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Selective Vulnerability of Hippocampal CA3 Neurones after Microinfusion of Paraquat into the Rat Substantia Nigra or into the Ventral Tegmental Area
j. Comp. Path. 1990 Vol. 103
Selective V u l n e r a b i l i t y of H i p p o c a m p a l CA3 N e u r o n e s after M i c r o i n f u s i o n of P a r a q u a t into the Rat S u b s t a n t i a N i g r a or into the V e n t r a l T e g m e n t a l Area M. Ca16", M. Iannone**, M. Passafaro** and G. Nistic6 t Institute of Pharmacology, Faculty of Medicine, Policlinico Mater Domini, Via 7-. Campanella, 88100, Catanzaro, University of Reggio Calabria, Italy and * *FIDIA-GRF Neuropharmacology Center, Girifalco, Catanzaro, Italy
Summary The neuropathological effects of various doses of paraquat, a widely used herbicide, given directly into different areas of the rat brain, were studied. Paraquat, microinfused into the pars compacta of the substantia nigra (3'2, 16, 32 and 160 nmol), i.e. concentrations of 3'2 to 160 mmol 1-t applied at 1 glmin -1 for l min, produced dose-dependent neuropathological lesions culminating in neuronal necrosis. A particular feature of paraquat neurotoxicity after its microinfusion into the substantia nigra (3"2 mmol per 1 at 1 gl per rain for 1 rain) or into the ventral tegmental area (1'6 mmol per 1 at 1 gl per rain for 1 rain), but not into other areas of the brain, was a selective vulnerability of hippocampal CA3 neurones consisting initially of a significant decrease of dendritic spines and later in neuronal degeneration and cell loss. No damage occurred after microinfusion ofparaquat into other areas of the brain near or distant from the infusion sites. In addition, similar neuropathological alterations occurred in other non-dopaminergic areas, such as the locus coeruleus and some raphe nuclei after direct microinfusion of paraquat into these sites. I n conclusion, the above neuropathological findings show that paraquat possesses marked neurotoxicity which, despite its chemical similarity to MPTP, is not selective for dopaminergic neurones.
Introduction P a r a q u a t is a widely used herbicide resembling in its chemical structure MPP + (N-methyl-4-phenyl-pyperidinium ion), the active metabolite of M P T P (Nmethyl-4-phenyl-l,2,3,6-tetrahydropyridine), a specific neurotoxin for the nigrostriatal dopaminergic (DA) p a t h w a y which produces, in man, monkeys, marmosets and mice, neuropathological and neurochemical lesions resembling those of Parkinson's disease (Jenner, Rupniak, Rose, Kelly, Kilpatrick, Lees and Marsden, 1984; Langston, 1985). It has been reported that, in some C a n a d i a n regions, a high correlation exists between Parkinson's disease t To whom correspondence should be addressed. * Research Fellow from the Istituto di Patologia Generale e Anatomia Patologica, Facolfft di Mediclna Veterinarla, Unlverslt~. degli Studi di Messina, Italy. 0021-9975/90/05073 + 06 $03.00/0
