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Intrastriatal quinolinic acid injections protect against 6-hydroxydopamine-induced lesions of the dopaminergic nigrostriatal system
BRAIN RESEARCH ELSEVIER
Brain Research 672 (1995) 153-158
Research report
Intrastriatal quinolinic acid injections protect against 6-hydroxydopamine-induced lesions of the dopaminergic nigrostriatal system Jose L. Venero, Marina Romero-Ramos, Mati Revuelta, A. Machado, Josefina Cano * Departamento de Bioqu{mica, Bromatolog[a y Toxicolog[a, Facultad de Farmacia, Universidad de Sevilla, c / . Prof. Garc[a Gonzdlez s/n, 41012 Sevilla, Spain
Accepted 16 November 1994
Abstract
We tested the effect of intrastriatal quinolinic acid (QA) injections 2 weeks before subsequent intrastriatal injections of 6-hydroxydopamine (6-OHDA). Levels of DA and its metabolites were measured 2 days and 21 days after lesioning the dopaminergic nigrostriatal system with 6-OHDA. Intrastriatal 6-OHDA injections in the absence of prior treatment of QA significantly decreased dopamine (DA) and its metabolite levels in striatum but not in substantia nigra at day 2, and in striatum and substantia nigra at day 21, a clear indication of a time-dependent retrograde axonal degeneration of substantia nigra cell bodies. Intrastriatal QA injections 2 weeks before subsequent intrastriatal injection of 6-OHDA partially prevented the 6-OHDA-depleting effect on DA and its metabolite levels in both striatum and substantia nigra 21 days after 6-OHDA injection. However, no statistically significant differences were found between QA + 6-OHDA- and 6-OHDA-treated animals at day 2. Our results suggest that intrastriatal QA injections partially prevent the naturally-occurring retrograde axonal degeneration of substantia nigra cell bodies caused by 6-OHDA, and illustrate a target-derived interaction between dopaminergic nerve endings and cell bodies. We suggest that the protective effect found in the QA-injected animals against the neurotoxic action of 6-OHDA is mediated by neurotrophic agents released by activated astroglia. Keywords: Quinolinic acid; 6-Hydroxydopamine; Striatum; Substantia nigra; Dopaminergic system; Neuroprotection
I. Introduction
The neurotoxicity elicited by the excitotoxin quinolinic acid (QA) resembles many of the neurochemical and pathological characteristics of Huntington's disease [4,5,17]. The excitotoxic actions of Q A in the striatum are mediated via the N-methyl-D-aspartate ( N M D A ) subtype of glutamate receptors. Intrastriatal injections of Q A induce a specific pattern of neuronal cell loss along with astroglia proliferation, as deduced by increased levels of glial fibrillary acidic protein [18]. Striatal medium-sized spiny neurons containing the neurochemical markers G A B A , substance P, dynorphin, and enkephalin are preferentially affected in the Q A model of Huntington's disease [5,9]. In contrast,
* Corresponding author. Fax: (34) (5) 423-3765; E-mail: [email protected] 0006-8993/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0006-8993 (94)01368-3
medium-sized aspiny neurons containing the neuropeptides somatostatin and neuropeptide Y are spared [5,9]. In addition, striatal dopaminergic nerve terminals are preserved following single intrastriatal injection of Q A [5,18]. The central nervous system responds to diverse neurologic injuries with a vigorous activation of astrocytes. At present, reactive astrocytosis is defined primarily as an increase in the number and size of cells expressing glial fibrillary acidic protein. U p o n activation, astrocytes increase the expression of a large number of molecules [12]. From this molecular profile it becomes apparent that reactive astrocytes may benefit the injured nervous system [8]. The Q A model of Huntington's disease, with its combination of neuronal cell loss and a pronounced reactive astrocytosis along with a high preservation of striatal dopamine nerve terminals, offers us an opportunity to investigate the potential role of astrocytosis in vivo in the effects of 6 - O H D A injections.
