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GMP protects against quinolinic acid-induced loss of NADPH-diaphorase-positive cells in the rat striatum
Neuroscience Letters 225 (1997) 145–148
GMP protects against quinolinic acid-induced loss of NADPH-diaphorasepositive cells in the rat striatum Ce´sar Malcona,*, Matilde Achaval b, Fa´bio Komlos a, Wania Partata c, Maurı´cio Sauressig a, Galo Ramı´rez d, Diogo O. Souza a a
Departamento de Bioquı´mica, Instituto de Biocieˆncias, Universidade Federal do Rio Grande do Sul (UFRGS), Rua Sarmento Leite 500, Porto Alegre, RS 90946-900, Brazil b Departamento de Cieˆncias Morfolo´gicas, Instituto de Biocieˆncias, Universidade Federal do Rio Grande do Sul (UFRGS), Rua Sarmento Leite 500, Porto Alegre, RS 90946-900, Brazil c Departamento de Fisiologia, Instituto de Biocieˆncias, Universidade Federal do Rio Grande do Sul (UFRGS), Rua Sarmento Leite 500, Porto Alegre, RS 90946-900, Brazil d Centro de Biologı´a Molecular, Universidad Auto´noma de Madrid, Cantoblanco, 28049 Madrid, Spain Received 16 October 1996; revised version received 20 February 1997; accepted 25 February 1997
Abstract When injected into the rat striatum, quinolinic acid causes dose-dependent widespread cell death. All cell types, including the NADPHdiaphorase-positive neurons appear to be sensitive to the toxin. The latter cells are destroyed by quinolinic acid injections of 180 nmol per striatum, this effect being blocked by the concomitant administration of 5 mg/kg of the non-competitive N-methyl-d-aspartate antagonist MK-801. We report that guanosine-5′-monophosphate (GMP), at a dose of 360 nmol, is equally effective in protecting the diaphorasepositive cells against quinolinate toxicity. 1997 Elsevier Science Ireland Ltd. Keywords: Excitotoxicity; Quinolinic acid; Huntington’s disease; Neuroprotection; Guanine nucleotides; GMP; Rat striatum.
Striatal injections of quinolinic acid in rats [2,17] induce a pattern of structural lesions and behavioral symptoms reminiscent of those seen in Huntington’s disease (HD), a dominantly inherited neurodegenerative disorder characterized by choreiform movements, emotional disturbance and cognitive impairment leading to dementia [11]. The action of quinolinic acid, an endogenous metabolite of tryptophan, which accumulates in the brain with aging [13], appears to be mediated by the N-methyl-d-aspartate (NMDA) subclass of glutamate receptors [14], either by presynaptically stimulating the release of the excitatory amino acid transmitter, or by direct postsynaptic action, and it has been shown that the non-competitive NMDA antagonist (+)-5-methyl-10,11dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801) effectively protects against quinolinic acid (QA)-induced lesions when administered concomitantly [7]. Besides their well-known role in the activation/inactiva-
* Corresponding author.
tion cycle of G-proteins, guanine nucleotides interfere with the binding of excitatory amino acids to several receptors, including the NMDA type, apparently by acting extracellularly on the receptor itself [1,8,12,15,18,19]. We have chosen guanosine-5′-monophosphate (GMP), that is inactive on G-proteins, but still an efficient displacer of excitatory amino acids [15,18], to check whether guanine nucleotides can possibly prevent QA-induced neurotoxicity in the rat striatal model by blocking the access of the excitotoxin to the striatal NMDA receptors. Also, the more phosphorylated guanine nucleotides appear to have some toxic effect of their own in some experiments involving excitotoxinmediated lipid peroxidation (our unpublished results). There has been some disagreement concerning the relative sensitivity of the various striatal cell populations to local injection of QA. Whereas a number of papers stress the selective sparing of somatostatin/neuropeptide Y-containing, NADPH-diaphorase-labeled neurons [21] as one of the key features that validate the QA model of HD [2,5,6,9], other authors contend that QA effectively destroys this neu-
0304-3940/97/$17.00 1997 Elsevier Science Ireland Ltd. All rights reserved PII S0304-3940 (97 )0 0225-5
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C. Malcon et al. / Neuroscience Letters 225 (1997) 145–148