© 1990 Academic Press Limited
74
M. Cal6 et al.
incidence and the a m o u n t of p a r a q u a t used as herbicide (Barbeau q u o t e d by Lewin, 1985). However, the problem still is controversial since in C57 black mice the subcutaneous administration of p a r a q u a t failed to d a m a g e DA nigrostriatal neurons (Perry, Yong, Wall and Jones, 1986). On the contrary, it has been reported that p a r a q u a t lowers brain dopamine content in the frog, Rana pipiens (Barbeau, Dallaire, Bun, Poirier and Rudinska, 1985). More recently, De Gori, Froio, Strongoli, De Francesco, Cal6 and Nistic6 (1988) have reported that following the intracerebro-ventricular a d m i n i s t r a t i o n of p a r a q u a t or after microinfusion into some specific areas of the r a t brain, i.e. substantia nigra, pars compacta where DA perikarya are located, locus coeruleus, where noradrenaline cell bodies are contained and some raphe nuclei, where serotonin cell bodies are located, the herbicide possesses marked neurotoxicity characterized by an intense pattern of behavioural stimulation, circling, wet-dog syndrome accompanied by high voltage electrocortical (ECoG) epileptogenic discharges. T h e present experiments were aimed to characterize further the neuropathological lesions induced by p a r a q u a t after its administration into several areas of the brain, as evaluated by classical histological techniques and Golgi's silver impregnation method. Materials and M e t h o d s Stainless steel guide cannulae, were stereotaxicaUy implanted in the substantia nigra, pars compacta or in the ventral tegmental area, locus coeruleus, raphe dorsalis or medianus of adult Wistar-Morini rats (180 to 200 g), under chloral hydrate anaesthesia, according to the atlas coordinates of Paxinos and Watson (1982). The microinfusion of paraquat into the brain areas studied was performed in anaesthetized animals at least 48 h after surgical procedures. The guide cannula was implanted so that the tip was maintained 2 mm from the pars compacta of the substantia nigra or 2 mm from the other brain nuclei (ventral tegmental area, locus coeruleus, nucleus raphe dorsalis or medianus) in order to avoid non-specific lesions, whereas the microcannula for the infusions reached the dorsal side of these nuclei. Injection ofparaquat was made with a I0 gl Hamilton syringe, the infusion rate being 1 Ixl per min and the infusion volume up to 1 gl. The position of the cannula was verified histologically at post-mortem. Each animal was treated only once. Control infusions were carried out with the same volume of pyrogen-free twice-distilled water that had been used to dissolve paraquat and did not produce any neuronal damage. At different times after paraquat microinfusion, the animals were anaesthetized with chloral hydrate and perfused intracardically with 40 ml of saline followed by 200 ml of a mixture containing g per cent paraformaldehyde diluted with phosphate buffer at pI-I 7'4. The brain was removed and fixed in the same solution. Four to five days after fixation at room temperature, the brain was embedded in paraffin wax. Serial coronal sections 10 gm thick were stained by the Nissl method (cresyl-violet). In addition, in other animals, the perfused brain was stained by Golgi's silver impregnation method. Doses ofparaquat were expressed as total amounts (1"6 to 160 nmol); these represent concentrations of 1"6 mmol per 1 applied at a rate of 1 gl per min for 1 min. Results
P a r a q u a t microinfused into the substantia nigra, pars compacta (3.2, 16, 32 a n d 160 nmol, at least six rats for each dose) or into the ventral t e g m e n t a l area (1.6, 3"2 and 16nmol, six rats for each dose) produced neuropathological lesions which varied in extent and intensity in a dose-dependent m a n n e r , and
Microin£usion of Paraquat into Rat Brain
75
culminated in neuronal necrosis. By 24 h after microin.fusion, oedema, cytoplasmic vacuolar degeneration, nuclear pyknosis and/or chromatolysis were observed. At later stages (3, 5 and 8 days) neuronal loss accompanied by prominent proliferative vascular changes with adventitial and endothelial hypertrophy and massive gliosis was evident (Fig. 1). The extent of the degenerative area in the substantia nigra and in the ventral tegmental area was between 0'5 and 3 m m according to the dose used. A particular feature after microinfusion of the lowest doses of paraquat (3'2 nmol into substantia nigra and l ' 6 n m o l into the ventral tegmental area), was a selective vulnerability of hippocampal CA3 neurones consisting initially (24h) of a significant decrease of dendritic spines and later (3, 5 and 8 days) in neuronal degeneration and cell loss (Fig. 2). No damage was observed in CA1 and CA2 neurones or in other areas of the brain near or distal to the mesencephalic sites where microinfusions were performed (e.g. thalamus, hypothalamus, cortex). Similar neuropathological findings were observed in the area around the microinfusion site when paraquat (3'2, 16, 32 and 160 nmol) was directly applied into the locus coeruleus or into the nucleus raphe dorsalis or nucleus raphe medians (four rats for each site and dose). However, the degenerative lesions were confined to an area of approximately 0"5 to 3 m m around the cannula and paraquat given into these sites did not produce any damage in the hippocampal CA3 neurones 1, 3 and 5 days later.
