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Histological and ultrastructural changes in the rat brain following systemic administration of picolinic acid
Exp Toxic Patho11995; 47: 25-30 Gustav Fischer Verlag Jena
Medical Centre of Postgraduate Education Laboratory of Histochemistry, Warsaw, Poland
Histological and ultrastructural changes in the rat brain following systemic administration of picolinic acid M. BESKID, J. JACHIMOWICZ, A. TARASZEWSKA and D. KUKULSKA With 6 figures Received: January 28, 1994; Revised: March 31, 1994; Accepted: May 2, 1994 Address for correspondence: Prof. M. BESKID, M. D., DSc., Laboratory of Histochemistry, Marymoncka 99, 01-813 Warsaw, Poland Key words: Picolinic acid; Brain; Substantia nigra; Hippocampus
Summary Picolinic acid was administered intraperitoneally in a dose of 30, 60, or 100 mmol, once every 24 h for 8 days. Histologically, under normal conditions as well as when picolinie acid was administered in a dose of 30 mmol the brain formations exhibited characteristic features. When picolinic acid was administered in a dose of 60 mmo1 or 100 mmol, the alterations were profound and developed selectively in hippocampus, being much less intense in the substantia nigra and striatum. In such cases, injuries of neuronal cell bodies were accompanied by symptoms of spongiosis. Within the hippocampus, the neuronal cell body injury was selectively restricted to the hilar and CA3 regions of stratum pyramidale. Tissue spongiosis was more intense at the granular layer, particularly within the hilus and in the mossy fiber area at CA3. Histochemically, a variable intensity of the reaction of succinic and alpha-glycerophosphate dehydrogenases was demonstrated. A decrease in their activities was observed in areas where the neuronal cell body injuries and spongiosis took place. No changes in the Ca-ATP-ase activity in brain formation after picolinic acid treatment were observed. Ultrastructurally, the changes within substantia nigra were manifested by neuronal cell bodies of the dark type and dendritic degenerations. Also less damaged neuronal cell bodies were seen. They were swollen, depleted of polyribosomes. with dilated elements of RER and altered mitochondria. Some of the dendritic profiles were swollen with lucent cytoplasm. Most of the boutons in synaptic contact zones were unchanged. Most presynaptic terminals which were in junction with dark dendrites were swollen with or without crystal-like aggregates of synaptic vesicles.
Introduction Picolinic acid (pyridine-2-carboxylic acid) is an endogenous metabolite of tryptophan, synthesized by the ky-
nurenine pathway, like quinolinic and nicotinic acids (STONE and CONNICK 1985). This compound was normally found in the liver, as well as in the kidney (DECKER et al. 1961). Moreover, the presence of kynurenine and related metabolites in the brain was demonstrated (GAL et al. 1966; GAL 1974; GAL and SHERMAN 1978; 1980; JoSEPH et al. 1978). Little work has been done on the participation of picolinic acid in neuronal cell body activity in spite of a vast literature on quinolinic acid properties. Brain injuries caused by quinolinic acid have been explored in detail, but the reports on the effect of picolinic acid on neuronal cell bodies are scarce and this prompted us to undertake this study.
