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Systemic injection of kainic acid: Gliosis in olfactory and limbic brain regions quantified with [3H]PK 11195 binding autoradiography
EXPERIMENTAL NEUROLOGY 1 0 9 , 333-341 (1990)
Systemic Injection of Kainic Acid: Gliosis in Olfactory and Limbic Brain
Regions Quantified with [3H]PK 11195 Binding Autoradiography C. ANTHONY ALTAR* AND MICHEL BAUDRY~ *Developmental Biology, Genentech, Inc., South San Francisco, California 94080; and ~fProgram in Neural, Informational, and Behavioral Sciences, University of Southern California, Los Angeles, California 90089-2520
Alzheimer's type has been carefully mapped (20, 30; for review, see 5). T h e most consistently affected areas include t he hippocampus; subiculum; d e n t a t e gyrus; amygdala; entorhinal, piriform and temporal cortices; and olfactory p a t h w a y s including the olfactory bulb. d e g e n er ativ e changes t h r o u g h o u t r a t b r a i n following Clearly, an animal model of Alzheimer's disease should the systemic administration of kainic acid (12 mg/kg) reproduce this distribution. One n e u r o t r a n s m i t t e r syswere mapped with quantitative autoradiography of tem t h a t is a candidate for mediating this p a t t e r n of [3H]PK 1 1 1 9 5 . T h i s r a d i o l i g a n d b i n d s to a m i t o c h o n neurodegeneration uses excitatory amino acids as endrial benzodiazepine binding site (MBBS) on mieroglia dogenous ligands. Alzheimer's disease has been proand astrocytes. Analysis of eight horizontal and four posed to result from excessive stimulation of excitatory c o r o n a l b r a i n l e v e l s r e v e a l e d up to 1 6 - f o l d i n c r e a s e s in [3H]PK 1 1 1 9 5 b i n d i n g f r o m 1 to 5 w e e k s b u t n o t 1 d a y amino acid receptors by glutamate, aspartate, or possia f t e r k a i n a t e i n j e c t i o n . I n c r e a s e s in [aH]PK 1 1 1 9 5 b i n d bly anot her endogenous agonist (42). Kainic acid, while i n g w e r e p r e d o m i n a n t l y in v e n t r a l l i m b i c b r a i n r e g i o n s not an endogenous ligand, is a rigid cyclic analogue of a n d olfactory projections to neocortical areas, w i t h t h e gl ut am at e t h a t is highly toxic to n e u r o n s in diverse olfactory cortex > subiculum/CA1 > a n t e r i o r olfactory brain regions (22, 27). Widespread damage to various nucleus, medial thalamic nucleus, a n d p i r i f o r m c ort ex areas of the limbic forebrain, including hippocampus, > cingulate cortex and rostral hippocampus > d e n t a t e occurs following systemic injections of kainic acid (6, 19, gyrus, septum, and amygdala > entorhinal cortex a n d 24, 35, 37) and following discrete injections of kainic temporal cortex. Little or no e nha nc e m ent of [sH]PK 11195 binding was observed in numerous regions in- acid into the neostriatum (34, 41), olfactory cortex (35), or amygdala (7). c l u d i n g the caudate-putamen, substantia nigra, nucleus Neurodegeneration is usually accompanied by numeraccumbens, o l f a c t o r y t uber c l e , c e r ebel l um , t h a l a m i e nuclei, ch o r o id plexus, medulla, p a r i e t a l or occipital ous glial reactions (31), and one marker for neurodegencortex, or ports. A 2-fold g r e a t e r e xt e nt of neurodegen- eration has recently been shown to be a brain binding eration was o b t a i n e d in v e n t r a l portions of the olfac- site t hat is located almost exclusively in glial cells (11, t o r y bulb, entorhinal cortex, temporal cortex, and den- 38, 39). Specifically, glial mitochondria contain a benzotate gyrus compared w i t h t h e dorsal portions of these diazepine binding site t h a t is pharmacologically diss t r u c t u r e s . T h e p a t t e r n of i n c r e a s e in [aH]PK 1 1 1 9 5 tinct from n e u r o n a l benzodiazepine recept ors (3, 21, b i n d i n g closely matched the p a t t e r n s of neuronal de- 39). T hi s m i t o c h o n d r i a l benzodiazepine binding site generation r ep o r ted following p a r e n t e r a l kainate injec- (MBBS) is a particularly sensitive marker for ischemic tion. These findings st r engt hen t h e n o t i o n t h a t q u a n t i and local excitotoxic neuronal injuries (9, 13, 34). Gliot a t i v e au to r ad io g r ap h y of [aH]PK 11195 is a valuable sis is a consistent feature of kainate toxicity (22, 35, 41) tool to quantify the e xt e nt of neuronal degeneration. and is a common finding in the brains of demented paF u r t h e r m o r e , t h e q u a n t i t a t i v e c h a n g e s in [aH]PK 11195 b i n d i n g in d i f f e r e n t l i m b i c structures parallel tients (5) and in Huntington's chorea (36). Elevations in M B B S density have been observed in the temporal cort h e i r r e l a t i v e v a r i a t i o n in neuropathology observed in tex of patients with Alzheimer's disease (28) and in the Alzheimer's disease b u t n o t Huntington's chorea. These c a u d a t e - p u t a m e n of patients with H u n t i n g t o n ' s chofindings ar e in agreement w i t h t h e i d e a t h a t excessive rea (34). stimulation of e x c i t a t o r y amino receptors may c o n t r i b If excessive stimulation of excitatory amino acid reu t e to t h e e t i o l o g y o f Alzheimer's disease. © 199o Academic Press, Inc. ceptors contributes to the neurodegeneration observed in H u n t i n g t o n ' s chorea (27) or Alzheimer's disease, t hen excitotoxin-induced changes in M B B S densities INTRODUCTION should resemble the clinical p a t t e r n of neurodegeneraT h e widespread and severe n e u r o d e g e n e r a t i o n ob- tion associated with one or the other disease. T h e purserved in the brains of patients with dementia of the pose of the present study was to map the distribution Neurodegenerative diseases may result from excessive stimulation of excitatory amino acid receptors by endogenous ligands. Because neuronal degeneration is associated with giial proliferation and h y p e r t r o p h y , t h e
