Epilepsy Res., 9 (1991) 79-85
79
Elsevier EPIRES 00412
Lesions of noradrenergic neurons in rats with spontaneous generalized non-convulsive epilepsy
Btatrice Lannes a, Marguerite Vergnes b, Christian Marescaux a, Antoine Depaulis b, Gabriel Micheletti a, Jean-Marie Warter a and Eliane Kempf b aClinique Neurologique H6pital Civil, bCentre de Neurochimie C.N.R.S., Strasbourg (France) (Received 30 October 1990; revision received 15 March 1991; accepted 2 April 1991)
Key words: Rat; Absence epilepsy; Neurotoxin; Noradrenaline; Locus coerulens; Lesion
The role of noradrenergic neurons in the control of a spontaneous generalized non-convulsive epilepsy (GNCE) was investigated. In rats with genetic spontaneous absence seizures, we produced lesions using 2 neurotoxins: 6-hydroxydopamine (6-OHDA) and N(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP4). Lesions of noradrenergic neurons were made either in pups by neonatal 60 H D A intraperitoneal (i.p.) injection (2 x 100 mg/kg) or in adult rats by i.p. administration of DSP4 (60 mg/kg) or bilateral microinjection of 6-OHDA in the locus coeruleus (LC) (4 #g//~l, 2 gl/side). Effectiveness of the lesions was controlled by measuring dopamine (DA) and noradrenaline (NA) contents in the brains. Neonatal 6-OHDA administration did not lead to any difference in seizures in adult animals, compared with control rats. DSP4 injections and LC lesions with local injections of 6-OHDA produced a transient increase of the seizures. Within one to two weeks, the seizure duration went back to prelesion levels. No seizure occurred when the same lesions were performed in non epileptic rats. These results suggest that NA is not involved in the genesis of this generalized non-convulsive epilepsy; they confirm that NA participates in the control of seizures in this model, but the rapid development of compensatory mechanisms shows that this control is not critical.
INTRODUCTION A role for catecholamines in human epilepsies has been suggested since the finding that administration of amphetamines suppresses petit real absence seizures, and that administration of reserpine aggravates convulsive and non-convulsive epilepsies 22. In experimental models of seizures, studies have shown that noradrenaline has mostly an inhibitory effect 3-6'8A1'12'35. In the spontaneous generalized non-convulsive epilepsy (GNCE) Correspondence to: Dr. B. Lannes, Cfinique Neurologique, H6pitai Civil, 67091 Strasbourg Cedex, France.
model in the Wistar rat, seizures are characterized by the occurrence of bilateral and synchronous spike and wave discharges (SWD) on the cortical electroencephalogram (EEG). During the SWD, the rats are motionless, presenting only some twitching of the vibrissae 2°'23'3a'34. A pure epileptic strain has been inbred from the initial colony at our laboratory, while a non-epileptic one has been outbred from the same colony. Previous pharmacological studies have shown inhibitory control of noradrenaline in this model. Activation of a-noradrenergic neurotransmission with a-1 agonists (cirazoline, ST 587) or an a-2 antagonist (yohimbine) reduces SWD duration; reduction of a-nor-
0920-1211/91/$03.50 © 1991 Elsevier Science Publishers B.V. All rights reserved
80 adrenergic neurotransmission with an a-1 antagonist (prazosin) or an a-2 agonist (clonidine) enhances seizure duration. Drugs interacting with flnoradrenergic neurotransmission have no effect on the seizures24. In order to confirm the role of noradrenaline in the control of absence seizures, lesions of noradrenergic systems were produced in rats with spontaneous SWD, using selective neurotoxins. N-(2chloroethyl)-N-ethyl-2-bromobenzylamine (DSP4) crosses the blood-brain barrier and produces a specific destruction of noradrenergic neurons in the central nervous system (CNS) 14. Subcutaneous injection of 6-hydroxydopamine (6-OHDA) in the newborn rat also leads to the degeneration of noradrenergic neurons in the CNS (at birth, the blood-brain barrier and the dopaminergic systems are not yet functional)2'31. In the adult rat, local injection of 6-OHDA into the locus coeruleus (LC), the mesencephalic nucleus which contains the main noradrenergic neurons projecting towards the forebrain, leads to the specific destruction of the noradrenergic neurons of this nucleus. Because output from the LC is mostly ipsilateral, the effect of unilateral 6-OHDA lesion of the LC on SWD was examined in rats in which the corpus callosum had been previously transected in order to avoid interhemispheric seizure propagation.
in the LC were performed in adult animals (5-6 months), under general anesthesia, by the mean of a microsyringe using stereotaxic methods (A/P, -1.5 mm; M/L, 1.2 mm; DV, 7 and 7.8 mm, with 3. as the reference). At each site 4 #g in 1 #1 was injected (8/~g per side). The injection lasted 1 min, and the needle was left in place for an additional 1 min.
Corpus callosum transection. Transection of the corpus callosum was carried out under general anesthesia, using a sharp blade fixed vertically on the stereotactic instrument and moved from back to front (AP, 2-10 ram; DV, 4-4.5 mm with ~ as the reference) through the medial suture of the skull, which had previously been removed with a dental drill, as described previously32. EEG recordings Recordings were made between 9:00 a.m. and 5:00 p.m. using an Alvar electroencephalograph (Minidix-TR). Derivations were fronto-parietal, left and right. After a habituation session of 15 min, the EEG was recorded for 1 h. On the days before lesion, in adult-lesioned animals, 3 recordings were made, and the cumulative SWD duration for each hour was measured. The average of these 3 recordings was used as the reference. In 6OHDA injected neonate rats, the EEG was recorded when they became adults.
