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Astroglial ablation prevents MPTP-induced nigrostriatal neuronal death
Brain Research, 509 (1990) 55-61
53
Elsevier BRES 15184
Astroglial ablation prevents MPTP-induced nigrostriatal neuronal death M. Takada, Z.K. Li and T. Hattori Department of Anatomy, University of Toronto, Toronto, Ont. (Canada) (Accepted 11 July 1989)
Key words: 1-Methyl-4-phenyl-l,2,3,6-tetrahydropyridine;a-Aminoadipic acid; Nigrostriatal dopamine system; Parkinsonism; Fluorescent retrograde axonal tracing; Rat
1-Methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP) is a potent neurotoxin which destroys nigrostriatal dopamine neurons, resulting in irreversible idiopathic parkinsonism. MPTP displays dopaminergic neurotoxicity to humans, monkeys, cats and rodents. The oxidative conversion of MPTP to 1-methyl-4-phenylpyridine (MPP +) is responsible for the generation of its neurotoxicity. This metabolism is mediated by the action of monoamine oxidase B, which in the substantia nigra pars compacta (SNc) is localized specifically in astroglia. Employing various combinations of intra-SNc injections of MPTP and the astroglia-specific toxin, L-a-aminoadipic acid (L-a-AA), we examined the effects of selective astroglial ablation on MPTP-induced nigrostriatal neuronal death in the rat. Varying nigrostriatal cell loss was assessed primarily by the aid of fluorescent retrograde axonal tracing. Treatment with MPTP alone caused tremendous nigrostriatal cell loss, while intra-SNc co-injections of MPTP and L-a-AA produced protection against MPTP neurotoxicity in a dose-dependent fashion. Similar effects of L-a-AA occurred in the SNc pretreated with the gliotoxin just prior to or 1 day before MPTP administration. However, this preventive action by L-a-AA was considerably reduced 3 days after its intra-SNc injection. Interestingly, 7 days following l_-a-AA pretreatment, nigrostriatal cell loss was even enhanced rather than attenuated by MPTP administered into the SNc. Thus, our data provide clear morphological evidence for the critical importance of the presence of astroglia in the onset of MPTP neurotoxicity.
INTRODUCTION The c o m p o u n d 1-methyl-4-phenyl-l,2,3,6-tetrahydropyridine ( M P T P ) is increasingly being recognized as a crucial n e u r o t o x i n which induces irreversible neurological s y m p t o m s similar to idiopathic parkinsonism. M P T P causes selective d e g e n e r a t i o n of nigrostriatal d o p a m i n e n e u r o n s in h u m a n s 12"27, m o n k e y s 4'28, cats 48 50, and mice 3" 14,16,54 Recently, both in vivo 13'53 and in vitro 35'37'46 studies have also r e p o r t e d that high concentrations of M P T P exhibit a certain neurotoxicity to rats. It is now indisputable that the oxidative metabolism converting M P T P to 1-methyl-4-phenylpyridine ( M P P ÷) is a critical step for the manifestation of the dopaminergic neurotoxic effect 7'29'32. This active m e t a b o l i t e is p r o d u c e d by the catalysis of m o n o a m i n e oxidase B ( M A O - B ) s, which in the substantia nigra pars c o m p a c t a (SNc) is localized specifically in astroglial cells 31'42'56. A l t h o u g h two recent in vitro studies 2°'43 have indeed implicated astroglia in the conversion of M P T P to M P P ÷, the c o m m i t m e n t of M P T P - i n d u c e d nigrostriatal neuronal death to astroglia has not directly b e e n tested yet. C o n s i d e r a b l e evidence from both in vivo 15"39"40"52 and in vitro 21"22 e x p e r i m e n t s has previously shown selective
toxicity of a - a m i n o a d i p i c acid ( a - A A ) , a 6-carbon chemical analogue of glutamate, to astroglia. These studies have indicated that the toxin displays no degenerative effects on all the other cell types in the central nervous system, including neurons, oligodendroglia, microglia and endothelial cells. In addition to the cellular specificity, comparison of the D- with L-isomer of a - A A has revealed r e m a r k a b l e stereospecificity~5"2~'22'39"52; the latter is the only isomer effective on the production of selective gliotoxicity. F u r t h e r m o r e , our recent work 53 has p r o v i d e d evidence that a c o m b i n a t i o n of r e t r o g r a d e axonal tracing and intracerebral M P T P injections is a successful tool to m o r p h o l o g i c a l l y identify MPTP-induced destruction of nigrostriatal d o p a m i n e neurons in the rat. In the present p a p e r , fluorescent r e t r o g r a d e axonal tracing following various combinations of intraSNc injections of M P T P and L - a - A A , was e m p l o y e d in the rat to examine w h e t h e r the presence of astrocytes is indeed a prerequisite to the generation of M P T P neurotoxicity. We r e p o r t here the varying effects (in both doseand t i m e - d e p e n d e n t manners) of selective astroglial ablation on the precipitation of M P T P - i n d u c e d nigrostriatal neuronal death.
