26
Brain Research, 578 (1992) 26-32 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00
BRES 17616
Distribution of [3H]clonidine binding sites in the brain of the convulsive mutant quaking mouse: a radioautographic analysis Nadia Mitrovic a'b, Frangoise Le Saux a'b, Henri Gioanni a, Yves Gioanni c, Marie Jo Besson a and Yves Maurin a aLaboratoire de Neurochirnie Anatomie, lnstitut des Neurosciences, UPMC-CNRS UA 1199, Paris (France) and bLaboratoire de Neurochimie, INSERM U 134, HOpital de la Salp~tri~re, Paris (France) and cUnit~ de recherche sur l'dpilepsie, INSERM U 97, Paris (France)
(Accepted 26 November 1991) Key words: Noradrenaline; [3H]Clonidine binding; Quaking mutant; Convulsion; Radioautography
The radioautographic analysis of [3H]clonidine binding was performed on brain slices from the convulsive mutant mice quaking and their controls of the same strain. In the quaking mice significant increases were observed mostly in the brainstem and the cerebellum, but also in a few regions of the forebrain, such as the lateral and medial thalamic nuclei, the medial geniculate nucleus, the amygdala and the hypothalamus. Other regions, such as the cerebral cortex and the hippocampus, which are classically involved in various models of epilepsy, but not in the quaking mice, did not show any modification of [3H]clonidine binding. A high degree of correlation was found between the structures with an increased density of a2-adrenoceptor binding sites and the distribution of regions from which seizures can be elicited by intracerebral electrical stimulation in head-restrained quaking mice. This comparison emphasizes the role of noradrenaline acting at the level of a2-adrenoceptors in the epileptic syndrome of the quaking mutants. INTRODUCTION The quaking mutation (recessive, autosomal, localized on c h r o m o s o m e 17) determines the susceptibility of affected mice to convulsions which occur either spontaneously (during the paradoxical sleep 29) or in response to tactile stimulations 9. It has been shown 4'5 that handlinginduced convulsions can be inhibited in a dose-dependent m a n n e r by yohimbine (an a2-adrenoceptor antagonist) and p o t e n t i a t e d by clonidine (an a2-adrenoceptor agonist). These behavioral effects are p r o b a b l y related to alterations of a2-adrenoceptor sites as shown by the different d e v e l o p m e n t a l profiles of [3H]clonidine binding sites in the brain of quaking and control mice 2°. Recently, G i o a n n i and coworkers 8 have d e t e r m i n e d various brain regions from which seizures can be elicited by intracranial stimulation in awake head-restrained quaking mice. In the present study, we have carried out a radioautographic study of [3H]clonidine binding on brain slices of quaking and control mice in o r d e r (i) to d e t e r m i n e the regional localization of the modifications of a2a d r e n o c e p t o r sites observed previously on cerebral membrane preparations using the same ligand for a2-site labeling and (ii) to search for an overlap b e t w e e n structures
with a modified density of a2-adrenoceptors and structures with a high sensitivity to electrical stimulation. O u r results extend our previous findings concerning the cerebral distribution of the increase of a : - a d r e n o c e p t o r binding sites in the brain of the quaking mutants 2°. M o r e o v e r , a large overlap was found between structures with an increased density of a2-adrenoceptors and those from which convulsions can be elicited by electrical stimulation 8. Therefore, these results emphasize the role of the noradrenergic neurotransmission in the inherited epilepsy of the quaking mutant mice. MATERIALS AND METHODS All experiments were carried out on male quaking mice (50 days old, weighing 18-23 g) and their controls of the same strains (C57BI/6J), raised in our colony under standard laboratory conditions (free access to food and water, and 7.00-19.00 light-dark cycles). Mice were killed by cervical dislocation. Their brains were rapidly removed, frozen in isopentane at -30°C, and kept frozen at -25°C until utilization. Serial cryostat sections (20 pm thick) were collected on cold gelatin-coated slides (1% gelatin in 0.05% chrome alum) and kept at -25°C until the day of the experiment. Slices were dried at room temperature under the hood, and used either for [3H]clonidine binding or Nissl staining. The [3H]clonidine binding was performed according to a slightly modified protocole described by Johnson et al. 13 and Unnerstall et al. 26 for [3H]p-aminoclonidine binding. Briefly sections were washed during 1 h at room temperature with Tris-HCl buffer (170 mM, pH 7.8) and then
Correspondence: M.J. Besson, Neurochimie Anatomie Lab., Institut des Neurosciences, UPMC-CNRS UA 1199, 9 Quai St. Bernard, 75005,
Paris, France.
27 incubated for 1 h at room temperature with [3H]clonidine (S.A. 828.8 GBq/mmol, Amersham) diluted in Tris-HCl buffer (170 mM, pH 7.8) containing 10 mM MgCI213'26.For non-specific binding, the incubation medium was supplemented with 10 gM phentolamine (Sandoz). At the end of the incubation, sections were washed twice during 2 min in ice-cold incubation buffer, quickly dipped into bidistilled water and dried under a cool air stream. Saturation curves of [3H]clonidine binding were first determined on adjacent sections incubated with varied concentrations of ligand (1,3,5,7,10,20 nM). At the end of the incubation, sections were carefully scraped and the radioactivity measured in a liquid scintillation counter following addition of 5 ml of Emulsifier Safe (Packard). The equilibrium constant (KD) of [3H]clonidine binding was calculated by computer-assisted non-linear regression analysis31. Radioautograms were obtained from sections incubated with 5 nM of [3H]clonidine, a concentration which corresponded to around two-fold the Ko value determined from saturation curves. After incubation, washing and drying sections and two types of [3H]microscale standard (Amersham P506 and P507) were simultaneously apposed (in X-ray cassettes) on Hyperfilm (Amersham) for 60 days. Films were developed with Kodak D19. The analysis of binding density was carried out by quantitative densitometry using computer-assisted image analysis (Imstar, France). Structures were defined according to the atlas by Sidman et al. 25. For each animal and on every structure examined, a mean optical density was calculated from the measurements done on 3-6 sections, depending on the rostrocaudal extension of the structure. In the cerebellum, we also measured the binding of [3H]clonidine in membranes according to a method previously described2° in order to verify that the increased binding observed in the quaking mice by radioautography was not due to 3H-quenching. The values presented in tables are the mean + S.E.M. of values obtained on 4-6 mice. Statistical analyses were done by the Student's t-test. RESULTS Fig. 1 shows the saturation curve of [3H]clonidine binding o b t a i n e d on coronal sections t a k e n at the level of the brainstem and cerebellum in control mice. The
$
10~
•
•
NS
S5o
1.1
3.2
5.3 7.4 10.6 3H-Cionidine (nM)
Fig. 1. Saturation curve of [3H]clonidine binding to slide-mounted and scraped sections of tissue from control mouse hindbrain corresponding to levels through the brainstem and the cerebellum. Sections were washed and incubated in buffer with various concentrations of [3H]clonidine. Non-specific binding (NS) was determined in the presence of 10 #M phentolamine, the specific binding (S) corresponds to the difference between total and non-specific binding. The KD value was calculated by non-linear regression analysis31.
