BEHAVIORAL BIOLOGY 18, 111-122 (1976), Abstract No. 6131
Macromolecules of the Central Nervous System and Acoustic Priming in C57BL/6Bg Mice
STEPHEN C. MAXSON, ANDREW C. TOWLE, and PAUL Y. SZE 1
Department o f Biobehavioral Sciences, The University o f Connecticut, Storrs, Connecticut 06268
Cycloheximide (30 mg/kg, ip) or puromycin (200/~g/mouse, intraventricular) was administered to C57BL/6Bg mice either before or after acoustic palming. Treatment with either of these inhibitors of protein synthesis significantly attenuates the acoustic priming. Although intraventricular injection of actinomycin D (2.5/~g/mouse) before and after the initial auditory stimulus completely blocks the acoustic priming, another inhibitor of RNA synthesis, diaminopurine (100 #g/mouse) had no effect. Thus, acoustic priming may be partially dependent upon protein synthesis in the brain either during and/or briefly (0.5 hr) after the initial auditory stimulus, and may not be dependent upon RNA synthesis. Also, acoustic priming was not "transferred" through brain extracts prepared from 19- or 28-day-old donors and injected into 19- or 27-day-old recipients.
Exposure to an initial auditory stimulus (IAS) during a sensitive period of postnatal development induces susceptibility to audiogenic seizures in otherwise resistant mice (Henry, 1967; Fuller and Collins, 1968; Iturrian and Fink, 1968; Jumonville, 1968; Sze, 1970). Several studies on the mechanisms of this acoustic priming effect support the hypothesis that the IAS damages the acoustic receptors and thereby causes partial deafness (Henry, 1972; Willott and Henry, 1974), that the partial hearing loss results in a disuse supersensitivity of the brain stem auditory system, and that at the subsequent exposure to the sound stimulus either an increase in amount of neural activity (Saunders et al., 1972) or a change in pattern of neural activity (WiUott et al., 1975) in the inferior colliculus elicits the brain and behavioral seizure. This theory is supported by evidence that tympanic rupture (Gates et al., 1973) or ear block (McGinn et al., 1973) also induces susceptibility to audiogenic 1This work was supported by NIH Grants RR 00602-04, AA 00297-03, and by a grant from The Grant Foundation, Inc. 111 Copyright © 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
112
MAXSON, TOWLE AND SZE
seizures in otherwise resistant mice. Thus, this theory suggests that the initial events in the chain of causes are peripheral and that the changes in the central nervous system are derivative. Also, this theory resembles those of disuse supersensitivity for epilepsies following depressant withdrawal (Sharpless, 1969; Freund and Walker, 1971; Gates and Chen, 1974). However, there is evidence that this may not be the only mechanism for the acoustic priming effect. Chen (1973) has demonstrated genetic heterogeneity,and Maxson et al. (1975) have provided evidence of pharmacogenetic differences among inbred strains of mice. More directly, Chen and Fuller (1975) reported that tympanic rupture does not induce susceptibility in a selected stock of acoustically primable mice. This suggests that some effects of the IAS may directly involve the central nervous system. A similar hypothesis has been proposed by Iturrian and Johnson (1971) on the basis of studies of development of unilateral susceptibility after the IAS. Some of these central effects of the IAS may be mediated by biochemical events. Sze (1970) has reported that the IAS causes a small and transient fall (within 0.5 hr) in the level of brain 7-aminobutyric acid (GABA) and that reversal of this fall by prior treatment with aminooxyacetic acid (AOAA) prevents the development of seizure susceptibility. Also, Sze and Maxson (1975) have shown that the acoustic priming effect in C57BL/6 mice is dependent on glucocorticoids. Adrenalectomy, inhibitors of glucocorticoid synthesis, and blockers of glucocorticoid receptors all prevent acoustic priming. Since GABA may regulate glutamic acid decarboxylase synthesis through feedback repression (Sze and Lovell, 1970) and since glucocorticoids may modulate protein synthesis (Thompson and Lippman, 1974), we have hypothesized that acoustic priming may, in part, be mediated by an effect on some aspects of protein synthesis in the brain. In order to explore this possibility, three experiments were conducted. In Experiment 1, protein synthesis was inhibited during and after the IAS with cycloheximide. The inhibitory effect of cycloheximide on the rate of protein synthesis in the brain of mice of this strain and age was monitored. In the second experiment, in order to ensure the specificity of the cycloheximide effect, puromycin, another inhibitor of protein synthesis, was used. Also, the possible involvement of RNA synthesis was examined by using actinomycin D and diaminopurine. These drugs were administered intraventricularly. In Experiment 3, a different approach to a role of macromolecules in acoustic priming was used. Here attempts to transfer the priming effect through brain extracts were made. EXPERIMENT 1 Method Subjects. A total of 227 male and female mice of the C57BL/6Bg strain were used. These mice were maintained in our specific-pathogen-free colony.
MACROMOLECULES AND ACOUSTIC PRIMING
113
Acidified (pH 2.5), chlorinated (12-15 ppm) water and pasteurized Charles River mouse chow were available ad lib. The water was acidified and chlorinated to prevent infection by Pseudomonas species. The colony room was on a 12hr light: 12hr dark cycle (lights on at 6 AM) with temperature kept at 22 +- I°C and relative humidity at 52 + 2%. Procedure. In the study of the effects of cycloheximide on acoustic priming, five groups (N= 203) were used. At 19 days of age, all except the no priming control group were exposed for 60 sec to an initial auditory stimulus (IAS) from an electric doorbell (95-105 dB; re 2 × 10-s dynes/cm2). The no priming control group was treated only with saline on Day 19 of age, and the acoustic priming control group was injected with saline 0.5 hr prior to IAS. The three drug-treated groups were injected with cycloheximide (30 mg/kg, ip) at either 0.5 hr prior to IAS or immediately after IAS or 0.5 hr after IAS. At 28 days of age, all mice were tested for audiogenic seizures by exposure, after a 60 sec wait in the testing chamber, to the same intense noise as the IAS for 90 sec or until the occurrence of a clonic-tonic convulsion. Seizure incidence was calculated as the percentage of mice that exhibited clonic-tonic seizures among the total number tested. The level of significance between proportions was used to analyze the data (Npq: Arkin and Colton, 1970). To monitor the effects of cycloheximide on the inhibition of brain protein synthesis, six groups with four animals per group were used. Brain protein synthesis was estimated by the rate of incorporation of [14C] leucine into proteins according to a modification of the method of Flexner et al. (1965). Cycloheximide (30 mg/kg, ip) was injected into 19 day old mice at 0.5, 1.0, 2.0, 3.0, or 4.0 hr before sacrifice. L-[U -14 C]-leucine (1/.t Ci/mouse, sc) was injected into all mice at 0.5 hr before sacrifice. The whole brain was removed, homogenized in 5 ml of 0 . 1 N NaOH, and 15 ml of ice-cold 10% trichoroacetic acid (TCA) was then added. After standing for 1 hr at 4°C, the TCA precipitate was collected by centrifugation at 11,000 g for 20 rain and washed three times with 5 ml of cold 10% TCA. The washed precipitate was dissolved in 1.0 ml of perchloric acid (PCA). After heating at 60°C overnight, the radioactivity of the PCA digest was measured in Bray's solution by liquid scintillation spectrometry. An aliquot (0.5 ml) of the TCA supernatant was also measured for radio-activity in order to correct for the variation of [14C]leucine taken up by the brain as described by Flexner et al. (1965). The degree of inhibition after cycloheximide was calculated by comparison with the rate of [14C]leucine incorporation into protein in mice not treated with cycloheximide. Results. As shown in Fig. 1, treatment with cycloheximide at 0.5 hr prior to the IAS markedly attenuates the acoustic priming ( P < 0 . 0 2 ) . Treatment with cycloheximide immediately after or at 0.5 hr after the IAS had no effect. Brain protein synthesis was inhibited by cycloheximide almost
114
MAXSON, TOWLE AND SZE
100 N=26
N=67
2
6O
u
40
N=36
N:54 c
2 2O N:20 n
I
-m-
"rrr
~-
Fig. 1. Effect of cycloheximide on acoustic priming of C57BL/6Bg mice. There were five groups of mice. These were no priming control (I); acoustic priming control (II); cycloheximide (30 mg/kg, ip 0.5 hr prior to acoustic priming (III); cycloheximide immediately after acoustic priming (IV); and cycloheximide 0.5 hr after acoustic priming (V). The control groups were injected with saline. The mice were acoustically primed on Day 19 of age. All mice were tested for audiogenic seizures on Day 29 of age. *p < 0.02 (NPQ test; Arkin and Colton, 1970) in comparison of groups II and III.