Correspondence: M. Takada, Department of Anatomy, University of Toronto, Medical Sciences Building, Toronto, Ontario M5S 1A8, Canada. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
~b MATERIALS AND METHODS
g r a d e l y labeled S N c cells w e r e e x a m i n e d on both sides f o l l o w i n g T B i n j e c t i o n s which s y m m e t r i c a l l y i n v o l v e d the
Under sodium pentobarbital anesthesia (65 mg/kg b. wt., i.p.), adult male albino rats (Wistar, 250-300 g b. wt.) were positioned in a David Kopf stereotaxic apparatus. Various combinations of unilateral injections of MPTP (Aldrich, MPTP-hydrochloride) and c-ct-AA (Sigma) were made into the SNc. Either or both of MPTP (25 ~tg)53 and L-a-AA (ranging from 10 to 50/~g)_~2 were dissolved in 4/d of 0.05 M phosphate buffer (pH 7.4), and each solution was infused into 2 different rostrocaudal portions of the SNc through a 104d Hamilton microsyringe. In order to avoid physical damages to the SNc, the injection needle was placed somewhat dorsally to the nucleus and the drug-containing solution was slowly delivered over 20 min. Thus, in two needle penetrations, the unilateral SNc of each rat received total doses of 50/~g of MPTP and/or 20-100/~g of L-a-AA. On the contralateral side, symmetrical vehicle (0.05 M phosphate buffer) injections into the SNc were usually carried out to serve as control. First, injections of MPTP alone were made for the estimate of its dopaminergic neurotoxicity yielded by the dose of 50/~g. Second, the dose-dependent effects of L-a-AA on MPTP neurotoxicity were examined by co-injecting increasing doses (20, 40, 60, 80 and 100 ¢tg) of L-a-AA and 50 ¢tg of MPTE Third, the time-dependent effects of L-a-AA (the dose of 80 ktg which had afforded maximal protection) on MPTP neurotoxicity were investigated by setting varying time intervals between the gliotoxin and neurotoxin injections; L-a-AA was pretreatcd just prior to or l, 3, 7 days before MPTP administration. Additionally, co-injections of 50 ktg of MPTP and 80 ¢tg of the D-isomer of a-AA were made to test the stereospecificity of the gliotoxin. Three weeks following the final drug injection, a fluorescent retrograde tracer, True blue (TB, 0.4 ~1 of a 5% aqueous suspension), was stereotaxically deposited bilaterally into the core of the striata through a 1-~1 Hamilton microsyringe. After a survival of 4 days, the animals were deeply reanesthetized and transcardially perfused with 300 ml of 10% formalin in 0.1 M phosphate buffer (pH 7.4). The brains were removed immediately, saturated with 25% sucrose in the same buffer at 4 °C overnight, and then cut serially into coronal sections of 40 ktm thickness on a cryostat. Every second section through the SNc was kept, while every fourth section containing the striatal TB injection site was kept. The sections were mounted onto clean slides and observed with a Leitz fluorescence microscope. An ultraviolet filter providing excitation light of approximately 360 nm wave-length was used to examine the blue-emitting TB-positive cells. In each case, the extent of striatal TB was verified and the number of retrogradely labeled nigrostriatal cells was counted throughout the SNc on both sides, The number of TB-labeled cells on the drug-injected side was expressed as a percentage (the mean _+ S.E.M. for 8 rats) of that on the vehicle-injected (contralateral) side. In some cases (especially in animals administered with MPTP alone or MPTP 7 days after L-a-AA pretreatment), the SNccontaining sections were Nissl-stained with Cresyl violet and/or processed for tyrosine hydroxylase(TH) immunohistochemistry to confirm cell degeneration. According to the peroxidase-antiperoxidase (PAP) technique of Sternberger 5~, the sections were incubated in rabbit antisera against TH (Eugene) for 48 h at 4 °C, followed by goat anti-rabbit IgG (Cappel, 1:50 dilution) for 4 h at room temperature. Then, after incubation with rabbit PAP (Dako, 1:50 dilution) overnight at 4 °C, the sections were reacted in 0.05 M Tris buffer (pH 7.6) containing 0.05% diaminobenzidine (Sigma) and 0.01% H202 for 5-10 min at room temperature. RESULTS T h e d a t a s h o w i n g t h e d o s e - o r t i m e - d e p e n d e n t effects o f L - a - A A o n M P T P - i n d u c e d nigrostriatal cell loss are s u m m a r i z e d in Table I and Fig. 1, r e s p e c t i v e l y . R e t r o -
m a j o r p o r t i o n s of the bilateral striata. In each case, the striatal T B i n j e c t i o n p r o d u c e d i n t e n s e r e t r o g r a d e perikaryal labeling in the SNc on the c o n t r o l side infused with vehicle. T h u s , a large n u m b e r ( a p p r o x i m a t e l y 120-140 cells p e r section) of T B - p o s i t i v e cells w e r e c o n s t a n t l y f o u n d o v e r the full size r a n g e of the S N c on this side (Fig. 2a). C o n v e r s e l y , i n t r a - S N c M P T P a d m i n i s t r a t i o n drastically d e c r e a s e d the n u m b e r of SNc cells r e t r o g r a d e l y l a b e l e d f r o m the striatum. T h e density of T B - c o n t a i n i n g ceils in the S N c i n j e c t e d with 5 0 / ~ g of M P T P was only 19.5 _+ 4 . 0 % of c o n t r o l (Fig. 2b). C o - i n j e c t i o n s of L - a - A A a n d M P T P r e s u l t e d in conspicuous p r o t e c t i o n against M P T P - i n d u c e d nigrostriatal cell loss in a d o s e - d e p e n d e n t fashion. T h e n u m b e r of
100
Q) t~
50
-~oo "Ot'-
rr
i
MPTP
Co
0
1
3
7
Treatment
Fig. 1. Time-dependent effects of L-a-AA on MPTP neurotoxicity. Total doses of 50 ktg of MPTP and/or 80 ktg of L-a-AA were administered into the unilateral SNc with different time intervals. The numbers of retrogradely labeled SNc cells on both sides following TB injections symmetrically involving the major portions of the bilateral striata were compared. Data are expressed as percentages of the control (contralateral) side infused with vehicle. Each bar represents the mean + S.E.M. for 8 rats (P < 0.05). MPTP, MPTP treatment alone; Co, co-injection of MPTP and L-a-AA; 0, 1, 3, 7, L-a-AA pretreatment just prior to, or 1, 3, 7 days before MPTP administration, respectively.
57 TB-labeled SNc cells was increased by the injection of increasing quantities of the gliotoxin, which afforded maximal protection at the dose of 80 ktg (Table I). Thus, after co-injecting MPTP (50 ~g) and 80/~g of L-a-AA, a multitude of SNc cells (as many as 85.0 + 4.2% of control) were labeled with TB retrogradely transported from the striatum (Fig. 2c). In remarkable contrast, the
same dose (80 /~g) of the D-isomer of a-AA did not display any protective effects on dopaminergic neurotoxicity of MPTP (50 ktg); in comparison with the control side, only 17.2 + 3.9% of SNc cells contained TB (Table I). Subsequently, in a second series of experiments, the time-dependent effects of L-a-AA (the dose of 80 /~g
4
Fig. 2. Photomicrographs of retrogradely labeled SNc neurons following TB injections into the striatum. Intra-SNc administration of 50 pg of MPTP and/or 80/~g of L-a-AA was made with different time intervals, a: the SNc on the control side infused with vehicle, b-f: the SNc on the experimental side administered with MPTP and/or L-a-AA. b, MPTP treatment alone; c, co-injection of MPTP and L-ct-AA; d,e,f, L-ct-AA pretreatment 1, 3, 7 days before MPTP administration, respectively. All the pictures show almost the same level of the Snc. An arrow in e indicates an injection needle track, a-f, x69.