specific binding was saturable. The KD value calculated from this saturation cure d o n e in triplicate was 2.4 nM. The non-specific binding m e a s u r e d in the presence of 10 /~M p h e n t o l a m i n e c o r r e s p o n d e d to a r o u n d 6% of the total binding at the KD value. Thus the r a d i o a u t o g r a p h i c analysis was p e r f o r m e d following incubation of brain sections with 5 nM of [3H]clonidine, a concentration corresponding to two K D values. Fig. 2 shows r a d i o a u t o g r a m s o b t a i n e d from hindbrain and forebrain sections of quaking and control mice. Densitometric m e a s u r e m e n t s were d e t e r m i n e d in various brain structures and values are indicated in Table I and II. Most of the structures in which [3H]clonidine binding was significantly higher in the quaking mice than in the controls were located within the brainstem where an increased binding of [3H]clonidine had been observed previously on m e m b r a n e p r e p a r a t i o n 2°. This radioautographic study shows that the density of a 2 receptors was increased in several brainstem nuclei. T h e y include the grey m a t t e r of the medulla oblongata, the nucleus of the solitary tract, the medial vestibular nucleus, the hypoglossal nucleus, the nucleus of the lateral lemniscus, the nucleus locus coeruleus, the cochlear nucleus, the m o t o r trigeminal nucleus, the mesencephalic grey matter, the pontine nuclei and the reticular formation. A m a r k e d increase of [3H]clonidine binding was also observed in the cerebeUar cortex (+109%) and the d e e p cerebellar nuclei (+308%). Since in the cerebeUar cortex, [3H]clonidine binding was detected at the level of myelinated fibers we verified that the increase density of a 2 sites measured by r a d i o a u t o g r a p h y could not be attributed to 3Hquenching. F o r this purpose we d e t e r m i n e d the binding of [3H]clonidine on m e m b r a n e s p r e p a r e d from cerebellum of controls and quaking mice. Expressed in fmol/mg protein, [3H]clonidine binding was 59.7 + 4.1 in controls (n = 3) and 87.7 + 2.3 in quaking mice (n = 3). Thus as c o m p a r e d to controls a highly significant increase ( + 4 7 % , P < 0.001 by the Student's t-test) was also obtained on m e m b r a n e s , a result in a g r e e m e n t with the e n h a n c e m e n t d e t e c t e d by r a d i o a u t o g r a p h y in the cerebellum of quaking mice. A few forebrain regions exhibited increased [3H]clonidine binding: they included the medial geniculate nuclei, the hypothalamus, the lateral and medial thalamic nuclei, the stria terminalis and the amygdala. Interestingly, [3H]clonidine binding was not modified in the hippocampus, the c a u d a t e / p u t a m e n and the neocortex. DISCUSSION Several lines of evidence have shown the involvement of the noradrenergic systems in the convulsions of the quaking mice: their seizures can be antagonized by the
28
CONTROL
Coch
/
.
~
QUAKING
'
Fig. 2. Dark-field illustrations of autoradiograms obtained in quaking and control mice following the incubation of brain slices with [3H]clonidine. Three different rostrocaudal levels are presented: medulla-pons (upper 4 pictures), and diencephalon/telencephalon (lower 2 pictures). Note in various structures the higher density of binding in the quaking mice than in the controls. Hy, Nucleus prepositus hypoglossi; Ccb, Cortex cerebelli; Ncb, Nuclei cerebelli; Coch, Nucleus cochlearis dorsalis; Vest, Nucleus vestibularis medialis; Solit, Nucleus tractus solitarii; Thml, Nucleus thalami (lateralis + medialis); Amyg, Nucleus amygdaloideus.
a2-adrenoceptor antagonist yohimbine and potentiated by the aE-adrenoceptor agonist clonidine4'5; the profile of aE-adrenoceptor appearance during brain maturation is modified when compared to control mice of the same strain2°; at adult age, the density of al-receptors is higher in the quaking mice than in controls whereas the density of fl-adrenoceptors is unchanged2°; the release of noradrenaline in brainstem slices is significantly enhanced x7 and the electrolytic destruction of the nucleus locus coeruleus blocks their convulsions 19. The experiments re-
ported here were designed to precisely determine the distribution of [3H]clonidine binding sites in brainstem nuclei, cerebellum and several forebrain structures of the quaking mice. The main finding of these radioautographic studies is the observation that in various forebrain areas, [3H]clonidine binding was higher in quaking mice than in their controls. Noradrenergic receptors of the ct2 type are present at the presynaptic 16 as well as at the postsynaptic levels 16"21'27'28. In the forebrain of the quaking mice,
29 TABLE I
Radioautographic analysis of [3H]clonidine binding in the hindbrain of quaking and control mice Results are means + S.E.M. of independent determinations performed on at least 4 qua ki ng mice and 4 controls.
Structures
Control mice (fmol[3H]Clo/mg tissue)
Quaking mice (fmol[3H]Clo/mg tissue)
% increase in quaking mice
82.9 125.7 52.8 44.8 44.8 70.4 59.3 83.1 51.5 101.5
118.8 169.7 105.2 106.1 98.7 131.9 89.1 126.6 114.5 97.1
5.9* 5.5** 4.5*** 11.9"* 7.8** 7.1"* 4.5* 14.2" 9.7** 11.3
43 35 99 137 120 87 50 52 122 -
83.1 + 5.5** 102.1 + 3.1"**
109 308
Medulla-pons Substantia grisea centralis Nucl. locus coeruleus Nucl. cochlearis dorsalis Nucl. centralis caudalis pontis Nucl. pontis Nucl. tractus solitarii Nucl. vestibularis medialis Nucl. prepositus hypoglossi Nucl. motorius trigemini Nucl. raph6 dorsalis
+ + + + + + + + + +
9.1 4.2 4.5 8.1 9.5 7.9 7.7 4.0 8.3 8.5
+ + + + + + + + + +
Cerebellum Cortex cerebelli Nucl. Cerebelli
39.8 + 7.6 25.0 + 3.4
Mesencephalon Substantia grisea centralis Nucl. lemniscus lateralis Colliculus posterior Colliculus anterior Nucl. intercollicularis
64.5 110.9 78.9 78.8 75.5
+ + + + +
6.2 7.9 10.1 7.4 11.5
97.6 143.9 103.9 86.6 110.9
+ + + + +
7.9* 5.3* 3.8 7.0 15.8
51 30 -
*P < 0.05, **P < 0.01, ***P < 0.001 when compared to the corresponding value in the control mice (Student's t-test). The percent increase in quaking is indicated only when the difference reaches statistical significance.
T A B L E II
Radioautographic analysis of [3H]clonidine binding on the forebrain of quaking and control mice Results are means + S.E.M. of independent determinations performed on at least 4 qua ki ng mice and 4 controls.