Material and methods Studies were carried out on 21 rats, divided in three groups of 7 animals each, which were given picolinic acid (Sigma, purity grade 99 %) in a dose of 30, 60 or 100 mmol in a volume of 1 ml saline (0.9 NaCl), intraperitioneally, once daily for 8 days. The control group consisted of 10 animals, which were given 1 ml of saline i.p. once daily for 8 days. Serial paraffin sections of the brain were stained with toluidine blue and with hematoxylin and eosin. In the infixed material cut on cryostat, the activities of succinic and alpha-glycerophosphate dehydrogenases were detected after PEARSE (1968), and by the calcium method for adenosine triphosphatase after PADYKULA and HERMAN (1955). For electron microscopic examinations, fresh substantia nigra was taken, fixed in buffered glutaraldehyde solution followed by osmium tetrooxide according to PALADE (1952). After dehydration, the material was embedded in Epon 812 by LUFf'S method (LUFf 1961). Ultrathin sections were stained with uranyl acetate and lead citrate according to REYNOLDS (1963). The material was examined in a JEM 100B electron microscope. Exp Toxic Pathol 47 (1995) 1
25
Results Histologically, under normal conditions, as well as when picolinic acid in a dose of 30 mmol was administered, the brain formations displayed characteristic features. In the experimental material, when picolinic acid in a dose of 60 or 100 mmol was administered, the alterations were more profound and developed selectively in hippocampus, whereas in the substantia nigra and striatum they were much less intense. In such cases, the injury of neuronal cell bodies was accompanied by symptoms of spongiosis. A more intense tissue spongiosis was seen when picolinic acid was administered in a dose of 100 mmol. Within the hippocampus formation, the neuronal cell body injury was selectively restricted to the hilar and CA3 regions of stratum pyramidale, whereas the CA 1 and CA2 regions were unaffected (fig. 1.). In such cases, neuronal cell bodies with shrunken dark cytoplasm and pyknotic nuclei were visible (fig. 2.). Moreover, a focal loss of neuronal cell bodies was observed. The architectonic composition of the pyramidal cell layer was irregular. As a rule, signs of spongiosis were detected, which
were more intense at the granular layer, within the hilus and in the mossy fiber area at CA3 (fig. 1.). In the substantia nigra, the density of neuronal cell cytoplasm was either increased, the cell bodies being condensed and dark or pale, indicating the swelling of perikarya. When picolinic acid was administered in a dose of 100 mmol, the neuronal cell bodies were pale, resembling cell shadows. Moreover, the symptoms of spongiosis, accompanied by neuronal injury were marked, especially when picolinic acid was applied in a dose of 100 mmol. In the striatum, dispersed neuronal cell bodies were pale, swollen, with pyknotic nuclei. Spongiosis was of focal character, its intensity being usually weak. Histochemical investigation revealed a variable intensity of the reaction of succinic and alpha-glycerophosphate dehydrogenases. A decrease in their activities was observed in areas where the neuronal cell body injuries and spongiosis took place. No changes of Ca-ATP-ase activity in the brain formations after picolinic acid treatment were observed. Ultrastructurally, in substantia nigra of animals treated with 30 mmol of picolinic acid, the most distinctive changes were the appearance of the dark type of neuronal cell bodies and dendritic degeneration.
Fig. 1. Picolinic acid. Neuronal cell body injury within CA3 region of stratum pyramidale. H.E. x 120.
Fig. 2. Picolinic acid. Neuronal cell body injury within stratum pyramidale. H.E. x 360.
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Exp Toxic Pathol47 (1995) 1
3
4 Fig. 3. Electron-dense pyramidal cell body with marked vacuolar degeneration of cytoplasm and clumped nuclear chromatin. x 10,750. Fig. 4. Slightly swollen neuronal perikaryon. In cytoplasm rarefaction of polyribosomes, enlarged empty-looking mitochondria with loss of cristae and vacuoles. x 10,750.
Exp Toxic Pathol47 (1995) 1
27
5
6 Fig. 5. Swollen electron-lucent cytoplasm of dendrites. Presynaptic axonal terminals are unchanged. x 13,450. Fig. 6. Enlarged axonal boutons with depletion of synaptic vesicles in junction with electron-dense dendrite. x 13,450.
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Exp Toxic Pathol 47 (1995) 1
The neuronal cell bodies were shrunken and showed electron-dense nucleus and cytoplasm, vacuolar disintegration of mitochondria, and strong condensation of all other cytoplasmic constituents (fig. 3.). In the neuropil, enlarged electron-lucent astrocytic processes accompanied the injured cells and numerous dark degenerated dendrites. Besides these degenerative changes, less damaged neuronal cell bodies were seen, characterized by slightly swollen perikaryal cytoplasm exhibiting depletion of polyribosomes, vacuolar dilatation along with lamellar arrangement of rough-surfaced reticulum elements, and alteration of mitochondria (fig. 4). In the neuropil, some dendritic profiles showed similar changes, characterized by swollen electron-lucent cytoplasm (fig. 5.). Most axonal boutons being in synaptic contact with dark, degenerated, or with electron-lucent dendrites, were unchanged. However, most presynaptic terminals in junction with dark dendrites, were swollen and exhibited marked depletion or crystal-like aggregation of synaptic vesicles (fig. 6.). Administration of 100 mmol of picolinic acid resulted in a more pronounced dark neuronal degeneration and increased swelling of astrocytic processes, leading to a loose vacuolar appearance of neuropil. Moreover, an increased frequency of a massive swelling of neuronal cell bodies was also observed.
