Neuroscience Letters, 131 (1991) 1-4 © 1991 Elsevier Scientific Publishers Ireland Ltd. 0304-3940/91/$ 03.50
ADONIS 0304394091005575 NSL 07905
Characteristics of muscarinic cholinergic, -aminobutyric acidA and phencyclidine receptors in spontaneously epileptic rats; in vitro quantitative autoradiographic analysis Kenshi Murase I , Toshitaka N a b e s h i m a ~'2, T s u t o m u K a m e y a m a ~, Masashi Sasa 3, Shuji Takaori 3 , Hisamitsu Ujihara 3, K u m a t o s h i Ishihara 3, Junzo Y a m a d a 4 and T a d a o Serikawa 4 1Department of ChemicalPharmacology, Faculty of PharmaceuticalSciences, Memo University, Nagoya (Japan), 2Departmentof Hospital Pharmacy, Nagoya University School of Medicine, Nagoya (Japan), 3Departmentof Pharmacology and 4Institute of Laboratory Animals, Faculty of Medicine, Kyoto University, Kyoto (Japan) (Received 21 January 1991; Revised version received 19 April 1991; Accepted 22 April 1991)
Key words: Spontaneously epileptic rat (SER); In vitro quantitative autoradiography; Muscarinic cholinergic (mACh) receptor; 7-Aminobutyric acid^ (GABAA) receptor; Phencyclidine (PCP) receptor Characteristics of muscarinic cholinergic (mACh), ?-aminobutyric acid^ (GABAA) and phencyclidine (PCP) receptors in the spontaneously epileptic rats (SER), which exhibit both absence-like seizures and tonic convulsion, were examined using in vitro quantitative autoradiography. Computer analysis using autoradiographic technique revealed that the amount of the specific binding of [3H]quinuclidinyl benzilate (QNB) to mACh receptors in the striatum of SER was more than that of zitter rats, not exhibiting both seizures and convulsion. However, the specific bindings of [3H]muscimol and [3H]N-(1-[2-thienyl]cyclohexyl)3,4-piperidine (TCP) to GABAA and PCP receptors, respectively, of SER were not different from those of zitter rats in various regions tested. These results suggest that hyperfunction of mACh receptors in the striatum is involved in the appearance of absencelike seizures and tonic convulsion of SER.
Spontaneously epileptic rats (SER) are the zi, tm double mutant rats obtained by mating the tremor heterozygous rat (tm/+) with the zitter homozygous rat (zi/zO [8, 10]. SER exhibits both absence-like seizures and tonic convulsion without external stimuli or when mildly stimulated by tapping or sound [9]. The absence-like seizures were inhibited by the treatment with trimetadione and ethosuximide, which are known to be effective for human absence seizures, whereas the tonic convulsion was not affected by these drugs. Conversely, the tonic convulsion was inhibited by phenitoin, which is known as a therapeutic drug for tonic-clonic convulsion. Moreover, both the absence-like seizures and the tonic convulsion were inhibited by phenobarbital and valproate. They are clinically used for generalized seizures, including convulsive and absence seizures. Therefore, the absence-like seizures and the tonic convulsion in SER were considered as petit mal and grand mal in human, respectively, and SER is considered as an advantageous animal model for evaluation of anti-epileptic drugs [9]. It has been disputed that seizures and convulsion result Correspondence: T. Nabeshima, Department of Hospital Pharmacy, Nagoya University School of Medicine, Showa-ku, Nagoya 466, Japan.