immediately and was maximally inhibited (95 to 100%) between 0.5 and 1 hr after injection of cycloheximide (Fig. 2). Thus, when cycloheximide was injected 0.5 hr prior to acoustic priming, there was almost total inhibition of protein synthesis during priming and for 0.5 hr after. Since injection of cycloheximide immediately after IAS had no effect on acoustic priming, protein synthesis was not required 0.5 hr or more after the IAS for the consequent development of susceptibility to audiogenic seizures. 100 ¢n
} 8o E Iv ~
6o
e
40
.1=
_~
2o
o 0.5
1
2
3
4
5
Hr. a f t e r C y c l o h e x l m l d e InJection
Fig. 2. Effect of cyclohexirnide on [C 14 ] leucine incorporation into brain protein. Day 19 C57BL/6 mice were injected with cycloheximide (30 mg/kg, ip) at the indicated time intervals before sacrifice. There were four mice per point; the SEM for all points was less than +5% of the mean.
MACROMOLECULES AND ACOUSTIC PRIMING
l 15
EXPERIMENT 2
Method Subjects. A total of 115 male and female mice of the C57BL/6Bg strain served as subjects. Colony maintenance and conditions were the same as those in Experiment 1. Procedure. In this study to compare the effects of various inhibitors of RNA and protein synthesis on acoustic priming, eight treatment groups were used. The drugs were injected intraventricularly as follows: A hole was drilled stereotaxically on the skull 2 mm posterior to Bregma and 2 mm lateral to the midline while the mouse was under ether anesthesia. The needle of a 10-/al Hamilton syringe was inserted to a depth o f 1.5 mm and the drug (dissolved in 5/ll o f 4% surcrose) was injected into the lateral ventricle. The controls were injected with 5/~1 of the sucrose vehicle. The needle was left in place for 1 min to prevent back leakage of the injected solution. After the needle was removed, the scalp incision was immediately dosed with a wound clip. All mice except the no priming control group were exposed at 19 days of age to IAS. The drugs and the time periods of treatment were: puromycin, 200/ag/mouse, 2 hr before IAS or immediately after IAS; acfinomycin D, 2.5/ag/mouse, 2.5 hr before IAS or immediately after or 8 days after IAS, diaminopurine, 100#g/mouse, 2 . 5 h r before IAS. At 28 days of age, all mice were tested for audiogenic seizures as described in Experiment 1. Results As shown in Table 1, when puromycin was administered 2 hr before IAS, seizure risk was reduced by 29% ( P < 0 . 0 1 ) . When it was injected TABLE 1 Effect ofPuromycin on Acou~ic Priming
Treatment No priming Acoustic priming and 5.0/~1 vehicle Puromycin (200/~g/mouse at 2 hr before acoustic priming) Puromycin (200/~g/mouse immediately after acoustic priming)
N
C-T seizures %
20 30
0 90
23
61"
8
100
Note. Acoustic priming was given at 19 days of age. All mice were tested for audiogenic seizures at 28 days of age. Injections of puromycin (200/~g/mouse in 5.0 ~1 of 4% sucrose) or vehicle (5.0 /A of 4% sucrose) were intraventricular. *P < 0.01.
116
MAXSON, TOWLE AND SZE TABLE 2 Effect of Actinomycin D and Diaminopurine on Acoustic Priming
Treatment
N
No priming and 5.0 #1 of 4% sucrose Acoustic priming and 5.0 #1 of 4% sucrose Actinomycin D (2.5 #g/mouse) at 2.5 hr before acoustic priming Actinomycin D (2.5 #g/mouse) immediately after acoustic priming Actinomycin D (2.5 #g/mouse) 8 days after acoustic priming Diaminopurine (100 gg/mouse) 2.5 hr before acoustic priming
20 30
C-T seizures (%) 0 90
10
0*
10
0*
7
0*
7
86
Note. Acoustic priming was given at 19 days of age. All mice were tested at 28 days. Injections of actinomycin D (2.5 #g/mouse in 5.0 #1 of 4% sucrose), diaminopurine (100 #g/mouse in 5.0 #1 of 4% sucrose) or vehicle (5.0 #1 of 4% sucrose) were intraventricular. *P < 0.01 (significance of difference bewteen actinomycin D groups and acoustic priming control group). immediately after IAS, there was no effect on seizure incidence. These effects of puromycin are similar to those found in Experiment 1 with cycloheximide. In contrast, as shown in Table 2, treatment with actinomycin D either 2.5 hr before IAS or immediately after IAS completely blocked acoustic priming (0% seizure incidence; P < 0.01). When actinomycin D was injected at 1 day before testing (8 days after IAS), the drug was found also effective in preventing the seizure incidence in the already primed mice. Diaminopurine, an in vivo inhibitor of R N A synthesis in brain (Hitchings and Elion, 1963; Kobiler and Allweis, 1974), had no effect on acoustic induction of seizure risk.
EXPERIMENT 3 Method Subjects. A total of 64 male and female C57BL/6Bg mice were used. Colony conditions have been described in Experiment 1. Procedure. In this study of "transfer" of acoustic priming, there were five treatment groups. The no priming control and the acoustic priming control were the same as those used in Experiment 1. Each mouse of the three experimental groups was injected with a brain extract obtained from
MACROMOLECULES AND ACOUSTIC PRIMING
117
TABLE 3 Brain Extract and "Transfer" of Acoustic Priming
Treatment No priming and saline Acoustic priming and saline 19D-19R 28D-19R 28D-27R
N
C-T seizures %
20 67 8 10 6
0 85 12 0 0
Note. Mice were acoustically primed on Day 19 and tested for audiogenic seizures on Day 28. Each brain extract was prepared from four donors and dissolved in 0.5 ml of saline; the brain extracts were injected ip. There were no significant differences between saline and brain extract no priming groups. XD is the age in days of mice donating (D) brain extract and YR is the age in days of mice receiving (R) the brain extract.
four donors which had been exposed on Day 19 to IAS. To prepare the extract (Ungar, 1971), the brains from four donors were homogenized in 2 volumes of ice-cold distilled water and the homogenate was centrifuged at 4 0 , 0 0 0 g for 20 rain. The supernatant was lyophilized and the residue was redissolved in 0.5 ml of saline. In the first experimental group, the brain extract was prepared from donors 0.5 hr after exposure to IAS and it was injected into the recipient at 19 days o f age. In the other two experimental groups, the brain extract was taken from donors 9 days after exposure to IAS and it was injected into either 19- or 27-day-old recipients. At 28 days o f age, all mice were tested for audiogenic seizures as described in Experiment 1. Results
Although one mouse did seize in the group receiving brain extract at 19 days of age, there were no significant differences between any of the experimentals and no priming control (Table 3).
