Ultrastructural alterations in caudate nucleus in aged cats

Brain Research, 4401"1988)267-279 Elsevier

267

BRE 13250

Ultrastructural alterations in caudate nucleus in aged cats M.S. Levine 1, A.M. Adinolfi 2, R.S. Fisher 1"2, C.D. Hull l, D. Guthrie I and N . A . Buchwald I 1Mental Retardation Research Center and 2Department of Anatomy, University of ()difornia at Los Angeles, Los Angeles. CA 90024 (U.S.A.)

(Accepted 21 July 1987) Key words: Aging: Caudate nucleus: Uitrastructure; Synaptic density: Synaptic apposition length: Cat

These studies provide information on the changes in the ultrastructure in the caudate nucleus of aged cats. The major finding was that there was a decrease in the density of synapses in caudate neuropil. This decrease occurred in animals after 3 years of age and remained relatively constant in older animals. In conjunction with this change a population of unusually long synapses also occurred. These larger synaptic appositions were associated with enlarged spine heads, The caudate also showed a number of qualitative ultrastructural alterations. Many neurons contained accumulations of lipofuscin or lipopigment granules in aged animals. These inclusions occurred in both soma and dendrites of neurons and all types of glial cells. A unique configuration of collapsed agranular cisterns also was observed in aged animals. The present results indicate that decreases in synaptic density may be one morphological event underlying functional alterations observed in eaudate neurons in aged cats.

INTRODUCTION In previous studies ~'e have described a series of anatomical and electrophysioiogicai changes that occur in neurons of the caudate nucleus of the cat throughout its lifespan 3°-33. Neurophysiological alterations consisted of decreases in excitability in caudate neurons. Morphological changes consisted of decreases in spine density and loss of dendritic segments in medium-sized spiny neurons in aged cats 3°. We have proposed that the decreases in excitability might be related to the decreases in spine density -''~. Activation of afferents to the caudate nucleus produces excitatory postsynaptic potentials ~'-'-''-'~'. Many of the afferents synapse on spines of medium-sized neurons -~4-'~4~. The decrease in spine density may be associated with a decrease in synaptic density in caudate neuropil. A decrease in the n u m b e r of synaptic contacts would contribute to the diminution in physiological responsiveness. The present ultrastructural studies were undertaken to provide quantitative estimates of the changes in synaptic density that occur as

a result of the aging process. In addition, these studies provide qualitative descriptive information on some of the ultrastructural changes observed in caudate neuropil. MATERIALS AND METHODS Fifteen cats ranging in age from 1 to 22 years were used. The animals were obtained from 3 sources: the cat breeding colony at the Mental Retardation Research Center ( M R R C ) at the University of California at Los Angeles ( U C L A ) (n = 10, 7 females and 3 males), local veterinarians (n = 2, 1 female and 1 male), and the Starkist Cat Food C o m p a n y (n = 3, all females). The ages of animals, their origins and gender are shown in Table I. With the exception of the cats obtained from the veterinarians, all animals were housed in relatively standard laboratory environments described in our previous publications 3t~-33. None of the animals had been neutered. At the time of sacrifice all animals were in good physical condition according to examinations by the U C L A veteri-

Correspondence: M.S. Lcvine, Mental Retardation Research Center, University of California at Los Angeles, 7hi| Wcstwood Plaza, Los Angeles, CA 9111124,U.S.A.

0006-8993/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

268 FABLE I Summary of quantitative data

Volume density = (areal density)/(apposition length + section thickness (100 nm)). The first letter after animal number is gender. The second letter is source. M, MRRC; S, Starkist; V, Veterinarians. Animal

Age lyears)

Total area ~!on2)

Number of synapses

Areal density (Syn/l~m 2)

Apposition length ( / ~ m )

