Age-related morphological changes in the hippocampus in two mouse strains

medwisms

of ageing

and davhpment Mechanisms

ELSEVIER

of Ageing

and Development

87 (1996) 155

Age-related

164

morphological changes in the hippocampus two mouse strains

in

Abstract The granule cell number (nGR) in the dentate gyms (DG) has been reported to vaq proof of some geneticall) considerably among inbred strains of mice, thus providing associated components to this variation. Furthermore, several authors have described age-related morphological changes in the DG in both humans and animals. but there is no general agreement in the literature about the occurrence of such changes. The purpose of this study was to investigate for strain differences in hippocampal structure changes in old C57BLj6J (B) and DBA/2J (D) mice as compared with younger ones. The nGR in the DG. as well as other structural parameters of the hippocampus. were determined in female B and D mice of 4 and 24 months. The two-way analysis of variance indicated a significant interaction between ‘strain’ and ‘age’ for the nGR. suggesting that this parameter changes differently with age in B and D mice. This finding indicates that these strains could present a differential susceptibility in granule cell aging raising the possibility that age effects on the granule cell population in the DG could be influenced by some hereditary factors. K~~~orr/.s: Aging; Granule

cells; Hippocampus;

Inbred

mice

I. Introduction Many brains

histopathological in old age and

changes in some

human

have

been

described

neurodegenerative

in human diseases

and

of aging.

animal These

*Corresponding author: Laboratoire de Genttique mokculaire de la Neurotransmission et des Processus NeurodCgtnPratifs (LGN). UMR 9923 CNRS. Bat. CERVI Hbpital de la Piti SalpPtriPre. 83 Bd de I’HGpital. 75013 Paris. France. Fa: + 33 I 42177533. 0047.6374’90/%15.00 P/I

(C 1996 Elsccier

s0047-6373(95)01702-2

Science

Ireland

Ltd. All rights

rrrerced

age-dependent alterations of the brain tissue include selective cell loss [l-7], decreased dendritic or synaptic density [&lO]. and the occurrence of specific histological brain lesions such as senile plaques, neurofibrillary tangles and granulovacuolar degenerations [l 1~ 131. When affecting the hippocampal formation, these age-related changes are considered to be involved in the memory disturbances common to old age. Indeed, an accumulating body of evidence shows that the hippocampus is critical to learning and memory in many mammalian species including humans [ 14 - 161. Furthermore, the cognitive changes generally occurring with age are similar to those observed after hippocampal damage [17719]. The cognitive capacity, however, of aged humans or animals varies markedly as some aged individuals present a severe age-related impairment while others fail to exhibit any memory disturbance [20,21]. Distinct neuroanatomical parameters in the hippocampus, supposed to be related to memory performance, might therefore be differentially affected by the aging process. Also, in animals the aging process exhibits a large strain dependency [22-251. In particular, it seems that differences exist between rodent strains as to whether, or how, some particular parameters of brain structure change with age [26,27]. Such individual or strain differences in aging would emphasize the possible role of genetic factors in the neurobehavioral sensitivity to aging. A comparative study of age-related hippocampal changes carried out in different inbred mouse strains therefore constitutes an experimental approach to examine the genetic basis which underlies individual differences in the neurobehavioral response to aging. Many authors have described age-related changes in the morphology of the dentate gyrus (DG) both in humans and animals. However, there is no general agreement in the literature about the extent of such age-related changes. Indeed, a number of morphological changes have been described: granule cell loss [2,6,28,29] or stability [30-321 accompanied by dendritic or synaptic growth [33,34] regression [31l36] or stability [2&37] in the molecular layer. Methodological artifacts (tissue processing, counting techniques, etc.) are often evoked to explain the uncertainties highlighted by these studies [38,39], but individual, species, or strain-related differences in aging could further explain these discrepancies. The purpose of this study is, therefore, to attempt to evaluate in the mouse the strain influence on possible age-related changes in some structural parameters of the hippocampus. To this end, we have investigated the number of dentate granule cells and other neuroanatomical parameters in the hippocampus of young and old mice from the C57BL/6J and DBA/U inbred strains. The dentate granule neurons are of particular interest because of their likely involvement in memory and especially in the processing of spatial information [40P421.

