Journal of Comparative and Physiological Psychology 1976, Vol. 90, No. 8, 773-779
Inhibition of Acoustic Priming in Mice Bernard D. Lieff, Seth K. Sharpless, and Kurt Schlesinger Department of Psychology and Institute for Behavioral Genetics, University of Colorado Mice of the C57BL/6J strain can be made susceptible to audiogenic seizures by a process known as acoustic priming. Acoustic priming can be blocked when the animals are injected either with puromycin or with puromycin aminonucleoside before the application of the priming stimulus. Cycloheximide, diphenylhydantoin, and d-amphetamine had little effect on priming-induced audiogenic seizures in these animals. All of these drugs, however, when given in combination with puromycin reversed the protective action of puromycin against audiogenic seizures. Puromycin administered to 19-day-old mice increased susceptibility to electroconvulsive seizures when the animals were tested at 22 days of age. It is suggested that puromycin is able to block priming-induced audiogenic seizures by producing abnormal electrical activity in the brain or through an interference with normal neurohumoral transmission by incomplete peptides.
Mice from strains that are resistant to audiogenic seizures can be rendered susceptible to sound-induced convulsions by being exposed to an intense acoustic stimulus during a critical period of neural development. This phenomenon was first described by Henry (1967) who termed it acoustic priming. Priming-induced susceptibility to audiogenic seizures is of considerable interest inasmuch as it can be thought of as a relatively simple instance of the plasticity of the central nervous system. The procedure used to induce acoustic priming involves a pretest during which the mouse is exposed to a test or priming stimulus and a subsequent retest during which the animal is again exposed to the test stimulus and its behavior is observed. Several parameters of acoustic priming have been investigated: These include studies on (a) the critical age at wrhich priming is most effective, (b) the optimal priming-retest interval, and (c) the optimal duration for which the priming stimulus is presented to produce the maximal effect. These experiments have been reported by Henry and Bowman (1970), Collins (1970), and Boggan,
Freedman, Lovell, and Schlesinger (1971). The results indicate that the critical period differs for mice of different genotypes, both in terms of the best age for effecting priming and in the duration of the critical period. The same phenomenon has been observed with respect to the priming-retest interval, which also differs for mice of different genotypes; it is of interest to note that in some animals increased susceptibility to seizures can be observed for periods up to 30 days after the exposure of the mouse to the test stimulus. Thirty to 60 sec of priming appear to be optimal; periods of longer duration appear to reduce priming efficacy. Exposure to the test stimulus for periods of only 1 sec has been shown to produce statistically significant priming effects. Some investigators have interpreted their data as suggesting that the development of susceptibility to audiogenic seizures requires protein synthesis. However, Maxson, Sze, and Cowen (1975) have reported that cycloheximide inhibits priming-induced seizures in C57BL/6/Bg, but not in DBA/1/Bg-asr, mice. The effects of various pharmacological treatments on priming efficacy can be diThis research was supported in part by Research Grant MH 13026 from the National In- vided into two classes depending on whether stitute For Mental Health. We wish to thank the treatments are given before the retest Christopher J. Froelich for technical assistance. or before the priming stimulus. With respect Requests for reprints should be sent to Bernard D. Lieff, Department of Psychology, University to the former, it has been observed that lowering levels of the biogenic amines enof Colorado, Boulder, Colorado 80302. 773
774
B. LIEFF, S. SHARPLESS, AND K. SCHLESINGER