Structures
Control mice (fmol[3H]Clo/mg tissue)
Quaking mice (fmol[3H]Clo/mg tissue)
% increase in quaking mice
69.7 105.3 75.4 48.5 101.6 114.1 62.8
+ + + + + + +
4.2 6.2 6.8 8.1 10.3 9.21 9.7
92.4 126.6 94.7 54.4 111.0 123.7 79.9
+ + + + + + +
6.9* 5.9* 3.9* 6.9 8.6 4.2 0.3
33 20 26 -
128.2 116.7 143.3 97.3 92.8 122.7 131.5 140.4 36.7 116.1 57.2 97.4
+ + + + + + + + + + + +
5.5 11.0 12.2 12.0 8.0 16.5 11.9 11.1 10.0 10.I 13.1 15.2
164.6 177.5 159.6 111.3 114.7 162.0 132.7 137.7 27.6 132.1 69.8 110.2
+ + + + + + + + + + + +
10.2" 11.0"* 9.2 4.7 7.2 12.5 12.5 8.8 6.8 12.2 4.1 9.6
28 52 -
Diencephalon Nucl. corporis geniculati medialis Hypothalamus Nucl. thalami (lateralis + medialis) Nucl. mamillaris Nucl. paraventricularis thalami Nuc|. preopticus Nucl. habenulae
Telencephalon Nucl. amygdaloideus Striae terminalis Nucl. interstitiae striae terminalis Claustrum Fibrae perforantes Hippocamp us pars anterior Nucl. septi Nucl. fimbrialis septi Nucl. caudatus/putamen Massa intercalata Cortex occipitalis Cortex entorhinalis
*P < 0.05; **P < 0.01; ***P < 0.001 when compared to the corresponding value in the control mice (Student's t-test). The percent increase in quaking is indicated only when the difference reaches statistical significance.
30 the uptake of [3H]noradrenaline is not significantly different from the uptake in the control mice 18. This suggests that the density of noradrenergic fibers is similar in the two strains of mice as well as the population of presynaptically located a2-adrenoceptor binding sites, is likely similar in the quaking mice and their controls. In the brainstem, although the regulation of the release of [3H]noradrenaline by presynaptic a2-adrenoceptors is similar in both strains, an increased density of noradrenergic fibers and likely of presynaptic a2-adrenoceptors is suggested by the 20% higher noradrenaline concentration of this brain structure in the quacking mice than in their controls 17. Therefore, the increase of [3H]clonidine binding observed in the present study can be due to a 2adrenoceptor sites located at the postsynaptic and presynaptic levels. Interestingly, the distribution of the modifications of [3H]clonidine binding fit in nicely with the recent results of Gioanni and coworkers 8 concerning the mapping of brain structures from which seizures can be elicited by electrical stimulation in head-restrained quaking mice. The comparison of their observations with the present results show that in the brainstem, most of the regions exhibiting an increased binding of [3H]clonidine also show a high sensitivity to seizures induced by electrical stimulation. Only two structures with a higher binding of [3H]clonidine did not verify this correlation. These are the hypoglossal nucleus (which innervates tongue muscles) and the cerebellar cortex, in which the electrical stimulation failed to produce seizures. In all other structures, a high level of [3H]clonidine binding was associated with a high seizure sensitivity. Some of these structures are known to have important sensory and motor functions. They include the trigeminal motor nucleus (which mediates cutaneous and proprioceptive sensations from skin and muscles of the face), the nucleus caudalis pontis (which belong to the reticular formation, a structure involved in sensory motor integration) and the pontine nuclei (a group of neurons which give rise to crossed fibers innervating the cerebellar hemisphere and which play an important role in the cerebellar control of movement and posture). The increasing binding of [3H]clonidine found in deep cerebellar nuclei could be in direct relationship with the increased binding of [3H]clonidine in the pontine nuclei which innervate them. Some regions in the brainstem, related to functions that are altered in the quaking mutants, have a higher density of [3H]clonidine binding: the vestibular nuclei, for instance, which relay information for the maintenance of body posture. Other regions also show a higher density of [3H]clonidine binding, although the significance of the alteration is unclear, as in the nucleus of the solitary tract. This nucleus is involved in the baroreceptor reflex
loop and a high density of adrenergic innervation is found in this region a6. Of interest, the nucleus of the solitary tract sends projections to the parabrachial nucleus, and then to the amygdala, where the density of [3H]clonidine binding is also increased. Finally, [3H]clonidine binding was increased in the locus coeruleus of the quaking mice. Since it has been shown previously that this nucleus had a greater number of NA neurons in the mutants than in the controls TM, it can be assumed that our present observation reflects the hyperplasia of the nucleus, the a2-adrenoceptors being located on noradrenergic neuronal cell bodies. The locus coeruleus plays a crucial role in the convulsive phenotype of the quaking mutants. In fact, convulsions evoked by tactile stimulation can be abolished by the electrolytic lesion of this nucleus 19. Furthermore, seizures can be easily elicited at a low threshold by electrical stimulation of the locus coeruleus in head-restrained quaking mice 8. Thus, in the brainstem, a close correlation seems to exist between the distribution of regions showing an increased binding of [3H]clonidine and a high susceptibility to electrically induced seizures. Besides tactile stimulation, the quaking mice are also sensitive to acoustic stimulation 3 and exhibit audiogenic seizures which are similar to the audiogenic seizures of DBA/2J mice ~°'3° or genetically epilepsy-prone (GEP) rats 12. Several structures involved in the auditory pathway exhibit an abnormally high level of [3H]clonidine binding. These structures include the cochlear nucleus (in which the auditory fibers terminate), the nucleus of the lateral lemniscus (on which some fibers of the cochlear nucleus project), and the medial geniculate body (the thalamic relay of the auditory pathway). It should be mentioned, however, that the inferior colliculus did not shown any modification of the binding of [3H]clonidine. The inferior colliculus plays an important role in the audiogenic seizures of GEP rats. In these animals, audiogenic seizures can be elicited by electrical or chemical stimulation of this structure ~. Audiogenic seizures can also be blocked by local microinjections of GABA agonists or excitatory amino acid antagonists 1"6'7'11. Finally, the inferior colliculus of GEP rats contains an increased number of GABAergic neurons 24. The lack of modification of [3H]clonidine binding in the inferior colliculus of the quaking mice, together with the observation that seizures could not be evoked by electrical stimulation of this structure 8, and that GABAergic receptors are not modified in the brain of the quaking mice 2 suggest that at variance with other audiogenic seizure sensitive strains, the inferior colliculus is unlikely to play a crucial role in the convulsions of the quaking mutants. In the forebrain, very few structures exhibited an in-
31 creased [3H]clonidine binding. A m o n g them, the amygdala and the hypothalamus two structures which are reciprocally connected 15.23. The higher density of a Eadrenoceptors in the quaking mice than in controls in these two structures could reflect an increased number of both a z presynaptic and postsynaptic sites. These two structures not only exhibit a high binding of [3H]clonidine, but are also sensitive to electrically evoked seizures 8. A n o t h e r region with an increased binding of [3H]clonidine corresponds to the intralaminar nuclei of the thalamus which are also sensitive to electrically evoked seizures. These nuclei could be involved in the triggering of seizures produced by the various sensory stimuli for which the quaking mice are susceptible. Other regions classically involved in the convulsions of numerous animal models of epilepsy, such as the cerebral cortex and the hippocampus, did not exhibit any modification of the binding of [3H]clonidine. The lack of changes of [3H]clonidine binding in the cerebral cortex (occipital and entorhinal parts) agrees with the identical values of noradrenaline release, uptake and endogenous content measured in the cerebral cortex of quaking mice 17. It also agrees with the observation that the neonatal administration of 6-hydroxdopamine which destroys the noradrenergic fibers innervating the forebrain does not block the convulsions of adult mutant mice 19. Finally, Gioanni and collaborators 8 were unable to provoke seizures by electrical stimulation of the cerebral cortex and have observed that the cortical E E G of quaking mice is devoid of any epileptic-like activity during the fits. Thus, our radioautographic results strengthen the hypothesis that the cerebral cortex is unlikely to participate in the epileptic syndrome of the quaking mice.