2. Materials

and methods

2.1. Anirnuls Twenty-two

female C57BLj6J

and DBA/2J

mice purchased

from IFFA-CREDO

(France) were used in this study. Animals were maintained in our animal house 22 + of the (12:‘12 h light-dark cycle, temperature _ 2°C) until the beginning experimentation (about 2 weeks after receipt). Half of the animals, considered to be ‘young’, were 4 months old (5 C57BL;6J, 6 DBA/2J); the other half, 24 months of age. were classified as ‘old’ (6 C56BL;6J, 5 DBA/2J).

Animals were anaethetized with an i.p. injection of pentobarbital and perfused transcardially with a first PBS solution followed by a second PBS solution containing 0.2% paraformaldehyde. Brains were then quickly removed and frozen in cooled isopentane. Serial 10 jlrn thick horizontal brain sections were cut on a motorized cryostat at the mid-septotemporal level of the hippocampus and thawed on uncoated slides. The sections were then placed for 10 min in a post-fixative PBS solution containing 3.7% paraformaldehyde. At the end of the post-fixation, the sections were washed and stained with a 0.01% aqueous solution of toluidine blue containing 0.01% di-sodium tetraborate and adjusted to pH 9 [43]. Finally, the sections were gradually dehydrated in ethanol and xylene, and mounted with Eukit under glass coverslips.

2.3. Morpllomrtry

Three horizontal sections from the left hippocampus were selected per animal and examined by quantitative image analysis (SAMBA image analyzer, Alcatel TITN). The sections were 40 pm apart, the first one corresponding to the horizontal plane located at 400 pm under the septal pole. Eight morphometrical parameters were determined in the hippocampus with the image analyzer connected via a TV camera to the microscope (Zeiss). The areas of the whole hippocampus (HPC) (hippocampus proper plus DC), the molecular and granular layers of the DG (respectively MOL and CR), the hilus of the fascia dentata (CA4) and the stratum pyramidale (SP), were delimited at x 78 magnification (Fig. 1A). The means of the relative areas, expressed as percentages of the whole hippocampus area to correct for unequal absolute sizes [44], were calculated for each animal. The dentate granule cells were counted at a final magnification of x 625. The cell density estimates (dGR) were obtained directly by counting the neuronal nuclei in three well-defined areas of the stratum granulosum. The final granule cell number (nGR) was then estimated by averaging the three estimates of density and multiplying the result by the granule cell layer area. The hilar cells were counted in the entire CA4 area at a x 625 magnification (nCA4).

2.4. Statistical nnalysis The results were analysed by a two-way analysis of variance (ANOVA), the main factors being ‘strain’ (C57BL/6J, DBA/2J) and ‘age’ (young, aged). Partial analyses were performed by means of the Student’s t-test.

Fig. 1. (A) Diagram of a cross-section of the mouse hippocampus (MOL. CR, granular layer of the DG; CA4, hilus of the fascia dentata: SP, stratum cross-section from the mid-septotemporal level of the mouse hippocampus

molecular layer of the DC;; pyramidale). (B) Horizontal stained with toluidine blue.

Table I Mean k SEM OFhippocampal variables measured in young and DBAZJ mace, and results of the statistical analysis Variable

DBA>ZJ

young 1,) = 5)

dGR

1878 73 5.6 5.x 7.5 47 964 91x3

Aged (II = 6)

* 5x * 1.2 F 0. I * 0.4 & 0.3 + 4 * 53 * 191

C57BL’hJ

young (I/ = 6)

Strain

Age

Strain x Age

P<0.0001 ns

ns ns ns

Aged (n = 5)

1591 f 21 23.4 _+ 0.4 5 f 0.3 9.1* 0.3 7.6 & 0.3 35 f 2

1677i 22.7 _+ 5.6 f 9.4 * 6.7 f 40 *

55 0.5 0.2 0.3 0.6 2

1437 * 2Y 23 * 0.9 5.8 + 0.4 8.7 kO.4 6.4 & 0.2 30* I

P
684 f 26 859Y + 338

808 * 49 5512 * 240

741*23 8989 _t 540

ns ns

HPC. whole hippocampus cell density: t&R, granular ns. non-significant results. HPC. dGR IS expressed in

3. Experimental

and aged (24 months)

