EXPERIMENTAL
Audiogenic
NEUROLOGY
56, 518-526 (1977)
Seizure Levels in C57BL/6J to the Priming and Testing ROBERT
Brain
Research
Institute,
A.
SCHREIBER
Mice Stimuli *
University of Tennessee Ceftter Memphis, Tennessee 38143 Received
November
as Related
for
the Health
Sciences,
24,1976
The effects of reversing two audiogenic priming stimuli during testing for audiogenic convulsions were examined in C57BL/6J mice not normally susceptible to audiogenic seizures on the first exposure to an acoustic stimulus. Mice were exposed to either a small (b) or a large (B) bell during audiogenie priming for either 10, 30, or 60 s at 16 days of age. They were then tested using either the same bell or under reversed conditions (bb, BB, bB, or Bb) at varying ages thereafter. Responses were graded as expected according to the duration of priming. The low incidences of audiogenic seizures observed during testing were similar for the bb and bB groups, and the high incidences were similar for the BB and Bb groups. Thus, priming conditions determine later seizure levels, not testing conditions.
INTRODUCTION Mice of appropriate genotypes, within appropriate age ranges, are subject to audiogenic priming, a phenomenon of seizures in response to a second exposure to a sufficient acoustic stimulus but not to the first (12, 14). The development of this response is best characterized for the C57BL/6J strain, in which the magnitude of the average seizure responseis dependent on the age at first exposure (during which no behavioral seizure occurs) and also on the duration in days between the first and secondacoustic challenges. Agreement is general that these mice are maximally sensitive to the effects 1 This research was supported by National Institutes of Health General Research Support Grant RR 54.23 to the College of Medicine, University of Tennessee Center for the Health Sciences. The aid of Dr. J. Studebaker, Memphis State University Speech and Hearing Center, in measuring the power spectra, and of Ms. Linda Craddock and Mr. Toa Nguyen in mouse testing is acknowledged. I also thank Ms. Mary Jackson for her excellent typing skills. Request reprints from Dr. Robert A. Schreiber, Brain Research Institute, Department of Biochemistry, University of Tennessee Center for the Health Sciences, Memphis, Tennessee 38163. 518 Copyright All rights
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
ISSN
0014-4886
A~DIOGENIC
SEIZURES
AND
STIMULI
519
of a first exposure to acoustic stimuli at a time coincident with the opening of the ear canal between 14 and 16 days of age, followed by decreasing sensitivity thereafter ($6, 12,21) . At present little is known about the cellular event(s) induced by an initial exposure to an acoustic stimulus which culminates in a convulsion upon a subsequent reexposure. Appropriate experiments on a cellular level, however, await a reduction in within-group variability brought about by knowledge and control of parameters which affect the magnitude and time course of induced susceptibility to audiogenic seizures. For example, even with use of the same strain of mouse (C57BL/6J), primed at the same age (16 days), gross disagreement exists as to both the levels of peak seizure risk at peak susceptibility (usually considered to be 21 days) and also the duration of induced susceptibility to audiogenic seizures (3, 6,21). Many investigations have long recognized that the intensity of the stimulus has been an important variable in determining levels of seizures ,in strains of mice susceptible on an initial exposure (1, 9, 23). More recently, others have shown that intensity of the stimulus during audiogenic priming also affects later seizure susceptibility (3, 6, 11) as does duration of exposure (3, 11). Recent data indicate that stimulus intensity and duration of priming interact to affect both the magnitude and the time course of induced susceptibility to seizures when the same stimulus complex used during audiogenic priming is used for testing (20). This study (i) examines whether the complex p&&g stimulus or the complex testing stimulus, or both, are important in determination of seizure levels, and (ii) determines whether or not the time course of the response is primarily influenced by the priming or testing stimuli. METHODS mice (Mus musculus) was used in Subjects. A total of 380 C57BL/6J these experiments. [The origins and inbreeding of these mice have been described by Jay (15).] A nimals were raised in our colony, and were derived from breeding stocks obtained from the Jackson Laboratory, Bar Harbor, Maine. Mice were maintained ad libituvpz on Wayne Mouse Breeder chow and tap water. An automatic switch turned on fluorescent lights in the colony room at 0700 h and off at 1900 h. The temperature was maintained at approximately 23 ’ C. Apparatus and Procedure. Mice were placed in a battery jar, 30 (outside diameter) x 46 cm high, enclosed in a sound-attenuating box with an observation window as previously described by Schlesinger et al. (19). Each mouse was allowed 15 s to explore during which time the lids to the jar and the box were put in place. An Edwards #13-3 bell was attached
520
ROBERT
A.
