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Aging reverts to juvenile conditions the synaptic connectivity of cerebral cortical pyramidal shafts
Developmenlal Brain Research, 69 (1992) 41-49 © 1992 Elsevier Science Publishers B.V. All rights reserved 0165-3806/92/$05.00
41
BRESD 51501
Aging reverts to juvenile conditions the synaptic connectivity of cerebral cortical pyramidal shafts A n t o n i o R u i z - M a r c o s , F e r n a n d o S a n c h e z - T o s c a n o a n d Jos6 A n t o n i o M u f i o z - C u e t o Unidad de Neuroanatomia, lnstituto Cajal, Madrid (Spain) (Accepted 19 May 1992)
Key words: Aging; Cerebral cortex; Development; Pyramidal cell; Dendritic spine
Quantitative analysis of the total number and distribution of dendritic spines along the apical shafts of layer V cerebral cortical pyramids has been performed on aging rats (90-120 to 1,135 days old) and on rats during the period of early and late development (10-80 days). As expected from previous works, present results show that the total number of dendritic spines along the shafts increase from 10 to 80 days, after which it starts to gradually decrease until the last age studied (1,135 days). The quantitative analysis of the effect of aging on the relative decrease of dendritic spines shows that this decrease starts being homogeneous along the whole length of the apical shafts and that from a certain age onwards, estimated according to present results in 400 days, this effect is significantly more pronounced in layers IV and l l l - l l than in deep layers. Furthermore, the comparison made between the distribution of dendritic spines along the apical shafts of pyramidal neurons of old and young animals has shown that aging produces a regression of this distribution to juvenile conditions.
INTRODUCTION
It has been widely proved that aging produces a progressive deterioration of learning and memory ability in animals 1,'~'12.a°'2~'22'2sand learning performance in man 42'44. In this respect and given the importance of cerebral cortex in cognitive and intelligent processes the question of how aging affects the development of its structure seems to be an important one. In recent years there has been an increasing number of studies dealing with the problems of how this natural condition affects different neuronal cortical elements. It has been described that aging produces neuronal loss 9'17'18'32, deterioration and changes in the structure of dendritic trees 6't3'4°'49, amongst other effects. Different authors have shown the importance of dendritic spines on the establisment of thalamo-corticai connections 7's'29'3~. It has also been proposed that these neuronal elements could be an important substrate for information storage t6. These findings and the one reported by Bertoni-Freddari et al. 4, according to which aging seriously impairs the morphological plasticity of synapses, led us to think that the study of how
aging affects the number and distribution of the dendritic spines which are present along the apical shafts of layer V cortical pyramids, could shed light on how this natural condition affects not only the neuronal connectivity in the cerebral cortex, but also the organization of its general structure. Feldman and Dowd 14 showed that aging produces a slow and progressive decrease in the number of spines from apical, basal, oblique and terminal tufts dendrites of layer V pyramids of the visual cortex of the rat. This finding has been confirmed by some other authors who have also found an age-dependent spine loss in pyramidal neurons of layer III and V of the visual cortex of mice 23, dog 25, monkey 45,4e and man 41'42. It has also been found that aging produces a distortion of the shape of the spines 2s'32. Although these studies have clearly established that aging affects the number of spines, none of them have taken into consideration the effect of aging on the distribution of these elements along the dendrites. In 1969 it was found 39'4s that dendritic spines are not distributed at random along the apical and basal dendrites of layer V and III pyramidal neurons of the
Correspondence: A. Ruiz-Marcos, Institute Cajal, c./Doctor Atce 37, 28002 Madrid, Spain.
42 visual cortex of mice, but are distributed according to a precise pattern which could be defined by a mathematical model. This model is formed by one principal equation which describes how these elements are distributed along the dendrites, and three parametrical ones which describe how the distribution is affected by development 3~. This model was found valid to describe the distribution of spines along the apical and basal dendrites of several cortical layers 34-3~''3'~'48 and in species such as mice, hamster, cat, monkey and even m a ~ 24'3"'48. The generality of this model allowed to take it as a definition of how dendritic spines are distributed along the dendrites in animals raised under normal conditions, and as a valid tool to study how several abnormal conditions of development affect cortical
TABLE !
Mean t'ah~es ( + S.E.M.) of the thickness of apical shafts selected for this study, measured at a distance of 100 l~m from the soma. Age (days) 10 16 20 30 40 60 80 90 120 215 540 1135
Mean tahoe (l~m) 3.40 3.58 3.72 3.78 3.83 3.80 3.85 3.79 3.83 3.88 3.36 3.51
S.E.M.
n
0.09 0.19 0.13 0.11 0.12 0.14 0.16 0.13 0.15 0.12 0.08 0.14
28 27 30 29 30 30 30 30 29 28 28 29
mat urat ion.~5-37.3t}. Furthermore, different authors have shown that some experimentally induced sensory deprivations 3'~'4x or pathological conditions 3~''~'-'~'~, which affect preferentially a certain region of the cerebral cortex (CC), also produce a concomitant derangement of the distribution of dendritic spines along the apical shafts of deep pyramidal neurons. These results, and the important role played by dendritic spines in the establishment of neuronal connectivity 7'N'2'~'~ led us to think that a careful study of how aging affects the distribution ot' dendritic spines along the apical shafts o[' layer
V pyramidal neurons could give deeper insight in how this natural condition affects the structure of the CC. In the present paper, we present the result obtained from the study of the distribution of dendritic spines,
along the apical shafts of layer V pyramidal neurons in young, adult and aging rats. MATERIALS AND METHODS Animals Wistar albino rats were used. The animals studied at 10, 16, 20, 30. 40. 60. 8[l, 9(). 120 and 215 days of age came from litters equalled on the day fl~llowing birth to 8 pups, irrespective of their sex. A total of 8 animals were used for each age, The number of males and females used for each age were not exactly equal, nevertheless and in order to avoid any possible bias introduced on the results by sex differences''~''~7, special care was taken to avoid any significant difference in the proportions of males and females used at each age. To study the effect of aging at 514 and 1,135 days of age, 6 and 5 rats, respectively, from the same animal house were used. These last animals ate the same stock diet as the rest, as this diet was not changed in the animal house during the whole period of their lives. All animals were raised on a 12 light/12 dark hours schedule. Following the routine of the animal house, the rats were separated by sexes when they were 25 days old. From this age onwards the animal~ were housed in cages in groups of 5-6 each to avoid the possible bia~ |hat could be introduced on the data by the social isolation.
