Brain Research, 526 (1990) 113-121
113
Elsevier
BRES 15799
Autoradiographical analysis of excitatory amino acid binding sites in rat hippocampus during the development of hippocampal kindling A. Vezzani 1, R. Serafini 1, R. Samanin I and A.C. Foster 2 l lstituto di Ricerche Farmacologiche 'Mario Negri', Milan (Italy) and 2Merck, Sharp and Dohme Ltd., Neuroscience Research Centre, Terlings Park, Harlow, Essex (U. K.)
(Accepted 27 February 1990) Key words: N-Methyl-D-aspartatereceptor; Quisqualate receptor; Kainate receptor; MK-801; Epilepsy
Binding sites for excitatory amino acids have been determined by autoradiographicalprocedures in the rat hippocampus and striatum during hippocampal kindling.The bindingsites measured were the N-methyl-D-aspartate(NMDA)-sensitivesites for L-[3H]glutamateand [3H]MK-801 sites (transmitter recognitionsite and ion channelof the NMDA receptor, respectively),[3H]a-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) sites (quisqualate receptor), [3H]kainatesites (kainate receptor) and NMDA-insensitivesites for L-[aH]glutamate.In general, little change was apparent in the hippocampus or striatum for any of these binding sites when assessed 48 h after attaining stages 1/2, 3 or 5 of kindling. These results suggest that hippocampal kindling does not bring about a change in the excitatory amino acid receptor binding sites examined, and that the appearance of an NMDA receptor-mediated component to synaptic responses in the hippocampus produced by kindling15, cannot be explained on this basis. INTRODUCTION Kindling is an animal model of chronic epilepsy which develops over time following repetitive high-frequency stimulation of various brain areas 9'26. The electrical stimulation is initially sub-convulsive and eventually leads to fully generalised seizures. This phenomenon is thought to be a consequence of some form of neuronal plasticity which underlies the enhanced excitability of the activated tissue ~4. Several lines of evidence suggest that the N-methylD-aspartate (NMDA) sub-type of excitatory amino acid receptors significantly contributes to the establishment of kindling. NMDA receptor antagonists retard the aquisition and block expression of the kindled seizures 2-4'24'25. In accordance with these results we have recently found that intrahippocampal injection of the NMDA receptor antagonist 2-amino-7-phosphonoheptanoate (AP7) at the early stages (1-2) of hippocampal kindling significantly delayed the occurrence of generalized convulsions without modifying the seizure after-discharge (AD) duration, whereas both behavioural seizures and AD were suppressed when the same dose of AP7 was administered after the induction of stage 5 seizures 33. These results suggest that the contribution of NMDA receptors to the generation of the AD in the hippocampus increases during kindling development.
Electrophysiological experiments using hippocampal slices have recently shown that NMDA receptors become involved in synaptic transmission after amygdaloid or hippocampal commisure kindling 15, and an enhanced post-synaptic sensitivity to NMDA has been shown following kindling-like activation of the hippocampal Schaffer collateral pathway in vitro al. In addition, an increase in NMDA receptor-mediated responses has been observed in hippocampus by extracellular calcium measurements 35 and inhibition of phosphoinositide hydrolysis2°, following hippocampal or amygdaloid kindling. An increased density of excitatory amino acid receptors (particularly NMDA receptors) in the hippocampus could be a plastic alteration contributing to these observations. In the present study we have examined binding sites for the NMDA, quisqualate and kainate sub-types of excitatory amino acid receptors in rat hippocampus by autoradiographical analysis during the development (stages 1/2 and 3) and following acquisition (stage 5) of hippocampal kindling in rats. A preliminary account of this work has appeared 34. MATERIALS AND METHODS Male Sprague-Dawleyrats (250-280 g, Charles River, Italy) were used. The animalswere housed at constant temperature (23 °C) and
Correspondence: A.C. Foster, Merck, Sharp and Dohme Research Laboratories, Neuroscience Research Centre, Terlings Park, Eastwick Road, Harlow, Essex, CM20 2QR, U.K.
0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
114 relative humidity (60%) with a fixed 12 h light-dark cycle and free access to food and water.
Implantation procedure The surgical procedure for electrode implantation in the dorsal hippocampus under Equithesin anaesthesia (9.7 mg/ml sodium pentobarbital in saline, 42.6 mg/ml chloral hydrate in propyleneglycol, 21.2 mg/ml MgSO4 in ethanol; 3.5 ml/kg, i.p.) was as previously described 32. For implantation, the co-ordinates from bregma were: (nose bar -2.5) AP 3.5, L 2.3, H 2.9. In addition, stainless-steel screw electrodes were placed bilaterally over the sensorimotor cortex and a ground lead screwed over the nasal sinus. The electrodes were connected to a multipin socket (March Electronics, New York, NY), and were secured to the skull by acrylic dental cement. Electroencephalographic (EEG) recordings were made in unanaesthetised, freely moving animals as previously described 32.
Kindling procedure Kindling was started after a postoperative period of 7 days, when no behavioural signs of pain or discomfort were noted in the operated animals. The animals were allowed to acclimatize in a Plexiglas cage and an EEG recording (4-channel EEG polygraph, model BBP, Battaglia Rangoni, Bologna, Italy) was made for at least 10 min to assess the spontaneous EEG pattern. Constant current stimuli were delivered to the left hippocampus (granule cells of the dentate gyrus) through a bipolar electrode (recording electrode) twice daily for 5 days and once daily for 2 days (weekend), at intervals of at least 6 h. The parameters of stimulation were 50 Hz, 2 ms monophasic rectangular wave pulses for 1 s, the current intensity ranging between 60 and 200/~V. Behaviour was observed and AD duration measured in the stimulated hippocampus after each stimulation, for every animal. Before starting electrical stimulation, the rats were randomly assigned to 3 groups of 5 animals per group and received 12 + 1, 19 _+ 0.6 and 24 + 2 stimuli (mean + S.E.M.) to reach stage 1/2, 3 or 5 of kindling, respectively, according to Racine's classification26. Previous experiments showed that after 12 stimuli the animals displayed stereotypies and occasional retraction of a forelimb (stage 2), while after 19 stimuli they had head nodding and muscle twitching (stage 3). Stage 5 was characterised by tonic-clonic seizures with rearing and failing. Animals were considered fully kindled when they experienced three consecutive stage 5 seizures. Control rats were implanted with electrodes and handled in the same way as the experimental rats but were not electrically stimulated. Each kindled rat and its respective control were killed 48 h after the last stimulation. This time was chosen since it falls in the interval in which an enhanced activation of NMDA receptors has been demonstrated by electrophysiological studies in kindled animals (i.e. 24 h to 50 days) 15. Whole brains were rapidly frozen on dry ice and stored at -80 °C.
