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Autoradiographic analysis of [35S]TBPS binding in entorhinal cortex-kindled rat brains
Brain Research, 570 (1991) 167-172 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00
167
BRES 17367
Autoradiographic analysis of [35S]TBPS binding in entorhinal cortex-kindled rat brains John S. Petrasek 1, Jos6 N. Nobrega 2, Stephen J. Kish 1'2 and W. Mclntyre Burnham 1 1Department of Pharmacology, University of Toronto, Toronto, Ont. (Canada) and 2Clarke Institute of Psychiatry, Toronto, Ont. (Canada) (Accepted 10 September 1991)
Key words: Kindling; Seizures; Quantitative autoradiography; y-Aminobutyric acidA receptor; Chloride ionophore; t-Butylbicyclophosphorothionate
A quantitative autoradiographic analysis of [35S]t-butylbicyclophosphorothionate (TBPS) binding to the 7-aminobutyric acid (GABA)-mediated chloride ionophore was carded out in 104 brain areas of entorhinal cortex-kindled and control rats. Subjects were sacrificed either 24 h or 28 days after the last kindled seizure. Kindled subjects in the 24 h group showed reductions in mean [35S]TBPS binding in the lateral nucleus of the amygdala (-31%), the infralimbic cortex (-14%), and the paracentral nucleus of the thalamus (-22%). At 28 days, reductions in binding were observed in the infralimbic cortex (-15%) and the paracentral nucleus of the thalamus (-18%). These data suggest that repeated seizures (kindling) modify the GABA-mediated chloride ionophore, and that in some brain areas related to seizure generalization the modifications are very long lasting. INTRODUCTION Kindling is an e x p e r i m e n t a l m o d e l of epilepsy in which the r e p e a t e d focal stimulation of forebrain structures leads to the progressive d e v e l o p m e n t of generalized seizure activity and the eventual a p p e a r a n c e of clonic convulsions 12'28. Interictal changes are also observed, both in learned and u n l e a r n e d behaviors 1'4'7'19'2°'27. The kindling process seems to involve p e r m a n e n t changes in the brain, since generalized convulsions are readily triggered in kindled animals even after several months without stimulation 12'38. The exact nature of the p e r m a n e n t changes underlying kindling, however, remains unclear. A n u m b e r of workers have recently r e p o r t e d longt e r m changes in the 7-aminobutyric acid ( G A B A ) system following the d e v e l o p m e n t of kindled seizures 6. These include Sneddon et al. 3° and Tietz and Chiu 35, who found long-lasting reductions in G A B A - s t i m u l a t e d chloride flux in the brainstem, cortex, h i p p o c a m p u s and cerebellum of kindled rats. These flux changes presumably reflect a kindling-induced alteration of the postsynaptic G A B A A r e c e p t o r / i o n o p h o r e complex 3°. Previous a u t o r a d i o g r a p h i c studies, however, have revealed no lasting changes in radioligand binding to either G A B A A 24'29 or b e n z o d i a z e p i n e 23'29 r e c e p t o r sites in the kindled brain. It seems possible, therefore, that kindling may induce a change in the chloride i o n o p h o r e compo-
nent of the G A B A A complex. This i o n o p h o r e can be r a d i o l a b e l e d with the cage convulsant ligand [35S]t-butylbicyclophosphorothionate ([355]TBPS)31. The present study was designed as an exhaustive examination of [35S]TBPS binding in the kindled rat brain. Binding in 104 brain regions was assessed via quantitative r e c e p t o r a u t o r a d i o g r a p h y in subjects sacrificed either 24 h or 28 days after the last seizure. The site of stimulation (entorhinal cortex), kindling p r o c e d u r e , and times of sacrifice were matched to those previously used in our studies of radioligand binding to the G A B A A and b e n z o d i a z a p i n e r e c e p t o r sites 23'24. MATERIALS AND METHODS
Materials [3sS]TBPS (103-108 Ci/mmol) was obtained from New England Nuclear (Boston, MA). [35S] (as inorganic sulfate in aqueous solution; 684 Ci/mmol at the time of use) was obtained from Amersham (Oakville, Ont.). Other chemicals and supplies were obtained from standard commercial sources. Subjects and kindling procedure Adult male Long Evans rats (Royal Victoria hooded strain, Charles River Canada, Montreal, Quebec), weighing 280-350 g at the time of surgery, served as subjects. Each subject was anesthetized with sodium pentobarbital (65 mg/kg i.p.) and implanted with a single, bipolar stimulating electrode (type MS 303/1, Plastics One, Roanoke, VA) in the right entorhinal cortex. The electrode was positioned using the following stereotaxic coordinates: 5.0 mm posterior to bregma, 4.7 mm right lateral to the midline, and
Correspondence: W.M. Burnham, Department of Pharmacology, Medical Sciences Building, University of Toronto, Toronto, Ont. M5S 1A8, Canada.
168 8.3 mm vertical from the skull surface. Following a two week post-surgical period for recovery and handling, subjects were randomly divided into two groups: 'kindled' and 'control'. Kindled subjects were stimulated once daily with a 2 s train of balanced, biphasic, 60 Hz, 1 ms square-wave pulses, delivered at a standard peak-to-peak intensity of 1000/~A. Stimulation was continued until each subject reached a criterion of at least 6 full-scale ('stage 5') 28 generalized seizures. Subjects in the control group received identical handling, but no stimulation. As each kindled subject reached criterion, it was withdrawn from stimulation along with a matched control for a period of either 24 h or 28 days.
Sectioning of brains At the end of the stimulation-free period, kindled and control subjects were sacrificed by decapitation, and their brains were rapidly removed and frozen on dry ice. Coronal sections of 20/~m thickness were cut at -15°C on a cryostat microtome and thawmounted on subbed, gelatin-coated microscope slides. Every 15th section was mounted, from the frontal pole to the caudal medulla. Slide-mounted sections were then stored at -70°C inside foilwrapped boxes before assay.
[3sS]TBPS binding assay On the day of assay, slide boxes containing kindled and control brain sections were removed from the freezer and brought to room temperature for 45-60 min. The slide-mounted sections were then assayed with [35S]TBPS according to the method of Edgar and Schwartz H. Briefly, brain sections were pre-incubated for 10 min at 22°C in buffer (comprised of 50 mM K2HPO4, 50 mM NaH2PO 4 and 200 mM NaC1, pH 7.4) containing 1 mM EDTA to facilitate removal of endogenous GABA from the tissue aLax. Sections were then transferred to polyethylene slide mailers for a 3 h incubation at 22°C in buffer containing 4 nM [35S]TBPS in the presence or absence of 100 /~M picrotoxin. Non-specific binding was defined as binding in the presence of picrotoxin, and was assayed using 6 representative sections from each brain. The incubation was terminated by two 15 min washes in buffer at 22°C, followed by a brief rinse in distilled water. The sections were then air-dried and apposed to Hyperfilm-fl max (Amersham, Oakville, Ont.) in tungsten cassettes. Sections from each matched pair of kindled and control brains were apposed to film together with a set of methylmethacrylate [~4C]standards, which were appropriately calibrated for [35S]TBPS binding (see below). Films were exposed at room temperature for a period of 3 days before being developed.