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6-OHDA dissolved in 2 /zl of a 0.9% saline solution containing 0.1% ascorbic acid in each striatum. The dose used in the present study has been shown to induce a near-total depletion of striatal dopaminergic terminals as deduced by DA and its metabolite levels and mazindol binding autoradiography [3]. The coordinates were the same as those used for the QA injections. Animals of group 2 received bilateral injections of 2 p,l of a 0.9% saline solution containing 0.1% ascorbic acid in each striatum under identical conditions to 6-OHDA-injected animals. Two different time points were evaluated following the last set of injections: half the animals of each group (n = 6) were killed at day 2 from the last injection, and the other half at day 21. This treatment paradigm gives 4 different experimental conditions, henceforth referred to as: (i) saline-treated animals (controls) (right striatum of group 2); (ii) QA-treated animals (left striaturn of group 2); (iii) 6-OHDA-treated animals (right striatum of group 1); and (iv) Q A + 6 - O H D A - t r e a t e d animals (left striatum of group 1). Experiments were carried out in accordance with the guidelines of the European Union Council ( 8 6 / 6 0 9 / E U ) and following the Spanish regulations (BOE 67/8509-12, 1988) for the use of laboratory animals. The experiments carried out in the present study
2. Material and methods 2.1. AnimaLs attd treatment
A total of 24 female Wistar rats (200-250 g) were used for these studies. The rats were anesthetized with chloral hydrate (400 mg/kg, i.p.) and positioned m a stereotaxic apparatus (Kopf Instruments, Tujunga, CA) to conform with the brain atlas of Paxinos and Watson [15]. All animals received unilateral left intrastriatal injections of 150 nmol of QA in 2 #1 of Ringer solution. The dose was chosen according to previously published reports [2,7,14,16]. The solution was injected with a Hamilton syringe positioned at 1").5 mm caudal. 2.5 mm lateral and 6.5 ventral to bregma at a rate of 0.5 # t / m i n . Thc right striatum was injected with 2 p.I of Ringer solution in the same coordinates. Animals were left for recovery and separated into two groups (12 animals each for group 1 and 2, respectively). Two weeks after the QA injectkms were given, animals were again anesthetized with chloral hydrate and positioned in a stereotaxic apparatus. Animals of group I received bilateral injections of 25 #g of 12000
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Fig. I. Concentration ol DA and metabolites in striatum of control animals (black-filled box), QA-treated animals (narrow-line box), 6-OHDA-treated animals (wide-line box) and QA + 6-OHDA-treated animals (dot-filled box). All animals received unilateral intrastriatal injections of QA (150 nmol). 2 weeks later, half of the animals were bilaterally injected with saline solution, and the other half were bilaterally injected with 25 p.g of 6-OHDA in the same coordinates as those used for the QA injections. Animals were killed 2 days and 21 days subsequent to the last set of injections. This treatment paradigm gives the four different experimental conditions above specified. Results are expressed in n g / g of wet tissue, and represent the mean + S.D. of 6 animals. Statistical significance: one-way analysis of variance followed by the Schefee test: *" P < 0.05 as compared with control animals; * b p < 0.05 as compared with QA-injected animals; *c p < 0.05 as compared with 6-OHDA-treated animals.
J.L. Venero et al. / Brain Research 672 (1995) 153-158 were approved by the Scientific Committee of the University of Seville.
2.2. Measurement of dopamine and its metabolites in rat striatum and substantia nigra At the appropiate time-points, animals were decapitated and the brains were quickly removed. T h e striatum was first dissected out on a cold plate. Additionally, the mesencephalon was divided into two parts with a cut from the ventral side perpendicular to the long axis of the mesencephalon exactly at the caudal border eminence. T h e two substantia nigrae were then easily identified and freely dissected on a cold plate. T h e ventral tegmental area was not included. The tissue was frozen at - 8 0 ° C until analysis. Neurochemical analyses were performed by H P L C with electrochemical detection. A Merck L-6200 p u m p was used in conjunction with a glassy carbon electrode ( A N T E C ) set at 0.8V (vs. A g / A g C 1 reference electrode). A Merck Lichrocart cartridge ( 1 2 5 x 4 mm) column filled with Lichrospher reverse-phase C18 5 / ~ m material was used. The mobile phase consisted of a mixture of 100 m M formic acid, 0.36 m M octane sulfonic acid, 1.0 m M citric acid, 0.1 m M E D T A , 5.0% (v/v) acetonitrile and 0.25% diethylamine (v/v) ad-
700
justed to pH 3.1 with K O H and was thoroughly degassed. Analyses were performed in the isocratic mode, at a flow rate of 1 m l / m i n at room temperature [19,20]. Standards were prepared in 0.1 M perchloric acid/1 m M sodium bisulfite and stored at + 4°C for up to 2 months. The detection limit of the assay was 50-100 p g / s a m p l e . T h e day of analysis, the brain tissue was defrosted and weighed and further homogenized in 8 vols. of 0.1 M perchloric acid containing 1 m M sodium bisulfite by ultrasonic disintegration over ice using a Labsonic 1510. Average weights for striatal and nigral tissue were 24 and 6 mg, respectively. Samples were centrifuged at 30,000 x g for 15 min at + 4°C and the supernatant was then filtered through a 0.2 /zm filter. Concentrations in brain samples were calculated with the aid of (linear) calibration curves obtained after the injection of pure standard.
2.3. Statistical analyses M e a n concentration of dopamine and its metabolites from the different experimental conditions were analyzed by one-way analysis of variance (ANOVA). Observed m e a n differences were evaluated using the parametric Schefee test.
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Fig. 2. Concentration of D A and metabolites in substantia nigra of control animals (black-filled box), QA-treated animals (narrow-line box), 6-OHDA-treated animals (wide-line box) and Q A + 6 - O H D A - t r e a t e d animals (dot-filled box). Results are expressed in n g / g of wet tissue, and represent the m e a n :t: S.D. of 6 animals. Statistical significance: one-way analysis of variance followed by the Schefee test: * a p < 0.05 as compared with control animals; * b p < 0.05 as compared with QA-injected animals; * c p < 0.05 as compared with 6-OHDA-treated animals. See legend for Fig. 1 for further details. N.D. = not detectable.