REFERENCES 1 Browning, R.A., Lanker, M.L. and Faingold, C.L., Injections of noradrenergic and GABAergic agonists into the inferior colliculus: effects on audiogenic seizures in genetically epilepsyprone rats, Epilepsy Res., 4 (1989) 119-125. 2 Caboche, J., Mitrovic, N., Le Saux, E, Besson, M.J., Sauter, A. and Maurin, Y., Postnatal evolution of the gamma-aminobutyric acid/benzodiazepine receptor complex in a model of inherited epilepsy: the quaking mouse, J. Neurochem., 52 (1989) 419-427. 3 Chauvel, P., Louvel, J., Kurcewicz, I. and Debono, M., Epileptic seizures of the quaking mouse: electroclinical correlations. In N. Baumann (Ed.), Neurological Mutations Affecting Myelination, Elsevier/North-Holland, Amsterdam, 1980, pp. 513-516. 4 Chermat, R., Doar& L., Lachapelle, E and Simon, P., Effects of drugs affecting the noradrenergic system on convulsions in the quaking mouse, Naunyn-Schmiedeberg's Arch. Pharmacol., 318 (1981) 91-99. 5 Chermat, R., Lachapelle, E, Baumann, N. and Simon, P., Anticonvulsant effect of yohimbine in quaking mice. Antagonism by clonidine and prazosin, Life Sci., 25 (1979) 71-74. 6 Duplisse, B.R., Picchioni, A.L., Chin, L. and Consroe, P.E,
In the hippocampus, [3H]clonidine binding was also not different in both species. The hippocampus receives a massive projection from the amygdala 14 where an increased density in aE-adrenoceptors was detected in the quaking mouse. Thus it can be postulated that the amygdalo-hippocampic pathway does not contribute to [3H]clonidine binding sites in the hippocampus. Furthermore it can be noted that in the coronal sections examined, corresponding to the anterior part of the hippocampus, ct2 binding sites were mainly located at the level of CA1 in the laculosum-molecular layer as this was reported in the rat E6. This hippocampic layer receiving a massive N A innervation EE, it is likely that in this area ct2 sites correspond to presynaptic sites. The similar density of [3H]clonidine binding sites in the hippocampus of the quaking mice and their controls supports the lack of N A hyperinnervation in the hippocampus of the quaking mouse. Furthermore it agrees with the results of Gioanni and coworkers s who reported that no seizures could be evoked by the intracerebral stimulation of this structure. Altogether, our data are consistent with previous observations suggesting that the convulsions of the quaking mice arise from non-cortical structures 3's. They provide additional support to the crucial role of brainstem structures in the seizures of these genetically epileptic mutants, and emphasize the role of the noradrenergic system (and particularly of aE-adrenoceptors ) in their convulsive phenotype. Acknowledgements. This study was supported by the University Pierre et Marie Curie, CNRS URA 1199, DRET (Grant 86/1502) and the Fondation pour la Recherche M6dicale. N.M. was a fellowship from the Fondation pour la Recherche M6dicale. We are very grateful to Dr. N. Baumann for her support.
Relationship of the inferior colliculus and gamma-aminobutyric acid (GABA) to audiogenic seizure in the rat, Fed. Proc., 33 (1974) 1452. 7 Faingold, C.L., Millan, M.H., Boersma, C.A. and Meldrum, B.S., Excitant amino acids and audiogenic seizures in the genetically epilepsy-prone rat. I. Afferent seizure initiation pathway, Exp. Neurol., 99 (1988) 678-686. 8 Gioanni, Y., Gioanni, H. and Mitrovic, N., Seizures can be triggered by stimulating non-cortical structures in the quaking mutant mouse, Epilepsy Res., 9 (1991) 19-31. 9 Goujet, M.A., Simon, E and Boissier, J.R., Modifications neurologiques et comportementales chez une souche de souris 'tremblantes', Therapie, XXVI (1971) 823-830. 10 Hall, C.S., Genetic differences in fatal audiogenic seizures, J. Hered., 38 (1947) 2-6. 11 Huxtable, R. and Laird, H., The prolonged anticonvulsant action of taurine on genetically determined seizure-susceptibility, Can. J. Neurol. Sci., 5 (1978) 215-221. 12 Jobe, RC., Picchioni, A.L. and Chin, L., Role of brain norepinephrine on audiogenic seizure in the rat, J. Pharmacol. Exp. Ther., 184 (1973) 1-10. 13 Johnson, A.E., Nock, B., McEwen, B.S. and Feder, H.J., a 1and aE-noradrenergic receptor binding in guinea pig brain: sex differences and effects of ovarian steroids, Brain Res., 442
32 (1988) 205-213. 14 Krettek, J.E. and Price, J.L., Projections from the amygdaloid complex and adjacent olfactory structures to the entorhinal cortex and to the subicu|um in the rat and cat, J. Comp. Neurol., 172 (1977) 723-752. 15 Krettek, J.E. and Price, J.L., Amygdaloid projections to subcortical structures within the basal forebrain and brainstem in the rat and cat, J. Comp. Neurol., 178 (1978) 225-254. 16 Langer, S.Z., Presynaptic regulation of the release of catecholamines, Pharmacol. Rev., 32 (1980) 337-362. 17 Maurin, Y., Arbilla, S., Dedek, J., Lee, C.R., Baumann, N. and Langer, S.Z., Noradrenergic neurotransmission in the brain of a convulsive mutant mouse. Differences between the cerebral cortex and the brain stem, Naunyn-Schmiedeberg's Arch. Pharamacol., 320 (1982) 26-33. 18 Maurin, Y., Berger, B., Le Saux, E , Gay, M. and Baumann, N., Increased number of locus coeruleus noradrenergic neurons in the convulsive mutant quaking mouse, Neurosci. Lett., 57 (1985) 313-318. 19 Maurin, Y., Enz, A., Le Saux, E and Besson, M.J., Supernumerary locus coeruleus neurons as a determinant of inherited epilepsy in the convulsive mutant mouse quaking, Brain Res., 366 (1986) 379-384. 20 Maurin, Y., Le Saux, E , Graillot, C. and Baumann, N., Altered postnatal ontogeny of a 1- and a2-adrenoceptor binding sites in the brain of a convulsive mutant mouse (quaking), Dev. Brain Res., 22 (1985) 229-235. 21 Morris, M.J., Elghozi, J.L., Dausse, J.P. and Meyer, P., a 1- and a2-adrenoceptors in rat cerebral cortex: effect of frontal lobotomy, Naunyn-Schmiedeberg's Arch. Pharrnacol., 316 (1981) 4244. 22 Oleskevich, S., Descarries, L. and Lacaille, J.C., Quantified distribution of the noradrenaline innervation in the hippocampus of adult rat, J. Neurosci., 9 (1989) 3803-3815.