SCHREIBER
to one jar lid and an Edwards #340 bell was attached to another. The voltage delivered to the #13-3 electric bell was set to generate a broadband output of 108 C 1 db at the level of the mouse ; the #340 bell generated a broad band output of 127 k 2 db. The small bell will be referred to as the b bell and the large bell as B. The power spectrum from 0 to 20 kHz for each bell was measured with a B & K type 4145 condenser microphone placed in the center of the testing jar and a B & K Microphone power supply type 2801. The output was fed into an SDC Model SD330A real time analyzer and the average of 512 passes through the spectrum was plotted for each bell on an H/P 7034A XY recorder. The power spectra of these bells are published (20). All mice were exposed to the noise from one of the two bells between 1100 and 1400 h to control for circadian rhythms (22). All mice were primed at 16 days of age (day of birth is day 0) and remained with their mothers until testing at a later date. The only observable response to the initial exposure to the bell in most mice was the normally observed Preyer reflex, in which the pinnae are reflected downward immediately after the onset of the bell. The very few mice which responded with a wild run or more severe seizure during the priming period were discarded. A number of priming-testing combinations were included in this experiment. Mice were either primed and tested using the same bell (bb or BB), or primed and tested under reversed conditions (bB or Bb). The duration of priming was either 10, 30, or 60 s ( 11,ZO). All mice were retested at a later date for as much as 30 s in the same apparatus under the same conditions as described above. The bell was turned off earlier if the mouse attained a tonic convulsion. [Turning off the bell during even a clonic convulsion does not influence the incidence of tonic convulsions or lethality]. No more than three animals from one litter were used in a single test-retest group. There were 20 mice per group. RESULTS This experiment was conducted during and shortly after an experiment investigating the effects of stimulus intensity and duration during audiogenie priming on susceptibility to seizure after testing with the same stimulus complex used for priming (20). The data for a significant number of bb and BB groups reported here have been published previously (20), and are indicated by the larger of the two types of circles used in the figures. To be considered as having had a seizure, a mouse must wild-run prior to entering into a clonic seizure and have a clonic prior to a tonic seizure ; death may or may not follow a tonic seizure. Table 1 is constructed to reflect this: The fact that an animal had a tonic seizure, for example, was
75
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0
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0
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Intensity
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of Bell
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to Seizuresa
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6.58
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on Susceptibility
TABLE
ii
a0
3.5
75
ii
25
40 -
30
15
T
20
?iti
15
5
D
4.5
20
20
60
‘30
Priming
90
85
95
40
100
70
70
WR
condition
C
2.5
75
55
30
90 -
55
2.5
75
55
2s
8.5 -
so
55
C+
b (reversed)
5.5
B
20
75
50
25
70 -
4.5
3.5
T
*
20
70
10
20
3.5 -
5
20
D
“.
a CS?BL/6J mice, at 16 days of age, were subjected to audiogenic priming for 10, 30, or 60 s using either a small (b) or a large (B) bell. They were then tested at ages indicated using either the same (bb or BB) or the other (bB or Bb) bell. See text for details. * Testing conditions. c WR-Wild run, C-clonic-flexion, C+-clonic-extension, T-tonic seizure, D--lethal seizure. d Percentages of mice tested (20 per group). e Underlined values in the BB column indicate statistically significant xa differences between bb and BB mice. There are no significant differences at any level between bb and bB mice. Significant differences at any level of convulsion between BB and Bb mice are indicated in the Bb column.
0
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0
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10 30
26 days
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10
22 days
30d
30
WRc
10
18 days
Test age (days) and duration of priming (s)
Effect
522
ROBERT
A.