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3. Results
3.1. Effect of quinolinic acid injections on DA and metabolite lecels in rat striatum and substantia nigra Intrastriatal QA injections induced modest changes in the dopaminergic nigrostriatal system. In striatum, no statistical differences were found in any of the components of the dopaminergic system except for a statistically significant decrease in the levels of 3methoxytyramine (3-MT) ( - 2 5 % control levels) at day 2 (Fig. 1). However, at day 21 there were overall decreases in the concentration of DA and the different DA metabolites: DA ( - 2 1 % control levels), homovanillic acid (HVA) ( - 28% control levels) and 3-MT ( - 3 4 % control levels) (Fig. 1). In substantia nigra, statistically significant increases were found in the concentration of DA at day 2 ( + 35% control levels) and at day 21 ( + 3 5 % control levels) and in the concentration of 3,4-dihydroxyphenylacetic acid (DOPAC) ( + 46% control levels) at day 21 (Fig. 2). 3.2. Effect of intrastriatal injections of 6-OHDA on DA and metabolite lecels in rat striatum and substantia nigra Intrastriatal injection of 6-OHDA alone had a pronounced effect on the dopaminergic nigrostriatal tract. Thus, at day 2, only striatum showed overall decreases in the different DA components (DA, - 8 4 % control levels; DOPAC, - 79%; HVA, - 70%; 3-MT, - 83%) (Fig. 1) with no change in substantia nigra (Fig. 2). However, at day 2t the dopaminergic system was highly affected in both striatum and substantia nigra, which strongly suggests a retrograde axonal degeneration of the cell bodies of the pars compacta of the substantia nigra. The declines in the concentration of DA and the different DA components were as follows: DA ( 98% and -71~/( control levels in striatum and substantia nigra respectively); DOPAC ( - 9 2 % and - 4 9 % in striatum and substantia nigra, respectively); HVA ( - 8 7 % in striatum with no statistically significant change in substantia nigra) and 3-MT ( - 97% in striaturn, and no detectable levels in substantia nigra) (Figs. 1 and 2).
3.3. Lf]ect of quinolinic acid lesions on 6-hydroxydopamine-induced toxicity Intrastriatal injections of QA 14 days before intrastriatal injections of 6-OHDA showed a very different pattern of dopaminergic integrity as deduced by DA and its metabolite levels in the nigrostriatal system, especially at day 21 when the 6-OHDA-induced neurotoxicity is fully completed. At this time-point, QA + 6OHDA-treated animals showed a much higher preservation of the dopaminergic system in both striatum and
substantia nigra as compared with animals receiving 6-OHDA alone (Figs. 1 and 2). Levels of DA in QA + 6-OHDA-treated animals were 17- and 2.1-fold higher in striatum and substantia nigra respectively than those receiving 6-OHDA alone. QA injections also protected against the 6-OHDA-induced decreases in DA metabolites, although to a lesser degree. Thus, striatal DOPAC and HVA levels were 4.2- and 2.7-fold higher in QA + 6-OHDA- than 6-OHDA-treated rats, while the nigral values of such metabolites were not different between both experimental conditions (Figs. 1 and 2). 3-MT was the only DA metabolite whose levels were altered in parallel fashion to those of DA by both QA + 6OHDA- and 6-OHDA-treated animals. In the former, striatal 3-MT levels were ll.4-fold higher than in the latter (Fig. 1). No direct comparison could be made in substantia nigra since 3-MT levels were not detectable in 6-OHDA-treated animals (Fig. 2). At the shorter 2 days time-point studied, no differences were detected between QA + 6-OHDA- and 6OHDA-treated animals in the striatum. However, nigral levels of DA, DOPAC and 3-MT were 1.5-, 1.7and 1.7-fold higher in Q A + 6 - O H D A - than in 6OHDA-treated rats (Fig. 2).
4. Discussion
Neurochemical analysis of the dopaminergic nigrostriatal system reveals that QA-lesioned animals are more resistant to the neurotoxicity induced by subsequent intrastriatal injections of 6-OHDA. In absence of QA lesions, intrastriatal 6-OHDA injections resulted in high reductions in striatal DA levels at both 2 days and 21 days following the neurotoxin administration ( - 8 4 % and - 9 8 % control values, respectively). The cell bodies of the substantia nigra showed, however, a different neurochemical profile. While no changes were detected 2 days following 6-OHDA injections, DA levels decreased ( - 7 1 % control values) at 21 days. This observation indicates a clear time-dependent retrograde axonal degeneration of substantia nigra cell bodies, which is in agreement with Ichitani et al. [11] who showed degeneration of nigral dopamine neurons 2 to 4 weeks after intrastriatal 6-OHDA injections. Comparisons of this treatment condition with animals previously lesioned with QA showed remarkable differences in terms of DA and its metabolite levels. Thus, in animals with combined lesions, while DA levels decreased ( - 7 2 % and - 6 2 % QA-treated animal levels) in striatum at days 2 and 21, respectively, in substantia nigra there were no change in DA levels at day 2 when compared to QA-treated animals, later decreasing at day 21 ( - 54%). It is particularly relevant that the overall protection induced by QA against the 6-OHDA-induced neurotoxicity of the nigrostriatal sys-
J.L. Venero et al. / Brain Research 672 (1995) 153-158