ronal subpopulation in an area that depends on the amount of QA injected [3,4]. Since these cells are easy to identify by use of specific staining, we thought it desirable to use them as markers of QA-induced cellular loss. After verifying in our laboratory (see below) that QA, in the usual concentration range, destroys diaphorase-positive neurons, we have chosen this well-characterized type of neurons to test our hypothesis that GMP may prevent the excitotoxic action of QA by direct competition at the receptor agonist site. Male Wistar rats (2–3 months-old, weighing 200–300 g) were used. The animals were anesthetized with thiopental (45 mg/kg, intraperitoneally) and placed in a David Kopf stereotaxic frame. Two holes were drilled in the skull and a 30-gauge Hamilton syringe needle was inserted into each striatum, according to the stereotaxic coordinates of Paxinos and Watson [16] (AP, 1.2 mm, L, ±2.6 mm, DV, −5.2 mm). QA and GMP were slowly injected in 1 ml of PBS, whereas MK-801 was injected intraperitoneally 30 min before intrastriatal injections. Control animals received 1 ml of PBS in each striatum. Six weeks after the treatment, the rats were anesthetized as before and, after a brief saline flush, intracardially perfused with a mixture of 4% paraformaldehyde and 0.2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4). The striatum was postfixed in a solution containing 4% paraformaldehyde in phosphate buffer at 4°C for 2 h, and cryoprotected by immersion in 15% and 30% sucrose solutions in phosphate buffer at 4°C, with continuous agitation, until sunk. Coronal serial sections (50 mm) were
Fig. 1. Dose-dependency of the effects of intrastriatal QA on diaphorasepositive neurons. Diaphorase-positive cells were stained specifically, as described in the text, and the sections were counterstained with hematoxylin–eosin to obtain a more general picture. (A) Intrastriatal injection of PBS only. (B) 100 nmol of QA in PBS, (C) 120 nmol of QA in PBS, (D) 180 nmol of QA in PBS. Scale bar 50 mm.
Table 1 NADPH-diaphorase-positive neurons in the rat striatum after different treatments Treatment (group)
Cells/field
Control (PBS) (A) QA (B) MK-801 (C) MK-801 + QA (D) GMP (E) GMP + QA (F)
10.4 3.6 10.0 8.2 9.6 8.4
± ± ± ± ± ±
3.3 1.6a 2.6 2.1 2.0 1.8
% of control 100 34 96 79 92 81
Treatments were as described in the text and in the legend to Fig. 2 (letters in parentheses, in the first column, refer to the homologous panels in Fig. 2). Cell counts were carried out in a field of 0.52 × 0.36 mm. Twenty different fields (2 fields in each of 10 different sections) were counted for each experimental condition (typically, a total of about 200 cells were counted in all cases), and the results were averaged and expressed as mean ± SD. Two-way ANOVA was used to assess the significance of the observed differences. a Different from all the other values (P , 0.001).
obtained with a freezing microtome and collected in cold phosphate buffer. Free-floating sections were stained for NADPH-diaphorase according to Valtschanoff et al. [20], using b-NADPH as substrate and nitro blue tetrazolium as electron acceptor. Control sections were incubated without substrate. Microscopic observations and cell counts were carried out in the middle third of the lesion (practically the middle third of the striatum since 80–90% of it was affected by QA 6 weeks after the injection), thus avoiding the more distorted area immediately adjacent to the injection site. QA-injected rats developed transient tonic-clonic convulsive episodes soon after the injection which were not observed in the other groups. This lack of abnormal movements was specially obvious in the GMP + QA rats since MK-801 produces at times ataxic/hyperkinetic phenomena that are not always easy to distinguish from true convulsions. Besides, QA rats (but not MK-801 + QA, or GMP + QA) showed increased nocturnal locomotor activity in the first 2 weeks, returning to a practically normal behavior by the time chosen for the anatomical studies. Fig. 1 illustrates the sensitivity of diaphorase-positive cells to increasing concentrations of QA. While 100 nmol (B) has little detectable effect, 120 nmol (C) causes blurring of the cell contours and disruption of some prolongations, and 180 nmol (D), the standard concentration used in our experiments, leaves only an accumulation of debris in place of the cell bodies. The background hematoxylin–eosin staining confirms that most striatal cells are sensitive to the same range of concentrations of QA. As stated, between 80 and 90% of the striatum was damaged at this 180 nmol QA dose. Gliosis was more marked in the inner third of the lesion but NADPH-positive neurons were affected all throughout the lesion, with only a few intact ones seen in the very periphery of the striatum. Although we did not undertake a quantitative estimation of the damage sustained by other types of neurons, it can be said that the distribution
C. Malcon et al. / Neuroscience Letters 225 (1997) 145–148
Fig. 2. NADPH-diaphorase positive cells in the rat striatum after different treatments with QA and prospective antagonists. (A) PBS, (B) QA, 180 nmol, (C) MK-801 (5 mg/kg), (D) MK-801 followed, after 30 min, by QA (180 nmol), (E) GMP, 360 nmol, (F) QA (180 nmol) together with GMP (360 nmol). Scale bar 50 mm.