SCHREIBER
FIG. 1. Effect of BB and Bb on susceptibility indicated by the larger circles.
to seizures. Data from Schreiber
(20)
reflected in the percentage of mice tested having a wild run and also in the percentage of clonic seizures. The same data as in Table 1 are plotted as “cumulative seizure score” data in Figs. 1 and 2. Each mouse was assigneda “seizure score” according to the most severe seizure attained : no response (NR) , 0 ; wild run (WR) , 1 ; clonic-flexion seizure (C) , 2 ; clonic-extension seizure (C + ) , 3 ; tonic seizure (T), 4, and lethal seizure (D), 5. The cumulative score for each group of 20 mice is plotted in the figures,
b-‘b
0 or 0
b-18
n
AGE
FIG. 2. Effect of bb and bB on susceptibility indicated by the larger circles.
to seizures. Data from Schreiber
(20)
AUDIOGENIC
SEIZURES
AND
STIMULI
523
Forty nonprimed mice were tested for the first time using the small bell at 18, 22, or 28 days of age ; none showed any seizure activity (n = 10 mice/point). Additional mice were tested at the same ages for the first time using the large bell ; none convulsed (20). Table 1 shows that the time course of susceptibility is directly determined by the quality of the priming stimulus. The mice primed with the small bell had very few clonic or more severe convulsions by 26 days of age, whereas this level was attained by at least 25% of the mice primed with the large bell as late as 30 days of age. Within b or B priming conditions, the duration of exposure to the complex priming stimulus also plays an important role in determining later seizure levels at peak susceptibility. In general the longer the exposure (to as much as 60 s), the greater the proportion of mice entering into seizures. The only exception to this was observed in the b30 groups. When they were retested at either 18 or 22 days using the b bell, the observed incidences of clonic or more’ severe seizures were significantly greater than those observed in the b&Lb groups (2 x 2 x2 test, P < 0.05). There are two possibilities : First, these particular data were generated after the b60 data. The quality of the stimulus may have subtly changed, resulting in greater effectiveness. Second, these statistics may be the outcome of random sampling from a large number of groups, resulting in spurious statistical significance in a few comparisons. In either case, it makes no significant difference because the bb and BB groups constituted the baseline data against which the bB and Bb groups could be compared. More importantly, it is evident from both Table 1 and the figures that within each priming-duration group the distributions of responses of the bb and bB mice are the same, and the distributions of responses of the BB and Bb mice are the same. The only deviation from the above observation was seen in the incidences of clonic or more severe seizures in the BlO-B and NO-B groups as compared to the BlO-b and B60-b groups, respectively. In both cases, the mice tested with the small bell showed significantly more seizures than those tested with the large one (2 x 2 x2 test, P < 0.05). The latter case is attributed to a lower incidence of seizures in the B&?-B group than expected, assuming an increasing severity with increasing duration of priming. DISCUSSION Audiogenic priming was previously shown in mice of a number of genotypes using acoustic stimuli which varied from pure tones (13) to frequency-modulated tones (2), small doorbells (14), and 6-inch fire alarms @,20). Exposure to a sufficient acoustic stimulus for an adequate length of
524
ROBERT
A.
SCHREIBER
time has been postulated to result in at least two, and perhaps more, physiological defense responses in these mice. First, central “awareness/ reactivity” systems appear to be activated, as indicated by heightened reactivity to above-threshold stimulation. Gross overstimulation, as presented by intense noise, results in massive central nervous system discharge and behavioral convulsion (12, 14, 21). Second, peripheral responses seem to be activated as indicated by the phenomenon of unilateral priming (7, 10). Other evidence for a peripheral involvement comes from data which indicate a temporary threshold shift, a raised auditory threshold with an as yet undetermined time course, which has been taken as presumptive evidence of altered hearing in these mice (12, 17, 18). This assumption of altered hearing, however, should be accepted with caution (4). The magnitude of temporary threshold shifts, peripheral in nature, is known to be intensity-dependent (16). Because the large bell used in this experiment is known to be almost four times as intense as the small bell (broad-band dbA scale), it should in theory induce a much larger threshold shift. If seizure susceptibility is dependent on adequate stimulation above the level of a temporary threshold shift, then one might expect that