Volume density (Syn/l~m 31

3157 F-M 262-2M-M 262-1F-M 262-4M-M 1539 F-M

1 I 1 1 3

1579 1170 1251 1194 1410

245 199 261 222 244

0.1552 0.1701 0.2086 0.1859 0.1730

0.477 0.391 0.365 0.392 0.331

0.269 0.346 0.449 0.378 0.401

004 F-M 007 F-M 203 F-M 211M-M

6 6 9 9

1969 1215 1098 933

191 123 99 59

0.0970 0.1012 0.0901 0.0632

0.494 0.426 0.519 0.627

0.163 0,192 0.146 0.087

7051 F-S 7053 F-S 7040 F-S 14 M-V 675 F-M 19 F-V

12 12 13 14 16 22

2727 1260 1838 1929 583 583

230 177 217 180 49 51

0.0843 0.1405 O.1180 0.0933 0.0840 0.0875

0.530 0.591 0.497 0.707 0 565 0.516

0.134 0.203 O.198 0.116 0.126 0.142

narv staff. Animals were divided into 3 groups: 1-3, 6-9 and 12-22 years. This division was based on the results of our previous morphological, electrophysiological and behavioral studies showing differential changes during these 3 periods3"-3k All animals were deeply anesthetized with sodium pentobarbital and sacrificed by vascular perfusion with 2% paraformaldehyde and 2% glutaraldehyde buffered to pH 7.4 with 0.1 M sodium cacodylate. The caudate nucleus was dissected, washed in the buffer, dehydrated rapidly through graded changes of acetone and embedded in Spurr's E R L 4206. Ultrathin sections were stained with lead citrate and examined with an Hitachi HS-8 electron microscope. In order to provide quantitative estimates of synaptic density, synapses were counted and measured in randomly chosen photomicrographs. All samples for quantification were taken from an area of the dorsolateral head of the caudate 1-2 mm from the internal capsule. Samples were taken from i - 3 blocks of tissue from each animal. Measures were obtained at the same magnification for all animals (x17,800). Micrographs were coded and counts made by individuals blind to the age group from which tissue had been obtained. Because micrographs were randomly chosen, synapses intercepting the borders of the micrographs were counted and measured from only

two sides of the photographs. For a synapse to be counted, a m e m b r a n e junctional apposition (synaptic contact length) had to be present in the micrograph and at least 3 synaptic vesicles had to be detectible in the presynaptic element. The total area analyzed consisted of neuropil, Photographs containing cell bodies, large dendrites or blood vessels were excluded. Serial sections were not obtained and no attempt was made to classify synapses according to junctional specialization or postsynaptic element (dendrite, spine or cell body). The apposition length of each counted synapse was traced o n t o an Apple graphics tablet interfaced with an Apple c o m p u t e r for measurement. The total area of each micrograph was also obtained. For each micrograph, synaptic number, synaptic density and length of each synapse were determined. After all micrographs had been digitized for each animal, the total n u m b e r of synapses, total area examined, density of synapses, average apposition length and distribution of apposition lengths were determined. These values were averaged for all tissue in each age group. Distributions of apposition lengths for all synapses measured in each age group were constructed. The n u m b e r of synapses per unit volume was calculated from the n u m b e r per area using an appropriate correction formula 34 (Table I).

269 TABLE

II

Group averages Values are the means + S.E.M.

Age (}'ears)

n

Total area (!on2;

Number of synapses

Areal density (Syntpm" l

Apposition length U~.'n)

Volume density (Synvmr~l

1-3 6-9 12-22

5 4 6

1321 +_ 77 1304 _+ 229 1487 + 344

234 _+ II 118 _+ 28 151 ___33

11.179 + (1.009 0.088 + O.t)08 O. I01 + 0.009

11.391 + t).024 0,516 + 0.042 0.5118 + 0.003

0.369 __+0.030 O, 147 + 0.022 O. 153 __+0.1) 15

Statistical differences among average areal and volume densities were assessed with one-way analyses of variance while group differences in apposition length were assessed with a nested design to account for within animal variance in apposition length since multiple measures were taken from each animal ~s. When differences among group means were statistically significant, the Bonfuroni correction was used for post-hoe comparisons between individual group means using appropriate error variance terms for measures of synaptic density 37. Differences bezween measures of apposition length for individual groups were assessed with t-tests using the appropriate error term from the nested analysis of variance 4s. For determination of relationships with age, Spearman rank order correlation coefficient~ were computed ~s. Differences among distributions of apposition lengths were assessed with the },-statistic, a measure of nominal association ~'~4. For this analysis, data were collapsed into 4 categories of length: <0.35, 0.35-(1.70, 0.70-1.00 and >l.(I Ftm to determine if there was a significant association between synaptic size and age. Subsequent assessments of statistical significance of differences between each pair of distributions were performed with ~-tests ~s.

two older groups were not significantly different from each other. In addition, there was a statistically significant increase in average apposition lengm in both groups of aged cats (F = 11.73, df = 2, 12, P < 0.01 ) (Table 1I). Post hoc analyses indicated average apposition lengths were significantly longer in both groups of older cats compared to l-3-year-old animals. The difference between average apposition length in the two older groups was also statistically significant. Increases in apposition length indicate that either smaller synapses were lost due to'the aging process or that more longer synaptic appositions occurred. Examination of the distributions indicated that there was an increase in the proportion of long synapses (>1 Itm in length) in both groups of aged cats (Fig. 1). This effect was evident in 6-9-year-old DISTRIBUTION OF SYNAPTIC LENGTHS I-3 YR 15 10

I

r..J

0

RESULTS

J

5

6-9

I

m I--'1

YR

10

g5 n~

o..

