Download Inhibition of cerebral protein synthesis impairs long

Braht Research, 97 (1975) 367-372
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367
Inhibition of cerebral protein synthesis impairs long-term habituation
L A R R Y R. SQUIRE AND C A R L K. BECKER
Veterans Admbdstration Hospital and Department of Psychiatry, University of California School of
Medicine, La Jolla, Salt Diego, Calif. 92161 (U.S.A.)
(Accepted July 1st, 1975)
When brain protein synthesis is inhibited shortly before or after training, retention is normal for a few hours but subsequently is severely impaired 1,7,1°. When inhibition is initiated 30 min or longer after training, memory is not affected. These
findings have been obtained with rodents, chicks and fish, using a variety of learning
tasks including discrimination problems, passive avoidance, and active avoidance.
Taken together, these results suggest that brain protein synthesis, during or shortly
after training, is required for the formation of 'long-term' memory. Recently, interest
has developed in the study of simpler forms of learning which, in suitable preparations, might be amenable to relatively direct neurophysiological and biochemical
analysis. Habituation, the gradual waning of a response to repeated presentations of
the same stimulus, is exhibited throughout the animal kingdom and is frequently
considered to be the simplest form of learning 5. Under favorable circumstances
habituation can persist for days or weeks~,6.
Recently, it was shown that the electrophysiological correlates of short-term
habituation recorded from motoneuron L-7 of Aplysia proceeded normally during
stimulation of the siphon nerve in spite of marked inhibition of ganglionic protein
synthesis 8. This result suggested that protein synthesis is not required for short-term
habituation, and is in keeping with evidence from fish, chicks, and rodents that memory can persist normally for many minutes and sometimes for hours when training
occurs during profound inhibition of brain protein synthesis 1,7,1°. We now report
that inhibition of cerebral protein synthesis by anisomycin impairs long-term habituation in mice.
Male hybrid mice (C3H × Balb/C) were obtained from Simonson Laboratories
(Gilroy, California) at 8 weeks of age and introduced to the experimental situation at
12 weeks of age when they weighed 23-25 g. Habituation was studied with an innate
suppression technique6,16. This technique assessed the responsiveness of mice to
presentations of loud auditory stimuli (conspecific distress cries), as indicated by the
capacity of these stimuli to interrupt drinking. Typically, stimuli given in a short
session become progressively less effective in interrupting drinking, and this reduction
in interruptability constitutes short-term habituation. Long-term habituation is evi-
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denced by the fact that this reduction in responsiveness to auditory stimuli can persist for several days.
Mice were fluid deprived for 48 h and then for the duration of the experiment
were permitted each day to drink water for 3 min in 12 cm × 13 cm × 19 cm high
chambers constructed of 3 mm white Plexiglas. To maintain body weight, mice were
given an additional 7 min access to water in their home cages at the end of each daily
3-min drinking period. Each lick of water emitted during the 3-min drinking period
activated a pen on an event recorder. The total fluid volume consumed during each
drinking period was also noted. A recording of a distress cry was obtained by exposing
a male hybrid mouse (C3H × Balb/C) to inescapable footshock (0.4 mA). For presentation, the cries were amplified to a volume of 106 dB (measured 3 cm above the
floor of the closed drinking chamber with a Brfiel and Kjaer sound level meter, Type
2209, C scale, slow ballistic response).
The mice drank undisturbed for 7 days while the fluid volume consumed during
each drinking period stabilized. Distress cries were first presented on the following
day (Day 1). The stimuli were 4 repetitions of a 4-sec conspecific distress cry, delivered
through a 10 cm round loudspeaker (Oaktron AA3C) mounted in the top of each
drinking chamber. The first distress cry occurred 15 sec after the beginning of the
3-min drinking period, and the 3 repetitions of this stimulus occurred at 15-sec intervals. To assess long-term habituation, mice heard the same recorded distress cry
during their daily drinking period 3 days later (Day 4). No distress cries were presented during the intervening days. On these days, mice drank for their usual 3-min
period without disturbance.
Anisomycin (30 mg/kg) and lithium chloride (150 mg/kg) were injected subcutaneously and intraperitoneally, respectively, in saline solution (0.3 ml/25 g body
weight). Statistical comparisons between groups were made with Mann-Whitney
U-tests 9.
