- Email: [email protected]
Age-related toxicity in prefrontal cortex and caudate-putamen complex of gerbils (Meriones unguiculatus) after a single dose of methamphetamine
Neurophavmacology Vol. 30, No. 7, pp. 733-743, 1991 Printed in Great Britain. All rights t~erved
0028-3908/91 $3.0~)+ 0.00 Copyright© 1991PergamonPress pie
AGE-RELATED TOXICITY IN PREFRONTAL CORTEX A N D C A U D A T E - P U T A M E N COMPLEX OF GERBILS (MERIONES UNGUICULATUS) AFTER A SINGLE DOSE OF METHAMPHETAMINE G. TEUCHERT-NOODTand R. R. D^wms Department of Neuroanatomy, Faculty of Biology, University of Bielefeld, 4800 Bielefeld, Germany (Accepted 15 January 1991)
Summa'y--Single, intermediate to large doses (6-60 mg/kg) of methamphetamine were applied to study the acute neurotoxic effects in developing male gerbils (up to 24 months). A sensitive silver-staining method was used to analyze the toxicity of methamphetamine by light and electron-microscopy. It was shown that treatment with the drug degraded synaptic components, as well as a small population of neurones in the caudate-putamen complex accompanied by accumulation of lysosomes in fibers and axon terminals. In juveniles, methamphetamine in doses of 25-60 mg/kg, resulted in accumulation of lysosomes, selectively in the prefrontal cortex. In young adults, only about half of these doses were sufficient to produce consistent and/or additional effects in the caudate-putamen complex. When the gerbils grew older than 8 months, treatment with drug led to accumulation of lysosomes, exclusivelyin the caudate-putamen, with acute doses ranging from 6 to 12 mg/kg. Acute neurotoxicity with methamphetamine has thus been induced by doses, which hitherto have been claimed to produce behavioural sensitization. Since dopamine (DA) seems the most likely transmitter to be affected, age-related differences in methamphetamine-induced neurotoxicity are discussed in relation to the background of developing DA-response systems, which are still changing in pattern during ageing. Key words--methamphetamine, neurotoxicity, gerbils, development.
"Amphetamine-neurotoxicity" has been described as neuromorphological and neurochemical changes in catecholaminergic systems in brain, with fairly selective effects on dopaminerglc nerve terminals. In that manner, amphetamines cause long-term depletion of both dopamine (DA) and the activity of tyrosine hydroxylase in brain (Ellison, Eison, Huberman and Daniel, 1978; Wagner, Ricaurte, Sciden, Schuster, Miller and Westley, 1980; Nwanze and Jonsson, 1981; Jonsson and Nwanze 1982; Ricaurte, Fuller, Perry, Sciden and Schuster, 1983; Trulson, Cannon, Faegg and Raese, 1987). Levels of metabolites of dopamine (Ricaurte, Guillery, Seiden Schuster and Moore, 1982; Steranke, 1982), populations of DA receptors (McCabe, Hanson, Dawson, Wamsley and Gibb, 1987) and numbers of synaptosomal uptake sites for DA become reduced (Wagner et al., 1980; Woolverton, Ricaurte, Forno and Seiden, 1989). Further, there is some morphological evidence, suggesting neurotoxic damage to DA nerve terminals (Ellison et al., 1978; Nwanze and Jonsson, 1981; Jonsson and Nwanze, 1982; Ricaurte et al., 1982; Recaurte, Guillery, Selden and Schuster, 1984; for further ref. see Robinson and Becker, 1986; Robinson and Camp. 1987), partly indicating spatial selectivity (Lorez, 1981). Neurotoxic responses to amphetamines were described in both the striatum (Ellison et al., 1978; Wagner et al., 1980; Nwanze and Jonsson, 1981; Ricaurte et al., 1982; Ricaurte et al., 1984)
and prefrontal cortex (Wahnschaffe and Esslen, 1985; Trulson et al., 1987). In additon to neurotoxicity, "behavioural sensitization" also occurs in response to administration of amphetamine. In that, animals reach a long-lasting state of supersensitivity to further challenges with amphetamine (Kashihara, Sato, Kazahaya and Otsuki, 1986; Fujiwara, Kazahaya, Nakashima, Sato and Otsuki, 1987; Kolta, Shrev¢ and Uretsky, 1989). Contrary to amphetamine-neurotoxicity, behavioural sensitization is caused by smaller doses, both after intermittent and single application of the drug (Robinson and Camp, 1987). Reports on amphetamine-neurotoxicity are frequently based on continuous administration of the drug through osmotic minipumps or subcutaneous silicone pellets (Ellison et al., 1978; Jonsson and Nwanze, 1982) and intermittent application of large to largest tolerable doses (Wagner et al., 1980; Lorez, 1981; Ricaurte et al., 1983; Trulson et al., 1987; for review see Ellison and Eison, 1983). However, a once-repeated large dose (Ricaurte, Schuster and Sciden, 1980; Wahnschaffe and Esslen, 1985) and even a single large dose after treatment with iprindole (Fuller and Hemrick-Leucke, 1980; Steranka, 1982; Ricaurte et ai., 1984) still results in brain damage. The present study was conducted to analyze the structural phenomena of methamphetamine-induced acute neurotoxicity in the brain of male gerbils, at 733
734
G. TEUCHERT-NOODTand R. R. DAWIRS
different stages of postnatal development. For that purpose, the animals received a single application of methamphetamine, once within their 2 years of postnatal life and morphological changes were studied by light and electron-microscopy. Since DA systems, mostly responsible for the effects of the drug, are under permanent development, starting early in prenatal life and still changing features throughout ageing (Seeman, 1981; Roubein, Embree and Jackson, 1986; O'Boyle and Waddington, 1986; Marshall and Altar, 1986; Ushijima, Mizuki, Soeda, Kishimoto, Hara and Yamada, 1987; Watanabe, 1987; Moretti, Carfagna and Trunzo, 1987; Noison and Thomas, 1988; Kalsbeek, Voorn, Buijs, Pool and Uylings, 1988; Hyttel, 1989), correlations between methamphetamine-induced neurotoxicity and the development of a responding transmitter system were being looked for. METHODS Male gerbils were kept under natural day/night cycles in home cages (30 x 40 cm), 2-6 animals each. Food and water were provided ad libitum. Corresponding to the annual reproductive cycle of the gerbil experiments were performed exclusively from February to November, during 3 subsequent years. The animals received an intermediate to large single dose of methamphetamine (i.p.) and were then housed individually for 3 days, which proved to be the adequate period of survival in all age-groups, in order to produce staining effects by the currently used silver-labelling technique (see below). For applications of methamphetamine, appropriate amounts of "Pervitin" (15 mg methamphetamine-hydrochloride per ml; Temmler-Factories, Marburg, Germany) were diluted in 0.3 ml saline immediately before injection. Control animals received equivalent volumes of saline. The study comprised 3 age-groups, confined to juveniles (1-2 months), young adults (2-6 months) and older adults (8-24 months). A sensitivity to methamphetamine, clearly increasing with age (Esslen and Teuchert-Noodt, 1987), was found. Thus, the doses had to be smaller in older animals, to prevent mortality (Table 1). For light-microscopic studies, the animals were transcardically peffused with 5% formalin or with 5% cacodylate buffered paraformaldehyde (pH 7.2). The brains were subsequently kept in the fixative for a minimum of 2 days Table I. Number of animals (n) treated with methamphetamineor saline (controls); doses were in different ranges (mg/kg) in subsequent age-groups(months) Animals (n) Age Dose-range Methampbetamine(months) (mg/kg) treated Controls 1-2 25-60 44 7 2-6 16-21 106 10 8-24 6-12 22 8
and a maximum of 3 weeks. Serial parasagittal sections (80/~m) were regularly performed from the right side of frozen brains. Additionally coronal sections were cut from the left hemisphere. Processing was according to a selective silver impregnation technique (Gallyas, Wolff, Brttcher and Zaborszky, 1980), highly sensitive for the specific labelling of accumulations of lysosomes accompanying degradation and/or degeneration of axon terminals (Wolff, Leutgeb Holzgraefe and Teuchert, 1989). In order to visualize degrading synaptic components and cells in selected striatal subfields, labelled parasagittal slices were embedded in Epon. A small sector was cut out from the rostral-central region of the striatum (Fig. la) and semi-thin sections were stained for light-microscopical evaluation of distribution patterns of silver stained precipitates. Further, ultra-thin sections were contrasted with uranylacetate and lead citrate and analyzed by electron-microscopy. Additional electron-microscopy was done with 4 methamphetamine-treated young adults, perfused with paraformaldehyde (2.5%) in glutaraldehyde (2.5%) and cacodylate buffer (pH 7.4). Dissected brains were kept in buffer overnight. Parasagittal slices of 1 mm were taken from the designed striatal subfield and further processed for embedding in Epon. Semi-thin sections were stained to look for dark neurones by light-microscopy (Holl/inder and Vaaland, 1968). Ultra-thin sections were provided for electron-microscopical analysis of both accumulation of lysosomes in degrading synaptic elements, as well as morphological correlates of cell death. RESULTS
Morphological features neurotoxicity
of
methamphetamine-
Silver impregnation, visualizing accumulation of lysosomes in an overview pattern throughout serial sections of brain, was detected light-microscopically in both subareas of caudate-putamen complex and prefrontal cortex (schematically in Fig. 1). Using dark-field illumination, the strongest silver labelling was always detected in the central striatum of medial, up to lateral parasagittal section planes (Figs 2 a--c). Labelling appeared either in diffuse or mosaic-like distribution patterns of accumulation of lysosomes (Fig. 3a). Disposed precipitates, covering the tissue on a level with fibre bundles and likewise with cell clusters, were obviously associated with somata and stringed up in fibres (Fig. 3b). Few neurones, exhibiting dark staining, due to increased cytoplasmatic and nuclear densities, bordered others with normal cytological characteristics (Fig. 3c). These dark neurones frequently had shrunken cell bodies, showing irregular contours and sometimes eccentric spherical nuclei. These features were completely absent in controls. Electron-microscopy revealed further details as to subcellular and cellular degradation. Labelled products in silver-impregnated slices accumulated in fibre
735
Age-related methamphetamine neurotoxicity
Table 2. Numberof individuals, in juveniles (I-2), young(2-6) and older adults(8-24)showingq~cificdiverlabelling,i.e. accumulation of lysosorneseither in prefrontal (PFC) or caudate-putamen (CP) subate~ o r in both of thcae regions
profiles (Figs 4 a-c), whereas degraded synaptic elements could hardly be detected because of tissue defects. Non-labelled slices showed lysosomes accumulating in fibres and additionally in degraded presynaptic profiles apposing postsynaptic densities (Fig. 4d). Further, axon terminals were found in the lamellar state of degradation (Fig. 4¢). Irregularly shaped and fairly electron lucent vesicles and multivisicular bodies, were also present. Postsynaptic profiles, associated with degrading terminals, were either electron lucent (Fig. 4d) or as a common mode of typical cell degradation, characterized by pronounced increases in matrix densities (Fig. 4f). It was found that it was a distinct enrichment of free ribosomes, that caused the dark appearance of degrading neurones (Fig. 4g). Some quite unusual amount of clear intracellular spaces represented cisternae of the ¢ndoplasmatic reticulum. The nuclear cromatin was clumped into irregular masses, while evenly dispersed in unaffected cells. Nevertheless, the nuclear membranes of degrading cells remained intact.