Quantitative densitometry [35S]TBPS films were developed in Kodak D-19 developer and fixed in Kodak Rapid Fixer solution. Computer-assisted densitometric analyses were performed with the MCID system (Imaging Research, St. Catharines, Ont.), with a resolution of 256 gray levels per image point. After a [35S] standard curve had been generated for each film, optical density values were automatically converted to [35S] tissue concentrations. Brain regions were anatomically defined according to Paxinos and Watson 26, and were measured by an investigator who was unaware of the individual identities of kindled and control animals (J.N.N.). For all subjects, densitometric analysis indicated that specific binding accounted for 99-100% of the total signal (non-specific binding was virtually non-detectable throughout the brain). In virtually all cases, a 4th degree polynomial function provided the best fit for [3SS] standard curves, with less than 1% error between predicted and observed values within the sampling range. [35S]TBPS binding was measured in a total of 104 brain regions.
Data analysis In each of the 104 brain areas examined, data were first analyzed in terms of right-left differences for bilateral brain structures (unpaired t-tests). If no significant right-left differences were found, right and left values were combined for each region. Subsequently, a 2 × 2 analysis of variance was performed on each area, with kindling treatment and time of sacrifice as factors. Orthogonal ANOVA contrasts were then conducted on areas which showed either a significant kindling effect or a significant interaction between kindling treatment and time of sacrifice.
Verification of electrode placements Since the entire brain was examined for [35S]TBPS binding, it was possible to verify electrode placements directly from autoradiographic images on film. All electrode tips were found to have been located within the right entorhinal cortical region, or in the boundary area between the entorhinal cortex and the most caudal aspect of the ventral subiculum. RESULTS S i n c e n o significant r i g h t - l e f t d i f f e r e n c e s w e r e f o u n d , d a t a f r o m r i g h t a n d left b r a i n a r e a s w e r e c o m b i n e d f o r s u b s e q u e n t analysis. Table I presents means
(+
S.E.M.)
f o r [35S]TBPS
b i n d i n g in e a c h o f t h e 104 b r a i n a r e a s . A t 24 h, k i n d l e d
Calibration of [14C]standards for [35S]TBPS binding
s u b j e c t s s h o w e d significantly l o w e r levels o f b i n d i n g in
A set of [35S]tissue paste standards was prepared and used to calibrate commercial [14C]standards for [35S]TBPS binding. A similar calibration technique has been described by Miller21. Briefly, tissue paste standards were prepared by adding varying amounts of [35S] to whole brain tissue which had been ground to a paste. Twenty-micron sections of frozen paste were subsequently cut at -15°C and thaw-mounted on microscope slides. Additional sections were cut, weighed and counted in a Phillips PW 4700 liquid scintillation counter to determine the concentration of [35S] in each sample. A total of 13 different concentrations was used, ranging from 0.028 to 110 pCi/g tissue. The slide-mounted tissue paste sections were then apposed to Hyperfilm-flmax, together with a set of methyl-methacrylate [14C] standards (American Radiolabeled Chemicals, St. Louis, MO), for a period of 3 days. A standard curve relating tissue paste concentrations of [35S] to optical density values was then plotted, and used to correlate optical densities of [14C] standards to [35S] concentrations. The degree of correlation between actual [aSs] tissue paste concentrations and those predicted from the [14C] standards which were co-exposed to the same film was found to be greater than r = 0.99 (data not shown).
the lateral nucleus of the amygdala (-31% below controls), the infralimbic cortex (-14%) and the paracentral n u c l e u s o f t h e t h a l a m u s ( - 2 2 % ) . A t 28 d a y s , t h e d e c r e a s e in b i n d i n g in t h e l a t e r a l n u c l e u s o f t h e a m y g d a l a o f kind l e d s u b j e c t s w a s n o l o n g e r significant ( - 1 2 % ) , b u t sign i f i c a n t b i n d i n g r e d u c t i o n s w e r e still o b s e r v e d i n t h e inf r a l i m b i c c o r t e x ( - 1 5 % ) a n d t h e p a r a c e n t r a l n u c l e u s of the thalamus (-18%). DISCUSSION T h e p r e s e n t s t u d y r e p r e s e n t s t h e first i n v e s t i g a t i o n o f [35S]TBPS b i n d i n g in t h e b r a i n s o f e l e c t r i c a l l y k i n d l e d s u b j e c t s . S i g n i f i c a n t b i n d i n g r e d u c t i o n s w e r e o b s e r v e d in 3 b r a i n a r e a s 24 h a f t e r e n t o r h i n a l k i n d l i n g , a n d in t w o b r a i n a r e a s 28 d a y s a f t e r e n t o r h i n a l k i n d l i n g . T h e s e d a t a
169 TABLE I [35S]TBPS binding 24 h and 28 days after kindled seizure
Values represent means (S.E.M.) in pmol/gm tissue. 24 Hours
28 Days
Controls (n = 7)
Kindled (n = 7)
Controls (n = 7)
Kindled (n = 7)
12.44 25.47 13.35 15.01 14.56 21.88 22.32 5.80 16.84 8.95 8.95 23.07 11.03 10.22 15.25 7.56 10.56 12.63
(0.82) (1.59) (0.56) (0.47) (1.04) (0.95) (1.53) (0.37) (0.90) (0.85) (0.91) (0.74) (0.83) (0.90) (0.72) (0.58) (0.77) (0.25)
13.10 25.30 12.96 14.72 14.46 21.92 21.31 5.64 12.62 8.36 7.45 21.59 9.32 9.25 14.53 8.59 9.86 12.20
(0.44) (2.26) (0.51) (0.55) (0.53) (0.98) (0.85) (0.25) (1.25) (0.75) (0.64) (1.21) (0.40) (0.44) (0.72) (0.40) (0.96) (0.83)
14.35 25.57 12.12 13.77 13.25 20.91 21.90 5.83 13.56 8.68 8.15 23.73 9.60 7.94 15.37 7.92 10.68 13.71
(0.78) (1.51) (0.77) (0.57) (1.08) (0.95) (1.33) (0.40) (1.33) (0.45) (0.67) (1.65) (0.88) (0.31) (1.07) (0.89) (1.31) (0.98)
15.12 26.09 12.58 13.78 14.75 20.97 23.52 6.79 14.64 9.33 9.94 24.65 8.88 8.84 15.27 8.04 10.64 12.64
(0.96) (1.58) (0.37) (0.30) (0.54) (1.46) (0.91) (0.86) (0.99) (0.70) (1.18) (1.42) (1.24) (0.29) (0.60) (0.45) (0.31) (0.97)
26.71 22.52 21.28 14.35 52.17 36.19 59.04 11.45 14.28 23.15 16.60 22.38
(1.36) (0.95) (1.09) (0.94) (4.79) (3.24) (6.89) (0.68) (0.76) (1.73) (1.03) (0.44)
26.93 22.83 21.60 13.23 58.96 30.34 52.56 11.34 13.23 24.72 16.13 22.44
(0.72) (1.55) (0.63) (0.96) (4.73) (1.06) (5.50) (0.58) (0.84) (1.34) (1.77) (0.38)
27.19 24.35 20.53 11.48 63.20 34.05 59.51 10.55 13.18 22.64 16.38 21.45
(1.05) (1.87) (1.11) (0.87) (5.05) (4.04) (3.23) (0.78) (0.72) (1.70) (1.02) (0.70)
27.67 26.30 21.11 13.86 57.50 29.67 60.25 11.44 13.67 23.64 15.65 21.42
(1.41) (2.01) (0.57) (0.78) (5.78) (1.44) (6.00) (0.44) (0.90) (1.17) (0.97) (0.76)
HINDBRAIN Cerebellum Granular 1. Molecular 1. Interpositus n. Lateral n. Medial n. Cuneiform n. Dorsal tegmental n. Gigantocellular n. Hypoglossal n. Inferior olive Inferior olive dorsal n. Laterodorsal tegmental n. Lateral reticular n. Raphe pallidus n. n. Solitary tract Spinal trigeminal n. Spinal trigeminal motor n. Vestibular n. MIDBRAIN Anterior pretectal area Central gray Deep mesencephalic n. Interpeduncular n. Inferior colliculus Mammillary n. Parabigeminal n. Pontine n. Pontine reticular n. Raphe dorsalis Raphe medialis Red n. Substantia nigra Pars compacta Pars reticulata Superior colliculus Deep 1. Superficial 1. Ventral tegmental area Ventral tegmental n.