DISCUSSION Both cycloheximide and puromycin attenuate acoustic priming if administered immediately before but not after the exposure to IAS. Since b o t h cycloheximide and puromycin almost completely inhibit protein synthesis, and since each does so by different mechanisms (Caskey, 1973), these results might be taken as evidence that acoustic priming requires, at least in part, brain protein synthesis. This dependence appeared to occur in a
118
MAXSON,TOWLEAND SZE
relatively brief period, extending from the presentation of IAS to 0.5 hr after IAS. Such an effect of cycloheximide parallels that on memory (Squire and Barondes, 1973); cycloheximide disrupts memory if given before or immediately after training. On the basis of their finding, Squire and Barondes (1973) concluded that normal memory storage may depend on protein synthesis within a few minutes of initial training. Our findings on the effects of puromycin on acoustic priming differ from those of puromycin on learning and memory, where puromycin is effective for 24 to 48 hr after training. Both drugs have other effects which might account for the attenuation of acoustic priming. For example, puromycin produces abnormal electrical activity in the hippocampus (Cohen et al., 1966) and decreases the threshold to pentylenetetrazol-induced seizures (Cohen and Barondes, 1967). Thus, brain seizures might be a cause in the attenuation of the acoustic priming effect. However, Henry and Bowman (1970) showed that electroshock convulsions, between 30 sec and 24 hr after IAS, had no effect on acoustic priming. Since the disruption of memory by cycloheximide can be partially reversed by foot shock, dextroamphetamine and corticosteroids (Barondes and Cohen, 1968), cycloheximide might attenuate acoustic priming by lowering the "level of arousal." However, Henry (1967) showed that mice anesthetized with ether or barbiturate could be acoustically primed. Also, Henry and Bowman (1970) reported that hypothermia stress had no effect on acoustic priming. Moreover, it has recently been demonstrated that the effects of acetoxycycloheximide (Serota et al., 1972) or puromycin (Roberts et al., 1970) on memory can be reversed by adrenergic drugs and it has been hypothesized that both drugs act on memory by decreasing the norepinephrine available at the synapse. However, reserpine and catron, which lower brain levels of norepinephrine, and pargyline, which blocks the oxidation of monoamines, did not antagonize acoustic priming (Boggan et al., 1971; Kellog, 1975). Similarly, no changes in brain level of norepinephrine or serotonin occurred after the IAS (Sze, 1970). Thus, the attenuation of the acoustic priming effect by cycloheximide and puromycin cannot be attributed to their effect on the levels of biogenic amines. In fact, by ruling out such major side effects of cycloheximide and puromycin, a strong case can be made that these drugs may indeed antagonize acoustic priming because of their inhibition of protein synthesis. If acoustic priming is in part dependent on protein synthesis, it might also require de novo synthesis of one or more types of RNA, at least in the period during and immediately after IAS. However, our findings on the effects of diaminopurine and actinomycin D do not sustain this hypothesis. Diaminopurine, which inhibits RNA synthesis probably by blocking the incorporation of purine nucleotides (Hitchings and Elion, 1963; Kobiler and Allweis, 1974), had no effect on acoustic priming. Actinomycin D, which inhibits RNA synthesis by preventing elongation of RNA chains (Sung, 1972), completely blocked acoustic priming when administered either 2.5 hr before,
MACROMOLECULES AND ACOUSTIC PRIMING
119
or immediately after, or even 8 days after the IAS. In contrast to the inhibitors of protein synthesis, actinomycin D totally blocks the acoustic priming effect both before and after the IAS. These results and that of diaminopurine suggest that the actinomycin D may be acting on acoustic priming by neurological damage (Appel, 1965) rather than specific inhibition of RNA synthesis. Several investigators have suggested that acoustic priming may be a model or type of simple learning. Such a proposal is based on observations of similarity between training and test stimulus (Henry et al., 1971) and of a critical period (Fjerdingstad, 1973; Corwin and Stanford, 1973). The latter studies involved the transfer of acoustic priming effect in brain extracts. Fjerdingstad (1973) reported that six out of 10 recipients of a brain extract prepared from primed C57BL/6 mice convulsed on initial exposure to an intense noise, and that none of the recipients of brain extract prepared from control mice convulsed. Corwin and Stanford (1973) reported that the recipients of brain extracts from C57BL/6 mice exposed to an IAS were not significantly different on the first day's test from recipients of brain extract from control mice; whereas they had a higher seizure incidence on a second exposure to the sound stimulus. Thus, the findings of Corwin and Stanford for first day test resemble ours, whereas those of Fjerdingstad do not. Since two out of three studies on transfer of acoustic priming in brain extract have not found an increase in seizure susceptibility on first day test, the evidence for the synthesis of an "informational" protein after exposure to the IAS is equivocal. However, differences in injection route may account for the discrepant findings; Fjerdingstad's was intraventricular, whereas the others were intraperitoneal. It should also be noted that acoustic priming differs from learning in the absence of the effect of electroshock convulsions (Henry and Bowman, 1970), and in the extreme brevity of the period requiring protein synthesis following presentation of the initial stimulus. In addition to protein synthesis, acoustic priming in C57BL/6 mice appears to be dependent also on GABA (Sze, 1970) and corticosteroids (Sze and Maxson, 1975). It also appears to be mediated, at least in part, by hearing loss and by disuse supersensitivity (Henry, 1972). All of these findings may not be contradictory and may rather be complementary. It is possible that there is more than a single mechanism of acoustic priming in these mice, and that the IAS leads to an increase in seizure susceptibility through several routes. In support of this hypothesis is the observation that neither AOAA nor inhibitors of protein synthesis totally block acoustic priming and that mechanical loss of hearing does not cause the same increment in susceptibility to seizures as does the exposure to the IAS. Thus, we suggest that there may be two mechanisms acting separately or synergistically to produce acoustic priming in the C57BL/6 mice. One of these is mediated by GABA levels and also requires protein synthesis, with both biochemical events occurring within
120
MAXSON, TOWLE AND SZE
0.5 hr of the IAS. The other is mediated by peripheral hearing loss. Since inhibitors of glucocorticoid synthesis can completely block acoustic priming (Sze and Maxson, 1975), both mechanisms may require glucocorticoids.