Discussion
The present experiments show that paraquat produces marked neuropathological effects when microinfused into different areas of the rat brain. Interestingly, the neurot0xicity of paraquat was similar to that evoked by putrescine when given directly in some specific areas of the rat brain (De Sarro, Ascioti, Bagetta, Libri and Nistic6, 1986). This is not surprising since it has been reported that these compounds share a common structure, having two charged nitrogen atoms separated by an intramolecular distance of approximately 6"6° A (Ross and Krieger, 1981). In contrast to the systemic administration of MPTP, which gives rise to the formation of MPP + and has been reported to produce a selective degeneration of the DA nigrostriatal pathway (Langston, 1985), in the present study, the direct microinfusion of paraquat in other non-DA areas, such as the locus coeruleus and the raphe nuclei, produced similar neuropathological lesions. However, such a lack of selective toxicity for DA nigrostriatal system is not surprising since M P T P neurotoxicity does not seem to be limited to the DA nigrostriatal pathway in the mouse, having been reported to cause extensive depletion of dopamine and its metabolites in the limbic system as well as of noradrenaline in the substantia nigra and striatum (Bradbury, Costall, Jenner, Kelly, Marsden and Naylor, 1986). In addition, our data are consistent with recent reports that, following the bilateral infusion of M P T P or MPP + into the rat substantia nigra, there is a significant fall not only in dopamine and its metabolite in the nigra itself, but also a significant fall of noradrenaline and serotonin indicating a non-specific action of this neurotoxin (Bradbury, Costall, Domeney, Jenner, Marsden, Naylor and Tan, 1985).
76
M . C a l 6 e t al.
Fig. 1. Neuropathologieal effects of a single unilateral microinfusion o f p a r a q u a t (3"2 nmol) into the rat s. nigra, pars compacta. (a) Substanfia nigra (pars compacta) from a control rat. (b) Substantia nigra (pars compacta) from a rat treated 24 h earlier with paraquat ( 1'6 nmol into the substantia nigra); there is cytoplasmic vacuolar degeneration, nuclear pyknosis, neuronal loss and gliosis. (c and d) Substantia nigra from two rats treated 5 and 8 days before with the same dose ofparaquat. There is a more prominent glial proliferation. Nissl stained, × 100.
M i c r o i n f u s i o n o f Paraquat into Rat Brain
77
Fig, 2. Selective vulnerability of hippocampal CA3 neurones after microinfusionof paraquat (3'2nmol) into tile s. nigra (pars compaeta). In comparisonwith a controls. nigra (a), paraquat produced 5 days later (b) marked lossin the CA3 neurones. Nisslstained, x 100. Such non-specific action is not surprising since paraquat is known to interfere with the first step of the electron transport chain of mitochondria, thus blocking respiration and killing the cells (Lewin, 1985; Kindt, Nickas, Sonsalle and Heikkila, 1986). Naturally, during the first stage following paraquat administration there is, as a consequence of the respiratory chain blockade, a dramatic neuronal depolarization culminating in some behavioural and electrocortical epileptic disorders (De Gori el al., 1988). The specific vulnerability of CA3 hippocampal neurones after intranigral or intra-ventral tegmental microinjection of paraquat, could be well due to an activation of pathways linking the A9 (substantia nigra)-A10 (ventral tegmental area) areas of the mesencephalon to the hippocampus (Bischoff, Scatton and Korf, 1979; Scatton, Simon, Le Moal and Bischoff, 1980). It m a y be that following activation of these pathways, the nature of which should be explored, a massive release of excitatory amino-acids occurs in the hippocampus, similar to that reported in other experimental conditions such as brain ischaemia, hypoglycaemia, etc., in which a massive release of glutamate and/or aspartate occurs. It is well documented, in fact, that neuronal death after abnormal stimulation ofN-methyl-D-aspartate (NMDA) receptors in the hippocampus is due to intracellular accumulation of Ca 2+ responsible for a cascade ofceUular events culminating in proteolytic activation and other lethal metabolic mechanisms (Cotman and Iversen, 1987). References
Barbeau, A., Dallaire, R., Bun, N. T., Poirier, J. and Rudinska, E. (1985). Comparative behavioural, biochemical and pigmentary effects of MPTP, MPP ÷ and paraquat in Rana pipiens. Life Sciences, 37, 1529-1538.