ANOVA

Strain C57BL:6J

HPC MOL GR SP CA4 nCA4 nGR

(4 months)

ns ns ns P
nr 11s ns P
area; MOL. stratum moleculare; CR. stratum granulosum: dGR, granular cell number; CA4. hilus; nCA4. hilar cell number: SP. stratum pyramidale: HPC is expressed in x IO’ Atm’. The other values presented are relatives to number of cells:mm’.

results

The mean values of the different morphometrical parameters, their standard errors of the mean, and the ANOVA results are summarized in Table 1. The area of the HPC was significantly larger in strain C57BL/6J as compared with strain DBA/2J. None of the other areas (MOL, GR, CA4, SP) significantly differed between strains. Furthermore, the hilar cell number (nCA4) was significantly higher in strain C57BL/6J, while no global strain effect was noticeable for the dentate nGR or dGR. A significant age effect was found for the HPC area as well as for the nCA4 and nGR when comparing aged versus young of both species. The mean values of these neuroanatomical parameters were always reduced with age. In contrast, no significant age-related difference was found either for the dGR or the relative sizes of the measured areas. Interestingly. a significant interaction between strain and age appeared for the dentate nGR. This interaction was highlighted by partial analyses showing an agerelated cell number decrease in the C57BLj6J strain (T= 5.04; ddl = 9: P < 0.001). without significative difference between age groups in the DBA;2J strain.

160

M. Barkars

rt al. I/)Mechanisms

of’ Age&

and Decelopmmi

87 (19%)

155. 164

4. Discussion Many studies have focused on age-related morphological changes in the rodent hippocampus. For several reasons, including methodological artifacts and individual or strain differences, these studies have often led to contradictory results. In this study, age-related histological changes have been examined in the hippocampus using two different mouse strains in order to test for possible strain-differences in hippocampal aging. 4.1. Strain dij+rences Our results indicate substantial strain differences in the size of HPC with it being larger in the strain C57BLj6J than in the strain DBA:‘2J. This finding is in agreement with several previous studies concerning the hippocampal regio irzftrior [45,46]. These differences could be compared to the marked differences in the learning performance exhibited by the two inbred strains (C57BLj6J showing better performances) [47,48]. Furthermore, strain differences in the dentate nGR profiles were observed in the young animals. Our finding of strain variability in nGR agrees with previous studies reporting a genetically-related strain variability in the dentate nGR in several inbred mouse strains [49,50]. For Wimer and Wimer [50]. 80 to 90% of the observed variation could be attributed to genetic factors (genes located upon autosomes). the remaining variation depending on ‘environmental’ sources. Similarly, strain differences were observed for the nCA4 but, to our knowledge. no experimental result is available for comparison. As with nGR, the strain-related variability of nCA4 could possess some genetically associated components. 4.2. Age-related

clamges

Global age-related changes in the HPC were further demonstrated by the two-way analysis of variance, this area being affected by age in both strains. Our results also indicate a global age-related decrease in the nGR at the mid-septotemporal level of the hippocampus without global reduction in the nerve cell density (dGR). Our findings of an age-dependent decline of granule cell profiles in the mouse hippocampus are in agreement with other studies showing a loss of this cell type in aged Sprague-Dawley rats [6,7,51]. In contrast, other studies indicate no loss of granule cells [32] or change of the granule cell volume with aging in the DG of F344 rats [52]. The reasons for these discrepancies may be related to a variety of different factors such as differences in counting techniques and tissue preparation, or in the strain of the animals used [53]. Our results argue for the latter hypothesis as the age-dependent decline in the nGR was not the same in the two strains used in this study. This remarkable difference in the effects of age between the two strains was demonstrated by the occurrence of a significant nGR reduction with age in the C57BL/6J mice, whereas this age-related decrease did not reach significance in the DBA/2J mice.

