Age-related changes in sulfide-silver staining in the rat neostriatum: A quantitative histochemical study

NeurobiologyofAging,Vol. 13, pp. 501-504, 1992

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Age-Related Changes in Sulfide-Silver Staining in the Rat Neostriatum: A Quantitative Histochemical Study MAURIZIO MANCINI, ALBERTO RICCI* AND FRANCESCO AMENTA l

Dipartimento di Sanitd Pubblica e Biologia Cellulare, Universitdt Tor Vergata and *Dipartimento di Scienze Cardiovascolari e Respiratorie, Universitd "La Sapienza, " R o m a Italy R e c e i v e d 2 A u g u s t 1991; A c c e p t e d 10 J a n u a r y 1992 MANCINI, M., A. RICCI AND F. AMENTA. Age-relatedchanges in sulfide-silver staining in the rat neostriatum:A quantitative histochemical study. NEUROBIOL AGING 13(4) 501-504, 1992.--The density and distribution of sulfide-silver staining in the neostriatum of 3-, 12-, and 24-month-old male Sprague-Dawley rats were analyzed using the neo-Timm sulfide-silver histochemicai technique associated with microdensitometry. This technique stains zinc-containing terminals in the striatum and the density of neo-Timm staining is considered to be parallel to the density of synaptic boutons containing zinc. In the neostriatum sulfide-silver, staining was intense in the matrix, although the striosomes did not show appreciable reactivity. The density of sulfide-silver staining was significantly reduced (p < 0.001) in the matrix of 12-month-old in comparison to 3-monthold rats. No further changes were noticeable between 24- and 12-month-old rats. In contrast, the area and the perimeter of neostriatum that were assessed by quantitative image analysis did not show age-related changes. The present results indicated that similar to the observations for a variety of neurochemical parameters of rat neostriatum such as local cerebral glucose utilization, cholinergic muscarinic receptors, and dopamine D-1 receptors, zinc-containing striatal terminal were primarily decreased between young and adult subjects but not between adult and aged animals. Neostriatum

Zinc

Matrix

Striosomes

Aging

S T R I A T A L matrix, like much of the neuropil in the telencephalon, stains intensely for zinc (6,8). The presence of zinc in the striatum has been demonstrated in several mammals and in lizards (4,8,13), primarily using histochemical techniques for the detection of zinc and heavy metals such as the T i m m ' s technique (4,8) as well as the modification of this technique allowing the selective demonstration of zinc tissue stores (5,6). In the striatum, zinc has been demonstrated in the caudateputamen complex (neostriatum) but not in the globus pallidus paleostriatum; for a review see (6). Moreover, it has been shown that most o f neostriatal zinc is contained in synaptic boutons (6). As to the functional role of zinc in the neostriatum, the existence of a zinc-containing cortico-striatal projection has been demonstrated (14). Moreover, a possible association between zinc-containing terminals and metenkephalin-, substance P- and dynorphin B-immunoreactive terminals has been suggested in the marginal division of the rat striatum (16). The marginal division is a disc-shaped portion of the neostriatum which is located postero-medially and surrounds the anterolateral edge of the globus pallidus (17). The occurrence o f developmental changes in the pattern of

Microdensitometry

distribution of zinc and heavy metals has been reported (19). However, no information is available so far, to our knowledge, regarding possible age-dependent changes in the density and pattern of zinc-containing fibers and/or terminals in the neostriatum. In view of this, we decided to investigate whether the intensity of sulfide-silver staining undergoes age-dependent changes in the rat neostriatum. Moreover, to assess whether age-related changes in the density of sulfide-silver staining were dependent on modifications in size of the striatum, both its area and perimeter were assessed by quantitative image analysis. METHOD Male Sprague-Dawley rats of 3 months of age (n = 8 - - c o n sidered to be young), 12 months of age (n = 10--considered to be adult) and 24 months of age (n = 10--considered to be old) were obtained from Charles River (Calco, Italy). The animals were kept under a constant 12L: 12D (light period 07:00 a.m.-07:00 p.m.) at an ambient temperature of 22 ° _+ loC. They had free access to water and laboratory chow (4 R F 18, Italmangimi, Italy).

~Requests for reprints should be addressed to Francesco Amenta, M.D., Dipartimento di Sani~ Pubblica e, Biologia Cellulare, Via O. Raimondo, 8, 00173 Roma Italy. 501

502

MANCINI 1:71 AI..

/°°

¢ FIG. 1. Section A 8380 according to KOnig and Klippel [10] showing the arbitrary divisions of'the neostriatum (L = lateral: I = middle: M =

medial) to perform microdensitometric analysis of the density of sulfide-silver staining.

