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NORMAL RETENTION DURING INHIBITION
8F PROTEIN SYNTHESIS INDUCED
BY CYCLOHEXIMIDE IN RATS

Dissertation for the Degree of Ph. 0..
MICHIGAN STATE UNIVERSITY
MAURLINE MURRAY PREACHE

1973 v '

   
   

  

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ABSTRACT

NORMAL RETENTION DURING INHIBITION OF PROTEIN SYNTHESIS
INDUCED BY CYCLOHEXIMIDE IN RATS

By

Maurline Murray Preache

As a test of the hypothesis that memory requires ongoing synthesis
of brain proteins, an inhibitor of protein synthesis, cycloheximide
(CXM), was evaluated for its effect on acquisition and retention of
brightness discriminated shock avoidance in rats and two strains of
mice. For rats both subcutaneous (3.0 mg/kg body weight) and intra—
cerebral (400 pg) treatments were explored, while for mice only subcu-
taneous injections (120 or 150 mg/kg) were used. Training was to a
criterion of five correct responses in six trials and retention was
measured as percent savings on trials to criterion 6 or 24 hr later.

To control for the possibility of overtraining, in one eXperiment
training was limited to a low fixed number of trials (2, 5, or 12) and
percent correct responses in 12 test trials was compared for CXM-
treated and control rats. Whole brain protein synthesis was also
determined in rats after subcutaneous CXM administration (0.45, 0.9,
1.8, and 2.4 mg/kg), with the result that all doses inhibited synthesis
by at least 40% and 2.4 mg/kg dose produced inhibition of 93.6%.

The major outcome of the behavioral experiments in rats and mice
was that in no case did CXM treatment produce statistically significant

impairment of retention as measured by percent savings or by percent

Maurline Murray Preache

correct responses after a fixed number of training trials. Intra—
cerebral injections of CXM (400 Hg) in rats resulted in treated-control
group differences in savings that were.relatively large compared to
those observed with subcutaneous injections, but these too fell short
of statistical significance (p>.10).

Variables which were secondary to the drug treatments were also
evaluated for their effect on brightness-discriminated avoidance and
for interaction with CXM treatments. These included an investigation
of the effect of retaining rats in the goal box for 10 vs 30 sec, the
effect of training mice during the light vs the dark part of their 12
hr light-dark.cycle, and the effect of housing mice in brighter or
dimmer environments prior to discrimination training. With these three
manipulations, the only effect to approach significance was that
animals trained in the dark part of the cycle tended to have higher
savings scores than those trained during the lights-on period (p<.10).

The results were examined in relation to reports which indicated
CXM-induced amnesia in mice (Baroudes, S. H., and Cohen, H. D.
Proceedings: Natl. Acad. of Science, 1968, 61, 923). It was tenta-
tively suggested that the failure to induce amnesia with CXM in mice
was due to the failure of both CXM-treated and control mice to learn
the discrimination during acquisition. Across the different experi-
ments, savings for control mice averaged less than 40%. This was in
contrast to the 70+Z savings in control mice observed by Barondes and
Cohen (1968) and was in spite of a replication of their procedures
to the extent that they were known. The assumption was made that the
difference in their results and those reported here must be attributed
to some difference in the pre-experimental experience of the mice, or

to differences in handling, or to some other methodological deviation

Maurline Murray Preache
not apparent in a comparison of the two procedures. With respect to
the results of the rat experiments. it was suggested that the observar
tions of normal retention in rats trained while greater than 90% of
brain protein synthesis was inhibited is contradictory to the hypothesis
that CXM impairs retention because it inhibits brain protein synthesis.
Literature related to other cerebral effects of the protein inhibitor,
puromycin, was offered as indirect support for the suggestion that the
mechanism of action in CXM induction of amnesia may be unrelated to the
protein inhibiting prOperties of the drug. It was further suggested
that whatever the mechanism, rats and mice were different in their
susceptibility to it. However, in the final analysis it could not be
conclusively stated that inadequate inhibition of protein synthesis in
the case of rats, or some undetermined procedural variation common to
both the rat and mouse experiments, accounted for the failure to induce
amnesia with CXM.

In summary, these experiments showed that amnesia did not obtain
in rats treated subcutaneously with CXM despite inhibition of protein
synthesis in excess of 90%, and despite comparability of procedures
with those that have resulted in amnesia in mice (Barondes and Cohen,
1968) as evidenced by comparability of control group performance. The
mice experiments reported here seemed to indicate that undertraining

may prevent the observation of CXM-induced amnesia.

NORMAL RETENTION DURING INHIBITION.OF PROTEIN SYNTHESIS

INDUCED BY CYCLOHEXIMIDE.IN RATS

By

Maurline.Murray Preache

A.DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

.DOCTOR OF PHILOSOPHY

Department of Psychology

1973

Dedication

This dissertation is dedicated to my parents,
Walter and Nellie Murray,
who have given loving and patient moral
support during my academic career
and throughout my life

ii

ACKNOWLEDGMENTS

The author wishes to thank the graduate committee members,
Drs. M. Ray Denny, Edward M. Eisenstein, James E. Gibson, Glenn
I. Hatton, and especially John I. Johnson, Jr., for their assistance
throughout this project. In addition to the guidance they provided,
they have generously contributed, laboratory space, equipment, and
supplies to make the project possible. Appreciation is also
expressed to the Upjohn Company for providing cycloheximide; to
Dr. John L. King for the use of equipment; to Gary Connors, Marvin
Little and Norman St. Pierre for assistance in constructing equip—
ment; to Andrew Harton for his procurement activities; to Robert
Canada, Joyce Pennington, and William Stevens for assistance in
carrying out experiments, and to Janice Fuller for typing the
dissertation. Finally, a very special thank you is due Cynthia
Keller, who from the beginning of this project enthusiastically
participated as though it were her own, and whose contribution in
terms of the number of hours she devoted to it could only be equaled
by the number of bad puns she made.