21.33 (1.54) 36.06 (1.83)
20.25 (0.94) 37.57 (2.12)
18.87 (0.72) 34.80 (0.58)
21.92 (1.78) 32.04 (1.01)
34.40 38.41 14.04 24.91
33.98 39.94 14.52 21.80
33.88 38.50 13.78 20.44
34.44 37.30 14.66 23.07
(2.36) (1.59) (0.52) (1.75)
(0.96) (2.18) (0.43) (0.86)
(1.23) (1.36) (0.97) (1.27)
(1.71) (1.89) (0.57) (1.20)
FOREBRAIN n. Accumbens Amygdala Basolateral n. Central n. Lateral n. Medial n. Bed n. stria terminalis Caudate-putamen Dorsolateral Dorsomedial Ventrolateral Claustrum
20.50 (0.89)
19.03 (0.59)
21.23 (0.90)
20.52 (1.42)
24.56 26.75 31.29 28.70 19.71
(1.53) (1.01) (1.73) (1.33) (1.25)
21.14 25.68 21.60 25.75 19.38
(1.15) (1.03) (1.42)*** (1.71) (1.57)
26.39 (1.14) 27.52 (1.86) 33.85 (1.41) 26.60(1.26) 20.45 (0.95)
23.86 24.77 29.68 27.48 20.46
(1.19) (0.90) (1.56) (2.46) (1.65)
15.15 12.96 19.19 32.43
(0.68) (0.63) (0.60) (2.57)
16.28 13.60 19.73 26.75
(0.49) (0.63) (0.33) (2.37)
16.25 13.22 21.06 40.33
16.13 13.01 19.77 36.36
(0.74) (0.60) (0.91) (1.26)
(0.76) (0.91) (0.87) (2.48)
(to be continued on p. 170)
170 TABLE I (continued) 24 Hours
Cerebral cortex Cingulate anterior Cingulate posterior Entorhinal Entorhinal layer I Frontal Infralimbic Occipital Pyriform SS Parietal Temporal Diagonal band horizontal Entopeduncular n. Globus pallidus Habenula lateral n. Habenula medial n. Hippocampus CA1 lac.-mol. I. oriens 1. radiatum I. CA3 lac.-mol. 1. oriens 1. radiatum 1. Dent. gyrus inf. blade Dent. gyrus sup. blade Dent. gyrus polym. 1. Subiculum Hypothalamus Anterior area Dorsomedial n. Lateral area Posterior area Paraventricular n. Ventromedial n. Islands of Calleja Olfactory tubercle Preoptic area lateral Preoptic area medial Septum lateral Septum medial Thalamus Anterodorsal n. Anteroventral n. Central lateral n. Central medial n. Laterodorsal n. Lateral geniculate n. Lateral posterior n. Mediodorsal n. Medial geniculate n. Paracentral n. Parafascicular n. Paratenial n. Posterior n. Reticular n. Subparafascicular n. Ventrolateral n. Ventromedial n. Ventroposterior n. Ventral pallidum Zona incerta Zona incerta ventral
28 Days
Con~o~ (n = 7)
Kind&d (n = 7)
Con~o~ (n = 7)
Kind~d (n = 7)
46.28 37.85 31.97 37.97 38.84 36.66 42.34 37.77 58.61 35.07 51.36 17.46 28.89 17.84 8.94
(2.16) (2.57) (2.34) (2.41) (1.26) (1.84) (2.73) (2.06) (3.63) (2.60) (2.56) (1.03) (1.55) (0.63) (1.08)
40.72 37.48 31.98 34.83 35.59 31.64 46.26 34.57 49.18 28.14 54.03 16.68 27.34 20.03 11.21
(3.12) (2.47) (2.43) (1.24) (1.10) (1.42)* (2.91) (2.86) (2.92) (1.15) (4.93) (1.24) (0.69) (0.69) (0.74)
41.02 32.92 36.53 37.40 46.77 42.79 41.39 40.91 54.67 35.10 49.75 18.86 28.97 16.68 10.27
(1.71) (0.98) (2.10) (2.44) (2.56) (2.27) (3.22) (2.60) (4.23) (1.40) (3.76) (1.19) (0.78) (0.90) (1.56)
41.54 33.65 35.85 42.15 44.94 36.40 41.08 36.77 52.07 35.74 49.31 15.61 27.93 17.64 10.64
(3.56) (2.78) (1.60) (2.33) (3.09) (1.41)* (2.53) (1.73) (3.36) (1.65) (3.58) (2.71) (1.28) (0.88) (2.14)
51.49 48.12 38.35 45.24 28.97 30.76 39.20 56.86 17.23 37.00
(2.16) (2.20) (1.02) (1.80) (0.59) (0.73) (1.97) (3.39) (0.73) (1.23)
51.77 48.02 36.93 43.06 27.01 28.98 41.50 64.43 18.62 35.12
(3.24) (2.96) (1.47) (2.20) (0.82) (0.93) (2.93) (4.68) (0.78) (1.98)
42.76 39.59 32.59 38.69 24.93 27.01 33.27 46.14 15.90 31.60
(1.33) (1.44) (0.81) (0.89) (0.49) (0.62) (1.50) (2.69) (0.50) (0.69)
41.87 38.34 30.93 37.06 24.40 25.72 34.79 48.81 16.83 30.52
(1.56) (1.18) (0.88) (1.12) (0.74) (0.75) (1.53) (1.92) (0.59) (1.16)
23.28 19.18 22.70 22.79 13.89 18.63 59.22 20.55 24.57 21.22 14.74 39.86
(1.57) (1.52) (0.40) (1.65) (0.57) (0.98) (6.88) (1.30) (0.99) (0.94) (0.99) (3.38)
20.49 18.79 23.30 21.51 13.30 17.11 53.04 18.15 24.63 18.60 14.56 38.78
(0.80) (1.34) (1.15) (1.32) (0.47) (0.67) (4.63) (0.81) (1.44) (0.84) (0.78) (4.33)
22.28 16.74 22.09 20.89 14.34 18.26 50.46 19.10 25.50 20.75 14.68 41.22
(1.00) (0.79) (0.88) (0.59) (0.51) (1.02) (4.34) (1.30) (1.77) (1.13) (0.46) (2.90)
22.01 16.57 21.30 19.03 13.26 17.52 53.07 20.54 22.65 19.77 14.07 41.02
(1.24) (0.86) (0.67) (1.01) (0.32) (1.22) (3.83) (2.91) (0.91) (0.92) (0.84) (4.36)
37.61 26.75 31.19 32.90 23.73 25.56 23.90 24.04 34.29 49.91 22.84 41.25 20.04 19.88 25.48 26.40 19.11 21.68 42.32 22.88 43.07
(4.63) (1.23) (2.08) (1.77) (1.25) (1.11) (0.64) (1.04) (1.27) (3.56) (1.22) (3.18) (0.52) (0.25) (1.80) (0.94) (0.54) (1.21) (3.32) (1.06) (5.14)
32.11 27.95 27.00 28.60 24.90 23.81 24.78 21.38 32.93 39.03 21.95 37.17 18.18 17.92 22.38 22.96 19.25 19.27 41.46 22.97 39.04
(1.54) (2.25) (1.10) (0.94) (1.09) (1.15) (1.07) (0.78) (1.03) (1.49)** (0.84) (2.16) (0.72) (0.78) (0.86) (0.60) (0.77) (1.02) (5.31) (0.73) (1.57)
40.70 29.34 31.75 30.79 23.30 26.60 26.44 23.46 36.01 47.56 22.41 41.19 21.43 19.57 26.38 26.34 19.10 24.81 45.59 21.77 44.77
(3.88) (2.51) (2.35) (1.29) (0.78) (1.63) (1.22) (1.24) (1.44) (2.61) (1.18) (1.84) (1.07) (0.66) (2.16) (1.73) (1.42) (1.63) (2.41) (0.88) (2.49)
32.96 26.14 30.13 31.29 22.84 26.94 27.94 23.04 34.98 38.79 23.77 41.34 21.53 20.34 27.43 24.57 19.66 23.38 43.84 20.94 39.80
(2.93) (2.08) (0.94) (1.86) (0.91) (1.42) (1.46) (0.80) (1.10) (1.66)* (1.39) (3.75) (0.71) (0.66) (0.52) (1.62) (1.20) (1.14) (3.79) (0.83) (2.04)
* P < 0.05, ** P < 0.01, *** P < 0.001, ANOVA contrasts kindled vs control subjects for each time period.