This analysis raises the possibility of a differential susceptibility to age-related cell death in the DG of the two strains. However, these results must be viewed with caution. Indeed, it has been demonstrated that some histological methods can lead to differential shrinkage of young or old brains [5]. However, our results indicate no significant interaction between strain and age for either the whole HPC surface area. or the GR area which seems to exclude strain differences in shrinkage in this study. The age-related decrease in the nGR could be related to the reduction with age of the mossy fiber synaptic fields observed in rats [54,55] and in inbred mice (Bertholet et al., unpublished data). Hypoplasia of the granule cell soma is also known to involve the regression of the mossy fiber innervation in the CA3 area [41,56]. Our present results can therefore be related to previously observed strain differences in C57BL/6J and DBAi2J aging for the size of the intra- and infrapyramidal mossy fiber (iipMF) synaptic fields in CA3 (dentate granule cell projections) (unpublished data). On the other hand, Boss et al., [.57]. demonstrated the occurrence of strain-differences in the effects of age on the total nGR in the Sprague-Dawley and Wistar rat strains. According to these authors, strain differences may be observed not only in the initial number of postnatally generated granule cells, but also in the generation and/or the survival of these cells during the first year of life. Finally, our present study also revealed a subtantial loss of neurons in the hilus of the gyrus dentatus. This result is in agreement with data presented by West which provided evidence for an age-related decline in the total number of neurons in the hilus of human DG [39]. According to West, the reduction in the number of these neurons might reduce the associative capacity of the dentate granule cells and compromise the processing of information within the hippocampal formation. The present observed age-related loss of neurons in the hippocampus could result in deficits in performance in various memory tasks. Further studies will be helpful to evaluate the correlations between these specific parameters in the hippocampus and performance in memory tasks. A major interest of this study is the demonstration of a differential sensibility to aging of the dentate granule cells in the C57BL;‘6J and DBA/2J mouse strains. The mechanisms underlying this strain-related differential susceptibility to aging have yet to be elucidated. These different patterns of granule cells aging may be influenced by genetic factors and/or environmental ones. With respect to the free radical theory of aging [58], a differential hippocampal aging could be related to possible interstrain differences in the effects of age on the protective systems against the free radicals toxicity. Such differences have already been described in mouse strains for age-related changes of catalase activity which appear to be dependent upon genotype [59]. Such variations in antioxidant levels or activities could have repercussions for the mossy fiber system (including the dentate granule cells) which seems to be particularly vulnerable to free radical-mediated oxidative damage [60.61]. This hypothesis could be tested by further genetical correlative analyses between antioxidant levels and hippocampal morphometry in different inbred mouse strains with aging.

Acknowledgements

The authors thank Mrs Catherine Marchaland and Mr. Fernando Perez-Diaz for their expert contribution to the statistical analysis. This research was supported by CNRS and Universitk Paris V.