tern was detected only at 21 days. At 2 days, prior QA lesions were ineffective against the 6-OHDA-depleting effect on striatal DA levels, thus suggesting that the neuroprotective effect found in the QA-injected animals at 21 days is not related either to inhibition of the direct neurotoxic action of 6-OHDA or to diffusion problems of 6-OHDA in reaching its targets in the striatum. The mechanism by which prior intrastriatal QA lesions protect against subsequent 6-OHDA injections has to be neccesarily associated to the typical pattern of neuronal degeneration induced by this neurotoxin. One of the best known features of QA, when applied to the striatum, is the destruction of neurons containing GABA and substance P as neurotransmitters as reflected by neuronal cell loss [5,9] and high reductions of the mRNA levels encoding glutamate decarboxylase [21]. It is reasonable to think that the destruction of the striatal GABA and substance P-descending axons to the substantia nigra [6,13] would release nigral dopamine cells from tonic inhibition and thus result in increased production of DA. This effect is particularly evident in nigral DA levels following QA lesions. In the absence of 6-OHDA injections, we found significant increases of nigral DA levels following intrastriatal QA injections at the two different time points studied (35% and 35% control levels at 2 days and 21 days respectively) without significant changes of the ratio of the acidic metabolites, DOPAC or HVA, to DA (as an index of DA turnover), which is in accordance with an increased production of nigral DA without major changes in DA metabolism. The question is whether an increased production of DA is enough to explain the higher levels of DA and its metabolites in QA + 6-OHDA-injected animals than in animals injected with 6-OHDA alone. There are, however, some observations difficult to reconcile with this possibility. A known feature of the lesioned dopaminergic system is its ability to compensate for the cell loss [22]. Thus, rats sustaining near-total (> 95%) depletions of striatal dopamine, similar to those studied here, demonstrated up to 10-fold elevation of dopamine metabolism within the surviving neurons as evidenced by D O P A C / D A or H V A / D A ratios [1]. In our experiments, both D O P A C / D A and H V A / D A were elevated in striatum (4.4- and 7.3-fold) and in substantia nigra (1.8- and 3.1-fold) 21 days following intrastriatal 6-OHDA injections in absence of QA injections for D O P A C / D A and H V A / D A , respectively. In QA + 6-OHDA-treated rats, however, neither the D O P A C / D A ratio nor the H V A / D A ratio varied to the same extent as in animals injected with 6-OHDA alone in either striatum or substantia nigra. If we consider that elevated D O P A C / D A or H V A / D A ratios are ascribed to rats with an extensive (> 90%) loss of striatal DA, the maintenance of such ratios in QA + 6-OHDA-lesioned
157
animals when compared with animals injected with 6-OHDA alone strongly suggest a higher density of residual striatal dopaminergic terminals and hence a higher dopaminergic cell body survival. The mechanism by which prior QA lesions protect against 6-OHDA-induced neurotoxicity is an open question. However, the typical pattern of neuronal cell degeneration along with astroglia proliferation (see section 1) induced by QA deserves special consideration. Thus, neurotrophic factors released by activated astroglia may be responsible for the protective effect found in QA + 6-OHDA-lesioned animals. It has to be that taken into account that the activation of astrocytes in culture preparations is small relative to increases observed during reactive gliosis in the adult rat brain [12]. It may be also worth considering a probable relationship between our results and the mild improvements seen in animals lesioned with 6-OHDA which underwent adrenal medulla autografting in the striatum. Such grafts have been shown effective in reducing amphetamine-induced rotations despite little evidence of fiber outgrowth [10]. It seems quite likely that a similar glial reaction to the surgery itself may have been responsible for the minor improvements observed.
Acknowledgements This work was supported by a grant from DGICYT PM91-0097. J.L.V. thanks to the Spanish Ministerio de Educaci6n y Ciencia for a Contrato de Incorporaci6n.
References [1] Altar, C.A., Marien, M.R. and Marshall J.F., Time course of adaptations in dopamine biosynthesis, metabolism, and release following nigrostriatal lesions: implications for behavioral recovery from brain injury, Z Neurochem., 48 (1987) 390-399. [2] Altar, C.A., Dugich-Djordjevic, M., Armanini, M. and Bakhit, C., Medial-to-lateral gradient of neostriatal NGF receptors: relationship to cholinergic neurons and NGF-like immunoreactivity, J. Neurosci., 11 (1991) 828-836. [3] Altar, C.A., Jakeman, L.B., Acworth, I.N., Soriano, R. and Dugich-Djordievic, M.M., Regionally restricted loss and partial recovery of nigrostriatal dopamine input folowing intrastriatal 6-bydroxydopamine, Neurodegeneration, 1 (1992) 123-133. [4] Beal, M.F., Kowall, N.W., Ellison, D.W., Mazurek, M.F., Swartz, K.J. and Martin, J.B., Replication of the neurochemical characteristics of Huntington's disease by quinolinic acid, Nature, 321 (1986) 168-171. [5] Beal, M.F., Ferrante, R.J., Swartz, K.J. and Kowall, N.W., Chronic quinolinic acid lesions in rats closely resemble Huntington's disease, J. NeuroscL, 11 (1991) 1649-1659. [6] Bolam, J.P. and Smith, Y., The GABA and substance P input to dopaminergic neurones in the substantia nigra of the rat, Brain Res., 529 (1990) 57-78.