REFERENCES Appel, S. H. (1965). Effect of inhibition of RNA synthesis on neural information storage. Nature (London) 207, 1163-1166. Arkin, H., and Colton, R. R. (1970). "Statistical Methods." New York: Barnes and Noble. Barondes, S. H., and Cohen, H. D. (1968). Arousal and the conversion of "short term" to "long term" memory. Proc. Nat. Acad. ScL U.S.A. 61, 923-929. Boggan, W. O., Freedman, D. X., and Lovell, R. A. (1971). Studies in audiogenic seizure susceptibility. Psychopharmacologia(Berlin) 20, 48-56. Caskey, C. T. (1973). Inhibitors of protein synthesis. In R. M. Hochster, M. Kates, and J. H. Quastel (Eds.), "Metabolic Inhibitors," Vol. 4, pp. 131-177. New York: Academic Press. Chert, C.-S. (1973). Sensitization of audiogenic seizures in two strains of mice and their F1 hybrids. Develop. Psychobiol. 6, 13t-138. Chen, C.-S. and Fuiler, J. L. (1975). Selection for genetically determined of priming induced audiogenic seizure susceptibility in mice. Behav. Genet. 5, 91-92. Cohen, H., Ervin, F., and Barondes, S. H. (1966). Puromyein and eydoheximide: Different effects on hippocampal electrical activity. Science 154, 1557-1558. Cohen, H. D., and Barondes, S. H. (1967). Purornycin effect on memory may be due to occult seizures. Science 157, 333-334. Corwin, J. M., and Stanford, A. L. (1973). Increased susceptibility to audiogenic seizures resulting from injection of brain extract from acoustically primed mice. Physiol. PsychoL 1, 324-326. Fjerdingstad, E. J. (1973). Chemical transfer of learned information in mammals and fish. In H. P. Zippel (Ed.), "Memory and Transfer of Information," pp. 429-449. New York: Plenum Press. Flexner, L. B., Flexner, J. B., de la Habo, G., and Roberts, R. B. (1965). Loss of memory as related to cerebral protein synthesis. J. Neurochem. 12, 505-541. Freund, G., and Walker, D. W. (1971). Sound-induced seizures during ethanol withdrawal in mice. Psychopharmacologia (Berlin) 22, 45-49. Fuller, J. L., and Collins, R. L. (1968). Temporal parameters of sensitization for audiogenic seizures. Develop. Psychobiol. 1, 185-188. Gates, G. R., Chen, C.-S., and Bock, G. R. (1973). Effects of monaural and binaural auditory deprivation on audicigenic seizure susceptibility in BALB/e mice. Exp. Neurol. 38, 488-493. Gates, G. R., and Chen, C.-S. (1974). Effects of barbiturate withdrawal on audiogenic seizure susceptibility in Balb/c mice. Nature (London) 244, 162-164. Henry, K. R. (1967). Audiogenic seizure susceptibility induced in C57BL/6J mice by prior auditory exposure. Science 158, 938-940. Henry, K. R., and Bowman, R. E. (1970). Acoustic priming of audiogenic seizures in mice. In B. E. Welch, and A. S. Welch (Eds.), "Physiological Effects of Noise," pp. 185-201. New York: Plenum Press. Henry, K. R., Thompson, K. A., and Bowman, R. E. (1971). Frequency characteristics of acoustic priming of audiogenic seizures in mice. Exp. NeuroL 31,402-407.
MACROMOLECULES AND ACOUSTIC PRIMING
121
Henry, K.R. (1972). Pinna reflex thresholds and audiogenic seizures: developmental changes after acoustic oriming. J. Comp. PhysioL Psychol. 79, 77-81. Hitchings, G. H., and Elion, G. B. (1963). Purine analogues. In R. M. Hochster, M. Kates, and J. H. Quastel (Eds.),"Metabolic Inhibitors," pp. 215-237. New York: Academic Press. Iturrian, W. B., and Fink, G. B. (1968). Effects of age and condition-test interval (days) on an audio conditioned convulsive response in CF#I mice. Develop. Psychobiol. 1, 230-235. Iturrian, W. B., and Johnson, H. D. (1971). Sound induced convulsions: Latency and severity in unilaterally audiosensitized mice. Pharmacology 5, 65-73. Jumonville, J. E. (1968). Influence o f genotype-environment interactions on studies o f "emotionality" in mice. Ph.D. dissertation, University of Chicago. Kellog, C. (1975). Audiogenic seizures: Relation to age and central monaminergic mechanisms. NeuroscL Abstr. 1, 793. Kobiler, D., and AUweis, C. (1974). The prevention of long-term memory formation by 2,6 diaminopurine. Pharmacol. Biochem. Behav. 2, 9-17. Maxson, S. C., Sze, P. Y., and Cowen, J.S. (1975). Pharmacogenetics of the sensory induction of audiogenic seizures in two strains of mice. Behav. Genet. 5, 102-103. McGinn, M. D., Willott, J. F., and Henry, K. R. (1973). Effects of conductive hearing loss on auditory evoked potentials and audiogenic seizures in mice. Nature (New Biol.) 244, 255-256. Roberts, R. B., Flexner, J. B., and Flexner, L. B. (1970). Some evidence for the involvement of adrenergic sites in the memory trace. Proc. Nat. Acad. Sci. U.S.A. 66, 3110-3113. Saunders, J. C., Bock, G. R., James, R., and Chen, C.-S. (1972). Effects of priming for audiogenic seizures on auditory responses in cochlear nucleus and inferior colliculus of BALB/c mice. Exp. Neurol. 37, 388-394. Scrota, R. G., Roberts, R. B., and Flexner, L. B. (1972). Acetoxycycloheximide-induced transient amnesia: Protective effects of adrenergic stimulation. Proe. Nat. Acad. Sci. U.S.A. 69, 340-342. Sharpless, S. K. (1969). Isolated and deafferented neurons: Disuse supersensitivity. In H. H. Jaspers, A. A. Ward, and A. Pope (Eds.), "Basic Mechanism of the Epilepsies," pp. 329-348. Boston: Little, Brown. Squire, L. R., and Barondes, S. H. (1973). Memory impairment during prolonged training in mice given inhibitors of cerebral protein synthesis. Brain Res. 56, 215-225. Sung, S. C. (1972). Inhibitors of RNA and DNA biosynthesis. In R. M. Hochster, M. Kates, and J. H. Quastel (Eds.), "Metabolic Inhibitors," Vol. 8, pp. 185-204. New York: Academic Press. Sze, P. Y. (1970). Neurochemical factors in auditory stimulation and development of susceptibility to audiogenic seizures. In B. E. Welch, and A. S. Welch (Eds.), "Physiological Effects of Noise," pp. 259-269. New York: Plenum Press. Sze, P. Y., and Lovell, R. A. (1970). Reduction of levels of L-glutamic acid decarboxylase by ~-aminobutyric acid in mouse brain. J. Neurochem. 17, 1657-1664. Sze, P. Y., and Maxson, S. C. (1975). Involvement of corticosteroids in acoustic induction of audiogenic seizure susceptibility in mice. Psychopharmacologia (Berl.) 45, 79-82,. Thompson, E. B., and Lippman, M. E. (1974). Mechanism of action of glucocorticoids. Metabolism 23, 159-202. Ungar, G. (1971). Bioassays for the chemical correlates of acquired information. In E. J. Fjerdingstad (Ed.), "Chemical Transfer of Learned Information," pp. 31-49. Amsterdam: North Holland.
122
MAXSON, TOWLE AND SZE
WiUott, J.F., and Henry, K. R. (1974). Auditory evoked potentials: Developmental changes of threshold and amplitude following early acoustic trauma. J. Comp. Physiol. Psyc'hol. 86, 1-7. Willott, J. F., Henry, K. R., and George, F. (1975). Noise-induced hearing loss, auditory evoked potentials and protection from audiogenie seizures. Exp. Neurol. 46, 542-553.