of damaged neurons was similar in different fields of the affected area. Having confirmed that diaphorase-positive cells are sensitive to QA, we have undertaken specific experiments to check the protective effect of MK-801 and GMP. Fig. 2 displays typical fields of diaphorase-positive neurons in rat striatal sections after injection of different combinations of QA and potential antagonists. Again, only stained cell debris can be seen after injecting 180 nmol of QA (B), in contrast with the excellent cell preservation in (A), where only the PBS vehicle has been injected. The other four panels in the figure clearly demonstrate that either MK801 (D) or GMP (F) block the effect of QA when injected with (or before) the toxin. Both substances fail to produce any effects when injected alone (C,E). Actually, however, some neurons in (D) and (F) (and even in (C) and (E)) show irregularities in the contour and varicosity-like changes in the prolongations. This may suggest incomplete protection by the dose of antagonist co-injected with QA (it takes more GMP to displace all QA). Also, some local damage due to the high concentration of drugs injected (osmotic effect)
147
cannot be totally excluded, although it would be probably limited to the inner core of the lesion. We have furthermore observed in our preparations that not only diaphorase-positive neurons are protected by MK-801 [7] and GMP. However, our present analysis is limited to this characteristic cell type. The quantitative evaluation of the effect of the different treatments is presented in Table 1. Cell counts of diaphorase-positive neurons confirm the microscopic picture in Fig. 2. We should add that even the 3–4 cells/field that could be tentatively identified in the QA sections (B) were heavily damaged. Only cell counts in QA sections were significantly different from all other cases (P , 0.001). MK-801 + QA (D) and GMP + QA (F) did not differ from their respective controls (without QA, (C) and (E)), but were somewhat lower than the absolute control with saline (A), although the statistical significance of this difference was less marked (P in the range of 0.05–0.02). The possibility of incomplete protection or the concurrence of osmotic damage has been mentioned in the previous paragraph. Excitatory amino acids are currently thought to play a significant role in most acute and chronic neurological disorders, as far as the direct causation of neuronal injury and death is concerned, even if quite often they just play an adjuvant or secondary role to a primary form of alteration or lesion that causes the uncontrolled release of the amino acids [10,22]. In the case of the Huntington’s disease, an endogenous excitotoxin, acting on the NMDA receptor, has been proposed as the specific pathogenic factor of the disease [2,13,17]. This hypothesis is largely based on the evidence obtained with the rat striatal experimental model where QA injections replicate to a variable extent the lesions and behavioral anomalies observed in the human disease. One of the salient features of this model was thought to be the apparent resistance of the peptide-containing, diaphorase-positive neurons to the injected QA since this population of neurons seems to be reasonably well preserved in HD [2,5,6,9,11], and in other metabolic/ toxic conditions. However, the work of Boegman et al. [3], Davies and Roberts [4], and our own results show that diaphorase-positive neurons are sensitive to QA. The longerthan-usual recovery time and the use of a sufficient QA dose of 180 nmol/striatum (but still well within the published dosage range of 60–600 nmol) in our work lead to a stabilized lesion that affects most of the striatum so that we can measure the effects of the different treatments at some distance from the mechanically damaged injection site. In any case, one should take into account the chronic and slow nature of the HD when comparing the pattern of lesions with the acute effects of massive local injections of the excitotoxin, and, in this context, the acute sensitivity of the diaphorase-positive cells should not invalidate some of the positive aspects of the QA model of HD. Actually, the sensitivity of the diaphorase-positive cells to excitotoxins affords an excellent test to assay for the ability of