Staining and quantification of destdritic sphzes At the age indicated above the animals were weighed, anesthetized with ether and dissected out. Their hmgs, liver and in-
testines were inspected, and there was not found anything from which it could be inferred that the health of the animals was impaired. The animals were then perfused through the left cardiac ventricle for 5 min with 0.9% NaCI and for 20 rain with 19% formaldehyde in PBS. The portions of the brain comprising the visual cortex were stained by the rapid Golgi procedure. After 2 days in PBS containing 10% formaldehyde, they were transferred for 7.2 h into a solution containing 2.4% potassium dichromate and 0.2% osmium tetroxide, in distilled water. They were then transferred for 24 h into 0.75% silver nitrate solution, also prepared in distilled water. Slices 200 g,m thick were prepared and mounted. For each animal 3-6 complete and well impregnated apical shafts of layer V pyramidal neurons from the primary visual cortex (area 17) were selected at low magnification (25 × ) to complete a total of 30 apical shafts fi~r each age studied. Since at this magnification it is not pt~ssihle to ~otic¢ either the totat number or the distribution of dendritic spines along the apical shafts, this procedure allows the s~:lection of well impregnated neurons avoiding possible bias, The cout~ting of the visible spines in each 50/~m long segment of apical shafts of th~se cells was then perh)rmed at higher magnification (500×), starting from the cell body. In aU the neurons selected for spine counting the thickness of their apical shafts was measured at a distance of I(10 ~tm from their soma. Table ! shows the mean value ( :l: S.E.M.) corresponding to this data. The value of b'tl I,> t:0)" 1,46, obtained fi'om the one-way ANOVA applied to this data, showed that there does not exist any significant difference among them. Taking into account this result, it has been assumed tha! the error induced in the total number and distribution of dendritic spines, by not considering the spines hidden by the thickness of the apical shafts, equally affects all counts obtained at different ages 48, Therefore it was not deemed neccesary to introduce the appropiate correction, to obtain the total number of spines present all around the apical shafts t5. The data obtained was stored on the permanent magnetic memory of an IBM PS2/60 computer for further study.
Data processh~g Total mmzber of sphws/shaft. A program was prepared for the computer which calculated the total number of visible spines for each neuronal shaft from the 50 #m segment data introduced into it. To calculate this variable attention was paid to the fact that a great proportion of the shafts chosen for the study appears to be cut by the microtome before the bifurcation point where it starts the apical tufts. To avoid the possible bias introduced in this variable by the different lengths of the shafts, the machine was instructed to reject from the calculation those neurons not having the cell body inside layer V and with a shaft not long enough to surpass a distance from the surface of the brain equal to one-quarter of the thickness of the cortex. To make this selection the program received, as initial data, the proportions attributed by Zilles et al? ° to each layer of the cortex and, for each apical shaft, the width of the cortex and the
43 A
respect the value of this parameter can be considered as a measurement of the 'statistical distance' between the two mean values considered. Requirements of normality and homogeneity of variances were assessed by the Koimogoroff-Smirnoff and Cochran's test, respectively Io,l l.
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Fig. 1. A: mean values (+S.E.M.) of the weigths of rats 10-1,135 days of age. B: development of the total number of spines (+ S.E.M.) along the apical shafts of layer V cortical pyramidal neurons of the visual cortex of rats 10-1,135 days of age.
distance from the soma to the pial surface of the brain. The few neurons eliminated, because they had shorter shafts or had part of their shafts cut off during sectioning, never represented more than 10% of the 30 neurons chosen for each of the ages studied. The value of n in Table I represents the final number of apical shafts entering on the present study. Distribution of spines along the apical shafts. A second program was used in order to assess whether or not the distribution of dendritic spines along the shafts of the neurons could be fitted to the already
mentioned mathematical model a'~. The mathematical procedure which was followed to find out the degree of fit of the mathematical model to the experimental distribution has been carefully explained elsewhere 4s. This procedure was later programmed. The computer following the instructions of this program found out the best possible fit of the mathematical model to the experimental distribution giving, as a final result, the corresponding value of the X2 parameter, which was taken as a measurement of the degree of fit. Statistical analysis. The test for difference in proportions described by Dixon and Massey 10 was used to asses the statistical significance of the relative decrease of spine number, distributed along apical shafts, with respect to the highest value reached at 80 days of age. In order to find out the statistical significance among mean values distributed along the apical shafts the one-way ANOVA was used. The factor considered in this type of study was the distance to the cell body. The two-way ANOVA has been used to find out the significance of the difference between distributions of spines corresponding to two different ages. The factors involved in this study were the age and the distance to the cell body. In the case the result of the ANOVA showed that there does not exist homogeneity among the mean value, the test of Q, described by Dixon and Massey is, was used to assess the statistical significance of the difference between two particular mean values. This test is essentially the same as the 'Least Significance Difference' test described by Snedecor and Cochran 43. The numerical value of Q is equal to the quotient between the difference of the two mean values contrasted and the residual error of the previous ANOVA. In this
In the upper part of Fig. 1 the body weights of the animals entering the present study are represented and at the bottom of this same figure the evolution with age of the total number of dendritic spines counted along the apical shafts of the pyramidal neurons selected for the study is also represented. As we expected from previous w o r k s 36'37 the total number of dendritic spines distributed along the apical shafts of layer V pyramidal ne:arons of the rat visual cortex increases continuously from 10 to 80 days 36, the age at which the number started to decrease 14 (Fig. 1B). The regression analysis applied to the data corresponding to the descendent part of this evolution (901,135 days) showed that this decrease was linear with a negative slope 9.37 spines/month and a degree of significance of P < 0.001. Fig. 2 shows the experimental distributions of dendritic spines, along the apical shafts of layer V pyramidal neurons of the cerebral cortex of animals 16-1,135 days of age. In this figure it can be appreciated how the distribution of dendritic spines gradually decrease from 80 days onwards, reaching at 1,135 days a level similar to the one it had at 16 days, a result which will be further discussed in detail. In order to find out if there was a region of the shafts which could have been more affected than the rest by spines loss, the differences between the mean number of the spines/50 ~m segments of apical shaft of rats older than 80 days, and the corresponding ones of animals 80 days old, were calculated. To evaluate properly the importance of this spine loss, the relative value of these differences with respect to the number of dendritic spines at 80 days were further calculated. In Table I the mean values (+S.E.M.) of the absolute differences obtained are given and in Fig. 3 the mean values (:t: S.E.M.) of the corresponding relative differences are represented. The test of significance for proportions I° applied to the mean values of the relative loss of dendritic spines showed that at 215 days this loss is already significant with P < 0.01. The one-way ANOVA applied to the data of Fig. 3 showed that, with the exception of the first segment not included in the study, the relative loss of dendritic spines is homogeneous along the whole length of the apical shafts of cortical pyramids of rats
44
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Fig. 2. Experimental distributions of dendritic spines along the apical shafts of layer V pyramidal neurons of the visual cortex of rats 16 and ~0-1,135 days of age. In the upper right corner of the figure the values of the X" coefficient obtained from the fit of the mathematical model of spines distribution to the experimental ones are shown. A value of X': > 4.57 indicates lack of it. Vertical bars represent the range of the S.E.M. of individual mean values.