333 0014-4886/90 $3.00 Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
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TABLE
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[~H]PK 11195 Binding 2 Weeks after Kainic Acid Injection B r a i n region Olfactory cortex Subiculum/CA1 Olfactory c o r t e x / v e n t r a l striatum A n t e r i o r nu. olfactory bulb Ventral 1 Ventral 2 Dorsal Medio/periventricular thalamus P i r i f o r m cortex Pretectal nu., t h a l a m u s Cingulate cortex Rostral hippocampus D e n t a t e gyrus Septum Amygdala E n t o r h i n a l cortex T e m p o r a l cortex (level 3) Caudal c a u d a t e - p u t a m e n Parietal cortex (caudal) Parietal cortex (rostral) S u b s t a n t i a nigra Rostral*caudate-putamen T r i g e m i n a l nucleus T h a l a m i c nuclei Cerebellar lobule
Vehicle (1 ml/kg)
Kainate (12 m g / k g , ip)
166 ± 95 ±
38 30
2665 ± 161 1257 + 126
16 13
238 ±
55
2521 +
11
242 ± 200 ± 269 ±
77 60 68
2468 ± 26 1006 ± 143"* 869 ± 94**
128 300 83 201 367 318 325 375 404 366 313 141 220 371 200 394 63 326
± 22 ± 64 ± 21 ± 60 ± 124 ± 50 ± 126 ± 70 ± 102 ± 47 ± 68 ± 25 _+ 40 ± 48 ± 30 ± 39 _+ 22 ± 71
4
1057 ± 56 2228 ± 62 580 ± 21 1006 ± 146 1663 ± 134 1260 ± 66 1171 ± 248 1423 ± 235 1288 ± 145 1172 ± 150 803 ± 322 293 ± 66 270 _+ 51 312 ± 34 299 ± 30 231 ± 59 57 ± 32 312 ± 35
Fold increase
10 5 3 8 7 5 5 5 4 4 4 3 3 3 2 1 1 1 1 1 1
ns
ns ns ns ns ns n~
Note. Horizontal b r a i n sections were analyzed for [~H]PK 11195 b i n d i n g as described. Values r e p r e s e n t specific binding (Total binding - binding in the p r e s e n c e of 10 ttM RO 5-4864), Values are m e a n s ± SEM; n = 4/group). All significance levels exceed P < 0.05 u n l e s s indicated by ns or by **P < 0.01 v e r s u s the m o s t v e n t r a l level s h o w n in Fig. 3.
and time course of changes in MBBS densities throughout the rat brain following systemic injection of kainic acid. The pattern of changes, assessed with quantitative receptor autoradiography, was compared first with the pattern of degeneration reported following parenteral kainate injections and then with those reported for the brains of patients with either neurodegenerative disease. MATERIALS
AND
METHODS
Male Sprague-Dawley rats (180-220 g) received a single ip injection of 12 mg/kg of kainic acid. Kainic acid (Sigma Chemical Co., St. Louis, MO) was dissolved in phosphate-buffered saline (pH adjusted to 6.6 with 1 N NaOH). Control rats were injected with the vehicle only (1 ml/kg). Animals were sacrificed 1 day or 1, 2, or 5 weeks later (n = 4 or 5 per group). Brains were removed and frozen in isopentane (-15°C). For coronal sections, 4 or 5 hemispheres were frozen adjacently in a brain matrix (1) and processed in parallel. Twelve-microme-
ter-thick sections were cut on a cryostat (Hacker Instruments, Clifton, NJ) and stored frozen (-70°C) for up to I month before binding assays. Sections were collected at eight horizontal levels (Fig. 1) and at four coronal levels which included the caudate-putamen, globus pallidus, rostral hippocampus, and caudal hippocampus. Sections were thawed and incubated in a 50 m M Tris-HC1 buffer, pH 7.4, t h a t included 120 m M NaC1 and a 3 n M concentration of the selective MBBS antagonist [aH]PK 11195 (sp act, 80 Ci/mmol; D u P o n t - N E N ) (8). RO 5-4864 (Generously supplied by Hoffman-LaRoche, Nutley, NJ) is a potent and selective MBBS agonist t h a t differs structurally from P K 11195 and was included at 10 #M in coplin jars containing adjacent sections to define nonspecific binding (21, 34). RO 5-4864 (10 m M ) was first dissolved in dimethyl sulfoxide (DMSO) and diluted dropwise in the assay buffer. The assay was conducted at room temperature for 45 min. Sections were washed for 2 × 1 min at 4°C in the~uffer, rinsed 3 × 1 sec in distilled water, and dried in 5 : : ~ by a stream of room temperature air. The dried sections a n d tritium-containing radioactivity standards (Amersham, Inc.) were exposed for 4 weeks to tritium-sensitive film (Hyperfilm, Amersham, Inc.). Following development in D-19 (Kodak), binding was quantified in each section by computer-assisted image analysis (2). Betweengroup differences in specific [3H]PK 11195 binding were determined with the Dunnet's t test following an analysis of variance (40). RESULTS Low levels of RO 5-4864-displaceable [3H]PK 11195 binding were present in many brain regions (Table 1) of animals injected with the vehicle. This binding ranged between 60 and 400 fmol/mg protein throughout the" brain parenchyma in the eight horizontal and the four coronal levels (Figs. 1 and 2; Table 1). Exceptions to this were as reported previously (8), with high densities (1000-2500 fmol/mg protein) of specific [3H]PK 11195 binding present on the choroid plexus and ependymal cells of the ventricular walls (Figs. 2 and 3). A survival period of 2 weeks following kainic acid was associated with large increases in [~H]PK 11195 binding in certain areas (Table 1). Most brain regions displaying any apparent increase in labeling following kainate, and many regions t h a t did not, were selected for quantitative analysis according to Fig. 3. Of these areas, the ventral portions of the limbic forebrain, including the olfact o r y cortex, the anterior nucleus of the olfactory bulb, the cingulate cortex, the rostral hippocampus, the entorhinal cortex, the ventral neocortex, the piriform cortex, and the central amygdaloid nuclei (Table 1), displayed the greatest increases in [~H]PK 11195 binding. In Niss!-counterstained sections (data not shown), ne-
KAINATE-INDUCED NEURODEGENERATION
337
FIG. 2. Ventral-to-dorsal (top to bottom panels) distribution of [3H]PK 11195 binding obtained in horizontal sections from vehicle-injected animals (left half of figure) and kainate-injected animals (right half of figure). The computer-generated bright-field photographs represent greater binding with increasing darkness.