This project was supported by a Biomedical Research Grant.

iii

TABLE OF CONTENTS

INTRODUCTION AND REVIEWLOF LITERATURE . . . . . . . . . . .

Conditions Which Attenuate or Eliminate Amnesia
Induced by AXM or CXM O O O O O O O O O O O O O O

RATIONALE AND PLAN‘OF EXPERIMENTS . . . . . . . . . . . . .

EXPERIMENTAL METHODS AND RESULTS. . . . . . . . . . . . . .

Experiments 1 and 2: CXM-Induced Inhibition of Rat
Brain Protein Synthesis . . . . . . . . . . . . .

Me thOd O O O O O O O O O O O O O O O O O O O 0
Results and.Discussion. . . . . . . . . . . .

Experiments 3 and 4: Subcutaneous CXM Treatments in
Rats Trained to Criterion . . . . . . . . . . . .

MethOd O O O O O O O O O O O O O 0 O O O O O O
Res-flts O O O O O O O O O O O I O O O O O O 0

Experiment 5: Subcutaneous CXM Treatments in Rats
Trained a Fixed Number of Trials. . . . . . . .

Method. . . . . . . . . . . . . . . . . . . .
Requ-ts O O O O O O O O O O O O O I O O 0 O 0

Experiments 6 and 7: Retention After Intracerebral
CXM.Treatments in Rats. . . . . . . . . . . . . .

MethOd O O C O O O O O O O O O O O O O O O 9
Results . . . . . . . . . . . . . . . . . .

Experiment 8: Retention after CXM Treatments in Swiss-

web star Mice O O O O O O O O O O O O O O O O O O O

Meth 0d 0 O O O O O O O O O O O O O O O O O O 0
Results 0 O O O O O O O O O O O O O O O O O 0

Experiments 9 and 10: Retention after CXM Treatments

in Charles River Swiss Albino Mice. . . . . . . .

MethOd O O O O O O O O O O O O O O O O O O O 0
Results 0 O O O O O O O O O O O O O O O O 0

iv

Page

11

15

15
15
17
20
20
23
25
26
27
29
29
31
31
32
34
34

35
36

Experiment 11: Retention in CXM-Treated
vs Ni 8h t Trainin g 0 O O O O O I O O 0

Method 0 O O O O O O O 0 O O O O O
Restllts O O O O O O O O O O O O 0

Experiment 12: CXM Treatments in Mice Trained

Black-White Discrimination. . . . . .

Method 0 O O O O O O O O O O I O O
Resu1ts O O I O O O O O O O O O 0

DISCUSSION OF BEHAVIORAL EXPERIMENTS. . . . . .

Is Inhibition of Protein Synthesis the Effective

Mechanism.of.Drug-Induced.Amnesia?. .

Retention by Rats After CXM Treatments . . . . .

Retention in Mice.After CXM.Treatments . . . . .

Brightness Discrimination Training After CXM Treatment .

Effect of Secondary Variables on Discriminated Avoidance

SUMRY O O O O O O O O O O O O O O O O O O O O

WENCES O O O O O O O O O O O O O O O O O O O

Page

36
37
4O
42

43
44

46

46
47
53
57
58
59

6O

Table

10

11

12

13

LIST OF TABLES

Summary from selected literature sources . . . . . .

Distribution of rats used in Experiments 3 and 4 and
their mm hady wej-ghts O O O O O O O O O O O O O O 0

Mean trials to criterion during training for CXM-
treated and control rats of Experiments 3 and 4. . .

Mean percent savings on.trials.to criterion for CXMr
treated and control rats of Experiments 3 and 4. . .

Within-group comparisons on trials to criterion in
training and testing (Experiments 3 and 4) . . . . .

Analysis of variance for percent.correct responses
(test trials 1-10, Experiment 5) . . . . . . . . . .

Summary of animals used and their mean.body weights
for Emeriments 6 and 7 O O O O O O O O O O O O O O 0

Median trials to training criterion and mean percent
savings (Experiments 6 and 7). . . . . . . . . . . .

Distribution of mice used in Experiments 9 and 10 and

their median trials to criterion in training . . . .

Mean percent savings for EXperiments 9 and 10. . . . .

Distribution of animals to subgroups of Experiment 11

with median trials to criterion and mean percent
saving for each subgroup . . . . . . . . . . . . . .

Median trials to criterion and mean percent savings
for composite groups of Experiment 11. . . . . . . .