Autoradiographical analysis Each brain was given a code number and the autoradiographical procedures performed blind to the experimental history of the animal. However, within each experiment (typically involving 6 brains), the corresponding control and experimental brains were always included to ensure that both received identical treatment. Ten/~m coronal sections of rat brain were cut on a cryostat and thaw-mounted onto subbed glass microscope slides. For each brain, sections were taken at 3 levels corresponding as closely as possible to 0.5 mm, 3.5 mm and 5 mm posterior to bregma, and a section
from each level placed on each individual slide. The slides were stored overnight at -20 °C. The methods employed for determination of the binding of acidic amino acid receptor ligands were based on those previously reported and validated by Monaghan et al. 17 (L_[3H]glutamate), Monaghan et al. 18 ([3H]AMPA) and Bowery et al. l ([3H]MK-801). The method for [3H]kainate binding was adapted from that of Monaghan and Cotman ~6, to include an incubation time of 30 min at 30 °C which in preliminary experiments was found to be sufficient for equilibrium to be achieved. The radioligands employed, and final concentrations in the assay were: L-[3H]glutamate (50 nM), [3H]amino-3-hydroxy-4-methyl-5-isoxazolepropionate (AMPA; 36 nM), [3H]kainate (16 nM) and [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo-[a,d]cyclohepten-5,10-imine maleate (MK-801; 2 nM). The assay buffer used for L-[3H]glutamate, [3H]AMPA and [3H]kainate binding was 50 mM Tris-acetate, pH 7.2 and for [3H]MK-801 binding was 50 mM Tris-HCl, pH 7.4. To define NMDA-sensitive L-[3H]glutamate binding, a parallel incubation in the presence of 300 pM NMDA was run. [3H]AMPA binding was performed in the presence of 100 mM KSCN 12, and [3H]MK-801 binding was carried out in the presence of 30/~M L-glutamate and 3/~M glycines36. Non-specific binding was defined by parallel incubations in the presence of 1 mM L-glutamate (in the case of L-[3H]glutamate, [3H]AMPA and [3H]kainate binding) or 100/~M MK-801 (in the case of [3H]MK-801 binding). Before exposure to radioligand, the slides were incubated for 2 h in a large volume (approx. ! liter for 50 slides) of 50 mM Tris-acetate (pH 7.2) at 4 °C to remove endogenous amino acids, and then for 10 min at 30 °C in the respective assay buffer. Incubation with radioligand was carried out for 30 min at 30 °C, after which each slide was immediately washed by 4 sequential 8 s immersions in 50 mM Tris-acetate (pH 7.2) at 4 °C (in the case of L-[3H]glutamate, [3H]AMPA and [3H]kainate), or 2 sequential 20 s immersions in 50 mM Tris-HCl (pH 7.4) at room temperature (in the case of [3H]MK-801). Excess buffer was removed from the slides by careful aspiration and the slides dried under a stream of cool air, mounted onto cardboard and apposed to tritium-sensitive film (Amersham) in cassettes and exposed for 6-8 weeks at 4 °C. Films were developed using standard procedures, dried, and quantified using a Cambridge Instruments Image Analyser and Amersham tritium standards. Optical density measurements were made in the hippocampus and striatum by defining the chosen portion of the section using a 'mouse' as part of the image analysis system. For measurements in the CA1 region an area was defined which included all layers from the hippocampal fissure to the alveus; in the dentate gyrus the area defined was the molecular layer of the lower blade. 'Anterior' hippocampal values were from the sections taken close to the injection site approximately 3.5 mm from bregma, and 'posterior' values were from the sections taken at 5 mm from bregma. Striatal values were from sections taken at 0.5 mm from bregma. A standard curve was constructed for each film using the Amersham standards and this was used to convert optical densities into amount of radioligand bound (nCi/mg tissue). Values are expressed in the Results section as specific binding i.e. total binding minus nonspecific binding. For L-[3H]glutamate, 'NMDA-sensitive' binding was calculated as total binding minus binding in the presence of 300 ~M NMDA, and 'NMDA-insensitive' binding as binding in the presence of 300/~M NMDA minus non-specific binding.
Fig. 1. Autoradiograms of L-[3H]glutamate binding to coronal sections from control and kindled (stage 5) rats. Experiments were carried out as described in the text. The images are negatives of the original autoradiograms and light areas represent the distribution of silver grains corresponding to binding sites on the original brain sections. A: control; B: kindling stage 5. Sections A1, A2, B1 and B2 are the total binding of L-[3H]glutamate, and sections A3, A4, B3 and B4 are L-Jail]glutamate binding in the presence of 300/~M NMDA (i.e. NMDA-insensitive binding). Sections A1, A3, B1 and B3 were taken at 3.5 mm posterior to bregma and sections A2, A4, B2, and B4 were taken at 5 mm posterior to bregma. Dark 'tracks' extending into the dorsal hippocampus through the overlying cerebral cortex in sections A1, A3, B1 and B3 represent the site of hippocampal electrodes.
115
116
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I Fig. 2. Autoradiograms of [3H]AMPA and [3H]kainate binding to coronal sections from control and kindled (stage 5) rats. Experiments were carried out as described in the text. Sections were taken from the same animals as those illustrated in Fig. 1. C: control; D: kindling stage 5. Sections C1, C2, D1 and D2 are the total binding of [3H]AMPA and sections C3, C4, D3 and D4 arc the total binding of [3H]kainate.
117 Materials
L-[3H]Glutamate (69.7 Ci/mmol), [3H]AMPA (27.6 Ci/mmol), [3H]vinylidenekainate (60 Ci/mmol) and [3H]MK-801 (29.4 Ci/ mmol) were purchased from NEN-Dupont. NMDA was from Cambridge Research Biochemicals. MK-801 was synthesised at Merck, Sharp and Dohme and all other chemicals were obtained from commercial sources at the highest purity available.
N M D A - I N S E N S I T I V E L-[3H]GI_LITAMATE B I N D I N G I
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RESULTS T h e r a d i o l i g a n d s e m p l o y e d label the different excita t o r y a m i n o acid r e c e p t o r sub-types as follows: N M D A sensitive L-[3H]glutamate binding and [3H]MK-801 binding - - t r a n s m i t t e r recognition site and ion channel, respectively, of the N M D A receptor; [ 3 H ] A M P A binding - - quisqualate receptor; [3H]kainate - - kainate receptor. T h e N M D A - i n s e n s i t i v e c o m p o n e n t of L-[3H]glutamate binding is not well characterised and m a y r e p r e s e n t binding to a c o m b i n a t i o n of quisqualate and kainate r e c e p t o r s 17 in a d d i t i o n to other, as yet undefined, sites. F o r a u t o r a d i o g r a p h i c a l analysis it was decided to conc e n t r a t e on the dorsal h i p p o c a m p u s since this was t h e kindling site in the present e x p e r i m e n t s and displays p r o f o u n d electrophysio!ogical changes as a result of the kindling process 15'31. To differentiate b e t w e e n effects caused by electrical stimulation p e r se, r a t h e r than by the
NMDA-SENSITIVE L-[3H]GLUTAMATE BINDING 10 t I con i T ~
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L
R
L
II
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R
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A N T CA1 A N T DG POST CAI POST DG
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Fig. 4. NMDA-insensitive binding of L-[3H]glutamate in control and kindled rats. Details as for Fig. 3. Values were obtained by subtracting binding in the presence of 1 mM L-glutamate (nonspecific binding) from binding in the presence of 300/tM NMDA. The NMDA-insensitive component typically comprised 21-44% of the total L-[3H]glutamate binding. Control: n = 14; stage 1/2: n = 5; stage 3: n = 5; stage 5: n = 5.
kindling process, m e a s u r e m e n t s were t a k e n on both the left and right sides of the brain (electrical stimulation was a d m i n i s t e r e d to the left side only), and at an anterior level, at the site o f e l e c t r o d e p l a c e m e n t , and a posterior level several m m distant. M e a s u r e m e n t s were also m a d e in the striatum, since this a r e a is not thought to be part of the p r i m a r y n e u r o n a l circuits involved in kindling, and possesses a high density of excitatory a m i n o acid binding sites. C o m p a r i s o n of the binding values for control animals (which received e l e c t r o d e p l a c e m e n t but no stimulation and were processed in parallel witti kindled animals)
[3H] MK-801 B I N D I N G 12
I con
1
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Fig. 3. NMDA-sensitive binding of L-[3H]glutamate in control and kindled rats. Quantitative measurements from the autoradiograms were made as described in the text for control (CON: n = 14), stage 1/2 (ST 1/2; n = 5), stage 3 (ST 3; n = 5) and stage 5 (ST 5; n = 5). Values are in nCi/mg tissue (mean + S.E.M.) and were calculated by subtracting binding in the presence of 300/tM NMDA from total binding. Typically, the NMDA-sensitive component comprised 65-70% of the total L-[3H]glutamate binding. ANT CA1 and ANT DG represent values from the CA1 region and lower blade of the dentate gyrus, respectively, in sections taken 3.5 mm posterior to bregma, and POST CA1 and POST DG represent equivalent values from sections taken 5 mm posterior to bregma. STR represents values from the striatum in sections taken 0.5 mm posterior to bregma. L, left (stimulated) side: R, right side. *P < 0.05 vs control (t-test).