338
ALTAR AND BAUDRY
tween 1 and 5 weeks after kainate injection. In contrast, no increase was observed in the parietal cortex or rostral caudate-putamen at any time after the kainate injection (Fig. 7). DISCUSSION
1
J
F I G . 3. I l l u s t r a t i o n s o f h o r i z o n t a l levels at w h i c h [3HIPK 11195 w a s q u a n t i f i e d in t h e p r e s e n t e x p e r i m e n t s . T h e s h a d e d a r e a s illust r a t e r e g i o n s of q u a n t i t a t i o n a n d t h e n u m b e r s w i t h i n t h e s h a d e d are a s c o r r e s p o n d to t h e c o n s e c u t i v e h o r i z o n t a l levels in w h i c h q u a n t i t a tion w a s p e r f o r m e d . T h e n u m b e r in t h e u p p e r r i g h t c o r n e r of e a c h region is t h e m m d o r s a l to t h e skull s u r f a c e at b r e g m a (29).
crosis and vacuolation were seen in the amygdala, piriform cortex, and ventral olfactory cortex, as reported following systemic injections of kainate (35, 37, 43). MBBS densities were not elevated in n u m e r o u s thalamic nuclei (except the medial/periventricular thalamic nucleus) or in the brain stem, choroid plexus, cerebellum, pons, trigeminal nucleus, nucleus accumbens, olfactory tubercle, and most portions of the caudate-putamen (Figs. 1, 2, and 6). A ventral to dorsal gradient of decreasing [3H]PK 11195 binding was observed in the dentate gyrus (Fig. 4) and along six horizontal levels of the entorhinal cortex (data not shown). MBBS levels were also increased in ventral but not dorsal regions of the temporal cortex (Fig. 5) and by five- to sixfold throughout seven levels of the rostral hippocampus (data not shown). In the caudal aspect of the caudate-putamen, only the most ventral and dorsal sections of this structure exhibited a significant increase in [3H]PK 11195 binding (Fig. 6). Little or no increases in [3H]PK 11195 binding were observed in any region at 1 day postinjection (Fig. 7), although seizure-like motor activities were evident from 1 h postinjection until this time. Increases in MBBS were observed at I and 2 weeks postinjection in the septum and rostral hippocampus and at 1, 2, and 5 weeks in the piriform cortex, dentate gyrus, and subiculum/CA1. The increase in binding remained about the same be-
Our results demonstrate t h a t the pattern of increase in [3H]PK 11195 binding t h a t we observed closely matches the pattern of neuronal degeneration and gliosis that occurs after systemic injections of kainic acid in the adult rat (6, 24, 37, 43). Elevations in [aH]PK 11195 binding were prevalent in ventral brain regions, as shown in the dentate gyrus and temporal lobe, and involved many limbic and olfactory portions of the brain. Similarly, the neuronal losses following systemic kainate are prevalent in the hippocampus, amygdala, piriform lobe, septum, medial thalamus, olfactory bulbs, and anterior olfactory nucleus. Systemic kainate injections do not produce neuronal damage in the midbrain, pons, or cerebellum (6, 24, 35, 37), and gliosis was not observed in these areas with MBBS autoradiography. Thus, the present findings confirm that quantitative autoradiography of [3H]PK 11195 binding can be used to study the topography and extent of neurodegeneration following neuronal lesions. It has been shown t h a t ligands which bind to the mitochondrial type of benzodiazepine receptor are good markers for degeneration resulting from ischemia or lo-
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F I G . 4. Q u a n t i t a t i o n of specific [~H]PK 11195 b i n d i n g to t h e d e n t a t e g y r u s of a n i m a l s injected 2 w e e k s before sacrifice w i t h vehicle (black c o l u m n s ) or k a i n a t e (gray c o l u m n s ) . Values are m e a n s _+ S E M (n = 5 / g r o u p ) . [SH]PK 11195 b i n d i n g is e l e v a t e d c o m p a r e d to c o n t r o l by k a i n a t e in all g r o u p s ( P < 0.01, D u n n e t ' s t test) a n d t h e elevation in b i n d i n g d e c r e a s e s f r o m t h e v e n t r a l to t h e dorsal d e n t a t e g y r u s (F(7,29) = 5.0; P < 0.002).
339
KAINATE-INDUCED NEURODEGENERATION
cal injections of excitotoxins (9, 13, 34). W e have also observed t h a t [3H]RO 5-4864 binding a n d [SH]PK 11195 b i n d i n g i n c r e a s e in r a t h i p p o c a m p a l h o m o g e n a t e s or tissue sections, respectively, following e n t o r h i n a l c o r t e x lesion a n d i n t r a h i p p o c a m p a l c o l c h i c i n e i n j e c t i o n ( B a u d r y a n d Altar, s u b m i t t e d ) . U n d e r e a c h of t h e s e conditions, it has b e e n shown t h a t the n e u r o n a l degeneration is a c c o m p a n i e d by a massive p r o l i f e r a t i o n of microglial cells a n d a h y p e r t r o p h y a n d proliferation of ast r o c y t e s (14, 16, 32). T h e close c o r r e l a t i o n b e t w e e n the regional distribution of n e u r o n a l d e a t h a n d of quantitative increases in M B B S densities r e p o r t e d here strongly s u p p o r t s the idea t h a t these sites are localized on glial cells and t h a t a u t o r a d i o g r a p h y of t h e i r distribution constitutes a q u a n t i t a t i v e m e t h o d with which to analyze p a t t e r n s of n e u r o d e g e n e r a t i o n . A definitive t e s t of this p r o p o s a l awaits double-labeling of single cells f r o m int a c t or injured b r a i n with glial-, astrocytic-, or o t h e r cell-specific m a r k e r s and [3H]PK 11195 radiolabel. S e v e r a l m e c h a n i s m s h a v e b e e n p r o p o s e d for t h e n e u r o d e g e n e r a t i o n p r o d u c e d by systemic injections of kainic acid. One possibility is t h a t kainic acid increases the permeability of brain to toxic molecules, cells, or o t h e r factors t h a t are n o r m a l l y excluded f r o m this comp a r t m e n t (43). N e u r a l i n j u r y i n d u c e d b y e x c i t a t o r y amino acids p r o m o t e s the influx f r o m the circulation of m a c r o p h a g e s (18) a n d increases the n u m b e r or size of brain a m o e b o i d microglia (15) and a s t r o c y t e s (31). Be-
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FIG. 5. Ventral to dorsal distribution of [3H]PK 11195binding in the temporal cortex of horizontal sections from animals injected with vehicle or kainate 2 weeks before sacrifice. Binding is decreased to a greater extent in ventral than in dorsal portions of the temporal cortex (F(7,29) - 2.8; P < 0.03). *P < 0.05, **P < 0.01 versus vehicle, Dunnett's t test.