Animals were anesthetized with ether and perfused, through the left ventricle, with a 0.9% NaCI solution containing 2 mg/Kg heparin and 10% polyvinylpyrrolydone. This first solution was replaced by a second one containing 0.1% Na2S. At the end of the perfusion the brain was removed and cut in coronal frontal sections corresponding to the coordinates A 8920-A 7890 according to the atlas of K6nig and Klippel (10). The sections were placed in a cryoprotectant medium and frozen in a dry ice-acetone mixture. Serial 10 um thick sections were mounted on gelatine-coated microscope slides and developed in a solution containing arabic gum (20%), silver lactate (10%), citric acid (5%), and hydroquinone (2%). Further details on histochemical procedures are reported elsewhere (1,3,15). Sections from each of the various animal age groups were incubated together to minimize the influence of different incubation conditions on the development of the histochemical reaction. The density of sulfide-silver staining within the matrix and striosomes of the neostriatum in the rats of the three age groups was assessed microdensitometrically according to the protocol detailed in our previous articles ( 1,3,12,15). Briefly, the striaturn was divided into three portions corresponding to the medial, middle and lateral portions as shown in Fig. 1. The density of sulfide-silver staining was measured on 15 different fields of the matrix or striosomes for each of these animal portions. The instrument was calibrated taking zero as the value of microscope slide areas free of tissue. Measurements were made separately for the matrix and for the striosomes in an area of 10 um in diameter in the center of an ocular using a X 25/0.95 planapochromatic objective and a X 10 ocular. The value of the density of staining of the corpus callosum, which is a brain

portion free of positive reaction was used as a covariate. Further details on microdensitometric procedures are reported elsewhere ( 1,3,12,15). The area and the perimeter of the neostriatum in the three age groups examined were assessed in five consecutive sulfide-silver stained sections per animal corresponding to the coordinates A 8920-A 7890. Measurements were made with a Videoplan image analyzer (Kontron-Zeiss, F. R. G.) connected via a TV camera to the microscope. Statistical analysis of the differences between the intensity of sulfide-silver staining in the three portions of the neostriatum investigated in 3-, 12-, and 24-month-old rats was performed by analysis ofcovariance (ANOCOVA) using the value of intensity of staining of corpus callosum as the covariate. The data obtained in single animals were tested as repeated values of dependent variables. The values of the area and the perimeter of neostriatum were analyzed statistically by analysis of variance (ANOVA). A probability at the 0.05 level was considered to be significant. The significance between pairs of the three age groups was evaluated with the Newman-Keuls Test. RESULTS

A dense dark brown staining was developed in the neostriatum processed with the neo-Timm sulfide-silver technique. The staining was dense in the matrix of the neostriatum (Fig. 2). In contrast, no differences in the density of sulfide-silver staining were noticeable between striosomes of the neostriaturn and the white matter of the corpus callosum which is known to have no positive sulfide-silver reactivity (data not shown). In view of this, the values of the intensity of staining of striosomes were not considered for our investigation. The intensity of sulfide-silver staining of neostriatal matrix

A G I N G OF THE STRIATUM

503

FIG. 2. Three month rat: Neostriatum (middle portion). Sulfide-silver staining. S = striosomes; M = matrix. In the matrix, a dense darkbrown staining could be observed. No reactivity is noticeable in the striosomes. Calibration bar: 50 um.

FIG. 4. Twelve month rat: Neostriatum (middle portion). Sulfide-silver staining. S = striosomes; M = matrix. In the matrix, a dark brown staining could be observed. Note the decrease, in comparison with Fig. 2, of the intensity of staining of the matrix. Calibration bar: 50 um.

was similar in the three different portions examined (medial, middle, and lateral portions, see Fig. 1 and Fig. 3) as well as in the superior or inferior fields of neostriatum (data not shown) or in different sections of the head of the neostriatum (from A 8920 to A 7890) considered (data not shown). A significant decrease (p < 0.001) in the density of sulfidesilver staining was noticeable in the matrix of the neostriatum in 12 month-old in comparison to 3-month-old rats (Figs. 3 and 4). No further decrease was observed in the intensity of the sulfide-silver staining in 24-month-old rats in comparison with 12-month-old animals (Figs. 3 and 5). The age-dependent reduction in the density of sulfide-silver staining was homogeneous in the different striatal portions examined without particular change in any specific area of the neostriatum (Fig. 3). The values of the morphometric analysis of neostriatum are summarized in Table 1. As shown, the values of the area or of the perimeter (of the striatum) did not show age-dependent modifications.