mice primed with the very intense large bell and tested with the small one would show a lesser seizure rate than the BB mice. Yet, this did not occur; there was no significant reduction in the incidence of seizures in the Bb mice relative to the BB mice. Conversely, no enhancement of seizure rate was observed in the bB groups compared to the bb ones, which would indicate that even additional stimulation well above that to which a temporary threshold shift should develop added little or nothing to susceptibility 1.0 seizures. The only reasonable explanation for the finding that b testing or B testing did not alter the seizure rate induced by b or B priming is that maximal threshold shifts must have occurred at stimulation levels lower than that generated by either bell, In that case, one might argue that both bells, being sufficiently intense during priming to induce a maximum threshold shift, should have resulted in the same high levels of seizures when confronted by a supramaximal challenge. This was obviously not the case in these experiments. The overall seizure data for the B priming were considerably greater than those for b priming; therefore, the b bell was not supramaximally intense. Therefore, these data are difficult to reconcile with a “flooding of peripheral defenses” hypothesis without extraordinarily complex mental gymnastics. In summary, C57BLJ6J mice were subjected to acoustic priming at 16 days of age, using either weak or strong complex priming stimuli which were known to result in disparate rates of later susceptibility to seizures. The mice were then tested at various intervals thereafter, using either th& salne bell used during priming, or else under reversed conditions. Data
AUDIOGENIC
SEIZURES
AND
STIMULI
525
show that the priming condition determines later susceptibility. The overall pattern of the data is difficult to reconcile with a “flooding of peripheral defense” theory of induction of seizures. REFERENCES J. J. 1954. Intensity of white noise and frequency of convulsive reactions in DBA/l mice. Science 120: 139-140. BOCK, G. R., AND C.-S. CHEN, 1972. Frequency-modulated tones as priming stimuli for audiogenic seizures in mice. Exp. Neural. 37: 124-130. BOGCAN, W. O., D. X. FREEDMAN, AND R. A. LOVELL. 1971. Studies in audiogenic seizures susceptibility. Psychobharmacologia 20, 48-56. BROWN, A. M., AND J. D. PYE. 1975. Auditory sensitivity at high frequencies in mammals. Adv. Corn). Physiol. Biochem. 6: l-73. CHEN, C.-S. 1973. Sensitization for audiogenic seizures in two strains of mice and their Fl hybrids. Devel. Psychobiol. 6: 131-138. CHEN, C.-S. 1973. Effects of priming for audiogenic seizures in mice as a function of genotype and sound intensity. J. Camp. Physiol. Psychol. 84 : 586592. CHEN, C.-S., G. R. GATES, AND G. R. BOCK. 1973. Effect of priming and tympanic membrane destruction on development of audiogenic seizures susceptibility in BALB/C mice. Exp. Neural. 39 : 277-288. FJERDINGSTAD, E. R. 1973. Transfer of learning in rodents and fish. Pages 73-98 in W. B. ESSMAN AND S. NAKAJIMA, Eds., Czwrent Biochemical Approaches to Learning and Memory. Spectrum Press, New York. FRINGS, H., AND M. FRINGS 1952. Acoustical determinants of audiogenic seizures in laboratory mice. 1. Acoust. Sot. Amer. 24: 163-169. FULLER, J. L., AND R. C. COLLINS. 1968. Mice unilaterally sensitized for audiogenie seizures. Science 162 : 1295. GATES, G. R., AND C.-S. CHEN. 1975. Priming for audiogenic seizures in BALB/c mice as a function of stimulus exposure duration and intensity. Exp. Nezcrol. 46: 209-214. HENRY, K. R. 1967. Audiogenic seizure susceptibility induced in C57BL/6J mice by prior auditory exposure. Science 158: 938-940. HENRY, K. R., K. A. THOMPSON, AND R. E. BOWMAN. 1971. Frequency characteristics of acoustic priming of audiogenic seizures in mice. Exp. Nezlrol. 31: 402-407. ITURRIAN, W. B., AND G. B. FINK. 1967. Conditioned convulsive reaction. Fed. Proc. 26 : 736 (Abst.). JAY, G. E., JR. 1963. Genetic strains and stocks. Pages 93-123 in W. J. BURDETTE, Ed., Methodology in Mammalian Genetics. Holden-Day, San Francisco. KRYTER, K. D. 1972. Non-auditory effects of environmental noise. Amer. J. Pub.
1. ANTONITIS,
2. 3. 4. 5. 6. 7. 8.
9. 10. 11. 12. 13. 14. 15. 16.
Health
62 : 389-392.
17. SAUNDERS, J. C., G. R. BOCK, C.-S. &EN, AND G. R. GATES. 1972. The effects of priming for audiogenic seizures on cochlear and behavioral responses in BALB/c mice. Exfi. Neural. 36 : 426-436. 18. SAUNDERS, J. C., G. R. BOCK, R. JAMES, AND C.-S. CHEN. 1972. Effects of priming for audiogenic seizure on auditory evoked responses in the cochlear nucleus and inferior colliculus of BALB/ c mice. En). Neural. 37, 388-394. 19. SCHLESINGER, K., W. 0. BOGGAN, AND D. X. FREEDMAN. 1965. Genetics of audiogenie seizures: I. Relation to brain serotonin and norepinephrine in mice. Life Sci. 4: 23452351.