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F I G . 6. [aH]PK 11195 binding at 6 levels of the rostral (top) and caudal (bottom) caudate-putamen. Binding was elevated in only the most ventral and dorsal portion of the caudal level (*P < 0.01, paired t
test).
cause the M B B S is also f o u n d on p o l y m o r p h o n u c l e a r m o n o c y t e s (C. A. Altar and A. Piranian, u n p u b l i s h e d observations) a n d l y m p h o c y t e s (23) as well as on astrocytes (10) a n d glioma cells (39), it is possible t h a t the M B B S increases m a y have resulted from a p e n e t r a t i o n into brain by m o n o c y t e s or l y m p h o c y t e s t h r o u g h a comp r o m i s e d b l o o d - b r a i n barrier. However, M B B S densities were not increased at 1 day a f t e r kainic acid, even t h o u g h the permeability of the b l o o d - b r a i n b a r r i e r is i n c r e a s e d to a m a x i m a l e x t e n t at this t i m e (25, 43). T h u s , kainate probably does not increase M B B S densities in brain simply by allowing m a c r o p h a g e s or o t h e r circulating cells t h a t contain the M B B S access to brain. T h e increase in M B B S is m o s t likely a result of glial h y p e r t r o p h y a n d proliferation in response to cellular necrosis, since these events, like the M B B S increases observed here, occur over 1-2 weeks b u t are not p r e s e n t at 1 day a f t e r n e u r o n a l injury (15, 18). It is g e n e r a l l y b e l i e v e d t h a t s y s t e m i c i n j e c t i o n s of kainic acid to rats provides a good model for t e m p o r a l lobe epilepsy (6, 24). T h e e x t r e m e s u s c e p t i b i l i t y to kainic acid o f i n h i b i t o r y i n t e r n e u r o n s in t h e d e n t a t e gyrus is believed to be p r o d u c e d by s y n c h r o n o u s discharges as a result of disinhibition, a n d most of the neuronal d e g e n e r a t i o n observed following systemic injections of kainic acid is due to disturbances associated with seizure activity. N e u r o d e g e n e r a t i o n in the hippoc a m p u s following amygdala injections of kainate can be
340
ALTAR AND BAUDRY
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dency and neurochemical toxicity of sustained treatments with kainic acid are investigating the relevance of this animal model to Alzheimer's disease. In conclusion, the present study with [~H]PK 11195 autoradiography has provided a quantitative, whole brain mapping of gliosis following systemic injections of kainic acid. Increases in [3H]PK 11195 binding, a marker for glial cells (9, 11, 13, 38), are present from I to at least 5 weeks postinjection. Increases in [3H]PK 11195 binding were greatest in ventral regions of olfactory and limbic pathways, including the olfactory bulb and cortex, hippocampus, piriform cortex, entorhinal cortex, septnm, and amygdala. The absence of quantitative changes in most striatal, cerebellar, pontine, or thalamic regions demonstrates a selectivity to the pattern of neurodegeneration that is reminiscent of the neurodegenerative topography of Alzheimer's disease. This pattern of neurodegeneration, and the loss of cholinergic cells following kainic acid (17, 33, 37), is consistent with the possible role for excessive excitatory amino acid transmission in the etiology of Alzheimer's disease (42).
**"
FIG. 7. [~H]PK 11195 binding to eight areas in coronal sections of rats treated with vehicle and killed 2 weeks later or with kainic acid (12 mg/kg) and killed I day or 1, 2, or 5 weeks later. *P < 0.05, **P < 0.01 versus vehicle. N = 5/group.
blocked by the antiepileptic drug diazepam (7). Because kainic acid toxicity to the hippocampus and other structures can also be blocked by glutamatergic denervation (22), excessive glutamate release may be a common p a t h w a y for diaschisis following seizures induced by kainic acid or other agents. In the neostriatum, systemic kainate injections produce neurodegeneration (35) and increases in M B B S densities (present findings) in dorsal and ventral caudal areasin a pattern that resembles the innervation of the caudoventral and dorsal neostriatum by amygdaloid afferents (12). Thus, kainic acid injections may promote tSis limited neostriatal neurodegeneration by seizure activity of amygdaloid afferents to neostriatum. However, these small and scattered increases in M B B S levels in the neostriatum, a region capable of producing extensive gliosis (27, 41) and increased M B B S densities (34) following local kainate, d e m o n s t r a t e that Huntington's chorea was not modeled in the present study. Instead, 'the increases in [3H]PK 11195 binding following systemic injections of kainic acid parallel the susceptibility of various ]imbic structures to neuropathological alterations in the braii~s of patients with ~Alzheimer's disease (5, 20, 30). Compared with temporal lobe epilepsy, it may be more difficult to link excitatory amino acid toxicity with a tardive neurodegenerative disease such as Alzheimer's disease. Further studies on t h e age-depen-
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KAINATE-INDUCED N E U R O D E G E N E R A T I O N of glial cells is associated with a calcium channel. Eur. J. Pharmacol. 110: 287-288.
27.
11.
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GIULIAN, D. 1987. Ameboid microglia as effectors of inflammation in the central nervous system. J. Neurosci. Res. 18: 155171.
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MARANGOS,P. J., J. PATEL, J. BOULENGER, AND R. CLARK-RoSENBERG. 1982. Characterization of peripheral-type benzodiazepine binding sites in brain using [3H]RO 5-4864. Mol. Pharmacol. 22: 26-32.
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NADLER, J. V. 1981. Kainic acid as a tool for the study of temporal lobe epilepsy. Life Sci. 29: 2031-2042.
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NITSCH,C., AND H. HUBAUER. 1986. Distant blood-brain barrier opening in subfields of the rat hippocampus after intrastriatal injections of kainic acid but not ibotenic acid. Neurosci. Lett. 64: 53-58.