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[7] Buisson, A., Pateau, V., Plotkine, M. and Boulu, R.G., Nigrostriatal pathway modulates striatum vulnerability to quinolinic acid, Neurosci. Lett., 131 (1991) 257-259. [8] Eddleston, M. and Mucke, L., Molecular profile of reactive astrocytes. Implications for their role in neurologic diseases, Neuroscience, 54 (1993) 15-36. [9] Ferrante, R.J., Kowall, N.W., Cipolloni, P.B., Storey, E. and Beal, M.F., Excitotoxin lesions in primates as a model for Huntington's disease: histopathologic and neurochemical characterization, Exp. Neurol., 119 (1993)46-71. [10] Hansen, J.T., Bing, G., Notter, M.F.D. and Gash, D.M., Paraneuronal grafts in unilateral 6-hydroxydopamine-lesioned rats: morphological aspects of adrenal chromaffin and carotid body glomus cell implants. In D.M. Gash and J.R. Sladek Jr. (Eds.), Transplantations Into the Mammalian CNS, Progress in Brain Res., Vol. 78, 1988, pp. 507-511. [11] Ichitani, Y., Okamura, H., Matsumoto, Y., Nagatsu, T. and Ibata, Y., Degeneration of nigral dopamine neurons after 6-hydroxydopamine injection into the rat striatum, Brain Res., 549 (1991) 350-353. [12] McMillian, M.K., Thai, L., Hong, J.S., O'Callaghan, J.P. and Pennypacker, K.R., Brain injury in a dish: a model for reactive gliosis, Trends Neurosci., 17 (1994) 138-142. [13] Mendez, I., Elisevich, K. and Flumerfelt, B., Substance P synaptic interactions with GABAergic and dopaminergic neurons in rat substantia nigra: an ultrastructural double-labeling immunocytochemical study, Brain Res. Bull,, 28 (1992) 557-563. [14] Norman, A.B., Ford, L.M. and Sanberg, P.R., Differential loss
of neurochemical markers following quinolinic acid-induced lesions of rat striatum, Exp. Neurol., 114 (1991) 132-135. [15] Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, 1986, Academic Press, London. [16] Sanberg, P.R., Zubrycki, E., Ragozzino, M.E., Giordano, M. and Shipley, M.T., Tyrosine hydroxylase-positive fibers and neurons in transplanted striatal tissue in rats with quinolinic acid lesions of the striatum, Brain Res. Bull., 25 (1990) 889-894. [17] Schwarcz, R., Whetsell, W.O. and Mangano, R.M., Quinolinic acid: an endogenous metabolite that produces axon-sparing lesions in rat brain, Science, 219 (1983) 316-318. [18] Schwarcz, R. and Shoulson, I., Excitotoxins and Huntington's disease. In J.T. Coyle (Ed.), Animal Models o f Dementia, 1987 pp. 39-68. [19] Venero, J.L., Machado, A. and Cano, J., Turnover of dopamine and serotonin and their metabolites in the striatum of aged rats, J. Neurochem., 56 (1991) 1940-1948. [20] Venero, J.L., Machado, A. and Cano, J., Age effects on monoamine turnover of the rat substantia nigra, Brain Res., 557 (1991) 109-114. [21] Venero, J.L., Beck, K.D. and Hefti, F., lntrastriatal infusion of nerve growth factor after quinolinic acid prevents reduction of cellular expression of choline acetyltransferase messenger RNA and trkA messenger RNA, but not glutamate decarboxylase messenger RNA, Neuroscience, 61 (1994) 257-268. [22] Zigmond, M.J. and Stricker, E.M., Animal models of Parkinsonism using selective neurotoxins: clinical and basic implications, Int. Reu. Neurobiol,, 31 (1989) 1-79.
Brain Research 672 (1995) 153-158
Research report
Intrastriatal quinolinic acid injections protect against 6-hydroxydopamine-induced lesions of the dopaminergic nigrostriatal system Jose L. Venero, Marina Romero-Ramos, Mati Revuelta, A. Machado, Josefina Cano * Departamento de Bioqu{mica, Bromatolog[a y Toxicolog[a, Facultad de Farmacia, Universidad de Sevilla, c / . Prof. Garc[a Gonzdlez s/n, 41012 Sevilla, Spain
Accepted 16 November 1994
Abstract
We tested the effect of intrastriatal quinolinic acid (QA) injections 2 weeks before subsequent intrastriatal injections of 6-hydroxydopamine (6-OHDA). Levels of DA and its metabolites were measured 2 days and 21 days after lesioning the dopaminergic nigrostriatal system with 6-OHDA. Intrastriatal 6-OHDA injections in the absence of prior treatment of QA significantly decreased dopamine (DA) and its metabolite levels in striatum but not in substantia nigra at day 2, and in striatum and substantia nigra at day 21, a clear indication of a time-dependent retrograde axonal degeneration of substantia nigra cell bodies. Intrastriatal QA injections 2 weeks before subsequent intrastriatal injection of 6-OHDA partially prevented the 6-OHDA-depleting effect on DA and its metabolite levels in both striatum and substantia nigra 21 days after 6-OHDA injection. However, no statistically significant differences were found between QA + 6-OHDA- and 6-OHDA-treated animals at day 2. Our results suggest that intrastriatal QA injections partially prevent the naturally-occurring retrograde axonal degeneration of substantia nigra cell bodies caused by 6-OHDA, and illustrate a target-derived interaction between dopaminergic nerve endings and cell bodies. We suggest that the protective effect found in the QA-injected animals against the neurotoxic action of 6-OHDA is mediated by neurotrophic agents released by activated astroglia. Keywords: Quinolinic acid; 6-Hydroxydopamine; Striatum; Substantia nigra; Dopaminergic system; Neuroprotection
I. Introduction
The neurotoxicity elicited by the excitotoxin quinolinic acid (QA) resembles many of the neurochemical and pathological characteristics of Huntington's disease [4,5,17]. The excitotoxic actions of Q A in the striatum are mediated via the N-methyl-D-aspartate ( N M D A ) subtype of glutamate receptors. Intrastriatal injections of Q A induce a specific pattern of neuronal cell loss along with astroglia proliferation, as deduced by increased levels of glial fibrillary acidic protein [18]. Striatal medium-sized spiny neurons containing the neurochemical markers G A B A , substance P, dynorphin, and enkephalin are preferentially affected in the Q A model of Huntington's disease [5,9]. In contrast,
* Corresponding author. Fax: (34) (5) 423-3765; E-mail: [email protected] 0006-8993/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0006-8993 (94)01368-3
medium-sized aspiny neurons containing the neuropeptides somatostatin and neuropeptide Y are spared [5,9]. In addition, striatal dopaminergic nerve terminals are preserved following single intrastriatal injection of Q A [5,18]. The central nervous system responds to diverse neurologic injuries with a vigorous activation of astrocytes. At present, reactive astrocytosis is defined primarily as an increase in the number and size of cells expressing glial fibrillary acidic protein. U p o n activation, astrocytes increase the expression of a large number of molecules [12]. From this molecular profile it becomes apparent that reactive astrocytes may benefit the injured nervous system [8]. The Q A model of Huntington's disease, with its combination of neuronal cell loss and a pronounced reactive astrocytosis along with a high preservation of striatal dopamine nerve terminals, offers us an opportunity to investigate the potential role of astrocytosis in vivo in the effects of 6 - O H D A injections.