0
L i
R
L i
R
L il
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L Ii
FI
L
R
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A N T CA1 A N T DG POST CA1 POST DG
1/2
i
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Fig. 5. [3H]MK-801 binding in control and kindled rats. Details as for Fig. 3. Values were obtained by subtracting binding in the presence of 100 /tM MK-801 (non-specific binding) from total binding, and typically comprised 21 (striatum)-59 (CA1 area)% of total [3H]MK-801 binding. Control: n = 9; stage 1/2: n = 3; stage 3: n = 3; stage 5; n = 3.
118
[3H] AMPA 10
BINDING
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L R L R / R II II II I DO POST CAIPOST DG STR
Fig. 6. [3H]AMPAbinding in control and kindled rats. Details as for Fig. 3. Values were obtained by subtracting binding in the presence of 1 mM L-glutamate(non-specificbinding) from total binding, and typically comprised 99% of total [3H]AMPA binding. Control: n = 13; stage 1/2: n = 4; stage 3: n = 5; stage 5: n = 5. *P < 0.05 vs control (t-test). indicated no statistically significant difference between controls prepared for stage 1/2, 3 or 5 (unpaired t-test), and consequently all control values were pooled. Examples of autoradiograms tor control and kindled animals are shown in Figs. 1 and 2, and Figs. 3-7 illustrate the quantified measurements for each binding site. Overall, few statistically significant changes of excitatory amino acid binding sites were apparent at any stage of kindling versus control. For NMDA-sensitive L[3H]glutamate binding, mean values for kindled animals tended to be lower than controls, although this only reached statistical significance in 2 areas at stage 1/2: the left posterior dentate gyrus and the right striatum (Fig. 3). The other NMDA receptor ligand employed, [3H]-
[3H] KAINATE
BINDING
12 I I
I
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, II
I con
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II
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ANT CA3 ANT DG POST CA3POST DG
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Fig. 7. [3H]kainatebinding in control and kindled rats. Details as for Fig. 3 except that 'CA3' represents measurements made in the stratum lucidum of the CA3 region. Values were obtained by subtracting binding in the presence of 1 mM L-glutamate (nonspecific binding) from total binding, and typically comprised 86-95% of total [3H]kainate binding. Control: n = 14; stage 1/2: n = 4; stage 3: n = 5; stage 5: n = 5.
MK-801, gave a similar result with a tendency to lower binding values in the kindled animals, but no statistically significant change from control was apparent (Fig. 5). The NMDA-insensitive binding of L-fall]glutamate was unchanged versus control in all areas measured at each kindling stage (Fig. 4). There appeared to be a general reduction of [3H]AMPA binding at stage 3 of kindling, however, this only reached statistical significance in two areas posterior to the stimulation site: the left CA1 region and right dentate gyrus (Fig. 6). At stages 1/2 and 5, no statistically significant difference from control was apparent. For [3H]kainate binding, measurements in the hippocampus were made in the two regions which possess a high density of sites: the stratum lucidum of CA3 and the inner molecular layer of the dentate gyrus. No significant change from control was apparent in these areas at any stage of kindling (Fig. 7). In addition to the quantitative measurements described above, close visual inspection of the autoradiograms failed to reveal any obvious change in the pattern of excitatory amino acid binding sites from control in any other brain region present in the sections taken from kindled animals at all stages. DISCUSSION The main conclusion to be drawn from the present study is that, in general, excitatory amino acid binding sites in the hippocampus (and striatum) are unchanged by the development and acquisition of hippocampal kindling. On the basis of previous experiments, a change in NMDA receptors might have been anticipated. NMDA receptor responses measured by changes in extracellular calcium 35 or inhibition of phosphoinositide hydrolysis2° are enhanced following in vivo kindling of the hippocampus or amygdala, respectively, and synaptic responses in the dentate gyrus evoked by low-frequency stimulation exhibit a large NMDA receptor-mediated component after amygdala or hippocampal commissure kindling in vivo 15. NMDA receptor responses are also increased in hippocampal CA1 neurones following kindling-like stimulation of the Schaeffer-collateral fibres in vitro. This increased excitability in the hippocampal formation may underlie the kindling phenomenon. If this were due to an increase in the number of N M D A receptors or an increase in their affinity for the neurotransmitter glutamate, an increase in the binding of NMDA receptor radioligands in the hippocampus would have been expected. The present results indicate that binding sites for both L-[3H]glutamate, which labels the transmitter recognition site of the NMDA receptor, and [3H]MK-801, which labels the receptor ion channel, are unchanged and
119 if anything tend to decrease during and after hippocampal kindling. Since a single radioligand concentration was used in the present experiments, it is possible that no change in binding for kindled vs. control animals was observed despite opposite effects on the affinity and number of binding sites (e.g. reduction in dissociation constant, increase in binding site density). However, two radioligands were used which bind to distinct sites within the NMDA receptor complex and although parallel changes in binding site density might occur, it is very unlikely that a similar change of affinity could be induced by kindling for both the transmitter recognition site and ion channel. It seems unlikely that the choice of hippocampus as the site of kindling can explain the lack of changes apparent in the present results, since alterations in functional NMDA receptor responses have been observed following kindling at several sites, including hippocampus (see above). A possibility which cannot be ruled out at present is that some change occurs in a regulatory site on the receptor whose influence was not apparent in the present radioligand binding experiments. Indeed, whilst this manuscript was in preparation, Yeh et al. 37 reported an increase in the number of binding sites in membranes for the competitive NMDA receptor antagonist [3H]3[(+) - 2 - (carboxypiperazin - 4- yl)]propyl - 1 - phosphonate (CPP), the co-agonist [3H]glycine and the ion channel blocker [3H]N-[(1-thienyl)cyclohexyl]piperidine (TCP) 1 month following kindling of the amygdala. Interestingly, no changes in [3H]glycine binding were observed 1 day after the last kindled seizure. Taken together with the results of the present study this may suggest that alterations in NMDA receptor binding site density are a long term 'plastic' consequence of the establishment of kindling, but such changes do not contribute to the kindling phenomenon itself. The present results are in agreement with two recent studies which have also found no increase in the binding of NMDA receptor ligands in the hippocampus following kindling. Thus, Okazaki et al. 22 observed no change in NMDA-sensitive L-[3H]glutamate binding sites in hippocampal areas 1 day after the last stage 4-5 seizure in rats kindled by stimulation of the angular bundle, whereas a small decrease was apparent 28 days later. Sircar et al. 3° have reported a 25% decrease in the maximum number of binding sites for [3H]TCP in the hippocampus following amygdala kindling in rats, 3 days after the last kindled seizure. The present results are consistent with a reduction of NMDA receptor parameters in the hippocampus since the values for NMDA-sensitive L-[3H]glutamate binding and [3H]MK-801 binding in kindled animals were lower than in controls, although this did not reach statistical significance in the majority of cases. Therefore
it is possible that, as suggested by others 22, a small down-regulation of hippocampal NMDA receptors occurs in kindling due to increased receptor activation. Binding sites for the quisqualate receptor ligand [3H]AMPA, for [3H]kainate and the NMDA-insensitive sites for L-[3H]glutamate were also unchanged following hippocampal kindling. To our knowledge, this is the first report of the status of [3H]AMPA binding sites during kindling. (A previous paper by Savage et al. 2s reported a transient increase of quisqualate-sensitive L-[3H]glutamate binding sites in the hippocampus following angular bundle kindling, but it is now clear that these binding sites do not represent a receptor - see ref. 7.) However, previous reports have appeared which indicate changes in [3H]kainate binding sites in the hippocampus as a result of kindling. Savage et al. 29 observed a 47-61% decrease of [3H]kainate binding sites in the stratum lucidum of CA3, and a smaller reduction in the inner molecular layer of the dentate gyrus, 1 day after amygdala or angular bundle kindling, but no change at 28 days22. On the other hand, Represa et al. 27 observed an increase of [3H]kainate binding sites in the supra-granular layer of the dentate gyrus and in the stratum infrapyramidale of CA3 following amygdala kindling, which was related to the sprouting of mossy fibres. The discrepancy between the present results and those of Savage et al. 29 may be due to the site of kindling, although the fact that the initial reduction of [3H]kainate binding sites observed by these authors reverts to normal by 28 days suggests that this phenomenon is not directly related to the kindling process. Similarly, the failure to observe an increase in [3H]kainate binding in the dentate gyrus or CA3 region may be due to the different site of kindling, but is more likely to be a result of the rat strain used (Sprague-Dawley), which already possesses a mossy fibre innervation in the infrapyramidal layer of the CA3 region27. Amongst neurotransmitter receptors, the NMDA receptor has some unique properties. The NMDA receptor-associated ion channel is blocked by Mg2+ in a voltage-dependent manner 13'21 and as a consequence NMDA receptor-mediated neuronal events are critically dependent on the local membrane potential. This property is important for current ideas about the role of NMDA receptors in neuronal plasticity, e.g. learning and memory19. It may also be important for pathological situations, since any factor which brings about a change in neuronal membrane potential can influence the extent of NMDA receptor activity. In the hippocampus, low frequency stimulation of excitatory pathways evokes an excitatory postsynaptic potential (EPSP) which is mediated by non-NMDA receptors, despite the presence of a high density of NMDA receptors on the target neurones5.
