Arch. Gerontol. Geriatr., 14 (1992) 175-182 175 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0167-4943/92/$05.00

AGG 00436

Age-related changes in sulfide-silver stainable fibres in the rat cerebral cortex

M a u r i z i o Manc in i a, Ca r lo Cava l lo t t i b, Ra f fae le M a n c i n o b,

A l b e r t o Ricc i b and F r a n c e s c o A m e n t a a

UDipartimento di Sanitfz Pubblica e Biologia Cellulare, Universit~ 'Tor Vergata' and hDipartimento di Science Cardiovascolari e Respiratorie, Universith 'La Sapienza', Rorna, hal),

(Received 30 July 1991; revised version received 28 November 1991; accepted 3 December 1991)

Summary

The present study was designed to assess the age-dependent changes in sulfide-silver stainable fibres in frontal, parietal and occipital cortex areas of the rat cerebral cortex. Male Sprague-Dawley rats of 2 months (young), 12 months (adult) and 24 months (aged) were used. Sulfide-silver stainable fibres, which represent predominantly zinc-containing associational fibres arising from local cortical interneurons, were visualized using the neo-Timm histochemical technique. The density of sulfide-silver stainable fibres within the neuropil of laminae I - I I I (upper zone) and of lamina V (lower zone) of the different cerebral cortex areas was assessed microdensitometrically. In the frontal and parietal cortices the density of sulfide-silver stainable fibres was higher in adult and in aged than in young rats both in the upper and in the lower zones. No significant differences were noticeable in the density of sulfide-silver stainable fibres between adult and old rats. In the occipital cortex the density of sulfide-silver stainable fibres was similar in young or adult rats, but was remarkably increased in old animals. The possibility that the in- crease in the density of sulfide-silver stainable fibres represents a compensatory mechanism in the cerebral cortex area showing the most consistent nerve cell loss is discussed.

Aging; Cerebral cortex; Sulfide-silver staining; Histochemistry; Microdensitometry; Rat

Introduction

Trace metals in general and zinc in particular are involved in a variety of brain functions (for a review see Frederickson, 1989). A possible functional role for zinc has been suggested in glutamatergic pathways where the metal is probably involved in the release and/or in the storage of glutamic acid (Pfeiffer, 1972; Dreosti, 1984; Frederickson, 1989). This seems to be true primarily in the hippocampus where high zinc concentrations are contained in synaptic buttons of the intrahippocampal

Correspondence to." Francesco Amenta, Dipartimento di Sanit:~ Pubblica e Biologia Cellulare, c/o Via A. Borelli, 50, 00161 Roma, Italy.

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glutamatergic pathway of mossy fibres (Crawford and Connor, 1972, 1973; Danscher, 1984; Dreosti, 1984).

Need for zinc during neurogenesis and for normal development of the central ner- vous system has been documented (for a review see Dreosti, 1984). The metal is re- quired by several enzymes involved in the process of transcription and translation (O'Dell, 1974). Abnormalities in brain morphogenesis have been shown in zinc defi- ciency including hydrocephalus and dysmorphogenetic changes in the brain, olfac- tory tract, spinal cord and eye (Hurley and Sharder, 1972; Hurley, 1974).

Little information is available concerning zinc neurobiology in aging. The occur- rence of an age-dependent loss in the availability of zinc for newly synthesized zinc metalloenzymes has been documented (Burnet, 1981). Moreover, a deficiency of zinc in the brain of Alzheimer's disease patients has been reported (Constantinidis and Tissot, 1982).

In the cerebral cortex histochemically reactive zinc pools are localized in the so called sulfide-silver stainable fibres (Haug, 1973, 1984). These fibres are predomin- antly associative and arise mainly from local cortical interneurons (Frederickson, 1989).

Since no information is yet available concerning the sensitivity to aging of cortical sulfide-silver stainable fibres, we decided to investigate the subject using a combined histochemical and microdensitometric technique. A preliminary report of the present study has been published (Amenta et al., 1991a).