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6-OHDA dissolved in 2 /zl of a 0.9% saline solution containing 0.1% ascorbic acid in each striatum. The dose used in the present study has been shown to induce a near-total depletion of striatal dopaminergic terminals as deduced by DA and its metabolite levels and mazindol binding autoradiography [3]. The coordinates were the same as those used for the QA injections. Animals of group 2 received bilateral injections of 2 p,l of a 0.9% saline solution containing 0.1% ascorbic acid in each striatum under identical conditions to 6-OHDA-injected animals. Two different time points were evaluated following the last set of injections: half the animals of each group (n = 6) were killed at day 2 from the last injection, and the other half at day 21. This treatment paradigm gives 4 different experimental conditions, henceforth referred to as: (i) saline-treated animals (controls) (right striatum of group 2); (ii) QA-treated animals (left striaturn of group 2); (iii) 6-OHDA-treated animals (right striatum of group 1); and (iv) Q A + 6 - O H D A - t r e a t e d animals (left striatum of group 1). Experiments were carried out in accordance with the guidelines of the European Union Council ( 8 6 / 6 0 9 / E U ) and following the Spanish regulations (BOE 67/8509-12, 1988) for the use of laboratory animals. The experiments carried out in the present study
2. Material and methods 2.1. AnimaLs attd treatment
A total of 24 female Wistar rats (200-250 g) were used for these studies. The rats were anesthetized with chloral hydrate (400 mg/kg, i.p.) and positioned m a stereotaxic apparatus (Kopf Instruments, Tujunga, CA) to conform with the brain atlas of Paxinos and Watson [15]. All animals received unilateral left intrastriatal injections of 150 nmol of QA in 2 #1 of Ringer solution. The dose was chosen according to previously published reports [2,7,14,16]. The solution was injected with a Hamilton syringe positioned at 1").5 mm caudal. 2.5 mm lateral and 6.5 ventral to bregma at a rate of 0.5 # t / m i n . Thc right striatum was injected with 2 p.I of Ringer solution in the same coordinates. Animals were left for recovery and separated into two groups (12 animals each for group 1 and 2, respectively). Two weeks after the QA injectkms were given, animals were again anesthetized with chloral hydrate and positioned in a stereotaxic apparatus. Animals of group I received bilateral injections of 25 #g of 12000
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Fig. I. Concentration ol DA and metabolites in striatum of control animals (black-filled box), QA-treated animals (narrow-line box), 6-OHDA-treated animals (wide-line box) and QA + 6-OHDA-treated animals (dot-filled box). All animals received unilateral intrastriatal injections of QA (150 nmol). 2 weeks later, half of the animals were bilaterally injected with saline solution, and the other half were bilaterally injected with 25 p.g of 6-OHDA in the same coordinates as those used for the QA injections. Animals were killed 2 days and 21 days subsequent to the last set of injections. This treatment paradigm gives the four different experimental conditions above specified. Results are expressed in n g / g of wet tissue, and represent the mean + S.D. of 6 animals. Statistical significance: one-way analysis of variance followed by the Schefee test: *" P < 0.05 as compared with control animals; * b p < 0.05 as compared with QA-injected animals; *c p < 0.05 as compared with 6-OHDA-treated animals.
J.L. Venero et al. / Brain Research 672 (1995) 153-158 were approved by the Scientific Committee of the University of Seville.
2.2. Measurement of dopamine and its metabolites in rat striatum and substantia nigra At the appropiate time-points, animals were decapitated and the brains were quickly removed. T h e striatum was first dissected out on a cold plate. Additionally, the mesencephalon was divided into two parts with a cut from the ventral side perpendicular to the long axis of the mesencephalon exactly at the caudal border eminence. T h e two substantia nigrae were then easily identified and freely dissected on a cold plate. T h e ventral tegmental area was not included. The tissue was frozen at - 8 0 ° C until analysis. Neurochemical analyses were performed by H P L C with electrochemical detection. A Merck L-6200 p u m p was used in conjunction with a glassy carbon electrode ( A N T E C ) set at 0.8V (vs. A g / A g C 1 reference electrode). A Merck Lichrocart cartridge ( 1 2 5 x 4 mm) column filled with Lichrospher reverse-phase C18 5 / ~ m material was used. The mobile phase consisted of a mixture of 100 m M formic acid, 0.36 m M octane sulfonic acid, 1.0 m M citric acid, 0.1 m M E D T A , 5.0% (v/v) acetonitrile and 0.25% diethylamine (v/v) ad-
700
justed to pH 3.1 with K O H and was thoroughly degassed. Analyses were performed in the isocratic mode, at a flow rate of 1 m l / m i n at room temperature [19,20]. Standards were prepared in 0.1 M perchloric acid/1 m M sodium bisulfite and stored at + 4°C for up to 2 months. The detection limit of the assay was 50-100 p g / s a m p l e . T h e day of analysis, the brain tissue was defrosted and weighed and further homogenized in 8 vols. of 0.1 M perchloric acid containing 1 m M sodium bisulfite by ultrasonic disintegration over ice using a Labsonic 1510. Average weights for striatal and nigral tissue were 24 and 6 mg, respectively. Samples were centrifuged at 30,000 x g for 15 min at + 4°C and the supernatant was then filtered through a 0.2 /zm filter. Concentrations in brain samples were calculated with the aid of (linear) calibration curves obtained after the injection of pure standard.
