r n a t e in regular fashion w i t h regions of n o r m a l spine density. F i n a l l y , old d e n d r i t e s h a v e surviving spines belonging to all of t h e morphological t y p e s p r e s e n t in y o u n g a d u l t animals. Thus t h e aging process does n o t a p p e a r to selectively l e a d to loss of spines of one p a r t i c u l a r form. The q u a l i t a t i v e impression of spine loss in visual cortex is borne o u t in d e a r - c u t fashion b y q u a n t i t a t i v e analysis. The p h e n o m e n o n of spine loss is observable in all of t h e older a n i m a l s used in t h e p r e s e n t study. Visible spines were c o u n t e d on a t o t a l of 1024 d e n d r i t i c segments. The q u a n t i t a t i v e d a t a are p r e s e n t e d in Fig. 10--13. As shown in these graphs, each of t h e four d e n d r i t i c loci exhibits a decrease ~n the m e a n d e n s i t y of visible spines over t h e age range studied. I n different

Spine Loss in Aging Cortex

283

Fig. 2 Figs. 2--4. Photomicrographs of portions of Golgi-impregnated pyramidal neurons in layer V of rat visual cortex at 3 months of age. Note the spine density present on the apical dendrite shafts (AP), the oblique dendrites (OB), and the basal dendrites (BA). Calibration lines, 25

a n h n a l s a t a n y given age, t h e r e is v e r y close a g r e e m e n t b e t w e e n t h e m e a n spine densities for p a r t i c u l a r locus. I t is i m p o r t a n t to p o i n t out, however, t h a t for a n y one of t h e four d e n d r i t i c loci t h e cell-to cell v a r i a b i l i t y in spine densities on the p y r a m i d a l neurons of a p a r t i c u l a r a n i m a l is large. Consider, for e x a m p l e , t h e d a t a

284

M.L. Feldman and C. Dowd

Fig. 3 from two 3 month old animals as plotted in Fig. 10. The spine counts for the 36 apical shaft segments of the first animal range from 44 to 79 spines. The counts for the 63 segments of the second animal range from 50 to 87. Variability of this same order is present in every animal in this study. Consistent with this is the finding that regardless of the age of an animal, at least some dendrites have normal spine densites, i.e., spine densities typical of 3 month old dendrites (Fig. 9 d).

Spine Loss in Aging Cortex

285

Fig. 4 The fact that every brain contains some pyramidal dendrites with high spine densities suggests t h a t the observed spine losses with age are not due simply to inability of the Golgi method to stain old spines. I n order to facilitate a direct comparison of the spine losses at the four dendritic loci examined, a part of the data from Fig. 10--13 is presented in different form in Table 1. I n the table, all spine densities have been expressed as spines per micron. This measure is derived by dividing the number of visible spines

286

M.L. Feldm~n and C. Dowd

Fig. 5 Figs. 5--7. Photomicrographs of portions of Golgi impregnated pyramidM neurons in layer V of rat visual cortex at 29.5 months of age. Note the decrease in visible spine density relative to the dendrites of the 3-month animals. The neurons shown in Fig. 5, 6, and 7 should not be considered "typical"; they were selected to illustrate the extensiveness of the spine loss obserwd on only some of the dendrites in the aging cortex. Abbreviations and calibration as in Figs. 2, 3, ~nd 4 co u n t ed along a g iv e n d e n d r i ti c s e g m e n t by the length of t h e s e g m e n t in microns. F r o m t h e table, it can be seen t h a t in t h e y o u a g a d u l t cortex t h e h i g h est av er ag e spine density, 1.43 spines/a, occurs on t h e m a i n shafts of t h e apical dendrites,

Spine Loss in Aging Cortex

287

followed in order by the oblique dendrites, the basal dendrites, and finally the dendrites of the terminal tuft, which have an average spine density of 0.74 spines/B. I n general, about one-third of the spines are lost in old animals. More precisely, the loss ranges from 24 % for the dendrites of the terminal tuft to 40 % for the oblique dendrites. These data are suggestive of a correlation between the normal spine density of a dendritic region and the magnitude of the age-related spine loss. 5 Anat. :Embryol.

