Mechanisms underlying modifications in the severity of audiogenic convulsions

Life Sciences Vol . 20, pp . 2047-2060, 1977 Printed in the U .S .A .

Pergamon Press

MECHANISMS UNDERLYING MODIFICATIONS IN,THE SEVERITY OF AUDIOGENIC CONVULSIONS Alice LEHMANN Laboratoire de Physiologie Acoustique, I .N .R .A ., E .P .H .E ., C .N .R .S . 78350 Jouy en Josas, France (Received in final form May 19, 1977) Summary Catecholamine levels were selectively decreased in peripheral sites or in brain of mice by intravenous or intraventricular injections of 6-hydroxydopamine . Brain dopamine alone was decreased by administering i . p . desmethylimipramine prior to 6 hydroxydopamine injections . A brain noradrenaline decrease does not induce mortality in a tonic strain of mice but can transform clonic audiogenic seizures into tonic seizures in a clonic mouse strain, while a dopamine depletion alone has no effect . However, a noradrenalins decrease at peripheral sites increases the number of lethal seizures in a tonic strain without modifying clonic seizures in a clonic strain . Catecholamine metabolism is neither involved in the onset of seizures in a non-sensitive strain nor involved in the change of clonus into tonus in unilaterally deafened tonic strain animals, which in this case present only clonic seizures . Many studies have shown that catecholamine metabolism does not control the onset of audiogenic seizures, but does modify their severity (1-12) . With minor variations related to the spe cies (rat or mouse), strains, or procedures used, all authors agree that a total body increase or decrease in catecholamine levels reduces or enhances, respectively, convulsions induced by sound stimulation (1-12), changing clonic into tonic seizures and tonic into lethal tonic seizures . But very few studies have considered the separate effects of noradrenaline (NA) and dopamine (DA) (4), their respective sites of action (9), or the defined phase of the seizure upon which they act . Working with mice genetically selected over 20 years for their high sensitivity or resistance to audiogenic seizures, we have been able, by drug induced modifications in biochemistry of the animals, to establish a link between some of these modifications and the corresponding behavior of mice subjected to sound stimulation . In previous studies (7-11) we demonstrated a close relationship between catecholamines and the severity of convulsions an increase of these amines reduces convulsion severity or even totally suppresses them . A decrease intensifies the seizures and induces mortality (11) . Furthermore, it appears that protection is related more to the rate of NA available at receptor si2047

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tes than to its total level (8), and that lethal seizures can even be induced after lowering the NA level at peripheral sites alone (9) . However, this peripheral decrease has no effect on clonic seizures . Other factors can also modify the severity of audiogenic convulsions . For example, in the tonic strain, unilaterally deafening the animal totally suppresses the tonic phase of the seizure . In such mice, clonus can be changed into tonus by drugs acting solely on central nervous system (C .N .S .) excitability . A decrease in catecholamine levels has no effect (13) . Considering these data, we have developed the following hypotheses 1)

The biochemical processes which lead to lethal tonic seizures are different from those which change clonic into tonic seizures in strains genetically selected for these specific reactions (tonus without death or clonus) . Both processes depend on NA depletion but involve different sites of action, and

2) The mechanisms by which clonus is converted to tonus differs between the clonic strain and the unilaterally deafened tonic strain of mice . We attempted to verify these hypotheses experimentally by pharmacological manipulations .

Materials and Methods Animals Three sublines of Swiss Rb mice were genetically selected over 20 years for their resistance or their various sensitivities to sound stimulation . One (14) subline is non-sensitive while incidence of seizure in the other two is 99,9% . Fig . 1 shows audiogenic seizure patterns in the two sensitive lines . In one subline, after a latent period and a running phase, the process ends in clonic seizure . In the other subline, the clonic seizure is followed by a tonic phase and the mouse recovers after a second clonic phase . These seizures are never lethal except through drug action decreasing NA (5,7-11) . Conversely, when mice of this tonic strain are unilaterally deafened before sound stimulation, by introducing wax or glycerine into the auditory duct or by destroying the middle ear ossicles, the convulsions stop at the first clonic phase . [131 . Two to three month old mice of both sexes were selected from the clonic and tonic sublines for our experiments . Three groups of these animals were used : clonic strain, tonic strain and unilaterally deafened tonic strain mice .

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LATENT PERIOD RUNNING PHASE ist CLONIC PHASE * TONIC PHASE \ 1--01, 2d CLONIC PHASE

DEATH

FIG .

