hippocampus by chelation of heavy metals

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the effect of metal chelation on CCK by in vivo treatment. of rats with sodium diethyldithiocarbamate (DEDTC), the ac- tive metabolite of disulfiram (Antabus).
Proc. Nail. Acad. Sci. USA Vol. 81, pp. 5876-5880, September 1984 Neurobiology

Modulation of cholecystokinin concentrations in the rat hippocampus by chelation of heavy metals (zinc/radlohmmunoasay/immunochemistry/chromategraphy/sulfide silver stain) K. STENGAARD-PEDERSEN*, L.-I. LARSSONt, K. FREDENSI, AND J. F. REHFELD§ *Institute of Medical Biochemistry and *Anatomy, University of Aarhus, DK-8000 Aarhus C, Denmark; tDepartment of Histochemistry, Institute of Pathology, University of Copenhagen, DK-2100 Copenhagen 0, Denmark; and §University Department of Clinical Chemistry, Rigshospitalet, DK-2100 Copenhagen 0, Denmark Communicated by Diter von Wettstein, May 21, 1984

ABSTRACT Previously, we have reported that enkephalins, cholecystokinin, and heavy metals show roughly parallel distributional patterns in the hippocampus. A substantial body of evidence indicates that cholecystokinin-octapeptide (CCK8) and enkephalins act as neurotransmnitters. A CCK-8 degrading enzyme was recently detected in brain synaptosomes. Its activity depended on free thiol groups and the presence of a heavy metal. Since the heavy metal-containing neuropil is closely related to CCK-immunoreactive nerve terminals, we have investigated the effect of metal chelation on CCK conmponents in the rat hippocampus. In vivo treatment of rats with a single dose of the chelating agent diethyldithiocarbamate caused a reversible chelation of heavy metals in the hippocampus. This effect was paralleled by a 3-fold increase in hippocampal content of CCK-8 and a smaller increase in the intermediate forms of CCK (CCK-58, CCK-39, CCK-33). Diethyldithiocarbamate also decreased the spontaneous motility and aggressiveness of the rats. These data show reversible changes of neuronal CCK processing by a drug, and hence they provide additional evidence that CCK is involved in the regulation of neuronal activities.

occur in all layers of the rat hippocampus, but'mostly in the stratum pyramidale, the mossy fiber zone, and the stratum radiatum (8, 17). The COOH-terminal octapeptide of 'CCK (CCK-8) appears to be a putative transmitter (18-30) with an excitatory effect on hippocampal pyramidal cells (31, 32). Moreover, in vitro studies have indicated that a CCK-8 degrading enzyme is likely to be a metalloenzyme (33). These observations indicate a functional significance of the close relationship between heavy metals and CCK in the rat hippocampus. It is well known that treatment of rats with chelating agents causes a transient change in the histochemical staining for zinc and other heavy metals (34), paralleled by behavioral effects (34, 35). The neuronal processes underlying these effects are unknown, and, hence, prompted us to study the effect of metal chelation on CCK by in vivo treatment. of rats with sodium diethyldithiocarbamate (DEDTC), the active metabolite of disulfiram (Antabus).

MATERIALS AND METHODS Animals. DEDTC [C5H1oNS2Na-3H20 (Merck)] was dissolved in saline at a concentration of 50 mg/ml and injected intraperitoneally in male Wistar rats (180-200 g) at 1 mg of DEDTC per g of body weight. Control animals were injected with saline; 1, 5, and 24 hr after the injection the animals were processed for immunocytochemistry and Timm's silver sulfide method, or the hippocampus was dissected out for extraction and measurement of CCK and zinc. Extraction of Tissue. The hippocampus from identically treated animals was frozen in liquid nitrogen and stored at -80°C until weighed and extracted. The frozen tissue was cut into small pieces, boiled for 25 min in distilled water (10 ml per g of tissue, pH 6.6), homogenized, and centrifuged. The pellets were reextracted in equal volumes of 0.5 M acetic acid, homogenized, and centrifuged. Both supernatants were frozen at -20°C until assayed by sequence-specific radioimmunoassays in appropriate dilutions (36). Extracts (1.5 ml) were applied to Sephadex G-50 superfine columns, (10 x 1000 mm) eluted at 4°C with 0.02 M barbital buffer (pH 8.4) containing 0.1% bovine serum albumin at a flow rate of 4 ml/hr. Fractions of 1 ml were collected. The columns were calibrated with I251-labeled albumin [for indication of void volume (V0)], 22NaCl [for indication of total volume (Vt)], and with highly purified porcine CCK-33, CCK-8, arnd CCK-4. The elutions were monitored by sequence-specific CCK radioimmunoassays (19, 20, 36). Radioimmunoassays. The extracts were measured by two sequence-specific assays using either antiserum 2609, which is specific for the sequence 29-33, or antiserum 4698, which is specific for the sequence 25-30 of CCK-33 (36). Thus, antiserum 2609 binds all peptides containing the biologically