hances seizure susceptibility on retest whereas drugs that raise the levels of 5-hydroxytryptamine (serotonin, 5-HT) and norepinephrine (NE) protect animals against audiogenic seizures on retest (Boggan et al., 1971). These results are similar to those reported for sound-induced convulsions in genetically susceptible animals, in which lowering levels of the amines increases seizure risk whereas raising levels of the amines protects animals against audiogenic seizures (see e.g., Lehman, 1970; Schlesinger & Uphouse, 1972). Similar treatments before the priming experience itself did not prevent or otherwise affect priming. Three types of drugs have been found to affect acoustic priming efficacy when the treatments are given before the priming stimulus. Maxson and Sze (Note 1) reported that treatment with cycloheximide 30 min before priming markedly reduced the incidence of seizures at retest. Maxson, Sze, and Towle (Note 2) reported similar results for cycloheximide and for puromycin. Aminooxyacetic acid (AOAA), a drug that raises levels of gamma-aminobutyric acid (GABA) in the brain, has also been shown to inhibit priming efficacy when administered before the priming stimulus to C57BL/6 mice (Sze, 1970). Maxson et al. (1975) reported that AOAA effectively blocks priming in DBA/1/Bg-asr, but not in C57BL/6/Bg, animals. Bobbin, Gonzalez, and Guth (1969) have shown that AOAA renders animals hard of hearing, affecting the Preyer reflex, and that the effect of this compound on priming can probably be explained on these grounds. Finally, Boggan, Steele, and Freedman (1973) have shown that A9-tetrahydrocannabinol administered before exposure of the animal to the priming stimulus protects against subsequent audiogenic seizures. In this article we will describe a series of experiments that indicate that puromycin and puromycin aminonucleoside when administered before the priming stimulus protect mice against subsequent soundinduced convulsions. Cycloheximide not only failed to protect mice against priminginduced seizures but reversed the effects of puromycin when both protein synthesis in-
hibitors were injected before exposure to the priming stimulus. Diphenylhydantoin and amphetamine, when given in combination with puromycin before priming, also reversed the protective effects produced by puromycin. METHOD Subjects. Mice of the C57BL/6J strain, bred in our laboratory from stocks obtained from Jackson Laboratory, Bar Harbor, Maine, were used as experimental subjects in these experiments. Jay (1963) has previously described the origin and degree of inbre'eding of these animals. Approximately equal numbers of mice of both sexes were used in all experiments. Prior to use in any experiment all animals were reared in standard laboratory conditions in which the average ambient noise level was measured to be 55 dB re 20 /aN/m 2 . They were maintained under constant conditions of temperature (74 ± 3 °F.; 23 ± .1 °C), and controlled lighting (12-hr light cycle, 7 a.m.-7 p.m.) with ad lib access to Purina Mouse Breeder Chow and tap water. All seizure tests were conducted between the fourth and eighth hour of the daily light cycle. Ages of the animals at the beginning of the experiments are given below. Acoustic priming. Experimental subjects were primed and tested for subsequent susceptibility to audiogenic seizures one at a time as follows: At the appropriate age the mice were removed from their home cage and placed into a large chromatography jar, 17.24 in. high and 11.33 in. in diameter (approximately 43.8 and 28.8 cm, respectively) and given 30 sec to adapt. Following this interval a4-in. (approximately 10-cm) electric bell, generating a peak intensity of 118 ± 1 dB of noise as measured by a General Radio sound level meter (Model 1551-C) at the level of the mouse, was sounded for 60 sec. Sham-primed control mice were treated identically except that the bell was never sounded. Both primed and control animals were then immediately returned to their home cages. After the appropriate interval of time, mice were replaced into the chromatography jar and retested. The behavioral responses of the animals were recorded, and each mouse was given a "seizure severity score" based on its response. Scoring of the responses were as follows: no response = 0; wild running = 1; clonic seizure = 2; tonic seizure = 3; and lethal seizure = 4. All animals were primed at 19 days of age and retested 3 days later. Most previous investigations have found that 19 days of age is the most sensitive age for priming C57BL/6J mice, and the 3-day priming-retest interval has been described as most effective in this genotype (Boggan et al., 1971). Recent observations in our laboratory have confirmed these intervals in C57BL/6J animals.