90, 120 (not shown on the figure) and 215 days old, F)t.> ~,()- 0.08, 1.41 and 0.81, respectively. The result of this same type of analysis applied to the relative differences corresponding to animals 540 and 1,135
days of age showed that the relative loss of dendritic spines was not homogeneous along the shafts of pyramidal neurons of these animals, Fnt,>n2,=4.13 and F~,> t.,()= %58, respectively, P < 0.005.
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Fig. 3. Mean relative differences between the mean number of dendritic spines/50 pm segment along the apical shafts of layer V pyramidal neurons of the visual cortex of rats 80 days old and rats 90, 215, 540 and !,135 days old with respect to the values at 80 days. Vertical bars represent the range of the S.E.M. of all mean values represented.
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Fig. 4. Schematic representation of the widths of the visual cortex of animals 10-1,135 days of age, measured at the sites where the apical shafts entering the present study were located. Dashed lines show the limits of layers I-VI calculated according the proportions attributed to them by Zilles et aLs°. Vertical double headed arrows indicate the range of positions of sixth segment inside the cortex. One asterik means that the difference in the thickness is significant with P < 0.05; two asterisks mean that the difference is significant with P < 0.01.
With the exception of the first segment, the simple inspection of Fig. 3 indicates that the maximum relative difference is reached at a distance of the soma equal to 300 pm or sixth segment and further. The fact that the relative difference corresponding to the first segment is greater than the rest could be an artificial result due to the low number of spines found at this level of the shafts, and for this reason it has not been taken into consideration in the present study. Represented in Fig. 4 are the mean values (:1: S.E.M.) of the widths of the cerebral cortex measured at the sites where the neurons chosen for this study were located. Dashed lines show the limits of layers I - V I , calculated according to the proportions attributed to them by ZiUes et al. "~°. In this same figure it can be observed how the sixth segment is always located either inside layer IV of the cortex, or in the lower part of layer III which according to Valverde 47
and Peters 29 receive 80% of the specific afferent fibers coming from the lateral geniculate body. In order to assess the significance of the differences between mean values of spines present in different segments of the apical shafts the test of Q was used H). The meaning of this parameter has been already explained in the Materials and Methods section. Fig. 5 shows the evolution, with the age of the animals, of the
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TABLE !I
Absolute differences between the mean number of dendritic spines/50 I~m, long segment ( ± S.E.M.) of the apical shafts of layer V cerebral cortical pyramids of 80 day old rats and animals of the indicated ages 5"
"
I
/~m 25 125 175 225 2~ 325 3~ 425 475 525 5~ 625
80- 90 days 80- 215 days
80- 540 days 80-1,135 days
1.1±0.9 2.9±2.2 3.1±1.5 4.1±1.4 2.9±1.3 3.1±1.8 3.4±1.6 2.4±1.3 2.3±2.2 1.7±2.5 3.8±3.6 3.3±4.1 1.7±0.8
1.8±1.0 6.2±1.5 9.6±1.1 12.0±1.2 17.5±1.1 18.6±1.2 18.6±1.2 17.5±1.2 14.8±1.6 12.3±1.6 14.3±1.5 7.7±1.7 8.7±1.8
2.2±1.1 4.6±2.0 5.7±1.7 8.9±1.2 11.8±2.3 11.9±1.7 12.2±2.1 11.1±1.7 10.2±1.9 9.1±2.1 9.2±1.9 10.3±!.3 11.5±5.5
1.3±1.1 7.9±1.8 16.0±1.8 23.6±2.2 28.7±1.7 30.8±2.1 20.2±1.7 27.5±1.8 25.5±2.0 22.2±2.0 17.8±2.2 -
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Fig. 5. Values of Q obtained from the comparison between the mean values corresponding to the second and sixth segments of the distributions shown in Fig. 3. The horizontal dashed line represents the value of Q beyond which the values of Q are statistically significant with P < 0.05. The values of/)max and of the Cochran coefficients, for the one-way ANOVA performed ranged from 0.15 to 0.18 with 20 d.f. and from 0.16 to 0.19 with k = l0 and 20 d.f., respectively. A value of Dmax < 0.19 indicates, with P < 0.05, that the data can be considered as pertaining to a normally distributed population. A value of C > 0.2 indicates lack of homogeneity amongst the variance analyzed.
46 values of Q obtained from the comparison made between the mean number of spines corresponding to the second and sixth segments. The observed increase of the value of Q with the age of the animal indicates that the loss of dendritic spines produced by aging is greater in layer IV and l l l - l l , than in deep layers. The horizontal dashed line represents the value of Q beyond which the difference between the mean values compared is statistically significant at the level of 0.05. The interpolation made graphically in Fig. 5 shows that this value is attained at approx. 400 days, indicating that the greater loss of spines, produced by aging in layers IV and 111-11 rather than in layer V, starts being statistically significant at approximately this age in the rat. b; ~
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Furthermore, the one-way ANOVA followed by the test of Q applied to mean values of cortical thickness shown in Fig. 4 showed that, from 16 to 1,135 days there exists only a slight significant difference in the mean values of this variable between 540 and 1,135 days of a g e (F4.> IE0= 2.95, Q3,27 = 4.06, P < 0.05), but not to such an extent that their influence on the length of the apical shafts of deep pyramidal neurons could influence the conclusions derived from the present study. In Fig. 6 the distributions of dendritic spines along the apical shafts of animals 1,135 and 16; 540 and 20 and 215 and 40 days of age is shown. The values of F obtained as the result of the two-way ANOVA applied to the comparison of these three pairs of distributions were Fn.< 120= 2.069, 0.02 and 0.46, respectively. The non-significance of these values indicates the similarity between these pairs of distributions. The greater value of F, obtained from the comparison between the first pair of distributions considered, could be a consequence of the distortion introduced by age on the distribution of spines. This greater distortion is reflected in the greater value of the X 2 parameter obtained as a result of the fit of the mathematical model to the experimental distribution corresponding to animals 1,135 days old rather than the one corresponding to animals 16 days old. (See Fig. 2.)
525
625
Distance from the cell body in pm
Fig. 6. Distributions of dendritic spines along the apical shafts of layer V pyramidal neurons of the cerebral cortex of rats 1,135 and 16:540 and 20 and 215 lind 4() days old. Vertical bars represent the range of the S.E,M. of individual mean values. The values of Din,,~ ranged from 0.14 to 0.18. A value of Din,,~ < 0.19 indicates, with P < 0.05, that the data can be considered as pertaining to a normally distributed population. The value of the Cochran coefficients (C) ranged from 0.06 to 0.07 with k = 28 and d.f. 20. A value of C > 0.08 indicates a lack of homogeneity amongts the variances analyzed. In the upper right corner of the figme the values of th~ X: coefficients obtained from the fit of the mathematical model of spines distribution to the experimental ones are shown. A value of X z > 4.57 indicates lack of it. Vertical bars represent the range of S.E.M. of individual mean values.