A g e in m o n t h s 1-2 2-6 8-24
PFC
CP
PCF and CP
44 49 --
-31 22
-26 --
number exhibited labels in both regions. Distribution patterns of precipitates in the prefrontal cortex of this age-group were in good accordance with those in juveniles. As to animals, with methamphetamineinduced toxic effects in the caudate-putamen, a moderate amount of precipitates became evident thoughout the medial to lateral parasagittal section planes, with strongest staining occurring in the central subfields (Figs 2 a-c). All of the methamphetamine-treated older adults (Table 2) showed toxic effects, solely in the caudateputamen, always sparing the prefrontal cortex. There was some indication that, with the ageing of older adults, the greatest concentrations of silver labelling further expanded to the rostral and dorsal striatal subfields, and the density of precipitates still increased regionally. Altogether, it became obvious that during ageing methamphetamine-induced regions of accumulation of lysosomes clearly shifted from the prefrontal to striatal subfields (Fig. 5).
Age-related methamphetamine neurotoxicity In 44 methamphetamine-treated juveniles (Table 2), typical accumulation of lysosomes occurred exclusively in prefrontal cortical subareas. Both layer II and III of the medial prefrontal cortex were consistently labelled, resulting in a continuous longitudinal strip of silver staining. Further staining occupied nearly all layers, within the dorsal and ventral agranular insular cortex. All of 106 young adults (Table 2), treated with methamphetamine, showed labelling either in prefrontal or caudate-putamen regions and a reasonable
DISCUSSION
The present study has demonstrated agedependent, selective methamphetamine-induced neurotoxicity in prefrontal and/or striatal subareas after
PFC
08
ab
c
!
!
Fig. !. Reconstruction of parasagittal sections of brain of young adult gerbils; level of section planes (a-c) are shown in frontal view of the brain (inset); silver labelling, i.e. accumulation of lysosomes is shown in prefrontal (PFC) and caudate-putamen (CP) subareas, olfactory bulb (OB). Scale: 5 mm.
736
G. TEUCFERT-NOODTand R. R. DAWIRS
applications of single doses. These rather striking effects direct attention to (1) the present methodological approach, investigating the quality of degeneration and further to (2) topographical and (3) agerelated characteristics of responses to single doses. Morphological features of effects of single doses
Various long-term effects of amphetamines on several dopamine markers are now well documented (Steranka, 1982). Acute effects of large doses of methamphetamine on the mammalian brain are described as massive release of DA from presynaptic pools and blockade of monoamine oxidase (MAO) (Ricaurte et al., 1982). Although DA might re-enter the terminals by high affinity re-uptake under these conditions (Steranka, 1982), it will rather be restored but immediately released again, due to persisting large effective concentrations of methamphetamine in the tissue. It has been shown that, when MAO is absent, DA might be metabolized nonenzymatically to 2,4,5-trihydroxy-phenethylamine (6-OHDA) (Senoh and Witkop, 1959). This has recently been verified under in vivo conditions by Seiden and Vosmer (1984), who found formation of 6-OHDA in the striatum of the rat after a single large dose of methamphetamine. This endogenously produced 6-OHDA is a very likely candidate, causing the well-described, long-term or even permanent neuronal damage, also shown when 6-OHDA was applied exogenously. From all this, there is now reasonable evidence to assume that the present results on the labelling of precipitates under light-microscope, as well as accumulation of lysosomes in degrading axon terminals, seen under the electron-microscope, should mainly represent DAergic fibres. At present, DAimmunoreactivity in degrading axon terminals of methamphetamine-treated gerbils is being looked for using antibodies directed against DA. The currently used silver staining technique (Gallyas et al., 1980) proved to be a sensitive method for analyzing responses of selectively affected presynapses (Wolff, Eins, Holzgraefe and Zaborszky, 1980; Holzgraefe, Teuchert-Noodt and Wolff, 1981; Leutgeb, 1986; Teuchert-Noodt, Reissmann and Vockel, 1986; Teuchert-Noodt, Breuker and SchulzeGross, 1987; Teuchert-Noodt, 1989; Wolff el al., 1989). Significant accumulation of silver labelling in prefrontal and striatal subareas were characterized by mostly diffuse distributions of very fine precipitates, resembling pictures after the dense anterograde degeneration of distal axon fibres, following axotomy (Wolff et al., 1980). The present ultrastructural results of methamphetamine-neurotoxicity in gerbils confirmed that degrading axon terminals, accumulating lysosomes, were associated with unaffected postsynapses. In several cases, however, striatal postsynaptic profiles and a small proportion of neurones, exhibited dark staining of the background cytoplasm, accompanied
by all the signs of acute cell degradation. It should not be ruled out that it was the methamphetamineaffected afferents that caused these processes in striatal target cells. There were indications that the cellular degradations were reversible since, in prefrontal areas, these symptoms completely disappeared 7-10 days after treatment with methamphetamine (Wahnschaffe and Esslen, 1985). Nevertheless it is necessary to look for possible metabolic changes and alterations in local neuronal interactions, persisting beyond this critical time, since recently methamphetamine-induced changes in dendritic spine-densities of prefrontal pyramidal cells were found (Dawirs, Teuchert-Noodt and Busse, 1991). The question is, whether or not a single pharmacological impact by methamphetamine may serve as a trigger for further synaptic remodelling, so that reversibility of degradative events would not necessarily mean reestablishment of the structural status quo ante. Since enduring sensitization occurs after treatment with a single dose (Robinson, Becket and Presty, 1982), the present data do not rule out functional changes, by means of compensatory neuroplasticity (Wolff and Wagner, 1983; Dammasch, Wagner and Wolff, 1986, 1988). Topographical pattern of responses to single doses
Obviously, the present methamphetamine-induced toxicity was confined to mesocortical and nigrostriatal DA-projection fields. This further supports assumptions that DA-terminals may be primarily affected. Nevertheless, not all of the well known DA-terminal fields were equally affected: in the cortex, significant occurrence of accumulation of lysosomes was limited to a longitudinal band of superficial layers II/III in the supragenual area (i.e. anterior cingulate cortex), spreading into the pregenual area and to all layers of the agranular insular cortex. These areas correspond very well with terminal fields of the mesocortical DA-projection (for recent review s¢¢: Descarries, Lemay, Doucet and Berger, 1987). However, as well developed deep band of medial DA-innervation, predominating in the prelimbic area of the anterior medial cortex but also extending caudally into supragenual areas of layers II/III, obviously was not affected by the treatment with methamphetamine, since no silver staining was observed. Equally, developmental, morphological and topographical evidence is accumulating that the two superimposed and partly overlapping logitudinal bands of DA-terminations in the pregenual and supragenual cortical areas represent two subsystems of the mesorcortical projection (Berger, Verney, Febvret, Vigny and Helle, 1985; Descarries et al., 1987). Under the assumption that axon terminals of primarily DAergic neurones were degraded by methamphetamine, further physiological differences between these two subsystems may be responsible for the localized toxicity.
• ; :::..