Mice exhibited considerable within-session habituation to the 4 distress cries
presented on Day 1. Fig. 1A indicates the median number of licks of water emitted
by mice during each distress cry. Mice not drinking during the 4 sec prior to the first
distress cry on Day 1 were eliminated from the experiment (N = 18/166 = 10.8 %).
Mice emitted a significantly greater number of licks during the second and third stimuli
than during the first one (P < 0.01). The low number of licks emitted during the fourth
stimulus was due to the fact that by this time many mice (54/148) had already stopped
drinking. Since the effect of a stimulus on drinking could not be determined for mice
not drinking at stimulus onset, including the data for all mice probably underestimates the extent of within-session habituation. Habituation was decidedly more pronounced if only those mice were considered that emitted/> one lick of water during the
4 sec prior to stimulus presentation (Fig. 1A).
For the same reason, the best measure of long-term habituation was the number
of licks of water emitted during the first distress cry of the retention test. Nearly all
mice (93 %) emitted /> 10 licks of water during the 4 sec prior to the first distress cry
of the retention test. With each successive stimulus, however, the probability increased
that a mouse had already stopped drinking well before stimulus onset. Long-term
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Fig. 1. Thirsty mice heard 4 distress cries (106 dB for 4 sec) at 15-sec intervals, during a 3-min drinking period. Successive stimuli were progressively less effective in interrupting drinking (A, above).
Data are shown for all mice tested, and also for those mice (percentages indicated) emitting /> one
water lick during the 4 sec prior to each stimulus. The median number of water licks during the 4 sec
prior to each stimulus was 34, 34, 29, 15. The distress cries on Day 1 were followed immediately by injections of saline, anisomycin (Ani-0 Hrs), lithium chloride (LiC1-0 Hrs), or by a delayed injection of
anisomycin (Ani-8 Hrs.) Three days later (B, above), a distress cry interrupted the fluid intake of the
Ani-0 Hrs group to a greater extent than it interrupted the fluid intake of each of the other 3 groups
(P < 0.01). During the 4 sec prior to the distress cry on Day 1, the median number of water licks was
33, 34, 32, and 34 for mice given saline, LiC1, Ani-8, or Ani-0, respectively. During the 4 sec prior to
the first distress cry on Day 4, the corresponding values were 34, 34, 34, and 32.
h a b i t u a t i o n was therefore assessed by c o m p a r i n g the n u m b e r of licks emitted during
the first distress cry on D a y 1 with the n u m b e r of licks emitted during the first distress cry on D a y 4 (Fig. 1B).
Mice were given saline, l i t h i u m chloride, or a n i s o m y c i n immediately after the
d r i n k i n g period o n D a y 1. Since a n i s o m y c i n itself could possibly have p r o d u c e d a
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TABLE I
MEAN FLUID INTAKE (ml) DURING DAILY 3-MINUTE DRINKING PERIODS
Mice were given the indicated d r u g s immediately or 8 h after the drinking period on D a y 1. N o n e o f
the differences between g r o u p s on D a y 1 or D a y 4 were significant (P ~ 0.1).
Day
Saline-O Hrs
LiCI-O Hrs
Ani-8 Hrs
Ani-O Hrs
1
2
3
4
1.0
0.9
1.0
1.2
1.0
0.7
0.9
1.2
1.0
0.6
0.9
1.1
1.0
0.5
0.7
1.0
persisting effect on startle, another group was given anisomycin 8 h after the drinking
period (Ani-8 Hrs) and tested again on Day 4. Mice given anisomycin immediately
after the drinking period on Day 1 emitted significantly fewer licks during the first
distress cry on Day 4 than each of the 3 control groups (P < 0.01). Taken together,
these results indicate that long-term habituation to the distress cries persisted for at
least 4 days; and that anisomycin given immediately after the distress cries on Day 1
impaired long-term habituation.