from the abnormality of catecholamines [3, 7]. With regard to SER, dopamine (DA) contents in the caudate nucleus, including DAergic neuronal terminal, are lower as compared with Kyoto-Wistar rats. However, DA contents in the caudate nucleus of zitter rat, not exhibiting both absence-like seizures and tonic convulsion are also lower than that of Kyoto-Wistar rats (personal communication). These findings have suggested that further factors would be closely involved in the appearance of the absence-like seizures and the tonic convulsion of SER. Recently, autoradiographic studies revealed that the density of DAergic receptors in the caudate nucleus of SER is lower than that of zitter rat. We paid attention to muscarinic cholinergic mACh-ergic receptors as excitatory receptors, and phencyclidine (PCP) and y-aminobutyric acid (GABA)-ergic receptors as inhibitory receptors, and elucidated the alterations of these receptors in SER. The regions investigated were the striatum, the frontal cortex and the hippocampus being rich in AChergic and PCP receptors, and the ventral posteromedial thalamus nucleus and the preculminate fissure being rich in GABAergic receptors. We used 3 SER (both male and female) and 3 zitter rats, weighing 190-280 g in these experiments. They were
sacrificed by decapitation and their brains were rapidly frozen at - 1 0 0 ° C . We prepared 12 sections at 2 0 / t m thick with cryostat from one animal; 6 successive sections containing the striatum and others containing the hippocampus. The sections were thaw-mounted on the gelatin-coated slide glass and stored in a refrigerator until binding assay.
[3H]QNB autoradiography was done by a slight modification of the method described by N o n a k a and Moroji [5]. Briefly, the sections on the slide glass were incubated in 50 m M Tris-HCl (pH 7.4) with 1.5 nM [3H]QNB (spec. act., 33.2 Ci/mmol, N E N Research Products) at 25°C for 120 min. After incubation, they were rinsed at 4°C for 10 s 3 times in ice-cold Tris-HC1 buffer and dried
Fig. 1. Color-imaged autoradiograms of [3H]QNBbinding sites in the coronal sections of SER (right side) and zitter rat (left side). Autoradiogram of [3H]micro-scalewhich is used for quantitative analysis also shows the standard tritium concentration sequences increasing blue to red. a, bregma 0.20 mm, b, bregma -3.80 ram.
by a stream of cold air. Adjacent sections were incubated with 1.5 nM [3H]QNB in the presence of 20 pM atropine to determine non-specific binding. [3H]Muscimol autoradiography was done by the method described by Palacios et al. [6] with a slight modification. The sections on the slides were preincubated in 50 mM Tris-HC1 buffer at 25°C for 30 min. After preincubation, they were incubated in 50 mM TrisHC1 buffer (pH 7.4) with 5.0 nM [3H]muscimol (spec. act., 20.0 Ci/mmol, NEN Research Products) at 4°C for 45 min. The sections were rinsed for 20 s 3 times in icecold Tris-HC1 buffer and dried by a stream of cold air. Adjacent sections were incubated with 5.0 nM [3H]muscimol in the presence of 1 mM GABA to determine the non-specific binding. [3H]TCP autoradiography was done by a slight modification of the method described by Maragos et al. [4]. The sections on the slides were preincubated in ice-cold 50 mM Tris-HC1 buffer for 30 min. After preincubation, they were incubated in 50 mM Tris-HCl buffer (pH 7.4) with 20 nM [3H]TCP (spec. act., 48.9 Ci/mmol, NEN Research Products) at 25°C for 45 min. The sections were rinsed for 10 s 3 times in ice-cold Tris-HC1 buffer and dried by a stream of cold air. Adjacent sections were incubated with 20 nM [3H]TCP in the presence of 20 pM PCP-HCI to determine the non-specific binding. After each binding assay, the slides were tightly apposed to hyperfilm [3H] (Amersham) with [3H] microscale (tissue equivalent values, 1.3-33.0 nCi/mg, Amersham) and stored at 4°C for 21 days. After the exposure, the film was developed in D-19 (Kodak) at 20°C for 5 min and fixed in GBX (Kodak) at 20°C for 10 min. The autoradiogram was placed in a photographic illumination apparatus (Northern Light, Imaging Research Inc.) and the optical density of the various regions of interest was measured using an image analyzer (MCID system, Imaging Research, Inc., St. Catharines, Canada). The amount of binding of [3H]-ligands was determined comparing autoradiogram of [3H] micro-scale and the specific binding was calculated by subtracting the non-specific binding from the total binding using a computer system. The specific bindings for [3H]QNB, [3H]muscimol and [3H]TCP were 80%, 73% and 52%, respectively of the total bindings. Fig. 1 shows color-imaged autoradiograms of [3H]QNB binding sites in coronal sections of SER (right side) and zitter rat (left side), mACh receptors were widely distributed throughout SER and zitter rat brain as described in previous reports using Wistar rats [5]. The highest densities of mACh receptors were found in the striatum, the frontal cortex and the olfactory tubercle. Quantitative computer analysis indicated that the specific binding of [3H]QNB to mACh receptors in