58 TABLE I Dose-dependent effects of c-ct-AA on M P T P neurotoxici(v a-AA (/~g):
MPTP* alone . -
%**
19.5+4.0
.
Co-injection of MPTP* and L-~t-AA . . . . . 20 40 60
35.4+8.3
59.0+4.8
.
73.2+5.8
80
100
Co-injection of MPTP* and D-ct-AA .... 80
85.0+4.2
83.8+6.1
17.2_+3.9
* A total dose of 50/ag of MPTP was administered into the unilateral SNc in each case. ** The number of retrogradely labeled nigrostriatal ceils on the drug-injected side is expressed as a percentage of that on the control (contralateral) side infused with vehicle. Each number represents the mean + S.E.M. for 8 rats. which had produced maximal protection) on MPTPinduced nigrostriatal cell loss were examined (Fig. 1). Pretreatment with L-a-AA just prior to or 1 day before MPTP administration appeared to prevent nigrostriatal cells from degenerating. Thus, in both cases TB-labeled cells were evident in the SNc (Fig. 2d). However, such protection against MPTP neurotoxicity by L - a - A A was considerably attenuated in the SNc with MPTP administration 3 days following L-a-AA pretreatment; the number of TB-containing nigrostriatal cells went down to 51.4 + 7.2% of control (Fig. 2e). Interestingly, intra-SNc MPTP administration 7 days after L - a - A A pretreatment did even enhance rather than reduce its dopaminergic neurotoxicity. Thus, severer nigrostriatal cell loss occurred in this case than in the case of MPTP treatment alone (11.5 + 2.6% compared with 19.5 + 4.0% of TB-positive cells of control) (Fig. 2f). In some cases (especially in animals administered with MPTP alone or MPTP 7 days after L - a - A A pretreatmerit), Nissl-staining and/or T H immunohistochemistry were performed to verify SNc cell loss caused by MPTP. Tissue from these animals showed a marked decrease in (Nissl-stained and/or TH-immunoreactive) cell number in the SNc as compared with the control side, and surviving cells largely overlapped TB-labeled ones (not illustrated). Additionally, many of these animals progressively lost body weight and manifested discernible abnormalities in ambulatory/rotational behavior. DISCUSSION The selective gliotoxic effect of L-a-AA, a 6-carbon chemical analogue of glutamate, has previously been shown both in v i v o 15'39'4°'52 and in vitro 21'2a. Upon systemic administration to the infant mouse, L - a - A A causes glial lesions in the arcuate nucleus of the hypothalamus and retina 39'4°. In vitro treatment of the dissociated postnatal mouse cerebellum with the toxin results in a rapid nuclear and cytoplasmic swelling of astroglia 21. Furthermore, L-a-AA which is selectively taken up by astroglia and probably kills the cell following its intracellular accumulation, affects astrocytes only 22.
We have also demonstrated that similar astroglia-specific lesions in the rat can be produced by the stereotaxic intracerebral injection of L-a-AA 15'52. The present model system is the first to postulate the use of L-a-AA as a tool for selective astrogtial ablation. There has been a controversy over the vulnerability of the rat brain to MPTP. The relatively slower metabolism rate (i.e. inefficient conversion) of M P T P and/or the considerably shorter biological half life (i,e. insufficient retention) of MPP ÷, may issue part of factors in the lesser sensitivity of the rat to MPTP as compared with monkey or mouse z6. Also, [3H]MPTP binding sites are concentrated in the SNc and striatum in the human brain. whereas these regions have substantially fewer binding sites in the rat brain 25. In fact, MPTP causes behavioral impairments (possibly due to striatat dopamine depletion) in the rat without conspicuous nigrostriatal lesions 3" 9.45 although MPP -~ is extremely toxic to rat dopamine n e u r o n s 1"3'18"37"46"47. However, recent available evidence has revealed that high concentrations of MPTP itself elicit a certain neurotoxicity to rat SNc cells (to a lesser extent ventral tegmental area and dorsal raphe cells as well) both in vivo 13 and in vitro 35'37'46. We have also provided anatomical evidence, with the aid of retrograde axonal tracing, that fairly large amounts (the same as used in the present study) of MPTP administered directly into the SNc or medial forebrain bundle (MFB) (but not systemically), are toxic to rat nigrostriatal neurons s3. It is obvious that MPTP is oxidized to the major active metabolite, MPP ~-, by MAO-B 8, which is localized predominantly in astrocytes 31'42'56 and serotonergic neurons 31'38"55'56 in the brain. These histochemical studies have also pointed out that dopamine cells in the SNc fail to possess MAO-B, thus suggesting that nigrostriatal neurons themselves do not display the capacity to oxidize MPTP to MPP + Therefore. it is most likely that astroglia in the SNc region must be involved in converting MPTP to MPP + for the onset of its dopaminergic neurotoxicity. Indeed. two recent in vitro experiments 2°.43 have indicated a critical role of astroglia in the generanon of MPTP neurotoxicity. Cultured astrocytes take u p [3H]MPTP 2° and promote the conversion of MPTP to MPP ÷
59 (ref. 43). Moreover, high-affinity binding sites for [3H]MPTP correspond to the localization of MAO-B (virtually [3H]pargyline binding) 2'41. Our recent investigation 5 has also shown heavy glial labeling as well as neuronal labeling in the SNc area following intraventricular injections of [3H]MPTP. In the present study, fluorescent retrograde axonal tracing after various combinations of intra-SNc injections of MPTP and L-a-AA, has been employed to examine the effects of selective astroglial ablation on MPTP-induced nigrostriatal neuronal death. Our results clearly demonstrate that L-a-AA confers protection against MPTP in a dose-dependent manner. MPTP-induced loss of retrogradely labeled nigrostriatal cells is dramatically reduced by increasing doses of the gliotoxin. However, it may be a little puzzling that L-a-AA is strikingly protective when co-administered with MPTP. One would presume that some time would be required for the toxic effects of this compound to almost completely disable astroglia to mediate the conversion of MPTP to MPP +. To our knowledge, no other actions of L-a-AA to prevent MPTP neurotoxicity via some different mechanism have hitherto intimated. At present, it is most reasonable to understand that La-AA-induced glial dysfunction might occur so promptly as to obstruct the oxidative metabolism of MPTP. Additonally, co-injections of MPTP and the D-isomer of a-AA fail to afford any preventive effects. This stereospecificity of the gliotoxin