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Bischoff, S., Scatton, B. and Korf, J. (1979). Biochemical evidence for a transmitter role ofdopamine in the rat hippocampus. Brain Research, 165, 161-165. Bradbury, A.J., Costall, B., Domeney, A., Jenner, P. G., Marsden, C. D., Naylor, R.J. and Tan, C. C. W. (1985). The neurotoxic actions o f M P P + in the rat are not confined to dopamine and the substantia nigra. British Journal of Pharrnacology, 86, 691P. Bradbury, A.J., Costall, B., Jenner, P. G., Kelly, M. E., Marsden, C. D. and Naylor, R. J. (1986). The effect of l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP) on striatal and limbic catecholamine neurones in white and black mice. Neuropharmacology, 25, 897-904. Cotman, C. W. and Iversen, L. L. (1987). Excitatory amino acids in the brain focus on NMDA receptors. Trends in Neurological Sciences, 10, 263-265. De Gori, N., Froio, F., Strongoli, M. C., De Francesco, A., Cal6, M. and Nistic6, G. (1988). Behavioural and electrocortical changes induced by paraquat after injection in specific areas of the brain of the rat. Neuropharmaeology, 27, 201-207. De Sarro, G. B., Ascioti, C., Bagetta, G., Libri, V. and Nistic6, G. (1986). Behavloural and electrocortical changes of polyamines after their infusion in several areas of the rat brain. In: Neurotransmitters, Seizures and Epilepsy IIL G. Nistic6, P. L. Morselli, K. G. Lloyd, R. G. Fariello and J. Engel, Eds. Raven Press, New York, pp. 423-438. Jenner, P., Rupniak, N. M. J., Rose, S., Kelly, E., Kilpatrick, G., Lees, A. and Marsden, C. D. (1984). 1-Methyl-4-phenyl- 1,2,3,6-tetrahydropyridine induced parkinsonism in the common marmoset. Neuroscience Letters, 50, 85-90. Kindt, M. V., Nickas, W.J., Sonsalle, P. K. and Heikkila, A. E. (1986). Mitochondria and the neurotoxicity of MPTP. Trends in Pharmacological Sciences, 7, 473-475. Langston, J. W. (1985). MPTP and Parkinson's disease. Trends in Neurological Sciences, 8, 79-83. Lewin, R. (1985). Parkinson's disease: an environmental cause? Science, 229, 257-258. Paxinos, G. and Watson, C. (1982). The Rat Brain in Stereotaxic Coordinates. Academic Press, London. Perry, T. L., Yong, V. W., Wall, R. A. and Jones, K. (1986). Paraquat and endogenous analogues of the neurotoxic substance N-methyl-4-phenyl-l,2,3tetrahydropyridine do not damage dopaminergic nigrostriatal neurons in the mouse. Neuroscience Letters, 69, 285-289. Ross, J. M. and Krieger, R. I. (1981). Structure activity correlation of amine inhibiting active uptake of paraquat into rat lung slices. Toxicology and Applied Pharmacology, 59, 238-249. Scatton, B., Simon, H., Le Moal, M. and Bischoff, S. (1980). Origin of dopaminergic innervation of the rat hippocampal formation. Neuroscience Letters, 18, 125-131.