MATERIALS AND METHODS
Neurotoxic substances Animals Newborn or adult Wistar rats from the strain with GNCE and from the control strain were used in this study.
DSP4 was dissolved in distilled water; 6-OHDA (Sigma, USA) was dissolved in saline with ascorbate (0.2%) as an antioxidant. The respective vehicles were used as controls.
Surgery
Biochemical assays
Implantation. In adult animals, under general pentobarbital anesthesia (40 mg/kg intraperitoneally, i.p.), 4 stainless steel electrodes were implanted into the skull in a bilaterally symmetrical position over the left and right, frontal and parietal cortex. The electrodes were connected to a microconnector fixed to the skull.
Lesion of the LC. Bilateral injections of 6-OHDA
At the end of the experiments, the rats were decapitated and their brains were immediately dissected at 4 °C. The anterior part of the brain, comprising telencephalon and diencephalon, was removed and NA and DA were assayed. NA and DA were determined simultaneously utilizing a reverse phase chromatography procedure coupled with an electrochemical detector (LCEC). On the day of analysis, frozen samples of the anterior part of brains were weighed and homogenized in
81 HCIO 4 0.1 N containing Na-metabisulfite 6 mM and EDTA 1 mM. The homogenates were centrifuged at 10 000 x g for 20 min at 4 °C. Aliquots of the supernatants were transferred into the LCEC system with a Wisp automatic injector (Waters). The LCEC system consisted of a Bioanalytical system LC4 amperometric detector with a glassy carbon working electrode and a pump (Waters). The potential was set at 800 mV (vs. Ag-AgC1 reference electrode). The column, a Bondapack phenyl column (10/zm particle size, 300 x 3.1 mm i.d.) was purchased from Waters Assoc. The flow rate was 1.4 ml/min, and the sensitivity was set at 5 nA/V (1 V full scale). The mobile phase consisted of 3% methanol in 0.1 M Na-phosphate buffer pH 2.5/Na2-EDTA 0.1 mM/1-octane sulfonic acid-Na salt (BDH) 2 mM. 3,4-Dihydroxyhydrocinnamic acid (Aldrich) was used as internal standard. Control rats of the same age were killed simultaneously and the same biochemical analyses were performed. Data analysis Results are given as the cumulative SWD duration, in seconds, during 1 h of recording. In the case of corpus callosum transection, the results were analyzed separately for each hemisphere. Comparison of the seizure duration was done for
each test day after treatment vs. the reference period, using non-parametric tests (Friedmann and Wilcoxon) 28. Each animal was thus used as its own control. Biochemical data were compared between groups using Student's t-test. RESULTS 6-OHDA in newborn rats Newborn animals were injected twice subcutaneously 12 h and 24 h after birth with 100 mg/kg of 6-OHDA in a volume of 2 ml/100 g. Eight rats from the epileptic strain and 4 from the non-epileptic strain received 6-OHDA, and 8 epileptic and 4 non-epileptic rats were injected with the vehicle. Control rats (injected with vehicle) were chosen among siblings of the lesioned rats. At the age of 5 months, all 24 rats were implanted, and 3 EEGs of 1 h each were recorded between the ages of 5 and 6 months. The animals were killed at 7 months, and brain NA and DA were measured. Within the epileptic rats, no difference was observed in the SWD duration between the group injected with 6-OHDA and the group injected with vehicle only (results not shown). In non-epileptic animals treated with 6-OHDA, no SWD were ever observed. Biochemical analysis showed an important decrease in NA content, in treated animals
TABLE I
Concentrations of brain NA and DA in the different experiments Concentrations are expressed in ng per g of brain tissue (mean + S.E.M.). Treated rats versus controls in each epileptic (Epi) and nonepileptic (Non-epi) group. *, P < 0.05; **, P < 0.001.
Experiment 6-OHDA in newborns
Epi Non-epi
DSP4
Epi Non-epi
Bilateral lesion of LC
Epi Non-epi
Animals
n
NA (ng/g)
DA (ng/g)
6-OHDA Controls 6-OHDA Controls
8 8 4 4
75 294 20 287
+ + + +
45** 34 40* 46
1302 + 1477 + 990 + 1264 +
139 321 263 150
DSP4 Controls DSP4 Controls
5 4 4 4
113 + 366 + 151 + 353 +
19"* 48 98* 23
1304 + 1046 + 1157 + 1115 +
258 212 295 114
6-OHDA Controls 6-OHDA Controls
6 6 3 4
109 + 316 + 179 + 327 +
92* 50 86* 47
1405 + 1256 + 1011 + 1359 +
319 206 359 110
82 compared to the control ones. D A remained unchanged in the 4 groups (Table I). DSP4 in adult rats Seven epileptic rats (5-6 months old) were injected (i.p.) with 60 mg/kg of DSP4 in a volume of 2 ml/kg. EEGs were recorded every day during the first week following injection of DSP4, and also on day 10. Four non-epileptic rats were also injected with DSP4. EEGs were recorded on days 1, 3, 6, 10 and 15 following the injection. The animals were killed on day 16 for measurement of NA and DA in their brains. In epileptic rats, injection of DSP4 induced an important increase of SWD duration, beginning from the very first day after the injection and being maximal on the second day. The SWD duration went back to the reference values between days 6 and 10 (Fig. 1). DSP4 in non-epileptic rats did not induce any SWD. The measurement of NA in the cortex revealed a significant decrease in both epileptic and non-epileptic rats compared with their respective controls. Measurement of DA showed no significant difference between lesioned and intact animals (Table I). Bilateral lesion of the L C Six epileptic and 4 non-epileptic adult animals were injected in the LC. EEGs were recorded on days 1, 3, 4, 5, 9, 12 and 21 following the lesion.