Macromolecules of the Central Nervous System and Acoustic Priming in C57BL/6Bg Mice
STEPHEN C. MAXSON, ANDREW C. TOWLE, and PAUL Y. SZE 1
Department o f Biobehavioral Sciences, The University o f Connecticut, Storrs, Connecticut 06268
Cycloheximide (30 mg/kg, ip) or puromycin (200/~g/mouse, intraventricular) was administered to C57BL/6Bg mice either before or after acoustic palming. Treatment with either of these inhibitors of protein synthesis significantly attenuates the acoustic priming. Although intraventricular injection of actinomycin D (2.5/~g/mouse) before and after the initial auditory stimulus completely blocks the acoustic priming, another inhibitor of RNA synthesis, diaminopurine (100 #g/mouse) had no effect. Thus, acoustic priming may be partially dependent upon protein synthesis in the brain either during and/or briefly (0.5 hr) after the initial auditory stimulus, and may not be dependent upon RNA synthesis. Also, acoustic priming was not "transferred" through brain extracts prepared from 19- or 28-day-old donors and injected into 19- or 27-day-old recipients.
Exposure to an initial auditory stimulus (IAS) during a sensitive period of postnatal development induces susceptibility to audiogenic seizures in otherwise resistant mice (Henry, 1967; Fuller and Collins, 1968; Iturrian and Fink, 1968; Jumonville, 1968; Sze, 1970). Several studies on the mechanisms of this acoustic priming effect support the hypothesis that the IAS damages the acoustic receptors and thereby causes partial deafness (Henry, 1972; Willott and Henry, 1974), that the partial hearing loss results in a disuse supersensitivity of the brain stem auditory system, and that at the subsequent exposure to the sound stimulus either an increase in amount of neural activity (Saunders et al., 1972) or a change in pattern of neural activity (WiUott et al., 1975) in the inferior colliculus elicits the brain and behavioral seizure. This theory is supported by evidence that tympanic rupture (Gates et al., 1973) or ear block (McGinn et al., 1973) also induces susceptibility to audiogenic 1This work was supported by NIH Grants RR 00602-04, AA 00297-03, and by a grant from The Grant Foundation, Inc. 111 Copyright © 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
112
MAXSON, TOWLE AND SZE
seizures in otherwise resistant mice. Thus, this theory suggests that the initial events in the chain of causes are peripheral and that the changes in the central nervous system are derivative. Also, this theory resembles those of disuse supersensitivity for epilepsies following depressant withdrawal (Sharpless, 1969; Freund and Walker, 1971; Gates and Chen, 1974). However, there is evidence that this may not be the only mechanism for the acoustic priming effect. Chen (1973) has demonstrated genetic heterogeneity,and Maxson et al. (1975) have provided evidence of pharmacogenetic differences among inbred strains of mice. More directly, Chen and Fuller (1975) reported that tympanic rupture does not induce susceptibility in a selected stock of acoustically primable mice. This suggests that some effects of the IAS may directly involve the central nervous system. A similar hypothesis has been proposed by Iturrian and Johnson (1971) on the basis of studies of development of unilateral susceptibility after the IAS. Some of these central effects of the IAS may be mediated by biochemical events. Sze (1970) has reported that the IAS causes a small and transient fall (within 0.5 hr) in the level of brain 7-aminobutyric acid (GABA) and that reversal of this fall by prior treatment with aminooxyacetic acid (AOAA) prevents the development of seizure susceptibility. Also, Sze and Maxson (1975) have shown that the acoustic priming effect in C57BL/6 mice is dependent on glucocorticoids. Adrenalectomy, inhibitors of glucocorticoid synthesis, and blockers of glucocorticoid receptors all prevent acoustic priming. Since GABA may regulate glutamic acid decarboxylase synthesis through feedback repression (Sze and Lovell, 1970) and since glucocorticoids may modulate protein synthesis (Thompson and Lippman, 1974), we have hypothesized that acoustic priming may, in part, be mediated by an effect on some aspects of protein synthesis in the brain. In order to explore this possibility, three experiments were conducted. In Experiment 1, protein synthesis was inhibited during and after the IAS with cycloheximide. The inhibitory effect of cycloheximide on the rate of protein synthesis in the brain of mice of this strain and age was monitored. In the second experiment, in order to ensure the specificity of the cycloheximide effect, puromycin, another inhibitor of protein synthesis, was used. Also, the possible involvement of RNA synthesis was examined by using actinomycin D and diaminopurine. These drugs were administered intraventricularly. In Experiment 3, a different approach to a role of macromolecules in acoustic priming was used. Here attempts to transfer the priming effect through brain extracts were made. EXPERIMENT 1 Method Subjects. A total of 227 male and female mice of the C57BL/6Bg strain were used. These mice were maintained in our specific-pathogen-free colony.
MACROMOLECULES AND ACOUSTIC PRIMING
113
Acidified (pH 2.5), chlorinated (12-15 ppm) water and pasteurized Charles River mouse chow were available ad lib. The water was acidified and chlorinated to prevent infection by Pseudomonas species. The colony room was on a 12hr light: 12hr dark cycle (lights on at 6 AM) with temperature kept at 22 +- I°C and relative humidity at 52 + 2%. Procedure. In the study of the effects of cycloheximide on acoustic priming, five groups (N= 203) were used. At 19 days of age, all except the no priming control group were exposed for 60 sec to an initial auditory stimulus (IAS) from an electric doorbell (95-105 dB; re 2 × 10-s dynes/cm2). The no priming control group was treated only with saline on Day 19 of age, and the acoustic priming control group was injected with saline 0.5 hr prior to IAS. The three drug-treated groups were injected with cycloheximide (30 mg/kg, ip) at either 0.5 hr prior to IAS or immediately after IAS or 0.5 hr after IAS. At 28 days of age, all mice were tested for audiogenic seizures by exposure, after a 60 sec wait in the testing chamber, to the same intense noise as the IAS for 90 sec or until the occurrence of a clonic-tonic convulsion. Seizure incidence was calculated as the percentage of mice that exhibited clonic-tonic seizures among the total number tested. The level of significance between proportions was used to analyze the data (Npq: Arkin and Colton, 1970). To monitor the effects of cycloheximide on the inhibition of brain protein synthesis, six groups with four animals per group were used. Brain protein synthesis was estimated by the rate of incorporation of [14C] leucine into proteins according to a modification of the method of Flexner et al. (1965). Cycloheximide (30 mg/kg, ip) was injected into 19 day old mice at 0.5, 1.0, 2.0, 3.0, or 4.0 hr before sacrifice. L-[U -14 C]-leucine (1/.t Ci/mouse, sc) was injected into all mice at 0.5 hr before sacrifice. The whole brain was removed, homogenized in 5 ml of 0 . 1 N NaOH, and 15 ml of ice-cold 10% trichoroacetic acid (TCA) was then added. After standing for 1 hr at 4°C, the TCA precipitate was collected by centrifugation at 11,000 g for 20 rain and washed three times with 5 ml of cold 10% TCA. The washed precipitate was dissolved in 1.0 ml of perchloric acid (PCA). After heating at 60°C overnight, the radioactivity of the PCA digest was measured in Bray's solution by liquid scintillation spectrometry. An aliquot (0.5 ml) of the TCA supernatant was also measured for radio-activity in order to correct for the variation of [14C]leucine taken up by the brain as described by Flexner et al. (1965). The degree of inhibition after cycloheximide was calculated by comparison with the rate of [14C]leucine incorporation into protein in mice not treated with cycloheximide. Results. As shown in Fig. 1, treatment with cycloheximide at 0.5 hr prior to the IAS markedly attenuates the acoustic priming ( P < 0 . 0 2 ) . Treatment with cycloheximide immediately after or at 0.5 hr after the IAS had no effect. Brain protein synthesis was inhibited by cycloheximide almost
114
MAXSON, TOWLE AND SZE
100 N=26
N=67
2
6O
u
40
N=36
N:54 c
2 2O N:20 n
I
-m-
"rrr
~-
Fig. 1. Effect of cycloheximide on acoustic priming of C57BL/6Bg mice. There were five groups of mice. These were no priming control (I); acoustic priming control (II); cycloheximide (30 mg/kg, ip 0.5 hr prior to acoustic priming (III); cycloheximide immediately after acoustic priming (IV); and cycloheximide 0.5 hr after acoustic priming (V). The control groups were injected with saline. The mice were acoustically primed on Day 19 of age. All mice were tested for audiogenic seizures on Day 29 of age. *p < 0.02 (NPQ test; Arkin and Colton, 1970) in comparison of groups II and III.