INHIBITION OF ACOUSTIC PRIMING Electroconvulsive seizure tests. For electroconvulsive seizures, animals were stimulated through earclips with a 833.3-cps (cycle per second) square wave of .2-sec duration, having a negative and positive pulse width of .3 msec and a delay of .6 msec between each cycle (total period = 1.2 msec). The current intensity was 6.56 mA. Incidence of clonic-tonic seizures was recorded. Drug regimen. All drugs were administered 5 hr prior to presentation of the priming stimulus. Intracranial injections. Puromycin dihydrochloride, cycloheximide, and puromycin aminonucleoside were all injected intracerebrally. Before being injected, all mice were lightly anesthetized with ether.The injections were bilaterally into the cerebral hemispheres at points 1 mm on either side of the midsagittal line and 5 mm rostral from a line drawn through the center of the two ears. All injections were to a depth of 2.5 mm, and the volumes of all injections were 5 pi/side. The drugs were dissolved in physiological saline and the pH was adjusted to approximately 7.0 with 1 N NaOH. Doses in all experiments except those in which dose-response curves were obtained were as follows: puromycin, 200 pg/mouse; puromycin aminonucleoside, 200 ^g/mouse; cycloheximide, 200 jug/mouse. When animals were injected with both puromycin and cycloheximide, they received 100 /«g of each drug/mouse. Control animals were injected intracranially with physiological saline, 5 jul/side. Intraperitoneal injections. All ip injections were given at the same time as the intracranial injections at a volume of .01 ml/g of body weight. All drugs were dissolved in physiological saline and the pH was adjusted to approximately 7.0 with 1 N NaOH. The doses of these drugs were diphenylhydantoin, 35 mg/kg, and d-amphetamine, 12/5 mg/kg. (It should be noted that diphenylhydantoin could not be dissolved at a pH below approximately 11.0, and this drug was injected slowly at this pH.) Control mice were injected ip with equivalent volumes of saline. Protein synthesis. The procedure for protein extraction for the purpose of measuring proteinsynthesis inhibition was that described by Randt, Barnett, McEwen, and Quartermain (1971). Mice were first injected with the appropriate protein-synthesis inhibitor as already described, and protein synthesis was measured 5 hr later. Thirty minutes before the animals were sacrificed, each animal was injected ip with 25 ^Ci of [3H]lysine (New England Nuclear). The animals were then sacrificed by decapitation. The brains were quickly dissected free and homogenized in 5 ml of ice-cold water; 1.2 ml of 50% trichloroacetic acid (TCA) were added and the brain was homogenized again. The homogenates were incubated for 60 min in an ice bath and centrifuged for 10 min at 10,000 X g. The supernatant was decanted and saved. The precipitate was suspended and washed twice with 2 ml of TCA, then centrifuged for 10 min at 10,000 X g; the supernatant was again decanted and saved. The pellet was resuspended and
775
washed with 2 ml of hot (90 °C) 10% TCA for 15 min, repelleted as before, and the supernatant was saved. All supernatants were then combined. The pellet was washed and centrifuged at 10,000 X g for 10 min three more times, first with 2 ml of 95% EtOH, then with 2 ml of absolute EtOH, and finally with 2 ml of diethyl ether. The supernatants were discarded and the remaining pellet was dissolved in 2 ml of 1 N NaOH. One milliliter of the solubilized pellet was placed into a scintillation vial and neutralized with glacial acetic acid (pH 7.0) and 10 ml of PPOdioxanaphthalene fluor were added. The contents were then analyzed for radioactivity in a Beckman LS 250 liquid scintillation counter. A 2.5-^1 aliquot was taken from the solubilized pellet, and protein determinations were performed by using the procedure described by Lowry, Rosenbrough, Farr, and Randall (1951). The results were expressed as counts per minute incorporated/mg of protein, and corrected for the radioactive pool by using the supernatants collected at the beginning of the procedure.