All animals were killed on day 22 for measurement of NA and DA in their brains. Four epileptic rats were sham-operated; the needle of the microsyringe was, under general anesthesia, lowered stereotactically above the LC, but nothing was injected. 6-OHDA lesion of the LC in epileptic rats resuited in a transient increase in SWD duration, beginning on the day following the operation and being maximal on day 4 (Fig. 2). On day 12, the SWD duration had returned to reference values. No SWD were observed in non-epileptic rats after this lesion. Biochemical analysis showed a significant decrease of NA levels in lesioned epileptic and non-epileptic rats when compared with their respective controls. DA levels were not significantly different within the same animals (Table I). The 4 sham-operated rats did not show any significant difference in SWD duration before and after the operation (results not shown). Unilateral lesion of L C after corpus callosum transection After a corpus callosum transection, seizures may develop independently on one hemisphere or the other, and often occur unilaterally and alternately on both sides 32. The cumulated seizure duration for each hemisphere was measured. The ratio between the two sides was close to 1 before the LC lesion. The transections were made one week before the neurotoxic lesion, which was per-
2000 2000 1500
~o
/
1000
1500 ~
t
IXT • T I\T •.~_ (3
•
~ 1 ~
o
1000
1
C23
500
500
REF 1
5
10
post lesion days
Fig. 1. Effect of DSP4 on SWD duration in rats (mean + S.E.M.). Injection was performed on day 0 (between the reference and day 1). *, P < 0.05 (Wilcoxon test).
REF 1
5
10 15 post lesion days
20
Fig. 2. Effect of a bilateral lesion of LC on SWD duration in rats (mean + S.E.M.). The lesion was performed on day 0 (between the reference and day 1). *, P < 0.05 (Wilcoxon test).
83
/\ j\
1000
o
0--0 0--0
right left
5 10 post lesion dcys
15
,o,
REF 1
Fig. 3. SWD durations in left and fight hemispheres following a 6 - O H D A lesion of the right LC in a rat with transected corpus callosum.
formed as described above, on the right side only. Two epileptic adult animals were operated upon and their EEGs were recorded during the 2 weeks following the neurotoxic lesion. In both rats, a transient increase in SWD duration was observed, which was maximal on the fourth day after the lesion and predominated on the lesioned side (right side). SWD duration returned to values preceding the neurotoxic lesion about 8 days after the lesion (Fig. 3 shows the results for I of the 2 rats). DISCUSSION The present results suggest the existence of an inhibitory control of NA over the SWD in this model of GNCE. The lesion of NA neurons, being either localized to the LC cell bodies (6-OHDA in the LC) or to their terminals (DSP4 injections, which has been shown to produce its neurotoxic action mainly on the terminals of NA neurons from the LC TMincreases SWD duration). This corroborates the conclusions of a previous pharmacological investigation 24. However, NA does not~ seem to be implicated in the genesis of SWD since seizures were never observed in non-epileptic rats, whatever the lesion, even when NA depletion was performed in newborns of the non-epileptic strain. At that time, SWD have not yet developed in animals from the epileptic strain 34.