RECOVERY

1

Audiogenic seizure

pattern

The arrows indicate in increasing order of severity the different stages where seizures can stop running or clonic phase, + tonus, death . * This pattern is shown whether seizure susceptibility is of genetic origin or induced by unilateral deafening . Sound stimulation a

Mice were placed sound proof room .

in

a wire-meshed cage

(10

x 10

x 10 :cm .) in

100 dB

Audiogenic seizures were induced by delivery of 10 S .P .L . signal (at the level of the mouse) lasting

death)

The final stage of seizure (clonus, was recorded for each individual .

tonus,

kHz and 1 min .

recovery or

+ In the Rb strain of mice, running phase and clonus cannot be genetically separated as they are phenotypic expressions of the same genotype (E . Boesiger and A . Lehmann : unpublished data) . For this reason, only clonua has been considered in our experimental results .

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Drugs To verify our hypothesis the catecholamine levels were selectively decreased in the brain and/or in peripheral sites in tonic and clonic strains mice by different pharmacological means : 11 Globally (whole body) by reserpins (1 mg/kg i .p .) administered 1 or 24 h . before the test, or by a short-acting reserpine-like catecholamine depleting agent RO 4-1284 (20mg/kg i .p .) (15), administered 1 h . before the test . 21 Selectively by 6-OHDA which decreases NA levels by chemical sympathectomy . This drug does not cross the blood brain barrier : injected i .v . at the doses of 23 mg/kg twice within 8 hours and 46 mg/kg twice within 8 hours one week later (16), it lowers the peripheral catecholamine level with only a slight depletion in the brains injected i .v .c . at the dose of 68 g per mouse in 25 Ail (17) saline ascorbic acid, it decreases cerebral NA and DA only (all doses are expressed in mg or jug of base) . If one hour before this last injection, animals receive an i .p . injection of 25 mg/kg of desmethylimipramine (OMI) (18) only brain DA is decreased . All tests were performed seven days after the last injection of 6-OHDA .

r

C .N .S . excitability was increased in the unilaterally deafened tonic strain by injections of one of the convulsants, Isoniazid (INH), pentylenetetrazol or picrotoxin . The doses of INH (100 mg/kg s .c .), pentylenetetrazol (30 mg/kg i .p .) or picroto xin (4 mg/kg i .p .) was selected as the dose of the convulsant which did not induce audiogenic seizures in the nonaudiogenic strain of mice . In all cases control animals received the same vehicle volume (0,5 ml) . N .A . assay NA assays were performed in mice not previously subjected to sound stimulation . They were killed by immersion in liquid nitrogen and brains and hearts were quickly removed . NA was evaluated in both tissues by e modification of Maickel's method (5) . Results were evaluated with the Student's t test . Results The differences in NA decrease due to the route of 6-OHDA injections can be seen in Fig . 2 in which brain and heart NA contents 7 days after i .v . or i .v .c . 6-OHDA injections are plot ted : i .v . injections slightly decrease brain NA with a high depletion in the heart, while i .v .c . injections deplete brain NA only . 1) change of tonus into lethal tonic seizures . As clonic strain and unilaterally deafened tonic strain animals show no tonic seizures without drugs, only non deafened tonic strain animals are considered here .

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Fig . 3 shows the mortality rate in tonic seizures against A brain NA decrease (by 6-OHOA i .v .c .) brain or heart NA level . induces no lethal seizures,while a heart NA decrease (by 6-OHDA i .v .) increases mortality rate to 100 ; . Drugs such as reserpine, which simultaneously decrease brain and heart NA, also increase the incidence of death in these animals .

BRAIN

IM.C .

HEART

FIG .

BRAIN

I.V .

HEART

2

Brain and heart noradrenaline levels after intravenous (i .v .) or intraventricular (i .v .c .) 6-OHDA injections . Each value represents the mean of 10 animals . 2)

Change

of clonus

a)

genetically

into

selected

tonus . clonic

strain .

Table 1 shows what changes in central and/or peripheral NA concentrations and increases in C .N .S . excitability have on audiogenic seizure intensity in clonic mice . A peripheral NA decrease due to 6-OHDA i .v . induces no tonic seizures . A central NA decrease due to 6-OHDA i .v .c . transforms 50% of clonic seizures into tonic seizures . A decrease in both brain and heart NA through administration of reserpine (24 h .after injection) or RO 4-1284 (1h . after injection) also transforms clonus into tonus but with a high mortality rate . The change of clonus into tonus can be related to NA decrease in the brain while

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NA O

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- _9 LETHALITY(96) 9 0---0 HEART NA LEVEL ( 0/6OFCONTROL)

BRAIN NA LEVEL (40OF CONTROL)

Lethality C

100

100

50

50 "

FIG . 3 Comparison between heart and brain NA levels and mortality 1h . after reserpine injection and 7 days after 6-OHDA injections in mice normally showing non-lethal tonic audiogenic seizures . (Number of animals : 10 per NA dosage, 50 for mortality) . the high rate of mortality can be attributed to the added effect of peripheral induced NA depletion . Besides acting on NA metabolism, tonic seizures can also be induced in this strain by increasing C .N .S . excitability Thus, in this strain, drug through low doses of INH (Table 1) . action on the C .N .S . alone (6-OHDA i .v .c . or INH) produces a relatively high mortality rate within tonic seizures .