the striatum was significantly higher, about 15%, in SER than in zitter rat (Fig. 2, upper). On the other hand, in various regions tested, the specific bindings of [3H]muscimol and [3H]TCP to GABAA and PCP receptors, respectively, showed no significant changes compared to zitter rat (Fig. 2, middle and lower). In the present study, we demonstrated an increase of [3H]QNB binding to mACh receptors in the striatum of SER. These results suggest that up-regulation of mACh receptors in the striatum may be involved in the appearance of absence-like seizures and tonic convulsion of SER. However, we did not determine whether the increase of [3H]QNB binding indicates an increase in the number of mACh receptors or an increase in binding affinity. Recently, Sasa et al. have shown that the electrical stimulation of the substantia nigra can inhibit absence-like seizures and tonic convulsion, and a decrease of DAergic receptors and DA contents in the caudate nucleus in SER (personal communication). Therefore, they have suggested that disruption of nigro-striatal DAergic neuronal system may be involved in an appearance of absence-like seizures and tonic convulsion in SER. It is well known that the nigro-striatal DAergic neuronal system interacts with the ACh neuronal system in the striatum [1]. All these results together suggest that
Striatum
Frontal cortex
cA11
.......... I
0
i
/: ,
i
,
200 400 600 800 [3H]QNB bound (fmol/mg protein)
1000
Striatum Frontal cortex Ventral posteromedialthai n. Simple Iobule Preculminate fissure 0
i
I
,
500
1000
1500
2000
[3H]Muscimolbound (fmoVmgprotein)
Striatum L : . : : _ _ : Frontal cortex [ cA,
,l~
L,.= I
,
,
0
100
200
300
[3H]TCP bound (fmol/mgprotein) Fig. 2. The specific bindings of [3H]QNB (upper), [3H]muscimol (middle) and [3H]TCP (lower) to the different brain regions in SER (dotted) and zitter rats (open). *P
ADONIS 0304394091005575 NSL 07905
Characteristics of muscarinic cholinergic, -aminobutyric acidA and phencyclidine receptors in spontaneously epileptic rats; in vitro quantitative autoradiographic analysis Kenshi Murase I , Toshitaka N a b e s h i m a ~'2, T s u t o m u K a m e y a m a ~, Masashi Sasa 3, Shuji Takaori 3 , Hisamitsu Ujihara 3, K u m a t o s h i Ishihara 3, Junzo Y a m a d a 4 and T a d a o Serikawa 4 1Department of ChemicalPharmacology, Faculty of PharmaceuticalSciences, Memo University, Nagoya (Japan), 2Departmentof Hospital Pharmacy, Nagoya University School of Medicine, Nagoya (Japan), 3Departmentof Pharmacology and 4Institute of Laboratory Animals, Faculty of Medicine, Kyoto University, Kyoto (Japan) (Received 21 January 1991; Revised version received 19 April 1991; Accepted 22 April 1991)
Key words: Spontaneously epileptic rat (SER); In vitro quantitative autoradiography; Muscarinic cholinergic (mACh) receptor; 7-Aminobutyric acid^ (GABAA) receptor; Phencyclidine (PCP) receptor Characteristics of muscarinic cholinergic (mACh), ?-aminobutyric acid^ (GABAA) and phencyclidine (PCP) receptors in the spontaneously epileptic rats (SER), which exhibit both absence-like seizures and tonic convulsion, were examined using in vitro quantitative autoradiography. Computer analysis using autoradiographic technique revealed that the amount of the specific binding of [3H]quinuclidinyl benzilate (QNB) to mACh receptors in the striatum of SER was more than that of zitter rats, not exhibiting both seizures and convulsion. However, the specific bindings of [3H]muscimol and [3H]N-(1-[2-thienyl]cyclohexyl)3,4-piperidine (TCP) to GABAA and PCP receptors, respectively, of SER were not different from those of zitter rats in various regions tested. These results suggest that hyperfunction of mACh receptors in the striatum is involved in the appearance of absencelike seizures and tonic convulsion of SER.