Quantitative alterations The major result of this analysis was a marked decrease in the density of synapses in all cats over 3 years of age. The differences among the 3 age groups in average synaptic density were statistically significant (Table II) (F = 27, df = 2, 12, P < 0.0001). Pos~ hoc analyses indicated that both groups of aged animals had significantly lower synaptic densities than 1-3-year-old cats. However, values for each of the

757

12-22 YR

5i

i:.:i:i: o'5

.~5

.z5

35

45

.55

.65

.75

as

~5

>to

APPOSITION LENGTH(urn) Fig. I. P e r c e n t a g e d i s t r i b u t i o n s o f a p p o s i l i o n l e n g t h . C o u n t s o f all s y n a p s e s g r e a t e r t h a n 1 !~m in length arc c o n t a i n e d in the overflow bin. Bin size is 11.1)5p m .

270 animals, but was most prominent in the 12-22-yearold group. T h e r e was also a decrease in the proportion of smaller synapses (0,25-0.50 .,m in length) in the aged cats. T h e r e was apparently no change in the proportion of very small synapses (0.10-0.20 y m ) . Analysis of the distributions indicated that there was a statistically significant association of age and synaptic apposition length (y = 0,305, df = ~ , t = 11,73,

P < 0.001). All comparisons of the differences among the distributions of apposition length for the 3 groups were statistically significant (X2 = 67, df = 3, P < 0.0001 for 1-3- vs 6-9-year-old groups; Z~ = 136, df = 3, P < 0.0001 for 1-3- vs 12-22-year-old groups; ~ = 7.83, df = 3, P < 0.05 for 6 - 9 - vs 12-22year-old groups). Since there was a change in apposition length with

Fig. 2. An example ef a medium-sized neuron from a 12-year-old cat in which the perikaryon contains lipofuscin granules (LP) accumulated at one pole of the cell. The neuron also contains collapsed cisterns of smooth endoplasmic reticulum (SER) which are found often among the lipofuscin granules and which are continuous with elements of rough endoplasmic reticulum. An astrocyte process (A) containing a lipofuscin granule is seen apposed to the neuronal cell body.

271

Fig. 3. Examples of ultrastructure of caudate neuropil at 3 (A). 9 (BL 12 (C) and 22 (D) years. Prominent asymmetrical axodendritic and axospinous synaptic contacts (arrows) are surrotmded by fine neuronal and glial processes at all ages. However. in older animals many dendritic spines (ds in C) and axonal terminals (at in D) are enlarged and irregul:~,r in contour. Calibration is 1 .ttm.

272 age, volumetric estimates of synaptic density were not directly proportional to areal estimates. Tables I and II show that when synaptic density was estimated volumetrically, the group differences were more pronounced than areal density estimates. Differences among the 3 groups were statistically significant ( F = 31, df = 2, 12, P < 0.001). Post-hoc analyses indicated that average synaptic densities for both groups of older cats were significantly different from that of l - 3 - y e a r - o l d animals. The differences be-

tween volumetric measures of synaptic densitv for the two groups of aged cats were not statistically significant. Correlations between age and quantitative measures of synaptic density were significantly different from zero. The correlation between age and areal estimates of synaptic density was - 0 . 7 4 4 (df = 13, P < 0.0015) wMle the correlation between age and estimates of volumetric density w a s - 0 . 7 5 0 (df = 13, P < 0.0013). The highest correlation was obtained be-

Fig. 4. The dendritic spines along dendrites are characterized often by thin stalks and large, irregular-shaped heads. This is an example of such a dendritic spine from a 12-year-old cat. It is contacted asymmetrically by two axon terminals (arrows). SA, spine apparatus.

