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Kainate-induced degeneration of neostriatal neurons: dependency upon corticostriatal tract
Brain Research, 139 (1978) 381-383 © Elsevier/North-Holland Biomedical Press
Kainate-induced degeneration of neostriatal neurons: corticostriatal tract
381
dependency upon
E. G. McGEER, P. L. McGEER and K. SINGH
Kinsmen Laboratory of Neurological Research, Department of Psychiatry, Faculty of Medicine, University of British Columbia, Vancouver, B. C., V6T 1 W5 (Canada) (Accepted August 12th, 1977)
Evidence now suggests that glutamate may be a transmitter in the corticostriatal pathway2,~, 7. Lesioning of this pathway brings about a sharp reduction of glutamate uptake in synaptosomal preparations of rat striatum, whereas lesioning of afferents from the thalamus brings about no change 5. Injection of the glutamate analog kainate into the rat striatum causes destruction of cholinergic and GABAnergic cells1,4, possibly because they are postsynaptic to the glutamate nerve endings of this corticostriatal pathway, and therefore possess high concentrations of glutamate receptors. The phenomenon is of great interest because of its resemblance to the biochemical pathology seen in Huntington's chorea. Olney et al. 6, who originally observed that intracerebral injections of kainate caused neuronal destruction, suggested that kainate and other glutamate analogs might be excitotoxic because of their direct action on glutamate receptors. We present evidence here that the toxic effects of small doses of kainate in the striatum are dependent upon the integrity of the corticostriatal pathway. This again raises the question as to whether overactive glutamate pathways can do tissue damage as originally proposed for Huntington's chorea 4. Stereotaxic injections of 2.5 nmoles of kainate into the neostriatum of male Wistar rats weighing approximately 300 g were done as previously described 4 and glutamic acid decarboxylase (GAD) and choline acetyltransferase (CAT) assays were used as measures of the neuronal damage. The kainate was always injected in 1 #l over a 5-min period, since both time and volume were found to influence the results. Surgical lesioning of the corticostriatal tract and electrolytic lesioning of the thalamus were done exactly as previously described for the studies ~ which demonstrated that the former lesion, but not the latter, caused a highly significant reduction (of about 40 ~ ) in high affinity glutamate uptake in the neostriatum on the lesioned side. All operations were done under Nembutal anesthesia. Ten to 14 days were allowed between lesioning and the kainate injection, and 7 days between the kainate injection and sacrifice. As is evident in Table I, the effect of this relatively small dose of kainate was markedly reduced (almost eliminated) in animals where the ipsilateral corticostriatal tract had been lesioned, but was not significantly changed in animals with an ipsilateral
382 TABLE I
Protein and enzyme levels in neostriata injected with 2.5 nmoles o f kainate as a per cent o f those on the contralateral side (mean ± S.E.) The absolute levels for the contralateral side were comparable in all groups with those found in rats not subjected to any manipulation. Control values were: protein, 114.2 ± 3.8 mg/g of tissue; G A D , 14.68 ± 0.38/~mole/h/100 mg protein; CAT, 34.08 ± 0.81/~mole/h/100 mg protein. Six rats per group.
Rats with lesions o f the
Protein GAD CAT
Corticostriatal tract
Thalamus
Unlesioned 92.8 ± 3.6 40.0 ± 0.7 43.8 ± 2.5
97.4 ± 1.9 95.7 ± 6.3* 86.5 ~ 5.3*
91.4 ± 5.0 40.7 ± 7.8 45.1 i 8.9
* Indicates P < 0.001 for comparison with data from unlesioned rats.