immediately and was maximally inhibited (95 to 100%) between 0.5 and 1 hr after injection of cycloheximide (Fig. 2). Thus, when cycloheximide was injected 0.5 hr prior to acoustic priming, there was almost total inhibition of protein synthesis during priming and for 0.5 hr after. Since injection of cycloheximide immediately after IAS had no effect on acoustic priming, protein synthesis was not required 0.5 hr or more after the IAS for the consequent development of susceptibility to audiogenic seizures. 100 ¢n
} 8o E Iv ~
6o
e
40
.1=
_~
2o
o 0.5
1
2
3
4
5
Hr. a f t e r C y c l o h e x l m l d e InJection
Fig. 2. Effect of cyclohexirnide on [C 14 ] leucine incorporation into brain protein. Day 19 C57BL/6 mice were injected with cycloheximide (30 mg/kg, ip) at the indicated time intervals before sacrifice. There were four mice per point; the SEM for all points was less than +5% of the mean.
MACROMOLECULES AND ACOUSTIC PRIMING
l 15
EXPERIMENT 2
Method Subjects. A total of 115 male and female mice of the C57BL/6Bg strain served as subjects. Colony maintenance and conditions were the same as those in Experiment 1. Procedure. In this study to compare the effects of various inhibitors of RNA and protein synthesis on acoustic priming, eight treatment groups were used. The drugs were injected intraventricularly as follows: A hole was drilled stereotaxically on the skull 2 mm posterior to Bregma and 2 mm lateral to the midline while the mouse was under ether anesthesia. The needle of a 10-/al Hamilton syringe was inserted to a depth o f 1.5 mm and the drug (dissolved in 5/ll o f 4% surcrose) was injected into the lateral ventricle. The controls were injected with 5/~1 of the sucrose vehicle. The needle was left in place for 1 min to prevent back leakage of the injected solution. After the needle was removed, the scalp incision was immediately dosed with a wound clip. All mice except the no priming control group were exposed at 19 days of age to IAS. The drugs and the time periods of treatment were: puromycin, 200/ag/mouse, 2 hr before IAS or immediately after IAS; acfinomycin D, 2.5/ag/mouse, 2.5 hr before IAS or immediately after or 8 days after IAS, diaminopurine, 100#g/mouse, 2 . 5 h r before IAS. At 28 days of age, all mice were tested for audiogenic seizures as described in Experiment 1. Results As shown in Table 1, when puromycin was administered 2 hr before IAS, seizure risk was reduced by 29% ( P < 0 . 0 1 ) . When it was injected TABLE 1 Effect ofPuromycin on Acou~ic Priming
Treatment No priming Acoustic priming and 5.0/~1 vehicle Puromycin (200/~g/mouse at 2 hr before acoustic priming) Puromycin (200/~g/mouse immediately after acoustic priming)
N
C-T seizures %
20 30
0 90
23
61"
8
100
Note. Acoustic priming was given at 19 days of age. All mice were tested for audiogenic seizures at 28 days of age. Injections of puromycin (200/~g/mouse in 5.0 ~1 of 4% sucrose) or vehicle (5.0 /A of 4% sucrose) were intraventricular. *P < 0.01.
116
MAXSON, TOWLE AND SZE TABLE 2 Effect of Actinomycin D and Diaminopurine on Acoustic Priming
Treatment
N
No priming and 5.0 #1 of 4% sucrose Acoustic priming and 5.0 #1 of 4% sucrose Actinomycin D (2.5 #g/mouse) at 2.5 hr before acoustic priming Actinomycin D (2.5 #g/mouse) immediately after acoustic priming Actinomycin D (2.5 #g/mouse) 8 days after acoustic priming Diaminopurine (100 gg/mouse) 2.5 hr before acoustic priming
20 30
C-T seizures (%) 0 90
10
0*
10
0*
7
0*
7
86
Note. Acoustic priming was given at 19 days of age. All mice were tested at 28 days. Injections of actinomycin D (2.5 #g/mouse in 5.0 #1 of 4% sucrose), diaminopurine (100 #g/mouse in 5.0 #1 of 4% sucrose) or vehicle (5.0 #1 of 4% sucrose) were intraventricular. *P < 0.01 (significance of difference bewteen actinomycin D groups and acoustic priming control group). immediately after IAS, there was no effect on seizure incidence. These effects of puromycin are similar to those found in Experiment 1 with cycloheximide. In contrast, as shown in Table 2, treatment with actinomycin D either 2.5 hr before IAS or immediately after IAS completely blocked acoustic priming (0% seizure incidence; P < 0.01). When actinomycin D was injected at 1 day before testing (8 days after IAS), the drug was found also effective in preventing the seizure incidence in the already primed mice. Diaminopurine, an in vivo inhibitor of R N A synthesis in brain (Hitchings and Elion, 1963; Kobiler and Allweis, 1974), had no effect on acoustic induction of seizure risk.
EXPERIMENT 3 Method Subjects. A total of 64 male and female C57BL/6Bg mice were used. Colony conditions have been described in Experiment 1. Procedure. In this study of "transfer" of acoustic priming, there were five treatment groups. The no priming control and the acoustic priming control were the same as those used in Experiment 1. Each mouse of the three experimental groups was injected with a brain extract obtained from
MACROMOLECULES AND ACOUSTIC PRIMING
117
TABLE 3 Brain Extract and "Transfer" of Acoustic Priming
Treatment No priming and saline Acoustic priming and saline 19D-19R 28D-19R 28D-27R
N
C-T seizures %
20 67 8 10 6
0 85 12 0 0
Note. Mice were acoustically primed on Day 19 and tested for audiogenic seizures on Day 28. Each brain extract was prepared from four donors and dissolved in 0.5 ml of saline; the brain extracts were injected ip. There were no significant differences between saline and brain extract no priming groups. XD is the age in days of mice donating (D) brain extract and YR is the age in days of mice receiving (R) the brain extract.
four donors which had been exposed on Day 19 to IAS. To prepare the extract (Ungar, 1971), the brains from four donors were homogenized in 2 volumes of ice-cold distilled water and the homogenate was centrifuged at 4 0 , 0 0 0 g for 20 rain. The supernatant was lyophilized and the residue was redissolved in 0.5 ml of saline. In the first experimental group, the brain extract was prepared from donors 0.5 hr after exposure to IAS and it was injected into the recipient at 19 days o f age. In the other two experimental groups, the brain extract was taken from donors 9 days after exposure to IAS and it was injected into either 19- or 27-day-old recipients. At 28 days o f age, all mice were tested for audiogenic seizures as described in Experiment 1. Results
Although one mouse did seize in the group receiving brain extract at 19 days of age, there were no significant differences between any of the experimentals and no priming control (Table 3).