is in full agreement with p r e v i o u s reports 15'21'22"39'52. Furthermore, our combined intra-SNc injections of both drugs with different time intervals respect the peculiarity of the time-dependent effects that L-a-AA exerts over MPTP. L-a-AA pretreatment just prior to or 1 day before MPTP administration, favors conspicuous protection against MPTP. Conversely, the neurotoxic potency of MPTP is considerably restored 3 days after L-a-AA pretreatment, and is finally even enhanced rather than attenuated by the gliotoxin pretreated 7 days before MPTP administration. Thus, as compared with the case of MPTP treatment alone, severer loss of cells retrogradely labeled from the striatum occurs in the SNc. Our previous findings on the fine structural changes after the intracerebral injection of L-ct-AA 15'52 might account for this reversed phenomenon. Tremendous invasion of reactive astrocytes starting 3 days after the gtiotoxin injection, indicative of the recovery of astroglial function, reached its peak 7 days after the injection 15,52. A large number of these recruited astrocytes may accumulate excessive MAO-B to oxidize more efficiently MPTP to MPP +, which even destroys surviving nigrostriatal cells under the toxic environment produced by regularly administered MPTP. To date, attempts to reduce or prevent MPTP neuro-
toxicity have been focussed on the interference, by specific inhibitors, with MAO-B function or dopamine transport system, both of which are believed to be instrumental for the generation of its toxic effect. Indeed, MAO-B inhibitors (including pargyline and deprenyl) prevent the conversion of MPTP to MPP* (refs. 8, 32), and administration of these inhibitors prior to MPTP treatment is protective against MPTP neurotoxicity as well m'17"3°'35'36. Moreover, the fact that dopamine uptake inhibitors (including mazindol and amfenolic acid) protect against striatal dopamine depletion 33"44 and dopaminergic cell loss 46 caused by MPP +, supports the notion that the uptake system of MPP + into dopaminergic neurons is responsible for selective neurotoxicity of MPTP 24. There continues to be a great deal of debate regarding the primary site of action of MPTP/MPP + over nigrostriatal neurons. While many investigators believe that the major active site is at the level of SNc cell bodies, some argue that the compound first acts at the striatal terminal field and consequentially affects parent cells via retrograde axonal transport 48or simply retrograde degeneration 19. The present data showing that astroglial ablation at the SNc level prevents nigrostriatal cell loss following intra-SNc MPTP injections (see also ref. 53), stand in favor of the former idea, even though the latter cannot thoroughly be excluded. In fact, our recent autoradiographic study 6 has successfully demonstrated the capability of MPTP (eventually MPP +) to be taken up by nigrostriatal terminals and transported retrogradely to SNc cell bodies. Furthermore, SNc cell elimination resulting from intrastriata123 or intra-MFB 53 MPTP infusion respects retrograde degeneration. Astroglial ablation at the SNc or striatal level prior to systemic MPTP administration would be strategic to definitively determine its primary active site. We now elucidate a pronounced involvement of astroglia in the precipitation of MPTP-induced nigrostriatal neuronal death in vivo. The presence of astroglia must be instrumental in the mediation of drug-induced parkinsonism. The eminent role of astroglia seems peculiar to this major brain disease as yet, and the link between the cell and other types of neurodegenerative disorders remains a mystery. In this context, we have previously reported that L-a-AA also protects against kainic acid neurotoxicity in the rat striatum is. However, the possible functional relationship of astroglia to kainic acidqnduced striatal cell death, which duplicates Huntington's chorea 11'34, still remains elusive. Thus, astroglia may subserve a so-far-unappreciated role in the brain's defense mechanism against neurotoxic insults.
Acknowledgements. Supported by the Medical Research Council of Canada. M.T. is an MRC Postprofessional Fellow.
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61 38 Nakamura, S. and Vincent, S.R., Histochemistry of MPTP oxidation in the rat brain: sites of synthesis of the parkinsonisminducing toxin MPP +, Neurosci. Lett., 65 (1986) 321-325. 39 Olney, J.W., de Gubareff, T. and Collins, J.F., Stereospecificity of the gliotoxic and anti-neurotoxic actions of alpha-aminoadipate, Neurosci. Lett., 19 (1980) 277-282. 40 Olney, J.W., Ho, O.L. and Rhee, V., Cytotoxic effects of acidic and sulphur containing aminoacids on the infant mouse central nervous system, Exp. Brain Res., 14 (1971) 61-76. 41 Parsons, B. and Rainbow, T.C., High-affinity binding sites for (3H)MPTP may correspond to monoamine oxidase, Eur. J. Pharmacol., 102 (1984) 375-377. 42 Pintar, J.E., Levitt, P., Salach, J.l., Weyler, W., Rosenberg, M.B. and Breakefield, X.O., Specificity of antisera prepared against pure bovine MAO-B, Brain Research, 276 (1983) 127-139. 43 Ransom, B.R., Kunis, D.M., Irwin, I. and Langston, J.W., Astrocytes convert the parkinsonism inducing neurotoxin, MPTP, to its active metabolite, MPP +, Neurosci. Lett., 75 (1987) 323-328. 44 Ricaurte, G.A., Langston, J.W., DeLanney, L.E., Irwin, I. and Brooks, J.D., Dopamine uptake blockers protect against the dopamine depleting effect of l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP) in the mouse striatum, Neurosci. Lett., 59 (1985) 259-264. 45 Sahgal, A., Andrews, J.S., Biggins, J.A., Candy, J.M., Edwardson, J.A., Keith, A.B., Turner, J.D. and Wright, C., N-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP) affects locomotor activity without producing a nigrostriatal lesion in the rat, Neurosci. Lett., 48 (1984) 179-184. 46 Sanchez-Ramos, J., Barrett, J.N., Goldstein, M., Weiner, W.J. and Hefti, F., 1-Methyl-4-phenylpyridinium (MPP+) but not 1-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP) selectively destroys dopaminergic neurons in cultures of dissociated rat mesencephalic neurons, Neurosci. Lett., 72 (1986) 215-220. 47 Sanchez-Ramos, J.R., Michel, P., Weiner, W.J. and Hefti, E, Selective destruction of cultured dopaminergic neurons from
48
49
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55 56