288

M.L. Feldman and C. Dowd

Fig, 7

Table 1. Loss of pyrimidal dendritic spines, expressed in spines per micron, from young adulthood (3 months of age) to old age (27 or 29.5 months of age) Dendrite segments

Apical shaft, Terminal tuft :Basal dendrite Oblique dendrite

NIean spine density

% loss

Young adulthood

Old age

1,43 0,74 0.82 1.26

0.9~ 0,56 0.56 0.76

spines/~m (0.t9) a Spines/gin (0.11) spines/fzm (0,13) spines/~xm (0.18)

a Figures in parentheses are standard deviations.

spines/~m spines/tam spines/tam spines/tam

(0.28) (0.14) (0.11) (0.20)

36% 24% 32% 40%

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Fig. 9 a--e. Illustrations of spines on pyramidal apical dendrites within layer IV, 29.5 month old rat. Note the general decrease in dendritic spines (a, b, e) compared to Fig. 8. Dendrite d is a thick dendrite showing minimal or no spine loss. Labelling and calibration as for Fig. 8

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During the course of this investigation, we gradually became aware of ~ rela~ tionship between the loss of visible spines on apical dendrite shafts and the diameters of the shafts. As illustrated in the dendritic profiles in Fig. 8 and 9, apical dendrites of relatively large diameter appear to have a larger complement of spines than those of relatively small diameter, regardless of the age of the animal. This point was eollfirmed by quantitative study of 343 pyramidal apical dendrite segments in layer IV (see Fig. 1r These dendritic segments were categorized as thin, medium, and thick according to the thickness of the dendritic shafts as examined in the Golgi preparations. Thin dendrites measure approximately 1 ~z, medium dendrites approximately 2 ~z, and thick dendrites approximately 3 ~ or more. At every age, the mean spine density for the thick dendrites is higher than that for the medium dendrites, and the mean spine density for the medium dendrites is higher than that for the thin dendrites. Also, for each of the three categories of dendrite diameter--thin, medium, and t h i c k - - t h e mean spine count per 50 segment decreases with advancing age. The mean spine count for thick dendritic segments in 3 month old animals is 70.2 spines, and this is reduced to 56.3 spines

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in 29.5 m o n t h old animals, a decrease of 20%. The same d a t a for t h i n d e n d r i t i c segments are 47.0 spines in 3 m o n t h old animals, a n d 32.5 spines in 29.5 m o n t h old animals, a decrease of 31%. The largest spine loss, 36 %, affects t h e m e d i u m dendrites. I n t e r p r e t i n g t h e significance of these figures requires analysis of t h e n u m b e r s of each k i n d of d e n d r i t e a t progressively older ages. This i n f o r m a t i o n is p l o t t e d

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in Fig. 15. From this figure, it can be seen that at each age (with the exception of the 27 month old animals) the medium dendrites are nmnerically the most prevalent in our Golgi material. Further, over the age range studied, the medium dendrites constitute an increasing proportion of the total number of apical dendrites present. At 3 months of age they comprise 56 % of the dendrite population; at 29.5 months of age they comprise 76 % of the dendrite population. The proportion of thin dendrites also increases with age, from 6 % of the dendrite population at 3 months of age to 16% of the dendrite population at 29.5 months of age. The proportion of thick dendrites, on the other hand, decreases, from 38% of the dendrite population at 3 months of age to 8% of the dendrite population at 29.5 months of age. A clear picture of progressive apical dendrite spine loss with advancing age emerges from the following integration of the above data (see Table 2). Thick dendrites bear large numbers of spines. But even though their spine loss with age is relatively small, the large reduction in the proportion of thick dendrites with age produces an overall loss of considerable magnitude in average spine number. Medium and thin dendrites bear fewer spines than thick dendrites, and increase in relative number with age. But in spite of this increase, because their spine loss is greater than that of thick dendrites, they actually contribute to the spine decrease seen in old animals. The net result is the observation of a spine loss with advancing age.

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M.L. Feldman and C. Dowd

Table 2. Summary of data on age-related spine losses on pyramidal apicaI dendrite segments of varying diameters Dendrite diameters

3 months

29.5 months

Spine loss

Thick

mean spine density 70.2 spines/50 Bm (4.55) a proportion of 38 % total dendrites

56.3 spines/50 Bm (8.66) 8%

20 %

Medium

mean spine density 66.2 spines/50Bm (6.16) proportion of 56 % total dendrites

-12.4 spines/50 Bm (8.80) 76 %

36%

Thin

mean spine density 47.0 spines/50 Bm (4.24) proportion of 6% total dendrites

32.5 spines/50 ~m. (11.21) 16 %

31%

a Figures in parentheses are standard deviations Table 3. Spine counts that would be obtained on hypothetical populations of i00 dendrites each in 3 and 29.5 month old animals. Calculated on the basis of the data in Table 2 a Dendrite diameter

3 months

29.5 months

Thick ~edium Thin

2 668 3 707 282

450 3 222 520

Total spines

6 657

4192

Spine loss

37 %

The hypothetical spine counts in this Table are obtained by multiplying the percentage figures i~ Table 2 by their appropriate spine densities. For example, 38 dendrites times 70.2 spines = 2668 spines.