171
indicate that electrical kindling modifies the G A B A related chloride ionophore, and that, in some brain areas, the modifications are very long lasting. They add to a growing collection of studies which have reported longlasting GABAergic changes following the development of electrically kindled seizures 6, and complement and extend recent reports of deficits in [3sS]TBPS binding following chemical kindling 9,~s. How do these changes relate to the kindling effect? Since kindling is permanent, only long-lasting alterations, observed 28 days or more after the last seizure, can contribute directly to the 'kindled state '5. One area of the brain which showed a long-term reduction in TBPS binding was the infralimbic cortex, which is located in the medial prefrontal cortical region. This region has efferent connections to the amygdala 8, the thalamus 17'36, and the brainstem 17'34'36. Crawley and co-workers 1°'33 have shown that the microinjection of a cholinergic agonist into this region can induce motor seizures which resemble those characteristic of limbic kindling. Furthermore, there is clinical evidence to suggest that the prefrontal cortex may play a primary role in the elicitation of generalized epileptic seizures in human patients 2,14. A second structure that exhibited a persistent reduction in TBPS binding was the paracentral nucleus of the thalamus. The paracentral nucleus is one of the intralaminar thalamic nuclei which project to the striatum and the frontal agranular neocortex 16. It has been suggested that these nuclei play an important role in seizure generalization 37 and/or regulation 22. Thus, the two brain areas which exhibited long-lasting changes in binding in the present study are regions thought to be involved in the neural circuitry underlying seizure generalization. Further studies will be required to determine the functional significance of these changes, and whether they play a role in the development of kindled seizures or the interictal behavioral abnormalities associated with kindling. It is possible, for instance, that bilateral injections of G A B A ionophore blockers (e.g. picrotoxin) into the infralimbic cortex and/or paracentral nucleus might accelerate the progress of kindling. Studies designed to test this possibility are currently in progress, as well as studies designed to determine
REFERENCES 1 Adamec, R.E., Behavioral and epileptic determinants of predatory attack behavior in the cat, Can. J. Neurol. Sci., 2 (1975) 457-466. 2 Bancaud, J., Talairach, J., Morel, P., Bresson, M., Bonis, A., Geier, S., Hereon, E. and Buser, P., 'Generalized' epileptic seizures elicited by electrical stimulation of the frontal lobe in man, Electroencheph. Clin. Neurophysiol., 37 (1974) 275-282.
whether the observed binding changes are due to alterations in binding affinity (Kd) or number of binding sites (Bmax). It is interesting to note that the pattern of regional changes observed in the present study was not the same as the pattern of changes reported in previous studies of binding to the two other components of the GABAA receptor complex 23'24'29. In those studies, the only alterations that were observed following kindling were shortterm binding increases restricted to the dentate gyrus. It is conceivable that kindling modifies the G A B A ionophore while sparing the extracellular sites related to G A B A and benzodiazepine binding. Such a selective effect might result from a perturbation of membrane phospholipids, which are believed to stabilize TBPS binding sites on the ionophore (cf. Refs. 15 and 32). It could also result from changes in phosphorylation processes affecting the ionophore (cf. Refs. 3, 13, 25 and 39). The fact that the TBPS binding decreases observed in the present study are not accompanied by changes in G A B A or benzodiazapine receptor binding suggests that these decreases are not likely to have been caused by a loss of postsynaptic cells. The current data do not provide a clear mechanism for the reductions in GABA-stimulated chloride flux which have been observed in the brainstem one month after kindling 3° and in the cortex, hippocampus and cerebellum one week after kindling 35. A clarification of the precise biochemical correlates of these chloride flux changes thus must await further investigation. In addition to the significant changes discussed above, there seemed to be a general trend toward decreased binding in the forebrain and increased binding in the hindbrain at 28 days. It might be fruitful to re-examine the effects of kindling on these areas in future studies involving the administration of increased numbers of seizures.
Acknowledgements. This study was supported by Grant MA 5611 from the Medical Research Council of Canada. J.S.P. was supported by a University of Toronto Open Fellowship. S.J.K. is a Career Scientist of the Ontario Ministry of Health. These data were presented in preliminary form at the 1990 Meeting of the Society for Neuroscience (St. Louis, MO).
