Chronic infusion ofl -glutamate causes neurotoxicity in rat striatum

372

Brain Research, 290 (1984) 372-375 Elsevier

Chronic infusion of L-glutamate causes neurotoxicity in rat striatum GETHIN J. McBEANand PETER J. ROBERTS* Department of Physiology and Pharmacology, School of Biochemtcal and Phystological Sciences, Umversity of Southampton, Southampton S09 3 TU (U. K. ) (Accepted September 13th, 1983) Key words: L-glutamate- - striatum - - neurotoxicity- - Huntington's disease

Chronic infusion of a high dose of monosodium glutamate (approximately 8 nmol/min) into the rat left stnatum, over a period of 1 week, caused degeneration of striatal neurones. This was accompanied by a significant loss of neurochemical markers for both GABAergic and cholinergic interneurones. These results indicate that the sustained presence of elevated concentrations of glutamate will, in time, give rise to changes similar to those seen m human neurodegenerative disorders, such as Huntington's disease. There has been much interest in the ability of certain excitatory amino acid analogues, notably kainate and ibotenate, to cause axon-sparing lesions following direct injection into various brain areas 4 and these substances are widely used as tools in the study of neurobiology. Kainate bears a close structural resemblance to the endogenous excitatory amino acid, glutamate (Glu), yet Glu itself is only minimally effective as a neurotoxic agent 13. It is quite probable that efficient removal of Glu from the site of injection by high- and low-affinity transport mechanisms may not allow sufficient time for the development of toxicity. It has been shown that removal of possible sites of uptake in the hippocampal formation following transection of the perforant pathway 7, or in the striatum following decortication 11, confers a greater vulnerability on hippocampal and striatal cells, respectively, to the toxic effects of Glu. A further indication that removal of Glu from the vicinity of its receptors may play a role in preventing neurotoxicity comes from the observation that the potent Glu uptake inhibitor, DL-threo-3-hydroxy-aspartate5, will cause lesions when injected directly into the striatum 11. The experiments described in this paper were designed to establish whether long-term administration of Glu into the intact striatum using Alzet osmotic

minipumps, might eventually lead to a degeneration of striatal neurones. Female Wistar rats (200-220g) were used throughout this study. One week before insertion of the Alzet (model 2002) minipumps, rats were implanted with striatal guide cannulae, made from 23gauge stainless steel tubing, which were secured to the skull using one small screw and acrylic dental cement. The co-ordinates used for the striatum, taken from K6nig and KlippelS, were AP +7.5, ML +2.2. Insertion of the minipumps was performed according to the instructions of the manufacturers. Rats were anaesthetized using sodium pentobarbitone (60 mg/ kg i.p.), and the pump was secured subcutaneously at the back of the neck, and connected by a short length of cannula tubing to a 29-gauge stainless steel cannula. The cannula was then inserted into the striatum through the guide cannula, such that the tip extended 1 mm beyond the level of the guide (5.3 mm deep from the level of the dura). The pump had previously been filled with a solution containing 1 mL-Glu (monosodium salt; BDH) in 0.1 M phosphate-buffered saline (PBS), which was neutralized to pH 7.4 using 2 M NaOH. Control pumps contained either 0.1 M PBS alone, or 1 M NaC1. The estimated flow rate for each pump is 0.5/A/h (manufacturer's specification),

* To whom reprint requests should be addressed. Correspondence: G. J. McBean, Dept. of Physiologyand Pharmacology,Schoolof Biochemical and PhysiologicalSciences, University of Southampton, Southampton S09 3TU, U.K. 0006-8993/84/$03.00© 1984Elsevier Science Publishers B.V.

373 and the pumps remained in situ for 1 week. For histological analysis, rats were anaesthetized and perfusion-fixed through the left (cardiac) ventricle with a solution containing 1% glutaraldehyde - 2% paraformaldehyde in 0.1 M PBS. The brains were removed, dehydrated, and embedded in paraffin. 0.6 ~m coronal sections were stained in thionin for examination by light microscopy, using a Leitz Ortholux microscope with a Leitz Vario-Orthomat R camera attachment. Assay of acetyl C o A : choline-O-acetyltransferase (cholineacetyltransferase; CHAT) activity was performed on striatal homogenates following the method of Fonnum 3. [ 3 H ] G A B A uptake into a crude synaptosomal pellet was measured in the presence and absence of sodium (choline chloride substituted) using 1 x 10-6 M [3H]GABA (spec. act. 50 Ci/mmol diluted to 0.1 Ci/mmol; Amersham), during a 4-min incubation at 25 °C. The reaction was terminated by placing the tubes on ice, followed by rapid centrifugation, The pellet was washed, then solubilized in 2% SDS. Radioactivity was determined by liquid scintillation counting. Infusion of 1 M L-Glu led to a significant reduction in both C h A T activity and sodium-dependent [ 3 H ] G A B A uptake in the ipsilateral (left) striatum, when compared with the contralateral (right) side (by 38% and 35%, respectively), as shown in Table I. Neither of these parameters showed a reduction following infusion of 0.1 M PBS (Table I) nor was C h A T activity reduced after 1 M NaC! infusion