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TABLE

Drugs

205 3

Severity of Audiogenic Convulsions

Route of injection

1

Interval between test and injection

ôof tonic seizures

Mortality rate within tonic seizures (%)

6-OHDA

i .v .

7 days

0

0

6-OHDA

i .v .c .

7 days

50

38

RO 4-1284

i .p .

1 h.

62

64

Reserpine

i .p .

24 h .

75

100

Isoniazid

B.C .

1 h.

62

44

Table 1 indicates the percent of the total number of seizures which are tonic and the percent of these tonic seizures which are lethal in the clonic strain under the influence of va rious drugs . Normally no tonic seizures are induced in clonic strain, without drugs (N a 50) . As 6-0HOA injected i .v .c . decreases NA and DA simultaneously,w ,.e tried to separate the effects of these two amines by selective depletion of DA . One hour before i .v .c . 6-OHDA injections, mice of the clonic strain received an i .p . injection of desmethylimipramine (DMI) (25 mg/kg) . This treatment decreased DA while allowing NA levels to remain almost normal . In the clonic strain of mice, i .v .c . 6-OHOA transforms 50% of clonic seizures into tonic seizures, but the association desmethylimipramine-6-OHDA does not modify clonic seizures (Fig .4) It appears that the change from clonus to tonus is related to a central NA decrease only and is independant of DA level . b)

Clonus induced strain .

by

unilateral deafening

in mice

of the tonic

Reserpine has no effect on seizure severity in this case (13) . However, an increase in C .N .S . excitability induced by non-convulsant doses of convulsants like INH, picrotoxin or pen tylenetetrazol (Fig .5) can change clonus into tonus . While drug action on the C .N .S . alone (6-OHDA i .v .c . or INH) produces a relatively high mortality rate in the clonic strain, a brain NA decrease or an artificial increase in C .N .S . excitability in the tonic strain induces no lethal tonic seizures at all (Table 2) . Table 2

summarizes the above results .

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Severity of Audiogenic Convulsions

60HDA

60HDA + DMI

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C

FIG . 4 % of seizures in the clonic strain after i .v .c . 6-OHDA injections alone or preceded by DMI injections . C : control DMI : desmethylimipramine .

Discussion The influence of catecholamines on experimentally induced convulsions has been widely studied . Numerous studies on electroshock or various chemically induced convulsions have shown a relationship between catecholamine levels and the decrease or increase of convulsion severity (1-2) . Few ty of the tions, or phases of

authors, however, have attempted to relate the severiseizures to the sites of catecholamine level variato determine their specific action on the different the convulsive sequence .

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Tonic seizures PE : PENTETRAZOL INH : ISONIAZID

100

PI : PICROTOXIN R : RESERPINE

50-

PE

INH

PI

R

FIG . 5 Percentage of tonic seizures in unilaterally deafened mice which show only clonic seizures without drugs . An arbitrary unit score can be given to the intensity of audiogenic seizures and most authors (19) evaluate drug effects by these increased or decreased values . The above data clearly show that different mechanisms are involved in provoking lethal tonic seizures and in changing clonus into tonus . Considering this, it might be useful to specify, when dealing with an increase in audiogenic seizure severity, whether it is a change of clonus into tonus or of tonic non-lethal seizures into lethal seizures . Fig . 6 illustrates schematically the different mechanisms influencing the severity of audiogenic seizures, based on our experimental results . In the tonic strain of mice, raising cate cholamine levels decreases the intensity of seizures (B) . changing tonus .into clonus . A peripheral NA depletion leads to lethal seizures, but a central NA decrease has no effect . Conversely, in the clonic strain, a peripheral NA decrease has no effect . The transformation of clonus into tonus in this case ac-

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Severity of Audiogenic Convulsions

DEATH

LONIC SEIZURES

CLONIC SEIZURE

ONIC SEIZURES

2057

CLONIC SEIZURES

TONIC SEIZURES FIG .