Discussion Picolinic acid, following its systemic administration, produced alterations in neuronal cell bodies. These alterations developed within selected regions of the brain, as demonstrated within the hippocampus, substantia nigra and striatum. The injuries of neuronal cell bodies within the hippocampus were restricted to the hilar and CA3 regions, whereas the CAl and CA2 regions of stratum pyramidale were unaffected. Furthermore, signs of spongiosis were observed, and were more pronounced in places where mossy fibres were situated. We speculate that these changes may be associated with the effects of picolinic acid on zinc metabolism as this region of the hippocampus contains a particularly high zinc ion concentration (IBATA and OTSUKA 1969). Moreover, it was reported that zinc was released from the granule cells endings into the extracellular space during excitation (ASSAF and CHUNG 1984). On the other hand, there is evidence that in peripheral tissues picolinic acid participates in mineral metabolism, including the zinc turnover (SEAL 1988; KRIEGER 1980). Therefore, we associate the effects of picolinic acid on zinc metabolism with the presence of injuries of neuronal cell bodies within those regions of hippocampus which are particularly rich in zinc. In our experiments, altered, dark, degenerated forms, and less altered swollen ones were observed among neuronal cell bodies within the substantia nigra. Moreover, in the neuropil some dendritic profiles were strongly swol-
len, with electron-lucent cytoplasm. Again, the presynaptic terminals in junction with dark dendrites were swollen and exhibited marked depletion or crystal-like aggregation of synaptic vesicles. Most of these ultrastructural changes following picolinic acid administration, mimick those resulting from the neurotoxic effect of quinolinic acid treatment, described elsewhere (TARASZEWSKA et al. 1991; KIDA and MATYJA 1990; KIDA et al. 1988). However, the observed injuries should not be regarded as characteristic for the excitotoxic effect. This conclusion has been drawn from experiments in which excitatory properties of quinolinic acid analogs were studied. In these experiments, picolinic acid affected none of the examined parameters (FOSTER et al. 1983; LEHMAN et al. 1985). Moreover, there is no unequivocal evidence that picolinic acid can act via the Nmethyl-D-aspartate (NMDA) receptor population. Nevertheless, there is a report on the effect of picolinic acid on felline spinal interneurons in the presence of glycine. These later results suggested that picolinic acid might act as glycine agonist upon strychnine-sensitive receptors (TONOHIRO et al. 1990). On the other hand, picolinic acid administration has not changed the inhibitory properties of indole-2-carboxylic acid derivatives which competitively inhibit the action of glycine on the NMDA receptor (HUTTNER 1989). The involvement of glycine in the NMDA response makes it difficult to explain the results of experiments in which NMDA has been applied to a neuron without a complete exclusion of the external milieu, since a possible release of glycine by surrouding cells cannot be ruled out (JOHNSON and ASCHER 1987). The possibility that the observed alterations of neuronal cell bodies were the result of non-specific toxic effects of picolinic acid cannot be ruled out. It has been pointed out that N-methyl-D-aspartate itself is also able to produce a non-selective degeneration of neurons, this being detected in the striatum of adult and young rats (FOSTER et al. 1983). On the other hand, there are indications of inhibitory properties of picolinic acid, which was observed to completely inhibit seizures induced in mice by intracerebroventricular injections of L-kynurenine (LAPIN 1983). Antikynurenine properties were demonstrated for other kynurenines, such as kynurenic, xanthurenic, anthranilic and nicotinic acids and nicotinamide, all of which can either delay the seizures produced by intracerebroventricular administration of L-kynurenine or reduce their incidence (LAPIN 1981; 1983). The delay effect produced by kynurenic acid injected intracerebroventricularly prior to L-kynurenine, appears to be more pronounced as compared to its intraperitoneal administration, while the effects of picolinic and anthranilic acids are less pronounced (LAPIN 1983). It is noteworthy that picolinic acid is active even when given orally or intraperitoneally (LAPIN 1983). It has been suggested that the kyrunines, which possess antikynurenine properties, may playa role as endogenous regulators of the physiological activity of L-kynurenine (LAPIN 1983). Picolinic acid, being an inhiExp Toxic Patho147 (1995) 1
29
bitor of honnonal induction of liver tryptophan pyrrolase, may affect the metabolism ofL-kynurenine (KISELEVA et al. 1980).