273 tween age and apposition length (r = 0.761,, df = 13, P < 0.001). Synaptic density and apposition were also inversely related (r = -0.789, dl = 13, P < 0.0005). This relationship indicates ,hat as the number of synapses decreases the comact length of the remaining synapses increases. Table I shows that not all a~fimals displayed the effects of aging equaW!y. In 1-3-year-old animals synaptic density rang¢O from 15-20 synapses/100t~m-' in different cats. Synaptic density ranged from 6 to 14 synapses/100~4m2 in all aged animals. In a few cases

several additional tissue blocks from other areas in the head of the caudate were available from the same anbnal. Density estimates were reliable when differcnl tissue samples were obtained from the same animal. For example, two samples of 10 photomicrographs were analyzed from a 1-year-old animal. Density estimates were 17.0 and 17.6 synapses/100 l l m 2 for each sample, respectively. In a 16-year-old animal similar measures (15 photomicrographs from each block) yielded 11.8 and 12.3 synapses/100 u m 2 for each sample, respectively.

Fig. 5. An example of an expanded dendritic spine head in a 16-year-oldcat that is contacted asymmetrically(arrows) by a single large axon terminal. SA. spine apparatus.

274

Qualitative alterations Qualitatively both medium and large neuronal cell bodies showed ultrastructural evidence of the aging process. Man3' neurons contained numerous lipofuscin or lipopigment bodies accumulated throughout the perinuclear cytoplasm and extending into the proximal dendrites (Fig. 2). These bodies were composed of a single large electron-dense granular component which varied in size and shape but was more often irregular than rounded in contour and numer-

ous smaller electron-lucent components which ranged from small vesicles to large round or oval vacuoles. These components were surrounded by a single limiting membrane. Often closely associated with these accumulations of lipofuscin inclusions in cell bodies and proximal dendrites were unique lamellar configurations of collapsed agranular cisterns (Figs. 2 and 6). These smooth membraneous configurations were composed of stacks of flattened cisterns separated by nar-

Fig. 6. E×amplc of lipofuscingranules (LP) and flattened cisterns of smooth cndoplasmic reticulum (SER) at the base of dendritic spines (S~ in the shaft of the dendrite (D) in ~ 16-year-oldcat.

275 row channels of electron-dense finely granular cytoplasm. The outermost cisterns were continuous with the membranes of the rough endoplasmic reticulum. Occasionally, small dense granules were observed within these flattened cisterns. Frequently lipofuscin bodies and flattened smooth membranous cisterns occurred together within the shafts of the smaller diameter more distal dendrites or at the bases of dendritic spines (Figs. 2 and 6). In low magnification electron micrographs, the neuropil appeared little affected by the aging process (Fig. 3). Axodendritic and axospinous synaptic contacts and numerous fine neuronal and glial processes were packed tightly and separated by narrow extracellular channels. Upon closer examination, a number of structural alterations were observed. Dendritic spines were often enlarged and irregular in contour (Figs. 4 and 5). Frequently, they contained an elaborate spine apparatus and vesicular or tubular membranous profiles embedded in a fine filamentous matrix (Fig. 4). These spinous processes were contacted asymmetrically and symmetrically by a single expanded axon terminal or by several axon terminals which ensheathed, in some instances, the expanded head of the dendritic spine (Figs. 4 and 5). All types of neuroglial cells contained accumulations of lipofuscin or lipopigment bodies. Astrocytes were filled with filaments and large heterogeneous lipopigment bodies. In oligodendrocytes, the pigment components of lipofuscin inclusions were composed of smaller dust-like and larger coarse granules. Both satellite and interfascicular oligodendrocytes contained lipofuscin bodies. DISCUSSION These results demonstrate major age-related changes in the uitrastructure of the caudate nucleus. Of primary importance is the decrease in synaptic density in the neuropil of aged cats. This decrease begins after 3 years of age and appears to remain relatively constant in older animals. Associated with the decrease in synaptic density is an increase in average synaptic apposition length and the appearance of a population of unusually !ong synaptic appositions. In addition, many caudate neurons contain accumulations of lipofuscin or lipopigment granules in aged cats. These inclusions are evident in both soma and