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excitatory amino acid antagonists to prevent the striatal lesions. The striking protective effect of GMP is most likely related to the ability of guanine nucleotides to displace excitatory amino acids from different receptors, especially the NMDA and kainate types [1,8,12,15,18,19]. In the present experimental model, a 2:1 excess of GMP over QA was seemingly enough to achieve a high level of protection, suggesting that GMP remains in the striatal extracellular space at least as long as the injected QA. We lack precise data on the physiological presence of GMP or other guanine nucleotides in the extracellular space in the CNS, as potential regulators of receptor activity, although they must of course be released by cellular injury in the same way as the amino acids themselves are released. In this case, the native nucleotides could help slow down the propagation of the excitotoxic wave to the neighboring cells. Additionally, guanine nucleotides could serve as structural models to design a new type of glutamate antagonists potentially effective in the management of the excitotoxic component of CNS diseases [10,22]. This work was supported by Contract CI1*-CT94-0116 of the European Commission (D.G. and G.R.), by the Direccio´n General de Investigacio´n Cientı´fica y Te´cnica (Grant PM95-0021 to G.R.), and by Fundacio´n Ramo´n Areces (G.R.). [1] Baron, B.N., Dudley, M.W., McCarty, D.R., Miller, F.P., Reynolds, I.J. and Schmidt, C.J., Guanine nucleotides are competitive inhibitors of N-methyl-D-aspartate at its receptor site both in vitro and in vivo, J. Pharmacol. Exp. Ther., 250 (1989) 162–169. [2] 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. Neurosci., 11 (1991) 1649–1659. [3] Boegman, R.J., Smith, Y. and Parent, A., Quinolinic acid does not spare striatal neuropeptide Y-immunoreactive neurons, Brain Res., 415 (1987) 178–182. [4] Davies, S.W. and Roberts, P.J., No evidence for preservation of somatostatin-containing neurons after intrastriatal injections of quinolinic acid, Nature, 327 (1987) 326–329. [5] Dawbarn, D., DeQuidt, M.E. and Emson, P.L., Survival of basal ganglia neuropeptide Y-somatostatin neurons in Huntington’s disease, Brain Res., 340 (1985) 251–260. [6] Ferrante, R.J., Kowall, N.W., Beal, M.F. and Richardson, E.P., Jr, Bird, E.D. and Martin, J.B., Selective sparing of a class of striatal neurons in Huntington’s disease, Science, 230 (1985) 561–563.
[7] Foster, A.C., Gill, R. and Woodruff, G.N., Neuroprotective effects of MK-801 in vivo: selectivity and evidence for delayed degeneration mediated by NMDA receptor activation, J. Neurosci., 8 (1988) 4745–4754. [8] Gorodinsky, A., Paas, Y. and Teichberg, V.I., A ligand binding study of the interactions of guanine nucleotides with non-NMDA receptors, Neurochem. Int., 23 (1993) 285–291. [9] Koh, J.Y., Peters, S. and Choi, D.W., Neurons containing NADPHdiaphorase are selective resistant to quinolinate toxicity, Science, 234 (1986) 73–76. [10] Lipton, S.A. and Rosenberg, P.A., Excitatory amino acids as a final common pathway for neurologic disorders, N. Engl. J. Med., 330 (1994) 613–622. [11] Martin, J.B., Huntington’s disease: new approaches to an old problem, Neurology, 4 (1984) 1059–1072. [12] Monahan, J.B., Hood, W.F., Michel, J. and Compton, R.P., Effects of guanine nucleotides on N-methyl-D-aspartate