If the observed spine densities a n d proportions (Table 2) are applied to hypothetical populations of 100 dendrites in 3 a n d 29.5 m o n t h old animals, one can calculate the absolute n u m b e r s oi spines t h a t would be observed. These figures are given in Table 3. I n the y o u n g a n i m a l the total calculated spine count would be 6657, a n d in the old a n i m a l it would be 4192. This a m o u n t s to a n overall spine loss of 37 %. One consideration t h a t m u s t be raised with respect to dendritic diameters is t h a t the observed changes i n the d i s t r i b u t i o n of diameters with age m a y involve a n artifact of the Golgi method. T h a t is, it m a y be t h a t large dendrites are harder to i m p r e g n a t e i n old animals while small dendrites are more readily impregnated. I n order to evaluate this possibility it is necessary to measure, b y a i n d e p e n d e n t method, p y r a m i d a l apical dendrite diameters in y o u n g a n d old animals. The m e t h o d employed here entails the e x a m i n a t i o n of t r a n s v e r s e l y sectioned profiles of apical dendrites as t h e y appear i n t a n g e n t i a l l y oriented 1 B plastic sections through layer IV. I t has previously been d e m o n s t r a t e d ( F e l d m a n a n d Peters,

Spine Loss in Aging Cortex N I00

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29.5 (months)

Fig. 16. The differential age distribution of 400 pyramidM apical dendrite shaft diameters examined in 1 > tangentially oriented plastic sections through layer IV of area 17. Compare with Fig. 15. N's as for l~'ig. 14

1974; Peters and Walsh, 1972) that in these preparations the pyramidal apical dendrites appear in readily discernible discrete clusters. Each cluster generally contains 6-8 dendrites and is separated from its nearest neighboring cluster by a center-to-center distance of 30-40 ~. For the present study, 1 F preparations were examined from area 17 of two 3 month old rats and two 29.5 month old rats. In each of the four animals the diameters of 100 dendrites situated within clusters were measured. Dendrites were classified as thin, medium, or thick on the same basis as described for the Golgi material. The data, presented in Fig. 16, show that at each age the medium dendrites comprise the largest category (compare Fig. 15). The major shift seen in the Golgi material (Fig. 15) is corroborated. Thick dendrites decrease and thin dendrites increase in relative frequency over the age range studied. The increase seen in medium dendrites in the Golgi material was, however, not confirmed in the study of the 1 F sections. General comparison of the data derived from the two methods leads us to believe that the reduced dendritic diameters seen in the Golgi preparations of the aged cortex are real, and not due to an age-dependent differential stMnability of dendrites by the Golgi technique.

Discussion In the present study, the "normM" baseline data against which aging changes were measured are the spine counts obtained at 3 months of age. These spine counts (see Table 1) generally run slightly higher than the presumably mature