[Received, July 28th, 1989] [Accepted, March 7th, 1990]
Selective V u l n e r a b i l i t y of H i p p o c a m p a l CA3 N e u r o n e s after M i c r o i n f u s i o n of P a r a q u a t into the Rat S u b s t a n t i a N i g r a or into the V e n t r a l T e g m e n t a l Area M. Ca16", M. Iannone**, M. Passafaro** and G. Nistic6 t Institute of Pharmacology, Faculty of Medicine, Policlinico Mater Domini, Via 7-. Campanella, 88100, Catanzaro, University of Reggio Calabria, Italy and * *FIDIA-GRF Neuropharmacology Center, Girifalco, Catanzaro, Italy
Summary The neuropathological effects of various doses of paraquat, a widely used herbicide, given directly into different areas of the rat brain, were studied. Paraquat, microinfused into the pars compacta of the substantia nigra (3'2, 16, 32 and 160 nmol), i.e. concentrations of 3'2 to 160 mmol 1-t applied at 1 glmin -1 for l min, produced dose-dependent neuropathological lesions culminating in neuronal necrosis. A particular feature of paraquat neurotoxicity after its microinfusion into the substantia nigra (3"2 mmol per 1 at 1 gl per rain for 1 rain) or into the ventral tegmental area (1'6 mmol per 1 at 1 gl per rain for 1 rain), but not into other areas of the brain, was a selective vulnerability of hippocampal CA3 neurones consisting initially of a significant decrease of dendritic spines and later in neuronal degeneration and cell loss. No damage occurred after microinfusion ofparaquat into other areas of the brain near or distant from the infusion sites. In addition, similar neuropathological alterations occurred in other non-dopaminergic areas, such as the locus coeruleus and some raphe nuclei after direct microinfusion of paraquat into these sites. I n conclusion, the above neuropathological findings show that paraquat possesses marked neurotoxicity which, despite its chemical similarity to MPTP, is not selective for dopaminergic neurones.
Introduction P a r a q u a t is a widely used herbicide resembling in its chemical structure MPP + (N-methyl-4-phenyl-pyperidinium ion), the active metabolite of M P T P (Nmethyl-4-phenyl-l,2,3,6-tetrahydropyridine), a specific neurotoxin for the nigrostriatal dopaminergic (DA) p a t h w a y which produces, in man, monkeys, marmosets and mice, neuropathological and neurochemical lesions resembling those of Parkinson's disease (Jenner, Rupniak, Rose, Kelly, Kilpatrick, Lees and Marsden, 1984; Langston, 1985). It has been reported that, in some C a n a d i a n regions, a high correlation exists between Parkinson's disease t To whom correspondence should be addressed. * Research Fellow from the Istituto di Patologia Generale e Anatomia Patologica, Facolfft di Mediclna Veterinarla, Unlverslt~. degli Studi di Messina, Italy. 0021-9975/90/05073 + 06 $03.00/0
© 1990 Academic Press Limited
74
M. Cal6 et al.
incidence and the a m o u n t of p a r a q u a t used as herbicide (Barbeau q u o t e d by Lewin, 1985). However, the problem still is controversial since in C57 black mice the subcutaneous administration of p a r a q u a t failed to d a m a g e DA nigrostriatal neurons (Perry, Yong, Wall and Jones, 1986). On the contrary, it has been reported that p a r a q u a t lowers brain dopamine content in the frog, Rana pipiens (Barbeau, Dallaire, Bun, Poirier and Rudinska, 1985). More recently, De Gori, Froio, Strongoli, De Francesco, Cal6 and Nistic6 (1988) have reported that following the intracerebro-ventricular a d m i n i s t r a t i o n of p a r a q u a t or after microinfusion into some specific areas of the r a t brain, i.e. substantia nigra, pars compacta where DA perikarya are located, locus coeruleus, where noradrenaline cell bodies are contained and some raphe nuclei, where serotonin cell bodies are located, the herbicide possesses marked neurotoxicity characterized by an intense pattern of behavioural stimulation, circling, wet-dog syndrome accompanied by high voltage electrocortical (ECoG) epileptogenic discharges. T h e present experiments were aimed to characterize further the neuropathological lesions induced by p a r a q u a t after