Moreover, despite the permanence of the NA deficit measured in the brains, increases of SWD were always transient. This result suggests the development of compensatory mechanisms, leading to the recovery of 'normal' SWD duration in spite of the remaining deficit in NA. The absence of difference observed in SWD duration in rats which had received subcutaneous 6-OHDA at birth could be explained by a similar compensation. One of these mechanisms could be an up-regulation of NA receptors as it has been described after such neurotoxic lesions 7'1°A7'29. Another possibility might be a compensation from the remaining NA neurons themselves (the depletion of NE is between 65 and 75% in epileptic animals), perhaps by the mean of sprouting collaterals. LC neurons send projections toward the neocortex, the hippocampus, the amygdala, the thalamus, the colliculi and the cerebellum 13. The predominance of ipsilateral projections may account for the predominant aggravation of SWD on the lesioned side after unilateral lesion. Noradrenergic neurons from the LC are known to participate in the control of vigilance levels, and more specifically, in the degree of awareness 1'9'15'27. On the one hand, multi-unit recordings of LC neurons in the rat have shown that their spontaneous activity varies with the vigilance levels, and is enhanced in case of sensory stimulations 1. On the other hand, in the GNCE rat, sensory stimulations block seizures as they do in human petit mal epilepsy 16. The LC may thus control seizures directly or indirectly via its action on vigilance levels. In some other models of absence epilepsy, lesions of NA neurons have also been described. In the pentylenetetrazol (PTZ) model in the rat, seizures are potentiated by injection of 6-OHDA into the dorsal bundle (ascending NA system) in the mesencephalon 2~. On the other hand, electrical stimulation of the LC suppresses SWD induced by subconvulsive dose of PTZ 19. In the ~,-butyrolactone model in the rat (GBL), neonatal treatment with 6-OHDA leads to more severe and prolonged seizures induced by acute administration of GBL in the adult animal 3°. However, these data contrast with previous data obtained in the Tottering mouse, another genetic model where mice display 3 abnormal behaviors: wobbly gait, motor seizures
84 a n d s u d d e n a r r e s t of m o v e m e n t c o n c o m i t a n t with S W D o n the E E G 25. This last b e h a v i o r was cons i d e r e d as a m a n i f e s t a t i o n o f a b s e n c e - l i k e seizures. I n t h e s e m i c e , an a b n o r m a l i n c r e a s e in locus c o e r u l e u s axons has b e e n d e s c r i b e d w i t h o u t a n y i n c r e a s e in the n u m b e r o f cell s o m a t a in t h e L C TM. A f t e r n e o n a t a l 6 - O H D A t r e a t m e n t , t h e m i c e fail to d e v e l o p S W D o r gait d i s t u r b a n c e . B i l a t e r a l inj e c t i o n o f 6 - O H D A in L C in a d u l t a n i m a l s l e a d s to an i m p o r t a n t d e c r e a s e in S W D , w h i l e u n i l a t e r a l i n j e c t i o n s u p p r e s s e s S W D o n t h e i p s i l a t e r a l cortex 26. Such o p p o s i n g findings in 2 a n i m a l m o d e l s o f p e t i t m a l e p i l e p s y suggest t h a t N A is n o t a critical n e u r o t r a n s m i t t e r i n v o l v e d in t h e c o n t r o l o f sei-
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zures, a n d that t h e c o n t r o l o f seizures m a y b e a c h i e v e d in d i f f e r e n t w a y s in 2 g e n e t i c m o d e l s of non-convulsive epilepsy. ACKNOWLEDGMENTS The authors thank Any Boehrer and Carmen S c h l e e f for their v a l u a b l e t e c h n i c a l assistance, a n d Sylvie P a n s i o t for t y p i n g t h e m a n u s c r i p t . B. L a n nes was t h e r e c i p i e n t o f a f e l l o w s h i p f r o m L a Fondation p o u r la R e c h e r c h e M~dicale. This w o r k was s u p p o r t e d by grants f r o m I N S E R M (Contrat de R e c h e r c h e Externe N o . 866017) a n d Fondation p o u r la R e c h e r c h e M~dicale.
cycle oscillation: reciprocal discharge by two brainstem neuronal groups, Science, 189 (1975) 55-58. 10 Janowsky, A., Labarca, R. and Paul, S.M., Noradrenergic denervation increases alpha 1-adrenoreceptor-mediated inositolphosphate accumulation in the hippocampus, Eur. J. Pharmacol., 102 (1984) 193-194. 11 Jobe, P.C., Picchioni, A.L. and Chin, L., Role of brain norepinephrine in audiogenic seizure in the rat, J. Pharmacol. Exp. Ther., 184 (1973) 1-10. 12 Jobe, P.C. and Laird, H.E., Neurotransmitters abnormalities as determinants of seizure susceptibility and intensity in the genetic models of epilepsy, Biochem. Pharmacol., 30 (1981) 3137-3144. 13 Jones, B.E., Halaris, A.E., Mcllhany, M. and Moore, R.Y., Ascending projections of the locus coeruleus in the rat. I. Axonal transport in central noradrenaline neurons, Brain Res., 127 (1977) 1-21. 14 Jonson, G., Hallman, H., Ponzio, F. and Ross, S., DSP4[N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine) - a useful denervation tool for central and peripheral noradrenaline neurons, Eur. J. Pharmacol., 72 (1981) 173-188. 15 Jouvet, M., Biogenic amines and the states of sleep, Science, 163 (1969) 31-41. 16 Jung, R., Blocking of petit mal attacks by sensory arousal and inhibition of attacks by an active change in attention during the epileptic aura, Epilepsia, 3 (1962) 435-437. 17 Kendall, D.A., Brown, E. and Nahorski, S.R., Alpha 1-adrenoreceptor-mediated inositol phospholipid hydrolysis in rat cerebral cortex: relationship between receptor occupancy and response and effect of denervation, Eur. J. Pharmacol., 114 (1985) 41-52. 18 Levitt, P. and Noebels, J.L., Mutant mouse tottering: selective increase of locus coeruleus axons in a defined singlelocus mutation, Proc. Natl. Acad. Sci. U.S.A., 78 (1981) 4630-4634. 19 Libet, B., Gleason, C.A., Wright, E.W. and Feinstein, B.,
85 Suppression of an epileptiform type of electrocortieal activity in the rat by stimulation in the vicinity of locus eoeruleus, Epilepsia, 18 (1977) 451-462. 20 Mareseaux, C., Micheletti, G., Vergnes, M., Depaulis, A., Rumbach, L. and Warter, J.M., A model of chronic spontaneous petit mal-like seizures in the rat: comparison with pentylenetetrazol-induced seizures, Epilepsia, 25 (1984) 326-331. 21 Mason, S.T. and Corcoran, M.E., Catecholamines and convulsions, Brain Res., 170 (1979) 497-507. 22 Maynert, E.W., Marczinski, T.J. and Browning, R.A., The role of the neurotransmitters in the epilepsies. In: W.J. Friedlander (Ed.), Advances in Neurology, Vol. 13, Raven Press, New York, 1975, pp. 79-147. 23 Micheletti, G., Vergnes, M., Marescaux, C., Reis, J., Depaulis, A., Rumbach, L. and Warter, J.M., Antiepileptic drug elvaluation in a new animal model: spontaneous petit mal-like epilepsy, Arzneim.-Forsch./Drug Res., 35 (1985) 483-485. 24 Micheletti, G., Warter, J.M., Marescaux, C., Depaulis, A., Tranchant, C., Rumbach, L. and Vergnes, M., Effects of drug affecting noradrenergic neurotransmission in rats with spontaneous petit real-like seizures, Eur. J. Pharmacol., 135 (1987) 397-402. 25 Noebels, J.L. and Sidman, R.M., Inherited epilepsy: spikewave and focal motor seizures in the mutant mouse Tottering, Science, 204 (1979) 1334-1336. 26 Noebels, J.L., A singel gene error of noradrenergic axon growth synchronizes central neurones, Nature, 310 (1984) 409-411. 27 Saper, C.B., Function of the locus coeruleus, Trends Neurosci., 10 (1987) 343-344.