thalamic lesion. Careful examination of the tissue was undertaken to verify from needle tract marks correct placement of the kainate. Thalamic, cortical and non-lesioned animals were run together so that each group would receive an identical kainate solution. 2.5 nmoles of kainate is a relatively small dose, but it normally produces visible cellular destruction in the neostriatum, and severe enzymic losses. The present data would suggest that much of the effect of these small doses of kainate is an indirect one, and probably depends upon the release of glutamate from glutamatergic nerve endings, although interaction with some other (unknown) component of the corticostriatal tract cannot be ruled out. In in vitro experiments, kainate was found to inhibit the sodium-depende,at TABLE 1I
Effect o f kainic acid on sodium-dependent accumulation o f [14C]glutamate, dopamine by synaptosomal fraction o f rat neostriatal homogenates
114C]GAB,4 or 13H /-
All accumulation studies were done as previously described 5 on the P2 fraction of rat neostriatal homogenates using 10 6 M of the radioactive material; the kainate solution was made up immediately before use and was present during the 5 min pre-incubation and the 5 min exposure to radioactive material. Control accumulations in/~moles/5 min/g protein were: 1.67 -4- 0.12 for glutamate, 1.34 ! 0.09 for G A B A and 0.11 ± 0.04 for dopamine.
Uptake as per cent o f that observed with same homogenates in the absence o f kainate Concentration o f kainate
Glutamate accumulation
GABA accumulation
Dopamine accumulation
10 - 3 M 3.16 X 1 0 - 4 M 10 4 M 3.16 x 10 5 M 10 5 M 10 -6 M
24 + 2 61 ± 2 74±4 79±9 94±5 93 ~ 7
92 ± 9 1122_8 1 1 9 ± 10 102 ± 7
109 ~ 11 104±6 1 1 4 ± 10 107 ± 8
383 accumulation of radioactive glutamate by the Pe fraction of rat striatal homogenates (Table II). The effect has some apparent specificity, since kainate does not inhibit the accumulation of either G A B A or dopamine under the same conditions. The effective concentrations are high, but are of the order that may well be obtained locally in the intracerebral injection studies. The inhibition of about 75 ~o at 10-3 M (Table II) is comparable to that reported by Lakshmanan and Padmanaban 8 for kainate in synaptosomal preparations from whole young rat brains. A possible alternative hypothesis is that degeneration of the corticostriatal tract causes membrane changes in the postsynaptic neurons in the striatum, which renders them subsensitive to kainate. This seems unlikely, particularly since denervation usually induces super- rather than subsensitivity. A non-specific sequel of neuronal degeneration is unlikely, since lesions of the thalamostriatal afferents had no effect on the kainate-induced neurotoxicity. The present data do not rule out some additional direct action of kainate, particularly at higher concentrations, but they do lend weight to the hypothesis that the corticostriatal glutamatergic systems may be critical for slow and progressive cell death, such as seen in Huntington's disease. We thank Elizabeth Sutherland for technical assistance and gratefully acknowledge the financial support of the Huntington's Chorea Foundation, the W. GarfieldWeston Foundation and the Medical Research Council of Canada.
1 Coyle, J. T., Jr. and Schwarcz, R., Lesion of striatal neurones with kainic acid provides a model for Huntington's chorea, Nature (Lond.), 263 (1976) 244-246. 2 Divac, I., Fonnum, F. and Storm-Mathisen, J., High affinity uptake of glutamate in terminals of corticostriatal axons, Nature (Lond.), 266 (1977) 377-378. 3 Lakshmanan, J. and Padmanaban, G., Effect of some 'strong' excitants of central neurones on the uptake of L-glutamateand L-aspartate by synaptosomes, Biochem. biophys. Res. Commun., 58 (1974) 690-698. 4 McGeer, E. G. and McGeer, P. L., Duplication of biochemical changes of Huntington's chorea by intrastriatal injections of glutamic and kainic acids, Nature (Lond.), 263 (1976) 517-519. 5 McGeer, P. L., McGeer, E. G., Scherer, U. and Singh, K., A glutamatergic cortico-striatal path?, Brain Research, 128 (1977) 369-373. 60lney, J. W., Sharpe, L. G. and de Gubareff, T., Excitotoxic amino acids, Neurosci. Abstr., 5 (1975) 371. 7 Spencer, H. J., Antagonism of cortical excitation of striatal neurons by glutamic acid diethyl ester: evidence for glutamic acid as an excitatory transmitter in the rat striatum, Brain Research, 102 (1976) 91-101.