RESULTS The dose-response curves for proteinsynthesis inhibition obtained with puromycin and with cycloheximide are given in Figures 1 and 2, respectively. Average seizure-severity scores obtained on retest with animals given varying doses of puromycin and cycloheximide are also given in these figures. These data indicate that doses of puromycin that inhibit protein synthesis by 70 % or more effectively block priming. These animals received seizureseverity scores of 1.0 or less, whereas control animals received seizure-severity scores of approximately 3.5. Cycloheximide, even at doses that inhibit protein synthesis by 93 %, failed to prevent priming. The effects of other drugs on priming efficacy are summarized in Table 1. Data are given as average seizure-severity scores at retest ±1 SE. As already indicated, puromycin at doses of 100 and 200 jug/mouse was effective in blocking acoustic priming, whereas cycloheximide was not. Further, administration of both antibiotics together reversed the effects produced when puromycin alone was injected. Both diphenylhydantoin and amphetamine, when administered concurrently with puromycin, tended to reverse the effects observed with puromycin alone. Neither diphenylhydan-
B. LIEFF, S. SHARPLESS, AND K. SCHLESINGER
776 100
!
,40
75-
30
50-
-120
25
l 10
300
200
00
c
500
400
Dose of Puromycin
FIGURE 1. Effects of varying doses of puromycin on protein-synthesis inhibition and on priminginduced audiogenic seizures in C57BL/6J mice.
toin nor amphetamine when given alone produced any noticeable effects on priming efficacy. Finally, puromycin aminonucleoside given before the pretest prevented the
development of subsequent seizure susceptibility. The effects of puromycin on electroconvulsive seizure (ECS) activity are sum-
100
40
I--'
-
75
30
50 -
20
25 -
10
20
30
40
50
60
70
80
90
100
Dose of Cycloheximide (/*gm)
FIGURE 2. Effects of varying doses of cycloheximide on protein-synthesis inhibition and on priminginduced audiogenic seizures in C57BL/6J mice.
777
INHIBITION OF ACOUSTIC PRIMING TABLE 1 EFFECTS or DRUGS ON PRIMING EFFICACY Drug
Seizure severity score
U
Saline Saline (sham-primed) Puromycin (100 ^g) Puromycin (200 Mg) Cycloheximide Puromycin and cycloheximide Dilantin Puromycin and dilantin Puromycin aminonucleoside d-Amphetamine Puromycin and amphetamine
SE
3.53 .33 1.27 .93 3.90
.14 .13 .27 .21 .11
—
Inhibition of Acoustic Priming in Mice Bernard D. Lieff, Seth K. Sharpless, and Kurt Schlesinger Department of Psychology and Institute for Behavioral Genetics, University of Colorado Mice of the C57BL/6J strain can be made susceptible to audiogenic seizures by a process known as acoustic priming. Acoustic priming can be blocked when the animals are injected either with puromycin or with puromycin aminonucleoside before the application of the priming stimulus. Cycloheximide, diphenylhydantoin, and d-amphetamine had little effect on priming-induced audiogenic seizures in these animals. All of these drugs, however, when given in combination with puromycin reversed the protective action of puromycin against audiogenic seizures. Puromycin administered to 19-day-old mice increased susceptibility to electroconvulsive seizures when the animals were tested at 22 days of age. It is suggested that puromycin is able to block priming-induced audiogenic seizures by producing abnormal electrical activity in the brain or through an interference with normal neurohumoral transmission by incomplete peptides.