120 However, a large N M D A receptor-mediated component to the E P S P is revealed when Mg 2÷ is excluded from the extracellular medium 11, or G A B A e r g i c inhibition is blocked pharmacologically 6. The change(s) induced by kindling which bring about an increased N M D A receptor involvement could, therefore, result from a variety of factors. These include: (1) increased transmitter (glutamate?) release (see ref. 10); (2) upregulation of N M D A receptors; (3) upregulation of n o n - N M D A receptors
(which would result in more depolarisation and facilitate N M D A receptor activity); (4) decreased inhibition (see ref. 31); (5) alterations in other postsynaptic conductances (receptor or non-receptor operated). The present results, and those of the other studies mentioned above, suggest that a n upregulation of hippocampal N M D A or n o n - N M D A excitatory amino acid receptors does not underlie the kindling p h e n o m e n o n .
ABBREVIATIONS
EEG EPSP MK-801
AD AMPA AP7 CPP
afterdischarge D,L-a-amino-3-hydroxy-5-methylisoxazole-4-propionate 2-amino-7-phosphonoheptanoate 3-[(+)-2-(carboxypiperazin-4-yi)]propyl- 1-phosphonate
REFERENCES 1 Bowery, N,G., Wong, E.H.E and Hudson, A.L., Quantitative autoradiography of [3H]MK-801 binding sites in mammalian brain, Br. J. Pharmacol., 93 (1988) 944-954. 2 Bowyer, J.E, Phencyclidine inhibition of the rate of development of amygdaloid kindled seizures, Exp. Neurol., 75 (1982) 173-183. 3 Cain, D.E, Desborough, K.A. and McKitrick, D.J., Retardation of amygdala kindling by antagonism of NMD-aspartate and muscarinic cholinergic receptors: evidence for the summation of excitatory mechanisms in kindling, Exp. Neurol., 100 (1988) 179-187. 4 Croucher, M.J., Bradford, H.E, Sunter, D.C. and Watkins, J.C., Inhibition of the development of electrical kindling of the prepyriform cortex by daily focal injections of excitatory amino acid antagonists, Eur. J. Pharmacol., 152 (1988) 29-38, 5 Dingledine, R., NMDA receptors: what do they do?, Trends Neurosci., 9 (1986) 47-49. 6 Dingledine, R., Hynes, M.A. and King, G.L., Involvement of N-methyl-D-aspartate receptors in epilepiform bursting in the rat hippocampal slice, J. Physiol., 380 (1986) 175-189. 7 Foster, A.C. and Fagg, G.E., Acidic amino acid binding sites in mammalian neuronal membranes: their characteristics and relationship to synaptic receptors, Brain Res. Rev., 7 (1984) 103-164. 8 Foster, A.C. and Wong, E.H.E, The novel anticonvulsant MK-801 binds to the activated state of the N-methyl-o-aspartate receptor, Br. J. Pharmacol., 91 (1987) 403-409. 9 Goddard, G.V., McIntyre, D.C. and Leech, C.K., A permanent change in brain function resulting from daily electrical stimulation, Exp. Neurol., 25 (1969) 295-330. 10 Guela, C., Jarvie, P.A., Logan, T.C. and Slevin, J.T., Long-term enhancement of K+-evoked release of L-glutamate in entorhinal kindled rats, Brain Research, 442 (1988) 368-372. 11 Herron, C.E., Lester, R.A.J., Coan, E.J. and Collingridge, G.L., Intracellular demonstration of an N-methyl-D-aspartate receptor-mediated component of synaptic transmission in the rat hippocampus, Neurosci. Lett., 60 (1985) 19-23. 12 Honore, T. and Nielson, M., Complex structure of quisqualatesensitive glutamate receptors in rat cortex, Neurosci. Lett., 54 (1985) 27-32. 13 Mayer, M., Westbrook, G.L. and Guthrie, EB., Voltagedependent block by Mg2÷ of NMDA responses in spinal cord neurones, Nature, 309 (1984) 261-263. 14 McNamara, J.O., Bonhaus, D.W., Shin, C., Crain, B.J., Gellman, R.L. and Giacchino, J.L., The kindling model of epilepsy: a critical review, Crit. Rev. Clin. Neurobiol., 1 (1985) 341-391.