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normal spine counts obtained by others in different areas of cortex or in different species (Globus and Scheibel, t967a, b, e, d; Kemper, Caveness, and Yakovlev, 1973; Chan-Palay, Palay, and Billings-Gagliardi, 1974; Marin-Padilla and Stibitz, 1968; Marin-Padilla et al., 1969; Peters and Kaiserman-Abramof, 1970; Rutledge, Duncan, and Cant, 1972; Valverde, 1967, 1971b; Valverde and Est6ban, 1968). The present counts are also slightly higher than the counts obtained by others (Fifkov~, 1970; Globus etal., 1973; Parnavelas, Globus, and Kaups, 1973)for dendrites of the same neurons studied here, the pyramidal neurons in rat visual cortex. The source of this discrepancy is difficult to specify. It may be a matter of counting criteria or loci, of staining variables, or of genuine differences in spine density. It may also reflect the fact that the present counts were done under oil immersion while certain of the counts in the literature were done at lower magnifications, magnifications which we found unreliable (see Globus and Seheibel, 1967b, footnote). In addition, it may be noteworthy that Fifkov~ (1970) used rats of the Lewis strain, and Globus et al. (1973) rats of the Berkeley S~ strain. In both of these studies, and in the Parnavelas, Globus and Kaups (1973) study, the animals were all younger (35 55 days old) than the rats used in the present study. Fifkov~ (1970) measured the effect of unilateral visual deprivation by lid suture in 14 day old Lewis rats. Golgi-Cox preparations of pyramidal apical dendrites on the control and deprived sides were examined 10 and 30 days after operation. The 20-day survivors (aged 44 days) showed a mean spine density, for the whole apical dendrite, of 1.05 spines/B on the control side and 0.76 spines/B on the deprived side, a spine loss of 28%. This compares with a spine density of 1.43 spines/~z on the apical dendrites of the young animals in the present study and a density of 0.92 spines/~z in the old animals, for a total spine loss with age of 36 %. These figures indicate that the pyramidal spine losses due to aging are at least as severe in this cortex as those due to sensory deprivation. This conclusion still holds true if one compares the present results with spine loss figures obtained in other speeies and other cortiees. The age-induced apical dendrite spine loss of 36% may be compared with spine loss percentages of approximately 26% in enneleated mouse peristriate cortex (VMverde and Est6ban, 1968), 39% in darkreared mouse visual cortex (Valverde, 1967), and 30 % in enucleated rabbit visual cortex (Globus and Scheibel, 1967b). The age-induced oblique branch spine loss of 40% may be compared with a loss of approximately 30% in eallosum-sectioned rabbit parietal cortex (Globus and Scheibel, 1967 a). With respect to the terminal tuft spines, on the other hand, the age-induced spine loss of 24% is significantly lower than the 50% loss reported for undercut cat cortex by Rutledge, Duncan, and Cant (1972). One of the impressive features of the aging changes reported here is their temporal regularity. The average spine counts decline monotonically over the whole period from young adulthood to advanced age, though it is important to bear in mind that the rate of change during the 3-12 month old period was not determined in the present study. There is in the data, however, the implication of a continuously changing spine population throughout adulthood. Based on the results of studies by others (Fifkovs 1968; Le Vay, 1973; Marin-Padilla, 1967; Valverde, 1971b; Valverde and Ruiz-Marcos, 1969), this continuous change may typify the developmental period as well. The age-dependent changes in the number

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of spines opens up the possibility that spine densities m a y be utilized as one index of mammalian biological age (gies, 1974). I t also, of course, means that our conceptions of "normal" spine populations must be explicitly framed within the context of an animal's age. The hypothesis of continuously varying spine populations, from birth to death, is inconsistent with the implications of the f r e q u e n t l y encountered statement to the effect that, in development, spine number increases up to a certain age and then "stabilizes", or "reaches adult values". I t m a y be true that there does indeed exist an age range during which spine counts are at a plateau. I-Iowever, this has not yet been systematically and unequivocally demonstrated, and additional quantitative studies are needed covering in particular the period between late development and early senescence. In the rat, such a period might correspond to the ages between 15 days and 12 months. I t m a y also be noted that the hypothesis of continuous variation in dendritic spine population implies a continuous modification of the functional properties of pyramidal neurons throughout the lifespan, a factor not usually explored in neurophysiologieal investigations of adult animals. Progressive spine loss in rat visual cortex is a cleareut phenomenon which seems to affect all aging rats. I t is, however, a change which is expressed in a highly variable way among the cells within a given brain. Why certain neurons appear to be ravaged by the passage of time while others remain relatively normal, and why certain individual spines along a particular dendrite degenerate while certain neighboring spines do not, are unanswered questions of potentially great practical significance. In considering this pyramidal cell variability, we believe it is important to note the parallel findings of Valverde (1971 a). His work on spine changes in young dark-reared mice strongly suggested the existence of more than one population of dendrite spines, presumably related to more than one type of pyramidal cell. The particular population of interest consists of those neurons whose spines develop independently of the presence or absence of visual stimulation. Valverde interpreted these spines as developing ~'through the induction of morphogenetie agencies" in contrast to spines whose normal development depends upon normal arrival of visual impulses. I t m a y be that the neurons in the former group are the same neurons as the ones found in the present study to be resistant to the spine depopulation tendency that accompanies advancing age. One factor relevant to the issue of cell-to-cell variability is the present demonstration of a relationship between spine density and apical dendrite diameter. In the cortices of all ages examined, thin, medium, and thick dendrites are present. Thicker dendrites, at all ages, are spinier than thinner dendrites. With aging, the proportion of thick dendrites is reduced in number while that of the thin dendrites increases (and see Valverde, 1967, Fig. 3). One hypothesis that would account for the latter observation is that larger dendrites shrink with age, and, in shrinking, lose their spines. Whether such a shrinkage actually triggers spine loss, or whether spine loss initiates shrinkage, or whether the two processes are concomitant but independent is not known. We incline toward the view that spine loss precedes, and therefore m a y induce, shrinkage, since progressive spine loss on apical dendrites is a continuous process over the age range studied (Fig. 10), whereas apical dendrite shrinkage, as observed in Golgi preparations, appears to commence somewhat abruptly at about 18 months of age. Consistent with the shrinkage hypothesis is