2.3. Statistical analyses M e a n concentration of dopamine and its metabolites from the different experimental conditions were analyzed by one-way analysis of variance (ANOVA). Observed m e a n differences were evaluated using the parametric Schefee test.
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Fig. 2. Concentration of D A and metabolites in substantia nigra of control animals (black-filled box), QA-treated animals (narrow-line box), 6-OHDA-treated animals (wide-line box) and Q A + 6 - O H D A - t r e a t e d animals (dot-filled box). Results are expressed in n g / g of wet tissue, and represent the m e a n :t: S.D. of 6 animals. Statistical significance: one-way analysis of variance followed by the Schefee test: * a p < 0.05 as compared with control animals; * b p < 0.05 as compared with QA-injected animals; * c p < 0.05 as compared with 6-OHDA-treated animals. See legend for Fig. 1 for further details. N.D. = not detectable.
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3. Results
3.1. Effect of quinolinic acid injections on DA and metabolite lecels in rat striatum and substantia nigra Intrastriatal QA injections induced modest changes in the dopaminergic nigrostriatal system. In striatum, no statistical differences were found in any of the components of the dopaminergic system except for a statistically significant decrease in the levels of 3methoxytyramine (3-MT) ( - 2 5 % control levels) at day 2 (Fig. 1). However, at day 21 there were overall decreases in the concentration of DA and the different DA metabolites: DA ( - 2 1 % control levels), homovanillic acid (HVA) ( - 28% control levels) and 3-MT ( - 3 4 % control levels) (Fig. 1). In substantia nigra, statistically significant increases were found in the concentration of DA at day 2 ( + 35% control levels) and at day 21 ( + 3 5 % control levels) and in the concentration of 3,4-dihydroxyphenylacetic acid (DOPAC) ( + 46% control levels) at day 21 (Fig. 2). 3.2. Effect of intrastriatal injections of 6-OHDA on DA and metabolite lecels in rat striatum and substantia nigra Intrastriatal injection of 6-OHDA alone had a pronounced effect on the dopaminergic nigrostriatal tract. Thus, at day 2, only striatum showed overall decreases in the different DA components (DA, - 8 4 % control levels; DOPAC, - 79%; HVA, - 70%; 3-MT, - 83%) (Fig. 1) with no change in substantia nigra (Fig. 2). However, at day 2t the dopaminergic system was highly affected in both striatum and substantia nigra, which strongly suggests a retrograde axonal degeneration of the cell bodies of the pars compacta of the substantia nigra. The declines in the concentration of DA and the different DA components were as follows: DA ( 98% and -71~/( control levels in striatum and substantia nigra respectively); DOPAC ( - 9 2 % and - 4 9 % in striatum and substantia nigra, respectively); HVA ( - 8 7 % in striatum with no statistically significant change in substantia nigra) and 3-MT ( - 97% in striaturn, and no detectable levels in substantia nigra) (Figs. 1 and 2).
3.3. Lf]ect of quinolinic acid lesions on 6-hydroxydopamine-induced toxicity Intrastriatal injections of QA 14 days before intrastriatal injections of 6-OHDA showed a very different pattern of dopaminergic integrity as deduced by DA and its metabolite levels in the nigrostriatal system, especially at day 21 when the 6-OHDA-induced neurotoxicity is fully completed. At this time-point, QA + 6OHDA-treated animals showed a much higher preservation of the dopaminergic system in both striatum and
substantia nigra as compared with animals receiving 6-OHDA alone (Figs. 1 and 2). Levels of DA in QA + 6-OHDA-treated animals were 17- and 2.1-fold higher in striatum and substantia nigra respectively than those receiving 6-OHDA alone. QA injections also protected against the 6-OHDA-induced decreases in DA metabolites, although to a lesser degree. Thus, striatal DOPAC and HVA levels were 4.2- and 2.7-fold higher in QA + 6-OHDA- than 6-OHDA-treated rats, while the nigral values of such metabolites were not different between both experimental conditions (Figs. 1 and 2). 3-MT was the only DA metabolite whose levels were altered in parallel fashion to those of DA by both QA + 6OHDA- and 6-OHDA-treated animals. In the former, striatal 3-MT levels were ll.4-fold higher than in the latter (Fig. 1). No direct comparison could be made in substantia nigra since 3-MT levels were not detectable in 6-OHDA-treated animals (Fig. 2). At the shorter 2 days time-point studied, no differences were detected between QA + 6-OHDA- and 6OHDA-treated animals in the striatum. However, nigral levels of DA, DOPAC and 3-MT were 1.5-, 1.7and 1.7-fold higher in Q A + 6 - O H D A - than in 6OHDA-treated rats (Fig. 2).