Present results obtained from the study of the effect of early development and aging, in the number and distribution of dendritic spines along the apical shafts of layer V cortical neurons, indicate that aging produces a reduction of the total number of spines and a progressive derangement of the distribution of spines and that 80 days can be taken as the age at which this degenerative process starts in the rat. The results concerning the effects of aging on the total number of spines coincide with those reported previously by Feldman and Dowd n4. We have found this coincidence even in the speed of reduction of the number of spines with age. From the regression analysis made by us, it has been derived that the total number of spines along the shafts decreases at a speed of 9.37 spines/month which corresponds to 0.85 spines/month/50 ~m segment, while the speed of the reduction found by Feldman and Dowd as a consequence of their above mentioned work is approximately 1.08 spines/month/50/zm segment, a value which can be easily derived from the data reported by the authors in Fig. 10 of their paper. This similarity of our own results, with those reported
47 by Feldman and Dowd 14, could be taken as one index of the degree of the reliability of the pre~ent data. In order to properly evaluate the present results, regarding the effects of aging on the distribution of dendritic spines along the apical shafts of layer V cortical pyramidal neurons, it is necessary to take into account some important properties of these shafts: as is well known, the apical shafts of deep pyramids run perpendicular to the pial surface from the soma of the neurons to the end on cortical layers III-II-I, and therefore they cross almost all the depth of the cerebral cortex. They are covered with dendritic spines, which are important in establishing thalamocortical connections, as has been widely proved and documented 7'8'29'3°. In this respect, it has been reported that 83% of the thalamic afferents to apical shafts on the primary visual cortex establish synaptic connections on the head of spines, 15% on the inter spinous part of the trunk and 2% on the soma of pyramidal neurons 29.30. These two important properties led us to think that the way dendritic spines are distributed along the apical shafts of layer V pyramidal r,curons could reflect the conditions of the structure of the cerebral cortex. This assumption is reinforced by the results obtained by different authors. In fact, Valverde 47 noticed that the decrease in the number of dendritic spines, produced by monocular enucleation on the layer IV portion of the apical shafts of layer V pyramidal neurons, came accompanied with a redistribution of the dendrites of stellate cells of this same layer IV. Later on Ruiz-Marcos et al. 38 found that neonatal hypothyroidism affects more deeply the development of dendritic spines in the superficial region of apical shafts of
layer V cortical pyramidal neurons, than in other regions of these shafts, and that this severe pathological condition also preferentially affects the dendritic arborization is'33, myelinated profiles 3 and the density and distribution of microtubules present on the superficial layers of the cerebral cortex 2. Present results concerning the effect of aging on the
distribution of dendritic spines along the shafts of layer V cortical neurons indicates that the progressive derangement produced by this natural condition on this distribution starts at 80 days, but is not statistically significant until an age between 120 and 215 days, in the rat. Furthermore, these results indicate that until a certain age, estimated by us to be 400 days, the derangement produced by aging in the number of spines distributed along the shafts is uniform along their whole length and that, at this age, this derangement starts being significantly greater in layers IV and III than in layer V. Given the specific functional signifi-
cance of layers IV and I11 of the visual cortex29'30'39'47'48, this result could be considered as being a consequence of the degeneration of the peripheral visual system, or even to a specific pathology of the eyes of the rats studied. Unfortunately, and not foreseen in these results, the eyes of the rats used for this study were not inspected, looking for some possible type of pathology, nor were any visual tests performed on them. Nevertheless, the monotonic increase of the values of Q obtained indicates the continuity of this process, something which leads us to conclude that aging itself, and not an extra pathological condition, is responsible for the differential effect of aging in the different regions of the cerebral cortex. The similarity obtained between the distributions of spines corresponding to 16 and 1,135; 20 and 540 and 40 and 215 day old animals, shown in Fig. 6, indicated that, as the animal gets older, the distribution of dendritic spines regresses to the level they were at younger ages. The values of X2, obtained from the fit of these experimental distributions to the principal equation of the mathematical model, shows that, while the distributions corresponding to 16, 20 and 40 day old young animals can be fitted by this equation, the distributions corresponding to old animals can no longer be fitted. Taking into consideration that, according to previous results 3t''a~'4s, this equation can be taken as a definition of the pattern followed by the distribution of spines in normal animals, the above mentioned results indicate that aging produces a progressive derangement on the distribution of spines along the apical shafts of cortical pyramids. This derangement, in turn, could explain the dissimilarity of shapes observed between the distributions of spines corresponding to the two extreme ages contrasted, 16 and 1,135 days, which still, and according to the value of F~,>120- 2.069 obtained from their comparison, cannot be considered as significantly different. Finally, taking into consideration the above mentioned results according to which different authors 2.3'9.aa'3t''3s'47 have found a relationship between a decrease in the number of spines in different regions of the apical shaft and a concomitant deterioration or change of the structure of the neurons in these same regions of the cerebral cortex, the observed deterioration of the distribution of dendritic spines along the apical shafts of layer V pyramidal neurons could be a consequence of a concomitant derangement of the structure of the cerebral cortex. The authors of the present work are aware that, in order to make the conclusions derived from the study of the effect of aging on spines distributed along the apical shafts of deep pyramids extensive to other structures of the
48 cerebral cortex, it is necessary to perform more experiments. Nevertheless, the already mentioned relationship found between damages produced on the number of spines and changes of cortical structures in the same region ~'3A'j'33"3t+'38'47, leads us to tentatively formulate the hypotheses that aging also produces a regression to a juvenile-like situation of some other structures of the cerebral cortex, such as the one formed by the specific thalamo-cortical projection fibers. Acknowledgements. We are grateful to C. GabrieI-Alvarez for technical assistance, to A. Hurtado-Cano for his art work and to E. Fernandez de Molina for typing. This work has been supported by Grant PM-0012 from the DGICYT to A.R.-M, J.A.M.-C. is a fellow of the Fondo de lnvestigaciones Sanitarias de la Seguridad Social.
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49
40
41 42 43 44
distribution of spines in the visual cortex of normal and dark-reared mice, Exp. Brain Res., 8 (1969) 284-294. Scheibel, M.E., Lindsay, R.D., Tomiyasy, U. and Scheibel, A.B., Progressive dendritic changes in aging human cortex, Exp. Neurol., 47 (1975) 392-403. Scheibe!, M.E., Tomiyasu, U. and Scheibel, A.B., The aging human Betz cell, Exp. Neurol., 56 (1977) 598-609. Scheibel, M.E., Lindsay, R.E., Tomiyasu, U. and Scheibel, A.B. Progressive dendritic changes in the aging human limbic system, Exp. Neurol., 53 (1976) 420-430. Snedecor, G.W. and Cochran, W.G., Statistical Methods, The Iowa State University Press, Ames, IA, 1974. Tomlinson, B.E. and Henderson, H. Some quantitative cerebral findings in normal and demented old people. In R.D. Terry and S. Gershon (Eds.), Neurobiology of Aging. Vol. 3, Raven, 1976, pp. 183-204.