Heavy metals occur in distinct layers of the rat hippocampus, associated with the terminal fields of certain neuronal systems, including the mossy fibers (1, 2). In the mossy fibers, there is ample evidence that most of the detectable metal is zinc (3, 4), which is located in the mossy fiber boutons (5, 6). Otherwise, the exact localization of specific metals is unknown (7). The role of zinc and other heavy metals in specific nerve terminals of the hippocampus is unknown. Recently, we have shown that both enkephalin-like molecules and heavy metals (mainly zinc) occur in the mossy fibers of rodents (8, 9). Subsequently, we have shown that zinc and other metal ions can down-regulate the binding capacity of opioid receptors (10, 11). These observations, together with the fact that endogenous opioid peptides excite hippocampal pyramidal cells via binding to opioid receptors (12), indicate that zinc, and possibly other neuronal metals, may regulate the binding capacity of opioid receptors (10, 11). The close relationship between enkephalin and zinc in the mossy fiber system may reflect that zinc is a cofactor for the recently discovered enkephalin degrading enzyme "enkephalinase" (13-16). Also, cholecystokinin (CCK) nerve terminals occur closely associated with the heavy metal-containing neuropil in the rat hippocampus (8, 17). Thus, CCK immunoreactivity is located in the terminal areas of the medial perforant path and the temporo-ammonic tracts from the medial entorhinal cortex, as well as in the terminal area of the commissural-associational path of area dentata. CCK-containing cell bodies The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Abbreviations: CCK, cholecystokinin; DEDTC, sodium diethyldithiocarbamate.

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Neurobiology: Stengaard-Pedersen et aL active COOH-terminal tetrapeptide amide common to CCK and gastrin. Antiserum 4698 binds sulfated CCK-8 and larger sulfated forms of CCK with equimolar potency, but it does not recognize any gastrins. Synthetic sulfated CCK-8 was used as standard and 1251-labeled CCK-33 was used as a tracer (36). Immunocytochemistry. Rats were anesthetized with diethyl ether and perfusion-fixed with 4% paraformaldehyde as described (20, 37, 38). Horizontal brain slices were prepared for cryostat sectioning and immunocytochemically stained with anti-CCK antiserum 4562, recognizing the COOH-terminal tetrapeptide portion of CCK and gastrin (39). The site of the antigen-antibody reaction was demonstrated by the peroxidase-antiperoxidase procedure of Sternberger (40). Conventional staining controls, as detailed by Sternberger (40), as well as absorption (specificity) controls were used. Antiserum 4562 has been characterized in detail previously and shown to react with the COOH-terminal tetrapeptide of CCK (CCK-4). It also reacts with larger molecular forms of CCK (CCK-8, CCK-12, and CCK-33), which all contain the CCK-4 sequence (39). Also, gastrin reacts with antiserum 4562 since the COOH-terminal pentapeptides of gastrin and CCK are identical. However, gastrins are not present in the hippocampus (19, 20). Histochemical Detection of Heavy Metals. The principal steps of the method are sulfide-precipitation of the metals in the tissue and subsequent visualization of the argyrophilic metal sulfides by physical development (7, 41). Cryostat sections from tissue perfused with the Na2S solution and fixed in 4% paraformaldehyde were thawed onto slides and air-dried for 2 hr. They were then hydrated and exposed to a physical developer in the dark for 60 min at 25°C. During the latter stage, the metal sulfides catalyze reduction of silver ions by a reducing agent. Determination of Zinc in Tissue Samples. The wet weight of the hippocampus was recorded. Thereafter, wet washing was carried out through boiling in concentrated nitric acid followed by about a 1:20 dilution with distilled water. The zinc concentration in the diluted tissue samples was determined with a Perkin-Elmer atomic absorption spectrophotometer (42).