'
Fig. 2. Dark-field photomicrographs (a-c) from parasagittal section planes (cf. Fig. 1) showing distribution patterns of silver labelled precipitates in young adults (2-6 months). Scale: 0.5/zm.
737
Fig. 3. Photomicrographs of silver labelling in a caudate-putamen subarea (cf. Fig. l(a) for designed sector); dark-field micrograph of disposed precipitates (a), light-field micrographs, showing precipitates partly stringed up in fibers (b; arrows) and single dark neurones (c). Scales: 15~tm (a), 0.3ram (b), 0.4 mm (c).
738
F
If libel .li..,,.
_
;!; " . '
:,~...
,~t~ i ~ ..,
:
I
..
J
'.4
: .~
Fig. 4. Electron-micrographs of accumulation of lysomes in a caudate-putamen subarea (cf. Fig. l(a) for designed sector); silver labelling of accumulation of lysosomes in fibre profiles (a--c); non-labelled specimen, showing degraded axon terminals (d-f) associated with either electron lucent (d) or dark profile (f) showing clear postsynaptic differentiation (arrow); degrading neurone (8). Scales: 0.3/lm (a--c), 0.2/ira (d-f), 5/ira (lg).
739
741
Age-related methamphetamine neurotoxicity 1oo
50
o "0
_
o
E 100 Z
I
I
50
0
2
4
6
8
10
12
14
16
Age (months)
Fig. 5. Number of individuals (%) showing age-related methamphetamine-induced silver-labelied precipitates, either in prefrontal (PFC), caudate-putamen subareas or in both; mean age of juveniles (1 month), young adults (4 months), and older adults (16 months).
In the caudate-putamen, too, structural responses to treatment with methamphetamine revealed some kind of spatial selectivity. The mosaic-like pattern in distribution of silver labels resembled the heterogenous representation of numerous parameters in the striatum, with two types of complementary topographical zones, defined as "patch" and "matrix" compartments (Gerfen, 1985; Fukui, Kariyama, Kashiba, Kato and Kimura, 1986). Recently, more detailed information was obtained showing that, although striatal DA innervations appeared to be quite homogenous, nevertheless, nigrostriatal DA projections, organized with dorsal and ventral tiers of mesencephalic DA neurones, were selectively directed to patch and matrix compartments, respectively (G-erfen, Herkenham and Thibault, 1987a). Further, these two topographically divided DA subsystems were found to be both developmentally and hiochemically distinct (Geffen, Baimbridge and Thibault, 1987b). Several pharmacological treatments resulted in selective effects on either DAergic patch or matrixterminal fields (Fukui et al., 1986; Gerfen et al., 1987b). Taken together, it is noteworthy that, as in the prefrontal cortex, also in the caudate-putamen complex, at least the structural responses to methamphetamine were confined to single DA-subsystems. Age-related neurotoxicity of methamphetamine
In adult mammals, toxic effects were observed in the striatum, but not in other regions of the brain (Ellison et al., 1978; Nwanze and Jonsson, 1981; Lorez, 1981; Jonsson and Nwanze, 1982). In contrast, juvenile gerbils exhibited neurotoxic effects solely in prefrontal subareas 0Vahnschaffe and Esslen, 1985). There is one biochemical study on methamphet-
amine-induced changes of the content of DA in young adult rats, which besides depressions in the striatum also reports on minor depressions in the cortex (Ricaurte et al., 1980). Since methamphetamine influences complex mechanisms, like release and reuptake of DA, and enzymeactivity, neurones differentially equipped with respect to these functions, most likely show different responses to the drug. In fact there is evidence that such differences are manifest during development and ageing and between various DA subsystems (Kolb and Nonneman, 1976; Shalaby, Dendel and Spear, 1981; Berger et al., 1985; Gazzara, Fisher and Howard, 1986; Kalsbeek et al., 1988; Voorn, Kalsbeek, Jorritsma-Byham and Groenewegen, 1988). During ageing, rather complex and partly controversial changes were detected in transmitter systems in brain (Moretti et al., 1987). Dopaminergic transmission especially is impaired in various functions, with reduction of the content of transmitter, related enzyme activity, turnover rates and densities of receptors (Carfagna, Trunzo and Moretti, 1986). Nevertheless, which different functional and/or biochemical regional parameters exactly may cause selective vulnerability to challenges with methamphetamine, during subsequent periods of development must be left to future investigations. In conclusion, both, age-related and spatialselective sensitivity of methamphetamine-induced neurotoxicity were found in prefrontal and striatal subareas of the brains of gerbils. It can be argued that primarily DA neurones may be affected by the drug, although this has to be confirmed by future ultrastructural and immunohistochemical studies. Since not all known DA terminal fields were equally affected, those neurones showing methamphetamineinduced terminal degradative processes, may be allocated to "methamphetamine-sensitive DA subsystems". What parameters do these neurones, more than others, make sensitive to pharmacological impacts by single large doses of methamphetamine and further determine the DA subsystems which change methamphetamine-sensitivity throughout life, must be left to future investigations. Acknowledgements--The authors are indebted to Mrs E. Kemming-Graebner and Mrs R. Schulze-Gross for technical assistance. Thanks are due to Dr J. Esslen and Mr M. Friede for their help rendered to us with regard to pharmacological applications and histological preparations. We are also grateful to Mrs Fay Misselbrook for correcting the English draft. REFERENCES
Ikrger B., Verney C., Febvret A., Vigny A. and Helle K. B. (1985) Postnatal ontogenesis of the dopamJnergic innerration in the rat anterior cingulate cortex (area 24). Immtmol~stochemical and catecholamine fluorescence histochemical analysis. Dev. Brain Res. 21: 31-47. Caffagna N., Trunzo F. and Moretti A. (1986) Brain dopamine autoreceptors in aging rats. Exp. Geront. 21: 169-175.
742
G. TEUCFIERT-NOODTand R. R. DAWIRS
Dammasch I. E., Wagner G. P. and Wolff J. R. (1986) Selfstabilization of neuronal networks 1: The compensation algorithm for synaptogenesis. Biol. Cybern. 54: 211-222. Dammasch I. E., Wagner G. P. and Wolff J. R. (1988) Selfstabilization of neuronal networks I1: Stability conditions for synaptogenesis. Biol. Cybern. 58: 149-158. Dawirs R. R., Teuchert-Noodt G. and Busse M. (1991) Single doses of methamphetamine cause changes in the density of dendritic spines in the prefrontal cortex of gerbils ( Meriones unguiculatus ). Neuropharmacology 30:. 275-282. Descarries L., Lemay B., Doucet G. and Berger B. (1987) Regional and laminar density of the dopaminergic innervation in adult rat cerebral cortex. Neuroscience 21: 807-824. Ellison G., Eison M. S., Huberman H. S. and Daniel F. (1978) Long-term changes in dopaminergic innervation of the caudate nucleus after continuous amphetamine administration. Science 201: 276-278. Ellison G. and Eison M. S. (1983) Continuous amphetamine intoxication: An animal model of the acute psychotic episode. Psychol. Med. 13: 751-762. Esslen J. and Teuchert-Noodt G. (1987) Neurotoxische Effecte von Methamphetamin im Gehirn yon Wiistenrennm~.usen (Meriones Unguiculatus) in Abh~ingigkeit vom Entwicklungsalter. In: Verhandlungen der Deutschen Zoologischen Gesellschaft (Barth F. G., Ed.), Vol. 80, pp. 144-155. Fischer, Stuttgart. Fujiwara Y., Kazahaya Y., Nakashima M., Sato M. and Otsuki S. (1987) Behavioral sensitization to methamphetamine in the rat: an ontogenetic study. Psychopharmacology 91:316 -319. Fukui K., Kariyama H., Kashiba A., Kato N. and Kimura H. (1986) Further confirmation of heterogenity of the rat striatum: different mosaic patterns of dopamine fibers after administration of methamphetamine or reserpine. Brain Res. 382:81-86. Fuller R. W. and Hemrick-Leucke S. (1980) Long-lasting depletion of striatal dopamine by a single injection of amphetamine in iprindole-treated rats. Science 209: 305-307. Gallyas F., Wolff J. R., B6ttcher H. and Zaborszky L. (1980) A reliable and sensitive method to localize terminal degeneration and lysosomes in the central nervous system. Stain Technol. 55: 299-306. Gazzara R. A., Fisher F. S. and Howard S. G. (1986) The ontogeny of amphetamine-induced dopamine release in the caudate-putamen of the rat. Dex,. Brain Res. 28: 213-220. Gerfen C. R. (1985) The neostriatal mosaic: I. Compartmental organization of projections from the striatum to the substantia nigra in the rat. J. comp. Neurol. 236: 454-476. Gerfen C. R., Herkenham M. and Thibault J. (1987a) The neostriatal mosaic: II. Patch- and matrix-directed mesostriatal dopaminergic and non-dopaminergic systems. J. Neurosci. 7: 3915-3934. Gerfen C. R., Baimbridge K. G. and Thibault J. (1987b) The neostriatal mosaic: III. Biochemical and developmental dissociation of patch-matrix mesostriatal systems. J. Neurosci. 7: 3935-3944. Hollander H. and Valland J. L. (1968) A reliable staining method for semi-thin sections in experimental neuroanatomy. Brain Res. 10: 120-126. Holzgraefe M., Teuchert-Noodt G. and Wolff J. R. (1981) Chronic isolation of visual cortex induces reorganization of cortico-cortical connections. In: Lesion-Induced Neuronal Plasticity in Sensory-Motor System. (Flohr H. and Precht W., Eds), pp. 351-359. Springer, Berlin. Hyttel J. (1989) Parallel decrease in the density of dopamine D1 and D2 receptors in corpus striatum of rats from 3-25 months of age. Pharmac. Toxic. 64: 55-57.