(results not shown). No alteration in the behaviour of the rats was seen during the course of L-Glu infusion. Examination of striatal sections under low power by light microscopy revealed an area of pronounced neuronal degeneration surrounding the cannula tract for those animals which had received L-Glu (Fig. 1A). Fig. 1B and C show the corresponding area of striatum which received 0.1 M PBS or 1 M NaCI, respectively. The cannula tract is clearly identifiable in each case, yet there is no evidence of the great spread of degeneration seen in Fig. 1A. The lesioned and control striata are shown under higher magnification in Fig. 1D-F. Here the L-Glu-lesioned striatum (Fig. 1D) show severe neuronal degeneration at a site approximately 0.5 mm away from the centre of the cannula tract. The neurones appear very dark and misshapen. Fig. 1E (PBS control) and 1F (1 M NaC1 control) show a large number of normal cells adjacent to the cannula tract, which is marked by an arrow in each photograph, although it is possible that the high concentration of NaCI used may have caused slight shrinkage of cells, especially those bordering the injection site. The results reported in this paper clearly demonstrate that continuous administration of L-Glu for one week leads to a degeneration of striatal cells and a corresponding decrease in markers for cholinergic and G A B A e r g i c neurones, which is not associated with the presence of excess sodium ions. The high dose of glutamate used in this study was necessary in order to overcome the combined capaci-

TABLE I Effect of glutamate infusion on strtatal ChA T and sodium-dependent [3H]GABA uptake ChAT activity was determined in a striatal homogenate following the method of Fonnum 3. [3H]GABA uptake into crude P2preps was determined in the presence and absence of sodium (choline chloride substituted) using 1 x l0-~ [3H]GABA (final spec. act. 0.1 Ci/mmoo during a 4 min incubation at 25 °C. Reaction was terminated by placing the tubes on ice followed by rapid microfugation. Radioactivity was measured by liquid scintillation counting. The results are the mean + S.E.M. of 3-6 separate determinations, measured in triplicate. Statistical analysis is by Student's t-test. LHS: left-hand side; striatum that received L-Glu; RHS: contralateral control. ChAT activity (nmol/mg tissue~h)

Sodium-dependent [3H]GA BA uptake (nmol/mg tissue~h)

0.1 M phosphate buffer

LHS RHS

28.56 + 4.86 31.53 + 4.80

0.722 _+0.022 0.701 _+0.009

1 M e-Glu

LHS RHS

12.53 4- 1.10',* 20.40 _+2.86

0.486 + 0.050*,** 0.743 _+0.102

* P < 0.05 compared to contralateral control. * P < 0.02 compared to 0.1 M phosphate buffer. ** P < 0.01 compared to 0.1 M phosphate buffer.

374 ties of the low- and high-affinity uptake systems within the striatum. The total Vma x of both transport systems in this tissue has been estimated to be in the order of 4 nmol/mg tissue/min9. Given an approximate

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Fig. 1. Microscopic examination of striatal sections following Glu infusion. Rats were anaesthetized and then perfusion-fixed by transcardml perfuslon of 1% glutaraldehyde-2% paraformaldehyde in 0.1 M PBS, pH 7.4. Brains were dehydrated and embedded in paraffin 0.6/~m coronal sections were stained in thionin; 2-3 brains were analysed in each group. A: low power photomicrograph showing extent of L-Glu lesion within the striatum. The arrows delineate the extent of the lesion. B: low power photomicrograph of striatum which received 0.1 M PBS, showing the cannula tract. C: low power photomicrograph showing the cannula tract within the striatum after infusion of 1 M NaCI. D: high power photomicrograph of A. note the darkened, shrunken cells with few normal neurones. Photo taken approximately 0 5 mm from injection tract. E: enlarged view of B. Note the high density of normal cells bordering the injection tract (arrow). F: NaCl-injected striatum under higher magmfication. Again the majority of cells bordering the injection tract (arrow) appear normal. Scale bar: 100 ~m, A-C; 50/~m, D-F.