Distribution of mice used in Experiment 12 and their
median trials to criterion . . . . . . . . . . . . .

vi

Page

13

21

24

24

25

27

30

32

36

37

39

42

43

Figure

LIST OF FIGURES
Page

Percent inhibition of protein synthesis ----- and
inhibition of incorporation of 14C-1eucine----- in

rat brain after subcutaneous injections of CXM. Each

point represents the mean of four determinations . . . . . 18

Percent correct responses in 12 test trials for Experi-

ment 5. Rats were trained 2, 5, or 12 trials 30 min

after injections of 0.9% saline -—-—-—-'or CXM

----- and were tested 6 hr later . . . . . . . . . . . 28

Percent correct responses for 10 test trials 6 hr

after mice were trained to a criterion of five correct
responses in six trials. Saline (S) or CXM (120 mg/kg)

was administered 30 min before training. PM - trained
between 9:00~p.m. and 9:00 a.m., AM - trained between

9:00 a.m. and 9:00 a.m., Brighter Environment - mainte-

nance environment = 9 to 10 fc; Dimmer Environment -
maintenance environment = 5 fc . . . . . . . . . . . . . . 41

Percent savings on trials to criterion for mice trained

to a criterion of five correct responses in six trials

after treatment with 0, 120, or 150 mg/kg CXM. They

were trained 30 min after injection and tested 24 hr

later. The numbers in parentheses are the number of

animals in each group. . . . . . . . . . . . . . . . . . . 45

vii

INTRODUCTION AND REVIEW OF LITERATURE

The fundamental importance of proteins in regulation of cell
functioning has led to the hypothesis that.brain proteins may be
involved in longrterm.memory processes (Barondes, 1970b). The early
conception of protein as a template molecule which encoded experience
in terms of molecular composition and configuration (Katz and Halstead,
1950) has been replaced by models in which proteins are assigned an
enzymatic function in the synthesis of new or altered synaptic proteins
(Barondes, 1965; 1970b) or are conceived as part of a.feedback loop
which is initiated as the result of experience and is maintained by
the synthesis of new proteins which in turn regulate synthesis of DNA
and messenger RNA (Halstead and Rucker, 1970). If memory processes
involve.the synthesis of new or altered brain proteins, it might be
expected that a drastic reduction in ongoing synthesis would impair
memory. One approach to this problem has been to experimentally
inhibit protein synthesis and look at the effect on retention. This
approach now has a history of approximately ten years of investigations,
and typically involves treating an animal with an inhibitor shortly
before or after the acquisition of a learned response and then at some
variable time later testing for retention of what was learned. The
drug-treated group is compared with a control group that has been
injected with the drug vehicle. If it can be shown that the drug-
treated group is impaired in retention and that this is not attributable

to some nonspecific effect of the drug, the impaired retention may be

1

taken as support for the hypothesis that brain protein is required for
some aspect of the memory process.

The problem is of course that none of the inhibitory agents are
completely free of side effects, some of which are secondary to the
inhibition of protein synthesis and some of which are independent of it.
Squire and Barondes (1972a) reviewed literature related to effects other
than inhibition of protein synthesis which resulted from treatment with
puromycin and the glutamaride derivatives, acetoxycyclohexide (AXM) and
cycloheximide (CXM). Their review indicated that effects common to
both puromycin and the glutamarides included inhibition of ganglioside
synthesis and abnormal electrical activity in isolated ganglia. Puro—
mycin also resulted in abnormal cerebral electrical activity, mito-
chondrial abnormalities, inhibition of respiration in cerebral cortex
slices and inhibition of 3'5' adenosine monophosphate phosphodiesterase
Data concerning the latter effect were not available for the glutamarides;
however, AXM did not result in mitochondrial abnormalities (Gambetti,
Gonatas, and Flexner, 1968), nor did<DQiaffect respiration in guinea
pig cerebral cortex slices (Jones and Banks, 1969). CXM had no effect
on the cerebral activity of otherwise untreated mice (Cohen, Ervin, and
Barondes, 1966). Although the duration of seizures induced by electro-
convulsive shock (ECS) was longer in CXMetreated mice than in those
injected with saline, large doses of CXM (160-200 mg/kg) actually
raised the threshold for ROS-initiated seizures while smaller doses
had no effect (Luttges, Andry and MacInness, 1972), nor did AXM treat-
ment affect susceptibility to pentylenetetrazol seizures (Cohen and
Barondes, 1967).

Other side effects of inhibitors of protein synthesis are related

to systemic illness and Changes in behavioral activity levels. Although

3
no systematic data.are available, when doses were equated for inhibi-
tion of protein synthesis, subcutaneous injections of AXM in mice
appeared to.resnlt in.more symptoms of illness (e.g., lethargy, diarrhea,
and death) than was observed with intracerebral injections of puromycin,
CXM, or AXM or with subcutaneous injections of CXM (Barondes and Cohen,
1967; 1968b; Flexner, Stellar, de la Babe, and ROberts, 1962; Squire
and Barondes, 197.0b; Swanson, McGaugh, and Cotman, 1969). Even with the
more toxic treatment (subcutaneous AXM), the resulting retention deficit
was not.readily explainable in terms of systemic illness (Barondes and
Cohen, 1968b). Activity measures indicated that CXM increased and then
decreased activity in mice but that this effect could be dissociated
from the retention.deficits produced by this agent (Segal, Squire, and
Barondes, 1971).