Spontaneously epileptic rats (SER) are the zi, tm double mutant rats obtained by mating the tremor heterozygous rat (tm/+) with the zitter homozygous rat (zi/zO [8, 10]. SER exhibits both absence-like seizures and tonic convulsion without external stimuli or when mildly stimulated by tapping or sound [9]. The absence-like seizures were inhibited by the treatment with trimetadione and ethosuximide, which are known to be effective for human absence seizures, whereas the tonic convulsion was not affected by these drugs. Conversely, the tonic convulsion was inhibited by phenitoin, which is known as a therapeutic drug for tonic-clonic convulsion. Moreover, both the absence-like seizures and the tonic convulsion were inhibited by phenobarbital and valproate. They are clinically used for generalized seizures, including convulsive and absence seizures. Therefore, the absence-like seizures and the tonic convulsion in SER were considered as petit mal and grand mal in human, respectively, and SER is considered as an advantageous animal model for evaluation of anti-epileptic drugs [9]. It has been disputed that seizures and convulsion result Correspondence: T. Nabeshima, Department of Hospital Pharmacy, Nagoya University School of Medicine, Showa-ku, Nagoya 466, Japan.
from the abnormality of catecholamines [3, 7]. With regard to SER, dopamine (DA) contents in the caudate nucleus, including DAergic neuronal terminal, are lower as compared with Kyoto-Wistar rats. However, DA contents in the caudate nucleus of zitter rat, not exhibiting both absence-like seizures and tonic convulsion are also lower than that of Kyoto-Wistar rats (personal communication). These findings have suggested that further factors would be closely involved in the appearance of the absence-like seizures and the tonic convulsion of SER. Recently, autoradiographic studies revealed that the density of DAergic receptors in the caudate nucleus of SER is lower than that of zitter rat. We paid attention to muscarinic cholinergic mACh-ergic receptors as excitatory receptors, and phencyclidine (PCP) and y-aminobutyric acid (GABA)-ergic receptors as inhibitory receptors, and elucidated the alterations of these receptors in SER. The regions investigated were the striatum, the frontal cortex and the hippocampus being rich in AChergic and PCP receptors, and the ventral posteromedial thalamus nucleus and the preculminate fissure being rich in GABAergic receptors. We used 3 SER (both male and female) and 3 zitter rats, weighing 190-280 g in these experiments. They were
sacrificed by decapitation and their brains were rapidly frozen at - 1 0 0 ° C . We prepared 12 sections at 2 0 / t m thick with cryostat from one animal; 6 successive sections containing the striatum and others containing the hippocampus. The sections were thaw-mounted on the gelatin-coated slide glass and stored in a refrigerator until binding assay.
[3H]QNB autoradiography was done by a slight modification of the method described by N o n a k a and Moroji [5]. Briefly, the sections on the slide glass were incubated in 50 m M Tris-HCl (pH 7.4) with 1.5 nM [3H]QNB (spec. act., 33.2 Ci/mmol, N E N Research Products) at 25°C for 120 min. After incubation, they were rinsed at 4°C for 10 s 3 times in ice-cold Tris-HC1 buffer and dried
Fig. 1. Color-imaged autoradiograms of [3H]QNBbinding sites in the coronal sections of SER (right side) and zitter rat (left side). Autoradiogram of [3H]micro-scalewhich is used for quantitative analysis also shows the standard tritium concentration sequences increasing blue to red. a, bregma 0.20 mm, b, bregma -3.80 ram.