RESULTS With the Timm's silver sulfide method for histochemical localization of heavy metals, the rat hippocampal formation shows a distinctly stratified staining pattern, suggesting large regional differences in the content of heavy metals. The granule cell layer is profusely stained with large black grains and the hilus and regio inferior are crowded with black grains corresponding to mossy fiber terminals (Fig. 1). The intense staining of the mossy fibers is thought to reflect a high content of zinc (3, 4), which we measured to be 237 + 4 nmol per g wet weight of the total rat hippocampus. The heavy metalcontaining neuropil, especially the zinc-enriched mossy fiber terminals, is in close contact with the CCK nerve cell bodies and terminals (compare Figs. 1 and 2). In the rat hippocampus, CCK is located in the terminal areas of the medial perforant path, the medial temporo-ammonic tract, and the commissural-associational path, as well as in cell bodies dispersed throughout all layers of the rat hippocampus (Fig. 2). The stratum radiatum, the stratum pyramidale, and, especially, the mossy fiber zone contained many CCK cells, and the processes of these cells seemed to be distributed around the somas and apical dendrites of the pyramidal cells (Fig. 2), a distribution which suggests a role for CCK in the regulation of pyramidal cell activity. Furthermore, these neurons are in close contact with the neuronal structures enriched with heavy metals (compare Figs. 1 and 2).

Proc. NatL Acad Sci. USA 81 (1984)

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'24 h4 Zinc FIG. 1. Horizontal cryostat sections of the rat hippocampal formation. The Timm silver sulfide method for detection of heavy metals displays a staining pattern related to the laminar subdivision of the hippocampus. The mossy fiber system stains black, because of its large well-stained boutons. The neuropil in the stratum radiatum, the outer zone of stratum oriens, the inner zone of the subiculum with a wedge extending into the molecular layer, and the presubiculum display a medium dense staining. On the whole, the pyramidal cell layer and the molecular layer are pale. The molecular layer of area dentata stains in the inner and outer zone with an unstained middle zone (cf. ref. 8). The stain is thought to reflect the localization of heavy metals (cf. refs. 3 and 7). Note that most CCK-immunoreactive terminals will be in close contact with the heavy metalcontaining neuropil (compare with Fig. 2). Treatment with the chelating agent DEDTC caused a reversible bleaching of the stained neuropil, as visualized here 1, 5. and 24 hr after injection of DEDTC. In the mossy fibers, the staining chiefly reflects a high concentration of zinc (cf. refs. 3 and 4). However, the total concentration of zinc did not change. Note that the reversible chelation of heavy metals is paralleled by an important increase in the concentration of cholecystokinin octapeptide (compare with Fig. 3). (x20.)

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Proc. NatL Acad Sd USA 81

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FIG. 2. Horizontal cryostat section of the rat hippocampal formation. CCK immunoreactivity is detected as two parallel bands in the molecular layer of area dentata. The inner band corresponds to the terminal zone for the commissural-associational fibers from the hilus of the ipsilateral and contralateral area dentata. The outer band is located in the middle one-third of the molecular layer and can be followed through regions CA3, CAl, and the subiculum, and this corresponds to the termination area of the medial perforant path and the medial temporoammonic tract. CCK-immunoreactive cell bodies are located in the hilus of area dentata, the stratum pyramidale (B), the stratum radiatum (A), the stratum oriens, and the cell-rich layer of subiculum (for details see refs. 8 and 17). Sections are highly counterstained with hematoxylin. gr, Granule cells; pyr, pyramidal cells; ac, associational and commissural fibers; mpp, medial perforant path; Ipp, lateral perforant path; or, stratum oriens; rad, stratum radiatum; mol, stratum moleculare; sub, subiculum. (Left, x40; Insets A and B, x350.)