Because the glutamarides have relative fewer cerebral side effects,
they have.been considered more effective in supporting the hypothesis
that synthesis of brain proteins has a specific role in memory processes.
The discussion below, which is limited to investigations in which AXM
or CXM was used as the inhibitor of protein synthesis, gives a brief
overview of the extent to which these compounds have been explored for
their effect an retention.

Retention deficits for animals treated with AXM or CXM have been
reported in several different species (e.g., chicks - watts and Marks,
1971; cockroaches - Brown and Noble, 1968; goldfish - Agranoff, Davis
and Drink, 1966; Agranoff, Davis, Casola, and Lim, 1967; rats - Daniels,
1971; 1972;.Serota, 1971; Serota, Roberts, and Flexner, 1972; and
mice - Andry and Luttges, 1972; Barondes and Cohen, 1967b; 1968a; 1968b;
Cohen and Barondes, 1968a; 1968b; Flexner, Flexner, and Roberts, 1966;

Flood, Bennett, Rosenzweig and Orne, 1972; Geller, Robustelli, Barondes,

4

Cohen, and Jarvik, 1969; Geller, Robustelli, and Jarvik, 1970; 1971;
Quartermain and McEwen, 1970; Quartermain, McEwen, and Azmita, 1970;
1972; Randt, Barnett, McEwen, and Quartermain, 1971; and Swanson,
McGaugh and Cotman, 1969). The majority of these investigations
involved some type of avoidance training including maze avoidance
discriminated by brightness (Barondes and Cohen, 1968a; 1968b; Cohen
and Barondes, 1968a; Daniels, 1971; Serota, 1971; Serota et al., 1972),
by position (Barondes and Cohen, 1967b), and by size (Squire and
Barondes, 1972); as well as passive avoidance (Andry and Luttges,
1970; Geller et al., 1969; 1970; 1971; Quartermain et al., 1970; 1971;
Randt et al., 1971; Swanson et al., 1970) and shuttlebox avoidance
(Agranoff.et al., 1966; 1967).. However, at least two investigations
(Cohen and Barondes, 1968b; Daniels, 1972) have explored the effect
of CXMélike inhibitors on positively-reinforced training and found
disruption of retention. In the early investigations where maze
discriminations were the to—be—remembered responses, no effect was
observed if.the retention interval was as short as 3 hr after training
(Barondes and.Cohen, 1967b; 1968b; Cohen and Barondes, 1968a). Frem
this and related evidence it was suggested that long-term memory was
the protein requiring process and explanations in terms of disruption
of consolidation were tentatively presented (Barondes and Cohen, 1967b;
Barondes, 1970b). However, when one-trial passive avoidance was
employed as the training procedure, retention intervals 2 hr after
training (Swanson et aZ., 1969) or even as short as 1 min after train-
ing (Quartermain and McEwen, 1970; Randt et al., 1971) have indicated
AXM- or CXMrinduced amnesia.

Thus, there is an extensive literature which indicates that the

glutamaride derivatives when administered at appropriate times and in

5

sufficient quantities result in amnesia or impaired retention. The
critical dose has been established as that which will result in approxi-
mately 90% inhibition of brain protein synthesis (Barondes, 1970a) and
of course.varies with the route of administration and the agent (AXM
or CXM) used. The critical time for inhibition of protein synthesis
with maze discriminations was established as during or within minutes
after training (Barondes and Cohen, 1968b). With passive avoidance,
however, post-training injections as late as 30 min (Geller et a1.,
1969) or 2 hr (Quartermain et al., 1970) have been effective.

Aside from the time and dose factors which limit the effect of
AXM or CXM, other variables that are.not so readily explainable within
the framework of the protein hypothesis have been found to reduce or
eliminate the effect on retention or to result in recovery from the
drug-induced amnesia. As a preface to reporting a series of experi-
ments in which CXM failed to affect retention, some of these variables
will be described.

Conditions Which Attenuate or Eliminate
Amnesia Induced by AXM or CXM

Overtraining was one of the first variables shown to prevent AXM
induction of amnesia in mice. Barondes and Cohen (1967a) trained mice
to a criterion.of 9 correct responses in 10 trials on a position
discrimination. The retention test showed no differences between the
drug-treated and control groups. Later they were able to show that
AXM-treated mice trained for a position discrimination to a criterion of
3 out of 4 had impaired retention while those trained to 9 out of 10 did
not (Barondes and Cohen, 1967b). The degree of training required to
prevent AXMéinduced amnesia depended upon the nature of the discrimina-

tion. With a brightness discrimination, AXM-treated mice that were

6

trained to either 5 out of 6 or to.9 out of 10 were impaired, while
those trained to 15 out of 16 were not (Cohen and Barondes, 1968a).

