Neurochemical lesioning in the rat brain with iontophoretic injection of the 1-methyl-4-phenylpyridinium ion (MPP+)

Neuroscience Letters, 141 (1992) 203 207 ,~ 1992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/92/$ 05.00

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Neurochemical lesioning in the rat brain with iontophoretic injection of the 1-methyl-4-phenylpyridinium ion (MPP +) G . J . T e r H o r s t . M.F. K n i g g e a n d A.Van der Wal Departments ol Biolo~,ical Psychiatry and Neurobiolo~y. and Oral Physiology. University ~/' Gronin~en. Gronin~,en : Netherland.~ J (Received 18 July 1991; Revised version 30 March 1992; Accepted 30 March 1992) K
MPP+: lontophoresis: Neurodegeneration; Dopamine; Lipid peroxidation, Rat

lontophoretic injections of the 1-methyl-4-phenylpyridinium ion (MPP +) were made in the dopaminergic part of the substamia nigra to see whether this injection technique could be used for inducing localized neurochemical lesions in dopaminergic cell groups and to assess the effects of M P P on non-dopaminergic neurons. Three days after the iontophoretic injection of M P P , a gliosis or necrotic hole was found in the dopaminergic and non-dopaminergic target areas. This effect depended on the injection parameters that were used; iontophoretic injections of short duration (<- 3 minutes) and low current strength ( 1.5 pA) caused the gliosis, higher injection parameters gave lesions. The estimated injected amount of MPP' was between 0.5 and 10.8 nmol. Control injections, with sodium iodide, sodium chloride or N-mcthylpyridinium iodide showed that the neurodegcneration is not a side-effect of the iontophoretic injection procedure. It is concluded that iontophoretically injected MPP' is toxic for all neurons, irrespective of the neurotransmitter used, and also for glia cells and Iibers of passage. Excessive l\)rmation of free radicals, causing induction of lipid peroxidation, may be involved in the neurodegenerative process observed.

Since Langston's discovery in 1982 that 1-methyl-4phenyl-l,2,3,6-tetrahydropyridine (MPTP) caused Parkinson-like symptoms in young drug addicts, much research has been done to understand the pharmacology of this drug. A morphological characteristic of M P T P neurotoxicity is the relatively selective destruction o f d o p a m ine-containing neurons in the substantia nigra pars compacta (SNc) [1, 15]. This neurodegeneration is not caused by M P T P itself but by a metabolite, the 1-methyl-4-phenylpyridinium ion (MPP-) [7], which is formed in the central nervous system [3] in glia cells and serotonergic neurons, in a reaction catalyzed by monoamine oxidase B (MAO-B) [2]. The MPP + is actively accumulated by the mesencephalic dopaminergic cells of the SNc and ventral tegmental area (VTA) [6] alter its release into the extracellular fluid. This active accumulation of MPP ~ in dopaminergic neurons probably involves the dopamine re-uptake system [7, 14]. A cellular energy crisis, due to M PP+ inhibition of the mitochondrial respiratory chain [9], may eventually cause the death of these dopaminergic neurons (see for review refs. 10 and 15). Stereotaxic pressure microinjections of M P T P or MPP + in the substantia nigra and medial forebrain bun('orrespondencc: G J . Ter Horst, Department of Biological Psychiatry, Bldg. 32, 7th Ilr, Oostersingel 59, 9700 RB Groningen, Netherlands.

die are frequently used for studying the functions of the dopaminergic system in the rat [5, 8]. Pressure microinjections, however, have disadvantages for example due to mechanical damage caused by insertion of the needle and the injection of a volume of neurotoxin solution. Iontophoretic application of the neurotoxin, injecting solely the MPP + ions, would be preferable because of the minimal mechanical damage and the possibilities for lesioning small, specific regions of the substantia nigra. Moreover, with this injection technique the unknown ell fects of MPW on non-dopaminergic cells can be studied. The aim of the present investigation is to see whether it is possible to make small, localized lesions in the dopaminergic part of the substantia nigra with iontopb'~retically injected MPW and to assess the effects of MFP + on non-dopaminergic neurons. 1-Methyl-4-phenylpyridine iodide (MPPI) (kindly provided by Dr. Neal Castagnoli) was dissolved in 0.01 M sodium chloride to a final concentration of 10 mmol. Male Wistar rats weighing 250-300 g were anesthetized with halothane and placed in a Kopf stereotaxic apparatus adjusted to the coordinate system of Paxinos and Watson [11]. Bevelled glass micropipettes with tip diameters of 15 20/am were filled [16] with the MPW solution and stereotaxically inserted into the brain. The target areas were the SNc, the VTA, the striatum, the lateral