NMDA TCP
electroencephalogram excitatory postsynaptic potential (+)-5-methyl-10,11-dihydro-SH-dibenzo[a,d]cyclohepten-5,10-imine maleate N-methyl-o-aspartate N-( 1-[2-thienyl]cyclohexyl)piperidine
15 Mody, I. and Heinemann, U., NMDA receptors of dentate gyrus granule cells participate in synaptic transmission following kindling, Nature, 326 (1987) 701-704. 16 Monaghan, D.T. and Cotman, C.W., The distribution of [3H]kainic acid binding sites in rat CNS as determined by autoradiography, Brain Research, 252 (1982) 91-100. 17 Monaghan, D.T., Holets, V.L., Toy, D.W. and Cotman, C.W., Anatomical distribution of four pharmacologically distinct L[3H]glutamate binding sites, Nature, 306 (1983) 176-179. 18 Monaghan, D.T., Yao, D. and Cotman, C.W., Distribution of 3H-AMPA binding sites in rat brain as determined by quantitative autoradiography, Brain Research, 324 (1985) 160-164. 19 Morris, R.G.M., Elements of a hypothesis concerning the participation of hippocampal NMDA receptors in learning. In D. Lodge (Ed.), Excitatory Amino Acids in Health and Disease, Wiley, Chichester, 1988, pp. 297-320. 20 Morrisett, R.A., Chow, C., Nadler, J.V. and McNamara, J.O., Biochemical evidence for enhanced sensitivity to N-methylD-aspartate in the hippocampal formation of kindled rats, Brain Research, 496 (1989) 25-28. 21 Nowak, L., Bregestovski, P., Ascher, P., Herbet, A. and Prochiantz, A., Magnesium gates glutamate-activated channels in mouse central neurones, Nature, 307 (1984) 462-465. 22 Okazaki, M.M., McNamara, J.O. and Nadler, J.V., Regionallyspecific reductions in kainate and NMDA receptor binding after angular bundle kindling, Soc. Neurosci. Abstr., 14 (1988) 345.11. 23 Okazaki, M.M., McNamara, J.O. and Nadler, J.V., N-MethylD-aspartate receptor autoradiography in rat brain after angular bundle kindling, Brain Research, 482 (1989) 359-364. 24 Peterson, D.W., Collins, J.F. and Bradford, H.E, The kindled amygdala model of epilepsy: anticonvulsant action of amino acid antagonists, Brain Research, 275 (1983) 169-172. 25 Peterson, D.W., Collins, J.E and Bradford, H.E, Anticonvulsant action of amino acid antagonist against kindled hippocampal seizures, Brain Research, 311 (1984) 176-180. 26 Racine, R.J., Modification of seizure activity by electrical stimulation. II. Motor seizure, Electroencephalogr. Clin. Neurophysiol., 32 (1972) 281-294. 27 Represa, A., Le Gall La Salle, G. and Ben-Ari, Y., Hippocampal plasticity in the kindling model of epilepsy in rats, Neurosci. Left., 99 (1989) 345-350. 28 Savage, D.D., Werling, L.L., Nadler, J.V. and McNamara, J.O., Selective increase in L-[3H]glutamate binding to a quisqualate-sensitive site on hippocampal synaptic membranes after angular bundle kindling, Eur. J. Pharmacol., 85 (1982) 255-256. 29 Savage, D.D., Nadler, J.V. and McNamara, J.O., Reduced kainic acid binding in rat hippocampal formation after limbic kindling, Brain Research, 323 (1984) 128-131. 30 Sircar, R., Ludvig, N., Zukin, S.R. and Moshe, S.L., Down-
121 regulation of hippocampal phencyclidine (PCP) receptors following amygdala kindling, Eur. J. Pharmacol., 141 (1987) 167-168. 31 Stelzer, A., Slater, N.T. and Bruggencate, G., Activation of NMDA receptors blocks GABAergic inhibition in an in vitro model of epilepsy, Nature, 326 (1987) 698-701. 32 Vezzani, A., Wu, H.Q., Tullii, M. and Samanin, R., Anticonvulsant drugs effective against human temporal lobe epilepsy prevent seizures but not neurotoxicity induced in rats by quinolinic acid: electroencephaiographic, behavioural and histological assessments, J. Pharmacol. Exp. Ther., 239 (1986) 256-263. 33 Vezzani, A., Wu, H.Q., Moneta, E. and Samanin, R., Role of the N-methyl-o-aspartate receptors in the development and maintenance of hippocampal kindling in rats, Neurosci. Lett., 87 (1988) 63-68.
34 Vezzani, A., Serafini, R., Samanin, R. and Foster, A.C., Autoradiographic studies of excitatory amino acid (EAA) receptor subtypes during the development of hippocampal kindling in rats, Eur. J. Neurosci., Suppl. 2 (1989) 37.3. 35 Wadman, W.J., Heinemann, U., Konnerth, A. and Neuhaus, S., Hippocampal slices of kindled rats reveal calcium involvement in epileptogenesis, Exp. Brain Res., 57 (1985) 404-407. 36 Wong, E.H.E, Knight, A.R. and Ransom, R., Glycine modulates [aH]MK-801 binding to the NMDA receptor in rat brain, Eur. J. Pharmacol., 142 (1987) 487-488. 37 Yeh, G.-C., Bonhaus, D.W., Nadler, J.V. and McNamara, J.O., N-Methyl-o-aspartate receptor plasticity in kindling: quantitative and qualitative alterations in the N-methyl-D-aspartate receptor-channel complex, Proc. Natl. Acad. Sci. U.S.A., 86 (1989) 8157-8160.
113
Elsevier
BRES 15799
Autoradiographical analysis of excitatory amino acid binding sites in rat hippocampus during the development of hippocampal kindling A. Vezzani 1, R. Serafini 1, R. Samanin I and A.C. Foster 2 l lstituto di Ricerche Farmacologiche 'Mario Negri', Milan (Italy) and 2Merck, Sharp and Dohme Ltd., Neuroscience Research Centre, Terlings Park, Harlow, Essex (U. K.)
(Accepted 27 February 1990) Key words: N-Methyl-D-aspartatereceptor; Quisqualate receptor; Kainate receptor; MK-801; Epilepsy
Binding sites for excitatory amino acids have been determined by autoradiographicalprocedures in the rat hippocampus and striatum during hippocampal kindling.The bindingsites measured were the N-methyl-D-aspartate(NMDA)-sensitivesites for L-[3H]glutamateand [3H]MK-801 sites (transmitter recognitionsite and ion channelof the NMDA receptor, respectively),[3H]a-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) sites (quisqualate receptor), [3H]kainatesites (kainate receptor) and NMDA-insensitivesites for L-[aH]glutamate.In general, little change was apparent in the hippocampus or striatum for any of these binding sites when assessed 48 h after attaining stages 1/2, 3 or 5 of kindling. These results suggest that hippocampal kindling does not bring about a change in the excitatory amino acid receptor binding sites examined, and that the appearance of an NMDA receptor-mediated component to synaptic responses in the hippocampus produced by kindling15, cannot be explained on this basis. INTRODUCTION Kindling is an animal model of chronic epilepsy which develops over time following repetitive high-frequency stimulation of various brain areas 9'26. The electrical stimulation is initially sub-convulsive and eventually leads to fully generalised seizures. This phenomenon is thought to be a consequence of some form of neuronal plasticity which underlies the enhanced excitability of the activated tissue ~4. Several lines of evidence suggest that the N-methylD-aspartate (NMDA) sub-type of excitatory amino acid receptors significantly contributes to the establishment of kindling. NMDA receptor antagonists retard the aquisition and block expression of the kindled seizures 2-4'24'25. In accordance with these results we have recently found that intrahippocampal injection of the NMDA receptor antagonist 2-amino-7-phosphonoheptanoate (AP7) at the early stages (1-2) of hippocampal kindling significantly delayed the occurrence of generalized convulsions without modifying the seizure after-discharge (AD) duration, whereas both behavioural seizures and AD were suppressed when the same dose of AP7 was administered after the induction of stage 5 seizures 33. These results suggest that the contribution of NMDA receptors to the generation of the AD in the hippocampus increases during kindling development.
Electrophysiological experiments using hippocampal slices have recently shown that NMDA receptors become involved in synaptic transmission after amygdaloid or hippocampal commisure kindling 15, and an enhanced post-synaptic sensitivity to NMDA has been shown following kindling-like activation of the hippocampal Schaffer collateral pathway in vitro al. In addition, an increase in NMDA receptor-mediated responses has been observed in hippocampus by extracellular calcium measurements 35 and inhibition of phosphoinositide hydrolysis2°, following hippocampal or amygdaloid kindling. An increased density of excitatory amino acid receptors (particularly NMDA receptors) in the hippocampus could be a plastic alteration contributing to these observations. In the present study we have examined binding sites for the NMDA, quisqualate and kainate sub-types of excitatory amino acid receptors in rat hippocampus by autoradiographical analysis during the development (stages 1/2 and 3) and following acquisition (stage 5) of hippocampal kindling in rats. A preliminary account of this work has appeared 34. MATERIALS AND METHODS Male Sprague-Dawleyrats (250-280 g, Charles River, Italy) were used. The animalswere housed at constant temperature (23 °C) and
Correspondence: A.C. Foster, Merck, Sharp and Dohme Research Laboratories, Neuroscience Research Centre, Terlings Park, Eastwick Road, Harlow, Essex, CM20 2QR, U.K.