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OLNEY,J. W., V. RHEE, ANDO. L. HO. 1974. Kainic acid: A powerful analogue of glutamate. Brain Res. 77" 507-512. OWEN,F., M. POULTER, J. L. WADDINGTON, R. D. MASHAL,AND T. J. CROW. 1983. [3H]Ro 5-4864 and [3H]fiunitrazepam binding in kainate-lesioned rat striatum and temporal cortex of brains from patients with senile dementia of the Alzheimer subtype. Brain Res. 2 7 8 : 373-375. P~XINOS,G., AND C. WATSON. 1986. The Rat Brain in Stereotaxic Coordinates, 2nd ed. Academic Press, New York. PEARSON,R. C. A., M. M. ESIRI, R. W. HIORNS, G. K. WILCOCK, AND T. P. S. POWELL. 1985. Anatomical correlates of the distribution of the pathological changes in the neocortex in Alzheimer disease. Proc. Natl. Acad. Sci. USA 82: 4531-4534. REIER, P. J., L. F. ENG, AND L. JAKEMAN. 1988. Reactive astrocyte and axonal outgrowth in the injured CNS: Is gliosis really an impediment to regeneration? In Reactive Astrocytes and Axonal Outgrowth, pp. 1-27. A. R. Liss, New York. ROSE, G., C. W. COTMAN, AND G. S. LYNCH. 1976. Hypertrophy and redistribution of astrocytes in the deafferented hippocampus. Brain Res. Bull. 1: 87-92. SCHLIEBS, R., M. ZIVIN, J . STEINBACH, AND W. ROTHE. 1989. Changes in cholinergic but not in GABAergic markers in amygdala, piriform cortex, and nucleus basalis of the rat brain following systemic administration of kainic acid. J. Neurochem. 53: 212-218. SCHOEMAKERIH., M. MORELLI, M. P. DESH, AND H. I. YAMAMURA. 1982. [3H]Ro 5-4864 benzodiazepine binding in the kainate lesioned striatum and Huntington's diseased basal ganglia. Brain Res. 248: 396-401. SCHWOB,J. E., T. FULLER, J. L. PRICE, AND J. W. OLNEY. 1980. Widespread patterns of neuronal damage following systemic or intracerebral injections of kainic acid: A histological study. Neuroscience 5: 991-1014. SELKOE,D. J., F. J. SALAZAR,C. ABRAHAM, AND K. S. KOSIK. 1982. Huntington's disease: Changes in striatal proteins reflect astrocytic gliosis. Brain Res. 2 4 5 : 117-125. SPERK, G., H. LASSMANN, H. BARAN, S. J. KISH, F. SEITELBERGER, AND O. HORNYKIEWICZ. 1983. Kalnic acid induced seizures: Neurochemlcal and histopathologlcal changes. Neuroscience 10: 1301-1315. STAROSTA-RUBENSTEIN, S., B. J. CILIAX, J. B. PENNEY: P. MCKEEVER, AND A. B. YOUNG. 1987. Imaging of a glioma using peripheral benzodiazepine receptor ligands. Proc. Natl. Acad. Sci. USA 84: 891-895. SYAPIN,P. J., AND P. SKOLNICK.1982. Characterization ofbenzodiazepine binding sites in cultured cells of neuronal origin. J. Neurochem. 32:1047 1051. WINER,B. J. 1971. Statistical Principles in Experimental Design. McGraw-Hill, New York. WUERTHELE, S. M., K. L. LOVELL, M. Z. JONES, AND K . E. MOORE. 1978. A histological study of kainic acid-induced lesions in the rat brain. Brain Res. 149: 489-497. YOUNG, A. B. 1986. Cortical amino acidergic pathways in Alzheimer's disease, d. Neural Transm. (Suppl) 24: 147-152. ZUCKER, D. K., C. F. WOOTEN, AND E. W. LOTHMAN. 1983. Blood-brain barrier changes with kainic acid-induced limbic seizures. Exp. Neurol. 79: 422-433.
Systemic Injection of Kainic Acid: Gliosis in Olfactory and Limbic Brain
Regions Quantified with [3H]PK 11195 Binding Autoradiography C. ANTHONY ALTAR* AND MICHEL BAUDRY~ *Developmental Biology, Genentech, Inc., South San Francisco, California 94080; and ~fProgram in Neural, Informational, and Behavioral Sciences, University of Southern California, Los Angeles, California 90089-2520
Alzheimer's type has been carefully mapped (20, 30; for review, see 5). T h e most consistently affected areas include t he hippocampus; subiculum; d e n t a t e gyrus; amygdala; entorhinal, piriform and temporal cortices; and olfactory p a t h w a y s including the olfactory bulb. d e g e n er ativ e changes t h r o u g h o u t r a t b r a i n following Clearly, an animal model of Alzheimer's disease should the systemic administration of kainic acid (12 mg/kg) reproduce this distribution. One n e u r o t r a n s m i t t e r syswere mapped with quantitative autoradiography of tem t h a t is a candidate for mediating this p a t t e r n of [3H]PK 1 1 1 9 5 . T h i s r a d i o l i g a n d b i n d s to a m i t o c h o n neurodegeneration uses excitatory amino acids as endrial benzodiazepine binding site (MBBS) on mieroglia dogenous ligands. Alzheimer's disease has been proand astrocytes. Analysis of eight horizontal and four posed to result from excessive stimulation of excitatory c o r o n a l b r a i n l e v e l s r e v e a l e d up to 1 6 - f o l d i n c r e a s e s in [3H]PK 1 1 1 9 5 b i n d i n g f r o m 1 to 5 w e e k s b u t n o t 1 d a y amino acid receptors by glutamate, aspartate, or possia f t e r k a i n a t e i n j e c t i o n . I n c r e a s e s in [aH]PK 1 1 1 9 5 b i n d bly anot her endogenous agonist (42). Kainic acid, while i n g w e r e p r e d o m i n a n t l y in v e n t r a l l i m b i c b r a i n r e g i o n s not an endogenous ligand, is a rigid cyclic analogue of a n d olfactory projections to neocortical areas, w i t h t h e gl ut am at e t h a t is highly toxic to n e u r o n s in diverse olfactory cortex > subiculum/CA1 > a n t e r i o r olfactory brain regions (22, 27). Widespread damage to various nucleus, medial thalamic nucleus, a n d p i r i f o r m c ort ex areas of the limbic forebrain, including hippocampus, > cingulate cortex and rostral hippocampus > d e n t a t e occurs following systemic injections of kainic acid (6, 19, gyrus, septum, and amygdala > entorhinal cortex a n d 24, 35, 37) and following discrete injections of kainic temporal cortex. Little or no e nha nc e m ent of [sH]PK 11195 binding was observed in numerous regions in- acid into the neostriatum (34, 41), olfactory cortex (35), or amygdala (7). c l u d i n g the caudate-putamen, substantia nigra, nucleus Neurodegeneration is usually accompanied by numeraccumbens, o l f a c t o r y t uber c l e , c e r ebel l um , t h a l a m i e nuclei, ch o r o id plexus, medulla, p a r i e t a l or occipital ous glial reactions (31), and one marker for neurodegencortex, or ports. A 2-fold g r e a t e r e xt e nt of neurodegen- eration has recently been shown to be a brain binding eration was o b t a i n e d in v e n t r a l portions of the olfac- site t hat is located almost exclusively in glial cells (11, t o r y bulb, entorhinal cortex, temporal cortex, and den- 38, 39). Specifically, glial mitochondria contain a benzotate gyrus compared w i t h t h e dorsal portions of these diazepine binding site t h a t is pharmacologically diss t r u c t u r e s . T h e p a t t e r n of i n c r e a s e in [aH]PK 1 1 1 9 5 tinct from n e u r o n a l benzodiazepine recept ors (3, 21, b i n d i n g closely matched the p a t t e r n s of neuronal de- 39). T hi s m i t o c h o n d r i a l benzodiazepine binding site generation r ep o r ted following p a r e n t e r a l kainate injec- (MBBS) is a particularly sensitive marker for ischemic tion. These findings st r engt hen t h e n o t i o n t h a t q u a n t i and local excitotoxic neuronal injuries (9, 13, 34). Gliot a t i v e au to r ad io g r ap h y of [aH]PK 11195 is a valuable sis is a consistent feature of kainate toxicity (22, 35, 41) tool to quantify the e xt e nt of neuronal degeneration. and is a common finding in the brains of demented paF u r t h e r m o r e , t h e q u a n t i t a t i v e c h