Variations in trace metal levels in rat hippocampus during ontogenetic development

Anat Embryol (1983) 167:141-149 Anatomy and Embryology �9 Springer-Verlag 1983

Variations in Trace Metal Levels in Rat Hippocampus during Ontogenetic Development

P~ter Szerdahelyi and P6ter Kfisa Central Research Laboratory, Medical University, Szeged, Hungary

Summary. The variations in trace metal (zinc, iron, copper and manganese) levels in rat hippocampus were followed by atomic absorption spectrophotometry from the 17th embryonic day to the 100th postnatal day. In agreement with histochemical observations, it was found that the greatest relative increase in the hippocampal zinc level occurs during the 11th-20th postnatal days, simultaneously with the morphological maturation of the mossy fibre terminals. The iron level falls dramatically from the 17th embryonic day to the 3rd postnatal day. Beginning from the l l th postnatal day, the iron level, similarly to the copper level, continuously increases with age. The manganese level rises up to the 20th postnatal day, and subsequently progressively decreases. It is suggested that all of these elements are indispensable for the normal development and functioning of the hippocampus and the mossy fibre terminals.

Key words: Trace metals - Ontogenetic development- Rat hippocampus

Introduction

A large number of autoradiographic (von Euler 1962), histochemical (Fleischhauer and Horstmann 1957; Timm 1958 a; Haug 1973 ; Frederickson et al. 1981 ; Kozma et al. 1981), quantitative atomic absorption spectrophotometric (Hu and Friede 1967; Fjerdingstad et al. 1974; Danscher et al. 1976) and X-ray emission spectrophotometric (Kemp and Danscher 1979) examinations prove that the hippocampal formation, and within this the mossy fibre system of the area dentata, contains a significantly higher quan- tity of trace metals, mainly zinc, than any other brain area. Electronmicro- scopic histochemical research (Haug 1967; Ibata and Otsuka 1968; Kozma et al. 1978) demonstrates that the presence of zinc is primarily connected

Offprint requests to. P6ter Szerdahelyi, Central Res. Lab., Medical University, H-6720 Szeged, Somogyi B. fit 4. Hungary

142 P. Szerdahelyi and P. K/tsa

with the synaptic vesicles of the mossy fibre boutons. It is highly probable that zinc plays a role in the synaptic transmission of the mossy fibre terminals (von Euler 1962; McLardy 1970; Crawford et al. 1973), and its presence is indispensable for undisturbed development of the hippocampus (Szerda- hetyi et al. 1982) and for its normal functioning (Hesse 1979).

It was found by Crawford and Connor (1972) and by Kozma and Ferke (1979) that the ontogenetic changes in the trace metal levels vary in character in the different brain areas. It has been shown that zinc plays a determining role in the development of the hippocampus. However, it has not been clarified whether some trace metal other than zinc is essential for normal development of the hippocampus and the mossy fibre system, nor how the hippocampal concentrations of other trace metals vary from the embryonic age up to adulthood. In this paper, therefore, our main goal was to shed light on how the trace metal levels vary during the pre- and postnatal ontogenetic development of the hippocampus.

Materials and Methods

Material

Albino rats (CFY strain) of both sexes were used. After decapitation without anaesthesia on embryonic days (E), 17 and 19, and postnatal days (P) 1, 3, 5, 7, 9, 11, 14, 17, 20, 23, 26, 29, 40, 70 or 100 (adult) the hippocampus was bilaterally dissected from the brain using a plastic-coated stainless steel forceps. The tissue samples were then weighed and stored at - 2 0 ~ C until processed for atomic absorption spectrophotometry. In every group of animals 4- t0 samples were examined.

Atomic Absorption Spectrophotometry ( AAS)

Frozen tissues were dried at 115~ for 20 h and weighed. Dry samples were then ashed in a quartz crucible by gradual heating up to 550~ for 24 h. The ash was dissolved in a variable volume of 3M nitric acid (Merck, Suprapur| to ensure optimum concentrations of analytes for AAS measurements (Zn and Fe: up to 0.5 ~tg/ml; Cu: up to 25 ng/ml and Mn: up to 5 ng/ml).