0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
114 relative humidity (60%) with a fixed 12 h light-dark cycle and free access to food and water.
Implantation procedure The surgical procedure for electrode implantation in the dorsal hippocampus under Equithesin anaesthesia (9.7 mg/ml sodium pentobarbital in saline, 42.6 mg/ml chloral hydrate in propyleneglycol, 21.2 mg/ml MgSO4 in ethanol; 3.5 ml/kg, i.p.) was as previously described 32. For implantation, the co-ordinates from bregma were: (nose bar -2.5) AP 3.5, L 2.3, H 2.9. In addition, stainless-steel screw electrodes were placed bilaterally over the sensorimotor cortex and a ground lead screwed over the nasal sinus. The electrodes were connected to a multipin socket (March Electronics, New York, NY), and were secured to the skull by acrylic dental cement. Electroencephalographic (EEG) recordings were made in unanaesthetised, freely moving animals as previously described 32.
Kindling procedure Kindling was started after a postoperative period of 7 days, when no behavioural signs of pain or discomfort were noted in the operated animals. The animals were allowed to acclimatize in a Plexiglas cage and an EEG recording (4-channel EEG polygraph, model BBP, Battaglia Rangoni, Bologna, Italy) was made for at least 10 min to assess the spontaneous EEG pattern. Constant current stimuli were delivered to the left hippocampus (granule cells of the dentate gyrus) through a bipolar electrode (recording electrode) twice daily for 5 days and once daily for 2 days (weekend), at intervals of at least 6 h. The parameters of stimulation were 50 Hz, 2 ms monophasic rectangular wave pulses for 1 s, the current intensity ranging between 60 and 200/~V. Behaviour was observed and AD duration measured in the stimulated hippocampus after each stimulation, for every animal. Before starting electrical stimulation, the rats were randomly assigned to 3 groups of 5 animals per group and received 12 + 1, 19 _+ 0.6 and 24 + 2 stimuli (mean + S.E.M.) to reach stage 1/2, 3 or 5 of kindling, respectively, according to Racine's classification26. Previous experiments showed that after 12 stimuli the animals displayed stereotypies and occasional retraction of a forelimb (stage 2), while after 19 stimuli they had head nodding and muscle twitching (stage 3). Stage 5 was characterised by tonic-clonic seizures with rearing and failing. Animals were considered fully kindled when they experienced three consecutive stage 5 seizures. Control rats were implanted with electrodes and handled in the same way as the experimental rats but were not electrically stimulated. Each kindled rat and its respective control were killed 48 h after the last stimulation. This time was chosen since it falls in the interval in which an enhanced activation of NMDA receptors has been demonstrated by electrophysiological studies in kindled animals (i.e. 24 h to 50 days) 15. Whole brains were rapidly frozen on dry ice and stored at -80 °C.
Autoradiographical analysis Each brain was given a code number and the autoradiographical procedures performed blind to the experimental history of the animal. However, within each experiment (typically involving 6 brains), the corresponding control and experimental brains were always included to ensure that both received identical treatment. Ten/~m coronal sections of rat brain were cut on a cryostat and thaw-mounted onto subbed glass microscope slides. For each brain, sections were taken at 3 levels corresponding as closely as possible to 0.5 mm, 3.5 mm and 5 mm posterior to bregma, and a section
from each level placed on each individual slide. The slides were stored overnight at -20 °C. The methods employed for determination of the binding of acidic amino acid receptor ligands were based on those previously reported and validated by Monaghan et al. 17 (L_[3H]glutamate), Monaghan et al. 18 ([3H]AMPA) and Bowery et al. l ([3H]MK-801). The method for [3H]kainate binding was adapted from that of Monaghan and Cotman ~6, to include an incubation time of 30 min at 30 °C which in preliminary experiments was found to be sufficient for equilibrium to be achieved. The radioligands employed, and final concentrations in the assay were: L-[3H]glutamate (50 nM), [3H]amino-3-hydroxy-4-methyl-5-isoxazolepropionate (AMPA; 36 nM), [3H]kainate (16 nM) and [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo-[a,d]cyclohepten-5,10-imine maleate (MK-801; 2 nM). The assay buffer used for L-[3H]glutamate, [3H]AMPA and [3H]kainate binding was 50 mM Tris-acetate, pH 7.2 and for [3H]MK-801 binding was 50 mM Tris-HCl, pH 7.4. To define NMDA-sensitive L-[3H]glutamate binding, a parallel incubation in the presence of 300 pM NMDA was run. [3H]AMPA binding was performed in the presence of 100 mM KSCN 12, and [3H]MK-801 binding was carried out in the presence of 30/~M L-glutamate and 3/~M glycines36. Non-specific binding was defined by parallel incubations in the presence of 1 mM L-glutamate (in the case of L-[3H]glutamate, [3H]AMPA and [3H]kainate binding) or 100/~M MK-801 (in the case of [3H]MK-801 binding). Before exposure to radioligand, the slides were incubated for 2 h in a large volume (approx. ! liter for 50 slides) of 50 mM Tris-acetate (pH 7.2) at 4 °C to remove endogenous amino acids, and then for 10 min at 30 °C in the respective assay buffer. Incubation with radioligand was carried out for 30 min at 30 °C, after which each slide was immediately washed by 4 sequential 8 s immersions in 50 mM Tris-acetate (pH 7.2) at 4 °C (in the case of L-[3H]glutamate, [3H]AMPA and [3H]kainate), or 2 sequential 20 s immersions in 50 mM Tris-HCl (pH 7.4) at room temperature (in the case of [3H]MK-801). Excess buffer was removed from the slides by careful aspiration and the slides dried under a stream of cool air, mounted onto cardboard and apposed to tritium-sensitive film (Amersham) in cassettes and exposed for 6-8 weeks at 4 °C. Films were developed using standard procedures, dried, and quantified using a Cambridge Instruments Image Analyser and Amersham tritium standards. Optical density measurements were made in the hippocampus and striatum by defining the chosen portion of the section using a 'mouse' as part of the image analysis system. For measurements in the CA1 region an area was defined which included all layers from the hippocampal fissure to the alveus; in the dentate gyrus the area defined was the molecular layer of the lower blade. 'Anterior' hippocampal values were from the sections taken close to the injection site approximately 3.5 mm from bregma, and 'posterior' values were from the sections taken at 5 mm from bregma. Striatal values were from sections taken at 0.5 mm from bregma. A standard curve was constructed for each film using the Amersham standards and this was used to convert optical densities into amount of radioligand bound (nCi/mg tissue). Values are expressed in the Results section as specific binding i.e. total binding minus nonspecific binding. For L-[3H]glutamate, 'NMDA-sensitive' binding was calculated as total binding minus binding in the presence of 300 ~M NMDA, and 'NMDA-insensitive' binding as binding in the presence of 300/~M NMDA minus non-specific binding.