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20. SCHREIBER, R. A. 1976. Effects of stimulus intensity and stimulus duration during acoustic priming on audiogenic seizures in C57BL/6J mice. Dev. Psychobiol. 10: 77-85. 21. SCHREIBER, R. A., AND J. M. GRAHAM, JR. 1976. Audiogenic priming in DBAJ2J and C57BL/6J mice: Interactions among age, prime-to-test interval, and index of seizure. Dev. Psychobiol. 9 : 57-66. 22. SCHREIBER, R. A., AND K. SCHLESINGER. 1971. Circadian rhythms and seizure susceptibility : Relation to Shydroxytryptamine and norepinephrine in brain. Physiol.
Behav.
6 : 635440.
23. SPROTT, R. L., AND J. STAATS. mice-A bibliography. Behav.
1975. Behavioral Genet.
5 : 27-82.
studies using genetically defined
Audiogenic
NEUROLOGY
56, 518-526 (1977)
Seizure Levels in C57BL/6J to the Priming and Testing ROBERT
Brain
Research
Institute,
A.
SCHREIBER
Mice Stimuli *
University of Tennessee Ceftter Memphis, Tennessee 38143 Received
November
as Related
for
the Health
Sciences,
24,1976
The effects of reversing two audiogenic priming stimuli during testing for audiogenic convulsions were examined in C57BL/6J mice not normally susceptible to audiogenic seizures on the first exposure to an acoustic stimulus. Mice were exposed to either a small (b) or a large (B) bell during audiogenie priming for either 10, 30, or 60 s at 16 days of age. They were then tested using either the same bell or under reversed conditions (bb, BB, bB, or Bb) at varying ages thereafter. Responses were graded as expected according to the duration of priming. The low incidences of audiogenic seizures observed during testing were similar for the bb and bB groups, and the high incidences were similar for the BB and Bb groups. Thus, priming conditions determine later seizure levels, not testing conditions.
INTRODUCTION Mice of appropriate genotypes, within appropriate age ranges, are subject to audiogenic priming, a phenomenon of seizures in response to a second exposure to a sufficient acoustic stimulus but not to the first (12, 14). The development of this response is best characterized for the C57BL/6J strain, in which the magnitude of the average seizure responseis dependent on the age at first exposure (during which no behavioral seizure occurs) and also on the duration in days between the first and secondacoustic challenges. Agreement is general that these mice are maximally sensitive to the effects 1 This research was supported by National Institutes of Health General Research Support Grant RR 54.23 to the College of Medicine, University of Tennessee Center for the Health Sciences. The aid of Dr. J. Studebaker, Memphis State University Speech and Hearing Center, in measuring the power spectra, and of Ms. Linda Craddock and Mr. Toa Nguyen in mouse testing is acknowledged. I also thank Ms. Mary Jackson for her excellent typing skills. Request reprints from Dr. Robert A. Schreiber, Brain Research Institute, Department of Biochemistry, University of Tennessee Center for the Health Sciences, Memphis, Tennessee 38163. 518 Copyright All rights
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
ISSN
0014-4886
A~DIOGENIC
SEIZURES
AND
STIMULI
519
of a first exposure to acoustic stimuli at a time coincident with the opening of the ear canal between 14 and 16 days of age, followed by decreasing sensitivity thereafter ($6, 12,21) . At present little is known about the cellular event(s) induced by an initial exposure to an acoustic stimulus which culminates in a convulsion upon a subsequent reexposure. Appropriate experiments on a cellular level, however, await a reduction in within-group variability brought about by knowledge and control of parameters which affect the magnitude and time course of induced susceptibility to audiogenic seizures. For example, even with use of the same strain of mouse (C57BL/6J), primed at the same age (16 days), gross disagreement exists as to both the levels of peak seizure risk at peak susceptibility (usually considered to be 21 days) and also the duration of induced susceptibility to audiogenic seizures (3, 6,21). Many investigations have long recognized that the intensity of the stimulus has been an important variable in determining levels of seizures ,in strains of mice susceptible on an initial exposure (1, 9, 23). More recently, others have shown that intensity of the stimulus during audiogenic priming also affects later seizure susceptibility (3, 6, 11) as does duration of exposure (3, 11). Recent data indicate that stimulus intensity and duration of priming interact to affect both the magnitude and the time course of induced susceptibility to seizures when the same stimulus complex used during audiogenic priming is used for testing (20). This study (i) examines whether the complex p&&g stimulus or the complex testing stimulus, or both, are important in determination of seizure levels, and (ii) determines whether or not the time course of the response is primarily influenced by the priming or testing stimuli. METHODS mice (Mus musculus) was used in Subjects. A total of 380 C57BL/6J these experiments. [The origins and inbreeding of these mice have been described by Jay (15).] A nimals were raised in our colony, and were derived from breeding stocks obtained from the Jackson Laboratory, Bar Harbor, Maine. Mice were maintained ad libituvpz on Wayne Mouse Breeder chow and tap water. An automatic switch turned on fluorescent lights in the colony room at 0700 h and off at 1900 h. The temperature was maintained at approximately 23 ’ C. Apparatus and Procedure. Mice were placed in a battery jar, 30 (outside diameter) x 46 cm high, enclosed in a sound-attenuating box with an observation window as previously described by Schlesinger et al. (19). Each mouse was allowed 15 s to explore during which time the lids to the jar and the box were put in place. An Edwards #13-3 bell was attached
520
ROBERT
A.