6

Various mechanisms modifying the severity of audiogenic convulsions curs through action at the C .N .S . level only, either by a cenWhen clonus is due tral NA depletion or by a small dose of INH . to unilateral deafness, it can be changed into tonus only by an increase in C .N .S . excitability . wit h small doses of INH or of others convulsants . In this last case, increased seizure severity is not linked to NA metabolism, but rather to a similar mechanism by which INH at higher doses (20) can induce audiogenic seizures in a genetically resistant mouse strain . In both cases change of clonus into tonus depends on variations in the C .N .S ., whereas change of tonic non-lethal seizures into fatal seizures depends on peripheral metabolic changes . These results are in agreement with Bourn's hypothesis (4) proposing a different mechanism responsible for the origin of audiogenic convulsions or for the severity of these seizures in the Rat . It must be noticed that in tonic seizures induced in the genetically clonic strain, either by an NA decrease or by INH, 38% are lethal . This differentiates them from tonic seizures due to the INH effect on unilaterally deafened mice and from the effect of a central NA decrease in the tonic strain, which lead to non-lethal tonic seizures . Thus, there is a different sensi-

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tivity threshold to respiratory failure during tonic audiogenic convulsions between the clonic and the tonic Rb strains of mice . Just as in other audiogenic seizure suceptible strains such as DBA (21 ) . The above data seem to indicate that, in the mouse strains studied, NA metabolism at least is invoked in the severity of audiogenic seizures . When reserpine is used, both NA and sero tonin (5-HT) are depleted . However, a reserpine-like effect can be induced in the clonic strain by a central NA decrease alone and in the tonic strain by a peripheral NA decrease alone, thus eliminating the necessity of 5-HT effect . Other evidence in favor of this argument is that a decrease in NA due to d methyl para tyrosine is much more effective than a decrease in 5-HT due to parachlorophenylalanine in indu cing lethal tonic seizures in the tonic strain (22) . As administration of desmethylimipramine prior to 6-UHDA injection can deplete DA without really affecting NA levels and without increasing clonic seizure severity, the DA effect is thus eliminated . These results are in agreement with Bourn's findings in the Rat (4), where NA has a functional role in the modulation of audiogenic seizures severity, while DA does not . Boggan (3) showed that a high level of DA protects mice or decreases the severity of audiogenic convulsions . These results are not opposed to our findings . As we previously demons trated, any increase in catecholamines or in 5-HT can protect mice, depending on the level of the mediator available at the receptors sites (8) . However, an increase in convulsion severity seems to depend more specifically on the NA level or on its site of depletion . References 1 . 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 .

S . BIELEC . Arch . Int . Pharmacodyn ., 119, 352-357 (1959) W . BEVAN . R .Mc .C . CHINN . J .Comp . Physiol . Psycho( ., _50, 311-314 (1957) . W .O . BOGGAN and L .S . SEIDEN . Physiol . Behav ., _6, 215-217 (1971) W .M . BOURN . Ph . 0 . Thesis, Univ . of Arizona 11974) . A . LEHMANN and Y . MOROT-GAUDRY . C .R . Soc . Biol ., 165 (1 , 14-18 11971) . K . SCHLESINGER, K .L . STAVNES and W .O . BOGGAN . Psychopharmacol ., 15 (3) 228-232 (1969) A . LEHMANN and R .G . BUSNEL . Colloque C .N .H .S . n ° 112 , 453-471 (1963) . A . LEHMANN . Life Sci ., 6, 1423-1431 (1967) A . LEHMANN . Life Sci ., _9, part 1, 251-257 (1970) . A . LEHMANN . B .N . HALPERN, R .G . BUSNEL . J .d e Physiol . . 49 265-268 (1957) . A . LEHMANN . J . d e Physiol ., 57 . 646 (1965) .

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12 . 13 . 14 . 15 . 16 . 17 . 18 . 19 . 20 . 21 . 22 .

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C . KELLOGG . Brain Res ., 106, 87-104 (19761 . A . LEHMANN . J . d e Physiol ., 65 (2) 258A-259A (1972) . Mouse News Letter, n ° 21, Companion issue 4Z (1959) . A . PLETSCHER, A . BROSSI and K .F . GEY . Int . Hev . .Neurobiol . 4, 275-306 (1962) . H . THUENEN and J .P . TRANZER . Naunyn Schmiedbergs Arch . Pharmak . U . Exp . Path ., 261, 271-288 (1968) . H .T . BRITTAIN and S .L . HANDLEY . J . Physiol . (Lond .), 192 805-813 (1967) . A . SCOTTI de CAROLIS, H . ZIEGLER, P . DEL BASSO and V .G . LONGO . Physial . Behav ., 7, 705-708 (1971) . K . SCHLESINGER, R .C . ELSTON and W .O . BOGGAN . Genetics , _54, 95-103 (1966) . A . LEHMANN . J . d e Physiol ., 55, 282-283 (1963) . C .S . HALL . J . Hered ., 38, 2-6 (1947) . A . LEHMANN . C .R . Soc . Biol ., 162, 24-27 (1968) .