In passive avoidance experiments where foot shock was the aversive
stimulus, it has been observed that the intensity and duration of shock
interacts with CXM with respect to retention. Quartermain and McEwen
(1970) compared groups of CXMktreated mice that received 1.6 or .16 ms
of shock when they entered a.darkened compartment with their respective
saline-indented controls. Subgroups of each major training group were
tested at 1.or 5 min or at 6, 24, or 48 hr after the training. Regard—
less of the test time the CXM-treated mice which received the lower
level of shock gave no evidence of retention, while for the saline-
injected mice in this group retention was an increasing function up to
24 hr. With the more intense shock, the interval had no effect for
control mice, i.e., all showed good retention. The only interval at
which CXM~treated mice trained with intense shock were different from
the controls was at 24 hr where they showed significantly poorer reten-
tion. These results suggest that at higher shock levels, CXM amnesia
develops more slowly and is more transient than at lower levels. Flood
et al. (1972) reported that the percentage of control animals that were
amnesic after one-trial passive avoidance training decreased as the
shock intensity increased from 0.27 (24% amnesia) to 0.36 ma (0%
amnesia) in .03 ma steps. At the lower intensities of shock (0.27-
0.33 ma), 80—85% of CXM mice were amnesic 24 hr after training, while
with 0.36 ms only 20% showed amnesia. In a second experiment, these
investigators used a constant intensity of shock (0.33 ma) and varied
shock duration (0.5 to 0.13 min) and the retention interval (1 day,

l, 2, or 3 weeks). This resulted in a three-way interaction between

"drug condition (CXM vs saline), shock duration, and retention interval.

For the saline mice retention was good regardless of the duration of
shock or the test interval. When the retention interval was 24 hr
only the .OSemin shock duration resulted in amnesia for CXM-treated
mice. For the other intervals, the percentage of amnesic mice
decreased as the shock duration increased. More animals were amnesic
for the 2- or 3-week test interval than for the l-week interval and
this effect (of interval) was greatest for the longer shock durations.

A manipulation that has been successful in stimulating recovery
from CXMrinduced amnesia is the use of a "reminder shock." The term
"reminder" may be a misnomer in that it is not clear exactly why this
manipulation.improves retention. In any event, a noncontingent foot
shock (administered in an apparatus other than the training apparatus)
improved retention of a brightness discrimination for CXMetreated mice
when administered 3, but not 6, hr after training (Barondes and Cohen,
1960a). Quartermain et al. (1970) tested for recovery of memory of a
passive avoidance response by giving their CXM-treated mice a nonrein-
forced trial (T1) 24 hr after the initial training trial (To). When
this was followed 1 hr later by a noncontingent foot shock in a dif-
ferent apparatus, good retention was evident in the second test trial
(T2) given 44hr after T1. In contrast to the results of Barondes and
Cohen (1968a), who did not employ an intermediate test procedure, the
reminder shock did not improve retentionwwhen T1 was omitted. In a
later experiment recovery from amnesia was achieved by omitting the
RS entirely (Quartermain et aZ., 1972). Recovery developed as the

1 and T2 increased from 4 to 24 hr; however, even

interval between T
with a 24-hr interval recovery was not as complete as when T1 plus
RS was given. The effect of re—exposure to training stimuli, with or

without RS, was abolished or attenuated by administering a second dose

8

of CXM 30 min before T1 or RS. It was also shown that once memory was
recovered, it was as durable as memory established in the absence of a
protein inhibitor.

Quartermain and his co-workers emphasized that it was the original
memory that was recovered, and not some nonspecific effect of RS, or
some delayed association between T1 and RS as these procedures were not
effective with untrained animals. Their interpretation was that CXM
acted upon retrieval of memory rather than on consolidation. Barondes
and Cohen (1968a) placed a somewhat different emphasis on the recovery
of a maze discrimination stimulated by a reminder shock. They suggested
that increased arousal during the time that short-term memory was present
may have resulted in stimulation of consolidation of memory into long-
term stores. To support this they employed pharmacological agents
(which will be discussed below) to counteract the CXM effect. It is
possible that the R8 in the two situations were effective for quite
different reasons. This is suggested by the fact that the interval
after training during which the reminder shock stimulated recovery was
much longer in the passive avoidance experiments, and secondly by the
fact that in the maze experiment no re-exposure to the training stimuli
was necessary.

Several pharmacological agents provide protection from amnesia
obtained with AXM or CXM. Barondes and Cohen (1968a) administered
amphetamine or a corticosteroid mixture, at the same time as CXM
treatment, or 3.or 6 hr after training. Both the early and late
administrations failed to reduce the amnesia. Mice that received CXM
before training and amphetamine or steroids 3 hr after training had
better memory than mice that received only CXM when tested 3 hr or 7

days later. Neither of these drugs had any significant effect on

9
the CXM-inhibited protein synthesis. Giving a second treatment of
CXM 30 min prior to injecting amphetamine counteracted the enhancement
in retention, as did giving pentobarbital concurrently with the
amphetamine. The investigators' interpretation was the same as that
given for the reminder shock, i.e., increased arousal during a time
when short-term memory was present led to consolidation. With a second
CXM injection, protein inhibition was reinstated and the increased
arousal had no effect. With pentobarbital arousal was counteracted by
its sedative action.

Serota et a1. (1972) observed AXMrinduced amnesia in rats 24 hr
after training them in a brightness discrimination. Amphetamine or
metaraminol given 0.5 hr before training or 0 to 2 hr before testing
counteracted the effect of AXM on retention. A model which accounted
for these results and for AXMPinduced amnesia in terms of greater or
lesser release of norepinephrine was proposed. This model departs from
the protein theories and is as follows:

"(a) norepinephrine released during the time of training and

consolidation leads to facilitory changes in the specific

network of synapses that are associated with the training

situation; (b) norepinephrine released at the time of test-

ing causes preferential reactivation of this previously

facilitated network; (c) injection of AXM leads to a fall

in the amount of norepinephrine released to a correlative

fall in the degree of facilitation; (d) at 24 hr, nor-

epinephrine is still not available in sufficient quantities

to reactivate the network, resulting in a 'failure of

expression of memory'; (e) at 6 days, the norepinephrine

system has recovered and the memory is expressed (Serota

at al., 1972, p. 342)."