receptor-ligand interactions, Mol. Pharmacol., 34 (1988) 111–116. [13] Moroni, F., Lombardi, G., Moneti, G. and Aldinio, C., The excitotoxin quinolinic acid is present in the brain of several mammals and its cortical content increases during the aging process, Neurosci Lett., 47 (1984) 51–55. [14] Nakanishi, S., Molecular diversity of glutamate receptors and implications for brain function, Science, 258 (1992) 597–603. [15] Paz, M.M., Ramos, M., Ramı´rez, G. and Souza, D., Differential effects of guanine nucleotides on kainic acid binding and on adenylate cyclase activity in chick optic tectum, FEBS Lett., 355 (1994) 205–208. [16] Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, 2nd edn., Academic Press, New York, 1986. [17] Popoli, P., Pezzola, A., Domenici, M.R., Sagratella, S., Diana, G., Caporale, M.G., Bronzetti, E., Vega, J. and Scotti de Carolis, A., Behavioral and electrophysiological correlates of the quinolinic acid rat model of Huntington’s disease, Brain Res. Bull., 35 (1994) 329–335. [18] Souza, D.O. and Ramı´rez, G., Effects of guanine nucleotides on kainic acid binding and on adenylate cyclase in chick optic tectum and cerebellum, J. Mol. Neurosci., 3 (1991) 39–45. [19] Tasca, C., Wofchuck, S., Souza, D., Ramı´rez, G. and Rodnight, R., Guanine nucleotides inhibit the stimulation of GFAP phosphorylation by glutamate, NeuroReport, 6 (1995) 249–252. [20] Valtschanoff, J.G., Weinberg, R.J., Kharazia, V.N., Nakane, M. and Schmidt, H.H.W., Neurons in rat hippocampus that synthesize nitric oxide, J. Comp. Neurol., 331 (1993) 111–121. [21] Vincent, S.R., Johansson, O., Ho¨kfelt, T. and Skirboll, C., Elde, R.P., Terenius, L., Kimmel, J. and Goldstein, M., NADPH-diaphorase: a selective histochemical marker for striatal neurons containing both somatostatin and avian pancreatic polypeptide (APP)-like immunoreactivities, J. Comp. Neurol., 217 (1983) 252–263. [22] Vornov, J.J., Tasker, R.C. and Coyle, J.T., The differential neurotoxicity of endogenous and exogenous excitatory neurotransmitter receptor agonists. In A. Harvey (Ed.), Natural and Synthetic Neurotoxins, Academic Press, London, 1993, pp. 187–224.
GMP protects against quinolinic acid-induced loss of NADPH-diaphorasepositive cells in the rat striatum Ce´sar Malcona,*, Matilde Achaval b, Fa´bio Komlos a, Wania Partata c, Maurı´cio Sauressig a, Galo Ramı´rez d, Diogo O. Souza a a
Departamento de Bioquı´mica, Instituto de Biocieˆncias, Universidade Federal do Rio Grande do Sul (UFRGS), Rua Sarmento Leite 500, Porto Alegre, RS 90946-900, Brazil b Departamento de Cieˆncias Morfolo´gicas, Instituto de Biocieˆncias, Universidade Federal do Rio Grande do Sul (UFRGS), Rua Sarmento Leite 500, Porto Alegre, RS 90946-900, Brazil c Departamento de Fisiologia, Instituto de Biocieˆncias, Universidade Federal do Rio Grande do Sul (UFRGS), Rua Sarmento Leite 500, Porto Alegre, RS 90946-900, Brazil d Centro de Biologı´a Molecular, Universidad Auto´noma de Madrid, Cantoblanco, 28049 Madrid, Spain Received 16 October 1996; revised version received 20 February 1997; accepted 25 February 1997
Abstract When injected into the rat striatum, quinolinic acid causes dose-dependent widespread cell death. All cell types, including the NADPHdiaphorase-positive neurons appear to be sensitive to the toxin. The latter cells are destroyed by quinolinic acid injections of 180 nmol per striatum, this effect being blocked by the concomitant administration of 5 mg/kg of the non-competitive N-methyl-d-aspartate antagonist MK-801. We report that guanosine-5′-monophosphate (GMP), at a dose of 360 nmol, is equally effective in protecting the diaphorasepositive cells against quinolinate toxicity. 1997 Elsevier Science Ireland Ltd. Keywords: Excitotoxicity; Quinolinic acid; Huntington’s disease; Neuroprotection; Guanine nucleotides; GMP; Rat striatum.