Zinc and iron analyses were performed by flame AAS on a Perkin-Elmer 306 double-beam atomic absorption spectrophotometer at wavelengths of 213.9 and 248.3 nm, respectively (slit widths of 0.7 and 0.2 nm, respectively). Copper and manganese were analysed by electrothermal atomization in a HGA-500 graphite furnace (coupled to the basic apparatus) at wavelengths of 324.8 and 279.5 nm, respectively (slit widths of 0.7 and 0.2 nm, respectively). The absorbance signal during the atomization step (at 2600 ~ C) was recorded on a Perkin-Elmer 56 recorder.

Working standards were prepared daily with 3M nitric acid (Merck, Suprapur| No matrix interferences were observed and the ealibartion curves were linear in the concentration range of samples (see above).

The accuracy of analyses was checked by using NBS standard reference material 1577 (bovine liver). The reproducibility of copper and manganese analyses by manual sampling was better than • 0.8%, and 1.9% (S.D.), respectively.

All plastic and quartz ware used in the preparation of samples was kept in diluted hydro- chloric acid overnight and rinsed with distilled deionized water.

Statistical Analysis

Results are expressed as the m e a n • (standard deviation). Statistical analysis of the differences between means of different age groups was made by Student's t-test.

Trace Metal Levels in Rat Hippocampus during Ontogenesis 143

Trace Metal Histochem&try

For histochemical examinations the animals were perfused transcardially with a solution of 9 g Na2S.9H20+3 g NaH2PO4-9H20 in 250 ml distilled water. The pH was adjusted to 7.3-7.4 with dilute orthophosphoric acid. Frozen eryostat sections 25 ~tm in thickness were prepared from the excised brain. After drying at room temperature, the sections were postfixed in 96% ethanol for 15 min and developed in Timm's reagent (pH 3.8) for 55 min via a slight modification (Kozma et al. 1978) of the method described by Timm (Timm 1958 b).

Results

Atomic Absorption Spectrophotometry

In our experiments the variations in the hippocampal levels of the essential trace metals occurring in the highest concentrations (zinc, iron, copper, manganese) were followed from day E17 to day P100.

In the normal hippocampus the zinc concentration (referred to wet tissue weight) fluctuates between days E19 and P5 (Table 1), the change not being significant. From day P5 the zinc level increases continuously up to adulthood. The most intensive elevation in the zinc level is observed between days P11 and P20 (+ 38.6%, p <0.001). Following an intermediate stagna- tion, the zinc level undergoes a further increase and on day P40 it approxi- mates to the value typical for the adult, 100-day-old rat.

The hippocampal iron level (Table 1) decreases dramatically between days El7 and P3 ( -68 .5%, p < 0.001). Up to day P9 the iron concentration is essentially unchanged, while between days P9 and P l l there is a further drop of 24%. From day P11 on, the iron level increases in a stepwise

Table 1. Variations in hippocampal fresh weight (mg), water content (%), and trace metal levels (p.g/g fresh weight) during ontogenetic development of the rat. Each value represents the mean _+ S.D.