dendrites and are also apparent in all types of glial cells. A unique iamellar configuration of collapsed agranular cisterns is also observed in aged animals. Our previous electrophysiological and morphological findings indicate that connectivity between afferents and caudate neurons is altered during the aging process 3°'32"33. A population of medium-sized spiny neurons displays loss of spines on distal d"ndritic segments by 6-9 years in the cat which becomes more severe as cats exceed 11 years of age. After 11 years, spine loss is accompanied by loss of distal dendritic segments. Electrophysiologicatly there is a decrease in the frequency of initially excitatory responses, the responses evoked primarily by activation of caudate inputs 32. Thresholds for producing these excitatory responses increase and spontaneous activity decreases. The decrease in synaptic density provides additional morphological verification of age-related changes in connectivity indicating that communication between neuronal processes in the caudate is ciisrupted. This decrease may in part be responsible for the change in electrophysiological responsiveness. Fewer synapses per neuron would lead to less efficient coupling of afferents with their targets. Changes in synaptic size are more difficult to interpret. Although there is little direct evidence, the appearance of unusually large synapses might reflect degenerative changes occurring in caudate neuropil. The enlarged spine head and the enlarged synaptic apposition length may be an indication that a spine is presently undergoing or will undergo a degenerative change. However, we found no evidence of a large population of degenerating endings in the caudate neuropil ~. In contrast, a recent report has demonstrated degener~:ting fibers in a number of areas in the aged rodent ~7. Another interpretation of the enlarged synapse and spine is that they represent a response to synaptic loss. Perhaps size of the synapse is an index of its ability to function and these larger synapses represent hyperfunctional connections formed because of decreases in synaptic density. It has been proposed that total synaptic area may represent an important invariant parameter in the nervous system ~'-'°. In the cerebellum and piriform cortex there is an inverse relationship between the number of synapses and contact area 1°'2". Similarly, studies directed at assessing plasticity have found such rela-

276 tionships ~z.~3.~7. Hillman ~'J proposed that a constant synaptic area could serve as a stabilizing relationship to maintain neuronal function. The observations obtained in the present study extend this relationship to the caudate nucleus of the aging cat and further indicate that this principle may be important in determining how the nervous system reacts to deterioration with age. There is now considerable evidence for plasticity in the aging nervous system 6s' ~1'~1.~6.2~. The present findings indicate that ultrastructural changes are occurring by 6-9 years of age in the cat. Unpublished studies from our laboratory indicate that spine density on distal segments of caudate medium-sized spiny neurons decreases in cats during this age period indicating that loss of synapses and loss of spines appear to covary -'y. Such spine density decreases are associated with increases in the number of branches on dendrites of these neurons. The increase in the number of branches observed on caudate neurons may represent a non-functional growth of this type of ,,'ell for two reasons. First, since most synapses occur on spine heads 24'25 and the density of these decrease, afferent information would not be as capable of changing synaptic activity. Second, our electrophysiological data indicate that functional chalices have already begun to occur in 6-9-year-old cats 32. At face value a decrease in the synaptic density in the neuropil is usually interpreted as a loss of synapses. However, if a synaptic density decrease is ~lssociated with an increase in length of dendritic segments then it is possible that synapses were not 'lost' during the aging process but were occurring over an expanded area of a neuron. Since we did not measure dendritic surface area in the present study we cannot determine with certainty the relationship between number of synapses and the dendritic membrane area. However, at least two changes might be independent of this relationship. First, proportionately more larger synapses occurred in the 6-9-year-old group, the group displaying increases in dendritic branching. The appearance of these larger synapses argues against a simple relationship between increases in dendritic length and decreases in synaptic density. Second, in the 12-22-year-old group a decrease in synaptic density occurred without the increase in dendritic length. Decreases in synaptic density could result from a

number of other changes in the caudate. For example, increases in number of glial elements or blood vessels could lead to decreases in synoptic density. In the present study we did not quantify changes in glial cells. We did, however, exclude samples containing large glial elements or blood vessels from the quantification procedure. Similarly, we did not determine neuronal density changes. In both rodents and human~ there is evidence for neuronal density decreases in the aging striatum 4"7. The observations that both neurons and neuroglial cells accumulate lipofuscin in the caudate of aged cats are similar to those obtained from other species and from other neural areas 3'4"45. All types of caudate neurons accumulated lipofuscin or lipopigment. While such accumulations were most apparent in the perikaryal cytoplasm they also extended into dendritic processes. Frequently, lipofuscin droplets were observed at the neck of spines where they made contact with dendrites. It is possible that accumulations of lipopigment at this point in the neuron could lead to a blockage of the spine and ultimate spine degeneration. It is of interest that spiny neurons in a number of areas appear to be most susceptible to the effects of aging -'7'2s'4~H2. The present observations indicate that a unique lamellar configuration of collapsed agranular cisterns was observed in caudate neurons in aged cats. In dendrites, a close association of these inclusions with the bases of dendritic spines was often observed. The functional change represented by this inclusion remains unclear. To our knowledge similar inclusions have not been described for other areas of the brain although intracellular neuronal ultrastructural changes in mitochondria, nuclear membranes, ribosomes, appearance of multivescicular bodies, accumulation of degeneration products and axonal swelling have been described -'3"3~'3~'46. Previous experimentation on morphological substrates of aging concentrated on the regressive and degenerative changes occurring in the brain as animals aged. A more recent hypothesis proposes that during aging degenerative changes are balanced by growth ¢''~. Such growth appears to occur in different brain sites and is associated with neuronal loss s. Sites that do not show decreases in neuronal density do not seem to display this dendritic growth. Our observations do not provide information on neuronal death