Mice from strains that are resistant to audiogenic seizures can be rendered susceptible to sound-induced convulsions by being exposed to an intense acoustic stimulus during a critical period of neural development. This phenomenon was first described by Henry (1967) who termed it acoustic priming. Priming-induced susceptibility to audiogenic seizures is of considerable interest inasmuch as it can be thought of as a relatively simple instance of the plasticity of the central nervous system. The procedure used to induce acoustic priming involves a pretest during which the mouse is exposed to a test or priming stimulus and a subsequent retest during which the animal is again exposed to the test stimulus and its behavior is observed. Several parameters of acoustic priming have been investigated: These include studies on (a) the critical age at wrhich priming is most effective, (b) the optimal priming-retest interval, and (c) the optimal duration for which the priming stimulus is presented to produce the maximal effect. These experiments have been reported by Henry and Bowman (1970), Collins (1970), and Boggan,
Freedman, Lovell, and Schlesinger (1971). The results indicate that the critical period differs for mice of different genotypes, both in terms of the best age for effecting priming and in the duration of the critical period. The same phenomenon has been observed with respect to the priming-retest interval, which also differs for mice of different genotypes; it is of interest to note that in some animals increased susceptibility to seizures can be observed for periods up to 30 days after the exposure of the mouse to the test stimulus. Thirty to 60 sec of priming appear to be optimal; periods of longer duration appear to reduce priming efficacy. Exposure to the test stimulus for periods of only 1 sec has been shown to produce statistically significant priming effects. Some investigators have interpreted their data as suggesting that the development of susceptibility to audiogenic seizures requires protein synthesis. However, Maxson, Sze, and Cowen (1975) have reported that cycloheximide inhibits priming-induced seizures in C57BL/6/Bg, but not in DBA/1/Bg-asr, mice. The effects of various pharmacological treatments on priming efficacy can be diThis research was supported in part by Research Grant MH 13026 from the National In- vided into two classes depending on whether stitute For Mental Health. We wish to thank the treatments are given before the retest Christopher J. Froelich for technical assistance. or before the priming stimulus. With respect Requests for reprints should be sent to Bernard D. Lieff, Department of Psychology, University to the former, it has been observed that lowering levels of the biogenic amines enof Colorado, Boulder, Colorado 80302. 773
774
B. LIEFF, S. SHARPLESS, AND K. SCHLESINGER
hances seizure susceptibility on retest whereas drugs that raise the levels of 5-hydroxytryptamine (serotonin, 5-HT) and norepinephrine (NE) protect animals against audiogenic seizures on retest (Boggan et al., 1971). These results are similar to those reported for sound-induced convulsions in genetically susceptible animals, in which lowering levels of the amines increases seizure risk whereas raising levels of the amines protects animals against audiogenic seizures (see e.g., Lehman, 1970; Schlesinger & Uphouse, 1972). Similar treatments before the priming experience itself did not prevent or otherwise affect priming. Three types of drugs have been found to affect acoustic priming efficacy when the treatments are given before the priming stimulus. Maxson and Sze (Note 1) reported that treatment with cycloheximide 30 min before priming markedly reduced the incidence of seizures at retest. Maxson, Sze, and Towle (Note 2) reported similar results for cycloheximide and for puromycin. Aminooxyacetic acid (AOAA), a drug that raises levels of gamma-aminobutyric acid (GABA) in the brain, has also been shown to inhibit priming efficacy when administered before the priming stimulus to C57BL/6 mice (Sze, 1970). Maxson et al. (1975) reported that AOAA effectively blocks priming in DBA/1/Bg-asr, but not in C57BL/6/Bg, animals. Bobbin, Gonzalez, and Guth (1969) have shown that AOAA renders animals hard of hearing, affecting the Preyer reflex, and that the effect of this compound on priming can probably be explained on these grounds. Finally, Boggan, Steele, and Freedman (1973) have shown that A9-tetrahydrocannabinol administered before exposure of the animal to the priming stimulus protects against subsequent audiogenic seizures. In this article we will describe a series of experiments that indicate that puromycin and puromycin aminonucleoside when administered before the priming stimulus protect mice against subsequent soundinduced convulsions. Cycloheximide not only failed to protect mice against priminginduced seizures but reversed the effects of puromycin when both protein synthesis in-