its administration into several areas of the brain, as evaluated by classical histological techniques and Golgi's silver impregnation method. Materials and M e t h o d s Stainless steel guide cannulae, were stereotaxicaUy implanted in the substantia nigra, pars compacta or in the ventral tegmental area, locus coeruleus, raphe dorsalis or medianus of adult Wistar-Morini rats (180 to 200 g), under chloral hydrate anaesthesia, according to the atlas coordinates of Paxinos and Watson (1982). The microinfusion of paraquat into the brain areas studied was performed in anaesthetized animals at least 48 h after surgical procedures. The guide cannula was implanted so that the tip was maintained 2 mm from the pars compacta of the substantia nigra or 2 mm from the other brain nuclei (ventral tegmental area, locus coeruleus, nucleus raphe dorsalis or medianus) in order to avoid non-specific lesions, whereas the microcannula for the infusions reached the dorsal side of these nuclei. Injection ofparaquat was made with a I0 gl Hamilton syringe, the infusion rate being 1 Ixl per min and the infusion volume up to 1 gl. The position of the cannula was verified histologically at post-mortem. Each animal was treated only once. Control infusions were carried out with the same volume of pyrogen-free twice-distilled water that had been used to dissolve paraquat and did not produce any neuronal damage. At different times after paraquat microinfusion, the animals were anaesthetized with chloral hydrate and perfused intracardically with 40 ml of saline followed by 200 ml of a mixture containing g per cent paraformaldehyde diluted with phosphate buffer at pI-I 7'4. The brain was removed and fixed in the same solution. Four to five days after fixation at room temperature, the brain was embedded in paraffin wax. Serial coronal sections 10 gm thick were stained by the Nissl method (cresyl-violet). In addition, in other animals, the perfused brain was stained by Golgi's silver impregnation method. Doses ofparaquat were expressed as total amounts (1"6 to 160 nmol); these represent concentrations of 1"6 mmol per 1 applied at a rate of 1 gl per min for 1 min. Results
P a r a q u a t microinfused into the substantia nigra, pars compacta (3.2, 16, 32 a n d 160 nmol, at least six rats for each dose) or into the ventral t e g m e n t a l area (1.6, 3"2 and 16nmol, six rats for each dose) produced neuropathological lesions which varied in extent and intensity in a dose-dependent m a n n e r , and
Microin£usion of Paraquat into Rat Brain
75
culminated in neuronal necrosis. By 24 h after microin.fusion, oedema, cytoplasmic vacuolar degeneration, nuclear pyknosis and/or chromatolysis were observed. At later stages (3, 5 and 8 days) neuronal loss accompanied by prominent proliferative vascular changes with adventitial and endothelial hypertrophy and massive gliosis was evident (Fig. 1). The extent of the degenerative area in the substantia nigra and in the ventral tegmental area was between 0'5 and 3 m m according to the dose used. A particular feature after microinfusion of the lowest doses of paraquat (3'2 nmol into substantia nigra and l ' 6 n m o l into the ventral tegmental area), was a selective vulnerability of hippocampal CA3 neurones consisting initially (24h) of a significant decrease of dendritic spines and later (3, 5 and 8 days) in neuronal degeneration and cell loss (Fig. 2). No damage was observed in CA1 and CA2 neurones or in other areas of the brain near or distal to the mesencephalic sites where microinfusions were performed (e.g. thalamus, hypothalamus, cortex). Similar neuropathological findings were observed in the area around the microinfusion site when paraquat (3'2, 16, 32 and 160 nmol) was directly applied into the locus coeruleus or into the nucleus raphe dorsalis or nucleus raphe medians (four rats for each site and dose). However, the degenerative lesions were confined to an area of approximately 0"5 to 3 m m around the cannula and paraquat given into these sites did not produce any damage in the hippocampal CA3 neurones 1, 3 and 5 days later.