In addition to the reports of stimulated recovery following AXM or
CXM amnesia, there have been several reports of spontaneous recovery
where the variables that accounted for recovery were not clear° Serota

(1971) reported that rats treated with AXM were amnesic when tested 24

hr after training but not when tested 7 days after training. Procedures

10
were similar to those that result in amnesia in mice following a bright—
ness discrimination (greater than 90% inhibition of protein synthesis,
0.5 ma foot shock, and training to a criterion of six correct responses
in seven trials). Mice do.not show spontaneous recovery at 7 days
under similar conditions (Barondes and Cohen, 1968a; Cohen and Barondes,
1968a), nor did AXMrtreated rats that were trained to a criterion of
four correct responses in five trials (Daniels, 1971). In the latter
experiment, retention tests given 6 or 24 hr or 7 days after training
resulted in significant differences between the drug-treated and saline
animals. It is not clear whether the difference between Serota's
results (as far as recovery) and those of Daniels and of Barondes and
Cohen was due to strain and species differences or to some other feature
of the experimental design.

Squire and Barondes (1972) reported recovery from CXM—induced
amnesia of a size discrimination that was trained in a Deutsch Carousel.
In these experiments a fixed—trial procedure with 15 or 21 trials was
used. Mice trained 15 trials were different from controls during a
retention test given 1 day after training but not for test given 3 or
5 days after training. Mice trained 21 trials were different from
controls for tests 1 or 2 days after training but not for a test given
3 or 7 days after training.

The literature reviewed above specified conditions which eliminated
or attenuated the effect of CXM on retention. From such evidence the
ideal conditions for obtaining a CXM effect may be derived. These
conditions include a substantial degree of inhibition of brain protein
synthesis, a moderate level of training, moderate shock levels, and an
absence of conditions that might simulate the training conditions either
by re-exposure to the training stimuli or by producing a state of

increased arousal.

RATIONALE AND PLAN OF EXPERIMENTS

The general.purpose of the investigations reported here was to
extend the information available concerning the effect of inhibitors
of protein synthesis on memory in rats. The compound AXM had been used
intracerebrally with rats and retention deficits observed (Daniels,
1971; Serota, 1971), but neither CXM nor subcutaneous treatments with
an inhibitor had been investigatedfom their effect on brain protein
synthesis or on retention in rats. Experiments 1 and 2 determined the
degree of inhibition of brain protein synthesis that resulted from each
of four doses of CXM administered subcutaneously, while Experiments 3
and 4 investigated the effect of subcutaneous CXM treatments on
retention in.rats that had been trained to a specified criterion of
performance. In Experiment 5 subcutaneous treatments of CXM were
combined with a fixed-trial training procedure to determine whether
there was a point early in training where memory process in rats was
susceptible to CXM treatment. The sixth and seventh experiments were
included to study the effect of intracerebral injections of CXM on
retention in rats.

When repeated experiments with rats failed to show a statistically
significant effect of CXM on retention, a second series of experiments
(8—12) with the mouse as the experimental animal was initiated. As
there was an abundant literature which indicated that mice were
susceptible to disruption of retention by CXM, the mouse experiments

were an effort to clarify whether the failure to show retention

11

12

impairments in rats represented a true species difference or whether
some other aspect of the experiments could account for the lack of
effect. In each experiment the primary issue was whether the group
treated with CXM was different from their saline control group on
retention of.brightness-discriminated avoidance training. However,

due to the problems encountered in obtaining good retention in control
mice, the manipulations made across successive experiments were largely
dictated by the objective of improving retention in the control animals
to provide an adequate baseline for CXM-saline comparisons. These
between-experiment manipulations included a change in strain, trials
supplemental to meeting criterion, controlling the time of day animals
were trained, and changing the nature of the positive and negative
stimuli.

Throughout the investigations with both rats and mice, procedures
were selected to optimize the probability of finding an effect of CXM
on retention. Five experiments from the literature were used in the
design and selection of parameters for the experiments reported hereo
Certain aspects of these five experiments are summarized in Table 1.
All five involved brightness—discriminated escape-avoidance from shock
in a Ydmaze (rats) or T-maze (mice) with light as the positive stimulus.
All included a S-sec interval between beginning of a trial and shock
onset. After a trial mice were leftin the maze for 10 sec, while in
the rat experiments goal-box times were 5 (Daniels, 1971) or 15 (Serota,
1971) sec. For the mice experiments, an intertrial interval of l min
was employed while in the two experiments with rats, the intertrial
interval was 30 sec. Shock levels for all but one experiment (Daniels,
1971) were .5 ma and in that experiment 1.5 ma were used. Table 1

indicates that both AXM and CXM impaired retention in mice, while for

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14
rats only the acetoxy derivative had been investigated and there too

impairments were observed.