45 Uemura, E. Age-related changes in prefrontal cortex of Macaca mulatta: synaptic density, Exp. Neurol., 29 (1980) 164-172. 46 Uemura, E., Age-related changes in subiculum of Macaca mulatta: synaptic density, Exp. Neurol., 87 (1985) 403-411. 47 Valverde, F. Structural changes in the area striata of the mouse after enucleation, Exp. Brain Res., 5 (1968) 274-292. 48 Valverde, F. and Ruiz Marcos, A., Dendritic spine in the visual cortex of the mouse. Introduction to a mathematical model, Exp. Brain Res., 8 (1969) 269-293. 49 Vaughan, D.W., Age-related deterioration of pyramidal basal dendrites in rat auditory cortex, J. Comp. Neurol., 171 (1977) 501-516. 50 Ziiles, K., Wree, A., Scheicher, A. and Divac, I., The monocular and binocular subfields of the rat's primary visual cortex: a quantitative morphological approach, J. Comp. Neurol., 226 (1984) 391- 402.
41
BRESD 51501
Aging reverts to juvenile conditions the synaptic connectivity of cerebral cortical pyramidal shafts A n t o n i o R u i z - M a r c o s , F e r n a n d o S a n c h e z - T o s c a n o a n d Jos6 A n t o n i o M u f i o z - C u e t o Unidad de Neuroanatomia, lnstituto Cajal, Madrid (Spain) (Accepted 19 May 1992)
Key words: Aging; Cerebral cortex; Development; Pyramidal cell; Dendritic spine
Quantitative analysis of the total number and distribution of dendritic spines along the apical shafts of layer V cerebral cortical pyramids has been performed on aging rats (90-120 to 1,135 days old) and on rats during the period of early and late development (10-80 days). As expected from previous works, present results show that the total number of dendritic spines along the shafts increase from 10 to 80 days, after which it starts to gradually decrease until the last age studied (1,135 days). The quantitative analysis of the effect of aging on the relative decrease of dendritic spines shows that this decrease starts being homogeneous along the whole length of the apical shafts and that from a certain age onwards, estimated according to present results in 400 days, this effect is significantly more pronounced in layers IV and l l l - l l than in deep layers. Furthermore, the comparison made between the distribution of dendritic spines along the apical shafts of pyramidal neurons of old and young animals has shown that aging produces a regression of this distribution to juvenile conditions.
INTRODUCTION
It has been widely proved that aging produces a progressive deterioration of learning and memory ability in animals 1,'~'12.a°'2~'22'2sand learning performance in man 42'44. In this respect and given the importance of cerebral cortex in cognitive and intelligent processes the question of how aging affects the development of its structure seems to be an important one. In recent years there has been an increasing number of studies dealing with the problems of how this natural condition affects different neuronal cortical elements. It has been described that aging produces neuronal loss 9'17'18'32, deterioration and changes in the structure of dendritic trees 6't3'4°'49, amongst other effects. Different authors have shown the importance of dendritic spines on the establisment of thalamo-corticai connections 7's'29'3~. It has also been proposed that these neuronal elements could be an important substrate for information storage t6. These findings and the one reported by Bertoni-Freddari et al. 4, according to which aging seriously impairs the morphological plasticity of synapses, led us to think that the study of how
aging affects the number and distribution of the dendritic spines which are present along the apical shafts of layer V cortical pyramids, could shed light on how this natural condition affects not only the neuronal connectivity in the cerebral cortex, but also the organization of its general structure. Feldman and Dowd 14 showed that aging produces a slow and progressive decrease in the number of spines from apical, basal, oblique and terminal tufts dendrites of layer V pyramids of the visual cortex of the rat. This finding has been confirmed by some other authors who have also found an age-dependent spine loss in pyramidal neurons of layer III and V of the visual cortex of mice 23, dog 25, monkey 45,4e and man 41'42. It has also been found that aging produces a distortion of the shape of the spines 2s'32. Although these studies have clearly established that aging affects the number of spines, none of them have taken into consideration the effect of aging on the distribution of these elements along the dendrites. In 1969 it was found 39'4s that dendritic spines are not distributed at random along the apical and basal dendrites of layer V and III pyramidal neurons of the
Correspondence: A. Ruiz-Marcos, Institute Cajal, c./Doctor Atce 37, 28002 Madrid, Spain.
42 visual cortex of mice, but are distributed according to a precise pattern which could be defined by a mathematical model. This model is formed by one principal equation which describes how these elements are distributed along the dendrites, and three parametrical ones which describe how the distribution is affected by development 3~. This model was found valid to describe the distribution of spines along the apical and basal dendrites of several cortical layers 34-3~''3'~'48 and in species such as mice, hamster, cat, monkey and even m a ~ 24'3"'48. The generality of this model allowed to take it as a definition of how dendritic spines are distributed along the dendrites in animals raised under normal conditions, and as a valid tool to study how several abnormal conditions of development affect cortical
TABLE !
Mean t'ah~es ( + S.E.M.) of the thickness of apical shafts selected for this study, measured at a distance of 100 l~m from the soma. Age (days) 10 16 20 30 40 60 80 90 120 215 540 1135
Mean tahoe (l~m) 3.40 3.58 3.72 3.78 3.83 3.80 3.85 3.79 3.83 3.88 3.36 3.51
S.E.M.
n
0.09 0.19 0.13 0.11 0.12 0.14 0.16 0.13 0.15 0.12 0.08 0.14
28 27 30 29 30 30 30 30 29 28 28 29
mat urat ion.~5-37.3t}. Furthermore, different authors have shown that some experimentally induced sensory deprivations 3'~'4x or pathological conditions 3~''~'-'~'~, which affect preferentially a certain region of the cerebral cortex (CC), also produce a concomitant derangement of the distribution of dendritic spines along the apical shafts of deep pyramidal neurons. These results, and the important role played by dendritic spines in the establishment of neuronal connectivity 7'N'2'~'~ led us to think that a careful study of how aging affects the distribution ot' dendritic spines along the apical shafts o[' layer
V pyramidal neurons could give deeper insight in how this natural condition affects the structure of the CC. In the present paper, we present the result obtained from the study of the distribution of dendritic spines,
along the apical shafts of layer V pyramidal neurons in young, adult and aging rats. MATERIALS AND METHODS Animals Wistar albino rats were used. The animals studied at 10, 16, 20, 30. 40. 60. 8[l, 9(). 120 and 215 days of age came from litters equalled on the day fl~llowing birth to 8 pups, irrespective of their sex. A total of 8 animals were used for each age, The number of males and females used for each age were not exactly equal, nevertheless and in order to avoid any possible bias introduced on the results by sex differences''~''~7, special care was taken to avoid any significant difference in the proportions of males and females used at each age. To study the effect of aging at 514 and 1,135 days of age, 6 and 5 rats, respectively, from the same animal house were used. These last animals ate the same stock diet as the rest, as this diet was not changed in the animal house during the whole period of their lives. All animals were raised on a 12 light/12 dark hours schedule. Following the routine of the animal house, the rats were separated by sexes when they were 25 days old. From this age onwards the animal~ were housed in cages in groups of 5-6 each to avoid the possible bia~ |hat could be introduced on the data by the social isolation.