Materials and Methods

Male Sprague-Dawley rats of 2 months (considered to be young), 12 months (considered to be adult) and of 24 months (considered to be aged) were used. Animals were kept under standardized humidity, temperature and lighting condi- tions (07:00-19:00 h l ight-dark cycle). They had free access to water and laboratory chow. The average lifespan of the rat colony used in our experiments was 24-26 months.

For the demonstration of histochemically reactive zinc tissue stores in the cerebral cortex a procedure detailed in earlier papers has been used (Amenta et al., 1991b; Niglio et al., 1991). Briefly, animals where anaesthetized with ether and perfused with a 0.9% NaC1 solution containing heparin and polyvinylpyrrolidone. This first solution was replaced by a second one of 0.1% Na~S. At the end of perfusion the brain was removed, the left hemisphere was dissected free, placed in a cryoprotectant solution and frozen in a dry ice-acetone mixture. Serial parasagittal (from the frontal to the occipital pole) 10-~m areas of the three age groups were performed (data not shown).

Statistical analysis of the differences between the intensity of the sulfide-silver staining in the upper and in the lower zones of the cerebral cortex areas investigated in the three age groups examined was performed by analysis of variance (ANOVA) and of covariance (ANOCOVA). For analysis of covariance the value of the density of the white matter of corpus catlosum which represents an area without positive sulfide-silver reaction was used as covariate. The data obtained in different cortical areas of single animals were tested as repeated values of dependent variables. A pro- bability at the 0.5 level was considered to be significant.

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Results

Sections processed for the demons t ra t ion o f sulfide-silver fibres developed a dark- brown staining in the cerebral cortex. A heavy staining is noticeable in two zones extended cont inuously f rom the frontal to the occipital pole. The upper zone in- cludes the neuropil o f the layers I - I I I o f the neocortex (Figs. 1-3). The lower zone includes the neuropil o f the Vth layer o f the neocortex in frontal (Fig. 1) and parietal (Fig. 2) cortices. In the occipital (Fig. 3) cortex the lower zone includes the neuropil o f layers V and VI (Fig. 3).

The values of the density of sulfide-silver staining in the neuropil o f the upper and lower zones in the three cerebral cortical areas investigated of the left hemisphere are summarized in Table I. As can be seen, no significant differences in the density of sulfide-silver staining were noticeable in 2-month-old animals between frontal, parietal and occipital cortex (Table I). In adult and aged rats the density of sulfide-

Fig. 1. Sections of rat left frontal cortex stained for the histochemical detection of sulfide-silver fibers. A dark-brown staining is noticeable both in the upper (U) and in the lower (L) zone of the cerebrocortical neuropil. A, young rat; B, adult rat; C, aged rat. Note the higher intensity of sulfide-silver staining in adult

and aged in comparison with young rats. ( x 120)

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Fig. 2. Sections of rat left parietal cortex stained for the histochemical detection of sulfide-silver positive fibers. Note the development of a dark-brown staining in the upper (U) and in the lower (L) zone of the cerebrocortical neuropil. A, young rat; B, adult rat; C, aged rat. As found similarly in the frontal cortex. the density of sulfide-silver staining was higher in adult and aged in comparison with young rats. ( x 951

silver staining was significantly higher both in the upper and in the lower zones of frontal and parietal cortex in comparison with young animals (Figs. 1 and 2 and Table I).

In the occipital cortex the density of sulfide-silver staining was significantly higher in the upper and lower layers of aged than they were in either young or adult rats (Fig. 3 and Table I). In the upper zone the density of staining was higher by about 30% in comparison with adult (P < 0.001) and by about 23% (P < 0.001) in com- parison with young animals (Fig. 3 and Table I). In the lower zone the density of sulfide-silver staining was higher by about 31"/,, (P < 0.001) and 32% (P < 0.001) in comparison with adult and young rats respectively (Table I). No significant dif- ferences in the density of sulfide-silver staining were seen in upper or lower occipital layers between young and adult rats (Fig. 3 and Table I).