Kainate-induced degeneration of neostriatal neurons: corticostriatal tract
381
dependency upon
E. G. McGEER, P. L. McGEER and K. SINGH
Kinsmen Laboratory of Neurological Research, Department of Psychiatry, Faculty of Medicine, University of British Columbia, Vancouver, B. C., V6T 1 W5 (Canada) (Accepted August 12th, 1977)
Evidence now suggests that glutamate may be a transmitter in the corticostriatal pathway2,~, 7. Lesioning of this pathway brings about a sharp reduction of glutamate uptake in synaptosomal preparations of rat striatum, whereas lesioning of afferents from the thalamus brings about no change 5. Injection of the glutamate analog kainate into the rat striatum causes destruction of cholinergic and GABAnergic cells1,4, possibly because they are postsynaptic to the glutamate nerve endings of this corticostriatal pathway, and therefore possess high concentrations of glutamate receptors. The phenomenon is of great interest because of its resemblance to the biochemical pathology seen in Huntington's chorea. Olney et al. 6, who originally observed that intracerebral injections of kainate caused neuronal destruction, suggested that kainate and other glutamate analogs might be excitotoxic because of their direct action on glutamate receptors. We present evidence here that the toxic effects of small doses of kainate in the striatum are dependent upon the integrity of the corticostriatal pathway. This again raises the question as to whether overactive glutamate pathways can do tissue damage as originally proposed for Huntington's chorea 4. Stereotaxic injections of 2.5 nmoles of kainate into the neostriatum of male Wistar rats weighing approximately 300 g were done as previously described 4 and glutamic acid decarboxylase (GAD) and choline acetyltransferase (CAT) assays were used as measures of the neuronal damage. The kainate was always injected in 1 #l over a 5-min period, since both time and volume were found to influence the results. Surgical lesioning of the corticostriatal tract and electrolytic lesioning of the thalamus were done exactly as previously described for the studies ~ which demonstrated that the former lesion, but not the latter, caused a highly significant reduction (of about 40 ~ ) in high affinity glutamate uptake in the neostriatum on the lesioned side. All operations were done under Nembutal anesthesia. Ten to 14 days were allowed between lesioning and the kainate injection, and 7 days between the kainate injection and sacrifice. As is evident in Table I, the effect of this relatively small dose of kainate was markedly reduced (almost eliminated) in animals where the ipsilateral corticostriatal tract had been lesioned, but was not significantly changed in animals with an ipsilateral
382 TABLE I
Protein and enzyme levels in neostriata injected with 2.5 nmoles o f kainate as a per cent o f those on the contralateral side (mean ± S.E.) The absolute levels for the contralateral side were comparable in all groups with those found in rats not subjected to any manipulation. Control values were: protein, 114.2 ± 3.8 mg/g of tissue; G A D , 14.68 ± 0.38/~mole/h/100 mg protein; CAT, 34.08 ± 0.81/~mole/h/100 mg protein. Six rats per group.
Rats with lesions o f the
Protein GAD CAT
Corticostriatal tract
Thalamus
Unlesioned 92.8 ± 3.6 40.0 ± 0.7 43.8 ± 2.5
97.4 ± 1.9 95.7 ± 6.3* 86.5 ~ 5.3*
91.4 ± 5.0 40.7 ± 7.8 45.1 i 8.9
* Indicates P < 0.001 for comparison with data from unlesioned rats.