28 Siegel, S. (Ed.), Nonparametric Statistics for the Behavioral Sciences, McGraw-Hill, New York, 1956. 29 Skolniek, P., Stalvey, L.P., Daly, J.W., Hoyler, E. and Davis, J.N., Binding of alpha- and beta-adrenergic ligands to cerebral cortical membranes: effect of 6-hydroxydopamine treatment and relationship to the responsiveness of cyclic AMP-generating systems in two rat strains, Eur. J. Pharmacol., 47 (1978) 201-210. 30 Snead, O.C. III, Noradrenergic mechanisms in gammahydroxybutyrate-induced seizure activity, Eur. J. Pharmacol., 136 (1987) 103-108. 31 Ungerstedt, U., Effect of 6-hydroxydopamine on the monoamine containing neurons in the CNS. In: T. Malfors and H. Thoenen (Eds.), 6-Hydroxydopamine and Catecholamine Neurons, Elsevier/North-Holland, Amsterdam, 1971, pp. 101-127. 32 Vergnes, M., Marescanx, C., Lannes, B., Depaulis, A., Micheletti, G. and Warter, J.M., Interhemispheric desynchronization of spontaneous spike-wave discharges by corpus callosum transection in rats with petit mal-like epilepsy, Epilepsy Res., 4 (1989) 8-13. 33 Vergnes, M., Marescaux, C., Micheletti, G., Reis, J., Depaulis, A., Rumbach, L. and Warter, J.M., Spontaneous paroxysmal electroclinicalpatterns in rat: a model of generalized non-convulsive epilepsy, Neurosci. Lett., 33 (1982) 97-101. 34 Vergnes, M., Marescaux, C., Depaulis, A., Micheletti, G. and Warter, J.M., Ontogeny of spontaneous petit real-like seizures in Wistar rats, Dev. Brain Res., 30 (1986) 85-87. 35 Woodbury, D.M., Neurotransmitters and epilepsy: distinguishing characteristics and unifying precepts, Fed. Proc., 43 (1984) 2529-2531.
79
Elsevier EPIRES 00412
Lesions of noradrenergic neurons in rats with spontaneous generalized non-convulsive epilepsy
Btatrice Lannes a, Marguerite Vergnes b, Christian Marescaux a, Antoine Depaulis b, Gabriel Micheletti a, Jean-Marie Warter a and Eliane Kempf b aClinique Neurologique H6pital Civil, bCentre de Neurochimie C.N.R.S., Strasbourg (France) (Received 30 October 1990; revision received 15 March 1991; accepted 2 April 1991)
Key words: Rat; Absence epilepsy; Neurotoxin; Noradrenaline; Locus coerulens; Lesion
The role of noradrenergic neurons in the control of a spontaneous generalized non-convulsive epilepsy (GNCE) was investigated. In rats with genetic spontaneous absence seizures, we produced lesions using 2 neurotoxins: 6-hydroxydopamine (6-OHDA) and N(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP4). Lesions of noradrenergic neurons were made either in pups by neonatal 60 H D A intraperitoneal (i.p.) injection (2 x 100 mg/kg) or in adult rats by i.p. administration of DSP4 (60 mg/kg) or bilateral microinjection of 6-OHDA in the locus coeruleus (LC) (4 #g//~l, 2 gl/side). Effectiveness of the lesions was controlled by measuring dopamine (DA) and noradrenaline (NA) contents in the brains. Neonatal 6-OHDA administration did not lead to any difference in seizures in adult animals, compared with control rats. DSP4 injections and LC lesions with local injections of 6-OHDA produced a transient increase of the seizures. Within one to two weeks, the seizure duration went back to prelesion levels. No seizure occurred when the same lesions were performed in non epileptic rats. These results suggest that NA is not involved in the genesis of this generalized non-convulsive epilepsy; they confirm that NA participates in the control of seizures in this model, but the rapid development of compensatory mechanisms shows that this control is not critical.