The lithium chloride groups were designed to evaluate the possibility that conditioned aversion to water was established by anisomycin on Day 1, and that this
aversion was subsequently responsible for the observed suppression of drinking on
Day 4. The present dose of lithium chloride (150 mg/kg) had been determined previously to produce as much conditioned gustatory aversion as the 30 mg/kg dose of
anisomycin1~. Mice given lithium chloride immediately after the drinking period on
Day 1 behaved on Day 4 similarly to control mice (P > 0.2 for each comparison), and
also drank more water during the first distress cry of the retention test than the Ani-0
Hrs group (P < 0.01). These results suggest that conditioned aversion produced by
anisomycin was not responsible for the impairment of long-term habituation.
Table I indicates the total fluid volume ingested by mice on Days 1-4. On Day 2
the saline group drank more than each of the other drug groups (P < 0.05), and the
drug groups did not differ from each other (P > 0.05). By Day 4 fluid intake had returned to the pre-injection, Day 1 level for all groups and none of the between-group
comparisons were significant (P > 0.1). An analysis of drinking during the 4 sec prior
to the distress cries also suggested that by Day 4 mice had recovered from effects of
the drugs on fluid intake. By Day 4 each group had returned to its Day 1 level and the
4 groups were drinking at the same median rate during the 4 sec prior to the first
distress cry (saline = 34; LiC1 = 34; Ani-8 = 34; Ani-0 = 32; P > 0.4).
Four conspecific distress cries produced within-session habituation, and between
session habituation that lasted at least 3 days. Long-term habituation was partially
disrupted by initiating a period of protein synthesis inhibition immediately after
presentation of the distress cries. It seems unlikely that anisomycin caused su.ppression
of fluid intake during the distress cry on Day 4 by causing greater conditioned gustatory aversion than the other drugs. First, during the 3-min retention sessions total
371
fluid intake was similar across drug groups. Second, during the retention sessions
on Day 4, mice in each drug group drank at about the same rate during the 4 sec prior
to the distress cry.
The present results differ from an earlier report that cycloheximide did not affect
long-term habituation of exploratory activity in mice 14. Since overtraining is known to
reduce markedly the susceptibility of mice to the amnesic effects of protein synthesis
inhibitors 2, it seems possible that mice were relatively overtrained in the earlier study.
It is also possible, of course, that there are other important, and as yet unidentified,
differences between exploratory habituation and the present habituation paradigm.
Anisomycin produced only partial amnesia for long-term habituation (Fig. 1B).
This finding is consistent with previous reports that anisomycin or cycloheximide can
produce partial amnesia for discrimination training11,12 or passive avoidance training 4. In the present study, considerable protein synthesis required for long-term retention could have occurred during the habituation session on Day 1 before the establishment of inhibition by the post-session injection. Approximately 3 min elapsed between
the first distress cry and the drug injection, and several more minutes must have elapsed before a substantial level of inhibition could have been achieved 4. It has been determined that the present dose of anisomycin (30 mg/kg) inhibits more than 90 % cerebral protein synthesis during the 15-45 min period after injection 12, and that proteinsynthesizing capacity gradually recovers during the subsequent 2-8 h (ref. 4).
The possible involvement of side effects of protein synthesis inhibitors has been
considered repeatedly; and known effects of anisomycin on locomotor behavior 11,
tyrosine hydroxylase activity15 and conditioned gustatory aversion 13 have been dissociated from amnesia. In addition, as discussed previously 11, anisomycin, cycloheximide,
and puromycin each inhibits cerebral protein synthesis and causes amnesia, but each
has a different mechanism of action and exerts different side effects. All available
evidence is, therefore, consistent with the hypothesis that anisomycin inhibits protein
synthesis required for the formation of 'long-term' memory. Nevertheless, it is always
difficult in behavioral pharmacological studies of this type to attribute definitively the
behavioral effects of a drug to its major known mode of action.
Previous work has suggested that cerebral protein synthesis is required for
'long-term' memory of discrimination training, and active and passive avoidance
learningS,7, ~0. The present results suggest that this conclusion can be extended to
habituation, a phylogenetically primitive form of long-lasting behavioral plasticity.
This research was supported by Grant MH24600 from the National Institute of
Mental Health and by a Clinical Investigatorship (8084C) from the Veterans Administration to the first author. Anisomycin was the generous gift of N. Belcher of Pfizer
Pharmaceuticals.
We thank Dr. H. Ruhm for assisting with the sound level determinations.
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