204 hypothalamic area, nuclei of the thalamus, the reticular formation, the locus coeruleus and the hippocampus. Iontophoresis was done with a Midgard Constant Current source using alternating positive currents of 1.5, 3 or 6 HA during 1.5, 3, 5, 10 or 20 min. Following iontophoresis the pipette was left in situ for 10 min to avoid the deposit of M P P ÷ in the pipette track. After post-operative survival times of 1 or 3 days, rats were deeply anesthetized with an intraperitoneal injection of I ml of a 6% sodium pentobarbital solution and perfused transcardially, after a short saline prerinse, with 500 ml of a 4% paraformaldehyde solution in 0.05 M phosphate buffer (pH 7.4). Brains were postfixed during 4-6 h and dehydrated overnight at 4°C in 30% sucrose solution in 0.05 M phosphate buffer (pH 7.4). Serial 40 Hm sections were cut on a cryostat microtome and collected in 0.05 M phosphate buffer (pH 7.4). Every second section was mounted onto gelatin coated slides, air dried, counterstained with Cresyl violet, dehydrated in graded solutions of ethanol, cleared in xylene and cover slipped. The adjacent sections were used for revealing tyrosine hydroxylase (TH) immunoreactivity. Briefly, free floating sections were placed 24 h in rabbit anti-TH solution (t: 2,000; Eugene Tech., Allendale, USA), 18 h in goat antirabbit I g G solution (1:250 ; Sigma) and 4 h in rabbit peroxidase-anti-peroxidase solution ( 1:800; Dakopatts). The presence of peroxidase was revealed with a 0.02% solution of diaminobenzidine (DAB) to which 0.8 ml H202 per 100 ml was added. The sections were mounted onto gelatin coated slides, air dried, counterstained with Cresyl violet, dehydrated in graded series of ethanol, cleared in xylene and cover slipped. Control iontophoretic injections were made with 10 m m o l solutions of sodium iodide, sodium chloride and N-methylpyridinium iodide (MW; kindly provided by Dr. H. Rollema). The iontophoretic injections of M P W i n t o the dopaminergic SNc and VTA caused either a gliosis (Fig. 1A) or necrotic hole (Fig. 1B) in the target area. In general, a loss of TH-positive neurons and gliosis was found when, for depositing of the M P P +, a 1.5 HA current strength

was used during 1.5 or 3 min. Longer iontophoresis times (up to 20 min), higher current strength (up to 6 HA) and combinations of a higher current strength and longer injection time all depositing more MPP + caused lesions in the target area. Such lesions were not confined to the dopaminergic area but fanned out into the surrounding structures, affecting TH-negative neurons, glia cells and fibers. These injections suggested that at higher doses M P P ÷ is a non-selective neurotoxin. A distinct gliosis or necrotic hole was found in the SNc 3 days after the iontophoretic M P P + injection. After 24 h, shrunken TH-positive cells with an excentrically located nucleus and vacuoles (Fig. 1H) (indications for an initiated neurodegenerative process) and swollen T H positive fibers (Fig. I G) were found in the SNc. These experiments showed that the degeneration has an early start and manifests itself both in the dopaminergic cell bodies and fibers. The effects of MPP + on non-dopaminergic neurons were studied by placing injections into the GABAergic substantia nigra pars reticulata (SNr) (Fig. I B), the lateral hypothalamic area (Fig. tC), the striatum, the hippocampus (Fig. 1D), the thalamus (Fig. 1E), the - noradrenergic locus coeruleus (Fig. IF) and the reticular formation. Also in these non-dopaminergic target areas a gliosis or lesion was found after 3 days and, analogous to the SNc injections, the appearance of the effect could be predicted. The determining factors were the current strength and duration of the iontophoretic injection. Noteworthy is that dopaminergic neurons in the SNc appeared healthy, 3 days after small and confined MPPinjections into the GABAergic SNr (Fig. IB). F r o m the number of electrons transferred during the iontophoresis we could estimate the injected amount of MPP + which was in the low nanomolar range. In experiments where a gliosis was seen in the target area, approximately 0.5-1.5 nmol M P W was deposited (this calculation is based on the following premises: complete dissociation of M P P I and 1 of every 7 positively charged ions in the 10 m m o l solution is MPP+). A dose- response-like relation was found between the estimated injected