EXPERIMENTAL METHODS AND RESULTS

Experiments 1 and 2
CXM-Induced Inhibition of Rat Brain Protein Synthesis

 

In order to determine the degree of inhibition of protein synthesis
that was possible with sublethal doses of CXM, four doses that ranged
from .45 to 2.4 mg/kg body weight were investigated. The rate of incor-
poration of 14C-leucine into whole brain protein was used as the basis
for determination of inhibition. The determinations were made in two

experiments with a separate saline control group for each experiment.
Method

Subjects (83). The 83 were 24 male, Sprague-Dawley rats (Spartan

 

Research Animal, Haslett, Michigan). Prior to the experiment they were
maintained in group cages where food and water were available at all
times. The 12 rats used in Experiment 1 weighed between 217 and 246 g
and those in Experiment 2 were between 195 and 230 g. In each experiment
four rats were used as saline-injected controls and four for each of

two dose levels of CXM.

Injection procedure. Cycloheximide (Acti—dione, Upjohn Co.,
Kalamazoo, Michigan) was prepared in 0.9% saline solution and adminis-
tered in a volume of 10 ml/kg body weight at doses of 0.0 (saline control),
0.45, 0.9, 1.8, or 2.4 mg/kg body weight. Thirty minutes later uniformly
labeled 14C-leucine (International Chemical and Nuclear Corp., Irvine,
Massachusetts) was administered intraperitoneally (100 uc/kg in a

15

16

volume of 10 ml/kg body weight prepared in 0.9% saline solution).

Protein extraction procedure. Thirty minutes after injection with
4C-leucine each rat was decapitated and the brain was removed. The brain

was exposed by clipping the skull along the midline and caudal sutures.
The tissue sample included the whole brain from the caudal part of the
cerebellum to the anterior part of the cerebral hemispheres. The olfac-
tory bulbs anterior to the hemispheres were severed, left in the skull,
and not included in the sample. Immediately following removal, the
tissue was weighed, placed in 10 ml 0.4 N perchloric acid (PCA) and
homogenized with a Potter Elvehjem homogenizer. This procedure, from
the time of decapitation to starting homogenization, required less than
1 min. During collection of the remaining samples, previously collected
samples were kept in an ice bath or were refrigerated. After all
samples were collected, they were mixed briefly on a vibrating mixer
(Curtin Scientific Co.) and were centrifuged for 10 min (International
Centrifuge, Model PR—2). The procedure of mixing, centrifuging, and
removing the supernatant from the samples will hereafter be referred to
as a wash. Unbound 14C-leucine was removed by washing the homogenate
first in 5 ml 0.2 N PCA.with 5 mg/ml L-leucine and then in 5 ml 0.2 N
PCA. This was followed by heating the precipitate at 70°C for 30 min
in 0.6 N PCA (5 ml) to remove RNA and DNA. Lipids were separated from
the protein by successive washes in 5 ml 95% ethanol (saturated with
sodium acetate, 5 ml ethanol:ether (3:1), and 5 ml of ether. Duplicates
of each sample precipitate were removed, dried in tared counting vials,
and weighed. With one exception (a 24 mg aliquot), the dry weights of
the aliquots ranged from 4 to 15 mg. The dried protein was solubilized
by heating it at 40°C in 1.01mLSolueneR(Packard Instrument Co.) for

6 hr. Fifteen milliliters toluene counting solution ( 5 g diphenyloxazole

17
[PPO]) plus 200 mg 1,4 bis[2-(4-methyl-5 phenyloxazolyl)]benzene/1iter
toluene) was added to the vials, radioactivity was determined by liquid
scintillation counting (Packard, Model 3380), and data expressed as
disintegrations per min (dpm) per mg protein. The PCA-soluble fraction
(supernatant from the first three washes) was recentrifuged for 10 min
for further removal of bound 14C-leucine. The supernatant was brought
to a volume of 20 ml, and from this a 1.0 ml aliquot was prepared for
liquid scintillation counting by the addition of 0.2 m1 1 N NaOH and 15
ml modified Bray's solution (100 g naphthalene, 6 g PPO in one liter
reagent grade dioxane). Radioactivity was determined as before and the
data expressed as dpm/ml PCA-soluble supernatant. Inhibition of protein
synthesis was calculated by the method of Barondes and Cohen (1967a).

By this method, inhibition for each treated animal (11 ) is defined by

J

the equation I - 100 x [l-(Xi /R.j]), where X is the ratio (dpm/mg

11 J 13
protein)/dpm/ml supernatant) for the specified treated animal and R.j

is the mean of the ratios for the control group.

Results and Discussion

Whole brain protein synthesis was inhibited by CXM at all dose
levels used (Figure 1). The inhibition (93.6%) obtained with 2.4 mg/kg
was approximately the same as that obtained by other investigators with
much larger doses of CXM in mice. For example, Barondes (1970b) reported
that in Swiss albino mice treated subcutaneously with CXM, whole brain
protein synthesis was inhibited by 95% for the period 30 to 60 min after
injection.

Some caution must be exercised in accepting the calculated inhibi-
tion as an absolute percentage, particularly at the lower dose levels.
According to Barondes and Cohen (1968a; Barondes, 1970b), their method

of calculation of inhibition assumes that the radioactivity in the

18

 

 

 

100r-
80"-
60"-
Percent
Inhibition
40L-
20--
0.45 0.90 1.80 3.60
Dose in mg/kg body weight
Log Scale
Figure 1. Percent inhibition of protein synthesis ----- and
inhibition of incorporation of 14C-leucine---------- in rat brain after

subcutaneous injections of CXM. Each point represents the mean of four
determinations.