References [I] M.J. Ball, Neuronal loss, ncurofibrillary tangles and granulovacuolar degeneration in the hippocampus with ageing and dementia: a quantitative study. .dc,ttrNwroprrth. (Bedirt). -37 ( 1977) III 118. [2] A. Mouritzen Dam, The density of neurons in the human hippocampus. flVr~r~~~p&~~~/. .dppl. .Veurohiol., 5 11979) 2499264. [3] P.W. Landfield. R.K. Baskin and T.A. Pitler. Brain aging correlates: retardation by hormonal-pharmacological treatments. Sciow. 114 ( I98I ) 58 I 584. [J] J.M. Anderson, B.M. Hubbard. G.R. Coghill and W. Slidders. The etfect of the advanced old age on the neurone content of the cerebral cortex. Observation with an automatic image analyser point counting method. J. IVeuroi. Sci.. .% (1983) 2333244. [S] P.D. Coleman and D.G. Flood. Neuron numbers and dendritic extent in normal aging and Alzheimer’s disease. :Veurohir,l. Ay@. 8 (1987) 521 545. [6] P. Napoleone. F. Ferrante. 0. Ghirardi. M.T. Ramacci and F. Amenta. Age-dependant nerve cell loss in the brain of long-term acetyl-L-carnitine treatment. A&r. G~~r~~rrol. Gcrrtrrj... /0 (1990) 173-185. [7] E. Bronzetti, W.L. Collier. L. Felici. D. Zacchco and F. Amenta, Long-term choline alfoscerate treatment counteracts age-dependent structural changes in the rat hippocampus. Drlr$ Dec.. Rm.. 26 (I 992) 449 -459. [8] A.B. Schiebel and U. Tomiyasu. Dendritic sprouting in Alzheimer’s pre-senile dementia. EXIT. Nrzrrol., 60 (1978) l-8. [9] Y. Geinisman. L. De Toledo-Morrell and F. Morrelf. Loss of perforated synapses in the dentate gyrus: Morphological substrate of memory deficit in aged rats. Proc.. V[lr/. .Accltl. %I. (;SA, S3 (1986) 3027 3031. [IO] Y. Geinisman. L. DeToledo-Morrell, F. MorreJJ. I.S. Persina and M. Rossi. Age-related loss of axospinous synapses formed by two afferent systems in the rat dentate gyrus as revealed by the unbiased stereological dissector technique. Hippmuym~~, 2 ( 1992) 437 444. [I I] B.E. Tomlinson, G. Blessed and M. Roth, Observation on the brains of non-demented old people. J. Nerrrol. Sci.. 7 (1968) 331-356. [I21 A.N. Davison, New concepts in the pathophysiology of Alzheimer‘s disease. In M. Briley. A. Kato and M. Weber (eds.). >‘v”enCo,~cept.s in Al-_lwirner :v Dixwr. Vol. I. Macmillan. London. 1986. pp. l-16. [I31 P. Delaere, C. Duyckaerts and J.-J. Hauw, Aspects morphologiques du vieillissement cerebral. In ‘I’. Lamour (ed.). Lr I’irillissrrnrrzt C’e%md. Presses Universitaires de France. Paris. 1990. pp. 61 -88. [I41 J. O’Keefe and L. Nadel. T/w hippoc~mpr.~ tu a oq’nctive rmrp. Oxlhrd University Press, London. 1978. 1151 D.S. Olton. J.T. Becker and G.E. Handelmann. Hippocampus. space. and memory. Bdu~. Brch Sci.. -7 (1979) 313365. 1161 S. Zota~Morgan, L.R. Squire and D.G. Amaral. Human amnesia and the medial temporal region: Enduring memory impairment following a bilateral lesion limited to the CA I field of the hippocampus. J. Xeurosci.. 6 (1986) 2950 2967. [I71 R.G.M. Morris. P. Garrud. J.N.P. Rawlins and J. O’Keefe. Place navigation impaired in rats with hippocampal lesions. jliclrurr. .?Y7 ( 1982) 68 l-683.