DISCUSSION The present results provide direct evidence that neostriatal zinc stores undergo age-dependent changes. These changes are noticeable between young and adult animals but do not occur between adult and aged rats. In view of the parallelism between the intensity of sulfide-silver staining and the density of zinccontaining synaptic boutons in several brain areas (for a review see 6), these data allow us to hypothesize the probable occurrence of a loss of zinc-containing synaptic boutons in the neostriatum between young and adult animals. This assumption is supported by the findings that the area and the perimeter of neostriatum did not undergo age-dependent changes. Hence, the decrease in the density o f n e o - T i m m staining in the striatal matrix reflects a real loss in the density of zinc-containingsynaptic boutons rather than their dilution caused by changes in the volume of the striatal tissue. A growing body of evidence suggests a possible role of zinc

lntgr~lty of staining (arbitrary units) 60

40 30

20

10

24 months mm 12 months I~ MEDIAL

MIDDLE

3 months

LATERAL

FIG. 3. Microdensitometric evaluation of the density of sulfide-silver staining in the three portions of the neostriatum examined in 3-, 12-, and 24-month-old rats. Values are the mean _+ SE of determinations performed as described in the Method section. The intensity of staining, which is expressed in arbitrary units, reflectsthe density of sulfidesilver reaction in the matrix of the striatum of different age groups. ap < 0.001 versus 12-month or 24-month-old.

FIG. 5. Twenty-four-month-old rat. Neostriatum (middle portion). Sulfide-silverstaining. S = striosomes; M = matrix. Note the reduced density in sulfide-silverstaining of the matrix in comparison with the 3 month rat (Fig. 2) and the lack of remarkable loss of staining in the matrix in comparison with the 12 month rat (Fig. 4). Calibration bar: 50 um.

MANCIN1 ET AL.

504 TABLE 1 MORPHOMETRYOF THE NEOSTRIATUMIN THE THREEAGE GROUPSINVESTIGATED

3 months(n = 8) 12 months(n = lO) 24 months(n = 10)

Area(mm2)

Perimeter(mm)

1037.5 + 69.2 1060.1 _+ 78.7 1103.6 _+ 88.3

122.7 + 5.6 132.4 _+ 3.9 118.6 _+ 7.3

Values are means +_ SE.

in brain function. In the central nervous system zinc is contained almost exclusively in three pools. The first pool, known as the vesicular zinc pool, is represented by the zinc contained in presynaptic vesicles (7). This pool is the only one that could be demonstrated easily using specific histochemical techniques such as the T i m m ' s technique and its recent modifications for increasing the sensitivity for zinc and avoiding the interference of other heavy metals (4,6,7). The second pool, known as the ionic pool, is constituted mainly by ionic zinc present in the interstitial fluid and in the cytosolic fraction of the nervous system. It is probably involved in the transduction of intercellular or intracellular signals (6). The third zinc pool, which is the largest one, is known as the enzymatic zinc pool and represents about the 85%-95% of the total zinc in the brain. It is constituted by the metal bound firmly into the molecules of the zinc-containingenzymes in the nervous tissue (6). Age-related changes in histochemically demonstrable zinc

stores have been described in the hippocampus, where the density of staining was the highest in adult rats followed in the descending order by young and old animals (3,15). In the cerebral cortex, where zinc is contained mainly in the neuropil of cerebrocortical circuitry, the density of staining was higher in adult and aged rats in comparison with young subjects in the frontal and in the parietal cortices but it is increased in the occipital cortex of old in comparison with young and adult rats (2). In the neostriatum, the age-dependent changes of zinccontaining stores were similar to those already described for local cerebral glucose utilization (l 1), for muscarinic cholinergic receptors (18) and for dopamine D- l receptors (9), with a loss between young age and maturity and no further important changes between maturity and senescence. In view of the lack of information about the functional role of zinc in striatal neurobiology (16) and of the unhomogeneous sensitivity to aging of the main zinc tissue stores (hippocampal and cerebrocortical) in the rat brain (2,3,15), it is difficult to postulate on the significance of the age-dependent changes in the density of sulfide-silver staining in the matrix of neostriatum observed in this study. These changes, however, show a behavior similar to that found in neurochemical and neurotransmitter-related parameters of neostriatum primarily affected in maturity rather than senescence. ACKNOWLEDGEMENTS The present study was supported in part by a grant of the Italian National Research Council (C.N.R., progetto finalizzato invecchiamento). We thank Dr. W. L. Collier for reviewingthis manuscript and Miss. P. Capuano for her invaluable secretarial assistance.

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