hibitors were injected before exposure to the priming stimulus. Diphenylhydantoin and amphetamine, when given in combination with puromycin before priming, also reversed the protective effects produced by puromycin. METHOD Subjects. Mice of the C57BL/6J strain, bred in our laboratory from stocks obtained from Jackson Laboratory, Bar Harbor, Maine, were used as experimental subjects in these experiments. Jay (1963) has previously described the origin and degree of inbre'eding of these animals. Approximately equal numbers of mice of both sexes were used in all experiments. Prior to use in any experiment all animals were reared in standard laboratory conditions in which the average ambient noise level was measured to be 55 dB re 20 /aN/m 2 . They were maintained under constant conditions of temperature (74 ± 3 °F.; 23 ± .1 °C), and controlled lighting (12-hr light cycle, 7 a.m.-7 p.m.) with ad lib access to Purina Mouse Breeder Chow and tap water. All seizure tests were conducted between the fourth and eighth hour of the daily light cycle. Ages of the animals at the beginning of the experiments are given below. Acoustic priming. Experimental subjects were primed and tested for subsequent susceptibility to audiogenic seizures one at a time as follows: At the appropriate age the mice were removed from their home cage and placed into a large chromatography jar, 17.24 in. high and 11.33 in. in diameter (approximately 43.8 and 28.8 cm, respectively) and given 30 sec to adapt. Following this interval a4-in. (approximately 10-cm) electric bell, generating a peak intensity of 118 ± 1 dB of noise as measured by a General Radio sound level meter (Model 1551-C) at the level of the mouse, was sounded for 60 sec. Sham-primed control mice were treated identically except that the bell was never sounded. Both primed and control animals were then immediately returned to their home cages. After the appropriate interval of time, mice were replaced into the chromatography jar and retested. The behavioral responses of the animals were recorded, and each mouse was given a "seizure severity score" based on its response. Scoring of the responses were as follows: no response = 0; wild running = 1; clonic seizure = 2; tonic seizure = 3; and lethal seizure = 4. All animals were primed at 19 days of age and retested 3 days later. Most previous investigations have found that 19 days of age is the most sensitive age for priming C57BL/6J mice, and the 3-day priming-retest interval has been described as most effective in this genotype (Boggan et al., 1971). Recent observations in our laboratory have confirmed these intervals in C57BL/6J animals.
INHIBITION OF ACOUSTIC PRIMING Electroconvulsive seizure tests. For electroconvulsive seizures, animals were stimulated through earclips with a 833.3-cps (cycle per second) square wave of .2-sec duration, having a negative and positive pulse width of .3 msec and a delay of .6 msec between each cycle (total period = 1.2 msec). The current intensity was 6.56 mA. Incidence of clonic-tonic seizures was recorded. Drug regimen. All drugs were administered 5 hr prior to presentation of the priming stimulus. Intracranial injections. Puromycin dihydrochloride, cycloheximide, and puromycin aminonucleoside were all injected intracerebrally. Before being injected, all mice were lightly anesthetized with ether.The injections were bilaterally into the cerebral hemispheres at points 1 mm on either side of the midsagittal line and 5 mm rostral from a line drawn through the center of the two ears. All injections were to a depth of 2.5 mm, and the volumes of all injections were 5 pi/side. The drugs were dissolved in physiological saline and the pH was adjusted to approximately 7.0 with 1 N NaOH. Doses in all experiments except those in which dose-response curves were obtained were as follows: puromycin, 200 pg/mouse; puromycin aminonucleoside, 200 ^g/mouse; cycloheximide, 200 jug/mouse. When animals were injected with both puromycin and cycloheximide, they received 100 /«g of each drug/mouse. Control animals were injected intracranially with physiological saline, 5 jul/side. Intraperitoneal injections. All ip injections were given at the same time as the intracranial injections at a volume of .01 ml/g of body weight. All drugs were dissolved in physiological saline and the pH was adjusted to approximately 7.0 with 1 N NaOH. The doses of these drugs were diphenylhydantoin, 