277 in the caudate during aging. However, others have indicated that aged rodents and humans show decreases in caudate neuronal density 4"7. If cats follow this pattern, the increased dendritic branching in caudate neurons at 6 - 9 years of age would be indicative of cell death. A technical problem in the interpretation of the present results concerns the degree to which changes observed in caudate neuropii are due to fixation or perfusion artifacts. One possibility is that age-related changes in brain vasculature slow or delay the tissue perfusion. There are very few published reports of age-related changes in cerebral blood vessels of old animals 36. Consistent findings have been that the mean capillary diameter decreases and the total capillary length per unit volume increases with age in thc cortex of rats and monkeys 36. Capillary diameter remains unchanged in aged human putamen but capillary length per unit volume increases, probably as a result of brain shrinkage 43. For example, cortical volume is reduced by 50% in old rats 36. Our qualitative observations of caudate neuropil indicated that there were no gross changes in the appearance of caudate microvasculature. Whether vascular alterations that we could not observe play a role in senescent changes in neuronal morphology cannot be determined from the present experiments. Such alterations, however, were of some concern to us in attempting to achieve optimum preservation of ultrastructure in our oldest animals. Here it was not uncommon to find scattered areas of less-well-fixed tissue within the head of the caudate nucleus. These tissues samples were excluded and our observations are based only on the best preserved regions of the caudate in the oldest cats. A related problem concerns tissue swelling. In ultrastructural studies tissue swelling is typically represented by large amounts of extracellular space, disrupted synaptic morphology and markedly fewer intracellular organelles. In developmental studies swelling along dendritic shafts has sometimes been interpreted as an artifact of the perfusion procedure 39. In the present experiments there were few large extracellular spaces, mitochondria and spine apparatuses were intact and intracellular organelles were present and numerous. Although spines were often enlarged and spine and dendritic contours were irregular (a possible indication of perfusion artifact) such spines had synapses with other elements that,

except for increased length, appeared normal. Our findings thus, do not appear to be a result simply of tissue swelling or inadequate fixation. In addition, the occurrence of collapsed agranular cisterns indicates further that there is disruption of intracellular events in caudate neurons apart from any perfusionrelated procedure. Finally, the fact that similar morphological changes have been observed using another method (Golgi staining) 3° provides additional validity to the present observations. A second technical problem is the degree to which our sampling procedures insure a reliable estimate of the true synaptic density changes in the aged cat. In our sampling we did not account for some of the known non-homogeneous aspects of the morphology of the caudate as exemplified by its organization into a patch and matrix tS. In addition, the area of neuropil sampled for each animal in the present study is somewhat low compared to other morphometric studies-"~l'2~L However, our estimates appeared reliable. When samples were available from multiple blocks or from multiple areas quantification revealed tba~ synaptic density for each animal did not vary markedly from area to area of the caudate as long as at least 500F~m2 was analyzed. This was the minimum area of neuropil analyzed. A major problem encountered by the use of animal models of aging is the interaction of experience and age. In our experiments no attempt was made to systematically alter experience. In rodents, a considerable literature demonstrates that differential housing and environmental conditions affect the morphology of many brain sites ''ll'~'. It is of interest to point out that our cats were obtained from 3 sources. If environmental variables are critical it might be expected that the brains of the animals obtained from the veterinarians would have been different from those of cats from the other populations. Examination of data from individual animals showed this was not the case. Thus, while environmental variables may have a role, it cannot be argued that they arc the major variable underlying the changes we ha~e observed.

ACKNOWLEDGEMENTS Supported by USPHS Grants AG 01558 and HD 05958.

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