23 Ottersen, O.E, Afferent connections to the amygdaloid complex of the rat and cat: II. Afferents from the hypothalamus and the basal telencephalon, J. Cornp. Neurol., 184 (1980) 267-289. 24 Roberts, R.C., Ribak, C.E. and Oertel, W.H., Increased number of GABAergic neurons occurs in the inferior colliculus of an audiogenic model of genetic epilepsy, Brain Res., 361 (1985) 324-338. 25 Sidman, R.L., Angevine Jr., J.B. and Pierce, E.T., Atlas of the Mouse Brain and Spinal Cord, Harvard University Press, Cambridge, MA, 1971. 26 Unnerstall, J.R., Kopajtic, T.A. and Kuhar, M.J., Distribution of a2 agonist binding sites in the rat and human central nervous system: analysis of some functional, anatomic correlates of the pharmacologic effects of clonidine and related adrenergic agents, Brain Res. Rev., 7 (1984) 69-101. 27 U'Prichard, D.C., Bechtel, W.D., Rouot, B.M. and Snyder, S.H., Multiple apparant alpha-noradrenergic receptor binding sites in rat brain: effect of 6-hydroxydopamine, Mol. Pharmacol., 16 (1979) 47-60. 28 U'Prichard, D.C., Reisine, T.D., Mason, S.T., Fibiger, H.C. and Yamamura, H.I., Modulation of rat brain a- and fl-adrenergic receptor populations by lesions of the dorsal noradrenergic bundle, Brain Res., 187 (1980) 143-154. 29 Valatx, J.L., Quaking mutation: relationship between sleep and seizures. In N. Baumann (Ed.), Neurological Mutations Affecting Myelination, Elsevier/North-Holland, Amsterdam, 1980, pp. 507-512. 30 Vicari, E., Fatal convulsive seizures in the DBA mouse strain, J. Psychol., 32 (1951) 79-97. 31 Vindimian, E., Robaut, C. and Fillion, G., A method for cooperative and non cooperative binding studies using non-linear regression analysis on a micro computer, J. Appl. Biochem., 5 (1983) 261-268.
Brain Research, 578 (1992) 26-32 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00
BRES 17616
Distribution of [3H]clonidine binding sites in the brain of the convulsive mutant quaking mouse: a radioautographic analysis Nadia Mitrovic a'b, Frangoise Le Saux a'b, Henri Gioanni a, Yves Gioanni c, Marie Jo Besson a and Yves Maurin a aLaboratoire de Neurochirnie Anatomie, lnstitut des Neurosciences, UPMC-CNRS UA 1199, Paris (France) and bLaboratoire de Neurochimie, INSERM U 134, HOpital de la Salp~tri~re, Paris (France) and cUnit~ de recherche sur l'dpilepsie, INSERM U 97, Paris (France)
(Accepted 26 November 1991) Key words: Noradrenaline; [3H]Clonidine binding; Quaking mutant; Convulsion; Radioautography
The radioautographic analysis of [3H]clonidine binding was performed on brain slices from the convulsive mutant mice quaking and their controls of the same strain. In the quaking mice significant increases were observed mostly in the brainstem and the cerebellum, but also in a few regions of the forebrain, such as the lateral and medial thalamic nuclei, the medial geniculate nucleus, the amygdala and the hypothalamus. Other regions, such as the cerebral cortex and the hippocampus, which are classically involved in various models of epilepsy, but not in the quaking mice, did not show any modification of [3H]clonidine binding. A high degree of correlation was found between the structures with an increased density of a2-adrenoceptor binding sites and the distribution of regions from which seizures can be elicited by intracerebral electrical stimulation in head-restrained quaking mice. This comparison emphasizes the role of noradrenaline acting at the level of a2-adrenoceptors in the epileptic syndrome of the quaking mutants. INTRODUCTION The quaking mutation (recessive, autosomal, localized on c h r o m o s o m e 17) determines the susceptibility of affected mice to convulsions which occur either spontaneously (during the paradoxical sleep 29) or in response to tactile stimulations 9. It has been shown 4'5 that handlinginduced convulsions can be inhibited in a dose-dependent m a n n e r by yohimbine (an a2-adrenoceptor antagonist) and p o t e n t i a t e d by clonidine (an a2-adrenoceptor agonist). These behavioral effects are p r o b a b l y related to alterations of a2-adrenoceptor sites as shown by the different d e v e l o p m e n t a l profiles of [3H]clonidine binding sites in the brain of quaking and control mice 2°. Recently, G i o a n n i and coworkers 8 have d e t e r m i n e d various brain regions from which seizures can be elicited by intracranial stimulation in awake head-restrained quaking mice. In the present study, we have carried out a radioautographic study of [3H]clonidine binding on brain slices of quaking and control mice in o r d e r (i) to d e t e r m i n e the regional localization of the modifications of a2a d r e n o c e p t o r sites observed previously on cerebral membrane preparations using the same ligand for a2-site labeling and (ii) to search for an overlap b e t w e e n structures
with a modified density of a2-adrenoceptors and structures with a high sensitivity to electrical stimulation. O u r results extend our previous findings concerning the cerebral distribution of the increase of a : - a d r e n o c e p t o r binding sites in the brain of the quaking mutants 2°. M o r e o v e r , a large overlap was found between structures with an increased density of a2-adrenoceptors and those from which convulsions can be elicited by electrical stimulation 8. Therefore, these results emphasize the role of the noradrenergic neurotransmission in the inherited epilepsy of the quaking mutant mice. MATERIALS AND METHODS All experiments were carried out on male quaking mice (50 days old, weighing 18-23 g) and their controls of the same strains (C57BI/6J), raised in our colony under standard laboratory conditions (free access to food and water, and 7.00-19.00 light-dark cycles). Mice were killed by cervical dislocation. Their brains were rapidly removed, frozen in isopentane at -30°C, and kept frozen at -25°C until utilization. Serial cryostat sections (20 pm thick) were collected on cold gelatin-coated slides (1% gelatin in 0.05% chrome alum) and kept at -25°C until the day of the experiment. Slices were dried at room temperature under the hood, and used either for [3H]clonidine binding or Nissl staining. The [3H]clonidine binding was performed according to a slightly modified protocole described by Johnson et al. 13 and Unnerstall et al. 26 for [3H]p-aminoclonidine binding. Briefly sections were washed during 1 h at room temperature with Tris-HCl buffer (170 mM, pH 7.8) and then
Correspondence: M.J. Besson, Neurochimie Anatomie Lab., Institut des Neurosciences, UPMC-CNRS UA 1199, 9 Quai St. Bernard, 75005,
Paris, France.