Jonsson G. and Nwanze E. (1982) Selective (+)-amphetamine neurotoxicity on striatal dopamine nerve terminals in the mouse. Br. J. Pharmac. 77: 335-345. Kalsbeek A., Voorn P., Buijs R. M. and Uylings H. B. M. (1988) Development of the dopaminergic innervation in the prefrontal cortex if the rat. J. comp. Neurol. 269: 58-72. Kashihara K., Sato M., Kazahaya Y. and Otsuki S. (1986) Behavioral hypersensitivity to apomorphine after chronic methamphetamine. Intermittent vs continuous regimen. Jap. J. Psychiat. Neurol. 40: 81--84. Kolb B. and Nonneman A. J. (1976) Functional development of prefrontal cortex in rats continues into adolescence. Science 193: 335-336. Kolta M. G., Shreve P. and Uretsky N. J. (1989) Effects of pretreatment with amphetamine on the interaction between amphetamine and dopamine neurons in the nucleus accumbens. Neuropharmacology 28: 9-14. Leutgeb U. (1986) Plastizit~it von synaptischen Verbindungen nach Kommissurotomien im Hippocampus der Ratte. Med. Inaug.-Diss. Universit~it Marburg. Lorez H. (1981) Fluorescence histochemistry indicates damage of striatal dopamine nerve terminals in rats after multiple dose of methamphetarnine. Life. Sci. 28: 911-916. Marshall J. R. and Altar A. (1986) Striatal dopamine uptake and swim performance of the aged rat. Brain Res. 379; 112-117. McCabe R. T., Hanson G. R. Dawson T. M., Wamsley J. K. and Gibb J. W. (1987) Methamphetamine-induced reduction in D1 and D2 dopamine receptors as evidenced by autoradiography: comparison with tryosine hydroxylase activity. Neuroscience 23: 253-261. Moretti A., Carfagna N. and Trunzo F. (1987) Effects of aging on monoamines and their metabolites in the rat brain. Neurochem Res. 12: 1035-1039. Noisin E. L. and Thomas W. E. (1988) Ontogeny of dopaminergic function in the rat midbrain tegmentum, corpus striatum and frontal cortex. Def. Brain Res. 41: 241--252. Nwanze E. and Jonsson G. (1981) Amphetamine neurotoxicity in dopamine nerve terminals in the caudate nucleus of the mice. Neurosci. Lett. 26: 163-168. O'Boyle K. M. and Waddington J. L. (1986) A re-evaluation of changes in rat striatal D2 dopamine receptors during development and aging. Neurobiol. Aging 7: 265-267. Ricaurte G. A., Schuster C. R. and Seiden L. S. (1980) Long-term effects of repeated methylamphetamine administration on dopamine and serotonin neurons in the rat brain: A regional study. Brain Res. 193: 153-163. Ricaurte G. A., Guillery R. W., Seiden L. S., Schuster C. R. and Moore R. Y. (1982) Dopamine nerve terminal degeneration produced by high doses of methamphetamine in the rat brain. Brain Res. 235: 93-103. Ricaurte G. A., Fuller R. W., Perry K. W., Seiden L. S. and Schuster C. R. (1983) Fluoxetine increases long-lasting neostriatal dopamine depletion after administration of d-methamphetamine and d-amphetamine. Neuropharmacology 22:1165-1169. Ricaurte G. A., Guillery R. W., Seiden L. S. and Schuster C. R. (1984) Nerve terminal degeneration after a single injection of d-amphetamine in iprindole-treated rats: Relation to selective long-lasting dopamine depletion. Brain Res. 191: 378-382. Robinson T. E., Becker J. B. and Presty S. K. (1982) Long-term facilitationof amphetamine-induced rotational behavior and striataldopamine release produced by a single exposure to amphetamine. Sex differences.Brain Res. 253: 231-241. Robinson T. E. and Becker J. B. (1986) Enduring changes in brain and behavior produced by chronic amphetamine administration:A review and evaluation of animal models of amphetamine psychosis. Brain Res. Rev. ll: 157-198.
Age-related methamphetamine neurotoxicity Robinson T. E. and Camp D. M. (1987) Long-lasting effects of escalating doses of d,amphetamine on brain monoamines, amphetamine-induced stereotyped behavior and spontaneous nocturnal locomotion. Pharmac. Biochem. Behv. 26: 821-827. Roubein I. F., Embree L. J. and Jackson D. W. (1986) Changes in catecholamine levels in descrete regions of rat brain during aging. Exp. Aging Res. 12: 193-196. Seeman P. (1981) Brain dopamine receptors. Pharmac. Rev. 32:229-313.
Sciden L. S. and Vosmer G. A. (1984) Formation of 6-hydroxydopamine in caudate nucleus of the rat brain after a single large dose of methamphetamine. Pharmac. Biochem. Behav. 21: 29-31. Senoh S. and Witkop B. (1959) Non-enzymatic conversions of dopamine to norepinephrine and trihydroxyphenethylamines. J. Am. Chem. Soc. 81: 6222-6235. Shalaby I. A., Dendel P. S. and Spear L. P. (1981) Differential functional ontogeny of dopamine presynaptic receptor regulation. Dev. Brain Res. 1: 434-439. Steranka L. R. (1982) Long-term decreases in striatal dopamine, 3,4-dihydroxyphenylacetic acid, and homovanillic acid after a single injection of amphetamine in iprindoletreated rats: time course and time-dependent interactions with amfonelic acid. Brain Res. 234: 123-136. Teuchert-Noodt G., Reismann T. and Vockel A. (1986) Olfaction in peking ducks (Anas platyrhynchos): A comparative study of centrifugal and centripetal olfactory connections in young ducks and in embryos and ducklings (ayes). Zoomorphology 106: 185-198. Teuchert-Noodt G., Breuker K. H. and Schulze-Gross R. (1987) Sequential degeneration processes during ontogeny of peking duck embryos and young ducklings: A light and electron microscopical study of structures in sensory, associative and higher motor brain centres (aves). In: New Frontiers in Brain Research (Eisner N. and Creutzfeldt O., Eds), Proceedings, p. 34. Thieme, Stuttgart. Teuchert-Noodt G. (1989) Central nervous system ontogeny: An evaluation of the recapitulation hypothesis. In: Progress in Zoology, Trends in Vertebrate Morphology (Splechtna H. and Hilgers H., Eds), Vol. 35, pp. 307-314. Fischer, Stuttgart. Trulson M. E., Cannon M. S., Faegg T. S. and Raese J. D. (1987) Tyrosine hydroxylase immunochemistry and
743
quantitative light microscopic study of the mesolimbic dopamine system in rat brain: Effects of chronic methamphetamine administration. Brain Res. Bull. lg: 269-277. Ushijima I., Mizuki Y., Soeda K., Kishimoto O., Hara T. and Yamada M. (1987) Effects of age on behavioral responses to dopamine agonists in the rat. Eur. J. Pharmac. 138: 101-106. Voorn P., Kalsbeek A., Jorritsma-Byham B. and Coroenewegen H. J. (1988) The pre- and postnatal development of the dopaminergic cell groups in the ventral mesencephalon and the dopaminergic innervation of the striatum of the rat. Neuroscience 25: 857-887. Wagner G. C., Ricaurte G. A., Selden L. S., Schuster C. R., Miller R. J. and Westley J. (1980) Long-lasting depletions of striatal dopamine and loss of dopamine uptake sites following repeated administration of methamphetamine. Brain Res. 181: 151-160. Wahnschaffe U. and Esslen J. (1985) Structural evidence for the neurotoxicity of methamphetamine in the frontal cortex of gerbils (Meriones unguiculat~): A light and electron microscopical study. Brain Res. 337: 299-310. Watanabe H. (1987) Differential decrease in the rate of dopamine synthesis in several dopaminergic neurons of aged rat brain. Expl Geront. 22: 17-25. Wolff, J. R., Eins S., Holzgraefe M. and Zaborszky L. (1980) The tempo-spatial course of degeneration after cutting-cortical connections in adult rats. Cell Tissue Res. 214: 303-321. Wolff J. R. and Wagner C. P. (1983) Self-organization in synaptogenesis: Interaction between the formation of excitatory and inhibitory synapses. In: Synergetics of the Brain. Proceedings of the International Symposium on Synergetics, SchloB Elmau, Bavaria (Basra E., Flohr H., Haken H. and Mandall A. J., Eds), pp. 50-59. Springer, Berlin. Wolff J. R., Leutgeb U., Holzgraefe M. and Teuchert G. (1989) Synaptic remodelling during primary and reactive synaptogenesis. In: Fundamentals of Memory Formation: Neuronal Plasticity and Brain Function (Rahman H., Ed.). Progress in Zoology, pp. 68-82. Fischer, Stuttgart. Woolverton W. L., Ricaurte G. A., Forno L. S. and Sciden L. S. (1989) Long-term effect of chronic methamphetamine administration in rhesus monkeys. Brain Res. 486: 73-78.