References ASSAF SY, CHUNG SH: Release of endogenous Zn2+ from brain tissue during activity. Nature 1984; 308: 734-736. DECKER RH, KANG HH, LEACH FR, et al.: Purification and properties of 3-hydroxuanthranilic acid axidase. J bioI chern 1961; 236: 3076-3082. FOSTER AC, COLLINS JF, SCHWARCZ R: On the excitotoxic properties of quinolinic acid, 2,3-piperidine dicarboxylic acids and structurally, related compounds. Neuropharmacology 1983; 22: 1331-1342. GAL EM: Cerebral tryptophan-2,3-dioxygenase (pyrrolase) and its induction in rat brian. J Neurochem 1974; 22: 861-863. GAL EM, SHERMAN, AD: Synthesis and metabolism of Lkynurenine in rat brain. J Neurochem 1978; 30: 607-613. GAL EM, ARMSTRONG JC, GINSBERG B: The nature of in vitro hydroxylation of L-tryptophan by brain tissue. J Neurochem 1966; 13: 643-654. GAL EM, SHERMAN AD: L-kynurenine: its synthesis and possible regulatory function in brain. Neurochem Res 1980; 5: 223-239. HUTTNER IE: Indole-2-carboxylic acid: a competitive antagonist of potentiation by glycine at the NMDA receptor. Science 1989; 243: 1611-1613. IBATA T, OTSUKA N: Electron microscopic demonstration of zinc in the hippocampal formation using Timm's sulphide silver technique. J Histochem Cytochem 1969; 17: 171-175. JOHNSON JW, ASCHER P: Glycine potentiates the NMDA responses in cultured mouse brain neurons. Nature 1987; 325: 529-531. JOSEPH MH, BAKER HF, LAWSON AM: Positive identification of kynurenine in rat and human brain. Biochem Soc Trans 1978; 6: 123-126. KIDA E, MATYJA E: Dynamics and pattern of ultrastructural alterations induced by quinolinic acid in organotypic culture of rat hippocampus. Neuropatol Pol 1990; 28: 67-82. KIDA E, MATYJA E, KHASPEROW L: The ultrastructure of rat hippocampal formation in organotypic tissue culture after exposure to quinolinic acid. Neuropato1 Pol 1988; 26: 507-520.