These observations pointed to a functional interaction between hippocampal CCK and heavy metals. Treating the rats with DEDTC, a chelating reagent, caused a reversible blocking of the Timm sulfide silver staining for heavy metals. The maximal effect was seen 1 hr after injection of DEDTC (1 mg per g of body weight). At that time, the neuropil became unreactive for heavy metals throughout the hippocampus (Fig. 1). Five hours after the injection, the reactivity reappeared (Fig. 1), and 24 hr after the injection, the pattern of neuropil staining appeared normal (Fig. 1). This reversible blocking of histochemically detectable heavy metals was not accompanied by changes in the total content of hippocampal zinc (Fig. 1). It was, however, paralleled by a characteristic change in the hippocampal content of CCK (Table 1). Table 1. Effect of DEDTC treatment on hippocampal content of CCK CCK in rat hippocampus, pmol/g wet

Treatment Control 1 hr after DEDTC 5 hr after DEDTC 24 hr after DEDTC

weight (CCK-8 equiv) Acetic acid Boiling water extraction of tissue extraction of tissue 23-46 93-115 38-45 242-361 145-191 39-40 20-47 101-129

In the boiling water and acetic acid extracts of the hippocampal tissue, different molecular forms of CCK occurred (Fig. 3): (i) a large component eluting with an elution constant (Kay) of 0.05 (CCK component I); (ii) intermediatesized components eluting with Ka, from 0.40 to 0.50, corresponding to CCK-58, CCK-39, and CCK-33; (iii) a predominant component eluting with a Kay of -1.10, corresponding to the COOH-terminal octapeptide of CCK-33 (CCK-8); and (iv) a small component eluting with a Ka, of -1.30, corresponding to a COOH-terminal tetrapeptide of CCK-33 (CCK-4-like). The CCK-8, the COOH-terminal fragment, and the largest molecular form of CCK (Ka,, -0.05) were extracted in the boiling water, whereas the intermediate size forms (CCK-58, CCK-33) were extracted most effectively by acetic acid. From the chromatograms, a relative concentration of the different CCK components was determined by integration of the peaks. In the control animals, the large CCK component I constituted =4% of the total hippocampal CCK immunoreactivity; the intermediate-sized components constituted -15% (including also the peak eluting just before CCK-33); the CCK-8-like component constituted ~=80%, and the CCK4-like component constituted =1%. Treatment of the rats with DEDTC increased the total amount of CCK in the hippocampus. The increase peaked 1 hr after the DEDTC treatment, and it reached normal levels within 24 hr (Table 1). Thus, the change in hippocampal CCK paralleled the chelat-

Neurobiology: Stenigaard-Pedersen et aL

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are indicated. Treatment with the chelating agent DEDTC caused a reversible increase in the hippocampal CCK immunoreactivity. 1 hr and 24 hr indicate the time after a single injection of DEDTC. The CCK-8-like component increased a25to above the normal concen-

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ing effect of DEDTC on the hippocampal heavy metals (Fig. 1). The CCK-8-like component =5250% increased above the normal concentration. The intermediate-sized CCK forms increased ~100% above the normal concentration, whereas the changes inthe remainingcomponents were insignificant. The DEDTC dose used caused astriking decrease in spontaneous motility of the rats. They were less aggressive and mostly

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DISCUSSION

Previously, there have beenonly afew reports ondrugs that can influence the peptide content ofneurons (43, 44). This work demonstratesreversible changes of neuronal CCK by a drug. Treatment of rats with a single dose of DEDTC caused a reversible blocking of Timm's sulfide silver stain for heavy metals and an increase in the hippocampal content of CCK,

Proc. NatL Acad Sci. USA 81 (1984)