0028-3908/91 $3.0~)+ 0.00 Copyright© 1991PergamonPress pie
AGE-RELATED TOXICITY IN PREFRONTAL CORTEX A N D C A U D A T E - P U T A M E N COMPLEX OF GERBILS (MERIONES UNGUICULATUS) AFTER A SINGLE DOSE OF METHAMPHETAMINE G. TEUCHERT-NOODTand R. R. D^wms Department of Neuroanatomy, Faculty of Biology, University of Bielefeld, 4800 Bielefeld, Germany (Accepted 15 January 1991)
Summa'y--Single, intermediate to large doses (6-60 mg/kg) of methamphetamine were applied to study the acute neurotoxic effects in developing male gerbils (up to 24 months). A sensitive silver-staining method was used to analyze the toxicity of methamphetamine by light and electron-microscopy. It was shown that treatment with the drug degraded synaptic components, as well as a small population of neurones in the caudate-putamen complex accompanied by accumulation of lysosomes in fibers and axon terminals. In juveniles, methamphetamine in doses of 25-60 mg/kg, resulted in accumulation of lysosomes, selectively in the prefrontal cortex. In young adults, only about half of these doses were sufficient to produce consistent and/or additional effects in the caudate-putamen complex. When the gerbils grew older than 8 months, treatment with drug led to accumulation of lysosomes, exclusivelyin the caudate-putamen, with acute doses ranging from 6 to 12 mg/kg. Acute neurotoxicity with methamphetamine has thus been induced by doses, which hitherto have been claimed to produce behavioural sensitization. Since dopamine (DA) seems the most likely transmitter to be affected, age-related differences in methamphetamine-induced neurotoxicity are discussed in relation to the background of developing DA-response systems, which are still changing in pattern during ageing. Key words--methamphetamine, neurotoxicity, gerbils, development.
"Amphetamine-neurotoxicity" has been described as neuromorphological and neurochemical changes in catecholaminergic systems in brain, with fairly selective effects on dopaminerglc nerve terminals. In that manner, amphetamines cause long-term depletion of both dopamine (DA) and the activity of tyrosine hydroxylase in brain (Ellison, Eison, Huberman and Daniel, 1978; Wagner, Ricaurte, Sciden, Schuster, Miller and Westley, 1980; Nwanze and Jonsson, 1981; Jonsson and Nwanze 1982; Ricaurte, Fuller, Perry, Sciden and Schuster, 1983; Trulson, Cannon, Faegg and Raese, 1987). Levels of metabolites of dopamine (Ricaurte, Guillery, Seiden Schuster and Moore, 1982; Steranke, 1982), populations of DA receptors (McCabe, Hanson, Dawson, Wamsley and Gibb, 1987) and numbers of synaptosomal uptake sites for DA become reduced (Wagner et al., 1980; Woolverton, Ricaurte, Forno and Seiden, 1989). Further, there is some morphological evidence, suggesting neurotoxic damage to DA nerve terminals (Ellison et al., 1978; Nwanze and Jonsson, 1981; Jonsson and Nwanze, 1982; Ricaurte et al., 1982; Recaurte, Guillery, Selden and Schuster, 1984; for further ref. see Robinson and Becker, 1986; Robinson and Camp. 1987), partly indicating spatial selectivity (Lorez, 1981). Neurotoxic responses to amphetamines were described in both the striatum (Ellison et al., 1978; Wagner et al., 1980; Nwanze and Jonsson, 1981; Ricaurte et al., 1982; Ricaurte et al., 1984)
and prefrontal cortex (Wahnschaffe and Esslen, 1985; Trulson et al., 1987). In additon to neurotoxicity, "behavioural sensitization" also occurs in response to administration of amphetamine. In that, animals reach a long-lasting state of supersensitivity to further challenges with amphetamine (Kashihara, Sato, Kazahaya and Otsuki, 1986; Fujiwara, Kazahaya, Nakashima, Sato and Otsuki, 1987; Kolta, Shrev¢ and Uretsky, 1989). Contrary to amphetamine-neurotoxicity, behavioural sensitization is caused by smaller doses, both after intermittent and single application of the drug (Robinson and Camp, 1987). Reports on amphetamine-neurotoxicity are frequently based on continuous administration of the drug through osmotic minipumps or subcutaneous silicone pellets (Ellison et al., 1978; Jonsson and Nwanze, 1982) and intermittent application of large to largest tolerable doses (Wagner et al., 1980; Lorez, 1981; Ricaurte et al., 1983; Trulson et al., 1987; for review see Ellison and Eison, 1983). However, a once-repeated large dose (Ricaurte, Schuster and Sciden, 1980; Wahnschaffe and Esslen, 1985) and even a single large dose after treatment with iprindole (Fuller and Hemrick-Leucke, 1980; Steranka, 1982; Ricaurte et ai., 1984) still results in brain damage. The present study was conducted to analyze the structural phenomena of methamphetamine-induced acute neurotoxicity in the brain of male gerbils, at 733
734
G. TEUCHERT-NOODTand R. R. DAWIRS
different stages of postnatal development. For that purpose, the animals received a single application of methamphetamine, once within their 2 years of postnatal life and morphological changes were studied by light and electron-microscopy. Since DA systems, mostly responsible for the effects of the drug, are under permanent development, starting early in prenatal life and still changing features throughout ageing (Seeman, 1981; Roubein, Embree and Jackson, 1986; O'Boyle and Waddington, 1986; Marshall and Altar, 1986; Ushijima, Mizuki, Soeda, Kishimoto, Hara and Yamada, 1987; Watanabe, 1987; Moretti, Carfagna and Trunzo, 1987; Noison and Thomas, 1988; Kalsbeek, Voorn, Buijs, Pool and Uylings, 1988; Hyttel, 1989), correlations between methamphetamine-induced neurotoxicity and the development of a responding transmitter system were being looked for. METHODS Male gerbils were kept under natural day/night cycles in home cages (30 x 40 cm), 2-6 animals each. Food and water were provided ad libitum. Corresponding to the annual reproductive cycle of the gerbil experiments were performed exclusively from February to November, during 3 subsequent years. The animals received an intermediate to large single dose of methamphetamine (i.p.) and were then housed individually for 3 days, which proved to be the adequate period of survival in all age-groups, in order to produce staining effects by the currently used silver-labelling technique (see below). For applications of methamphetamine, appropriate amounts of "Pervitin" (15 mg methamphetamine-hydrochloride per ml; Temmler-Factories, Marburg, Germany) were diluted in 0.3 ml saline immediately before injection. Control animals received equivalent volumes of saline. The study comprised 3 age-groups, confined to juveniles (1-2 months), young adults (2-6 months) and older adults (8-24 months). A sensitivity to methamphetamine, clearly increasing with age (Esslen and Teuchert-Noodt, 1987), was found. Thus, the doses had to be smaller in older animals, to prevent mortality (Table 1). For light-microscopic studies, the animals were transcardically peffused with 5% formalin or with 5% cacodylate buffered paraformaldehyde (pH 7.2). The brains were subsequently kept in the fixative for a minimum of 2 days Table I. Number of animals (n) treated with methamphetamineor saline (controls); doses were in different ranges (mg/kg) in subsequent age-groups(months) Animals (n) Age Dose-range Methampbetamine(months) (mg/kg) treated Controls 1-2 25-60 44 7 2-6 16-21 106 10 8-24 6-12 22 8
and a maximum of 3 weeks. Serial parasagittal sections (80/~m) were regularly performed from the right side of frozen brains. Additionally coronal sections were cut from the left hemisphere. Processing was according to a selective silver impregnation technique (Gallyas, Wolff, Brttcher and Zaborszky, 1980), highly sensitive for the specific labelling of accumulations of lysosomes accompanying degradation and/or degeneration of axon terminals (Wolff, Leutgeb Holzgraefe and Teuchert, 1989). In order to visualize degrading synaptic components and cells in selected striatal subfields, labelled parasagittal slices were embedded in Epon. A small sector was cut out from the rostral-central region of the striatum (Fig. la) and semi-thin sections were stained for light-microscopical evaluation of distribution patterns of silver stained precipitates. Further, ultra-thin sections were contrasted with uranylacetate and lead citrate and analyzed by electron-microscopy. Additional electron-microscopy was done with 4 methamphetamine-treated young adults, perfused with paraformaldehyde (2.5%) in glutaraldehyde (2.5%) and cacodylate buffer (pH 7.4). Dissected brains were kept in buffer overnight. Parasagittal slices of 1 mm were taken from the designed striatal subfield and further processed for embedding in Epon. Semi-thin sections were stained to look for dark neurones by light-microscopy (Holl/inder and Vaaland, 1968). Ultra-thin sections were provided for electron-microscopical analysis of both accumulation of lysosomes in degrading synaptic elements, as well as morphological correlates of cell death. RESULTS
Morphological features neurotoxicity
of
methamphetamine-
Silver impregnation, visualizing accumulation of lysosomes in an overview pattern throughout serial sections of brain, was detected light-microscopically in both subareas of caudate-putamen complex and prefrontal cortex (schematically in Fig. 1). Using dark-field illumination, the strongest silver labelling was always detected in the central striatum of medial, up to lateral parasagittal section planes (Figs 2 a--c). Labelling appeared either in diffuse or mosaic-like distribution patterns of accumulation of lysosomes (Fig. 3a). Disposed precipitates, covering the tissue on a level with fibre bundles and likewise with cell clusters, were obviously associated with somata and stringed up in fibres (Fig. 3b). Few neurones, exhibiting dark staining, due to increased cytoplasmatic and nuclear densities, bordered others with normal cytological characteristics (Fig. 3c). These dark neurones frequently had shrunken cell bodies, showing irregular contours and sometimes eccentric spherical nuclei. These features were completely absent in controls. Electron-microscopy revealed further details as to subcellular and cellular degradation. Labelled products in silver-impregnated slices accumulated in fibre
735
Age-related methamphetamine neurotoxicity
Table 2. Numberof individuals, in juveniles (I-2), young(2-6) and older adults(8-24)showingq~cificdiverlabelling,i.e. accumulation of lysosorneseither in prefrontal (PFC) or caudate-putamen (CP) subate~ o r in both of thcae regions
profiles (Figs 4 a-c), whereas degraded synaptic elements could hardly be detected because of tissue defects. Non-labelled slices showed lysosomes accumulating in fibres and additionally in degraded presynaptic profiles apposing postsynaptic densities (Fig. 4d). Further, axon terminals were found in the lamellar state of degradation (Fig. 4¢). Irregularly shaped and fairly electron lucent vesicles and multivisicular bodies, were also present. Postsynaptic profiles, associated with degrading terminals, were either electron lucent (Fig. 4d) or as a common mode of typical cell degradation, characterized by pronounced increases in matrix densities (Fig. 4f). It was found that it was a distinct enrichment of free ribosomes, that caused the dark appearance of degrading neurones (Fig. 4g). Some quite unusual amount of clear intracellular spaces represented cisternae of the ¢ndoplasmatic reticulum. The nuclear cromatin was clumped into irregular masses, while evenly dispersed in unaffected cells. Nevertheless, the nuclear membranes of degrading cells remained intact.