Age No. of Fresh Water Zinc Iron Copper Manganese (days) animals weight content

El7 4 9.3_+1.1 88.4_+0.3 9.06_+0.78 24.65+2.29 0.372___0.035 0.224+0.005 E19 10 15.7+_2.2 88.3_+0.7 11.46_+0.70 20.01_+1.98 0.296___0.032 0.188___0.008 P1 7 16.4___2.1 89.4_+0.3 9.69_+0.79 16.22_+2.54 0.458___0.072 0.290_+0.024 P3 5 24.8_+4.4 88.3+0.2 10.27_+0.20 7.76_+0.58 0.892___0.068 0.268_+0.005 P5 8 32.8_+0.7 88.7_+0.3 8.59_+0.40 8.40_+0.56 0.548___0,021 0.244+0.017 P7 8 39.5_+1.9 87.7-+0.9 9.22-+0.54 8.59___0.98 0.773+0.054 0.268-+0.034 P9 8 45.0__.1.2 87.7_+0.2 10.00_+0.40 8.46_+0.50 0.954___0.022 0.360___0,013 P l l 7 51.4_+2.9 87.3_+0.2 10.48_+0.56 6.44_+0.71 0.936_+0.085 0.312_+0.015 PI4 8 59.4_+5.5 85.9_+0.3 11.13___0.91 6.78___0.17 1.081_+0.070 0.411_+0.023 P17 9 62.7_+5.6 84.6_+0.8 12.89+0.69 9.73_+1.26 1.192+_0.100 0.459_+0.036 P20 7 72.2_+4.2 83.1 _+0.2 14.52-+0.27 10.19_+0.40 1.475_+0.034 0.471 _+0.042 P23 6 76.6_+3.5 82.1 _+0.1 14.71 _+0.42 10.50_+0.34 1.346_+0.110 0.442_+0.007 P26 5 80.0_+3.2 81.7_+0.1 15.55_+0.22 10.03_+0.65 1.403_+0.048 0.412_+0.013 P29 7 85.3_+4.5 81.8-+0.3 14.72_+0.39 9.94___0.37 1.630-+0.196 0.401_+0.040 P40 7 86.2_+7.2 80.5-+0.2 16.31_+0.58 12.61_+0.77 1.763_+0.123 0.295-+0.007 P70 6 87.5_+4.8 79.5_+0.5 16,28_+0.49 13.21-+0.77 1.767-+0.068 0.293-+0.020 P100 4 95.0-+4.6 79.9+__0.2 16.83_+0.36 13.55_+0.26 2.122-+0.120 0.287-+0.002

144 P. Szerdahelyi and P. K~sa

Fig. 1. a On day P3 the granule (G) and the pyramidal (P) cells of the regio inferior and the hilar mossy fibres (H) yielded a faint staining, x 52 b The staining of the layers in the normal hippocampus on day P l l is very faint. The hilar (H) and suprapyramidal (SP) mossy fibres are well stained. Timm's method, x 52


Fig. 2. a The staining intensity of the mossy fibres (M) in the hippocampus on day P20 approaches that of adulthood, x 58. b The mossy fibres (M), pyramidal cells (P) and granule ceils (G) of the adult hippocampus stain intensely. Timm's method, x 62


manner to attain the value observed in the adult hippocampus. The rate of increase is highest between days P14-P17 (+43.5%) and P29-P40 (+26.9%), respectively.

The copper level (Table 1) rises progressively from day E17 to adulthood. On day El7 the copper level of the hippocampus is merely 17.5% of that in adulthood. There are outstandingly high tissue copper contents on days P3 and P20.

In contrast with other trace metals examined, the variations in the hippocampal level of manganese are characterized by a maximum curve (Table 1). The manganese concentration rises continuously from day E19 to day P20, when its value is 64% higher (p<0.001) than the level for adults. From day P20 on, the manganese level steadily falls and on day P40 it approxi- mates to that typical for adulthood.

Histochemistry

When sections from day P3 (Fig. 1 a) are stained by Timm's method, trace metals are clearly revealed in the granule cells of the area dentata and in the pyramidal cells of the regio inferior. The pyramidal cells display weaker staining in the regio superior, as do the mossy fibre terminals in the hilus of the area dentata. Staining indicative of trace metals can likewise be demonstrated in the stratum oriens of the regio superior. On day P11 (Fig. I b) one can clearly observe the laminar structure of the hippocampus and the different intensities of staining of the layers. However, the staining of the pyramidal and granule cell layers and also the mossy fibres is substan- tially weaker than in adulthood. The hilar and the suprapyramidal mossy fibres are stained more strongly than the infrapyramidal ones. On day P20 (Fig. 2 a) the intensity of staining of all the layers of the hippocampus ap- proximates to (but does not attain) that in adulthood. The hilar, the supra- and the infrapyramidal mossy fibres are stained extremely strongly. With the same incubation time, the staining of the layers in the hippocampus of the adult animal (Fig. 2b) is even more intense than on day P20. The staining of the granule and pyramidal cell layers is also strikingly intensified.