Staining and quantification of destdritic sphzes At the age indicated above the animals were weighed, anesthetized with ether and dissected out. Their hmgs, liver and in-
testines were inspected, and there was not found anything from which it could be inferred that the health of the animals was impaired. The animals were then perfused through the left cardiac ventricle for 5 min with 0.9% NaCI and for 20 rain with 19% formaldehyde in PBS. The portions of the brain comprising the visual cortex were stained by the rapid Golgi procedure. After 2 days in PBS containing 10% formaldehyde, they were transferred for 7.2 h into a solution containing 2.4% potassium dichromate and 0.2% osmium tetroxide, in distilled water. They were then transferred for 24 h into 0.75% silver nitrate solution, also prepared in distilled water. Slices 200 g,m thick were prepared and mounted. For each animal 3-6 complete and well impregnated apical shafts of layer V pyramidal neurons from the primary visual cortex (area 17) were selected at low magnification (25 × ) to complete a total of 30 apical shafts fi~r each age studied. Since at this magnification it is not pt~ssihle to ~otic¢ either the totat number or the distribution of dendritic spines along the apical shafts, this procedure allows the s~:lection of well impregnated neurons avoiding possible bias, The cout~ting of the visible spines in each 50/~m long segment of apical shafts of th~se cells was then perh)rmed at higher magnification (500×), starting from the cell body. In aU the neurons selected for spine counting the thickness of their apical shafts was measured at a distance of I(10 ~tm from their soma. Table ! shows the mean value ( :l: S.E.M.) corresponding to this data. The value of b'tl I,> t:0)" 1,46, obtained fi'om the one-way ANOVA applied to this data, showed that there does not exist any significant difference among them. Taking into account this result, it has been assumed tha! the error induced in the total number and distribution of dendritic spines, by not considering the spines hidden by the thickness of the apical shafts, equally affects all counts obtained at different ages 48, Therefore it was not deemed neccesary to introduce the appropiate correction, to obtain the total number of spines present all around the apical shafts t5. The data obtained was stored on the permanent magnetic memory of an IBM PS2/60 computer for further study.
Data processh~g Total mmzber of sphws/shaft. A program was prepared for the computer which calculated the total number of visible spines for each neuronal shaft from the 50 #m segment data introduced into it. To calculate this variable attention was paid to the fact that a great proportion of the shafts chosen for the study appears to be cut by the microtome before the bifurcation point where it starts the apical tufts. To avoid the possible bias introduced in this variable by the different lengths of the shafts, the machine was instructed to reject from the calculation those neurons not having the cell body inside layer V and with a shaft not long enough to surpass a distance from the surface of the brain equal to one-quarter of the thickness of the cortex. To make this selection the program received, as initial data, the proportions attributed by Zilles et al? ° to each layer of the cortex and, for each apical shaft, the width of the cortex and the
43 A
respect the value of this parameter can be considered as a measurement of the 'statistical distance' between the two mean values considered. Requirements of normality and homogeneity of variances were assessed by the Koimogoroff-Smirnoff and Cochran's test, respectively Io,l l.
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Fig. 1. A: mean values (+S.E.M.) of the weigths of rats 10-1,135 days of age. B: development of the total number of spines (+ S.E.M.) along the apical shafts of layer V cortical pyramidal neurons of the visual cortex of rats 10-1,135 days of age.
distance from the soma to the pial surface of the brain. The few neurons eliminated, because they had shorter shafts or had part of their shafts cut off during sectioning, never represented more than 10% of the 30 neurons chosen for each of the ages studied. The value of n in Table I represents the final number of apical shafts entering on the present study. Distribution of spines along the apical shafts. A second program was used in order to assess whether or not the distribution of dendritic spines along the shafts of the neurons could be fitted to the already
mentioned mathematical model a'~. The mathematical procedure which was followed to find out the degree of fit of the mathematical model to the experimental distribution has been carefully explained elsewhere 4s. This procedure was later programmed. The computer following the instructions of this program found out the best possible fit of the mathematical model to the experimental distribution giving, as a final result, the corresponding value of the X2 parameter, which was taken as a measurement of the degree of fit. Statistical analysis. The test for difference in proportions described by Dixon and Massey 10 was used to asses the statistical significance of the relative decrease of spine number, distributed along apical shafts, with respect to the highest value reached at 80 days of age. In order to find out the statistical significance among mean values distributed along the apical shafts the one-way ANOVA was used. The factor considered in this type of study was the distance to the cell body. The two-way ANOVA has been used to find out the significance of the difference between distributions of spines corresponding to two different ages. The factors involved in this study were the age and the distance to the cell body. In the case the result of the ANOVA showed that there does not exist homogeneity among the mean value, the test of Q, described by Dixon and Massey is, was used to assess the statistical significance of the difference between two particular mean values. This test is essentially the same as the 'Least Significance Difference' test described by Snedecor and Cochran 43. The numerical value of Q is equal to the quotient between the difference of the two mean values contrasted and the residual error of the previous ANOVA. In this
In the upper part of Fig. 1 the body weights of the animals entering the present study are represented and at the bottom of this same figure the evolution with age of the total number of dendritic spines counted along the apical shafts of the pyramidal neurons selected for the study is also represented. As we expected from previous w o r k s 36'37 the total number of dendritic spines distributed along the apical shafts of layer V pyramidal ne:arons of the rat visual cortex increases continuously from 10 to 80 days 36, the age at which the number started to decrease 14 (Fig. 1B). The regression analysis applied to the data corresponding to the descendent part of this evolution (901,135 days) showed that this decrease was linear with a negative slope 9.37 spines/month and a degree of significance of P < 0.001. Fig. 2 shows the experimental distributions of dendritic spines, along the apical shafts of layer V pyramidal neurons of the cerebral cortex of animals 16-1,135 days of age. In this figure it can be appreciated how the distribution of dendritic spines gradually decrease from 80 days onwards, reaching at 1,135 days a level similar to the one it had at 16 days, a result which will be further discussed in detail. In order to find out if there was a region of the shafts which could have been more affected than the rest by spines loss, the differences between the mean number of the spines/50 ~m segments of apical shaft of rats older than 80 days, and the corresponding ones of animals 80 days old, were calculated. To evaluate properly the importance of this spine loss, the relative value of these differences with respect to the number of dendritic spines at 80 days were further calculated. In Table I the mean values (+S.E.M.) of the absolute differences obtained are given and in Fig. 3 the mean values (:t: S.E.M.) of the corresponding relative differences are represented. The test of significance for proportions I° applied to the mean values of the relative loss of dendritic spines showed that at 215 days this loss is already significant with P < 0.01. The one-way ANOVA applied to the data of Fig. 3 showed that, with the exception of the first segment not included in the study, the relative loss of dendritic spines is homogeneous along the whole length of the apical shafts of cortical pyramids of rats
44
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90, 120 (not shown on the figure) and 215 days old, F)t.> ~,()- 0.08, 1.41 and 0.81, respectively. The result of this same type of analysis applied to the relative differences corresponding to animals 540 and 1,135
days of age showed that the relative loss of dendritic spines was not homogeneous along the shafts of pyramidal neurons of these animals, Fnt,>n2,=4.13 and F~,> t.,()= %58, respectively, P < 0.005.