Discussion
The present experiments show that paraquat produces marked neuropathological effects when microinfused into different areas of the rat brain. Interestingly, the neurot0xicity of paraquat was similar to that evoked by putrescine when given directly in some specific areas of the rat brain (De Sarro, Ascioti, Bagetta, Libri and Nistic6, 1986). This is not surprising since it has been reported that these compounds share a common structure, having two charged nitrogen atoms separated by an intramolecular distance of approximately 6"6° A (Ross and Krieger, 1981). In contrast to the systemic administration of MPTP, which gives rise to the formation of MPP + and has been reported to produce a selective degeneration of the DA nigrostriatal pathway (Langston, 1985), in the present study, the direct microinfusion of paraquat in other non-DA areas, such as the locus coeruleus and the raphe nuclei, produced similar neuropathological lesions. However, such a lack of selective toxicity for DA nigrostriatal system is not surprising since M P T P neurotoxicity does not seem to be limited to the DA nigrostriatal pathway in the mouse, having been reported to cause extensive depletion of dopamine and its metabolites in the limbic system as well as of noradrenaline in the substantia nigra and striatum (Bradbury, Costall, Jenner, Kelly, Marsden and Naylor, 1986). In addition, our data are consistent with recent reports that, following the bilateral infusion of M P T P or MPP + into the rat substantia nigra, there is a significant fall not only in dopamine and its metabolite in the nigra itself, but also a significant fall of noradrenaline and serotonin indicating a non-specific action of this neurotoxin (Bradbury, Costall, Domeney, Jenner, Marsden, Naylor and Tan, 1985).
76
M . C a l 6 e t al.
Fig. 1. Neuropathologieal effects of a single unilateral microinfusion o f p a r a q u a t (3"2 nmol) into the rat s. nigra, pars compacta. (a) Substanfia nigra (pars compacta) from a control rat. (b) Substantia nigra (pars compacta) from a rat treated 24 h earlier with paraquat ( 1'6 nmol into the substantia nigra); there is cytoplasmic vacuolar degeneration, nuclear pyknosis, neuronal loss and gliosis. (c and d) Substantia nigra from two rats treated 5 and 8 days before with the same dose ofparaquat. There is a more prominent glial proliferation. Nissl stained, × 100.
M i c r o i n f u s i o n o f Paraquat into Rat Brain
77
Fig, 2. Selective vulnerability of hippocampal CA3 neurones after microinfusionof paraquat (3'2nmol) into tile s. nigra (pars compaeta). In comparisonwith a controls. nigra (a), paraquat produced 5 days later (b) marked lossin the CA3 neurones. Nisslstained, x 100. Such non-specific action is not surprising since paraquat is known to interfere with the first step of the electron transport chain of mitochondria, thus blocking respiration and killing the cells (Lewin, 1985; Kindt, Nickas, Sonsalle and Heikkila, 1986). Naturally, during the first stage following paraquat administration there is, as a consequence of the respiratory chain blockade, a dramatic neuronal depolarization culminating in some behavioural and electrocortical epileptic disorders (De Gori el al., 1988). The specific vulnerability of CA3 hippocampal neurones after intranigral or intra-ventral tegmental microinjection of paraquat, could be well due to an activation of pathways linking the A9 (substantia nigra)-A10 (ventral tegmental area) areas of the mesencephalon to the hippocampus (Bischoff, Scatton and Korf, 1979; Scatton, Simon, Le Moal and Bischoff, 1980). It m a y be that following activation of these pathways, the nature of which should be explored, a massive release of excitatory amino-acids occurs in the hippocampus, similar to that reported in other experimental conditions such as brain ischaemia, hypoglycaemia, etc., in which a massive release of glutamate and/or aspartate occurs. It is well documented, in fact, that neuronal death after abnormal stimulation ofN-methyl-D-aspartate (NMDA) receptors in the hippocampus is due to intracellular accumulation of Ca 2+ responsible for a cascade ofceUular events culminating in proteolytic activation and other lethal metabolic mechanisms (Cotman and Iversen, 1987). References
Barbeau, A., Dallaire, R., Bun, N. T., Poirier, J. and Rudinska, E. (1985). Comparative behavioural, biochemical and pigmentary effects of MPTP, MPP ÷ and paraquat in Rana pipiens. Life Sciences, 37, 1529-1538.
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[Received, July 28th, 1989] [Accepted, March 7th, 1990]