A g e in m o n t h s 1-2 2-6 8-24
PFC
CP
PCF and CP
44 49 --
-31 22
-26 --
number exhibited labels in both regions. Distribution patterns of precipitates in the prefrontal cortex of this age-group were in good accordance with those in juveniles. As to animals, with methamphetamineinduced toxic effects in the caudate-putamen, a moderate amount of precipitates became evident thoughout the medial to lateral parasagittal section planes, with strongest staining occurring in the central subfields (Figs 2 a-c). All of the methamphetamine-treated older adults (Table 2) showed toxic effects, solely in the caudateputamen, always sparing the prefrontal cortex. There was some indication that, with the ageing of older adults, the greatest concentrations of silver labelling further expanded to the rostral and dorsal striatal subfields, and the density of precipitates still increased regionally. Altogether, it became obvious that during ageing methamphetamine-induced regions of accumulation of lysosomes clearly shifted from the prefrontal to striatal subfields (Fig. 5).
Age-related methamphetamine neurotoxicity In 44 methamphetamine-treated juveniles (Table 2), typical accumulation of lysosomes occurred exclusively in prefrontal cortical subareas. Both layer II and III of the medial prefrontal cortex were consistently labelled, resulting in a continuous longitudinal strip of silver staining. Further staining occupied nearly all layers, within the dorsal and ventral agranular insular cortex. All of 106 young adults (Table 2), treated with methamphetamine, showed labelling either in prefrontal or caudate-putamen regions and a reasonable
DISCUSSION
The present study has demonstrated agedependent, selective methamphetamine-induced neurotoxicity in prefrontal and/or striatal subareas after
PFC
08
ab
c
!
!
Fig. !. Reconstruction of parasagittal sections of brain of young adult gerbils; level of section planes (a-c) are shown in frontal view of the brain (inset); silver labelling, i.e. accumulation of lysosomes is shown in prefrontal (PFC) and caudate-putamen (CP) subareas, olfactory bulb (OB). Scale: 5 mm.
736
G. TEUCFERT-NOODTand R. R. DAWIRS
applications of single doses. These rather striking effects direct attention to (1) the present methodological approach, investigating the quality of degeneration and further to (2) topographical and (3) agerelated characteristics of responses to single doses. Morphological features of effects of single doses
Various long-term effects of amphetamines on several dopamine markers are now well documented (Steranka, 1982). Acute effects of large doses of methamphetamine on the mammalian brain are described as massive release of DA from presynaptic pools and blockade of monoamine oxidase (MAO) (Ricaurte et al., 1982). Although DA might re-enter the terminals by high affinity re-uptake under these conditions (Steranka, 1982), it will rather be restored but immediately released again, due to persisting large effective concentrations of methamphetamine in the tissue. It has been shown that, when MAO is absent, DA might be metabolized nonenzymatically to 2,4,5-trihydroxy-phenethylamine (6-OHDA) (Senoh and Witkop, 1959). This has recently been verified under in vivo conditions by Seiden and Vosmer (1984), who found formation of 6-OHDA in the striatum of the rat after a single large dose of methamphetamine. This endogenously produced 6-OHDA is a very likely candidate, causing the well-described, long-term or even permanent neuronal damage, also shown when 6-OHDA was applied exogenously. From all this, there is now reasonable evidence to assume that the present results on the labelling of precipitates under light-microscope, as well as accumulation of lysosomes in degrading axon terminals, seen under the electron-microscope, should mainly represent DAergic fibres. At present, DAimmunoreactivity in degrading axon terminals of methamphetamine-treated gerbils is being looked for using antibodies directed against DA. The currently used silver staining technique (Gallyas et al., 1980) proved to be a sensitive method for analyzing responses of selectively affected presynapses (Wolff, Eins, Holzgraefe and Zaborszky, 1980; Holzgraefe, Teuchert-Noodt and Wolff, 1981; Leutgeb, 1986; Teuchert-Noodt, Reissmann and Vockel, 1986; Teuchert-Noodt, Breuker and SchulzeGross, 1987; Teuchert-Noodt, 1989; Wolff el al., 1989). Significant accumulation of silver labelling in prefrontal and striatal subareas were characterized by mostly diffuse distributions of very fine precipitates, resembling pictures after the dense anterograde degeneration of distal axon fibres, following axotomy (Wolff et al., 1980). The present ultrastructural results of methamphetamine-neurotoxicity in gerbils confirmed that degrading axon terminals, accumulating lysosomes, were associated with unaffected postsynapses. In several cases, however, striatal postsynaptic profiles and a small proportion of neurones, exhibited dark staining of the background cytoplasm, accompanied
by all the signs of acute cell degradation. It should not be ruled out that it was the methamphetamineaffected afferents that caused these processes in striatal target cells. There were indications that the cellular degradations were reversible since, in prefrontal areas, these symptoms completely disappeared 7-10 days after treatment with methamphetamine (Wahnschaffe and Esslen, 1985). Nevertheless it is necessary to look for possible metabolic changes and alterations in local neuronal interactions, persisting beyond this critical time, since recently methamphetamine-induced changes in dendritic spine-densities of prefrontal pyramidal cells were found (Dawirs, Teuchert-Noodt and Busse, 1991). The question is, whether or not a single pharmacological impact by methamphetamine may serve as a trigger for further synaptic remodelling, so that reversibility of degradative events would not necessarily mean reestablishment of the structural status quo ante. Since enduring sensitization occurs after treatment with a single dose (Robinson, Becket and Presty, 1982), the present data do not rule out functional changes, by means of compensatory neuroplasticity (Wolff and Wagner, 1983; Dammasch, Wagner and Wolff, 1986, 1988). Topographical pattern of responses to single doses
Obviously, the present methamphetamine-induced toxicity was confined to mesocortical and nigrostriatal DA-projection fields. This further supports assumptions that DA-terminals may be primarily affected. Nevertheless, not all of the well known DA-terminal fields were equally affected: in the cortex, significant occurrence of accumulation of lysosomes was limited to a longitudinal band of superficial layers II/III in the supragenual area (i.e. anterior cingulate cortex), spreading into the pregenual area and to all layers of the agranular insular cortex. These areas correspond very well with terminal fields of the mesocortical DA-projection (for recent review s¢¢: Descarries, Lemay, Doucet and Berger, 1987). However, as well developed deep band of medial DA-innervation, predominating in the prelimbic area of the anterior medial cortex but also extending caudally into supragenual areas of layers II/III, obviously was not affected by the treatment with methamphetamine, since no silver staining was observed. Equally, developmental, morphological and topographical evidence is accumulating that the two superimposed and partly overlapping logitudinal bands of DA-terminations in the pregenual and supragenual cortical areas represent two subsystems of the mesorcortical projection (Berger, Verney, Febvret, Vigny and Helle, 1985; Descarries et al., 1987). Under the assumption that axon terminals of primarily DAergic neurones were degraded by methamphetamine, further physiological differences between these two subsystems may be responsible for the localized toxicity.
• ; :::..
'
Fig. 2. Dark-field photomicrographs (a-c) from parasagittal section planes (cf. Fig. 1) showing distribution patterns of silver labelled precipitates in young adults (2-6 months). Scale: 0.5/zm.