27 incubated for 1 h at room temperature with [3H]clonidine (S.A. 828.8 GBq/mmol, Amersham) diluted in Tris-HCl buffer (170 mM, pH 7.8) containing 10 mM MgCI213'26.For non-specific binding, the incubation medium was supplemented with 10 gM phentolamine (Sandoz). At the end of the incubation, sections were washed twice during 2 min in ice-cold incubation buffer, quickly dipped into bidistilled water and dried under a cool air stream. Saturation curves of [3H]clonidine binding were first determined on adjacent sections incubated with varied concentrations of ligand (1,3,5,7,10,20 nM). At the end of the incubation, sections were carefully scraped and the radioactivity measured in a liquid scintillation counter following addition of 5 ml of Emulsifier Safe (Packard). The equilibrium constant (KD) of [3H]clonidine binding was calculated by computer-assisted non-linear regression analysis31. Radioautograms were obtained from sections incubated with 5 nM of [3H]clonidine, a concentration which corresponded to around two-fold the Ko value determined from saturation curves. After incubation, washing and drying sections and two types of [3H]microscale standard (Amersham P506 and P507) were simultaneously apposed (in X-ray cassettes) on Hyperfilm (Amersham) for 60 days. Films were developed with Kodak D19. The analysis of binding density was carried out by quantitative densitometry using computer-assisted image analysis (Imstar, France). Structures were defined according to the atlas by Sidman et al. 25. For each animal and on every structure examined, a mean optical density was calculated from the measurements done on 3-6 sections, depending on the rostrocaudal extension of the structure. In the cerebellum, we also measured the binding of [3H]clonidine in membranes according to a method previously described2° in order to verify that the increased binding observed in the quaking mice by radioautography was not due to 3H-quenching. The values presented in tables are the mean + S.E.M. of values obtained on 4-6 mice. Statistical analyses were done by the Student's t-test. RESULTS Fig. 1 shows the saturation curve of [3H]clonidine binding o b t a i n e d on coronal sections t a k e n at the level of the brainstem and cerebellum in control mice. The
$
10~
•
•
NS
S5o
1.1
3.2
5.3 7.4 10.6 3H-Cionidine (nM)
Fig. 1. Saturation curve of [3H]clonidine binding to slide-mounted and scraped sections of tissue from control mouse hindbrain corresponding to levels through the brainstem and the cerebellum. Sections were washed and incubated in buffer with various concentrations of [3H]clonidine. Non-specific binding (NS) was determined in the presence of 10 #M phentolamine, the specific binding (S) corresponds to the difference between total and non-specific binding. The KD value was calculated by non-linear regression analysis31.
specific binding was saturable. The KD value calculated from this saturation cure d o n e in triplicate was 2.4 nM. The non-specific binding m e a s u r e d in the presence of 10 /~M p h e n t o l a m i n e c o r r e s p o n d e d to a r o u n d 6% of the total binding at the KD value. Thus the r a d i o a u t o g r a p h i c analysis was p e r f o r m e d following incubation of brain sections with 5 nM of [3H]clonidine, a concentration corresponding to two K D values. Fig. 2 shows r a d i o a u t o g r a m s o b t a i n e d from hindbrain and forebrain sections of quaking and control mice. Densitometric m e a s u r e m e n t s were d e t e r m i n e d in various brain structures and values are indicated in Table I and II. Most of the structures in which [3H]clonidine binding was significantly higher in the quaking mice than in the controls were located within the brainstem where an increased binding of [3H]clonidine had been observed previously on m e m b r a n e p r e p a r a t i o n 2°. This radioautographic study shows that the density of a 2 receptors was increased in several brainstem nuclei. T h e y include the grey m a t t e r of the medulla oblongata, the nucleus of the solitary tract, the medial vestibular nucleus, the hypoglossal nucleus, the nucleus of the lateral lemniscus, the nucleus locus coeruleus, the cochlear nucleus, the m o t o r trigeminal nucleus, the mesencephalic grey matter, the pontine nuclei and the reticular formation. A m a r k e d increase of [3H]clonidine binding was also observed in the cerebeUar cortex (+109%) and the d e e p cerebellar nuclei (+308%). Since in the cerebeUar cortex, [3H]clonidine binding was detected at the level of myelinated fibers we verified that the increase density of a 2 sites measured by r a d i o a u t o g r a p h y could not be attributed to 3Hquenching. F o r this purpose we d e t e r m i n e d the binding of [3H]clonidine on m e m b r a n e s p r e p a r e d from cerebellum of controls and quaking mice. Expressed in fmol/mg protein, [3H]clonidine binding was 59.7 + 4.1 in controls (n = 3) and 87.7 + 2.3 in quaking mice (n = 3). Thus as c o m p a r e d to controls a highly significant increase ( + 4 7 % , P < 0.001 by the Student's t-test) was also obtained on m e m b r a n e s , a result in a g r e e m e n t with the e n h a n c e m e n t d e t e c t e d by r a d i o a u t o g r a p h y in the cerebellum of quaking mice. A few forebrain regions exhibited increased [3H]clonidine binding: they included the medial geniculate nuclei, the hypothalamus, the lateral and medial thalamic nuclei, the stria terminalis and the amygdala. Interestingly, [3H]clonidine binding was not modified in the hippocampus, the c a u d a t e / p u t a m e n and the neocortex. DISCUSSION Several lines of evidence have shown the involvement of the noradrenergic systems in the convulsions of the quaking mice: their seizures can be antagonized by the
28
CONTROL
Coch
/
.
~
QUAKING
'
Fig. 2. Dark-field illustrations of autoradiograms obtained in quaking and control mice following the incubation of brain slices with [3H]clonidine. Three different rostrocaudal levels are presented: medulla-pons (upper 4 pictures), and diencephalon/telencephalon (lower 2 pictures). Note in various structures the higher density of binding in the quaking mice than in the controls. Hy, Nucleus prepositus hypoglossi; Ccb, Cortex cerebelli; Ncb, Nuclei cerebelli; Coch, Nucleus cochlearis dorsalis; Vest, Nucleus vestibularis medialis; Solit, Nucleus tractus solitarii; Thml, Nucleus thalami (lateralis + medialis); Amyg, Nucleus amygdaloideus.
a2-adrenoceptor antagonist yohimbine and potentiated by the aE-adrenoceptor agonist clonidine4'5; the profile of aE-adrenoceptor appearance during brain maturation is modified when compared to control mice of the same strain2°; at adult age, the density of al-receptors is higher in the quaking mice than in controls whereas the density of fl-adrenoceptors is unchanged2°; the release of noradrenaline in brainstem slices is significantly enhanced x7 and the electrolytic destruction of the nucleus locus coeruleus blocks their convulsions 19. The experiments re-
ported here were designed to precisely determine the distribution of [3H]clonidine binding sites in brainstem nuclei, cerebellum and several forebrain structures of the quaking mice. The main finding of these radioautographic studies is the observation that in various forebrain areas, [3H]clonidine binding was higher in quaking mice than in their controls. Noradrenergic receptors of the ct2 type are present at the presynaptic 16 as well as at the postsynaptic levels 16"21'27'28. In the forebrain of the quaking mice,
29 TABLE I
Radioautographic analysis of [3H]clonidine binding in the hindbrain of quaking and control mice Results are means + S.E.M. of independent determinations performed on at least 4 qua ki ng mice and 4 controls.