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16
20
40
215
540
1155
AGE IN DAYS
Fig. 4. Schematic representation of the widths of the visual cortex of animals 10-1,135 days of age, measured at the sites where the apical shafts entering the present study were located. Dashed lines show the limits of layers I-VI calculated according the proportions attributed to them by Zilles et aLs°. Vertical double headed arrows indicate the range of positions of sixth segment inside the cortex. One asterik means that the difference in the thickness is significant with P < 0.05; two asterisks mean that the difference is significant with P < 0.01.
With the exception of the first segment, the simple inspection of Fig. 3 indicates that the maximum relative difference is reached at a distance of the soma equal to 300 pm or sixth segment and further. The fact that the relative difference corresponding to the first segment is greater than the rest could be an artificial result due to the low number of spines found at this level of the shafts, and for this reason it has not been taken into consideration in the present study. Represented in Fig. 4 are the mean values (:1: S.E.M.) of the widths of the cerebral cortex measured at the sites where the neurons chosen for this study were located. Dashed lines show the limits of layers I - V I , calculated according to the proportions attributed to them by ZiUes et al. "~°. In this same figure it can be observed how the sixth segment is always located either inside layer IV of the cortex, or in the lower part of layer III which according to Valverde 47
and Peters 29 receive 80% of the specific afferent fibers coming from the lateral geniculate body. In order to assess the significance of the differences between mean values of spines present in different segments of the apical shafts the test of Q was used H). The meaning of this parameter has been already explained in the Materials and Methods section. Fig. 5 shows the evolution, with the age of the animals, of the
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TABLE !I
Absolute differences between the mean number of dendritic spines/50 I~m, long segment ( ± S.E.M.) of the apical shafts of layer V cerebral cortical pyramids of 80 day old rats and animals of the indicated ages 5"
"
I
/~m 25 125 175 225 2~ 325 3~ 425 475 525 5~ 625
80- 90 days 80- 215 days
80- 540 days 80-1,135 days
1.1±0.9 2.9±2.2 3.1±1.5 4.1±1.4 2.9±1.3 3.1±1.8 3.4±1.6 2.4±1.3 2.3±2.2 1.7±2.5 3.8±3.6 3.3±4.1 1.7±0.8
1.8±1.0 6.2±1.5 9.6±1.1 12.0±1.2 17.5±1.1 18.6±1.2 18.6±1.2 17.5±1.2 14.8±1.6 12.3±1.6 14.3±1.5 7.7±1.7 8.7±1.8
2.2±1.1 4.6±2.0 5.7±1.7 8.9±1.2 11.8±2.3 11.9±1.7 12.2±2.1 11.1±1.7 10.2±1.9 9.1±2.1 9.2±1.9 10.3±!.3 11.5±5.5
1.3±1.1 7.9±1.8 16.0±1.8 23.6±2.2 28.7±1.7 30.8±2.1 20.2±1.7 27.5±1.8 25.5±2.0 22.2±2.0 17.8±2.2 -
0
I00
w
w 0
4O0
v
5O0 Age in doys
;
'l
I
w
I000
Fig. 5. Values of Q obtained from the comparison between the mean values corresponding to the second and sixth segments of the distributions shown in Fig. 3. The horizontal dashed line represents the value of Q beyond which the values of Q are statistically significant with P < 0.05. The values of/)max and of the Cochran coefficients, for the one-way ANOVA performed ranged from 0.15 to 0.18 with 20 d.f. and from 0.16 to 0.19 with k = l0 and 20 d.f., respectively. A value of Dmax < 0.19 indicates, with P < 0.05, that the data can be considered as pertaining to a normally distributed population. A value of C > 0.2 indicates lack of homogeneity amongst the variance analyzed.
46 values of Q obtained from the comparison made between the mean number of spines corresponding to the second and sixth segments. The observed increase of the value of Q with the age of the animal indicates that the loss of dendritic spines produced by aging is greater in layer IV and l l l - l l , than in deep layers. The horizontal dashed line represents the value of Q beyond which the difference between the mean values compared is statistically significant at the level of 0.05. The interpolation made graphically in Fig. 5 shows that this value is attained at approx. 400 days, indicating that the greater loss of spines, produced by aging in layers IV and 111-11 rather than in layer V, starts being statistically significant at approximately this age in the rat. b; ~
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Furthermore, the one-way ANOVA followed by the test of Q applied to mean values of cortical thickness shown in Fig. 4 showed that, from 16 to 1,135 days there exists only a slight significant difference in the mean values of this variable between 540 and 1,135 days of a g e (F4.> IE0= 2.95, Q3,27 = 4.06, P < 0.05), but not to such an extent that their influence on the length of the apical shafts of deep pyramidal neurons could influence the conclusions derived from the present study. In Fig. 6 the distributions of dendritic spines along the apical shafts of animals 1,135 and 16; 540 and 20 and 215 and 40 days of age is shown. The values of F obtained as the result of the two-way ANOVA applied to the comparison of these three pairs of distributions were Fn.< 120= 2.069, 0.02 and 0.46, respectively. The non-significance of these values indicates the similarity between these pairs of distributions. The greater value of F, obtained from the comparison between the first pair of distributions considered, could be a consequence of the distortion introduced by age on the distribution of spines. This greater distortion is reflected in the greater value of the X 2 parameter obtained as a result of the fit of the mathematical model to the experimental distribution corresponding to animals 1,135 days old rather than the one corresponding to animals 16 days old. (See Fig. 2.)