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the well-established finding that partial isolation of a nerve cell from its afferent supply leads to subsequent shrinkage of the neuron (see, e.g., West and Harrison, 1973). I t is interesting to note that m a n y of the types of conditions which have been demostrated to induce spine loss also lead to alterations of gross dendritic morphology (Coleman and Riesen, 1968; Jones and Thomas, 1962; Schad6 and Cavehess, 1968; Valverde, 1968, 1970; Volkmar and Greenough, 1972). Detailed Golgi observations of dendritic extents and branching patterns were not undertaken in the present study. Aside from the observation of apical dendrite thinning, no other striking changes were noted. Certainly all of the major dendritic systems persist with age. Our impression is that whatever changes in dendrite extent or branching m a y occur are of relatively small magnitude. Since the spines receive the vast majority of the synapsing axon terminals on the pyramidal neuron surface, the loss of spines entails the loss of synaptic input. Therefore, compared to a similar neuron in a young brain, a pyramidal neuron in an aging brain is likely to be operating on the basis of a seriously reduced input. J u s t how much of a functional impairment this actually represents will depend upon such factors as the colleetive and individual influence of axospinous synapses. At the present time, the nature and extent of the influence of postsynaptic potentials generated in individual spines is problematic, and the subject of considerable speculation (Chow and Leiman, 1970; Globus and Seheibel, 1967e; Seheibel and Seheibel, 1968). The idea that this influence m a y vary among spines located on different dendrites of the same neuron is suggested by the results of degeneration experiments in primary sensory cortices. These experiments reveal that afferent axons from different sources may synapse preferentially with the spines on particular portions of the cell's dendritic tree. For example, Valverde's (1968) and Globus and Scheibel's (1966, 1967a) data indicate that primary thalamoeortical afferents synapse on the spines of the apical dendrite whereas eallosal afferents synapse on the spines of the oblique branches of the apical dendrite. Basal dendrites receive most of their afferents from axonal branches of other cortical neurons (Globus and Seheibel, 1967e; Globus, 1971). The fact that the spine populations on all of these dendritic systems are reduced with age means that the loss of information to the pyramidal neuron is not confined to any one particular type of input. The above discussion presumes the integrity of the presynaptie axon at the time of spine loss. In fact, this is an assumption for which we have no evidence. It may be that the observed spine losses on aging dendrites represent a transneuronal atrophy, being the consequence of age-related degeneration of the presynaptic component of a normally functioning axospinous junction. But whichever process is operative--loss of a viable synapse through intrinsic spine degeneration or transneuronal degeneration of the spine--the progressive nature of the spine loss, affecting all the major pyramidal dendritic systems, poses a serious threat to the pyramidal neuron. The reality of this threat follows from the results of experiments which have shown cell degeneration consequent upon large-scale deafferentation (Cowan, 1970; Jones and Thomas, 1962; Liu and Liu, 1971 ; Matthews and Powell, 1962 ; Penman and Smith, 1950). The synaptic depopulation implied by the present results may thus be proposed as a possible precipitating factor or early stage in

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the process of n e u r o n a l loss with age, which has been reported for several cortices (Brody, 1955). The present s t u d y d e m o n s t r a t e s t h a t a n a t u r a l l y - o c c u r r i n g variable, the passage of time, alters the form of the cortical p y r a m i d a l neuron. Although the scope of the present s t u d y is limited, the results o b t a i n e d p r o m p t the speculation t h a t the general p h e n o m e n o n of progressive p y r a m i d a l spine loss m a y occur in the cortices of all m a m m a l i a n species. This speculation is based u p o n the u n i v e r s a l i t y of the aging process a n d u p o n the fact t h a t the cortical areas which we have e x a m i n e d have s t r u c t u r a l a n d f u n c t i o n a l homologues in the brains of most m a m mals, i n c l u d i n g man. W i t h i n those areas p y r a m i d a l neurons with the same general morphology as in the r a t are both readily identifiable a n d n u m e r i c a l l y p r o m i n e n t (see, for example, g a m d n y Cajal, 1911 ; Shkol'nik-Yarros, 1971). If f u r t h e r research should in fact support the idea of the u n i v e r s a l i t y of the aging changes reported here, we ~dll need to e x p a n d our t h i n k i n g a b o u t age-related sensory deficits such as presbyeusis a n d loss of visual a c u i t y (Corso, 1971). We will have to consider m u c h more seriously t h a n heretofore changes t a k i n g place in the central nervous system, as well as those which have long been recognized at the level of the peripheral receptor.

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