Fig. 1. Autoradiograms of L-[3H]glutamate binding to coronal sections from control and kindled (stage 5) rats. Experiments were carried out as described in the text. The images are negatives of the original autoradiograms and light areas represent the distribution of silver grains corresponding to binding sites on the original brain sections. A: control; B: kindling stage 5. Sections A1, A2, B1 and B2 are the total binding of L-[3H]glutamate, and sections A3, A4, B3 and B4 are L-Jail]glutamate binding in the presence of 300/~M NMDA (i.e. NMDA-insensitive binding). Sections A1, A3, B1 and B3 were taken at 3.5 mm posterior to bregma and sections A2, A4, B2, and B4 were taken at 5 mm posterior to bregma. Dark 'tracks' extending into the dorsal hippocampus through the overlying cerebral cortex in sections A1, A3, B1 and B3 represent the site of hippocampal electrodes.
115
116
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I Fig. 2. Autoradiograms of [3H]AMPA and [3H]kainate binding to coronal sections from control and kindled (stage 5) rats. Experiments were carried out as described in the text. Sections were taken from the same animals as those illustrated in Fig. 1. C: control; D: kindling stage 5. Sections C1, C2, D1 and D2 are the total binding of [3H]AMPA and sections C3, C4, D3 and D4 arc the total binding of [3H]kainate.
117 Materials
L-[3H]Glutamate (69.7 Ci/mmol), [3H]AMPA (27.6 Ci/mmol), [3H]vinylidenekainate (60 Ci/mmol) and [3H]MK-801 (29.4 Ci/ mmol) were purchased from NEN-Dupont. NMDA was from Cambridge Research Biochemicals. MK-801 was synthesised at Merck, Sharp and Dohme and all other chemicals were obtained from commercial sources at the highest purity available.
N M D A - I N S E N S I T I V E L-[3H]GI_LITAMATE B I N D I N G I
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RESULTS T h e r a d i o l i g a n d s e m p l o y e d label the different excita t o r y a m i n o acid r e c e p t o r sub-types as follows: N M D A sensitive L-[3H]glutamate binding and [3H]MK-801 binding - - t r a n s m i t t e r recognition site and ion channel, respectively, of the N M D A receptor; [ 3 H ] A M P A binding - - quisqualate receptor; [3H]kainate - - kainate receptor. T h e N M D A - i n s e n s i t i v e c o m p o n e n t of L-[3H]glutamate binding is not well characterised and m a y r e p r e s e n t binding to a c o m b i n a t i o n of quisqualate and kainate r e c e p t o r s 17 in a d d i t i o n to other, as yet undefined, sites. F o r a u t o r a d i o g r a p h i c a l analysis it was decided to conc e n t r a t e on the dorsal h i p p o c a m p u s since this was t h e kindling site in the present e x p e r i m e n t s and displays p r o f o u n d electrophysio!ogical changes as a result of the kindling process 15'31. To differentiate b e t w e e n effects caused by electrical stimulation p e r se, r a t h e r than by the
NMDA-SENSITIVE L-[3H]GLUTAMATE BINDING 10 t I con i T ~
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Fig. 4. NMDA-insensitive binding of L-[3H]glutamate in control and kindled rats. Details as for Fig. 3. Values were obtained by subtracting binding in the presence of 1 mM L-glutamate (nonspecific binding) from binding in the presence of 300/tM NMDA. The NMDA-insensitive component typically comprised 21-44% of the total L-[3H]glutamate binding. Control: n = 14; stage 1/2: n = 5; stage 3: n = 5; stage 5: n = 5.
kindling process, m e a s u r e m e n t s were t a k e n on both the left and right sides of the brain (electrical stimulation was a d m i n i s t e r e d to the left side only), and at an anterior level, at the site o f e l e c t r o d e p l a c e m e n t , and a posterior level several m m distant. M e a s u r e m e n t s were also m a d e in the striatum, since this a r e a is not thought to be part of the p r i m a r y n e u r o n a l circuits involved in kindling, and possesses a high density of excitatory a m i n o acid binding sites. C o m p a r i s o n of the binding values for control animals (which received e l e c t r o d e p l a c e m e n t but no stimulation and were processed in parallel witti kindled animals)
[3H] MK-801 B I N D I N G 12
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1
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Fig. 3. NMDA-sensitive binding of L-[3H]glutamate in control and kindled rats. Quantitative measurements from the autoradiograms were made as described in the text for control (CON: n = 14), stage 1/2 (ST 1/2; n = 5), stage 3 (ST 3; n = 5) and stage 5 (ST 5; n = 5). Values are in nCi/mg tissue (mean + S.E.M.) and were calculated by subtracting binding in the presence of 300/tM NMDA from total binding. Typically, the NMDA-sensitive component comprised 65-70% of the total L-[3H]glutamate binding. ANT CA1 and ANT DG represent values from the CA1 region and lower blade of the dentate gyrus, respectively, in sections taken 3.5 mm posterior to bregma, and POST CA1 and POST DG represent equivalent values from sections taken 5 mm posterior to bregma. STR represents values from the striatum in sections taken 0.5 mm posterior to bregma. L, left (stimulated) side: R, right side. *P < 0.05 vs control (t-test).
0
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A N T CA1 A N T DG POST CA1 POST DG
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Fig. 5. [3H]MK-801 binding in control and kindled rats. Details as for Fig. 3. Values were obtained by subtracting binding in the presence of 100 /tM MK-801 (non-specific binding) from total binding, and typically comprised 21 (striatum)-59 (CA1 area)% of total [3H]MK-801 binding. Control: n = 9; stage 1/2: n = 3; stage 3: n = 3; stage 5; n = 3.
118
[3H] AMPA 10
BINDING
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Fig. 6. [3H]AMPAbinding in control and kindled rats. Details as for Fig. 3. Values were obtained by subtracting binding in the presence of 1 mM L-glutamate(non-specificbinding) from total binding, and typically comprised 99% of total [3H]AMPA binding. Control: n = 13; stage 1/2: n = 4; stage 3: n = 5; stage 5: n = 5. *P < 0.05 vs control (t-test). indicated no statistically significant difference between controls prepared for stage 1/2, 3 or 5 (unpaired t-test), and consequently all control values were pooled. Examples of autoradiograms tor control and kindled animals are shown in Figs. 1 and 2, and Figs. 3-7 illustrate the quantified measurements for each binding site. Overall, few statistically significant changes of excitatory amino acid binding sites were apparent at any stage of kindling versus control. For NMDA-sensitive L[3H]glutamate binding, mean values for kindled animals tended to be lower than controls, although this only reached statistical significance in 2 areas at stage 1/2: the left posterior dentate gyrus and the right striatum (Fig. 3). The other NMDA receptor ligand employed, [3H]-
[3H] KAINATE
BINDING
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Fig. 7. [3H]kainatebinding in control and kindled rats. Details as for Fig. 3 except that 'CA3' represents measurements made in the stratum lucidum of the CA3 region. Values were obtained by subtracting binding in the presence of 1 mM L-glutamate (nonspecific binding) from total binding, and typically comprised 86-95% of total [3H]kainate binding. Control: n = 14; stage 1/2: n = 4; stage 3: n = 5; stage 5: n = 5.