30
Exp Toxic Pathol47 (1995) 1
KISELEVA IP, LAPIN IP, PRAKHIE IB, et al.: Dissimilar effect ofkynurenines on rat liver tryptophan pyrrolase activity. Vopr med khim (Moscov). 1980; 26: 458-461. KRIEGER I: Picolinic acid in the treatment of disorders requiring zinc supplementation. Nutr. Rev. 1980; 38: 148-150. LAPIN IP: Kynurenines and seizures. Epilepsia 1981; 22: 257-265. LAPIN IP: Nicotinamide, ionsine and hypoxanthine, putative endogenous ligand of the benzodiazepine receptor, opposite to diazepam are much more effective against kynurenine-induced seizure than against leptazol-induced seizures. Pharmac Biochem Behav 1981; 14: 589-593. LAPIN IP: Antagonism of kynurenine-induced seizure by picolinic, kynurenic and yanthurenic acids. J Neural Transmission 1983; 56: 177-185. LEHMANN J, FERKANY JW, SCHAEFFER P, et al.: Dissociation between the excitatory and "excitotoxic" effects of quinolinic acid analogs on the striatal holinergic interneuron. J Pharmacol exp Therap 1985; 232: 873-882. LUFT JH: Improvements in epoxy resin embedding methods. J Biophys Biochem Cytol1961; 9: 409-414. PADYKULA HA, HERMAN E: Factors affecting the acivity of adenosine triphosphatase and other phosphatases as measured by histochemical techniques. J Histochem Cytochem 1955; 3: 170-175. PALADE CE: A study of fixation for electron microscopy. J Exp Med 1952; 95: 285-298. PEARSE ACE: Histochemistry theoretical and applied. Third Ed. Churchill Ltd. London 1968. REYNOLDS, ES: The use oflead citrate at high pH as on electron-opaque stain in electron microscopy. J Cell BioI 1963; 17:208-212. SEAL CJ: Influence of dietary picolinic acid on mineral metabolism in the rat. Ann Nutr Metab 1988; 32: 186191. STONE TW, CONNICK JH: Quinolinic acid and other kynurenines in the central nervous system. Neurosci 1985; 15: 597-617. T ARASZEWSKA A, ZELMAN IB, SZMIELEW A: Development of selective neuronal loss in the rat hippocampus after injection of quinolinic acid. Light- and electron microscopic studies. Neuropat Pol 1991; 29: 157-170. TONOHIRO T, TANABE M, KANEKO T, et al.: Is picolinic acid a glycine agonist at strychnine-sensitive receptors? Brain Res 1990; 516: 332-334.
Medical Centre of Postgraduate Education Laboratory of Histochemistry, Warsaw, Poland
Histological and ultrastructural changes in the rat brain following systemic administration of picolinic acid M. BESKID, J. JACHIMOWICZ, A. TARASZEWSKA and D. KUKULSKA With 6 figures Received: January 28, 1994; Revised: March 31, 1994; Accepted: May 2, 1994 Address for correspondence: Prof. M. BESKID, M. D., DSc., Laboratory of Histochemistry, Marymoncka 99, 01-813 Warsaw, Poland Key words: Picolinic acid; Brain; Substantia nigra; Hippocampus
Summary Picolinic acid was administered intraperitoneally in a dose of 30, 60, or 100 mmol, once every 24 h for 8 days. Histologically, under normal conditions as well as when picolinie acid was administered in a dose of 30 mmol the brain formations exhibited characteristic features. When picolinic acid was administered in a dose of 60 mmo1 or 100 mmol, the alterations were profound and developed selectively in hippocampus, being much less intense in the substantia nigra and striatum. In such cases, injuries of neuronal cell bodies were accompanied by symptoms of spongiosis. Within the hippocampus, the neuronal cell body injury was selectively restricted to the hilar and CA3 regions of stratum pyramidale. Tissue spongiosis was more intense at the granular layer, particularly within the hilus and in the mossy fiber area at CA3. Histochemically, a variable intensity of the reaction of succinic and alpha-glycerophosphate dehydrogenases was demonstrated. A decrease in their activities was observed in areas where the neuronal cell body injuries and spongiosis took place. No changes in the Ca-ATP-ase activity in brain formation after picolinic acid treatment were observed. Ultrastructurally, the changes within substantia nigra were manifested by neuronal cell bodies of the dark type and dendritic degenerations. Also less damaged neuronal cell bodies were seen. They were swollen, depleted of polyribosomes. with dilated elements of RER and altered mitochondria. Some of the dendritic profiles were swollen with lucent cytoplasm. Most of the boutons in synaptic contact zones were unchanged. Most presynaptic terminals which were in junction with dark dendrites were swollen with or without crystal-like aggregates of synaptic vesicles.
Introduction Picolinic acid (pyridine-2-carboxylic acid) is an endogenous metabolite of tryptophan, synthesized by the ky-
nurenine pathway, like quinolinic and nicotinic acids (STONE and CONNICK 1985). This compound was normally found in the liver, as well as in the kidney (DECKER et al. 1961). Moreover, the presence of kynurenine and related metabolites in the brain was demonstrated (GAL et al. 1966; GAL 1974; GAL and SHERMAN 1978; 1980; JoSEPH et al. 1978). Little work has been done on the participation of picolinic acid in neuronal cell body activity in spite of a vast literature on quinolinic acid properties. Brain injuries caused by quinolinic acid have been explored in detail, but the reports on the effect of picolinic acid on neuronal cell bodies are scarce and this prompted us to undertake this study.