Discussion

Our atomic absorption spectrophotometric examinations show that the most rapid increase in the zinc level of the hippocampus occurs in the period Pl l -20 , which coincides with the morphological (Amaral and Dent 1981) and physiological (Bliss et al. 1974) maturation of the mossy fibre terminals. The histochemical results demonstrate that the staining intensity of the mossy fibre neuropil increases significantly in the same period, but it does not attain the level for adulthood. This is in full accordance with the findings of Zimmer and Haug (1978) that the number, size and staining intensity of the mossy fibre terminals increase enormously between days P12 and P18. However, it has also been suggested that there is no absolute correlation


between trace metal staining and trace metal content, since the chemical structure and character of the metal binding proteins, alter during the ontogenetic development and so the possibility of histochemical detection may alter, too (Crawford and Connor 1972; Zimmer and Haug 1978; Kozma and Ferke 1979).

Crawford and Connor (1972) observed the greatest increase in the hippocampal zinc level compared to the protein content between days P18 and P22, and they could demonstrate no further change in the zinc concentration up to adulthood.

However, our results indicate a slow rise in the hippocampal zinc level after day P20, which can be explained by the continuous increase in the number of granule cells (Bayer 1982) and their terminals, and the expansion of the mossy fibre terminals (Amaral and Dent 1981).

We observed a certain degree of coincidence between the ontogenetic variation in the zinc level and the cholinergic synaptogenesis (unpublished) in the rat hippocampus. The choline acetyltransverase activity on day P l l is low in every layer of the gyrus dentatus, and the adult level is approached with a rapid increase around days P16-17; however, this is not followed by an increase of similar extent in the acetylcholinesterase activity (Nadler et al. 1974).

In parallel with the disappearance of the germinal matrix and the differ- entiation of its cells (Bayer and Altman 1974), the tissue iron level decreases to about one-third between days El7 and P3. The extremely high embryonal iron level is probably connected with the enhanced, favoured cerebral 02 delivery in the foetus (Jones et al. 1982). Timm's reaction is not suitable for the faithful demonstration of the embryonal and perinatal iron levels (even higher than the adult levels) since the iron in the haemoglobin and in the mitochondria cannot be revealed with the sulphide-silver impregna- tion technique (Danscher and Zimmer 1978).

The copper level of the hippocampus progressively increases during ontogenetic development. Kofod (1970) observed a similar tendency with regard to the variation of the copper level of rat brain with age. The endogenous noradrenaline level (Loy and Moore 1979) rises at a rate similar to that of the copper level in the hippocampus in the course of ontogenesis, which may be explained by the role of copper in the metabolism of the catechol- amines (Morgan and O'Dell 1977).

Manganese besides its role in the metabolism of the biogenic amines (Papavasiliou et al. 1968), probably participates in the glutamate metabolism too, as a constituent of glutamine synthetase (Balakrishnan and Villafranca 1978). According to Crawford and Connor (1973) and Storm-Mathisen and Iversen (1979), glutamate is a transmitter substance of some of the mossy fibre terminals. A role of manganese may also be conjectured in other axon terminals mediating with glutamate [e.g. in the Schaffer collateral, the per- forant tract, and the commissural and association fibres (Fonnum et al. 1980)]. Further research is required, however, to elicit how the decreasing manganese level from day P20 on wards will affect the functioning these fibres.


Acknowledgements. The excellent technical assistance of Mrs. Susan Zab and the secretarial assistance of Mrs. G. Ugrin are gratefully acknowledged. This work was supported by the Scientific Research Council, Ministry of Health, Hungary (06/4-20/457).

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Accepted February 17, 1983

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Variations in trace metal levels in rat hippocampus during ontogenetic development