Discussion

The above results provide evidence that neocortical sulfide-silver stainable fibres, which represent mainly associational fibre systems arising from cortical interneurons

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Fig. 3. Sections of rat left occipital cortex stained for the histochemical detection of sulfide-silver positive fibers. These pictures show sulfide-silver staining of the upper (U) and of the lower (L) zone of cerebrocor- tical neuropil. A, young rat; B, adult rat; C, aged rat. The density of staining was similar in young and

adult animals, but it was significantly higher in aged subjects. (x 95)

(Frederickson, 1989), undergo age-dependent changes. Increasing evidence is sug- gestive of a possible role of zinc in central nervous system function. In brain zinc is contained mainly in three pools. The first one, called the vesicular zinc pool, is constituted by the zinc contained in presynaptic vesicles (Frederickson and Danscher, 1988). The role of the vesicular zinc pool is not yet understood (Frederickson and Danscher, 1988). However, it is known that this pool of zinc, which is the only pool which could be demonstrated with specific histochemical techniques such as the Timm sulfide-silver method and its modifications (Danscher, 1981; Frederickson and Danscher, 1988) is located primarily in the limbic system pathways, in cerebrocortical circuits and in corticofugal fibres (Haug, 1973; Dreosti, t984; Frederickson, 1989). The second zinc pool is represented by free ionic zinc and is likely to be present in the cytosolic fraction of neural tissue and in the interstitial fluid. It has been hypothesized that the zinc of the second pool is involved in the transduction of intercellular or intracellular signals (Frederickson, 1989). The third zinc pool, which is the largest one, is constituted by zinc bound to the zinc-

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TABLE I

Density of sulfide-silver staining in the neuropil of the cerebral cortex areas investigated in the three age groups examined

Upper zone Lower zone

Frontal eorte):

Young (n = 8) 45.68 ± 2.80 41.48 + 3.04 Adult (n = 8) 62.26 ± 4.70 a 53.98 ± 4.08 ~L Aged (n = 10) 56.30 ± 4.47 a 57.70 ± 4.98 ~t

Parietal cortex

Young (n = 8) 46.33 + 3.01 40.91 ± 3.15 Adult (n = 8) 57.06 ± 5.30 a 55.27 + 3.37 a Aged (n = 10) 52.27 + 3.65 b 49.35 m 3.24"

Occipital cortex

Young (n = 8) 49.98 + 3.01 42.38 ± 3.72 Adult (n = 8) 45.75 4- 3.97 43.01 + 4.21 Aged (n = 10) 64.91 ± 3.14 c 62.64 ± 4.31 c

The values are expressed in arbitrary units and were obtained by measuring the intensity of the sultide- silver staining in the areas FL of the frontal cortex, PAR 1 of the parietal cortex and OCI M and OC2M of the occipital cortex as described in Materials and Methods section. Each value is the mean ± S.E. of determinations per animal of different age groups. ap < 0.001 vs, young. hp < 0.05 vs. young. Cp < 0.001 vs. young or adult.

c o n t a i n i n g e n z y m e s p r e s e n t in t he b r a i n (Pfe i f fer , 1972: Dreos t i , 1984: F r e d e r i c k s o n ,

1989).