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graphic analysis of benzodiazepine binding in entorhinal-kindled rat brains, Brain Research, 498 (1989) 315-322. Nobrega, J.N., Kish, S.J. and Burnham, W.M., Regional brain [3H]muscimol binding in kindled rat brain: a quantitative autoradiographic examination, Epilepsy Res., 6 (1990) 102-109. Patel, J., Marangos, P.J., Contel, N., Gardner, G. and Post, R.M., Increased phosphorylation of a membrane protein consequent to amygdaloid kindling, J. Neurochem., 43 (1984) 169173. Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, 2nd edn., Academic Press, Sydney, 1986. Pinel, J.P.J., Treit, D. and Rovner, L.I., Temporal lobe aggression in rats, Science, 197 (1977) 1088-1089. Racine, R.J., Modification of seizure activity by electrical stimulation. II. Motor seizure, Electroenceph. Clin. Neurophysiol., 32 (1972) 281-294. Shin, C., Pedersen, H.B. and McNamara, J.O., Gammaaminobutyric acid and benzodiazepine receptors in the kindling model of epilepsy: a quantitative radiohistochemical study, J. Neurosci., 5 (1985) 2696-2701. Sneddon, W.B., Burnham, W.M., Prowse, S., Cheng, J. and Kish, S.J., Reduced GABA-mediated chloride flux in entorhinal cortex-kindled rat brains, Epilepsy Res., 6 (1990) 18-22. Squires, R.F., Casida, J.E., Richardson, M. and Saederup, E., [35S]t-Butylbicyclophosphorothionate binds with high affinity to brain-specific sites coupled to gamma-aminobutyric acid-A and ion recognition sites, Mol. Pharmacol., 23 (1983) 326-336. Stephenson, EA., Mamalaki, C., Casalotti, S.O. and Barnard, E.A., The GABA A receptor and its antibodies, Biochem. Soc. Syrup., 52 (1986) 33-40. Stivers, J.A., Skirboll, L.R., Long, R. and Crawley, J.N., Anatomical analysis of frontal cortex sites at which carbachol induces motor seizures in the rat, Pharmacol. Biochem. Behav., 30 (1988) 129-136. Terreberry, R.R. and Neafsey, E.J., Rat medial frontal cortex: a visceral motor region with a direct projection to the solitary nucleus, Brain Research, 278 (1983) 245-249. Tietz, E.I. and Chiu, T.H., Regional GABA-stimulated chloride uptake in amygdala kindled rats, Neurosci. Lea., 123 (1991) 269-272. Van der Kooy, D., Koda, L.Y., McGinty, J.F., Gerfen, C.R. and Bloom, EE., The organization of projections from the cortex, amygdala, and hypothalamus to the nucleus of the solitary tract in rat, J. Comp. Neurol., 224 (1984) 1-24. Velasco, M., Velasco, E, Velasco, A.L., Luj~ln, M. and del Mercado, J.V., Epileptiform EEG activities of the centromedian thalamic nuclei in patients with intractable partial motor, complex partial, and generalized seizures, Epilepsia, 30 (1989) 295306. Wada, J.A., Sato, M. and Corcoran, M.E., Persistent seizure susceptibility and recurrent spontaneous seizures in kindled cats, Epilepsia, 15 (1974) 465-478. Wu, K., Wasterlain, C., Sachs, L. and Siekevitz, P., Effect of septal kindling on glutamate binding and calcium/calmodulindependent phosphorylation in a postsynaptic density fraction isolated from rat cerebral cortex, Proc. Natl. Acad. Sci. U.S.A., 87 (1990) 5298-5302.
167
BRES 17367
Autoradiographic analysis of [35S]TBPS binding in entorhinal cortex-kindled rat brains John S. Petrasek 1, Jos6 N. Nobrega 2, Stephen J. Kish 1'2 and W. Mclntyre Burnham 1 1Department of Pharmacology, University of Toronto, Toronto, Ont. (Canada) and 2Clarke Institute of Psychiatry, Toronto, Ont. (Canada) (Accepted 10 September 1991)
Key words: Kindling; Seizures; Quantitative autoradiography; y-Aminobutyric acidA receptor; Chloride ionophore; t-Butylbicyclophosphorothionate
A quantitative autoradiographic analysis of [35S]t-butylbicyclophosphorothionate (TBPS) binding to the 7-aminobutyric acid (GABA)-mediated chloride ionophore was carded out in 104 brain areas of entorhinal cortex-kindled and control rats. Subjects were sacrificed either 24 h or 28 days after the last kindled seizure. Kindled subjects in the 24 h group showed reductions in mean [35S]TBPS binding in the lateral nucleus of the amygdala (-31%), the infralimbic cortex (-14%), and the paracentral nucleus of the thalamus (-22%). At 28 days, reductions in binding were observed in the infralimbic cortex (-15%) and the paracentral nucleus of the thalamus (-18%). These data suggest that repeated seizures (kindling) modify the GABA-mediated chloride ionophore, and that in some brain areas related to seizure generalization the modifications are very long lasting. INTRODUCTION Kindling is an e x p e r i m e n t a l m o d e l of epilepsy in which the r e p e a t e d focal stimulation of forebrain structures leads to the progressive d e v e l o p m e n t of generalized seizure activity and the eventual a p p e a r a n c e of clonic convulsions 12'28. Interictal changes are also observed, both in learned and u n l e a r n e d behaviors 1'4'7'19'2°'27. The kindling process seems to involve p e r m a n e n t changes in the brain, since generalized convulsions are readily triggered in kindled animals even after several months without stimulation 12'38. The exact nature of the p e r m a n e n t changes underlying kindling, however, remains unclear. A n u m b e r of workers have recently r e p o r t e d longt e r m changes in the 7-aminobutyric acid ( G A B A ) system following the d e v e l o p m e n t of kindled seizures 6. These include Sneddon et al. 3° and Tietz and Chiu 35, who found long-lasting reductions in G A B A - s t i m u l a t e d chloride flux in the brainstem, cortex, h i p p o c a m p u s and cerebellum of kindled rats. These flux changes presumably reflect a kindling-induced alteration of the postsynaptic G A B A A r e c e p t o r / i o n o p h o r e complex 3°. Previous a u t o r a d i o g r a p h i c studies, however, have revealed no lasting changes in radioligand binding to either G A B A A 24'29 or b e n z o d i a z e p i n e 23'29 r e c e p t o r sites in the kindled brain. It seems possible, therefore, that kindling may induce a change in the chloride i o n o p h o r e compo-
nent of the G A B A A complex. This i o n o p h o r e can be r a d i o l a b e l e d with the cage convulsant ligand [35S]t-butylbicyclophosphorothionate ([355]TBPS)31. The present study was designed as an exhaustive examination of [35S]TBPS binding in the kindled rat brain. Binding in 104 brain regions was assessed via quantitative r e c e p t o r a u t o r a d i o g r a p h y in subjects sacrificed either 24 h or 28 days after the last seizure. The site of stimulation (entorhinal cortex), kindling p r o c e d u r e , and times of sacrifice were matched to those previously used in our studies of radioligand binding to the G A B A A and b e n z o d i a z a p i n e r e c e p t o r sites 23'24. MATERIALS AND METHODS
Materials [3sS]TBPS (103-108 Ci/mmol) was obtained from New England Nuclear (Boston, MA). [35S] (as inorganic sulfate in aqueous solution; 684 Ci/mmol at the time of use) was obtained from Amersham (Oakville, Ont.). Other chemicals and supplies were obtained from standard commercial sources. Subjects and kindling procedure Adult male Long Evans rats (Royal Victoria hooded strain, Charles River Canada, Montreal, Quebec), weighing 280-350 g at the time of surgery, served as subjects. Each subject was anesthetized with sodium pentobarbital (65 mg/kg i.p.) and implanted with a single, bipolar stimulating electrode (type MS 303/1, Plastics One, Roanoke, VA) in the right entorhinal cortex. The electrode was positioned using the following stereotaxic coordinates: 5.0 mm posterior to bregma, 4.7 mm right lateral to the midline, and
Correspondence: W.M. Burnham, Department of Pharmacology, Medical Sciences Building, University of Toronto, Toronto, Ont. M5S 1A8, Canada.
168 8.3 mm vertical from the skull surface. Following a two week post-surgical period for recovery and handling, subjects were randomly divided into two groups: 'kindled' and 'control'. Kindled subjects were stimulated once daily with a 2 s train of balanced, biphasic, 60 Hz, 1 ms square-wave pulses, delivered at a standard peak-to-peak intensity of 1000/~A. Stimulation was continued until each subject reached a criterion of at least 6 full-scale ('stage 5') 28 generalized seizures. Subjects in the control group received identical handling, but no stimulation. As each kindled subject reached criterion, it was withdrawn from stimulation along with a matched control for a period of either 24 h or 28 days.