INTRODUCTION A role for catecholamines in human epilepsies has been suggested since the finding that administration of amphetamines suppresses petit real absence seizures, and that administration of reserpine aggravates convulsive and non-convulsive epilepsies 22. In experimental models of seizures, studies have shown that noradrenaline has mostly an inhibitory effect 3-6'8A1'12'35. In the spontaneous generalized non-convulsive epilepsy (GNCE) Correspondence to: Dr. B. Lannes, Cfinique Neurologique, H6pitai Civil, 67091 Strasbourg Cedex, France.
model in the Wistar rat, seizures are characterized by the occurrence of bilateral and synchronous spike and wave discharges (SWD) on the cortical electroencephalogram (EEG). During the SWD, the rats are motionless, presenting only some twitching of the vibrissae 2°'23'3a'34. A pure epileptic strain has been inbred from the initial colony at our laboratory, while a non-epileptic one has been outbred from the same colony. Previous pharmacological studies have shown inhibitory control of noradrenaline in this model. Activation of a-noradrenergic neurotransmission with a-1 agonists (cirazoline, ST 587) or an a-2 antagonist (yohimbine) reduces SWD duration; reduction of a-nor-
0920-1211/91/$03.50 © 1991 Elsevier Science Publishers B.V. All rights reserved
80 adrenergic neurotransmission with an a-1 antagonist (prazosin) or an a-2 agonist (clonidine) enhances seizure duration. Drugs interacting with flnoradrenergic neurotransmission have no effect on the seizures24. In order to confirm the role of noradrenaline in the control of absence seizures, lesions of noradrenergic systems were produced in rats with spontaneous SWD, using selective neurotoxins. N-(2chloroethyl)-N-ethyl-2-bromobenzylamine (DSP4) crosses the blood-brain barrier and produces a specific destruction of noradrenergic neurons in the central nervous system (CNS) 14. Subcutaneous injection of 6-hydroxydopamine (6-OHDA) in the newborn rat also leads to the degeneration of noradrenergic neurons in the CNS (at birth, the blood-brain barrier and the dopaminergic systems are not yet functional)2'31. In the adult rat, local injection of 6-OHDA into the locus coeruleus (LC), the mesencephalic nucleus which contains the main noradrenergic neurons projecting towards the forebrain, leads to the specific destruction of the noradrenergic neurons of this nucleus. Because output from the LC is mostly ipsilateral, the effect of unilateral 6-OHDA lesion of the LC on SWD was examined in rats in which the corpus callosum had been previously transected in order to avoid interhemispheric seizure propagation.
in the LC were performed in adult animals (5-6 months), under general anesthesia, by the mean of a microsyringe using stereotaxic methods (A/P, -1.5 mm; M/L, 1.2 mm; DV, 7 and 7.8 mm, with 3. as the reference). At each site 4 #g in 1 #1 was injected (8/~g per side). The injection lasted 1 min, and the needle was left in place for an additional 1 min.
Corpus callosum transection. Transection of the corpus callosum was carried out under general anesthesia, using a sharp blade fixed vertically on the stereotactic instrument and moved from back to front (AP, 2-10 ram; DV, 4-4.5 mm with ~ as the reference) through the medial suture of the skull, which had previously been removed with a dental drill, as described previously32. EEG recordings Recordings were made between 9:00 a.m. and 5:00 p.m. using an Alvar electroencephalograph (Minidix-TR). Derivations were fronto-parietal, left and right. After a habituation session of 15 min, the EEG was recorded for 1 h. On the days before lesion, in adult-lesioned animals, 3 recordings were made, and the cumulative SWD duration for each hour was measured. The average of these 3 recordings was used as the reference. In 6OHDA injected neonate rats, the EEG was recorded when they became adults.
MATERIALS AND METHODS
Neurotoxic substances Animals Newborn or adult Wistar rats from the strain with GNCE and from the control strain were used in this study.
DSP4 was dissolved in distilled water; 6-OHDA (Sigma, USA) was dissolved in saline with ascorbate (0.2%) as an antioxidant. The respective vehicles were used as controls.
Surgery
Biochemical assays
Implantation. In adult animals, under general pentobarbital anesthesia (40 mg/kg intraperitoneally, i.p.), 4 stainless steel electrodes were implanted into the skull in a bilaterally symmetrical position over the left and right, frontal and parietal cortex. The electrodes were connected to a microconnector fixed to the skull.