737
Fig. 3. Photomicrographs of silver labelling in a caudate-putamen subarea (cf. Fig. l(a) for designed sector); dark-field micrograph of disposed precipitates (a), light-field micrographs, showing precipitates partly stringed up in fibers (b; arrows) and single dark neurones (c). Scales: 15~tm (a), 0.3ram (b), 0.4 mm (c).
738
F
If libel .li..,,.
_
;!; " . '
:,~...
,~t~ i ~ ..,
:
I
..
J
'.4
: .~
Fig. 4. Electron-micrographs of accumulation of lysomes in a caudate-putamen subarea (cf. Fig. l(a) for designed sector); silver labelling of accumulation of lysosomes in fibre profiles (a--c); non-labelled specimen, showing degraded axon terminals (d-f) associated with either electron lucent (d) or dark profile (f) showing clear postsynaptic differentiation (arrow); degrading neurone (8). Scales: 0.3/lm (a--c), 0.2/ira (d-f), 5/ira (lg).
739
741
Age-related methamphetamine neurotoxicity 1oo
50
o "0
_
o
E 100 Z
I
I
50
0
2
4
6
8
10
12
14
16
Age (months)
Fig. 5. Number of individuals (%) showing age-related methamphetamine-induced silver-labelied precipitates, either in prefrontal (PFC), caudate-putamen subareas or in both; mean age of juveniles (1 month), young adults (4 months), and older adults (16 months).
In the caudate-putamen, too, structural responses to treatment with methamphetamine revealed some kind of spatial selectivity. The mosaic-like pattern in distribution of silver labels resembled the heterogenous representation of numerous parameters in the striatum, with two types of complementary topographical zones, defined as "patch" and "matrix" compartments (Gerfen, 1985; Fukui, Kariyama, Kashiba, Kato and Kimura, 1986). Recently, more detailed information was obtained showing that, although striatal DA innervations appeared to be quite homogenous, nevertheless, nigrostriatal DA projections, organized with dorsal and ventral tiers of mesencephalic DA neurones, were selectively directed to patch and matrix compartments, respectively (G-erfen, Herkenham and Thibault, 1987a). Further, these two topographically divided DA subsystems were found to be both developmentally and hiochemically distinct (Geffen, Baimbridge and Thibault, 1987b). Several pharmacological treatments resulted in selective effects on either DAergic patch or matrixterminal fields (Fukui et al., 1986; Gerfen et al., 1987b). Taken together, it is noteworthy that, as in the prefrontal cortex, also in the caudate-putamen complex, at least the structural responses to methamphetamine were confined to single DA-subsystems. Age-related neurotoxicity of methamphetamine
In adult mammals, toxic effects were observed in the striatum, but not in other regions of the brain (Ellison et al., 1978; Nwanze and Jonsson, 1981; Lorez, 1981; Jonsson and Nwanze, 1982). In contrast, juvenile gerbils exhibited neurotoxic effects solely in prefrontal subareas 0Vahnschaffe and Esslen, 1985). There is one biochemical study on methamphet-
amine-induced changes of the content of DA in young adult rats, which besides depressions in the striatum also reports on minor depressions in the cortex (Ricaurte et al., 1980). Since methamphetamine influences complex mechanisms, like release and reuptake of DA, and enzymeactivity, neurones differentially equipped with respect to these functions, most likely show different responses to the drug. In fact there is evidence that such differences are manifest during development and ageing and between various DA subsystems (Kolb and Nonneman, 1976; Shalaby, Dendel and Spear, 1981; Berger et al., 1985; Gazzara, Fisher and Howard, 1986; Kalsbeek et al., 1988; Voorn, Kalsbeek, Jorritsma-Byham and Groenewegen, 1988). During ageing, rather complex and partly controversial changes were detected in transmitter systems in brain (Moretti et al., 1987). Dopaminergic transmission especially is impaired in various functions, with reduction of the content of transmitter, related enzyme activity, turnover rates and densities of receptors (Carfagna, Trunzo and Moretti, 1986). Nevertheless, which different functional and/or biochemical regional parameters exactly may cause selective vulnerability to challenges with methamphetamine, during subsequent periods of development must be left to future investigations. In conclusion, both, age-related and spatialselective sensitivity of methamphetamine-induced neurotoxicity were found in prefrontal and striatal subareas of the brains of gerbils. It can be argued that primarily DA neurones may be affected by the drug, although this has to be confirmed by future ultrastructural and immunohistochemical studies. Since not all known DA terminal fields were equally affected, those neurones showing methamphetamineinduced terminal degradative processes, may be allocated to "methamphetamine-sensitive DA subsystems". What parameters do these neurones, more than others, make sensitive to pharmacological impacts by single large doses of methamphetamine and further determine the DA subsystems which change methamphetamine-sensitivity throughout life, must be left to future investigations. Acknowledgements--The authors are indebted to Mrs E. Kemming-Graebner and Mrs R. Schulze-Gross for technical assistance. Thanks are due to Dr J. Esslen and Mr M. Friede for their help rendered to us with regard to pharmacological applications and histological preparations. We are also grateful to Mrs Fay Misselbrook for correcting the English draft. REFERENCES
Ikrger B., Verney C., Febvret A., Vigny A. and Helle K. B. (1985) Postnatal ontogenesis of the dopamJnergic innerration in the rat anterior cingulate cortex (area 24). Immtmol~stochemical and catecholamine fluorescence histochemical analysis. Dev. Brain Res. 21: 31-47. Caffagna N., Trunzo F. and Moretti A. (1986) Brain dopamine autoreceptors in aging rats. Exp. Geront. 21: 169-175.
742
G. TEUCFIERT-NOODTand R. R. DAWIRS
Dammasch I. E., Wagner G. P. and Wolff J. R. (1986) Selfstabilization of neuronal networks 1: The compensation algorithm for synaptogenesis. Biol. Cybern. 54: 211-222. Dammasch I. E., Wagner G. P. and Wolff J. R. (1988) Selfstabilization of neuronal networks I1: Stability conditions for synaptogenesis. Biol. Cybern. 58: 149-158. Dawirs R. R., Teuchert-Noodt G. and Busse M. (1991) Single doses of methamphetamine cause changes in the density of dendritic spines in the prefrontal cortex of gerbils ( Meriones unguiculatus ). Neuropharmacology 30:. 275-282. Descarries L., Lemay B., Doucet G. and Berger B. (1987) Regional and laminar density of the dopaminergic innervation in adult rat cerebral cortex. Neuroscience 21: 807-824. Ellison G., Eison M. S., Huberman H. S. and Daniel F. (1978) Long-term changes in dopaminergic innervation of the caudate nucleus after continuous amphetamine administration. Science 201: 276-278. Ellison G. and Eison M. S. (1983) Continuous amphetamine intoxication: An animal model of the acute psychotic episode. Psychol. Med. 13: 751-762. Esslen J. and Teuchert-Noodt G. (1987) Neurotoxische Effecte von Methamphetamin im Gehirn yon Wiistenrennm~.usen (Meriones Unguiculatus) in Abh~ingigkeit vom Entwicklungsalter. In: Verhandlungen der Deutschen Zoologischen Gesellschaft (Barth F. G., Ed.), Vol. 80, pp. 144-155. Fischer, Stuttgart. Fujiwara Y., Kazahaya Y., Nakashima M., Sato M. and Otsuki S. (1987) Behavioral sensitization to methamphetamine in the rat: an ontogenetic study. Psychopharmacology 91:316 -319. Fukui K., Kariyama H., Kashiba A., Kato N. and Kimura H. (1986) Further confirmation of heterogenity of the rat striatum: different mosaic patterns of dopamine fibers after administration of methamphetamine or reserpine. Brain Res. 382:81-86. Fuller R. W. and Hemrick-Leucke S. (1980) Long-lasting depletion of striatal dopamine by a single injection of amphetamine in iprindole-treated rats. Science 209: 305-307. Gallyas F., Wolff J. R., B6ttcher H. and Zaborszky L. (1980) A reliable and sensitive method to localize terminal degeneration and lysosomes in the central nervous system. Stain Technol. 55: 299-306. Gazzara R. A., Fisher F. S. and Howard S. G. (1986) The ontogeny of amphetamine-induced dopamine release in the caudate-putamen of the rat. Dex,. Brain Res. 28: 213-220. Gerfen C. R. (1985) The neostriatal mosaic: I. Compartmental organization of projections from the striatum to the substantia nigra in the rat. J. comp. Neurol. 236: 454-476. Gerfen C. R., Herkenham M. and Thibault J. (1987a) The neostriatal mosaic: II. Patch- and matrix-directed mesostriatal dopaminergic and non-dopaminergic systems. J. Neurosci. 7: 3915-3934. Gerfen C. R., Baimbridge K. G. and Thibault J. (1987b) The neostriatal mosaic: III. Biochemical and developmental dissociation of patch-matrix mesostriatal systems. J. Neurosci. 7: 3935-3944. Hollander H. and Valland J. L. (1968) A reliable staining method for semi-thin sections in experimental neuroanatomy. Brain Res. 10: 120-126. Holzgraefe M., Teuchert-Noodt G. and Wolff J. R. (1981) Chronic isolation of visual cortex induces reorganization of cortico-cortical connections. In: Lesion-Induced Neuronal Plasticity in Sensory-Motor System. (Flohr H. and Precht W., Eds), pp. 351-359. Springer, Berlin. Hyttel J. (1989) Parallel decrease in the density of dopamine D1 and D2 receptors in corpus striatum of rats from 3-25 months of age. Pharmac. Toxic. 64: 55-57.