Sectioning of brains At the end of the stimulation-free period, kindled and control subjects were sacrificed by decapitation, and their brains were rapidly removed and frozen on dry ice. Coronal sections of 20/~m thickness were cut at -15°C on a cryostat microtome and thawmounted on subbed, gelatin-coated microscope slides. Every 15th section was mounted, from the frontal pole to the caudal medulla. Slide-mounted sections were then stored at -70°C inside foilwrapped boxes before assay.
[3sS]TBPS binding assay On the day of assay, slide boxes containing kindled and control brain sections were removed from the freezer and brought to room temperature for 45-60 min. The slide-mounted sections were then assayed with [35S]TBPS according to the method of Edgar and Schwartz H. Briefly, brain sections were pre-incubated for 10 min at 22°C in buffer (comprised of 50 mM K2HPO4, 50 mM NaH2PO 4 and 200 mM NaC1, pH 7.4) containing 1 mM EDTA to facilitate removal of endogenous GABA from the tissue aLax. Sections were then transferred to polyethylene slide mailers for a 3 h incubation at 22°C in buffer containing 4 nM [35S]TBPS in the presence or absence of 100 /~M picrotoxin. Non-specific binding was defined as binding in the presence of picrotoxin, and was assayed using 6 representative sections from each brain. The incubation was terminated by two 15 min washes in buffer at 22°C, followed by a brief rinse in distilled water. The sections were then air-dried and apposed to Hyperfilm-fl max (Amersham, Oakville, Ont.) in tungsten cassettes. Sections from each matched pair of kindled and control brains were apposed to film together with a set of methylmethacrylate [~4C]standards, which were appropriately calibrated for [35S]TBPS binding (see below). Films were exposed at room temperature for a period of 3 days before being developed.
Quantitative densitometry [35S]TBPS films were developed in Kodak D-19 developer and fixed in Kodak Rapid Fixer solution. Computer-assisted densitometric analyses were performed with the MCID system (Imaging Research, St. Catharines, Ont.), with a resolution of 256 gray levels per image point. After a [35S] standard curve had been generated for each film, optical density values were automatically converted to [35S] tissue concentrations. Brain regions were anatomically defined according to Paxinos and Watson 26, and were measured by an investigator who was unaware of the individual identities of kindled and control animals (J.N.N.). For all subjects, densitometric analysis indicated that specific binding accounted for 99-100% of the total signal (non-specific binding was virtually non-detectable throughout the brain). In virtually all cases, a 4th degree polynomial function provided the best fit for [3SS] standard curves, with less than 1% error between predicted and observed values within the sampling range. [35S]TBPS binding was measured in a total of 104 brain regions.
Data analysis In each of the 104 brain areas examined, data were first analyzed in terms of right-left differences for bilateral brain structures (unpaired t-tests). If no significant right-left differences were found, right and left values were combined for each region. Subsequently, a 2 × 2 analysis of variance was performed on each area, with kindling treatment and time of sacrifice as factors. Orthogonal ANOVA contrasts were then conducted on areas which showed either a significant kindling effect or a significant interaction between kindling treatment and time of sacrifice.
Verification of electrode placements Since the entire brain was examined for [35S]TBPS binding, it was possible to verify electrode placements directly from autoradiographic images on film. All electrode tips were found to have been located within the right entorhinal cortical region, or in the boundary area between the entorhinal cortex and the most caudal aspect of the ventral subiculum. RESULTS S i n c e n o significant r i g h t - l e f t d i f f e r e n c e s w e r e f o u n d , d a t a f r o m r i g h t a n d left b r a i n a r e a s w e r e c o m b i n e d f o r s u b s e q u e n t analysis. Table I presents means
(+
S.E.M.)
f o r [35S]TBPS
b i n d i n g in e a c h o f t h e 104 b r a i n a r e a s . A t 24 h, k i n d l e d
Calibration of [14C]standards for [35S]TBPS binding
s u b j e c t s s h o w e d significantly l o w e r levels o f b i n d i n g in
A set of [35S]tissue paste standards was prepared and used to calibrate commercial [14C]standards for [35S]TBPS binding. A similar calibration technique has been described by Miller21. Briefly, tissue paste standards were prepared by adding varying amounts of [35S] to whole brain tissue which had been ground to a paste. Twenty-micron sections of frozen paste were subsequently cut at -15°C and thaw-mounted on microscope slides. Additional sections were cut, weighed and counted in a Phillips PW 4700 liquid scintillation counter to determine the concentration of [35S] in each sample. A total of 13 different concentrations was used, ranging from 0.028 to 110 pCi/g tissue. The slide-mounted tissue paste sections were then apposed to Hyperfilm-flmax, together with a set of methyl-methacrylate [14C] standards (American Radiolabeled Chemicals, St. Louis, MO), for a period of 3 days. A standard curve relating tissue paste concentrations of [35S] to optical density values was then plotted, and used to correlate optical densities of [14C] standards to [35S] concentrations. The degree of correlation between actual [aSs] tissue paste concentrations and those predicted from the [14C] standards which were co-exposed to the same film was found to be greater than r = 0.99 (data not shown).
the lateral nucleus of the amygdala (-31% below controls), the infralimbic cortex (-14%) and the paracentral n u c l e u s o f t h e t h a l a m u s ( - 2 2 % ) . A t 28 d a y s , t h e d e c r e a s e in b i n d i n g in t h e l a t e r a l n u c l e u s o f t h e a m y g d a l a o f kind l e d s u b j e c t s w a s n o l o n g e r significant ( - 1 2 % ) , b u t sign i f i c a n t b i n d i n g r e d u c t i o n s w e r e still o b s e r v e d i n t h e inf r a l i m b i c c o r t e x ( - 1 5 % ) a n d t h e p a r a c e n t r a l n u c l e u s of the thalamus (-18%). DISCUSSION T h e p r e s e n t s t u d y r e p r e s e n t s t h e first i n v e s t i g a t i o n o f [35S]TBPS b i n d i n g in t h e b r a i n s o f e l e c t r i c a l l y k i n d l e d s u b j e c t s . S i g n i f i c a n t b i n d i n g r e d u c t i o n s w e r e o b s e r v e d in 3 b r a i n a r e a s 24 h a f t e r e n t o r h i n a l k i n d l i n g , a n d in t w o b r a i n a r e a s 28 d a y s a f t e r e n t o r h i n a l k i n d l i n g . T h e s e d a t a