Material and methods Studies were carried out on 21 rats, divided in three groups of 7 animals each, which were given picolinic acid (Sigma, purity grade 99 %) in a dose of 30, 60 or 100 mmol in a volume of 1 ml saline (0.9 NaCl), intraperitioneally, once daily for 8 days. The control group consisted of 10 animals, which were given 1 ml of saline i.p. once daily for 8 days. Serial paraffin sections of the brain were stained with toluidine blue and with hematoxylin and eosin. In the infixed material cut on cryostat, the activities of succinic and alpha-glycerophosphate dehydrogenases were detected after PEARSE (1968), and by the calcium method for adenosine triphosphatase after PADYKULA and HERMAN (1955). For electron microscopic examinations, fresh substantia nigra was taken, fixed in buffered glutaraldehyde solution followed by osmium tetrooxide according to PALADE (1952). After dehydration, the material was embedded in Epon 812 by LUFf'S method (LUFf 1961). Ultrathin sections were stained with uranyl acetate and lead citrate according to REYNOLDS (1963). The material was examined in a JEM 100B electron microscope. Exp Toxic Pathol 47 (1995) 1
25
Results Histologically, under normal conditions, as well as when picolinic acid in a dose of 30 mmol was administered, the brain formations displayed characteristic features. In the experimental material, when picolinic acid in a dose of 60 or 100 mmol was administered, the alterations were more profound and developed selectively in hippocampus, whereas in the substantia nigra and striatum they were much less intense. In such cases, the injury of neuronal cell bodies was accompanied by symptoms of spongiosis. A more intense tissue spongiosis was seen when picolinic acid was administered in a dose of 100 mmol. Within the hippocampus formation, the neuronal cell body injury was selectively restricted to the hilar and CA3 regions of stratum pyramidale, whereas the CA 1 and CA2 regions were unaffected (fig. 1.). In such cases, neuronal cell bodies with shrunken dark cytoplasm and pyknotic nuclei were visible (fig. 2.). Moreover, a focal loss of neuronal cell bodies was observed. The architectonic composition of the pyramidal cell layer was irregular. As a rule, signs of spongiosis were detected, which
were more intense at the granular layer, within the hilus and in the mossy fiber area at CA3 (fig. 1.). In the substantia nigra, the density of neuronal cell cytoplasm was either increased, the cell bodies being condensed and dark or pale, indicating the swelling of perikarya. When picolinic acid was administered in a dose of 100 mmol, the neuronal cell bodies were pale, resembling cell shadows. Moreover, the symptoms of spongiosis, accompanied by neuronal injury were marked, especially when picolinic acid was applied in a dose of 100 mmol. In the striatum, dispersed neuronal cell bodies were pale, swollen, with pyknotic nuclei. Spongiosis was of focal character, its intensity being usually weak. Histochemical investigation revealed a variable intensity of the reaction of succinic and alpha-glycerophosphate dehydrogenases. A decrease in their activities was observed in areas where the neuronal cell body injuries and spongiosis took place. No changes of Ca-ATP-ase activity in the brain formations after picolinic acid treatment were observed. Ultrastructurally, in substantia nigra of animals treated with 30 mmol of picolinic acid, the most distinctive changes were the appearance of the dark type of neuronal cell bodies and dendritic degeneration.
Fig. 1. Picolinic acid. Neuronal cell body injury within CA3 region of stratum pyramidale. H.E. x 120.
Fig. 2. Picolinic acid. Neuronal cell body injury within stratum pyramidale. H.E. x 360.
26
Exp Toxic Pathol47 (1995) 1
3
4 Fig. 3. Electron-dense pyramidal cell body with marked vacuolar degeneration of cytoplasm and clumped nuclear chromatin. x 10,750. Fig. 4. Slightly swollen neuronal perikaryon. In cytoplasm rarefaction of polyribosomes, enlarged empty-looking mitochondria with loss of cristae and vacuoles. x 10,750.