Jonsson G. and Nwanze E. (1982) Selective (+)-amphetamine neurotoxicity on striatal dopamine nerve terminals in the mouse. Br. J. Pharmac. 77: 335-345. Kalsbeek A., Voorn P., Buijs R. M. and Uylings H. B. M. (1988) Development of the dopaminergic innervation in the prefrontal cortex if the rat. J. comp. Neurol. 269: 58-72. Kashihara K., Sato M., Kazahaya Y. and Otsuki S. (1986) Behavioral hypersensitivity to apomorphine after chronic methamphetamine. Intermittent vs continuous regimen. Jap. J. Psychiat. Neurol. 40: 81--84. Kolb B. and Nonneman A. J. (1976) Functional development of prefrontal cortex in rats continues into adolescence. Science 193: 335-336. Kolta M. G., Shreve P. and Uretsky N. J. (1989) Effects of pretreatment with amphetamine on the interaction between amphetamine and dopamine neurons in the nucleus accumbens. Neuropharmacology 28: 9-14. Leutgeb U. (1986) Plastizit~it von synaptischen Verbindungen nach Kommissurotomien im Hippocampus der Ratte. Med. Inaug.-Diss. Universit~it Marburg. Lorez H. (1981) Fluorescence histochemistry indicates damage of striatal dopamine nerve terminals in rats after multiple dose of methamphetarnine. Life. Sci. 28: 911-916. Marshall J. R. and Altar A. (1986) Striatal dopamine uptake and swim performance of the aged rat. Brain Res. 379; 112-117. McCabe R. T., Hanson G. R. Dawson T. M., Wamsley J. K. and Gibb J. W. (1987) Methamphetamine-induced reduction in D1 and D2 dopamine receptors as evidenced by autoradiography: comparison with tryosine hydroxylase activity. Neuroscience 23: 253-261. Moretti A., Carfagna N. and Trunzo F. (1987) Effects of aging on monoamines and their metabolites in the rat brain. Neurochem Res. 12: 1035-1039. Noisin E. L. and Thomas W. E. (1988) Ontogeny of dopaminergic function in the rat midbrain tegmentum, corpus striatum and frontal cortex. Def. Brain Res. 41: 241--252. Nwanze E. and Jonsson G. (1981) Amphetamine neurotoxicity in dopamine nerve terminals in the caudate nucleus of the mice. Neurosci. Lett. 26: 163-168. O'Boyle K. M. and Waddington J. L. (1986) A re-evaluation of changes in rat striatal D2 dopamine receptors during development and aging. Neurobiol. Aging 7: 265-267. Ricaurte G. A., Schuster C. R. and Seiden L. S. (1980) Long-term effects of repeated methylamphetamine administration on dopamine and serotonin neurons in the rat brain: A regional study. Brain Res. 193: 153-163. Ricaurte G. A., Guillery R. W., Seiden L. S., Schuster C. R. and Moore R. Y. (1982) Dopamine nerve terminal degeneration produced by high doses of methamphetamine in the rat brain. Brain Res. 235: 93-103. Ricaurte G. A., Fuller R. W., Perry K. W., Seiden L. S. and Schuster C. R. (1983) Fluoxetine increases long-lasting neostriatal dopamine depletion after administration of d-methamphetamine and d-amphetamine. Neuropharmacology 22:1165-1169. Ricaurte G. A., Guillery R. W., Seiden L. S. and Schuster C. R. (1984) Nerve terminal degeneration after a single injection of d-amphetamine in iprindole-treated rats: Relation to selective long-lasting dopamine depletion. Brain Res. 191: 378-382. Robinson T. E., Becker J. B. and Presty S. K. (1982) Long-term facilitationof amphetamine-induced rotational behavior and striataldopamine release produced by a single exposure to amphetamine. Sex differences.Brain Res. 253: 231-241. Robinson T. E. and Becker J. B. (1986) Enduring changes in brain and behavior produced by chronic amphetamine administration:A review and evaluation of animal models of amphetamine psychosis. Brain Res. Rev. ll: 157-198.
Age-related methamphetamine neurotoxicity Robinson T. E. and Camp D. M. (1987) Long-lasting effects of escalating doses of d,amphetamine on brain monoamines, amphetamine-induced stereotyped behavior and spontaneous nocturnal locomotion. Pharmac. Biochem. Behv. 26: 821-827. Roubein I. F., Embree L. J. and Jackson D. W. (1986) Changes in catecholamine levels in descrete regions of rat brain during aging. Exp. Aging Res. 12: 193-196. Seeman P. (1981) Brain dopamine receptors. Pharmac. Rev. 32:229-313.
Sciden L. S. and Vosmer G. A. (1984) Formation of 6-hydroxydopamine in caudate nucleus of the rat brain after a single large dose of methamphetamine. Pharmac. Biochem. Behav. 21: 29-31. Senoh S. and Witkop B. (1959) Non-enzymatic conversions of dopamine to norepinephrine and trihydroxyphenethylamines. J. Am. Chem. Soc. 81: 6222-6235. Shalaby I. A., Dendel P. S. and Spear L. P. (1981) Differential functional ontogeny of dopamine presynaptic receptor regulation. Dev. Brain Res. 1: 434-439. Steranka L. R. (1982) Long-term decreases in striatal dopamine, 3,4-dihydroxyphenylacetic acid, and homovanillic acid after a single injection of amphetamine in iprindoletreated rats: time course and time-dependent interactions with amfonelic acid. Brain Res. 234: 123-136. Teuchert-Noodt G., Reismann T. and Vockel A. (1986) Olfaction in peking ducks (Anas platyrhynchos): A comparative study of centrifugal and centripetal olfactory connections in young ducks and in embryos and ducklings (ayes). Zoomorphology 106: 185-198. Teuchert-Noodt G., Breuker K. H. and Schulze-Gross R. (1987) Sequential degeneration processes during ontogeny of peking duck embryos and young ducklings: A light and electron microscopical study of structures in sensory, associative and higher motor brain centres (aves). In: New Frontiers in Brain Research (Eisner N. and Creutzfeldt O., Eds), Proceedings, p. 34. Thieme, Stuttgart. Teuchert-Noodt G. (1989) Central nervous system ontogeny: An evaluation of the recapitulation hypothesis. In: Progress in Zoology, Trends in Vertebrate Morphology (Splechtna H. and Hilgers H., Eds), Vol. 35, pp. 307-314. Fischer, Stuttgart. Trulson M. E., Cannon M. S., Faegg T. S. and Raese J. D. (1987) Tyrosine hydroxylase immunochemistry and
743
quantitative light microscopic study of the mesolimbic dopamine system in rat brain: Effects of chronic methamphetamine administration. Brain Res. Bull. lg: 269-277. Ushijima I., Mizuki Y., Soeda K., Kishimoto O., Hara T. and Yamada M. (1987) Effects of age on behavioral responses to dopamine agonists in the rat. Eur. J. Pharmac. 138: 101-106. Voorn P., Kalsbeek A., Jorritsma-Byham B. and Coroenewegen H. J. (1988) The pre- and postnatal development of the dopaminergic cell groups in the ventral mesencephalon and the dopaminergic innervation of the striatum of the rat. Neuroscience 25: 857-887. Wagner G. C., Ricaurte G. A., Selden L. S., Schuster C. R., Miller R. J. and Westley J. (1980) Long-lasting depletions of striatal dopamine and loss of dopamine uptake sites following repeated administration of methamphetamine. Brain Res. 181: 151-160. Wahnschaffe U. and Esslen J. (1985) Structural evidence for the neurotoxicity of methamphetamine in the frontal cortex of gerbils (Meriones unguiculat~): A light and electron microscopical study. Brain Res. 337: 299-310. Watanabe H. (1987) Differential decrease in the rate of dopamine synthesis in several dopaminergic neurons of aged rat brain. Expl Geront. 22: 17-25. Wolff, J. R., Eins S., Holzgraefe M. and Zaborszky L. (1980) The tempo-spatial course of degeneration after cutting-cortical connections in adult rats. Cell Tissue Res. 214: 303-321. Wolff J. R. and Wagner C. P. (1983) Self-organization in synaptogenesis: Interaction between the formation of excitatory and inhibitory synapses. In: Synergetics of the Brain. Proceedings of the International Symposium on Synergetics, SchloB Elmau, Bavaria (Basra E., Flohr H., Haken H. and Mandall A. J., Eds), pp. 50-59. Springer, Berlin. Wolff J. R., Leutgeb U., Holzgraefe M. and Teuchert G. (1989) Synaptic remodelling during primary and reactive synaptogenesis. In: Fundamentals of Memory Formation: Neuronal Plasticity and Brain Function (Rahman H., Ed.). Progress in Zoology, pp. 68-82. Fischer, Stuttgart. Woolverton W. L., Ricaurte G. A., Forno L. S. and Sciden L. S. (1989) Long-term effect of chronic methamphetamine administration in rhesus monkeys. Brain Res. 486: 73-78.