a n g e s in [aH]PK 11195 b i n d i n g in d i f f e r e n t l i m b i c structures parallel tients (5) and in Huntington's chorea (36). Elevations in M B B S density have been observed in the temporal cort h e i r r e l a t i v e v a r i a t i o n in neuropathology observed in tex of patients with Alzheimer's disease (28) and in the Alzheimer's disease b u t n o t Huntington's chorea. These c a u d a t e - p u t a m e n of patients with H u n t i n g t o n ' s chofindings ar e in agreement w i t h t h e i d e a t h a t excessive rea (34). stimulation of e x c i t a t o r y amino receptors may c o n t r i b If excessive stimulation of excitatory amino acid reu t e to t h e e t i o l o g y o f Alzheimer's disease. © 199o Academic Press, Inc. ceptors contributes to the neurodegeneration observed in H u n t i n g t o n ' s chorea (27) or Alzheimer's disease, t hen excitotoxin-induced changes in M B B S densities INTRODUCTION should resemble the clinical p a t t e r n of neurodegeneraT h e widespread and severe n e u r o d e g e n e r a t i o n ob- tion associated with one or the other disease. T h e purserved in the brains of patients with dementia of the pose of the present study was to map the distribution Neurodegenerative diseases may result from excessive stimulation of excitatory amino acid receptors by endogenous ligands. Because neuronal degeneration is associated with giial proliferation and h y p e r t r o p h y , t h e
333 0014-4886/90 $3.00 Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
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[~H]PK 11195 Binding 2 Weeks after Kainic Acid Injection B r a i n region Olfactory cortex Subiculum/CA1 Olfactory c o r t e x / v e n t r a l striatum A n t e r i o r nu. olfactory bulb Ventral 1 Ventral 2 Dorsal Medio/periventricular thalamus P i r i f o r m cortex Pretectal nu., t h a l a m u s Cingulate cortex Rostral hippocampus D e n t a t e gyrus Septum Amygdala E n t o r h i n a l cortex T e m p o r a l cortex (level 3) Caudal c a u d a t e - p u t a m e n Parietal cortex (caudal) Parietal cortex (rostral) S u b s t a n t i a nigra Rostral*caudate-putamen T r i g e m i n a l nucleus T h a l a m i c nuclei Cerebellar lobule
Vehicle (1 ml/kg)
Kainate (12 m g / k g , ip)
166 ± 95 ±
38 30
2665 ± 161 1257 + 126
16 13
238 ±
55
2521 +
11
242 ± 200 ± 269 ±
77 60 68
2468 ± 26 1006 ± 143"* 869 ± 94**
128 300 83 201 367 318 325 375 404 366 313 141 220 371 200 394 63 326
± 22 ± 64 ± 21 ± 60 ± 124 ± 50 ± 126 ± 70 ± 102 ± 47 ± 68 ± 25 _+ 40 ± 48 ± 30 ± 39 _+ 22 ± 71
4
1057 ± 56 2228 ± 62 580 ± 21 1006 ± 146 1663 ± 134 1260 ± 66 1171 ± 248 1423 ± 235 1288 ± 145 1172 ± 150 803 ± 322 293 ± 66 270 _+ 51 312 ± 34 299 ± 30 231 ± 59 57 ± 32 312 ± 35
Fold increase
10 5 3 8 7 5 5 5 4 4 4 3 3 3 2 1 1 1 1 1 1
ns
ns ns ns ns ns n~
Note. Horizontal b r a i n sections were analyzed for [~H]PK 11195 b i n d i n g as described. Values r e p r e s e n t specific binding (Total binding - binding in the p r e s e n c e of 10 ttM RO 5-4864), Values are m e a n s ± SEM; n = 4/group). All significance levels exceed P < 0.05 u n l e s s indicated by ns or by **P < 0.01 v e r s u s the m o s t v e n t r a l level s h o w n in Fig. 3.
and time course of changes in MBBS densities throughout the rat brain following systemic injection of kainic acid. The pattern of changes, assessed with quantitative receptor autoradiography, was compared first with the pattern of degeneration reported following parenteral kainate injections and then with those reported for the brains of patients with either neurodegenerative disease. MATERIALS
AND
METHODS
Male Sprague-Dawley rats (180-220 g) received a single ip injection of 12 mg/kg of kainic acid. Kainic acid (Sigma Chemical Co., St. Louis, MO) was dissolved in phosphate-buffered saline (pH adjusted to 6.6 with 1 N NaOH). Control rats were injected with the vehicle only (1 ml/kg). Animals were sacrificed 1 day or 1, 2, or 5 weeks later (n = 4 or 5 per group). Brains were removed and frozen in isopentane (-15°C). For coronal sections, 4 or 5 hemispheres were frozen adjacently in a brain matrix (1) and processed in parallel. Twelve-microme-
ter-thick sections were cut on a cryostat (Hacker Instruments, Clifton, NJ) and stored frozen (-70°C) for up to I month before binding assays. Sections were collected at eight horizontal levels (Fig. 1) and at four coronal levels which included the caudate-putamen, globus pallidus, rostral hippocampus, and caudal hippocampus. Sections were thawed and incubated in a 50 m M Tris-HC1 buffer, pH 7.4, t h a t included 120 m M NaC1 and a 3 n M concentration of the selective MBBS antagonist [aH]PK 11195 (sp act, 80 Ci/mmol; D u P o n t - N E N ) (8). RO 5-4864 (Generously supplied by Hoffman-LaRoche, Nutley, NJ) is a potent and selective MBBS agonist t h a t differs structurally from P K 11195 and was included at 10 #M in coplin jars containing adjacent sections to define nonspecific binding (21, 34). RO 5-4864 (10 m M ) was first dissolved in dimethyl sulfoxide (DMSO) and diluted dropwise in the assay buffer. The assay was conducted at room temperature for 45 min. Sections were washed for 2 × 1 min at 4°C in the~uffer, rinsed 3 × 1 sec in distilled water, and dried in 5 : : ~ by a stream of room temperature air. The dried sections a n d tritium-containing radioactivity standards (Amersham, Inc.) were exposed for 4 weeks to tritium-sensitive film (Hyperfilm, Amersham, Inc.). Following development in D-19 (Kodak), binding was quantified in each section by computer-assisted image analysis (2). Betweengroup differences in specific [3H]PK 11195 binding were determined with the Dunnet's t test following an analysis of variance (40). RESULTS Low levels of RO 5-4864-displaceable [3H]PK 11195 binding were present in many brain regions (Table 1) of animals injected with the vehicle. This binding ranged between 60 and 400 fmol/mg protein throughout the" brain parenchyma in the eight horizontal and the four coronal levels (Figs. 1 and 2; Table 1). Exceptions to this were as reported previously (8), with high densities (1000-2500 fmol/mg protein) of specific [3H]PK 11195 binding present on the choroid plexus and ependymal cells of the ventricular walls (Figs. 2 and 3). A survival period of 2 weeks following kainic acid was associated with large increases in [~H]PK 11195 binding in certain areas (Table 1). Most brain regions displaying any apparent increase in labeling following kainate, and many regions t h a t did not, were selected for quantitative analysis according to Fig. 3. Of these areas, the ventral portions of the limbic forebrain, including the olfact o r y cortex, the anterior nucleus of the olfactory bulb, the cingulate cortex, the rostral hippocampus, the entorhinal cortex, the ventral neocortex, the piriform cortex, and the central amygdaloid nuclei (Table 1), displayed the greatest increases in [~H]PK 11195 binding. In Niss!-counterstained sections (data not shown), ne-
KAINATE-INDUCED NEURODEGENERATION
337
FIG. 2. Ventral-to-dorsal (top to bottom panels) distribution of [3H]PK 11195 binding obtained in horizontal sections from vehicle-injected animals (left half of figure) and kainate-injected animals (right half of figure). The computer-generated bright-field photographs represent greater binding with increasing darkness.