Lesion of the LC. Bilateral injections of 6-OHDA
At the end of the experiments, the rats were decapitated and their brains were immediately dissected at 4 °C. The anterior part of the brain, comprising telencephalon and diencephalon, was removed and NA and DA were assayed. NA and DA were determined simultaneously utilizing a reverse phase chromatography procedure coupled with an electrochemical detector (LCEC). On the day of analysis, frozen samples of the anterior part of brains were weighed and homogenized in
81 HCIO 4 0.1 N containing Na-metabisulfite 6 mM and EDTA 1 mM. The homogenates were centrifuged at 10 000 x g for 20 min at 4 °C. Aliquots of the supernatants were transferred into the LCEC system with a Wisp automatic injector (Waters). The LCEC system consisted of a Bioanalytical system LC4 amperometric detector with a glassy carbon working electrode and a pump (Waters). The potential was set at 800 mV (vs. Ag-AgC1 reference electrode). The column, a Bondapack phenyl column (10/zm particle size, 300 x 3.1 mm i.d.) was purchased from Waters Assoc. The flow rate was 1.4 ml/min, and the sensitivity was set at 5 nA/V (1 V full scale). The mobile phase consisted of 3% methanol in 0.1 M Na-phosphate buffer pH 2.5/Na2-EDTA 0.1 mM/1-octane sulfonic acid-Na salt (BDH) 2 mM. 3,4-Dihydroxyhydrocinnamic acid (Aldrich) was used as internal standard. Control rats of the same age were killed simultaneously and the same biochemical analyses were performed. Data analysis Results are given as the cumulative SWD duration, in seconds, during 1 h of recording. In the case of corpus callosum transection, the results were analyzed separately for each hemisphere. Comparison of the seizure duration was done for
each test day after treatment vs. the reference period, using non-parametric tests (Friedmann and Wilcoxon) 28. Each animal was thus used as its own control. Biochemical data were compared between groups using Student's t-test. RESULTS 6-OHDA in newborn rats Newborn animals were injected twice subcutaneously 12 h and 24 h after birth with 100 mg/kg of 6-OHDA in a volume of 2 ml/100 g. Eight rats from the epileptic strain and 4 from the non-epileptic strain received 6-OHDA, and 8 epileptic and 4 non-epileptic rats were injected with the vehicle. Control rats (injected with vehicle) were chosen among siblings of the lesioned rats. At the age of 5 months, all 24 rats were implanted, and 3 EEGs of 1 h each were recorded between the ages of 5 and 6 months. The animals were killed at 7 months, and brain NA and DA were measured. Within the epileptic rats, no difference was observed in the SWD duration between the group injected with 6-OHDA and the group injected with vehicle only (results not shown). In non-epileptic animals treated with 6-OHDA, no SWD were ever observed. Biochemical analysis showed an important decrease in NA content, in treated animals
TABLE I
Concentrations of brain NA and DA in the different experiments Concentrations are expressed in ng per g of brain tissue (mean + S.E.M.). Treated rats versus controls in each epileptic (Epi) and nonepileptic (Non-epi) group. *, P < 0.05; **, P < 0.001.
Experiment 6-OHDA in newborns
Epi Non-epi
DSP4
Epi Non-epi
Bilateral lesion of LC
Epi Non-epi
Animals
n
NA (ng/g)
DA (ng/g)
6-OHDA Controls 6-OHDA Controls
8 8 4 4
75 294 20 287
+ + + +
45** 34 40* 46
1302 + 1477 + 990 + 1264 +
139 321 263 150
DSP4 Controls DSP4 Controls
5 4 4 4
113 + 366 + 151 + 353 +
19"* 48 98* 23
1304 + 1046 + 1157 + 1115 +
258 212 295 114
6-OHDA Controls 6-OHDA Controls
6 6 3 4
109 + 316 + 179 + 327 +
92* 50 86* 47
1405 + 1256 + 1011 + 1359 +
319 206 359 110
82 compared to the control ones. D A remained unchanged in the 4 groups (Table I). DSP4 in adult rats Seven epileptic rats (5-6 months old) were injected (i.p.) with 60 mg/kg of DSP4 in a volume of 2 ml/kg. EEGs were recorded every day during the first week following injection of DSP4, and also on day 10. Four non-epileptic rats were also injected with DSP4. EEGs were recorded on days 1, 3, 6, 10 and 15 following the injection. The animals were killed on day 16 for measurement of NA and DA in their brains. In epileptic rats, injection of DSP4 induced an important increase of SWD duration, beginning from the very first day after the injection and being maximal on the second day. The SWD duration went back to the reference values between days 6 and 10 (Fig. 1). DSP4 in non-epileptic rats did not induce any SWD. The measurement of NA in the cortex revealed a significant decrease in both epileptic and non-epileptic rats compared with their respective controls. Measurement of DA showed no significant difference between lesioned and intact animals (Table I). Bilateral lesion of the L C Six epileptic and 4 non-epileptic adult animals were injected in the LC. EEGs were recorded on days 1, 3, 4, 5, 9, 12 and 21 following the lesion.
All animals were killed on day 22 for measurement of NA and DA in their brains. Four epileptic rats were sham-operated; the needle of the microsyringe was, under general anesthesia, lowered stereotactically above the LC, but nothing was injected. 6-OHDA lesion of the LC in epileptic rats resuited in a transient increase in SWD duration, beginning on the day following the operation and being maximal on day 4 (Fig. 2). On day 12, the SWD duration had returned to reference values. No SWD were observed in non-epileptic rats after this lesion. Biochemical analysis showed a significant decrease of NA levels in lesioned epileptic and non-epileptic rats when compared with their respective controls. DA levels were not significantly different within the same animals (Table I). The 4 sham-operated rats did not show any significant difference in SWD duration before and after the operation (results not shown). Unilateral lesion of L C after corpus callosum transection After a corpus callosum transection, seizures may develop independently on one hemisphere or the other, and often occur unilaterally and alternately on both sides 32. The cumulated seizure duration for each hemisphere was measured. The ratio between the two sides was close to 1 before the LC lesion. The transections were made one week before the neurotoxic lesion, which was per-
2000 2000 1500
~o
/
1000
1500 ~
t
IXT • T I\T •.~_ (3
•
~ 1 ~
o
1000
1
C23
500
500
REF 1
5
10
post lesion days
Fig. 1. Effect of DSP4 on SWD duration in rats (mean + S.E.M.). Injection was performed on day 0 (between the reference and day 1). *, P < 0.05 (Wilcoxon test).