SCHREIBER
to one jar lid and an Edwards #340 bell was attached to another. The voltage delivered to the #13-3 electric bell was set to generate a broadband output of 108 C 1 db at the level of the mouse ; the #340 bell generated a broad band output of 127 k 2 db. The small bell will be referred to as the b bell and the large bell as B. The power spectrum from 0 to 20 kHz for each bell was measured with a B & K type 4145 condenser microphone placed in the center of the testing jar and a B & K Microphone power supply type 2801. The output was fed into an SDC Model SD330A real time analyzer and the average of 512 passes through the spectrum was plotted for each bell on an H/P 7034A XY recorder. The power spectra of these bells are published (20). All mice were exposed to the noise from one of the two bells between 1100 and 1400 h to control for circadian rhythms (22). All mice were primed at 16 days of age (day of birth is day 0) and remained with their mothers until testing at a later date. The only observable response to the initial exposure to the bell in most mice was the normally observed Preyer reflex, in which the pinnae are reflected downward immediately after the onset of the bell. The very few mice which responded with a wild run or more severe seizure during the priming period were discarded. A number of priming-testing combinations were included in this experiment. Mice were either primed and tested using the same bell (bb or BB), or primed and tested under reversed conditions (bB or Bb). The duration of priming was either 10, 30, or 60 s ( 11,ZO). All mice were retested at a later date for as much as 30 s in the same apparatus under the same conditions as described above. The bell was turned off earlier if the mouse attained a tonic convulsion. [Turning off the bell during even a clonic convulsion does not influence the incidence of tonic convulsions or lethality]. No more than three animals from one litter were used in a single test-retest group. There were 20 mice per group. RESULTS This experiment was conducted during and shortly after an experiment investigating the effects of stimulus intensity and duration during audiogenie priming on susceptibility to seizure after testing with the same stimulus complex used for priming (20). The data for a significant number of bb and BB groups reported here have been published previously (20), and are indicated by the larger of the two types of circles used in the figures. To be considered as having had a seizure, a mouse must wild-run prior to entering into a clonic seizure and have a clonic prior to a tonic seizure ; death may or may not follow a tonic seizure. Table 1 is constructed to reflect this: The fact that an animal had a tonic seizure, for example, was
75
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15 35
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of Bell
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0
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10 1.5
10
D
806060 -----
?s
50
2% -----
45
3.5
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25 ?o
5s
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25
C
45
35
75
‘70
25
5.5 -
50
20
C+
B (same)*
to Seizuresa
100
90 -
?o
6.58
WR
on Susceptibility
TABLE
ii
a0
3.5
75
ii
25
40 -
30
15
T
20
?iti
15
5
D
4.5
20
20
60
‘30
Priming
90
85
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70
WR
condition
C
2.5
75
55
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90 -
55
2.5
75
55
2s
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so
55
C+
b (reversed)
5.5
B
20
75
50
25
70 -
4.5
3.5
T
*
20
70
10
20
3.5 -
5
20
D
“.
a CS?BL/6J mice, at 16 days of age, were subjected to audiogenic priming for 10, 30, or 60 s using either a small (b) or a large (B) bell. They were then tested at ages indicated using either the same (bb or BB) or the other (bB or Bb) bell. See text for details. * Testing conditions. c WR-Wild run, C-clonic-flexion, C+-clonic-extension, T-tonic seizure, D--lethal seizure. d Percentages of mice tested (20 per group). e Underlined values in the BB column indicate statistically significant xa differences between bb and BB mice. There are no significant differences at any level between bb and bB mice. Significant differences at any level of convulsion between BB and Bb mice are indicated in the Bb column.