fetal rat mesencephalon by 1-methyl-4-phenylpyridinium: cytochemical and morphological evidence, J. Neurochem., 50 (1988) 1934-1944. Schneider, J.S., MPTP parkinsonism in the cat: pattern of neuronal loss may partially be explained by the distribution of MAO-B in the brain. In M.B. Carpenter and A. Jayaraman (Eds.), The Basal Ganglia. H. Structure and Function- Current Concepts, Advances in Behavioral Biology, Vol. 32, Plenum, New York, 1987, pp. 405-413. Schneider, J.S. and Markham, C.H., Neurotoxic effects of Nomethyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP) in the cat. Tyrosine hydroxylase immunohistochemistry, Brain Research, 373 (1986) 258-267. Schneider, J.S., Yuwiler, A. and Markham, C.H., Production of a Parkinson-like syndrome in the cat with N-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP): behavior, histology, and biochemistry, Exp. Neurol., 91 (1986) 293-307. Sternberger, L.A., Immunocytochemistry, 2nd edn., Wiley, New York, 1979. Takada, M. and Hattori, T., Fine structural changes in the rat brain after local injections of gliotoxin, alpha-aminoadipic acid, Histol. Histopathol., 1 (1986) 271-275. Takada, M., Li, Z.K. and Hattori, T, Intracerebral MPTP injections in the rat cause cell loss in the substantia nigra, ventral tegmental area and dorsal raphe, Neurosci. Lett., 78 (1987) 145-150. Wallace, R.A., Boldry, R., Schmittgen, T., Miller, D. and Uretsky, N., Effect of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on monoamine neurotransmitters in mouse brain and heart, Life Sci., 35 (1984) 285-291. Westlund, K.N., Denney, R.M. Kochersperger, L.M., Rose, R.M. and Abell, C.W., Distinct monoamine oxidase A and B populations in primate brain, Science, 230 (1985) 181-183. Westlund, K.N., Denney, R.M., Rose, R.M. and Abell, C.W., Localization of distinct monoamine oxidase A and monoamine oxidase B cell populations in human brainstem, Neuroscience, 25 (1988) 439-456.
53
Elsevier BRES 15184
Astroglial ablation prevents MPTP-induced nigrostriatal neuronal death M. Takada, Z.K. Li and T. Hattori Department of Anatomy, University of Toronto, Toronto, Ont. (Canada) (Accepted 11 July 1989)
Key words: 1-Methyl-4-phenyl-l,2,3,6-tetrahydropyridine;a-Aminoadipic acid; Nigrostriatal dopamine system; Parkinsonism; Fluorescent retrograde axonal tracing; Rat
1-Methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP) is a potent neurotoxin which destroys nigrostriatal dopamine neurons, resulting in irreversible idiopathic parkinsonism. MPTP displays dopaminergic neurotoxicity to humans, monkeys, cats and rodents. The oxidative conversion of MPTP to 1-methyl-4-phenylpyridine (MPP +) is responsible for the generation of its neurotoxicity. This metabolism is mediated by the action of monoamine oxidase B, which in the substantia nigra pars compacta (SNc) is localized specifically in astroglia. Employing various combinations of intra-SNc injections of MPTP and the astroglia-specific toxin, L-a-aminoadipic acid (L-a-AA), we examined the effects of selective astroglial ablation on MPTP-induced nigrostriatal neuronal death in the rat. Varying nigrostriatal cell loss was assessed primarily by the aid of fluorescent retrograde axonal tracing. Treatment with MPTP alone caused tremendous nigrostriatal cell loss, while intra-SNc co-injections of MPTP and L-a-AA produced protection against MPTP neurotoxicity in a dose-dependent fashion. Similar effects of L-a-AA occurred in the SNc pretreated with the gliotoxin just prior to or 1 day before MPTP administration. However, this preventive action by L-a-AA was considerably reduced 3 days after its intra-SNc injection. Interestingly, 7 days following l_-a-AA pretreatment, nigrostriatal cell loss was even enhanced rather than attenuated by MPTP administered into the SNc. Thus, our data provide clear morphological evidence for the critical importance of the presence of astroglia in the onset of MPTP neurotoxicity.
INTRODUCTION The c o m p o u n d 1-methyl-4-phenyl-l,2,3,6-tetrahydropyridine ( M P T P ) is increasingly being recognized as a crucial n e u r o t o x i n which induces irreversible neurological s y m p t o m s similar to idiopathic parkinsonism. M P T P causes selective d e g e n e r a t i o n of nigrostriatal d o p a m i n e n e u r o n s in h u m a n s 12"27, m o n k e y s 4'28, cats 48 50, and mice 3" 14,16,54 Recently, both in vivo 13'53 and in vitro 35'37'46 studies have also r e p o r t e d that high concentrations of M P T P exhibit a certain neurotoxicity to rats. It is now indisputable that the oxidative metabolism converting M P T P to 1-methyl-4-phenylpyridine ( M P P ÷) is a critical step for the manifestation of the dopaminergic neurotoxic effect 7'29'32. This active m e t a b o l i t e is p r o d u c e d by the catalysis of m o n o a m i n e oxidase B ( M A O - B ) s, which in the substantia nigra pars c o m p a c t a (SNc) is localized specifically in astroglial cells 31'42'56. A l t h o u g h two recent in vitro studies 2°'43 have indeed implicated astroglia in the conversion of M P T P to M P P ÷, the c o m m i t m e n t of M P T P - i n d u c e d nigrostriatal neuronal death to astroglia has not directly b e e n tested yet. C o n s i d e r a b l e evidence from both in vivo 15"39"40"52 and in vitro 21"22 e x p e r i m e n t s has previously shown selective
toxicity of a - a m i n o a d i p i c acid ( a - A A ) , a 6-carbon chemical analogue of glutamate, to astroglia. These studies have indicated that the toxin displays no degenerative effects on all the other cell types in the central nervous system, including neurons, oligodendroglia, microglia and endothelial cells. In addition to the cellular specificity, comparison of the D- with L-isomer of a - A A has revealed r e m a r k a b l e stereospecificity~5"2~'22'39"52; the latter is the only isomer effective on the production of selective gliotoxicity. F u r t h e r m o r e , our recent work 53 has p r o v i d e d evidence that a c o m b i n a t i o n of r e t r o g r a d e axonal tracing and intracerebral M P T P injections is a successful tool to m o r p h o l o g i c a l l y identify MPTP-induced destruction of nigrostriatal d o p a m i n e neurons in the rat. In the present p a p e r , fluorescent r e t r o g r a d e axonal tracing following various combinations of intraSNc injections of M P T P and L - a - A A , was e m p l o y e d in the rat to examine w h e t h e r the presence of astrocytes is indeed a prerequisite to the generation of M P T P neurotoxicity. We r e p o r t here the varying effects (in both doseand t i m e - d e p e n d e n t manners) of selective astroglial ablation on the precipitation of M P T P - i n d u c e d nigrostriatal neuronal death.