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Fig. 1. Photomicrographs showing neurodegenerative effects of iontophoretic MPP÷ injections in the rat brain. Gliosis in the medial part of the SNc and the VTA (1.5 HA during 1.5 min; bar=300,um) is shown in A. The arrows point at unaffectedTH-positive neurons. B-F: necrotic holes in the SNr (B: 1.5/,tA/5 min; bar=350/,tm), the lateral hypothalamic area (C: 3/.tA/5 min; bar=750/lm), the dentate gyrus (D: 3/,tA/3 min; bar=l ram), the ventrobasal thalamic nuclei (E: 1.5/,tA/l0 min; bar=750 ,urn) and the ventral part of the locus coeruleus (F: 1.5/.tA/5 rain; bar-- 120 Hm). G,H: l-day effects of MPP+ injections on dopaminergic neurons. A swollen TH-positive fiber is shown in G (open arrow points at a normal-sized varicosity (bar= 10/lm)) and a shrunken SNc TH-positiveneuron with two vacuoles(open arrows) and membrane irregularities in H (bar= 15,um). ca 1,2, 3, area I, 2 and 3 of the cornu ammonis; cai, internal capsule; ce, central amygdaloid nucleus; cp, cerebral peduncle; ep, entopeduncular nucleus; ft, fimbria hippocampus; lc, locus coeruleus; ld, laterodorsal thalamic nucleus; m5, mesencephalic trigeminal nucleus; ml, medial lemniscus; rap, mammillary peduncle; mr, mammillothalamic tract; or, optic tract; pb, parabrachial nucleus; sc, subcoeruleus nucleus; snr, reticular part of substantia nigra; vmh, ventromedial hypothalamic nucleus; vpl, lateral ventroposterior thalamic nucleus: vta, ventral tegmental area; zi, zona incerta

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amount of MPP + and the diameter of the lesion (Fig. 2). Comparison of thalamic and nigral lesion sizes, after using the same injection parameters, however, showed that the affected areas in the substantia nigra were always, but non significantly, smaller (not illustrated). Locally, the possibilities for diffusion of MPP ÷ are different; unlike thalamic nuclei, the SN is surrounded by fiber tracts, limiting the diffusion distance. The neurodegeneration observed was not a side effect of the iontophoretic injection procedure, causing for example a pH or osmotic shift in the target area. Control injections with the solvent, 10 mmol sodium iodide or 10 mmol MP ÷ solution (a non-neurotoxic compound structurally related to MPP ÷ [12]) did not induce a gliosis or lesion in the dopaminergic and non-dopaminergic target areas. These iontophoretic injection experiments show that MPP ÷ can be used for making small localized neurochemical lesions in the rat brain but the neurotoxicity is aselective. Iontophoretically injected MPP + destroys dopaminergic and non-dopaminergic neurons, glia cells and fibers of passage. Some indications for a dopamine selective neurotoxicity of low dose MPP + was found in the short time/low current strength experiments where less than 0,5 nmot MPP ÷ was deposited. In these experiments TH-positive neurons could not be identified in the injected part of the SNc. Lipid peroxidation, a process destroying cell membranes after formation of free radicals, is an attractive explanation for the neurodegeneration seen after the iontophoretic injections of MPP +. The rapid breakdown of cell membranes (4-24 h) after infusion [17] or iontophoretic injection of MPP + could not be caused by its 4.0. 3.5"