338
ALTAR AND BAUDRY
tween 1 and 5 weeks after kainate injection. In contrast, no increase was observed in the parietal cortex or rostral caudate-putamen at any time after the kainate injection (Fig. 7). DISCUSSION
1
J
F I G . 3. I l l u s t r a t i o n s o f h o r i z o n t a l levels at w h i c h [3HIPK 11195 w a s q u a n t i f i e d in t h e p r e s e n t e x p e r i m e n t s . T h e s h a d e d a r e a s illust r a t e r e g i o n s of q u a n t i t a t i o n a n d t h e n u m b e r s w i t h i n t h e s h a d e d are a s c o r r e s p o n d to t h e c o n s e c u t i v e h o r i z o n t a l levels in w h i c h q u a n t i t a tion w a s p e r f o r m e d . T h e n u m b e r in t h e u p p e r r i g h t c o r n e r of e a c h region is t h e m m d o r s a l to t h e skull s u r f a c e at b r e g m a (29).
crosis and vacuolation were seen in the amygdala, piriform cortex, and ventral olfactory cortex, as reported following systemic injections of kainate (35, 37, 43). MBBS densities were not elevated in n u m e r o u s thalamic nuclei (except the medial/periventricular thalamic nucleus) or in the brain stem, choroid plexus, cerebellum, pons, trigeminal nucleus, nucleus accumbens, olfactory tubercle, and most portions of the caudate-putamen (Figs. 1, 2, and 6). A ventral to dorsal gradient of decreasing [3H]PK 11195 binding was observed in the dentate gyrus (Fig. 4) and along six horizontal levels of the entorhinal cortex (data not shown). MBBS levels were also increased in ventral but not dorsal regions of the temporal cortex (Fig. 5) and by five- to sixfold throughout seven levels of the rostral hippocampus (data not shown). In the caudal aspect of the caudate-putamen, only the most ventral and dorsal sections of this structure exhibited a significant increase in [3H]PK 11195 binding (Fig. 6). Little or no increases in [3H]PK 11195 binding were observed in any region at 1 day postinjection (Fig. 7), although seizure-like motor activities were evident from 1 h postinjection until this time. Increases in MBBS were observed at I and 2 weeks postinjection in the septum and rostral hippocampus and at 1, 2, and 5 weeks in the piriform cortex, dentate gyrus, and subiculum/CA1. The increase in binding remained about the same be-
Our results demonstrate t h a t the pattern of increase in [3H]PK 11195 binding t h a t we observed closely matches the pattern of neuronal degeneration and gliosis that occurs after systemic injections of kainic acid in the adult rat (6, 24, 37, 43). Elevations in [aH]PK 11195 binding were prevalent in ventral brain regions, as shown in the dentate gyrus and temporal lobe, and involved many limbic and olfactory portions of the brain. Similarly, the neuronal losses following systemic kainate are prevalent in the hippocampus, amygdala, piriform lobe, septum, medial thalamus, olfactory bulbs, and anterior olfactory nucleus. Systemic kainate injections do not produce neuronal damage in the midbrain, pons, or cerebellum (6, 24, 35, 37), and gliosis was not observed in these areas with MBBS autoradiography. Thus, the present findings confirm that quantitative autoradiography of [3H]PK 11195 binding can be used to study the topography and extent of neurodegeneration following neuronal lesions. It has been shown t h a t ligands which bind to the mitochondrial type of benzodiazepine receptor are good markers for degeneration resulting from ischemia or lo-
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F I G . 4. Q u a n t i t a t i o n of specific [~H]PK 11195 b i n d i n g to t h e d e n t a t e g y r u s of a n i m a l s injected 2 w e e k s before sacrifice w i t h vehicle (black c o l u m n s ) or k a i n a t e (gray c o l u m n s ) . Values are m e a n s _+ S E M (n = 5 / g r o u p ) . [SH]PK 11195 b i n d i n g is e l e v a t e d c o m p a r e d to c o n t r o l by k a i n a t e in all g r o u p s ( P < 0.01, D u n n e t ' s t test) a n d t h e elevation in b i n d i n g d e c r e a s e s f r o m t h e v e n t r a l to t h e dorsal d e n t a t e g y r u s (F(7,29) = 5.0; P < 0.002).