Striatal injections of quinolinic acid in rats [2,17] induce a pattern of structural lesions and behavioral symptoms reminiscent of those seen in Huntington’s disease (HD), a dominantly inherited neurodegenerative disorder characterized by choreiform movements, emotional disturbance and cognitive impairment leading to dementia [11]. The action of quinolinic acid, an endogenous metabolite of tryptophan, which accumulates in the brain with aging [13], appears to be mediated by the N-methyl-d-aspartate (NMDA) subclass of glutamate receptors [14], either by presynaptically stimulating the release of the excitatory amino acid transmitter, or by direct postsynaptic action, and it has been shown that the non-competitive NMDA antagonist (+)-5-methyl-10,11dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801) effectively protects against quinolinic acid (QA)-induced lesions when administered concomitantly [7]. Besides their well-known role in the activation/inactiva-
* Corresponding author.
tion cycle of G-proteins, guanine nucleotides interfere with the binding of excitatory amino acids to several receptors, including the NMDA type, apparently by acting extracellularly on the receptor itself [1,8,12,15,18,19]. We have chosen guanosine-5′-monophosphate (GMP), that is inactive on G-proteins, but still an efficient displacer of excitatory amino acids [15,18], to check whether guanine nucleotides can possibly prevent QA-induced neurotoxicity in the rat striatal model by blocking the access of the excitotoxin to the striatal NMDA receptors. Also, the more phosphorylated guanine nucleotides appear to have some toxic effect of their own in some experiments involving excitotoxinmediated lipid peroxidation (our unpublished results). There has been some disagreement concerning the relative sensitivity of the various striatal cell populations to local injection of QA. Whereas a number of papers stress the selective sparing of somatostatin/neuropeptide Y-containing, NADPH-diaphorase-labeled neurons [21] as one of the key features that validate the QA model of HD [2,5,6,9], other authors contend that QA effectively destroys this neu-
0304-3940/97/$17.00 1997 Elsevier Science Ireland Ltd. All rights reserved PII S0304-3940 (97 )0 0225-5
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C. Malcon et al. / Neuroscience Letters 225 (1997) 145–148
ronal subpopulation in an area that depends on the amount of QA injected [3,4]. Since these cells are easy to identify by use of specific staining, we thought it desirable to use them as markers of QA-induced cellular loss. After verifying in our laboratory (see below) that QA, in the usual concentration range, destroys diaphorase-positive neurons, we have chosen this well-characterized type of neurons to test our hypothesis that GMP may prevent the excitotoxic action of QA by direct competition at the receptor agonist site. Male Wistar rats (2–3 months-old, weighing 200–300 g) were used. The animals were anesthetized with thiopental (45 mg/kg, intraperitoneally) and placed in a David Kopf stereotaxic frame. Two holes were drilled in the skull and a 30-gauge Hamilton syringe needle was inserted into each striatum, according to the stereotaxic coordinates of Paxinos and Watson [16] (AP, 1.2 mm, L, ±2.6 mm, DV, −5.2 mm). QA and GMP were slowly injected in 1 ml of PBS, whereas MK-801 was injected intraperitoneally 30 min before intrastriatal injections. Control animals received 1 ml of PBS in each striatum. Six weeks after the treatment, the rats were anesthetized as before and, after a brief saline flush, intracardially perfused with a mixture of 4% paraformaldehyde and 0.2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4). The striatum was postfixed in a solution containing 4% paraformaldehyde in phosphate buffer at 4°C for 2 h, and cryoprotected by immersion in 15% and 30% sucrose solutions in phosphate buffer at 4°C, with continuous agitation, until sunk. Coronal serial sections (50 mm) were
Fig. 1. Dose-dependency of the effects of intrastriatal QA on diaphorasepositive neurons. Diaphorase-positive cells were stained specifically, as described in the text, and the sections were counterstained with hematoxylin–eosin to obtain a more general picture. (A) Intrastriatal injection of PBS only. (B) 100 nmol of QA in PBS, (C) 120 nmol of QA in PBS, (D) 180 nmol of QA in PBS. Scale bar 50 mm.