DISCUSSION Both cycloheximide and puromycin attenuate acoustic priming if administered immediately before but not after the exposure to IAS. Since b o t h cycloheximide and puromycin almost completely inhibit protein synthesis, and since each does so by different mechanisms (Caskey, 1973), these results might be taken as evidence that acoustic priming requires, at least in part, brain protein synthesis. This dependence appeared to occur in a
118
MAXSON,TOWLEAND SZE
relatively brief period, extending from the presentation of IAS to 0.5 hr after IAS. Such an effect of cycloheximide parallels that on memory (Squire and Barondes, 1973); cycloheximide disrupts memory if given before or immediately after training. On the basis of their finding, Squire and Barondes (1973) concluded that normal memory storage may depend on protein synthesis within a few minutes of initial training. Our findings on the effects of puromycin on acoustic priming differ from those of puromycin on learning and memory, where puromycin is effective for 24 to 48 hr after training. Both drugs have other effects which might account for the attenuation of acoustic priming. For example, puromycin produces abnormal electrical activity in the hippocampus (Cohen et al., 1966) and decreases the threshold to pentylenetetrazol-induced seizures (Cohen and Barondes, 1967). Thus, brain seizures might be a cause in the attenuation of the acoustic priming effect. However, Henry and Bowman (1970) showed that electroshock convulsions, between 30 sec and 24 hr after IAS, had no effect on acoustic priming. Since the disruption of memory by cycloheximide can be partially reversed by foot shock, dextroamphetamine and corticosteroids (Barondes and Cohen, 1968), cycloheximide might attenuate acoustic priming by lowering the "level of arousal." However, Henry (1967) showed that mice anesthetized with ether or barbiturate could be acoustically primed. Also, Henry and Bowman (1970) reported that hypothermia stress had no effect on acoustic priming. Moreover, it has recently been demonstrated that the effects of acetoxycycloheximide (Serota et al., 1972) or puromycin (Roberts et al., 1970) on memory can be reversed by adrenergic drugs and it has been hypothesized that both drugs act on memory by decreasing the norepinephrine available at the synapse. However, reserpine and catron, which lower brain levels of norepinephrine, and pargyline, which blocks the oxidation of monoamines, did not antagonize acoustic priming (Boggan et al., 1971; Kellog, 1975). Similarly, no changes in brain level of norepinephrine or serotonin occurred after the IAS (Sze, 1970). Thus, the attenuation of the acoustic priming effect by cycloheximide and puromycin cannot be attributed to their effect on the levels of biogenic amines. In fact, by ruling out such major side effects of cycloheximide and puromycin, a strong case can be made that these drugs may indeed antagonize acoustic priming because of their inhibition of protein synthesis. If acoustic priming is in part dependent on protein synthesis, it might also require de novo synthesis of one or more types of RNA, at least in the period during and immediately after IAS. However, our findings on the effects of diaminopurine and actinomycin D do not sustain this hypothesis. Diaminopurine, which inhibits RNA synthesis probably by blocking the incorporation of purine nucleotides (Hitchings and Elion, 1963; Kobiler and Allweis, 1974), had no effect on acoustic priming. Actinomycin D, which inhibits RNA synthesis by preventing elongation of RNA chains (Sung, 1972), completely blocked acoustic priming when administered either 2.5 hr before,
MACROMOLECULES AND ACOUSTIC PRIMING
119
or immediately after, or even 8 days after the IAS. In contrast to the inhibitors of protein synthesis, actinomycin D totally blocks the acoustic priming effect both before and after the IAS. These results and that of diaminopurine suggest that the actinomycin D may be acting on acoustic priming by neurological damage (Appel, 1965) rather than specific inhibition of RNA synthesis. Several investigators have suggested that acoustic priming may be a model or type of simple learning. Such a proposal is based on observations of similarity between training and test stimulus (Henry et al., 1971) and of a critical period (Fjerdingstad, 1973; Corwin and Stanford, 1973). The latter studies involved the transfer of acoustic priming effect in brain extracts. Fjerdingstad (1973) reported that six out of 10 recipients of a brain extract prepared from primed C57BL/6 mice convulsed on initial exposure to an intense noise, and that none of the recipients of brain extract prepared from control mice convulsed. Corwin and Stanford (1973) reported that the recipients of brain extracts from C57BL/6 mice exposed to an IAS were not significantly different on the first day's test from recipients of brain extract from control mice; whereas they had a higher seizure incidence on a second exposure to the sound stimulus. Thus, the findings of Corwin and Stanford for first day test resemble ours, whereas those of Fjerdingstad do not. Since two out of three studies on transfer of acoustic priming in brain extract have not found an increase in seizure susceptibility on first day test, the evidence for the synthesis of an "informational" protein after exposure to the IAS is equivocal. However, differences in injection route may account for the discrepant findings; Fjerdingstad's was intraventricular, whereas the others were intraperitoneal. It should also be noted that acoustic priming differs from learning in the absence of the effect of electroshock convulsions (Henry and Bowman, 1970), and in the extreme brevity of the period requiring protein synthesis following presentation of the initial stimulus. In addition to protein synthesis, acoustic priming in C57BL/6 mice appears to be dependent also on GABA (Sze, 1970) and corticosteroids (Sze and Maxson, 1975). It also appears to be mediated, at least in part, by hearing loss and by disuse supersensitivity (Henry, 1972). All of these findings may not be contradictory and may rather be complementary. It is possible that there is more than a single mechanism of acoustic priming in these mice, and that the IAS leads to an increase in seizure susceptibility through several routes. In support of this hypothesis is the observation that neither AOAA nor inhibitors of protein synthesis totally block acoustic priming and that mechanical loss of hearing does not cause the same increment in susceptibility to seizures as does the exposure to the IAS. Thus, we suggest that there may be two mechanisms acting separately or synergistically to produce acoustic priming in the C57BL/6 mice. One of these is mediated by GABA levels and also requires protein synthesis, with both biochemical events occurring within
120
MAXSON, TOWLE AND SZE
0.5 hr of the IAS. The other is mediated by peripheral hearing loss. Since inhibitors of glucocorticoid synthesis can completely block acoustic priming (Sze and Maxson, 1975), both mechanisms may require glucocorticoids.