339
KAINATE-INDUCED NEURODEGENERATION
cal injections of excitotoxins (9, 13, 34). W e have also observed t h a t [3H]RO 5-4864 binding a n d [SH]PK 11195 b i n d i n g i n c r e a s e in r a t h i p p o c a m p a l h o m o g e n a t e s or tissue sections, respectively, following e n t o r h i n a l c o r t e x lesion a n d i n t r a h i p p o c a m p a l c o l c h i c i n e i n j e c t i o n ( B a u d r y a n d Altar, s u b m i t t e d ) . U n d e r e a c h of t h e s e conditions, it has b e e n shown t h a t the n e u r o n a l degeneration is a c c o m p a n i e d by a massive p r o l i f e r a t i o n of microglial cells a n d a h y p e r t r o p h y a n d proliferation of ast r o c y t e s (14, 16, 32). T h e close c o r r e l a t i o n b e t w e e n the regional distribution of n e u r o n a l d e a t h a n d of quantitative increases in M B B S densities r e p o r t e d here strongly s u p p o r t s the idea t h a t these sites are localized on glial cells and t h a t a u t o r a d i o g r a p h y of t h e i r distribution constitutes a q u a n t i t a t i v e m e t h o d with which to analyze p a t t e r n s of n e u r o d e g e n e r a t i o n . A definitive t e s t of this p r o p o s a l awaits double-labeling of single cells f r o m int a c t or injured b r a i n with glial-, astrocytic-, or o t h e r cell-specific m a r k e r s and [3H]PK 11195 radiolabel. S e v e r a l m e c h a n i s m s h a v e b e e n p r o p o s e d for t h e n e u r o d e g e n e r a t i o n p r o d u c e d by systemic injections of kainic acid. One possibility is t h a t kainic acid increases the permeability of brain to toxic molecules, cells, or o t h e r factors t h a t are n o r m a l l y excluded f r o m this comp a r t m e n t (43). N e u r a l i n j u r y i n d u c e d b y e x c i t a t o r y amino acids p r o m o t e s the influx f r o m the circulation of m a c r o p h a g e s (18) a n d increases the n u m b e r or size of brain a m o e b o i d microglia (15) and a s t r o c y t e s (31). Be-
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FIG. 5. Ventral to dorsal distribution of [3H]PK 11195binding in the temporal cortex of horizontal sections from animals injected with vehicle or kainate 2 weeks before sacrifice. Binding is decreased to a greater extent in ventral than in dorsal portions of the temporal cortex (F(7,29) - 2.8; P < 0.03). *P < 0.05, **P < 0.01 versus vehicle, Dunnett's t test.
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F I G . 6. [aH]PK 11195 binding at 6 levels of the rostral (top) and caudal (bottom) caudate-putamen. Binding was elevated in only the most ventral and dorsal portion of the caudal level (*P < 0.01, paired t
test).
cause the M B B S is also f o u n d on p o l y m o r p h o n u c l e a r m o n o c y t e s (C. A. Altar and A. Piranian, u n p u b l i s h e d observations) a n d l y m p h o c y t e s (23) as well as on astrocytes (10) a n d glioma cells (39), it is possible t h a t the M B B S increases m a y have resulted from a p e n e t r a t i o n into brain by m o n o c y t e s or l y m p h o c y t e s t h r o u g h a comp r o m i s e d b l o o d - b r a i n barrier. However, M B B S densities were not increased at 1 day a f t e r kainic acid, even t h o u g h the permeability of the b l o o d - b r a i n b a r r i e r is i n c r e a s e d to a m a x i m a l e x t e n t at this t i m e (25, 43). T h u s , kainate probably does not increase M B B S densities in brain simply by allowing m a c r o p h a g e s or o t h e r circulating cells t h a t contain the M B B S access to brain. T h e increase in M B B S is m o s t likely a result of glial h y p e r t r o p h y a n d proliferation in response to cellular necrosis, since these events, like the M B B S increases observed here, occur over 1-2 weeks b u t are not p r e s e n t at 1 day a f t e r n e u r o n a l injury (15, 18). It is g e n e r a l l y b e l i e v e d t h a t s y s t e m i c i n j e c t i o n s of kainic acid to rats provides a good model for t e m p o r a l lobe epilepsy (6, 24). T h e e x t r e m e s u s c e p t i b i l i t y to kainic acid o f i n h i b i t o r y i n t e r n e u r o n s in t h e d e n t a t e gyrus is believed to be p r o d u c e d by s y n c h r o n o u s discharges as a result of disinhibition, a n d most of the neuronal d e g e n e r a t i o n observed following systemic injections of kainic acid is due to disturbances associated with seizure activity. N e u r o d e g e n e r a t i o n in the hippoc a m p u s following amygdala injections of kainate can be
340
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dency and neurochemical toxicity of sustained treatments with kainic acid are investigating the relevance of this animal model to Alzheimer's disease. In conclusion, the present study with [~H]PK 11195 autoradiography has provided a quantitative, whole brain mapping of gliosis following systemic injections of kainic acid. Increases in [3H]PK 11195 binding, a marker for glial cells (9, 11, 13, 38), are present from I to at least 5 weeks postinjection. Increases in [3H]PK 11195 binding were greatest in ventral regions of olfactory and limbic pathways, including the olfactory bulb and cortex, hippocampus, piriform cortex, entorhinal cortex, septnm, and amygdala. The absence of quantitative changes in most striatal, cerebellar, pontine, or thalamic regions demonstrates a selectivity to the pattern of neurodegeneration that is reminiscent of the neurodegenerative topography of Alzheimer's disease. This pattern of neurodegeneration, and the loss of cholinergic cells following kainic acid (17, 33, 37), is consistent with the possible role for excessive excitatory amino acid transmission in the etiology of Alzheimer's disease (42).
**"
FIG. 7. [~H]PK 11195 binding to eight areas in coronal sections of rats treated with vehicle and killed 2 weeks later or with kainic acid (12 mg/kg) and killed I day or 1, 2, or 5 weeks later. *P < 0.05, **P < 0.01 versus vehicle. N = 5/group.
blocked by the antiepileptic drug diazepam (7). Because kainic acid toxicity to the hippocampus and other structures can also be blocked by glutamatergic denervation (22), excessive glutamate release may be a common p a t h w a y for diaschisis following seizures induced by kainic acid or other agents. In the neostriatum, systemic kainate injections produce neurodegeneration (35) and increases in M B B S densities (present findings) in dorsal and ventral caudal areasin a pattern that resembles the innervation of the caudoventral and dorsal neostriatum by amygdaloid afferents (12). Thus, kainic acid injections may promote tSis limited neostriatal neurodegeneration by seizure activity of amygdaloid afferents to neostriatum. However, these small and scattered increases in M B B S levels in the neostriatum, a region capable of producing extensive gliosis (27, 41) and increased M B B S densities (34) following local kainate, d e m o n s t r a t e that Huntington's chorea was not modeled in the present study. Instead, 'the increases in [3H]PK 11195 binding following systemic injections of kainic acid parallel the susceptibility of various ]imbic structures to neuropathological alterations in the braii~s of patients with ~Alzheimer's disease (5, 20, 30). Compared with temporal lobe epilepsy, it may be more difficult to link excitatory amino acid toxicity with a tardive neurodegenerative disease such as Alzheimer's disease. Further studies on t h e age-depen-
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