169 TABLE I [35S]TBPS binding 24 h and 28 days after kindled seizure
Values represent means (S.E.M.) in pmol/gm tissue. 24 Hours
28 Days
Controls (n = 7)
Kindled (n = 7)
Controls (n = 7)
Kindled (n = 7)
12.44 25.47 13.35 15.01 14.56 21.88 22.32 5.80 16.84 8.95 8.95 23.07 11.03 10.22 15.25 7.56 10.56 12.63
(0.82) (1.59) (0.56) (0.47) (1.04) (0.95) (1.53) (0.37) (0.90) (0.85) (0.91) (0.74) (0.83) (0.90) (0.72) (0.58) (0.77) (0.25)
13.10 25.30 12.96 14.72 14.46 21.92 21.31 5.64 12.62 8.36 7.45 21.59 9.32 9.25 14.53 8.59 9.86 12.20
(0.44) (2.26) (0.51) (0.55) (0.53) (0.98) (0.85) (0.25) (1.25) (0.75) (0.64) (1.21) (0.40) (0.44) (0.72) (0.40) (0.96) (0.83)
14.35 25.57 12.12 13.77 13.25 20.91 21.90 5.83 13.56 8.68 8.15 23.73 9.60 7.94 15.37 7.92 10.68 13.71
(0.78) (1.51) (0.77) (0.57) (1.08) (0.95) (1.33) (0.40) (1.33) (0.45) (0.67) (1.65) (0.88) (0.31) (1.07) (0.89) (1.31) (0.98)
15.12 26.09 12.58 13.78 14.75 20.97 23.52 6.79 14.64 9.33 9.94 24.65 8.88 8.84 15.27 8.04 10.64 12.64
(0.96) (1.58) (0.37) (0.30) (0.54) (1.46) (0.91) (0.86) (0.99) (0.70) (1.18) (1.42) (1.24) (0.29) (0.60) (0.45) (0.31) (0.97)
26.71 22.52 21.28 14.35 52.17 36.19 59.04 11.45 14.28 23.15 16.60 22.38
(1.36) (0.95) (1.09) (0.94) (4.79) (3.24) (6.89) (0.68) (0.76) (1.73) (1.03) (0.44)
26.93 22.83 21.60 13.23 58.96 30.34 52.56 11.34 13.23 24.72 16.13 22.44
(0.72) (1.55) (0.63) (0.96) (4.73) (1.06) (5.50) (0.58) (0.84) (1.34) (1.77) (0.38)
27.19 24.35 20.53 11.48 63.20 34.05 59.51 10.55 13.18 22.64 16.38 21.45
(1.05) (1.87) (1.11) (0.87) (5.05) (4.04) (3.23) (0.78) (0.72) (1.70) (1.02) (0.70)
27.67 26.30 21.11 13.86 57.50 29.67 60.25 11.44 13.67 23.64 15.65 21.42
(1.41) (2.01) (0.57) (0.78) (5.78) (1.44) (6.00) (0.44) (0.90) (1.17) (0.97) (0.76)
HINDBRAIN Cerebellum Granular 1. Molecular 1. Interpositus n. Lateral n. Medial n. Cuneiform n. Dorsal tegmental n. Gigantocellular n. Hypoglossal n. Inferior olive Inferior olive dorsal n. Laterodorsal tegmental n. Lateral reticular n. Raphe pallidus n. n. Solitary tract Spinal trigeminal n. Spinal trigeminal motor n. Vestibular n. MIDBRAIN Anterior pretectal area Central gray Deep mesencephalic n. Interpeduncular n. Inferior colliculus Mammillary n. Parabigeminal n. Pontine n. Pontine reticular n. Raphe dorsalis Raphe medialis Red n. Substantia nigra Pars compacta Pars reticulata Superior colliculus Deep 1. Superficial 1. Ventral tegmental area Ventral tegmental n.
21.33 (1.54) 36.06 (1.83)
20.25 (0.94) 37.57 (2.12)
18.87 (0.72) 34.80 (0.58)
21.92 (1.78) 32.04 (1.01)
34.40 38.41 14.04 24.91
33.98 39.94 14.52 21.80
33.88 38.50 13.78 20.44
34.44 37.30 14.66 23.07
(2.36) (1.59) (0.52) (1.75)
(0.96) (2.18) (0.43) (0.86)
(1.23) (1.36) (0.97) (1.27)
(1.71) (1.89) (0.57) (1.20)
FOREBRAIN n. Accumbens Amygdala Basolateral n. Central n. Lateral n. Medial n. Bed n. stria terminalis Caudate-putamen Dorsolateral Dorsomedial Ventrolateral Claustrum
20.50 (0.89)
19.03 (0.59)
21.23 (0.90)
20.52 (1.42)
24.56 26.75 31.29 28.70 19.71
(1.53) (1.01) (1.73) (1.33) (1.25)
21.14 25.68 21.60 25.75 19.38
(1.15) (1.03) (1.42)*** (1.71) (1.57)
26.39 (1.14) 27.52 (1.86) 33.85 (1.41) 26.60(1.26) 20.45 (0.95)
23.86 24.77 29.68 27.48 20.46
(1.19) (0.90) (1.56) (2.46) (1.65)
15.15 12.96 19.19 32.43
(0.68) (0.63) (0.60) (2.57)
16.28 13.60 19.73 26.75
(0.49) (0.63) (0.33) (2.37)
16.25 13.22 21.06 40.33
16.13 13.01 19.77 36.36
(0.74) (0.60) (0.91) (1.26)
(0.76) (0.91) (0.87) (2.48)
(to be continued on p. 170)
170 TABLE I (continued) 24 Hours
Cerebral cortex Cingulate anterior Cingulate posterior Entorhinal Entorhinal layer I Frontal Infralimbic Occipital Pyriform SS Parietal Temporal Diagonal band horizontal Entopeduncular n. Globus pallidus Habenula lateral n. Habenula medial n. Hippocampus CA1 lac.-mol. I. oriens 1. radiatum I. CA3 lac.-mol. 1. oriens 1. radiatum 1. Dent. gyrus inf. blade Dent. gyrus sup. blade Dent. gyrus polym. 1. Subiculum Hypothalamus Anterior area Dorsomedial n. Lateral area Posterior area Paraventricular n. Ventromedial n. Islands of Calleja Olfactory tubercle Preoptic area lateral Preoptic area medial Septum lateral Septum medial Thalamus Anterodorsal n. Anteroventral n. Central lateral n. Central medial n. Laterodorsal n. Lateral geniculate n. Lateral posterior n. Mediodorsal n. Medial geniculate n. Paracentral n. Parafascicular n. Paratenial n. Posterior n. Reticular n. Subparafascicular n. Ventrolateral n. Ventromedial n. Ventroposterior n. Ventral pallidum Zona incerta Zona incerta ventral
28 Days
Con~o~ (n = 7)
Kind&d (n = 7)
Con~o~ (n = 7)
Kind~d (n = 7)
46.28 37.85 31.97 37.97 38.84 36.66 42.34 37.77 58.61 35.07 51.36 17.46 28.89 17.84 8.94
(2.16) (2.57) (2.34) (2.41) (1.26) (1.84) (2.73) (2.06) (3.63) (2.60) (2.56) (1.03) (1.55) (0.63) (1.08)
40.72 37.48 31.98 34.83 35.59 31.64 46.26 34.57 49.18 28.14 54.03 16.68 27.34 20.03 11.21
(3.12) (2.47) (2.43) (1.24) (1.10) (1.42)* (2.91) (2.86) (2.92) (1.15) (4.93) (1.24) (0.69) (0.69) (0.74)
41.02 32.92 36.53 37.40 46.77 42.79 41.39 40.91 54.67 35.10 49.75 18.86 28.97 16.68 10.27
(1.71) (0.98) (2.10) (2.44) (2.56) (2.27) (3.22) (2.60) (4.23) (1.40) (3.76) (1.19) (0.78) (0.90) (1.56)
41.54 33.65 35.85 42.15 44.94 36.40 41.08 36.77 52.07 35.74 49.31 15.61 27.93 17.64 10.64
(3.56) (2.78) (1.60) (2.33) (3.09) (1.41)* (2.53) (1.73) (3.36) (1.65) (3.58) (2.71) (1.28) (0.88) (2.14)
51.49 48.12 38.35 45.24 28.97 30.76 39.20 56.86 17.23 37.00
(2.16) (2.20) (1.02) (1.80) (0.59) (0.73) (1.97) (3.39) (0.73) (1.23)
51.77 48.02 36.93 43.06 27.01 28.98 41.50 64.43 18.62 35.12
(3.24) (2.96) (1.47) (2.20) (0.82) (0.93) (2.93) (4.68) (0.78) (1.98)
42.76 39.59 32.59 38.69 24.93 27.01 33.27 46.14 15.90 31.60
(1.33) (1.44) (0.81) (0.89) (0.49) (0.62) (1.50) (2.69) (0.50) (0.69)
41.87 38.34 30.93 37.06 24.40 25.72 34.79 48.81 16.83 30.52
(1.56) (1.18) (0.88) (1.12) (0.74) (0.75) (1.53) (1.92) (0.59) (1.16)
23.28 19.18 22.70 22.79 13.89 18.63 59.22 20.55 24.57 21.22 14.74 39.86
(1.57) (1.52) (0.40) (1.65) (0.57) (0.98) (6.88) (1.30) (0.99) (0.94) (0.99) (3.38)