525
625
Distance from the cell body in pm
Fig. 6. Distributions of dendritic spines along the apical shafts of layer V pyramidal neurons of the cerebral cortex of rats 1,135 and 16:540 and 20 and 215 lind 4() days old. Vertical bars represent the range of the S.E,M. of individual mean values. The values of Din,,~ ranged from 0.14 to 0.18. A value of Din,,~ < 0.19 indicates, with P < 0.05, that the data can be considered as pertaining to a normally distributed population. The value of the Cochran coefficients (C) ranged from 0.06 to 0.07 with k = 28 and d.f. 20. A value of C > 0.08 indicates a lack of homogeneity amongts the variances analyzed. In the upper right corner of the figme the values of th~ X: coefficients obtained from the fit of the mathematical model of spines distribution to the experimental ones are shown. A value of X z > 4.57 indicates lack of it. Vertical bars represent the range of S.E.M. of individual mean values.
Present results obtained from the study of the effect of early development and aging, in the number and distribution of dendritic spines along the apical shafts of layer V cortical neurons, indicate that aging produces a reduction of the total number of spines and a progressive derangement of the distribution of spines and that 80 days can be taken as the age at which this degenerative process starts in the rat. The results concerning the effects of aging on the total number of spines coincide with those reported previously by Feldman and Dowd n4. We have found this coincidence even in the speed of reduction of the number of spines with age. From the regression analysis made by us, it has been derived that the total number of spines along the shafts decreases at a speed of 9.37 spines/month which corresponds to 0.85 spines/month/50 ~m segment, while the speed of the reduction found by Feldman and Dowd as a consequence of their above mentioned work is approximately 1.08 spines/month/50/zm segment, a value which can be easily derived from the data reported by the authors in Fig. 10 of their paper. This similarity of our own results, with those reported
47 by Feldman and Dowd 14, could be taken as one index of the degree of the reliability of the pre~ent data. In order to properly evaluate the present results, regarding the effects of aging on the distribution of dendritic spines along the apical shafts of layer V cortical pyramidal neurons, it is necessary to take into account some important properties of these shafts: as is well known, the apical shafts of deep pyramids run perpendicular to the pial surface from the soma of the neurons to the end on cortical layers III-II-I, and therefore they cross almost all the depth of the cerebral cortex. They are covered with dendritic spines, which are important in establishing thalamocortical connections, as has been widely proved and documented 7'8'29'3°. In this respect, it has been reported that 83% of the thalamic afferents to apical shafts on the primary visual cortex establish synaptic connections on the head of spines, 15% on the inter spinous part of the trunk and 2% on the soma of pyramidal neurons 29.30. These two important properties led us to think that the way dendritic spines are distributed along the apical shafts of layer V pyramidal r,curons could reflect the conditions of the structure of the cerebral cortex. This assumption is reinforced by the results obtained by different authors. In fact, Valverde 47 noticed that the decrease in the number of dendritic spines, produced by monocular enucleation on the layer IV portion of the apical shafts of layer V pyramidal neurons, came accompanied with a redistribution of the dendrites of stellate cells of this same layer IV. Later on Ruiz-Marcos et al. 38 found that neonatal hypothyroidism affects more deeply the development of dendritic spines in the superficial region of apical shafts of
layer V cortical pyramidal neurons, than in other regions of these shafts, and that this severe pathological condition also preferentially affects the dendritic arborization is'33, myelinated profiles 3 and the density and distribution of microtubules present on the superficial layers of the cerebral cortex 2. Present results concerning the effect of aging on the
distribution of dendritic spines along the shafts of layer V cortical neurons indicates that the progressive derangement produced by this natural condition on this distribution starts at 80 days, but is not statistically significant until an age between 120 and 215 days, in the rat. Furthermore, these results indicate that until a certain age, estimated by us to be 400 days, the derangement produced by aging in the number of spines distributed along the shafts is uniform along their whole length and that, at this age, this derangement starts being significantly greater in layers IV and III than in layer V. Given the specific functional signifi-
cance of layers IV and I11 of the visual cortex29'30'39'47'48, this result could be considered as being a consequence of the degeneration of the peripheral visual system, or even to a specific pathology of the eyes of the rats studied. Unfortunately, and not foreseen in these results, the eyes of the rats used for this study were not inspected, looking for some possible type of pathology, nor were any visual tests performed on them. Nevertheless, the monotonic increase of the values of Q obtained indicates the continuity of this process, something which leads us to conclude that aging itself, and not an extra pathological condition, is responsible for the differential effect of aging in the different regions of the cerebral cortex. The similarity obtained between the distributions of spines corresponding to 16 and 1,135; 20 and 540 and 40 and 215 day old animals, shown in Fig. 6, indicated that, as the animal gets older, the distribution of dendritic spines regresses to the level they were at younger ages. The values of X2, obtained from the fit of these experimental distributions to the principal equation of the mathematical model, shows that, while the distributions corresponding to 16, 20 and 40 day old young animals can be fitted by this equation, the distributions corresponding to old animals can no longer be fitted. Taking into consideration that, according to previous results 3t''a~'4s, this equation can be taken as a definition of the pattern followed by the distribution of spines in normal animals, the above mentioned results indicate that aging produces a progressive derangement on the distribution of spines along the apical shafts of cortical pyramids. This derangement, in turn, could explain the dissimilarity of shapes observed between the distributions of spines corresponding to the two extreme ages contrasted, 16 and 1,135 days, which still, and according to the value of F~,>120- 2.069 obtained from their comparison, cannot be considered as significantly different. Finally, taking into consideration the above mentioned results according to which different authors 2.3'9.aa'3t''3s'47 have found a relationship between a decrease in the number of spines in different regions of the apical shaft and a concomitant deterioration or change of the structure of the neurons in these same regions of the cerebral cortex, the observed deterioration of the distribution of dendritic spines along the apical shafts of layer V pyramidal neurons could be a consequence of a concomitant derangement of the structure of the cerebral cortex. The authors of the present work are aware that, in order to make the conclusions derived from the study of the effect of aging on spines distributed along the apical shafts of deep pyramids extensive to other structures of the
48 cerebral cortex, it is necessary to perform more experiments. Nevertheless, the already mentioned relationship found between damages produced on the number of spines and changes of cortical structures in the same region ~'3A'j'33"3t+'38'47, leads us to tentatively formulate the hypotheses that aging also produces a regression to a juvenile-like situation of some other structures of the cerebral cortex, such as the one formed by the specific thalamo-cortical projection fibers. Acknowledgements. We are grateful to C. GabrieI-Alvarez for technical assistance, to A. Hurtado-Cano for his art work and to E. Fernandez de Molina for typing. This work has been supported by Grant PM-0012 from the DGICYT to A.R.-M, J.A.M.-C. is a fellow of the Fondo de lnvestigaciones Sanitarias de la Seguridad Social.
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49
40
41 42 43 44
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