by a stream of cold air. Adjacent sections were incubated with 1.5 nM [3H]QNB in the presence of 20 pM atropine to determine non-specific binding. [3H]Muscimol autoradiography was done by the method described by Palacios et al. [6] with a slight modification. The sections on the slides were preincubated in 50 mM Tris-HC1 buffer at 25°C for 30 min. After preincubation, they were incubated in 50 mM TrisHC1 buffer (pH 7.4) with 5.0 nM [3H]muscimol (spec. act., 20.0 Ci/mmol, NEN Research Products) at 4°C for 45 min. The sections were rinsed for 20 s 3 times in icecold Tris-HC1 buffer and dried by a stream of cold air. Adjacent sections were incubated with 5.0 nM [3H]muscimol in the presence of 1 mM GABA to determine the non-specific binding. [3H]TCP autoradiography was done by a slight modification of the method described by Maragos et al. [4]. The sections on the slides were preincubated in ice-cold 50 mM Tris-HC1 buffer for 30 min. After preincubation, they were incubated in 50 mM Tris-HCl buffer (pH 7.4) with 20 nM [3H]TCP (spec. act., 48.9 Ci/mmol, NEN Research Products) at 25°C for 45 min. The sections were rinsed for 10 s 3 times in ice-cold Tris-HC1 buffer and dried by a stream of cold air. Adjacent sections were incubated with 20 nM [3H]TCP in the presence of 20 pM PCP-HCI to determine the non-specific binding. After each binding assay, the slides were tightly apposed to hyperfilm [3H] (Amersham) with [3H] microscale (tissue equivalent values, 1.3-33.0 nCi/mg, Amersham) and stored at 4°C for 21 days. After the exposure, the film was developed in D-19 (Kodak) at 20°C for 5 min and fixed in GBX (Kodak) at 20°C for 10 min. The autoradiogram was placed in a photographic illumination apparatus (Northern Light, Imaging Research Inc.) and the optical density of the various regions of interest was measured using an image analyzer (MCID system, Imaging Research, Inc., St. Catharines, Canada). The amount of binding of [3H]-ligands was determined comparing autoradiogram of [3H] micro-scale and the specific binding was calculated by subtracting the non-specific binding from the total binding using a computer system. The specific bindings for [3H]QNB, [3H]muscimol and [3H]TCP were 80%, 73% and 52%, respectively of the total bindings. Fig. 1 shows color-imaged autoradiograms of [3H]QNB binding sites in coronal sections of SER (right side) and zitter rat (left side), mACh receptors were widely distributed throughout SER and zitter rat brain as described in previous reports using Wistar rats [5]. The highest densities of mACh receptors were found in the striatum, the frontal cortex and the olfactory tubercle. Quantitative computer analysis indicated that the specific binding of [3H]QNB to mACh receptors in
the striatum was significantly higher, about 15%, in SER than in zitter rat (Fig. 2, upper). On the other hand, in various regions tested, the specific bindings of [3H]muscimol and [3H]TCP to GABAA and PCP receptors, respectively, showed no significant changes compared to zitter rat (Fig. 2, middle and lower). In the present study, we demonstrated an increase of [3H]QNB binding to mACh receptors in the striatum of SER. These results suggest that up-regulation of mACh receptors in the striatum may be involved in the appearance of absence-like seizures and tonic convulsion of SER. However, we did not determine whether the increase of [3H]QNB binding indicates an increase in the number of mACh receptors or an increase in binding affinity. Recently, Sasa et al. have shown that the electrical stimulation of the substantia nigra can inhibit absence-like seizures and tonic convulsion, and a decrease of DAergic receptors and DA contents in the caudate nucleus in SER (personal communication). Therefore, they have suggested that disruption of nigro-striatal DAergic neuronal system may be involved in an appearance of absence-like seizures and tonic convulsion in SER. It is well known that the nigro-striatal DAergic neuronal system interacts with the ACh neuronal system in the striatum [1]. All these results together suggest that
Striatum
Frontal cortex
cA11
.......... I
0
i
/: ,
i
,
200 400 600 800 [3H]QNB bound (fmol/mg protein)
1000
Striatum Frontal cortex Ventral posteromedialthai n. Simple Iobule Preculminate fissure 0
i
I
,
500
1000
1500
2000
[3H]Muscimolbound (fmoVmgprotein)
Striatum L : . : : _ _ : Frontal cortex [ cA,
,l~
L,.= I
,
,
0
100
200
300
[3H]TCP bound (fmol/mgprotein) Fig. 2. The specific bindings of [3H]QNB (upper), [3H]muscimol (middle) and [3H]TCP (lower) to the different brain regions in SER (dotted) and zitter rats (open). *P