Structures
Control mice (fmol[3H]Clo/mg tissue)
Quaking mice (fmol[3H]Clo/mg tissue)
% increase in quaking mice
82.9 125.7 52.8 44.8 44.8 70.4 59.3 83.1 51.5 101.5
118.8 169.7 105.2 106.1 98.7 131.9 89.1 126.6 114.5 97.1
5.9* 5.5** 4.5*** 11.9"* 7.8** 7.1"* 4.5* 14.2" 9.7** 11.3
43 35 99 137 120 87 50 52 122 -
83.1 + 5.5** 102.1 + 3.1"**
109 308
Medulla-pons Substantia grisea centralis Nucl. locus coeruleus Nucl. cochlearis dorsalis Nucl. centralis caudalis pontis Nucl. pontis Nucl. tractus solitarii Nucl. vestibularis medialis Nucl. prepositus hypoglossi Nucl. motorius trigemini Nucl. raph6 dorsalis
+ + + + + + + + + +
9.1 4.2 4.5 8.1 9.5 7.9 7.7 4.0 8.3 8.5
+ + + + + + + + + +
Cerebellum Cortex cerebelli Nucl. Cerebelli
39.8 + 7.6 25.0 + 3.4
Mesencephalon Substantia grisea centralis Nucl. lemniscus lateralis Colliculus posterior Colliculus anterior Nucl. intercollicularis
64.5 110.9 78.9 78.8 75.5
+ + + + +
6.2 7.9 10.1 7.4 11.5
97.6 143.9 103.9 86.6 110.9
+ + + + +
7.9* 5.3* 3.8 7.0 15.8
51 30 -
*P < 0.05, **P < 0.01, ***P < 0.001 when compared to the corresponding value in the control mice (Student's t-test). The percent increase in quaking is indicated only when the difference reaches statistical significance.
T A B L E II
Radioautographic analysis of [3H]clonidine binding on the forebrain of quaking and control mice Results are means + S.E.M. of independent determinations performed on at least 4 qua ki ng mice and 4 controls.
Structures
Control mice (fmol[3H]Clo/mg tissue)
Quaking mice (fmol[3H]Clo/mg tissue)
% increase in quaking mice
69.7 105.3 75.4 48.5 101.6 114.1 62.8
+ + + + + + +
4.2 6.2 6.8 8.1 10.3 9.21 9.7
92.4 126.6 94.7 54.4 111.0 123.7 79.9
+ + + + + + +
6.9* 5.9* 3.9* 6.9 8.6 4.2 0.3
33 20 26 -
128.2 116.7 143.3 97.3 92.8 122.7 131.5 140.4 36.7 116.1 57.2 97.4
+ + + + + + + + + + + +
5.5 11.0 12.2 12.0 8.0 16.5 11.9 11.1 10.0 10.I 13.1 15.2
164.6 177.5 159.6 111.3 114.7 162.0 132.7 137.7 27.6 132.1 69.8 110.2
+ + + + + + + + + + + +
10.2" 11.0"* 9.2 4.7 7.2 12.5 12.5 8.8 6.8 12.2 4.1 9.6
28 52 -
Diencephalon Nucl. corporis geniculati medialis Hypothalamus Nucl. thalami (lateralis + medialis) Nucl. mamillaris Nucl. paraventricularis thalami Nuc|. preopticus Nucl. habenulae
Telencephalon Nucl. amygdaloideus Striae terminalis Nucl. interstitiae striae terminalis Claustrum Fibrae perforantes Hippocamp us pars anterior Nucl. septi Nucl. fimbrialis septi Nucl. caudatus/putamen Massa intercalata Cortex occipitalis Cortex entorhinalis
*P < 0.05; **P < 0.01; ***P < 0.001 when compared to the corresponding value in the control mice (Student's t-test). The percent increase in quaking is indicated only when the difference reaches statistical significance.
30 the uptake of [3H]noradrenaline is not significantly different from the uptake in the control mice 18. This suggests that the density of noradrenergic fibers is similar in the two strains of mice as well as the population of presynaptically located a2-adrenoceptor binding sites, is likely similar in the quaking mice and their controls. In the brainstem, although the regulation of the release of [3H]noradrenaline by presynaptic a2-adrenoceptors is similar in both strains, an increased density of noradrenergic fibers and likely of presynaptic a2-adrenoceptors is suggested by the 20% higher noradrenaline concentration of this brain structure in the quacking mice than in their controls 17. Therefore, the increase of [3H]clonidine binding observed in the present study can be due to a 2adrenoceptor sites located at the postsynaptic and presynaptic levels. Interestingly, the distribution of the modifications of [3H]clonidine binding fit in nicely with the recent results of Gioanni and coworkers 8 concerning the mapping of brain structures from which seizures can be elicited by electrical stimulation in head-restrained quaking mice. The comparison of their observations with the present results show that in the brainstem, most of the regions exhibiting an increased binding of [3H]clonidine also show a high sensitivity to seizures induced by electrical stimulation. Only two structures with a higher binding of [3H]clonidine did not verify this correlation. These are the hypoglossal nucleus (which innervates tongue muscles) and the cerebellar cortex, in which the electrical stimulation failed to produce seizures. In all other structures, a high level of [3H]clonidine binding was associated with a high seizure sensitivity. Some of these structures are known to have important sensory and motor functions. They include the trigeminal motor nucleus (which mediates cutaneous and proprioceptive sensations from skin and muscles of the face), the nucleus caudalis pontis (which belong to the reticular formation, a structure involved in sensory motor integration) and the pontine nuclei (a group of neurons which give rise to crossed fibers innervating the cerebellar hemisphere and which play an important role in the cerebellar control of movement and posture). The increasing binding of [3H]clonidine found in deep cerebellar nuclei could be in direct relationship with the increased binding of [3H]clonidine in the pontine nuclei which innervate them. Some regions in the brainstem, related to functions that are altered in the quaking mutants, have a higher density of [3H]clonidine binding: the vestibular nuclei, for instance, which relay information for the maintenance of body posture. Other regions also show a higher density of [3H]clonidine binding, although the significance of the alteration is unclear, as in the nucleus of the solitary tract. This nucleus is involved in the baroreceptor reflex
loop and a high density of adrenergic innervation is found in this region a6. Of interest, the nucleus of the solitary tract sends projections to the parabrachial nucleus, and then to the amygdala, where the density of [3H]clonidine binding is also increased. Finally, [3H]clonidine binding was increased in the locus coeruleus of the quaking mice. Since it has been shown previously that this nucleus had a greater number of NA neurons in the mutants than in the controls TM, it can be assumed that our present observation reflects the hyperplasia of the nucleus, the a2-adrenoceptors being located on noradrenergic neuronal cell bodies. The locus coeruleus plays a crucial role in the convulsive phenotype of the quaking mutants. In fact, convulsions evoked by tactile stimulation can be abolished by the electrolytic lesion of this nucleus 19. Furthermore, seizures can be easily elicited at a low threshold by electrical stimulation of the locus coeruleus in head-restrained quaking mice 8. Thus, in the brainstem, a close correlation seems to exist