Exp Toxic Pathol47 (1995) 1
27
5
6 Fig. 5. Swollen electron-lucent cytoplasm of dendrites. Presynaptic axonal terminals are unchanged. x 13,450. Fig. 6. Enlarged axonal boutons with depletion of synaptic vesicles in junction with electron-dense dendrite. x 13,450.
28
Exp Toxic Pathol 47 (1995) 1
The neuronal cell bodies were shrunken and showed electron-dense nucleus and cytoplasm, vacuolar disintegration of mitochondria, and strong condensation of all other cytoplasmic constituents (fig. 3.). In the neuropil, enlarged electron-lucent astrocytic processes accompanied the injured cells and numerous dark degenerated dendrites. Besides these degenerative changes, less damaged neuronal cell bodies were seen, characterized by slightly swollen perikaryal cytoplasm exhibiting depletion of polyribosomes, vacuolar dilatation along with lamellar arrangement of rough-surfaced reticulum elements, and alteration of mitochondria (fig. 4). In the neuropil, some dendritic profiles showed similar changes, characterized by swollen electron-lucent cytoplasm (fig. 5.). Most axonal boutons being in synaptic contact with dark, degenerated, or with electron-lucent dendrites, were unchanged. However, most presynaptic terminals in junction with dark dendrites, were swollen and exhibited marked depletion or crystal-like aggregation of synaptic vesicles (fig. 6.). Administration of 100 mmol of picolinic acid resulted in a more pronounced dark neuronal degeneration and increased swelling of astrocytic processes, leading to a loose vacuolar appearance of neuropil. Moreover, an increased frequency of a massive swelling of neuronal cell bodies was also observed.
Discussion Picolinic acid, following its systemic administration, produced alterations in neuronal cell bodies. These alterations developed within selected regions of the brain, as demonstrated within the hippocampus, substantia nigra and striatum. The injuries of neuronal cell bodies within the hippocampus were restricted to the hilar and CA3 regions, whereas the CAl and CA2 regions of stratum pyramidale were unaffected. Furthermore, signs of spongiosis were observed, and were more pronounced in places where mossy fibres were situated. We speculate that these changes may be associated with the effects of picolinic acid on zinc metabolism as this region of the hippocampus contains a particularly high zinc ion concentration (IBATA and OTSUKA 1969). Moreover, it was reported that zinc was released from the granule cells endings into the extracellular space during excitation (ASSAF and CHUNG 1984). On the other hand, there is evidence that in peripheral tissues picolinic acid participates in mineral metabolism, including the zinc turnover (SEAL 1988; KRIEGER 1980). Therefore, we associate the effects of picolinic acid on zinc metabolism with the presence of injuries of neuronal cell bodies within those regions of hippocampus which are particularly rich in zinc. In our experiments, altered, dark, degenerated forms, and less altered swollen ones were observed among neuronal cell bodies within the substantia nigra. Moreover, in the neuropil some dendritic profiles were strongly swol-
len, with electron-lucent cytoplasm. Again, the presynaptic terminals in junction with dark dendrites were swollen and exhibited marked depletion or crystal-like aggregation of synaptic vesicles. Most of these ultrastructural changes following picolinic acid administration, mimick those resulting from the neurotoxic effect of quinolinic acid treatment, described elsewhere (TARASZEWSKA et al. 1991; KIDA and MATYJA 1990; KIDA et al. 1988). However, the observed injuries should not be regarded as characteristic for the excitotoxic effect. This conclusion has been drawn from experiments in which excitatory properties of quinolinic acid analogs were studied. In these experiments, picolinic acid affected none of the examined parameters (FOSTER et al. 1983; LEHMAN et al. 1985). Moreover, there is no unequivocal evidence that picolinic acid can act via the Nmethyl-D-aspartate (NMDA) receptor population. Nevertheless, there is a report on the effect of picolinic acid on felline spinal interneurons in the presence of