0
0
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30 10
0
2.5
5.5
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C
10 30
26 days
60
5
40 4.5
10
22 days
30d
30
WRc
10
18 days
Test age (days) and duration of priming (s)
Effect
522
ROBERT
A.
SCHREIBER
FIG. 1. Effect of BB and Bb on susceptibility indicated by the larger circles.
to seizures. Data from Schreiber
(20)
reflected in the percentage of mice tested having a wild run and also in the percentage of clonic seizures. The same data as in Table 1 are plotted as “cumulative seizure score” data in Figs. 1 and 2. Each mouse was assigneda “seizure score” according to the most severe seizure attained : no response (NR) , 0 ; wild run (WR) , 1 ; clonic-flexion seizure (C) , 2 ; clonic-extension seizure (C + ) , 3 ; tonic seizure (T), 4, and lethal seizure (D), 5. The cumulative score for each group of 20 mice is plotted in the figures,
b-‘b
0 or 0
b-18
n
AGE
FIG. 2. Effect of bb and bB on susceptibility indicated by the larger circles.
to seizures. Data from Schreiber
(20)
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Forty nonprimed mice were tested for the first time using the small bell at 18, 22, or 28 days of age ; none showed any seizure activity (n = 10 mice/point). Additional mice were tested at the same ages for the first time using the large bell ; none convulsed (20). Table 1 shows that the time course of susceptibility is directly determined by the quality of the priming stimulus. The mice primed with the small bell had very few clonic or more severe convulsions by 26 days of age, whereas this level was attained by at least 25% of the mice primed with the large bell as late as 30 days of age. Within b or B priming conditions, the duration of exposure to the complex priming stimulus also plays an important role in determining later seizure levels at peak susceptibility. In general the longer the exposure (to as much as 60 s), the greater the proportion of mice entering into seizures. The only exception to this was observed in the b30 groups. When they were retested at either 18 or 22 days using the b bell, the observed incidences of clonic or more’ severe seizures were significantly greater than those observed in the b&Lb groups (2 x 2 x2 test, P < 0.05). There are two possibilities : First, these particular data were generated after the b60 data. The quality of the stimulus may have subtly changed, resulting in greater effectiveness. Second, these statistics may be the outcome of random sampling from a large number of groups, resulting in spurious statistical significance in a few comparisons. In either case, it makes no significant difference because the bb and BB groups constituted the baseline data against which the bB and Bb groups could be compared. More importantly, it is evident from both Table 1 and the figures that within each priming-duration group the distributions of responses of the bb and bB mice are the same, and the distributions of responses of the BB and Bb mice are the same. The only deviation from the above observation was seen in the incidences of clonic or more severe seizures in the BlO-B and NO-B groups as compared to the BlO-b and B60-b groups, respectively. In both cases, the mice tested with the small bell showed significantly more seizures than those tested with the large one (2 x 2 x2 test, P < 0.05). The latter case is attributed to a lower incidence of seizures in the B&?-B group than expected, assuming an increasing severity with increasing duration of priming. DISCUSSION Audiogenic priming was previously shown in mice of a number of genotypes using acoustic stimuli which varied from pure tones (13) to frequency-modulated tones (2), small doorbells (14), and 6-inch fire alarms @,20). Exposure to a sufficient acoustic stimulus for an adequate length of
524
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time has been postulated to result in at least two, and perhaps more, physiological defense responses in these mice. First, central “awareness/ reactivity” systems appear to be activated, as indicated by heightened reactivity to above-threshold stimulation. Gross overstimulation, as presented by intense noise, results in massive central nervous system discharge and behavioral convulsion (12, 14, 21). Second, peripheral responses seem to be activated as indicated by the phenomenon of unilateral priming (7, 10). Other evidence for a peripheral involvement comes from data which indicate a temporary threshold shift, a raised auditory threshold with an as yet undetermined time course, which has been taken as presumptive evidence of altered hearing in these mice (12, 17, 18). This assumption of altered hearing, however, should be accepted with caution (4). The magnitude of temporary threshold shifts, peripheral in nature, is known to be intensity-dependent (16). Because the large bell used in this experiment is known to be almost four times as intense as the small bell (broad-band dbA scale), it should in theory induce a much larger threshold shift. If seizure susceptibility is dependent on adequate stimulation above the level of a temporary threshold shift, then one might expect that mice primed with the very intense large bell and tested with the small one would show a lesser seizure rate than the BB mice. Yet, this did not occur; there was no significant reduction in the incidence of seizures in the Bb mice relative to the BB mice. Conversely, no enhancement of seizure rate was observed in the bB groups compared to the bb ones, which would indicate that even additional stimulation well above that to which a temporary threshold shift should develop added little or nothing to susceptibility 1.0 seizures. The only reasonable explanation for the finding that b testing or B testing did not alter the seizure rate induced by b or B priming is that maximal threshold shifts must have occurred at stimulation levels lower than that generated by either bell, In that case, one might argue that both bells, being sufficiently intense during priming to induce a maximum threshold shift, should have resulted in the same high levels of seizures when confronted by a supramaximal challenge. This was obviously not the case in these experiments. The overall seizure data for the B priming were considerably greater than those for b priming; therefore, the b bell was not supramaximally intense. Therefore, these data are difficult to reconcile with a “flooding of peripheral defenses” hypothesis without extraordinarily complex mental gymnastics. In summary, C57BLJ6J mice were subjected to acoustic priming at 16 days of age, using either weak or strong complex priming stimuli which were known to result in disparate rates of later susceptibility to seizures. The mice were then tested at various intervals thereafter, using either th& salne bell used during priming, or else under reversed conditions. Data
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show that the priming condition determines later susceptibility. The overall pattern of the data is difficult to reconcile with a “flooding of peripheral defense” theory of induction of seizures. REFERENCES J. J. 1954. Intensity of white noise and frequency of convulsive reactions in DBA/l mice. Science 120: 139-140. BOCK, G. R., AND C.-S. CHEN, 1972. Frequency-modulated tones as priming stimuli for audiogenic seizures in mice. Exp. Neural. 37: 124-130. BOGCAN, W. O., D. X. FREEDMAN, AND R. A. LOVELL. 1971. Studies in audiogenic seizures susceptibility. Psychobharmacologia 20, 48-56. BROWN, A. M., AND J. D. PYE. 1975. Auditory sensitivity at high frequencies in mammals. Adv. Corn). Physiol. Biochem. 6: l-73. CHEN, C.-S. 1973. Sensitization for audiogenic seizures in two strains of mice and their Fl hybrids. Devel. Psychobiol. 6: 131-138. CHEN, C.-S. 1973. Effects of priming for audiogenic seizures in mice as a function of genotype and sound intensity. J. Camp. Physiol. Psychol. 84 : 586592. CHEN, C.-S., G. R. GATES, AND G. R. BOCK. 1973. Effect of priming and tympanic membrane destruction on development of audiogenic seizures susceptibility in BALB/C mice. Exp. Neural. 39 : 277-288. FJERDINGSTAD, E. R. 1973. Transfer of learning in rodents and fish. Pages 73-98 in W. B. ESSMAN AND S. NAKAJIMA, Eds., Czwrent Biochemical Approaches to Learning and Memory. Spectrum Press, New York. FRINGS, H., AND M. FRINGS 1952. Acoustical determinants of audiogenic seizures in laboratory mice. 1. Acoust. Sot. Amer. 24: 163-169. FULLER, J. L., AND R. C. COLLINS. 1968. Mice unilaterally sensitized for audiogenie seizures. Science 162 : 1295. GATES, G. R., AND C.-S. CHEN. 1975. Priming for audiogenic seizures in BALB/c mice as a function of stimulus exposure duration and intensity. Exp. Nezcrol. 46: 209-214. HENRY, K. R. 1967. Audiogenic seizure susceptibility induced in C57BL/6J mice by prior auditory exposure. Science 158: 938-940. HENRY, K. R., K. A. THOMPSON, AND R. E. BOWMAN. 1971. Frequency characteristics of acoustic priming of audiogenic seizures in mice. Exp. Nezlrol. 31: 402-407. ITURRIAN, W. B., AND G. B. FINK. 1967. Conditioned convulsive reaction. Fed. Proc. 26 : 736 (Abst.). JAY, G. E., JR. 1963. Genetic strains and stocks. Pages 93-123 in W. J. BURDETTE, Ed., Methodology in Mammalian Genetics. Holden-Day, San Francisco. KRYTER, K. D. 1972. Non-auditory effects of environmental noise. Amer. J. Pub.
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studies using genetically defined