Correspondence: M. Takada, Department of Anatomy, University of Toronto, Medical Sciences Building, Toronto, Ontario M5S 1A8, Canada. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
~b MATERIALS AND METHODS
g r a d e l y labeled S N c cells w e r e e x a m i n e d on both sides f o l l o w i n g T B i n j e c t i o n s which s y m m e t r i c a l l y i n v o l v e d the
Under sodium pentobarbital anesthesia (65 mg/kg b. wt., i.p.), adult male albino rats (Wistar, 250-300 g b. wt.) were positioned in a David Kopf stereotaxic apparatus. Various combinations of unilateral injections of MPTP (Aldrich, MPTP-hydrochloride) and c-ct-AA (Sigma) were made into the SNc. Either or both of MPTP (25 ~tg)53 and L-a-AA (ranging from 10 to 50/~g)_~2 were dissolved in 4/d of 0.05 M phosphate buffer (pH 7.4), and each solution was infused into 2 different rostrocaudal portions of the SNc through a 104d Hamilton microsyringe. In order to avoid physical damages to the SNc, the injection needle was placed somewhat dorsally to the nucleus and the drug-containing solution was slowly delivered over 20 min. Thus, in two needle penetrations, the unilateral SNc of each rat received total doses of 50/~g of MPTP and/or 20-100/~g of L-a-AA. On the contralateral side, symmetrical vehicle (0.05 M phosphate buffer) injections into the SNc were usually carried out to serve as control. First, injections of MPTP alone were made for the estimate of its dopaminergic neurotoxicity yielded by the dose of 50/~g. Second, the dose-dependent effects of L-a-AA on MPTP neurotoxicity were examined by co-injecting increasing doses (20, 40, 60, 80 and 100 ¢tg) of L-a-AA and 50 ¢tg of MPTE Third, the time-dependent effects of L-a-AA (the dose of 80 ktg which had afforded maximal protection) on MPTP neurotoxicity were investigated by setting varying time intervals between the gliotoxin and neurotoxin injections; L-a-AA was pretreatcd just prior to or l, 3, 7 days before MPTP administration. Additionally, co-injections of 50 ktg of MPTP and 80 ¢tg of the D-isomer of a-AA were made to test the stereospecificity of the gliotoxin. Three weeks following the final drug injection, a fluorescent retrograde tracer, True blue (TB, 0.4 ~1 of a 5% aqueous suspension), was stereotaxically deposited bilaterally into the core of the striata through a 1-~1 Hamilton microsyringe. After a survival of 4 days, the animals were deeply reanesthetized and transcardially perfused with 300 ml of 10% formalin in 0.1 M phosphate buffer (pH 7.4). The brains were removed immediately, saturated with 25% sucrose in the same buffer at 4 °C overnight, and then cut serially into coronal sections of 40 ktm thickness on a cryostat. Every second section through the SNc was kept, while every fourth section containing the striatal TB injection site was kept. The sections were mounted onto clean slides and observed with a Leitz fluorescence microscope. An ultraviolet filter providing excitation light of approximately 360 nm wave-length was used to examine the blue-emitting TB-positive cells. In each case, the extent of striatal TB was verified and the number of retrogradely labeled nigrostriatal cells was counted throughout the SNc on both sides, The number of TB-labeled cells on the drug-injected side was expressed as a percentage (the mean _+ S.E.M. for 8 rats) of that on the vehicle-injected (contralateral) side. In some cases (especially in animals administered with MPTP alone or MPTP 7 days after L-a-AA pretreatment), the SNccontaining sections were Nissl-stained with Cresyl violet and/or processed for tyrosine hydroxylase(TH) immunohistochemistry to confirm cell degeneration. According to the peroxidase-antiperoxidase (PAP) technique of Sternberger 5~, the sections were incubated in rabbit antisera against TH (Eugene) for 48 h at 4 °C, followed by goat anti-rabbit IgG (Cappel, 1:50 dilution) for 4 h at room temperature. Then, after incubation with rabbit PAP (Dako, 1:50 dilution) overnight at 4 °C, the sections were reacted in 0.05 M Tris buffer (pH 7.6) containing 0.05% diaminobenzidine (Sigma) and 0.01% H202 for 5-10 min at room temperature. RESULTS T h e d a t a s h o w i n g t h e d o s e - o r t i m e - d e p e n d e n t effects o f L - a - A A o n M P T P - i n d u c e d nigrostriatal cell loss are s u m m a r i z e d in Table I and Fig. 1, r e s p e c t i v e l y . R e t r o -
m a j o r p o r t i o n s of the bilateral striata. In each case, the striatal T B i n j e c t i o n p r o d u c e d i n t e n s e r e t r o g r a d e perikaryal labeling in the SNc on the c o n t r o l side infused with vehicle. T h u s , a large n u m b e r ( a p p r o x i m a t e l y 120-140 cells p e r section) of T B - p o s i t i v e cells w e r e c o n s t a n t l y f o u n d o v e r the full size r a n g e of the S N c on this side (Fig. 2a). C o n v e r s e l y , i n t r a - S N c M P T P a d m i n i s t r a t i o n drastically d e c r e a s e d the n u m b e r of SNc cells r e t r o g r a d e l y l a b e l e d f r o m the striatum. T h e density of T B - c o n t a i n i n g ceils in the S N c i n j e c t e d with 5 0 / ~ g of M P T P was only 19.5 _+ 4 . 0 % of c o n t r o l (Fig. 2b). C o - i n j e c t i o n s of L - a - A A a n d M P T P r e s u l t e d in conspicuous p r o t e c t i o n against M P T P - i n d u c e d nigrostriatal cell loss in a d o s e - d e p e n d e n t fashion. T h e n u m b e r of
100
Q) t~
50
-~oo "Ot'-
rr
i
MPTP
Co
0
1
3
7
Treatment
Fig. 1. Time-dependent effects of L-a-AA on MPTP neurotoxicity. Total doses of 50 ktg of MPTP and/or 80 ktg of L-a-AA were administered into the unilateral SNc with different time intervals. The numbers of retrogradely labeled SNc cells on both sides following TB injections symmetrically involving the major portions of the bilateral striata were compared. Data are expressed as percentages of the control (contralateral) side infused with vehicle. Each bar represents the mean + S.E.M. for 8 rats (P < 0.05). MPTP, MPTP treatment alone; Co, co-injection of MPTP and L-a-AA; 0, 1, 3, 7, L-a-AA pretreatment just prior to, or 1, 3, 7 days before MPTP administration, respectively.