4. Discussion
Neurochemical analysis of the dopaminergic nigrostriatal system reveals that QA-lesioned animals are more resistant to the neurotoxicity induced by subsequent intrastriatal injections of 6-OHDA. In absence of QA lesions, intrastriatal 6-OHDA injections resulted in high reductions in striatal DA levels at both 2 days and 21 days following the neurotoxin administration ( - 8 4 % and - 9 8 % control values, respectively). The cell bodies of the substantia nigra showed, however, a different neurochemical profile. While no changes were detected 2 days following 6-OHDA injections, DA levels decreased ( - 7 1 % control values) at 21 days. This observation indicates a clear time-dependent retrograde axonal degeneration of substantia nigra cell bodies, which is in agreement with Ichitani et al. [11] who showed degeneration of nigral dopamine neurons 2 to 4 weeks after intrastriatal 6-OHDA injections. Comparisons of this treatment condition with animals previously lesioned with QA showed remarkable differences in terms of DA and its metabolite levels. Thus, in animals with combined lesions, while DA levels decreased ( - 7 2 % and - 6 2 % QA-treated animal levels) in striatum at days 2 and 21, respectively, in substantia nigra there were no change in DA levels at day 2 when compared to QA-treated animals, later decreasing at day 21 ( - 54%). It is particularly relevant that the overall protection induced by QA against the 6-OHDA-induced neurotoxicity of the nigrostriatal sys-
J.L. Venero et al. / Brain Research 672 (1995) 153-158
tern was detected only at 21 days. At 2 days, prior QA lesions were ineffective against the 6-OHDA-depleting effect on striatal DA levels, thus suggesting that the neuroprotective effect found in the QA-injected animals at 21 days is not related either to inhibition of the direct neurotoxic action of 6-OHDA or to diffusion problems of 6-OHDA in reaching its targets in the striatum. The mechanism by which prior intrastriatal QA lesions protect against subsequent 6-OHDA injections has to be neccesarily associated to the typical pattern of neuronal degeneration induced by this neurotoxin. One of the best known features of QA, when applied to the striatum, is the destruction of neurons containing GABA and substance P as neurotransmitters as reflected by neuronal cell loss [5,9] and high reductions of the mRNA levels encoding glutamate decarboxylase [21]. It is reasonable to think that the destruction of the striatal GABA and substance P-descending axons to the substantia nigra [6,13] would release nigral dopamine cells from tonic inhibition and thus result in increased production of DA. This effect is particularly evident in nigral DA levels following QA lesions. In the absence of 6-OHDA injections, we found significant increases of nigral DA levels following intrastriatal QA injections at the two different time points studied (35% and 35% control levels at 2 days and 21 days respectively) without significant changes of the ratio of the acidic metabolites, DOPAC or HVA, to DA (as an index of DA turnover), which is in accordance with an increased production of nigral DA without major changes in DA metabolism. The question is whether an increased production of DA is enough to explain the higher levels of DA and its metabolites in QA + 6-OHDA-injected animals than in animals injected with 6-OHDA alone. There are, however, some observations difficult to reconcile with this possibility. A known feature of the lesioned dopaminergic system is its ability to compensate for the cell loss [22]. Thus, rats sustaining near-total (> 95%) depletions of striatal dopamine, similar to those studied here, demonstrated up to 10-fold elevation of dopamine metabolism within the surviving neurons as evidenced by D O P A C / D A or H V A / D A ratios [1]. In our experiments, both D O P A C / D A and H V A / D A were elevated in striatum (4.4- and 7.3-fold) and in substantia nigra (1.8- and 3.1-fold) 21 days following intrastriatal 6-OHDA injections in absence of QA injections for D O P A C / D A and H V A / D A , respectively. In QA + 6-OHDA-treated rats, however, neither the D O P A C / D A ratio nor the H V A / D A ratio varied to the same extent as in animals injected with 6-OHDA alone in either striatum or substantia nigra. If we consider that elevated D O P A C / D A or H V A / D A ratios are ascribed to rats with an extensive (> 90%) loss of striatal DA, the maintenance of such ratios in QA + 6-OHDA-lesioned
157
animals when compared with animals injected with 6-OHDA alone strongly suggest a higher density of residual striatal dopaminergic terminals and hence a higher dopaminergic cell body survival. The mechanism by which prior QA lesions protect against 6-OHDA-induced neurotoxicity is an open question. However, the typical pattern of neuronal cell degeneration along with astroglia proliferation (see section 1) induced by QA deserves special consideration. Thus, neurotrophic factors released by activated astroglia may be responsible for the protective effect found in QA + 6-OHDA-lesioned animals. It has to be that taken into account that the activation of astrocytes in culture preparations is small relative to increases observed during reactive gliosis in the adult rat brain [12]. It may be also worth considering a probable relationship between our results and the mild improvements seen in animals lesioned with 6-OHDA which underwent adrenal medulla autografting in the striatum. Such grafts have been shown effective in reducing amphetamine-induced rotations despite little evidence of fiber outgrowth [10]. It seems quite likely that a similar glial reaction to the surgery itself may have been responsible for the minor improvements observed.
Acknowledgements This work was supported by a grant from DGICYT PM91-0097. J.L.V. thanks to the Spanish Ministerio de Educaci6n y Ciencia for a Contrato de Incorporaci6n.
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