[IX] P.W. Landfield, T.A. Pitler and M.D. Applegatc. The aged hippocampus, a model system for studies on mechanisms of behavioral plasticity and brain aging. In R.L. Isaacson and K.H. Pribram (cds.). r/lc Wippo~~arq~r.v. Vol. 3. Plenum. New York. 1986. pp. 321-367. 1191 M.L. Smith. Recall of spatial location by the amnesic patient H.M. Brrrir~ C‘O~~~I.. 7(198X) 17X IX?. [20] A.L. Markowska. W.S. Stone. D.K. Ingram. J. Reynolds, P.E. Gold. L.H. Conti. M.J. Pontecorco. G.L. Wrnk and D.S. Elton. Individual differences in agins: Behavioral and neurobiologic;rl correlates. :Vn/rohi0/. A,cing. /0 (1989)3 l-13. (II] P.R. Rapp and D.G. Amaral. Individual differences in the cognitive and neurobiological conscquences of normal agin. TIVS, IS (1991) 340 -345. 1221 J.T. Willott. Efects 01‘ aging. hearing loss. and anatomical location on thresholds of int’eriol. colliculus neurons in C57BL,6 and CBA mice. J. .Veu~~~/,lf~,.\io/...% (1986) 391-408. [23] A. Ebrl. M.T. Strosser and E. Kempf. Genotypic ditTerences in central neurotransmitter response\ to aging in mice. :Vr~roh/ol. .-lgirrg. X ( 1987) 417 327. [24] D.K. Ingram and M.A. Reynolds. The relationship of hod) Lveight to longevity within laborator! rodent species. Btrsic, Lift .%i.. 32 ( 1987) 247 282. [I!51 R.L. Sprott and C..4. Combs. Genetic aspects of aging in .1/~,.\ rr~~is~~~lu.s. Ret,. Bicd Rr.5. .-l,rinc. -/ ( 1990) .73- 80. J.W. Hinds and N.A. McNelly. Aging of the rat olfactor! gstem: Correlation of changes in the olfactor) epithelium and olfactory bulb. J. C’o,~p. Ve~r.01.. 20.: I I YXI1 441 -453. 1’71 B. Boss. G.M. Peterson and W.M. Cowan. On the number of neurons in the dentate pyrus of the rat. Bruirr Rex.. 3.18 (1985) 144 150. [7X] C.A. Curcio and J.W. Hinds, Stability of synaptic denhity and spine volume in dentate syrus 01 aged rats. !V~z(r~hio/. .lg/q. 3 (19X3) 77 X7. [2Y] A. RICCI. E. Bronzetti. J.A. Vega and F. Amenta, Oral choline althsccrate counteracts age-depeindent loss of mossy fibres 111 the rat hippocampus. \fr&. .4:e. De.. 66 (1992) Sl 91. [.?()I W. BonJareK and Y. Geinl. V~rol.. 53 (lY76) 420 430. atrophy in the dentate gyruc of the [32] Y. Geinisman. W. Bondaretr and J.T. Dodge. Dendritic senescent rat. .4r,1. J. .-lr~r.. li2 (1’278) 321 330. [37] D G. Flood. S.J. Buell. C.H. DeFiorr. G.J. Hoi-witz and P.11. Coleman. Age-related dendritic growth in dentate gyrus of humanhrain is foiiowcd by regression in the ‘oldest old’. &c/in RN.. .%A ( 1985) -366 368. [34] D.G. Flood. S.J. Buell. G.J. tlorwitz and P D. Coleman. Dendrltic extent in human dentate gyru, granule cells in normal aging and senile dementia. Bruin Rc.x.. 402 (lY87) 205 216 1351 Y. Geinisman and W. BondaretT. Decrease in the number ol‘ synapses in the senescent brain: a quantitative electron microscopic analysis of the dentate gyrus molecular layer in the rat. .tfc&. .4g[, D,T.. 5 (1976) 1I 23. 1361J.R. Mc William:. and G. Lynch. Synaptic density and axonal sprouting 111 rat hippocampus: stability in adulthood and decline In late adulthood. Bruir~ RCA.. 29-l ( 1984) l52- 156. 1.371 C.W. Cotman and S.W. Scheff, Compensatory sgnapse cgTroc\th in aged animals after neuronal death. i\lr< /I. ilge rl~.. 0 (I Y79) I03 I IS. [M] P.W. Landfield. 0. Thibault, M.L. Mazzanti. N.M. Porter and D.S. Kerr. Mechanisms of ncuronal death in brain aging and Alzheimer’s discrise: role of endocrille-inediiited calcium dyshomet,stasiz. ./. ,Vc,1r,r&icr/.. 2.1 ( 1992) I147 I xx. [?9] M.J. West. Regionall) specific Iosb of neurons in the aging human hippocampus. ,I~c~rr~hio/. dvi/,p. IJ (199:;) 287 793. /30] C‘. Wimcr. R.E. Wimer ,nd J.S. Wimcr. An association between granule cell density in the dentate gyrus a,ld two-way avoidance conditionninz in the house mouse. Bd~cri-. vrfr/-cJ.\~~i.. 9’ ( 198.7) X44 X561. 1411 G.A. Mickley. J.L. Fcrguson and T.J. Nemeth. Serial injection< of MK 801 (Dizocilpine) in neonatal rats reduce behaciordl defcita assc,ciated with X-rays-Induced hippocampal granule cell hypoplasia. P/fcr~?~~o/. BuI(&,~I. Bdm.. -1.7 ( 1991) 785 7Y3. [16]