35 mg/kg, and d-amphetamine, 12/5 mg/kg. (It should be noted that diphenylhydantoin could not be dissolved at a pH below approximately 11.0, and this drug was injected slowly at this pH.) Control mice were injected ip with equivalent volumes of saline. Protein synthesis. The procedure for protein extraction for the purpose of measuring proteinsynthesis inhibition was that described by Randt, Barnett, McEwen, and Quartermain (1971). Mice were first injected with the appropriate protein-synthesis inhibitor as already described, and protein synthesis was measured 5 hr later. Thirty minutes before the animals were sacrificed, each animal was injected ip with 25 ^Ci of [3H]lysine (New England Nuclear). The animals were then sacrificed by decapitation. The brains were quickly dissected free and homogenized in 5 ml of ice-cold water; 1.2 ml of 50% trichloroacetic acid (TCA) were added and the brain was homogenized again. The homogenates were incubated for 60 min in an ice bath and centrifuged for 10 min at 10,000 X g. The supernatant was decanted and saved. The precipitate was suspended and washed twice with 2 ml of TCA, then centrifuged for 10 min at 10,000 X g; the supernatant was again decanted and saved. The pellet was resuspended and
775
washed with 2 ml of hot (90 °C) 10% TCA for 15 min, repelleted as before, and the supernatant was saved. All supernatants were then combined. The pellet was washed and centrifuged at 10,000 X g for 10 min three more times, first with 2 ml of 95% EtOH, then with 2 ml of absolute EtOH, and finally with 2 ml of diethyl ether. The supernatants were discarded and the remaining pellet was dissolved in 2 ml of 1 N NaOH. One milliliter of the solubilized pellet was placed into a scintillation vial and neutralized with glacial acetic acid (pH 7.0) and 10 ml of PPOdioxanaphthalene fluor were added. The contents were then analyzed for radioactivity in a Beckman LS 250 liquid scintillation counter. A 2.5-^1 aliquot was taken from the solubilized pellet, and protein determinations were performed by using the procedure described by Lowry, Rosenbrough, Farr, and Randall (1951). The results were expressed as counts per minute incorporated/mg of protein, and corrected for the radioactive pool by using the supernatants collected at the beginning of the procedure.
RESULTS The dose-response curves for proteinsynthesis inhibition obtained with puromycin and with cycloheximide are given in Figures 1 and 2, respectively. Average seizure-severity scores obtained on retest with animals given varying doses of puromycin and cycloheximide are also given in these figures. These data indicate that doses of puromycin that inhibit protein synthesis by 70 % or more effectively block priming. These animals received seizureseverity scores of 1.0 or less, whereas control animals received seizure-severity scores of approximately 3.5. Cycloheximide, even at doses that inhibit protein synthesis by 93 %, failed to prevent priming. The effects of other drugs on priming efficacy are summarized in Table 1. Data are given as average seizure-severity scores at retest ±1 SE. As already indicated, puromycin at doses of 100 and 200 jug/mouse was effective in blocking acoustic priming, whereas cycloheximide was not. Further, administration of both antibiotics together reversed the effects produced when puromycin alone was injected. Both diphenylhydantoin and amphetamine, when administered concurrently with puromycin, tended to reverse the effects observed with puromycin alone. Neither diphenylhydan-
B. LIEFF, S. SHARPLESS, AND K. SCHLESINGER
776 100
!
,40
75-
30
50-
-120
25
l 10
300
200
00
c
500
400
Dose of Puromycin
FIGURE 1. Effects of varying doses of puromycin on protein-synthesis inhibition and on priminginduced audiogenic seizures in C57BL/6J mice.
toin nor amphetamine when given alone produced any noticeable effects on priming efficacy. Finally, puromycin aminonucleoside given before the pretest prevented the
development of subsequent seizure susceptibility. The effects of puromycin on electroconvulsive seizure (ECS) activity are sum-
100
40
I--'
-
75
30
50 -
20
25 -
10
20
30
40
50
60
70
80
90
100
Dose of Cycloheximide (/*gm)
FIGURE 2. Effects of varying doses of cycloheximide on protein-synthesis inhibition and on priminginduced audiogenic seizures in C57BL/6J mice.
777
INHIBITION OF ACOUSTIC PRIMING TABLE 1 EFFECTS or DRUGS ON PRIMING EFFICACY Drug
Seizure severity score
U
Saline Saline (sham-primed) Puromycin (100 ^g) Puromycin (200 Mg) Cycloheximide Puromycin and cycloheximide Dilantin Puromycin and dilantin Puromycin aminonucleoside d-Amphetamine Puromycin and amphetamine
SE
3.53 .33 1.27 .93 3.90
.14 .13 .27 .21 .11
—