57 TB-labeled SNc cells was increased by the injection of increasing quantities of the gliotoxin, which afforded maximal protection at the dose of 80 ktg (Table I). Thus, after co-injecting MPTP (50 ~g) and 80/~g of L-a-AA, a multitude of SNc cells (as many as 85.0 + 4.2% of control) were labeled with TB retrogradely transported from the striatum (Fig. 2c). In remarkable contrast, the
same dose (80 /~g) of the D-isomer of a-AA did not display any protective effects on dopaminergic neurotoxicity of MPTP (50 ktg); in comparison with the control side, only 17.2 + 3.9% of SNc cells contained TB (Table I). Subsequently, in a second series of experiments, the time-dependent effects of L-a-AA (the dose of 80 /~g
4
Fig. 2. Photomicrographs of retrogradely labeled SNc neurons following TB injections into the striatum. Intra-SNc administration of 50 pg of MPTP and/or 80/~g of L-a-AA was made with different time intervals, a: the SNc on the control side infused with vehicle, b-f: the SNc on the experimental side administered with MPTP and/or L-a-AA. b, MPTP treatment alone; c, co-injection of MPTP and L-ct-AA; d,e,f, L-ct-AA pretreatment 1, 3, 7 days before MPTP administration, respectively. All the pictures show almost the same level of the Snc. An arrow in e indicates an injection needle track, a-f, x69.
58 TABLE I Dose-dependent effects of c-ct-AA on M P T P neurotoxici(v a-AA (/~g):
MPTP* alone . -
%**
19.5+4.0
.
Co-injection of MPTP* and L-~t-AA . . . . . 20 40 60
35.4+8.3
59.0+4.8
.
73.2+5.8
80
100
Co-injection of MPTP* and D-ct-AA .... 80
85.0+4.2
83.8+6.1
17.2_+3.9
* A total dose of 50/ag of MPTP was administered into the unilateral SNc in each case. ** The number of retrogradely labeled nigrostriatal ceils on the drug-injected side is expressed as a percentage of that on the control (contralateral) side infused with vehicle. Each number represents the mean + S.E.M. for 8 rats. which had produced maximal protection) on MPTPinduced nigrostriatal cell loss were examined (Fig. 1). Pretreatment with L-a-AA just prior to or 1 day before MPTP administration appeared to prevent nigrostriatal cells from degenerating. Thus, in both cases TB-labeled cells were evident in the SNc (Fig. 2d). However, such protection against MPTP neurotoxicity by L - a - A A was considerably attenuated in the SNc with MPTP administration 3 days following L-a-AA pretreatment; the number of TB-containing nigrostriatal cells went down to 51.4 + 7.2% of control (Fig. 2e). Interestingly, intra-SNc MPTP administration 7 days after L - a - A A pretreatment did even enhance rather than reduce its dopaminergic neurotoxicity. Thus, severer nigrostriatal cell loss occurred in this case than in the case of MPTP treatment alone (11.5 + 2.6% compared with 19.5 + 4.0% of TB-positive cells of control) (Fig. 2f). In some cases (especially in animals administered with MPTP alone or MPTP 7 days after L - a - A A pretreatmerit), Nissl-staining and/or T H immunohistochemistry were performed to verify SNc cell loss caused by MPTP. Tissue from these animals showed a marked decrease in (Nissl-stained and/or TH-immunoreactive) cell number in the SNc as compared with the control side, and surviving cells largely overlapped TB-labeled ones (not illustrated). Additionally, many of these animals progressively lost body weight and manifested discernible abnormalities in ambulatory/rotational behavior. DISCUSSION The selective gliotoxic effect of L-a-AA, a 6-carbon chemical analogue of glutamate, has previously been shown both in v i v o 15'39'4°'52 and in vitro 21'2a. Upon systemic administration to the infant mouse, L - a - A A causes glial lesions in the arcuate nucleus of the hypothalamus and retina 39'4°. In vitro treatment of the dissociated postnatal mouse cerebellum with the toxin results in a rapid nuclear and cytoplasmic swelling of astroglia 21. Furthermore, L-a-AA which is selectively taken up by astroglia and probably kills the cell following its intracellular accumulation, affects astrocytes only 22.