[42] Z.J. Sienkiewicz. R.D. Saunders and B.K. Butland. Prenatal irradiation and spatial memory in mice: investigation of critical period. Im. J. Rdiut. Bid.. 62 (1992) 21 lP219. [43] P.B. Bradley. hfet/ro& ijt Brclbl Resrtrrch, Wiley. London. 1975. [44] H. Schwegler and H.-P. Lipp. Hereditary covariations of neuronal circuitry and behavior: correlations between the proportions of hippocampal synaptic fields in the regio inferior and two-way avoidance in mice and rats. Bd~ur-. Bruin Res.. 7 11983) l-38. [45] H.P. Lipp and H. Schwegler, Hippocampal mossy fibers and avoidance learning. In I. Lieblich ted.). Grrwrics of r/w Brcrin. Elsevier, Amsterdam, 1983, pp. 3266364. [46] W.E. Crusio. G. Genthner-Grimm and H. Schwegler. A quantitative-genetic analysis of hippocampal variation in the mouse. J. ~Veuro~m.. .i (1986) 203~214. [47] C. Rossi-Arnaud. S. Fagioli and M. Ammassari-Teule. Spatial learning in two inbred strains of mice: genotype-dependent effect of amygdaloid and hippocampal lesions. Behtrr. Brrrirt Raps.. 35 (1991) 9-- 16. [48] D.E. Fordyce and J.M. Wehner, Physical activity enhances spatial learning performance with an associated alteration in hippocampal protein kinasr C activity in C57BL:6 and DBA.2 mice. Brcrh Res.. 619tlY93) 111~119. [49] R.E. Wimcr, C.C. Wimer. J.E. Vaughn. R.P. Barber. B.A. Balvanz and C.R. Chernow. The genetic organization of neuron number in the granule cell layer of the area drntata in house mise. Bruk Ra.. ii7 (1978) 105~122. [St)] R.E. Wtmer and C.C. Wimer. A biometrical-genetic analysis of granule cell number in the area dentata of house mice. DPP. Bruirr Rr,\., 2 (1982) I79 140. [Sl] E. Bronzetti, L. Felici. D. Zaccheo and F. Amenta. Age-related anatomical changes in the rat hippocampus: retardation by choline alfoscerate treatment. ;frh. Gw~u~d. G~v%r~r.. 1.7 i I991 ) I67 17s. [52] P.D. Coleman, D.G. Flood and M.J. West. Volumes of the components of the hippocampus in the aging F344 rat. ./. Co,,~p. 4’e~rol.. 366 (1987) 300~306. [53] S.W. Schetf. K.J. Anderson and S.T. DeKosky. Strain comparison of synaptic density in hippocampal CAI of aged rats. ~Ve~,ohi~l. .1@qe. 6 (1985) 29- 34. [54] W.G.M. Raaijmakers. F.J. Van der Staay. T.H. Collijn and J.G. Veening, Hippocampal morphometry in the rat: age-related changes and modulation by chronic dietary choline enrichment, In A. Fisher, I. Hanin and C. Lachman (eds.). .4/sheir,1rr :s umf P~rX/rrso/r :\ Di.w~rs: S~rutegies to, RCSL’LII.C/Iund Drrchpnwu, Vol. 29. Plenum. New York. 1986. pp. hII9 -hIi. [55] A. Ricci. M.T. Ramacci. 0. Ghirardi and F. Amenta. Age-related changes of the mossy fibre system in rat hippocampus: effect of long term actyl-L-carnitinc treatment. .-lrc+. Ger~~r~rol. Gerrrrrr.. 8 (1989) 63 -71. [56] J.L. Gaiarsa, M. Beaudoin and Y. Ben-Ari, Etfect of neonatal degranulation on the morphological development of rat CA3 pyramidal neurons: inductive role of mossy fibers on the formation of thorny excrescences. J. Corqj. h’rnrul., 321 (1992) 613 625. [57] C.C. Wimer and R.E. Wimer. On the sources of strain and sex dtlfcrences in granule cell number in the dentate area of house mice. Drr,. Bruit RN.. 38 (1989) I67 176. [58] D. Harman. Free radical theory of aging. h4irftr/. Rex.. 275 (1992) 757 266. [59] N.J. Schisler and S.M. Singh. Inheritance and expression of tissue-specific catalase activity during development and aging in mice. Germrue. -79 (1987) 7488760. [60] M. Barkats. J.-Y. Bertholet. P. Venault. I. Ceballos-Picot, A. Nicole, J. Phillips. R. Moutier. P. Roubertoux. P.-M. Sinet and C. Cohen-Salmon, Hippocampal mossy fiber changes in mice transgenic for the human copper-zinc superoxide dismutase gene. ,Yerro.wi. Lrrt.. 160 ( 1993) 24 28. [6l] M. Barkats. P. Venault, Y. Chrtsten and C. Cohen-Salmon. Etfect of long-term EGb761 treatment on age-dependent structut-al changes in the hippocampus of three inbred mouse strains. L!lc, SU.. Ch (1994) 113

222.