375 from the region of the cannula tip, there must have been a gradual accumulation of Glu and a resultant hyperexcitation of post-synaptic cells. The striatum is one of the primary sites of n e u r o d e generation in H u n t i n g t o n ' s disease 6, yet the possible importance of Glu as a potential endogenous toxin in Huntington's disease (originally p r o p o s e d by Coyle and Schwarcz 2 and M c G e e r and McGeer12), has been difficult to prove. This is because of the ineffectiveness of Glu in causing lesions after direct injections into the adult rat striatum 13, except in cases of impaired uptake 11. F u r t h e r m o r e , there is, to date, little evidence of a readily-identifiable m a j o r defect in Glu-transmitter or r e c e p t o r function recognized in Huntington's disease patients which might lead to excess quantities of Glu in the region of the synaptic cleft or a b e r r a n t responses of target cells. The advantage of using the osmotic mini-pumps is that, apart from keeping surgery to a minimum, they permit the continuous long-term administration of Glu, hence allowing time for toxicity to develop in an otherwise normal striatum, without having to severely alter G l u - u p t a k e mechanisms. The inference from this work is that the changes which take place in the Huntington's disease brain might need to be only very slight to cause the onset of n e u r o d e g e n e r a t i o n . There is some evidence of a deficiency in glutamate

1 Carter, C J., Glutamme synthetase activity m Huntington's disease, Ltfe Sct, 31 (1982) 1151-1159. 2 Coyle, J. T. and Schwarcz, R., Lesion of striatal neurones with kalnic acid provides a model for Huntington's chorea, Nature (Lond.), 263 (1976) 244-246. 3 Fonnum, F., A rapid radlochemlcal method for the deterruination of cholineacetyltransferase activity, J. Neurochem., 24 (1975) 401-409. 4 Fuxe, K., Roberts, P. J. and Schwarcz, R (Eds.), Excitotoxins, Macmillan, London, 1983, m press. 5 Johnston, G. A. R , Lodge, D., Bornstem, J. C, and Curtis, D. R., Potentiation of L-glutamate and L-aspartate excitation of cat spinal neurones by the stereo ~somers of threo-3hydroxyaspartate, J. Neurochem, 34 (1980) 241-243. 6 Klawans, H. L, and Welner, W. J., The pharmacology of choreatlc movement disorders, Progr Neurobiol., 6 (1976) 49-80. 7 Kohler, C. and Schwarcz, R,, Monosodlum glutamate: increased neurotoxicity after removal of neuronal re-uptake sites, Brain Research. 211 (1981) 485--491 8 Konig. J. F. R. and Klippel, R. A., The Rat Brain, A Stereo-

synthetase in certain areas of the brain (including the caudate and p u t a m e n ) of H u n t i n g t o n ' s disease patients 1, and this, coupled with a possible a g e - d e p e n dent increase in Glu release (found in samples of human neocortex14) which, if generalized, could be sufficient to increase Glu concentrations b e y o n d the critical level for the onset of n e u r o d e g e n e r a t i o n . Since the start of this work, M a n g a n o and Schwarczl0 have published results on a similar study of long term Glu infusion into the rat brain, also using Alzet osmotic mini-pumps. A l t h o u g h this group used a more concentrated solution of Glu, and continued the infusion for two weeks, they failed to obtain any evidence for a G l u - m e d i a t e d lesion in either the striaturn or the h i p p o c a m p a l formation. R e p e a t e d acute manual injections (1.8/~mol Glu/0.5/~1 every 12 h for 2 weeks) were, on the other hand, m o d e r a t e l y effective in producing neuronal d e g e n e r a t i o n in the striatum. There is no a p p a r e n t explanation for the discrepancy between their results with the minipumps and our results r e p o r t e d here. This work was s u p p o r t e d by a W e l l c o m e Trust major award to P.J.R. We thank Drs. R o b e r t N a y l o r and B r e n d a Costall (University of B r a d f o r d ) for their guidance in the construction and use of the striatal cannulae.

taxic Atlas, Kreiger, New York, 1970. 9 Mangano, R. M. and Schwarcz, R., The human platelet as a model for the glutamate neuron: platelet uptake of glutamate, J. Neurochem, 36 (1981) 1067-1076. 10 Mangano, R. M. and Schwarcz, R., Chronic infusion of endogenous excitatory amino acids into rat striatum and hippocampus, Bram Res Bull,, 10 (1983) 47-51. 11 McBean, G J. and Roberts, P. J., Neurotoxicity of L-glutamate and DL-threo-3-hydroxyaspartate in the rat striatum. submitted. 12 McGeer, E. G and McGeer, P. L., Duplication of biochemical changes of Huntington's chorea by intrastriatal injections of glutamic and kamlc acids, Nature (Lond.), 263 (1976) 517-519. 13 Olney, J. W. and de Gubareff, T., Glutamate neurotoxlcity and Huntington's chorea, Nature (Lond.), 271 (1978) 557-559. 14 Smith, C. C. T., Bowen, D. M. and Davison, A, N., The evoked release of endogenous amino acids from tissue prisms of human neocortex, Bram Research, 269 (1983) 103-109.