A s m e n t i o n e d in the I n t r o d u c t i o n , su l f ide - s i lve r s t a i n e d f ibres o f the f o r e b r a i n

n e u r o p i l r e p r e s e n t p r i m a r i l y a s s o c i a t i o n a l f ib res o r i g i n a t i n g f r o m c e r e b r o c o r t i c a l

z i n c - c o n t a i n i n g o r c h e m o s e n s i t i v e n e u r o n s ( F r e d e r i c k s o n , 1989). It h a s b e e n d e m -

o n s t r a t e d t h a t in the c e r e b r a l c o r t e x the d e n s i t y o f su l f ide - s i lve r s t a i n i n g ref lec ts the

d e n s i t y o f z i n c - c o n t a i n i n g s y n a p t i c b u t t o n s ( H a u g , 1973, 1974; M a r t i n e z - G u i j a r r o et

al., 1987). H e n c e , o n t he bas i s o f o u r resul t s , we c o u l d h y p o t h e s i z e t h a t in the f r o n t a l

a n d in t he p a r i e t a l co r t i c a l a r e a s i n v e s t i g a t e d a h i g h e r d e n s i t y o f z i n c - c o n t a i n i n g

s y n a p t i c b u t t o n s o c c u r s in a d u l t as c o m p a r e d to y o u n g sub jec t s . O u r f i n d i n g s a re

c o n s i s t e n t w i t h the v iew t h a t t he m a t u r a t i o n p r o c e s s o f s o m e ra t b r a i n a r e a s m a y n o t

be c o m p l e t e u n t i l 1 y e a r o f age (see N a p o l e o n e et al. , 1990). T h e i n c r e a s e d d e n s i t y

o f su l f ide - s i lve r s t a i n i n g in the n e u r o p i l o f f r o n t a l a n d pa r i e t a l c o r t e x o f a d u l t r a t s

l ikely r e p r e s e n t s t he e x p r e s s i o n o f i n c r e a s e d n e u r o n a l c o n n e c t i o n s in c o m p a r i s o n

w i t h y o u n g a n i m a l s , r a t h e r t h a n a c o m p e n s a t o r y m e c h a n i s m . In fact , n o d i f f e r ences

in n e r v e cell d e n s i t y w e r e a p p a r e n t o n a q u a l i t a t i v e bas i s b e t w e e n y o u n g a n d a d u l t

m a l e S p r a g u e - D a w l e y r a t s in the t w o a r e a s i n v e s t i g a t e d w i th su l f ide - s i lve r

m i c r o d e n s i t o m e t r y ( N a p o l e o n e et al. , 1990). In aged ra t s in w h i c h the d e n s i t y o f

181

nerve cells in FL and PAR 1 areas is slightly reduced in comparison with adult animals (Napoleone et al., 1990), the density of sulfide-silver staining is not significantly reduced in comparison with adult rats. This suggests that the parameter investigated, after the maturational changes observed in adult subjects, is no longer sensitive to aging.

Different results were obtained in the two occipital cortex areas examined, which are the primary (OC1M) and the accessory (OC2L) visual cortex areas (Zilles and Wree, 1985). In these cortical regions the density of sulfide-silver positive fibres was unchanged between young and adult rats, but it was significantly increased in aged rats. The density of nerve cells in the two occipital areas was more remarkably reduc- ed in aged Sprague-Dawley rats than in the other cerebral regions investigated in the present study (Napoleone et al., 1990). It cannot be excluded that the increased density of sulfide-silver stainable fibres observed in the occipital cortex may repre- sent a compensatory phenomenon to counter the loss of nerve cells occurring with age in this part of the cerebral cortex. Further studies are necessary to evaluate whether changes in sulfide-silver stainable fibres may occur in the frontal or parietal cortices of rats older than 24 months.

In summary, we have shown that sulfide-silver stainable fibres, which represent a developed associative system in the cerebral cortex (Haug, 1973, 1984; Martinez- Guijarro et al., 1987), undergo age-dependent changes. These changes affect the cerebral cortex areas investigated to a different extent. The functional significance of the different sensitivity to aging of the various cortical areas should be in- vestigated in future studies.

Acknowledgements

The present study was supported in part by grant from Italian National Research (C.N.R., progetto finalizzato invecchiamento). The kind help of Dr. W.L. Collier in the revision of the manuscript and the expert secretarial assistance of Miss P. Capuano are gratefully acknowledged.

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