glycine. These later results suggested that picolinic acid might act as glycine agonist upon strychnine-sensitive receptors (TONOHIRO et al. 1990). On the other hand, picolinic acid administration has not changed the inhibitory properties of indole-2-carboxylic acid derivatives which competitively inhibit the action of glycine on the NMDA receptor (HUTTNER 1989). The involvement of glycine in the NMDA response makes it difficult to explain the results of experiments in which NMDA has been applied to a neuron without a complete exclusion of the external milieu, since a possible release of glycine by surrouding cells cannot be ruled out (JOHNSON and ASCHER 1987). The possibility that the observed alterations of neuronal cell bodies were the result of non-specific toxic effects of picolinic acid cannot be ruled out. It has been pointed out that N-methyl-D-aspartate itself is also able to produce a non-selective degeneration of neurons, this being detected in the striatum of adult and young rats (FOSTER et al. 1983). On the other hand, there are indications of inhibitory properties of picolinic acid, which was observed to completely inhibit seizures induced in mice by intracerebroventricular injections of L-kynurenine (LAPIN 1983). Antikynurenine properties were demonstrated for other kynurenines, such as kynurenic, xanthurenic, anthranilic and nicotinic acids and nicotinamide, all of which can either delay the seizures produced by intracerebroventricular administration of L-kynurenine or reduce their incidence (LAPIN 1981; 1983). The delay effect produced by kynurenic acid injected intracerebroventricularly prior to L-kynurenine, appears to be more pronounced as compared to its intraperitoneal administration, while the effects of picolinic and anthranilic acids are less pronounced (LAPIN 1983). It is noteworthy that picolinic acid is active even when given orally or intraperitoneally (LAPIN 1983). It has been suggested that the kyrunines, which possess antikynurenine properties, may playa role as endogenous regulators of the physiological activity of L-kynurenine (LAPIN 1983). Picolinic acid, being an inhiExp Toxic Patho147 (1995) 1
29
bitor of honnonal induction of liver tryptophan pyrrolase, may affect the metabolism ofL-kynurenine (KISELEVA et al. 1980).
References ASSAF SY, CHUNG SH: Release of endogenous Zn2+ from brain tissue during activity. Nature 1984; 308: 734-736. DECKER RH, KANG HH, LEACH FR, et al.: Purification and properties of 3-hydroxuanthranilic acid axidase. J bioI chern 1961; 236: 3076-3082. FOSTER AC, COLLINS JF, SCHWARCZ R: On the excitotoxic properties of quinolinic acid, 2,3-piperidine dicarboxylic acids and structurally, related compounds. Neuropharmacology 1983; 22: 1331-1342. GAL EM: Cerebral tryptophan-2,3-dioxygenase (pyrrolase) and its induction in rat brian. J Neurochem 1974; 22: 861-863. GAL EM, SHERMAN, AD: Synthesis and metabolism of Lkynurenine in rat brain. J Neurochem 1978; 30: 607-613. GAL EM, ARMSTRONG JC, GINSBERG B: The nature of in vitro hydroxylation of L-tryptophan by brain tissue. J Neurochem 1966; 13: 643-654. GAL EM, SHERMAN AD: L-kynurenine: its synthesis and possible regulatory function in brain. Neurochem Res 1980; 5: 223-239. HUTTNER IE: Indole-2-carboxylic acid: a competitive antagonist of potentiation by glycine at the NMDA receptor. Science 1989; 243: 1611-1613. IBATA T, OTSUKA N: Electron microscopic demonstration of zinc in the hippocampal formation using Timm's sulphide silver technique. J Histochem Cytochem 1969; 17: 171-175. JOHNSON JW, ASCHER P: Glycine potentiates the NMDA responses in cultured mouse brain neurons. Nature 1987; 325: 529-531. JOSEPH MH, BAKER HF, LAWSON AM: Positive identification of kynurenine in rat and human brain. Biochem Soc Trans 1978; 6: 123-126. KIDA E, MATYJA E: Dynamics and pattern of ultrastructural alterations induced by quinolinic acid in organotypic culture of rat hippocampus. Neuropatol Pol 1990; 28: 67-82. KIDA E, MATYJA E, KHASPEROW L: The ultrastructure of rat hippocampal formation in organotypic tissue culture after exposure to quinolinic acid. Neuropato1 Pol 1988; 26: 507-520.
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