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Fig. 2. Histogram showing a dose-response-like relation between the estimated iontophoretically injected amount of MPW (nmol) and the size of the lesioned area (mm2). The number of cases used is given between brackets. Each column represents the mean_+S.E.M.

inhibition of the respiratory chain [9] alone but must evolve from a more severe mechanism, like lipid peroxidation. Theoretically, radicals can be produced by MPP' interference with electron transport [9]. Biochemical evidence for MPP ÷ induced lipid peroxidation was found in submitochondrial particles isolated from bovine heart. In this preparation, NADH-dependent formation of malondialdehyde - a product of lipid peroxidation and superoxide increased in the presence of MPW [4]. Moreover, with in vitro spin trapping techniques formation of free radicals was shown when the metabolites of MPTE MPDP- and MPW were incubated together in the absence of submitochondrial particles [ 13]. In summary, the present experiments showed that the 1-methyl-4-phenylpyridinium ion can be injected into the brain iontophoretically and used for neurochemical lesioning. However, after intracerebral iontophoretic injection, MPP ÷ is not a specific dopaminergic neurotoxin. The formation of free radicals and induction of lipid peroxidation may be involved in the neurodegenerative process observed. The authors wish to thank Prof. Dr. N. Castagnoli Jr., Dr. H. Rollema, Dr. M.H.J. Ruiters, Dr. R de Boer and Drs. W. Verhagen-Kamerbeek for their comments on the manuscript and Nieske Brouwer for the histological assistance. 1 Burns, R.S., Chiueh, C.C., Markey, S.R, Ebert, M.H., Jakobowitz, D.M. and Kopin, I.J., A primate model of parkinsonism: selective destruction of dopaminergic neurons in the pars compacta of the substantia nigra by N-methy!-4-phenyl-l,2,3,6-tetrahydropyrimidine, Proc. Natl. Acad. Sci. USA, 80 (1983) 4546-4550. 2 Castagnoli, N., Chiba, K. and Trevor, A.J., Potential bioactivation pathways for the neurotoxin, l-methyl-4-phenyl-l,2,3,6-tetrahydropyrimidine (MPTP), Life Sci., 36 (1985) 225 230. 3 Fuller, R.W. and Hemrick-Luecke, S.K., Tissue concentrations of MPTP and MPP* after administration of lethal and sublethal doses of MPTP to mice, Toxicol. Lett., 54 (1990) 253.262. 4 Hasegawa, E., Takeshige, K., Oishi, T., Mural, Y. and Minakami. S., l-Methyl-4-phenylpyrimidinium (MPP ÷) induces NADH-dependent superoxide formation and enhances NADH-dependent lipidperoxidation in bovine heart submitochondrial particles, Biochem. Biophys. Res. Commun., 170 (1990) 1049-1055. 5 Heikilla, R,E., Nicklas, W.J,, Vyas, t. and Duvoisin, R.C., Dopaminergic toxicity of rotenone and the 1-methyl-4-phenylpyrimidinium ion after their stereotaxic administration to rats: implications for the mechanism of l-methyl-4-phenyl-l,2,3,6-tetrahydropyrimidine toxicity, Neurosci. Lett., 62 (1985) 389-394. 6 Herkenham, M., Little, M.D., Bankiewicz, K., Yang, S.C., Markey, S.P. and Johannessen, J.N., Selective retention of MPP* within the monoaminergic systems of the primate brain following MPTP administration: an in vivo autoradiographic study, Neuroscience, 40 (1991) 133 158. 7 .lavitch, J.A., D'Amato, R.J., Strittmatter, S.M. and Snyder, S.H., Parkinsonism inducing neurotoxin, N-methyl-4-phenyl-l,2,3,6tetrahydropyridine: uptake of the metabolite N-methyl-4-phenyl-

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