between the distribution of regions showing an increased binding of [3H]clonidine and a high susceptibility to electrically induced seizures. Besides tactile stimulation, the quaking mice are also sensitive to acoustic stimulation 3 and exhibit audiogenic seizures which are similar to the audiogenic seizures of DBA/2J mice ~°'3° or genetically epilepsy-prone (GEP) rats 12. Several structures involved in the auditory pathway exhibit an abnormally high level of [3H]clonidine binding. These structures include the cochlear nucleus (in which the auditory fibers terminate), the nucleus of the lateral lemniscus (on which some fibers of the cochlear nucleus project), and the medial geniculate body (the thalamic relay of the auditory pathway). It should be mentioned, however, that the inferior colliculus did not shown any modification of the binding of [3H]clonidine. The inferior colliculus plays an important role in the audiogenic seizures of GEP rats. In these animals, audiogenic seizures can be elicited by electrical or chemical stimulation of this structure ~. Audiogenic seizures can also be blocked by local microinjections of GABA agonists or excitatory amino acid antagonists 1"6'7'11. Finally, the inferior colliculus of GEP rats contains an increased number of GABAergic neurons 24. The lack of modification of [3H]clonidine binding in the inferior colliculus of the quaking mice, together with the observation that seizures could not be evoked by electrical stimulation of this structure 8, and that GABAergic receptors are not modified in the brain of the quaking mice 2 suggest that at variance with other audiogenic seizure sensitive strains, the inferior colliculus is unlikely to play a crucial role in the convulsions of the quaking mutants. In the forebrain, very few structures exhibited an in-
31 creased [3H]clonidine binding. A m o n g them, the amygdala and the hypothalamus two structures which are reciprocally connected 15.23. The higher density of a Eadrenoceptors in the quaking mice than in controls in these two structures could reflect an increased number of both a z presynaptic and postsynaptic sites. These two structures not only exhibit a high binding of [3H]clonidine, but are also sensitive to electrically evoked seizures 8. A n o t h e r region with an increased binding of [3H]clonidine corresponds to the intralaminar nuclei of the thalamus which are also sensitive to electrically evoked seizures. These nuclei could be involved in the triggering of seizures produced by the various sensory stimuli for which the quaking mice are susceptible. Other regions classically involved in the convulsions of numerous animal models of epilepsy, such as the cerebral cortex and the hippocampus, did not exhibit any modification of the binding of [3H]clonidine. The lack of changes of [3H]clonidine binding in the cerebral cortex (occipital and entorhinal parts) agrees with the identical values of noradrenaline release, uptake and endogenous content measured in the cerebral cortex of quaking mice 17. It also agrees with the observation that the neonatal administration of 6-hydroxdopamine which destroys the noradrenergic fibers innervating the forebrain does not block the convulsions of adult mutant mice 19. Finally, Gioanni and collaborators 8 were unable to provoke seizures by electrical stimulation of the cerebral cortex and have observed that the cortical E E G of quaking mice is devoid of any epileptic-like activity during the fits. Thus, our radioautographic results strengthen the hypothesis that the cerebral cortex is unlikely to participate in the epileptic syndrome of the quaking mice.
REFERENCES 1 Browning, R.A., Lanker, M.L. and Faingold, C.L., Injections of noradrenergic and GABAergic agonists into the inferior colliculus: effects on audiogenic seizures in genetically epilepsyprone rats, Epilepsy Res., 4 (1989) 119-125. 2 Caboche, J., Mitrovic, N., Le Saux, E, Besson, M.J., Sauter, A. and Maurin, Y., Postnatal evolution of the gamma-aminobutyric acid/benzodiazepine receptor complex in a model of inherited epilepsy: the quaking mouse, J. Neurochem., 52 (1989) 419-427. 3 Chauvel, P., Louvel, J., Kurcewicz, I. and Debono, M., Epileptic seizures of the quaking mouse: electroclinical correlations. In N. Baumann (Ed.), Neurological Mutations Affecting Myelination, Elsevier/North-Holland, Amsterdam, 1980, pp. 513-516. 4 Chermat, R., Doar& L., Lachapelle, E and Simon, P., Effects of drugs affecting the noradrenergic system on convulsions in the quaking mouse, Naunyn-Schmiedeberg's Arch. Pharmacol., 318 (1981) 91-99. 5 Chermat, R., Lachapelle, E, Baumann, N. and Simon, P., Anticonvulsant effect of yohimbine in quaking mice. Antagonism by clonidine and prazosin, Life Sci., 25 (1979) 71-74. 6 Duplisse, B.R., Picchioni, A.L., Chin, L. and Consroe, P.E,
In the hippocampus, [3H]clonidine binding was also not different in both species. The hippocampus receives a massive projection from the amygdala 14 where an increased density in aE-adrenoceptors was detected in the quaking mouse. Thus it can be postulated that the amygdalo-hippocampic pathway does not contribute to [3H]clonidine binding sites in the hippocampus. Furthermore it can be noted that in the coronal sections examined, corresponding to the anterior part of the hippocampus, ct2 binding sites were mainly located at the level of CA1 in the laculosum-molecular layer as this was reported in the rat E6. This hippocampic layer receiving a massive N A innervation EE, it is likely that in this area ct2 sites correspond to presynaptic sites. The similar density of [3H]clonidine binding sites in the hippocampus of the quaking mice and their controls supports the lack of N A hyperinnervation in the hippocampus of the quaking mouse. Furthermore it agrees with the results of Gioanni and coworkers s who reported that no seizures could be evoked by the intracerebral stimulation of this structure. Altogether, our data are consistent with previous observations suggesting that the convulsions of the quaking mice arise from non-cortical structures 3's. They provide additional support to the crucial role of brainstem structures in the seizures of these genetically epileptic mutants, and emphasize the role of the noradrenergic system (and particularly of aE-adrenoceptors ) in their convulsive phenotype. Acknowledgements. This study was supported by the University Pierre et Marie Curie, CNRS URA 1199, DRET (Grant 86/1502) and the Fondation pour la Recherche M6dicale. N.M. was a fellowship from the Fondation pour la Recherche M6dicale. We are very grateful to Dr. N. Baumann for her support.
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