Table 1 NADPH-diaphorase-positive neurons in the rat striatum after different treatments Treatment (group)
Cells/field
Control (PBS) (A) QA (B) MK-801 (C) MK-801 + QA (D) GMP (E) GMP + QA (F)
10.4 3.6 10.0 8.2 9.6 8.4
± ± ± ± ± ±
3.3 1.6a 2.6 2.1 2.0 1.8
% of control 100 34 96 79 92 81
Treatments were as described in the text and in the legend to Fig. 2 (letters in parentheses, in the first column, refer to the homologous panels in Fig. 2). Cell counts were carried out in a field of 0.52 × 0.36 mm. Twenty different fields (2 fields in each of 10 different sections) were counted for each experimental condition (typically, a total of about 200 cells were counted in all cases), and the results were averaged and expressed as mean ± SD. Two-way ANOVA was used to assess the significance of the observed differences. a Different from all the other values (P , 0.001).
obtained with a freezing microtome and collected in cold phosphate buffer. Free-floating sections were stained for NADPH-diaphorase according to Valtschanoff et al. [20], using b-NADPH as substrate and nitro blue tetrazolium as electron acceptor. Control sections were incubated without substrate. Microscopic observations and cell counts were carried out in the middle third of the lesion (practically the middle third of the striatum since 80–90% of it was affected by QA 6 weeks after the injection), thus avoiding the more distorted area immediately adjacent to the injection site. QA-injected rats developed transient tonic-clonic convulsive episodes soon after the injection which were not observed in the other groups. This lack of abnormal movements was specially obvious in the GMP + QA rats since MK-801 produces at times ataxic/hyperkinetic phenomena that are not always easy to distinguish from true convulsions. Besides, QA rats (but not MK-801 + QA, or GMP + QA) showed increased nocturnal locomotor activity in the first 2 weeks, returning to a practically normal behavior by the time chosen for the anatomical studies. Fig. 1 illustrates the sensitivity of diaphorase-positive cells to increasing concentrations of QA. While 100 nmol (B) has little detectable effect, 120 nmol (C) causes blurring of the cell contours and disruption of some prolongations, and 180 nmol (D), the standard concentration used in our experiments, leaves only an accumulation of debris in place of the cell bodies. The background hematoxylin–eosin staining confirms that most striatal cells are sensitive to the same range of concentrations of QA. As stated, between 80 and 90% of the striatum was damaged at this 180 nmol QA dose. Gliosis was more marked in the inner third of the lesion but NADPH-positive neurons were affected all throughout the lesion, with only a few intact ones seen in the very periphery of the striatum. Although we did not undertake a quantitative estimation of the damage sustained by other types of neurons, it can be said that the distribution
C. Malcon et al. / Neuroscience Letters 225 (1997) 145–148
Fig. 2. NADPH-diaphorase positive cells in the rat striatum after different treatments with QA and prospective antagonists. (A) PBS, (B) QA, 180 nmol, (C) MK-801 (5 mg/kg), (D) MK-801 followed, after 30 min, by QA (180 nmol), (E) GMP, 360 nmol, (F) QA (180 nmol) together with GMP (360 nmol). Scale bar 50 mm.
of damaged neurons was similar in different fields of the affected area. Having confirmed that diaphorase-positive cells are sensitive to QA, we have undertaken specific experiments to check the protective effect of MK-801 and GMP. Fig. 2 displays typical fields of diaphorase-positive neurons in rat striatal sections after injection of different combinations of QA and potential antagonists. Again, only stained cell debris can be seen after injecting 180 nmol of QA (B), in contrast with the excellent cell preservation in (A), where only the PBS vehicle has been injected. The other four panels in the figure clearly demonstrate that either MK801 (D) or GMP (F) block the effect of QA when injected with (or before) the toxin. Both substances fail to produce any effects when injected alone (C,E). Actually, however, some neurons in (D) and (F) (and even in (C) and (E)) show irregularities in the contour and varicosity-like changes in the prolongations. This may suggest incomplete protection by the dose of antagonist co-injected with QA (it takes more GMP to displace all QA). Also, some local damage due to the high concentration of drugs injected (osmotic effect)