REF 1
5
10 15 post lesion days
20
Fig. 2. Effect of a bilateral lesion of LC on SWD duration in rats (mean + S.E.M.). The lesion was performed on day 0 (between the reference and day 1). *, P < 0.05 (Wilcoxon test).
83
/\ j\
1000
o
0--0 0--0
right left
5 10 post lesion dcys
15
,o,
REF 1
Fig. 3. SWD durations in left and fight hemispheres following a 6 - O H D A lesion of the right LC in a rat with transected corpus callosum.
formed as described above, on the right side only. Two epileptic adult animals were operated upon and their EEGs were recorded during the 2 weeks following the neurotoxic lesion. In both rats, a transient increase in SWD duration was observed, which was maximal on the fourth day after the lesion and predominated on the lesioned side (right side). SWD duration returned to values preceding the neurotoxic lesion about 8 days after the lesion (Fig. 3 shows the results for I of the 2 rats). DISCUSSION The present results suggest the existence of an inhibitory control of NA over the SWD in this model of GNCE. The lesion of NA neurons, being either localized to the LC cell bodies (6-OHDA in the LC) or to their terminals (DSP4 injections, which has been shown to produce its neurotoxic action mainly on the terminals of NA neurons from the LC TMincreases SWD duration). This corroborates the conclusions of a previous pharmacological investigation 24. However, NA does not~ seem to be implicated in the genesis of SWD since seizures were never observed in non-epileptic rats, whatever the lesion, even when NA depletion was performed in newborns of the non-epileptic strain. At that time, SWD have not yet developed in animals from the epileptic strain 34.
Moreover, despite the permanence of the NA deficit measured in the brains, increases of SWD were always transient. This result suggests the development of compensatory mechanisms, leading to the recovery of 'normal' SWD duration in spite of the remaining deficit in NA. The absence of difference observed in SWD duration in rats which had received subcutaneous 6-OHDA at birth could be explained by a similar compensation. One of these mechanisms could be an up-regulation of NA receptors as it has been described after such neurotoxic lesions 7'1°A7'29. Another possibility might be a compensation from the remaining NA neurons themselves (the depletion of NE is between 65 and 75% in epileptic animals), perhaps by the mean of sprouting collaterals. LC neurons send projections toward the neocortex, the hippocampus, the amygdala, the thalamus, the colliculi and the cerebellum 13. The predominance of ipsilateral projections may account for the predominant aggravation of SWD on the lesioned side after unilateral lesion. Noradrenergic neurons from the LC are known to participate in the control of vigilance levels, and more specifically, in the degree of awareness 1'9'15'27. On the one hand, multi-unit recordings of LC neurons in the rat have shown that their spontaneous activity varies with the vigilance levels, and is enhanced in case of sensory stimulations 1. On the other hand, in the GNCE rat, sensory stimulations block seizures as they do in human petit mal epilepsy 16. The LC may thus control seizures directly or indirectly via its action on vigilance levels. In some other models of absence epilepsy, lesions of NA neurons have also been described. In the pentylenetetrazol (PTZ) model in the rat, seizures are potentiated by injection of 6-OHDA into the dorsal bundle (ascending NA system) in the mesencephalon 2~. On the other hand, electrical stimulation of the LC suppresses SWD induced by subconvulsive dose of PTZ 19. In the ~,-butyrolactone model in the rat (GBL), neonatal treatment with 6-OHDA leads to more severe and prolonged seizures induced by acute administration of GBL in the adult animal 3°. However, these data contrast with previous data obtained in the Tottering mouse, another genetic model where mice display 3 abnormal behaviors: wobbly gait, motor seizures
84 a n d s u d d e n a r r e s t of m o v e m e n t c o n c o m i t a n t with S W D o n the E E G 25. This last b e h a v i o r was cons i d e r e d as a m a n i f e s t a t i o n o f a b s e n c e - l i k e seizures. I n t h e s e m i c e , an a b n o r m a l i n c r e a s e in locus c o e r u l e u s axons has b e e n d e s c r i b e d w i t h o u t a n y i n c r e a s e in the n u m b e r o f cell s o m a t a in t h e L C TM. A f t e r n e o n a t a l 6 - O H D A t r e a t m e n t , t h e m i c e fail to d e v e l o p S W D o r gait d i s t u r b a n c e . B i l a t e r a l inj e c t i o n o f 6 - O H D A in L C in a d u l t a n i m a l s l e a d s to an i m p o r t a n t d e c r e a s e in S W D , w h i l e u n i l a t e r a l i n j e c t i o n s u p p r e s s e s S W D o n t h e i p s i l a t e r a l cortex 26. Such o p p o s i n g findings in 2 a n i m a l m o d e l s o f p e t i t m a l e p i l e p s y suggest t h a t N A is n o t a critical n e u r o t r a n s m i t t e r i n v o l v e d in t h e c o n t r o l o f sei-
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