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especially CCK-8. These observations suggest that heavy metals may be involved in the processing of CCK. DEDTC is the active metabolite of disulfiram (Antabus), a drug used in the treatment of alcoholism (45). Disulfiram and DEDTC have complex biochemical effects (34). In the present context it is of interest that DEDTC is a chelating agent (34). Moreover, it has been shown that DEDTC in vivo can inactivate the copper enzyme dopamine /8-hydroxylase, which converts dopamine to noradrenaline (46). Disulfiram and DEDTC seem to interact also with the sulfhydryl groups of various enzymes (47-49). Both chelation of ions and blocking of sulfhydryl groups could inactivate enzymes involved in the biosynthesis and/or degradation of CCK and, thus, explain otir data. Recently, a CCK-8 and CCK-4 degrading enzyme was detected in both the cytosol and the synaptic membranes of brain tissue (33). Moreover, in vitro experiments showed that the degradation of CCK-8 and CCK-4 could be markedly inhibited by both chelating and sulfhydryl-blocking agents, suggesting that their degradation required the presence of a metal ion probably linked to a thiol group (33). Our results could be explained by reversible inactivation of such a CCK degrading enzyme. It is well known that DEDTC can chelate heavy metals (Zn, Cu, Fe, Co, and others) (7, 34), which may function as cofactors for the CCK degrading enzyme and, in addition, DEDTC may also be capable of inactivating the enzyme by blocking the thiol groups (47). Such a mechanism could explain the observed accumulation of CCK-8. The close relationship between CCK nerves and neuronal structures enriched with heavy metals further strengthens the hypothesis that a metalloenzyme is involved in the processing and/or degradation of CCK. Such a mechanism is, however, difficult to reconcile with the long half-life of 16 hr reported for the degradation of CCK-8 (50). Another possible explanation for our findings is that the increase in CCK after DEDTC administration may be a consequence of altered dopamine release (46). This idea is in agreement with the coexistence of dopamine and CCK in several neuronal systems (51). Neuronal CCK occurs in several molecular forms, of which the smaller form, CCK-8, predominates (19, 20). For both the cerebral cortex and the hippocampus, CCK precursors are rapidly synthesized and processed to the small molecular forms (21, 22). The present results confirm these findings. The small forms of CCK are concentrated in synaptic vesicles (23), from which they are released by depolarization in a calcium-dependent manner (24, 25). Specific receptors for CCK have been detected in neuronal membranes of the hippocampus (28-30), and physiological data indicate that CCK-8 and CCK-4 can depolarize hippocampal pyramidal neurons (31, 32). In view of this, the processing and degrading of hippocampal CCK are important in controlling the level and use of CCK as a transmitter. In the liver, disulfiram is immediately converted into DEDTC; the complex actions of disulfiram are generally considered to occur after its conversion to DEDTC (45). Previously, we have found disulfiram and DEDTC to exert similar effects on the Timm's silver sulfide stainability of neuronal structures (34). The effect of disulfiram on CCK, however, has not been investigated. Based on our previous experiments, we chose to use a high dose (1 mg DEDTC per g of body weight), because it could completely block the Timm silver sulfide reaction in the neuropil, whereas lowering the dose to 0.1 mg/g led to a less complete blocking of the neuropil staining (34). Other chelating agents, such as dithizone and oxine, could also prevent the Timm sulfide silver reaction, but because of their greater toxicity, we preferred to use DEDTC (34). The CCK immunoreactive nerve terminals are widespread in the central nervous system (37, 52-59), and in most regions they display such striking overlaps with the enkephalin

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immunoreactive nerves (37) that functional interactions must be considered. Moreover, the degradation of the enkephalins is dependent on a metalloenzyme that uses Zn2' as a cofactor and that has been claimed to exhibit a distribution similar to that of the enkephalins (14, 16). The observation that the degradation of CCK is also dependent on a metalloenzyme makes it tempting to speculate that the metabolism of CCK and the enkephalins is dependent on a common metalloenzyme. 1. Haug, F.-M. S. (1974) Z. Anat. Entwicklungsgesch. 145, 1-27. 2. Haug, F.-M. S. (1973) Adv. Anat. Embryol. Biol. 47, 1-71. 3. Danscher, G., Fjerdingstad, E. J., Fjerdingstad, E. & Fredens, K. (1976) Brain Res. 112, 422-446. 4. Fjerdingstad, E., Danscher, G. & Fjerdingstad, E. J. (1974) Brain Res. 79, 338-342. 5. Haug, F.-M. S. (1967) Histochemie 8, 355-368. 6. Ibata, Y. & Otsuka, N. (1969) J. Histochem. Cytochem. 17,

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