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cannot be totally excluded, although it would be probably limited to the inner core of the lesion. We have furthermore observed in our preparations that not only diaphorase-positive neurons are protected by MK-801 [7] and GMP. However, our present analysis is limited to this characteristic cell type. The quantitative evaluation of the effect of the different treatments is presented in Table 1. Cell counts of diaphorase-positive neurons confirm the microscopic picture in Fig. 2. We should add that even the 3–4 cells/field that could be tentatively identified in the QA sections (B) were heavily damaged. Only cell counts in QA sections were significantly different from all other cases (P , 0.001). MK-801 + QA (D) and GMP + QA (F) did not differ from their respective controls (without QA, (C) and (E)), but were somewhat lower than the absolute control with saline (A), although the statistical significance of this difference was less marked (P in the range of 0.05–0.02). The possibility of incomplete protection or the concurrence of osmotic damage has been mentioned in the previous paragraph. Excitatory amino acids are currently thought to play a significant role in most acute and chronic neurological disorders, as far as the direct causation of neuronal injury and death is concerned, even if quite often they just play an adjuvant or secondary role to a primary form of alteration or lesion that causes the uncontrolled release of the amino acids [10,22]. In the case of the Huntington’s disease, an endogenous excitotoxin, acting on the NMDA receptor, has been proposed as the specific pathogenic factor of the disease [2,13,17]. This hypothesis is largely based on the evidence obtained with the rat striatal experimental model where QA injections replicate to a variable extent the lesions and behavioral anomalies observed in the human disease. One of the salient features of this model was thought to be the apparent resistance of the peptide-containing, diaphorase-positive neurons to the injected QA since this population of neurons seems to be reasonably well preserved in HD [2,5,6,9,11], and in other metabolic/ toxic conditions. However, the work of Boegman et al. [3], Davies and Roberts [4], and our own results show that diaphorase-positive neurons are sensitive to QA. The longerthan-usual recovery time and the use of a sufficient QA dose of 180 nmol/striatum (but still well within the published dosage range of 60–600 nmol) in our work lead to a stabilized lesion that affects most of the striatum so that we can measure the effects of the different treatments at some distance from the mechanically damaged injection site. In any case, one should take into account the chronic and slow nature of the HD when comparing the pattern of lesions with the acute effects of massive local injections of the excitotoxin, and, in this context, the acute sensitivity of the diaphorase-positive cells should not invalidate some of the positive aspects of the QA model of HD. Actually, the sensitivity of the diaphorase-positive cells to excitotoxins affords an excellent test to assay for the ability of
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excitatory amino acid antagonists to prevent the striatal lesions. The striking protective effect of GMP is most likely related to the ability of guanine nucleotides to displace excitatory amino acids from different receptors, especially the NMDA and kainate types [1,8,12,15,18,19]. In the present experimental model, a 2:1 excess of GMP over QA was seemingly enough to achieve a high level of protection, suggesting that GMP remains in the striatal extracellular space at least as long as the injected QA. We lack precise data on the physiological presence of GMP or other guanine nucleotides in the extracellular space in the CNS, as potential regulators of receptor activity, although they must of course be released by cellular injury in the same way as the amino acids themselves are released. In this case, the native nucleotides could help slow down the propagation of the excitotoxic wave to the neighboring cells. Additionally, guanine nucleotides could serve as structural models to design a new type of glutamate antagonists potentially effective in the management of the excitotoxic component of CNS diseases [10,22]. This work was supported by Contract CI1*-CT94-0116 of the European Commission (D.G. and G.R.), by the Direccio´n General de Investigacio´n Cientı´fica y Te´cnica (Grant PM95-0021 to G.R.), and by Fundacio´n Ramo´n Areces (G.R.). [1] Baron, B.N., Dudley, M.W., McCarty, D.R., Miller, F.P., Reynolds, I.J. and Schmidt, C.J., Guanine nucleotides are competitive inhibitors of N-methyl-D-aspartate at its receptor site both in vitro and in vivo, J. Pharmacol. Exp. Ther., 250 (1989) 162–169. [2] 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. Neurosci., 11 (1991) 1649–1659. [3] Boegman, R.J., Smith, Y. and Parent, A., Quinolinic acid does not spare striatal neuropeptide Y-immunoreactive neurons, Brain Res., 415 (1987) 178–182. [4] Davies, S.W. and Roberts, P.J., No evidence for preservation of somatostatin-containing neurons after intrastriatal injections of quinolinic acid, Nature, 327 (1987) 326–329. [5] Dawbarn, D., DeQuidt, M.E. and Emson, P.L., Survival of basal ganglia neuropeptide Y-somatostatin neurons in Huntington’s disease, Brain Res., 340 (1985) 251–260. [6] Ferrante, R.J., Kowall, N.W., Beal, M.F. and Richardson, E.P., Jr, Bird, E.D. and Martin, J.B., Selective sparing of a class of striatal neurons in Huntington’s disease, Science, 230 (1985) 561–563.
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