MK-801, gave a similar result with a tendency to lower binding values in the kindled animals, but no statistically significant change from control was apparent (Fig. 5). The NMDA-insensitive binding of L-fall]glutamate was unchanged versus control in all areas measured at each kindling stage (Fig. 4). There appeared to be a general reduction of [3H]AMPA binding at stage 3 of kindling, however, this only reached statistical significance in two areas posterior to the stimulation site: the left CA1 region and right dentate gyrus (Fig. 6). At stages 1/2 and 5, no statistically significant difference from control was apparent. For [3H]kainate binding, measurements in the hippocampus were made in the two regions which possess a high density of sites: the stratum lucidum of CA3 and the inner molecular layer of the dentate gyrus. No significant change from control was apparent in these areas at any stage of kindling (Fig. 7). In addition to the quantitative measurements described above, close visual inspection of the autoradiograms failed to reveal any obvious change in the pattern of excitatory amino acid binding sites from control in any other brain region present in the sections taken from kindled animals at all stages. DISCUSSION The main conclusion to be drawn from the present study is that, in general, excitatory amino acid binding sites in the hippocampus (and striatum) are unchanged by the development and acquisition of hippocampal kindling. On the basis of previous experiments, a change in NMDA receptors might have been anticipated. NMDA receptor responses measured by changes in extracellular calcium 35 or inhibition of phosphoinositide hydrolysis2° are enhanced following in vivo kindling of the hippocampus or amygdala, respectively, and synaptic responses in the dentate gyrus evoked by low-frequency stimulation exhibit a large NMDA receptor-mediated component after amygdala or hippocampal commissure kindling in vivo 15. NMDA receptor responses are also increased in hippocampal CA1 neurones following kindling-like stimulation of the Schaeffer-collateral fibres in vitro. This increased excitability in the hippocampal formation may underlie the kindling phenomenon. If this were due to an increase in the number of N M D A receptors or an increase in their affinity for the neurotransmitter glutamate, an increase in the binding of NMDA receptor radioligands in the hippocampus would have been expected. The present results indicate that binding sites for both L-[3H]glutamate, which labels the transmitter recognition site of the NMDA receptor, and [3H]MK-801, which labels the receptor ion channel, are unchanged and
119 if anything tend to decrease during and after hippocampal kindling. Since a single radioligand concentration was used in the present experiments, it is possible that no change in binding for kindled vs. control animals was observed despite opposite effects on the affinity and number of binding sites (e.g. reduction in dissociation constant, increase in binding site density). However, two radioligands were used which bind to distinct sites within the NMDA receptor complex and although parallel changes in binding site density might occur, it is very unlikely that a similar change of affinity could be induced by kindling for both the transmitter recognition site and ion channel. It seems unlikely that the choice of hippocampus as the site of kindling can explain the lack of changes apparent in the present results, since alterations in functional NMDA receptor responses have been observed following kindling at several sites, including hippocampus (see above). A possibility which cannot be ruled out at present is that some change occurs in a regulatory site on the receptor whose influence was not apparent in the present radioligand binding experiments. Indeed, whilst this manuscript was in preparation, Yeh et al. 37 reported an increase in the number of binding sites in membranes for the competitive NMDA receptor antagonist [3H]3[(+) - 2 - (carboxypiperazin - 4- yl)]propyl - 1 - phosphonate (CPP), the co-agonist [3H]glycine and the ion channel blocker [3H]N-[(1-thienyl)cyclohexyl]piperidine (TCP) 1 month following kindling of the amygdala. Interestingly, no changes in [3H]glycine binding were observed 1 day after the last kindled seizure. Taken together with the results of the present study this may suggest that alterations in NMDA receptor binding site density are a long term 'plastic' consequence of the establishment of kindling, but such changes do not contribute to the kindling phenomenon itself. The present results are in agreement with two recent studies which have also found no increase in the binding of NMDA receptor ligands in the hippocampus following kindling. Thus, Okazaki et al. 22 observed no change in NMDA-sensitive L-[3H]glutamate binding sites in hippocampal areas 1 day after the last stage 4-5 seizure in rats kindled by stimulation of the angular bundle, whereas a small decrease was apparent 28 days later. Sircar et al. 3° have reported a 25% decrease in the maximum number of binding sites for [3H]TCP in the hippocampus following amygdala kindling in rats, 3 days after the last kindled seizure. The present results are consistent with a reduction of NMDA receptor parameters in the hippocampus since the values for NMDA-sensitive L-[3H]glutamate binding and [3H]MK-801 binding in kindled animals were lower than in controls, although this did not reach statistical significance in the majority of cases. Therefore
it is possible that, as suggested by others 22, a small down-regulation of hippocampal NMDA receptors occurs in kindling due to increased receptor activation. Binding sites for the quisqualate receptor ligand [3H]AMPA, for [3H]kainate and the NMDA-insensitive sites for L-[3H]glutamate were also unchanged following hippocampal kindling. To our knowledge, this is the first report of the status of [3H]AMPA binding sites during kindling. (A previous paper by Savage et al. 2s reported a transient increase of quisqualate-sensitive L-[3H]glutamate binding sites in the hippocampus following angular bundle kindling, but it is now clear that these binding sites do not represent a receptor - see ref. 7.) However, previous reports have appeared which indicate changes in [3H]kainate binding sites in the hippocampus as a result of kindling. Savage et al. 29 observed a 47-61% decrease of [3H]kainate binding sites in the stratum lucidum of CA3, and a smaller reduction in the inner molecular layer of the dentate gyrus, 1 day after amygdala or angular bundle kindling, but no change at 28 days22. On the other hand, Represa et al. 27 observed an increase of [3H]kainate binding sites in the supra-granular layer of the dentate gyrus and in the stratum infrapyramidale of CA3 following amygdala kindling, which was related to the sprouting of mossy fibres. The discrepancy between the present results and those of Savage et al. 29 may be due to the site of kindling, although the fact that the initial reduction of [3H]kainate binding sites observed by these authors reverts to normal by 28 days suggests that this phenomenon is not directly related to the kindling process. Similarly, the failure to observe an increase in [3H]kainate binding in the dentate gyrus or CA3 region may be due to the different site of kindling, but is more likely to be a result of the rat strain used (Sprague-Dawley), which already possesses a mossy fibre innervation in the infrapyramidal layer of the CA3 region27. Amongst neurotransmitter receptors, the NMDA receptor has some unique properties. The NMDA receptor-associated ion channel is blocked by Mg2+ in a voltage-dependent manner 13'21 and as a consequence NMDA receptor-mediated neuronal events are critically dependent on the local membrane potential. This property is important for current ideas about the role of NMDA receptors in neuronal plasticity, e.g. learning and memory19. It may also be important for pathological situations, since any factor which brings about a change in neuronal membrane potential can influence the extent of NMDA receptor activity. In the hippocampus, low frequency stimulation of excitatory pathways evokes an excitatory postsynaptic potential (EPSP) which is mediated by non-NMDA receptors, despite the presence of a high density of NMDA receptors on the target neurones5.
120 However, a large N M D A receptor-mediated component to the E P S P is revealed when Mg 2÷ is excluded from the extracellular medium 11, or G A B A e r g i c inhibition is blocked pharmacologically 6. The change(s) induced by kindling which bring about an increased N M D A receptor involvement could, therefore, result from a variety of factors. These include: (1) increased transmitter (glutamate?) release (see ref. 10); (2) upregulation of N M D A receptors; (3) upregulation of n o n - N M D A receptors
(which would result in more depolarisation and facilitate N M D A receptor activity); (4) decreased inhibition (see ref. 31); (5) alterations in other postsynaptic conductances (receptor or non-receptor operated). The present results, and those of the other studies mentioned above, suggest that a n upregulation of hippocampal N M D A or n o n - N M D A excitatory amino acid receptors does not underlie the kindling p h e n o m e n o n .
ABBREVIATIONS
EEG EPSP MK-801
AD AMPA AP7 CPP
afterdischarge D,L-a-amino-3-hydroxy-5-methylisoxazole-4-propionate 2-amino-7-phosphonoheptanoate 3-[(+)-2-(carboxypiperazin-4-yi)]propyl- 1-phosphonate
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NMDA TCP
electroencephalogram excitatory postsynaptic potential (+)-5-methyl-10,11-dihydro-SH-dibenzo[a,d]cyclohepten-5,10-imine maleate N-methyl-o-aspartate N-( 1-[2-thienyl]cyclohexyl)piperidine
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