20.49 18.79 23.30 21.51 13.30 17.11 53.04 18.15 24.63 18.60 14.56 38.78
(0.80) (1.34) (1.15) (1.32) (0.47) (0.67) (4.63) (0.81) (1.44) (0.84) (0.78) (4.33)
22.28 16.74 22.09 20.89 14.34 18.26 50.46 19.10 25.50 20.75 14.68 41.22
(1.00) (0.79) (0.88) (0.59) (0.51) (1.02) (4.34) (1.30) (1.77) (1.13) (0.46) (2.90)
22.01 16.57 21.30 19.03 13.26 17.52 53.07 20.54 22.65 19.77 14.07 41.02
(1.24) (0.86) (0.67) (1.01) (0.32) (1.22) (3.83) (2.91) (0.91) (0.92) (0.84) (4.36)
37.61 26.75 31.19 32.90 23.73 25.56 23.90 24.04 34.29 49.91 22.84 41.25 20.04 19.88 25.48 26.40 19.11 21.68 42.32 22.88 43.07
(4.63) (1.23) (2.08) (1.77) (1.25) (1.11) (0.64) (1.04) (1.27) (3.56) (1.22) (3.18) (0.52) (0.25) (1.80) (0.94) (0.54) (1.21) (3.32) (1.06) (5.14)
32.11 27.95 27.00 28.60 24.90 23.81 24.78 21.38 32.93 39.03 21.95 37.17 18.18 17.92 22.38 22.96 19.25 19.27 41.46 22.97 39.04
(1.54) (2.25) (1.10) (0.94) (1.09) (1.15) (1.07) (0.78) (1.03) (1.49)** (0.84) (2.16) (0.72) (0.78) (0.86) (0.60) (0.77) (1.02) (5.31) (0.73) (1.57)
40.70 29.34 31.75 30.79 23.30 26.60 26.44 23.46 36.01 47.56 22.41 41.19 21.43 19.57 26.38 26.34 19.10 24.81 45.59 21.77 44.77
(3.88) (2.51) (2.35) (1.29) (0.78) (1.63) (1.22) (1.24) (1.44) (2.61) (1.18) (1.84) (1.07) (0.66) (2.16) (1.73) (1.42) (1.63) (2.41) (0.88) (2.49)
32.96 26.14 30.13 31.29 22.84 26.94 27.94 23.04 34.98 38.79 23.77 41.34 21.53 20.34 27.43 24.57 19.66 23.38 43.84 20.94 39.80
(2.93) (2.08) (0.94) (1.86) (0.91) (1.42) (1.46) (0.80) (1.10) (1.66)* (1.39) (3.75) (0.71) (0.66) (0.52) (1.62) (1.20) (1.14) (3.79) (0.83) (2.04)
* P < 0.05, ** P < 0.01, *** P < 0.001, ANOVA contrasts kindled vs control subjects for each time period.
171
indicate that electrical kindling modifies the G A B A related chloride ionophore, and that, in some brain areas, the modifications are very long lasting. They add to a growing collection of studies which have reported longlasting GABAergic changes following the development of electrically kindled seizures 6, and complement and extend recent reports of deficits in [3sS]TBPS binding following chemical kindling 9,~s. How do these changes relate to the kindling effect? Since kindling is permanent, only long-lasting alterations, observed 28 days or more after the last seizure, can contribute directly to the 'kindled state '5. One area of the brain which showed a long-term reduction in TBPS binding was the infralimbic cortex, which is located in the medial prefrontal cortical region. This region has efferent connections to the amygdala 8, the thalamus 17'36, and the brainstem 17'34'36. Crawley and co-workers 1°'33 have shown that the microinjection of a cholinergic agonist into this region can induce motor seizures which resemble those characteristic of limbic kindling. Furthermore, there is clinical evidence to suggest that the prefrontal cortex may play a primary role in the elicitation of generalized epileptic seizures in human patients 2,14. A second structure that exhibited a persistent reduction in TBPS binding was the paracentral nucleus of the thalamus. The paracentral nucleus is one of the intralaminar thalamic nuclei which project to the striatum and the frontal agranular neocortex 16. It has been suggested that these nuclei play an important role in seizure generalization 37 and/or regulation 22. Thus, the two brain areas which exhibited long-lasting changes in binding in the present study are regions thought to be involved in the neural circuitry underlying seizure generalization. Further studies will be required to determine the functional significance of these changes, and whether they play a role in the development of kindled seizures or the interictal behavioral abnormalities associated with kindling. It is possible, for instance, that bilateral injections of G A B A ionophore blockers (e.g. picrotoxin) into the infralimbic cortex and/or paracentral nucleus might accelerate the progress of kindling. Studies designed to test this possibility are currently in progress, as well as studies designed to determine
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whether the observed binding changes are due to alterations in binding affinity (Kd) or number of binding sites (Bmax). It is interesting to note that the pattern of regional changes observed in the present study was not the same as the pattern of changes reported in previous studies of binding to the two other components of the GABAA receptor complex 23'24'29. In those studies, the only alterations that were observed following kindling were shortterm binding increases restricted to the dentate gyrus. It is conceivable that kindling modifies the G A B A ionophore while sparing the extracellular sites related to G A B A and benzodiazepine binding. Such a selective effect might result from a perturbation of membrane phospholipids, which are believed to stabilize TBPS binding sites on the ionophore (cf. Refs. 15 and 32). It could also result from changes in phosphorylation processes affecting the ionophore (cf. Refs. 3, 13, 25 and 39). The fact that the TBPS binding decreases observed in the present study are not accompanied by changes in G A B A or benzodiazapine receptor binding suggests that these decreases are not likely to have been caused by a loss of postsynaptic cells. The current data do not provide a clear mechanism for the reductions in GABA-stimulated chloride flux which have been observed in the brainstem one month after kindling 3° and in the cortex, hippocampus and cerebellum one week after kindling 35. A clarification of the precise biochemical correlates of these chloride flux changes thus must await further investigation. In addition to the significant changes discussed above, there seemed to be a general trend toward decreased binding in the forebrain and increased binding in the hindbrain at 28 days. It might be fruitful to re-examine the effects of kindling on these areas in future studies involving the administration of increased numbers of seizures.
Acknowledgements. This study was supported by Grant MA 5611 from the Medical Research Council of Canada. J.S.P. was supported by a University of Toronto Open Fellowship. S.J.K. is a Career Scientist of the Ontario Ministry of Health. These data were presented in preliminary form at the 1990 Meeting of the Society for Neuroscience (St. Louis, MO).
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