We have also demonstrated that similar astroglia-specific lesions in the rat can be produced by the stereotaxic intracerebral injection of L-a-AA 15'52. The present model system is the first to postulate the use of L-a-AA as a tool for selective astrogtial ablation. There has been a controversy over the vulnerability of the rat brain to MPTP. The relatively slower metabolism rate (i.e. inefficient conversion) of M P T P and/or the considerably shorter biological half life (i,e. insufficient retention) of MPP ÷, may issue part of factors in the lesser sensitivity of the rat to MPTP as compared with monkey or mouse z6. Also, [3H]MPTP binding sites are concentrated in the SNc and striatum in the human brain. whereas these regions have substantially fewer binding sites in the rat brain 25. In fact, MPTP causes behavioral impairments (possibly due to striatat dopamine depletion) in the rat without conspicuous nigrostriatal lesions 3" 9.45 although MPP -~ is extremely toxic to rat dopamine n e u r o n s 1"3'18"37"46"47. However, recent available evidence has revealed that high concentrations of MPTP itself elicit a certain neurotoxicity to rat SNc cells (to a lesser extent ventral tegmental area and dorsal raphe cells as well) both in vivo 13 and in vitro 35'37'46. We have also provided anatomical evidence, with the aid of retrograde axonal tracing, that fairly large amounts (the same as used in the present study) of MPTP administered directly into the SNc or medial forebrain bundle (MFB) (but not systemically), are toxic to rat nigrostriatal neurons s3. It is obvious that MPTP is oxidized to the major active metabolite, MPP ~-, by MAO-B 8, which is localized predominantly in astrocytes 31'42'56 and serotonergic neurons 31'38"55'56 in the brain. These histochemical studies have also pointed out that dopamine cells in the SNc fail to possess MAO-B, thus suggesting that nigrostriatal neurons themselves do not display the capacity to oxidize MPTP to MPP + Therefore. it is most likely that astroglia in the SNc region must be involved in converting MPTP to MPP + for the onset of its dopaminergic neurotoxicity. Indeed. two recent in vitro experiments 2°.43 have indicated a critical role of astroglia in the generanon of MPTP neurotoxicity. Cultured astrocytes take u p [3H]MPTP 2° and promote the conversion of MPTP to MPP ÷
59 (ref. 43). Moreover, high-affinity binding sites for [3H]MPTP correspond to the localization of MAO-B (virtually [3H]pargyline binding) 2'41. Our recent investigation 5 has also shown heavy glial labeling as well as neuronal labeling in the SNc area following intraventricular injections of [3H]MPTP. In the present study, fluorescent retrograde axonal tracing after various combinations of intra-SNc injections of MPTP and L-a-AA, has been employed to examine the effects of selective astroglial ablation on MPTP-induced nigrostriatal neuronal death. Our results clearly demonstrate that L-a-AA confers protection against MPTP in a dose-dependent manner. MPTP-induced loss of retrogradely labeled nigrostriatal cells is dramatically reduced by increasing doses of the gliotoxin. However, it may be a little puzzling that L-a-AA is strikingly protective when co-administered with MPTP. One would presume that some time would be required for the toxic effects of this compound to almost completely disable astroglia to mediate the conversion of MPTP to MPP +. To our knowledge, no other actions of L-a-AA to prevent MPTP neurotoxicity via some different mechanism have hitherto intimated. At present, it is most reasonable to understand that La-AA-induced glial dysfunction might occur so promptly as to obstruct the oxidative metabolism of MPTP. Additonally, co-injections of MPTP and the D-isomer of a-AA fail to afford any preventive effects. This stereospecificity of the gliotoxin is in full agreement with p r e v i o u s reports 15'21'22"39'52. Furthermore, our combined intra-SNc injections of both drugs with different time intervals respect the peculiarity of the time-dependent effects that L-a-AA exerts over MPTP. L-a-AA pretreatment just prior to or 1 day before MPTP administration, favors conspicuous protection against MPTP. Conversely, the neurotoxic potency of MPTP is considerably restored 3 days after L-a-AA pretreatment, and is finally even enhanced rather than attenuated by the gliotoxin pretreated 7 days before MPTP administration. Thus, as compared with the case of MPTP treatment alone, severer loss of cells retrogradely labeled from the striatum occurs in the SNc. Our previous findings on the fine structural changes after the intracerebral injection of L-ct-AA 15'52 might account for this reversed phenomenon. Tremendous invasion of reactive astrocytes starting 3 days after the gtiotoxin injection, indicative of the recovery of astroglial function, reached its peak 7 days after the injection 15,52. A large number of these recruited astrocytes may accumulate excessive MAO-B to oxidize more efficiently MPTP to MPP +, which even destroys surviving nigrostriatal cells under the toxic environment produced by regularly administered MPTP. To date, attempts to reduce or prevent MPTP neuro-
toxicity have been focussed on the interference, by specific inhibitors, with MAO-B function or dopamine transport system, both of which are believed to be instrumental for the generation of its toxic effect. Indeed, MAO-B inhibitors (including pargyline and deprenyl) prevent the conversion of MPTP to MPP* (refs. 8, 32), and administration of these inhibitors prior to MPTP treatment is protective against MPTP neurotoxicity as well m'17"3°'35'36. Moreover, the fact that dopamine uptake inhibitors (including mazindol and amfenolic acid) protect against striatal dopamine depletion 33"44 and dopaminergic cell loss 46 caused by MPP +, supports the notion that the uptake system of MPP + into dopaminergic neurons is responsible for selective neurotoxicity of MPTP 24. There continues to be a great deal of debate regarding the primary site of action of MPTP/MPP + over nigrostriatal neurons. While many investigators believe that the major active site is at the level of SNc cell bodies, some argue that the compound first acts at the striatal terminal field and consequentially affects parent cells via retrograde axonal transport 48or simply retrograde degeneration 19. The present data showing that astroglial ablation at the SNc level prevents nigrostriatal cell loss following intra-SNc MPTP injections (see also ref. 53), stand in favor of the former idea, even though the latter cannot thoroughly be excluded. In fact, our recent autoradiographic study 6 has successfully demonstrated the capability of MPTP (eventually MPP +) to be taken up by nigrostriatal terminals and transported retrogradely to SNc cell bodies. Furthermore, SNc cell elimination resulting from intrastriata123 or intra-MFB 53 MPTP infusion respects retrograde degeneration. Astroglial ablation at the SNc or striatal level prior to systemic MPTP administration would be strategic to definitively determine its primary active site. We now elucidate a pronounced involvement of astroglia in the precipitation of MPTP-induced nigrostriatal neuronal death in vivo. The presence of astroglia must be instrumental in the mediation of drug-induced parkinsonism. The eminent role of astroglia seems peculiar to this major brain disease as yet, and the link between the cell and other types of neurodegenerative disorders remains a mystery. In this context, we have previously reported that L-a-AA also protects against kainic acid neurotoxicity in the rat striatum is. However, the possible functional relationship of astroglia to kainic acidqnduced striatal cell death, which duplicates Huntington's chorea 11'34, still remains elusive. Thus, astroglia may subserve a so-far-unappreciated role in the brain's defense mechanism against neurotoxic insults.
Acknowledgements. Supported by the Medical Research Council of Canada. M.T. is an MRC Postprofessional Fellow.
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