REFERENCES Appel, S. H. (1965). Effect of inhibition of RNA synthesis on neural information storage. Nature (London) 207, 1163-1166. Arkin, H., and Colton, R. R. (1970). "Statistical Methods." New York: Barnes and Noble. Barondes, S. H., and Cohen, H. D. (1968). Arousal and the conversion of "short term" to "long term" memory. Proc. Nat. Acad. ScL U.S.A. 61, 923-929. Boggan, W. O., Freedman, D. X., and Lovell, R. A. (1971). Studies in audiogenic seizure susceptibility. Psychopharmacologia(Berlin) 20, 48-56. Caskey, C. T. (1973). Inhibitors of protein synthesis. In R. M. Hochster, M. Kates, and J. H. Quastel (Eds.), "Metabolic Inhibitors," Vol. 4, pp. 131-177. New York: Academic Press. Chert, C.-S. (1973). Sensitization of audiogenic seizures in two strains of mice and their F1 hybrids. Develop. Psychobiol. 6, 13t-138. Chen, C.-S. and Fuiler, J. L. (1975). Selection for genetically determined of priming induced audiogenic seizure susceptibility in mice. Behav. Genet. 5, 91-92. Cohen, H., Ervin, F., and Barondes, S. H. (1966). Puromyein and eydoheximide: Different effects on hippocampal electrical activity. Science 154, 1557-1558. Cohen, H. D., and Barondes, S. H. (1967). Purornycin effect on memory may be due to occult seizures. Science 157, 333-334. Corwin, J. M., and Stanford, A. L. (1973). Increased susceptibility to audiogenic seizures resulting from injection of brain extract from acoustically primed mice. Physiol. PsychoL 1, 324-326. Fjerdingstad, E. J. (1973). Chemical transfer of learned information in mammals and fish. In H. P. Zippel (Ed.), "Memory and Transfer of Information," pp. 429-449. New York: Plenum Press. Flexner, L. B., Flexner, J. B., de la Habo, G., and Roberts, R. B. (1965). Loss of memory as related to cerebral protein synthesis. J. Neurochem. 12, 505-541. Freund, G., and Walker, D. W. (1971). Sound-induced seizures during ethanol withdrawal in mice. Psychopharmacologia (Berlin) 22, 45-49. Fuller, J. L., and Collins, R. L. (1968). Temporal parameters of sensitization for audiogenic seizures. Develop. Psychobiol. 1, 185-188. Gates, G. R., Chen, C.-S., and Bock, G. R. (1973). Effects of monaural and binaural auditory deprivation on audicigenic seizure susceptibility in BALB/e mice. Exp. Neurol. 38, 488-493. Gates, G. R., and Chen, C.-S. (1974). Effects of barbiturate withdrawal on audiogenic seizure susceptibility in Balb/c mice. Nature (London) 244, 162-164. Henry, K. R. (1967). Audiogenic seizure susceptibility induced in C57BL/6J mice by prior auditory exposure. Science 158, 938-940. Henry, K. R., and Bowman, R. E. (1970). Acoustic priming of audiogenic seizures in mice. In B. E. Welch, and A. S. Welch (Eds.), "Physiological Effects of Noise," pp. 185-201. New York: Plenum Press. Henry, K. R., Thompson, K. A., and Bowman, R. E. (1971). Frequency characteristics of acoustic priming of audiogenic seizures in mice. Exp. NeuroL 31,402-407.
MACROMOLECULES AND ACOUSTIC PRIMING
121
Henry, K.R. (1972). Pinna reflex thresholds and audiogenic seizures: developmental changes after acoustic oriming. J. Comp. PhysioL Psychol. 79, 77-81. Hitchings, G. H., and Elion, G. B. (1963). Purine analogues. In R. M. Hochster, M. Kates, and J. H. Quastel (Eds.),"Metabolic Inhibitors," pp. 215-237. New York: Academic Press. Iturrian, W. B., and Fink, G. B. (1968). Effects of age and condition-test interval (days) on an audio conditioned convulsive response in CF#I mice. Develop. Psychobiol. 1, 230-235. Iturrian, W. B., and Johnson, H. D. (1971). Sound induced convulsions: Latency and severity in unilaterally audiosensitized mice. Pharmacology 5, 65-73. Jumonville, J. E. (1968). Influence o f genotype-environment interactions on studies o f "emotionality" in mice. Ph.D. dissertation, University of Chicago. Kellog, C. (1975). Audiogenic seizures: Relation to age and central monaminergic mechanisms. NeuroscL Abstr. 1, 793. Kobiler, D., and AUweis, C. (1974). The prevention of long-term memory formation by 2,6 diaminopurine. Pharmacol. Biochem. Behav. 2, 9-17. Maxson, S. C., Sze, P. Y., and Cowen, J.S. (1975). Pharmacogenetics of the sensory induction of audiogenic seizures in two strains of mice. Behav. Genet. 5, 102-103. McGinn, M. D., Willott, J. F., and Henry, K. R. (1973). Effects of conductive hearing loss on auditory evoked potentials and audiogenic seizures in mice. Nature (New Biol.) 244, 255-256. Roberts, R. B., Flexner, J. B., and Flexner, L. B. (1970). Some evidence for the involvement of adrenergic sites in the memory trace. Proc. Nat. Acad. Sci. U.S.A. 66, 3110-3113. Saunders, J. C., Bock, G. R., James, R., and Chen, C.-S. (1972). Effects of priming for audiogenic seizures on auditory responses in cochlear nucleus and inferior colliculus of BALB/c mice. Exp. Neurol. 37, 388-394. Scrota, R. G., Roberts, R. B., and Flexner, L. B. (1972). Acetoxycycloheximide-induced transient amnesia: Protective effects of adrenergic stimulation. Proe. Nat. Acad. Sci. U.S.A. 69, 340-342. Sharpless, S. K. (1969). Isolated and deafferented neurons: Disuse supersensitivity. In H. H. Jaspers, A. A. Ward, and A. Pope (Eds.), "Basic Mechanism of the Epilepsies," pp. 329-348. Boston: Little, Brown. Squire, L. R., and Barondes, S. H. (1973). Memory impairment during prolonged training in mice given inhibitors of cerebral protein synthesis. Brain Res. 56, 215-225. Sung, S. C. (1972). Inhibitors of RNA and DNA biosynthesis. In R. M. Hochster, M. Kates, and J. H. Quastel (Eds.), "Metabolic Inhibitors," Vol. 8, pp. 185-204. New York: Academic Press. Sze, P. Y. (1970). Neurochemical factors in auditory stimulation and development of susceptibility to audiogenic seizures. In B. E. Welch, and A. S. Welch (Eds.), "Physiological Effects of Noise," pp. 259-269. New York: Plenum Press. Sze, P. Y., and Lovell, R. A. (1970). Reduction of levels of L-glutamic acid decarboxylase by ~-aminobutyric acid in mouse brain. J. Neurochem. 17, 1657-1664. Sze, P. Y., and Maxson, S. C. (1975). Involvement of corticosteroids in acoustic induction of audiogenic seizure susceptibility in mice. Psychopharmacologia (Berl.) 45, 79-82,. Thompson, E. B., and Lippman, M. E. (1974). Mechanism of action of glucocorticoids. Metabolism 23, 159-202. Ungar, G. (1971). Bioassays for the chemical correlates of acquired information. In E. J. Fjerdingstad (Ed.), "Chemical Transfer of Learned Information," pp. 31-49. Amsterdam: North Holland.
122
MAXSON, TOWLE AND SZE
WiUott, J.F., and Henry, K. R. (1974). Auditory evoked potentials: Developmental changes of threshold and amplitude following early acoustic trauma. J. Comp. Physiol. Psyc'hol. 86, 1-7. Willott, J. F., Henry, K. R., and George, F. (1975). Noise-induced hearing loss, auditory evoked potentials and protection from audiogenie seizures. Exp. Neurol. 46, 542-553.