thalamic lesion. Careful examination of the tissue was undertaken to verify from needle tract marks correct placement of the kainate. Thalamic, cortical and non-lesioned animals were run together so that each group would receive an identical kainate solution. 2.5 nmoles of kainate is a relatively small dose, but it normally produces visible cellular destruction in the neostriatum, and severe enzymic losses. The present data would suggest that much of the effect of these small doses of kainate is an indirect one, and probably depends upon the release of glutamate from glutamatergic nerve endings, although interaction with some other (unknown) component of the corticostriatal tract cannot be ruled out. In in vitro experiments, kainate was found to inhibit the sodium-depende,at TABLE 1I
Effect o f kainic acid on sodium-dependent accumulation o f [14C]glutamate, dopamine by synaptosomal fraction o f rat neostriatal homogenates
114C]GAB,4 or 13H /-
All accumulation studies were done as previously described 5 on the P2 fraction of rat neostriatal homogenates using 10 6 M of the radioactive material; the kainate solution was made up immediately before use and was present during the 5 min pre-incubation and the 5 min exposure to radioactive material. Control accumulations in/~moles/5 min/g protein were: 1.67 -4- 0.12 for glutamate, 1.34 ! 0.09 for G A B A and 0.11 ± 0.04 for dopamine.
Uptake as per cent o f that observed with same homogenates in the absence o f kainate Concentration o f kainate
Glutamate accumulation
GABA accumulation
Dopamine accumulation
10 - 3 M 3.16 X 1 0 - 4 M 10 4 M 3.16 x 10 5 M 10 5 M 10 -6 M
24 + 2 61 ± 2 74±4 79±9 94±5 93 ~ 7
92 ± 9 1122_8 1 1 9 ± 10 102 ± 7
109 ~ 11 104±6 1 1 4 ± 10 107 ± 8
383 accumulation of radioactive glutamate by the Pe fraction of rat striatal homogenates (Table II). The effect has some apparent specificity, since kainate does not inhibit the accumulation of either G A B A or dopamine under the same conditions. The effective concentrations are high, but are of the order that may well be obtained locally in the intracerebral injection studies. The inhibition of about 75 ~o at 10-3 M (Table II) is comparable to that reported by Lakshmanan and Padmanaban 8 for kainate in synaptosomal preparations from whole young rat brains. A possible alternative hypothesis is that degeneration of the corticostriatal tract causes membrane changes in the postsynaptic neurons in the striatum, which renders them subsensitive to kainate. This seems unlikely, particularly since denervation usually induces super- rather than subsensitivity. A non-specific sequel of neuronal degeneration is unlikely, since lesions of the thalamostriatal afferents had no effect on the kainate-induced neurotoxicity. The present data do not rule out some additional direct action of kainate, particularly at higher concentrations, but they do lend weight to the hypothesis that the corticostriatal glutamatergic systems may be critical for slow and progressive cell death, such as seen in Huntington's disease. We thank Elizabeth Sutherland for technical assistance and gratefully acknowledge the financial support of the Huntington's Chorea Foundation, the W. GarfieldWeston Foundation and the Medical Research Council of Canada.
1 Coyle, J. T., Jr. and Schwarcz, R., Lesion of striatal neurones with kainic acid provides a model for Huntington's chorea, Nature (Lond.), 263 (1976) 244-246. 2 Divac, I., Fonnum, F. and Storm-Mathisen, J., High affinity uptake of glutamate in terminals of corticostriatal axons, Nature (Lond.), 266 (1977) 377-378. 3 Lakshmanan, J. and Padmanaban, G., Effect of some 'strong' excitants of central neurones on the uptake of L-glutamateand L-aspartate by synaptosomes, Biochem. biophys. Res. Commun., 58 (1974) 690-698. 4 McGeer, E. G. and McGeer, P. L., Duplication of biochemical changes of Huntington's chorea by intrastriatal injections of glutamic and kainic acids, Nature (Lond.), 263 (1976) 517-519. 5 McGeer, P. L., McGeer, E. G., Scherer, U. and Singh, K., A glutamatergic cortico-striatal path?, Brain Research, 128 (1977) 369-373. 60lney, J. W., Sharpe, L. G. and de Gubareff, T., Excitotoxic amino acids, Neurosci. Abstr., 5 (1975) 371. 7 Spencer, H. J., Antagonism of cortical excitation of striatal neurons by glutamic acid diethyl ester: evidence for glutamic acid as an excitatory transmitter in the rat striatum, Brain Research, 102 (1976) 91-101.