19
acid-soluble fraction at the time of decapitation reflects the specific
activity of the radioactive amino acid in the brain throughout the 30
min period for which inhibition is calculated. It further assumes that
the radioactive pool is not affected by the drug treatment. With AXM
as the inhibitor, they showed that for several minutes after administra-
tion of l4C-valine, the radioactivity in the acid soluble fraction was
not affected by the drug treatment, but as time passed more radioactivity
was observed in the fractions taken from treated animals. This resulted
in overestimation of inhibition of protein synthesis and of course this
overestimation would also obtain in the experiments reported here. They
noted, however, that the overestimation is small when calculated inhi-
bition is in the 90% range. To make the data reported here comparable
to theirs, the same method of calculation has been used. However, to
facilitate comparisons with data not handled by this method the results
have also been shown as percent inhibition of incorporation of 14C-
leucine (Figure l).

The rate of incorporation of 14C-leucine (dpm/mg protein) for
control groups from the two experiments were significantly different from
one another (Experiment 1 - 311.3, Experiment 2 - 512.2, t = 2.54,
df - 6, p<.05). Rats from the two control groups differed in age,
weight, and length of time in the animal room and these variables could
account for the differences. No determination was made, however, of
the basis for the differences.

The greater sensitivity of rats to the protein inhibiting proper-
ties of CXM parallels their greater sensitivity to the lethal effects
of this agent. An LD-50 of 160 mg/kg has been reported for subcutaneous
administration.of CXM in mice (Ford and Klomparens, 1960). The eXperi-

ments reported here do not include a determination of the LD-50 for rats,

20
but observational data are available. No deaths occurred in rats that
were injeCted with 2.1 or 2.5 mg/kg CXM and were otherwise untreated.
In the course of pilot studies and the behavioral experiments reported
later in this paper, approximately 30% of the 75 rats injected with
3.0 mg/kg died. .At 3.2 and 3.3 mg/kg, two of the three rats injected
with each of these doses died. A dose of.3.6 mg/kg was lethal for five
of six animals so treated. Thus, under the conditions of the experi—
ments in.which these animals were used (all of which included training
in shock avoidance), it would appear that the LD-50 for subcutaneous
administration of CXM in rats was between 3.0 and 3.6 mg/kg. There
are apparently also strain differences in the toxic effects of CXM in
rats as Rahwan (1971) noted from observational data that Harlan-Wistar
rats "cannot tolerate more than 0.5 mg/kg of cycloheximide when the
drug is administered subcutaneously (p. 93)."

Experiments 3 and 4
Subcutaneous CXM Treatments in Rats Trained to Criterion

 

The purpose of these experiments was to determine whether conditions
similar to those where CXM produced amnesia in mice (Barondes and
Cohen, 1968b) would also result in CXM-induced amnesia in rats. However,
in contrast to the T—maze used for the mouse experiments, the current

experiments employed a Ydmaze.

Method

Ss, For each experiment, 25 male Sprague-Dawley rats (Spartan
Research Animal) were housed in group cages (15 x 13 x 6-1/2") located
on open shelves in the colony room. A light-dark cycle of 12 hr light

and 12 hr dark was employed and food and water were available at all

21
times. The mean.body weights for the 14 animals treated with CXM and

the 11 injected with saline are shown in Table 2.

Table 2. Distribution of rats used in Experiments 3 and 4 and their
mean body weights

_A

 

 

 

Experiment 3 Experiment 4
Category Saline CXM Saline CXM
a a
No. trained.‘ ll 12 11 10
No. that met training 11 11 11 10
criterion-,
No. with zero savings 3 1 1 4
Mean body wt. (g) 345 336 267 273

 

aTwo rats in Experiment 3 and four in EXperiment 4 died within 72
hours after injection and have not been included.

Eguipmen . The training apparatus was a Y-maze with a stem that
measured 32 x.5 x 5" and arms (goal boxes) that were 24 x 5 x 5". The
rear wall of each box was 1/4" clear plexiglass and was lined with white
contact paper. The remaining walls were 1/2" grey plywood. The top was
clear plexiglass with slits over the entrance to each goal box for a
guillotine door that was used to confine the animal in the goal box for
a few seconds after each trial. The grid floor, wired to deliver foot
shock, was made from 1/8" stainless steel rods spaced 1/2" apart. The
grid bars of the stem and about 2—1/2" into the goal boxes were wired
into a single circuit. Depending on which arm of the maze was incorrect,
the remaining.grid bars in one or the other of the goal boxes were
connected into this circuit by cable connectors. High intensity lamps

(Everready, Model EV-202, 12 v. bulb) were located behind the rear wall

22

of each goal box“ One or the other of the lamps was on for each trial
and thus.denoted.the correct goal box. The lamp for the incorrect box
was off and the back wall of this arm was covered by a black cardboard
insert.

A shock.generator (Applegate and Co.) and a Grason—Stadler shock
scrambler were used to deliver continuous foot-shock (.4 to .5 ma) through
the grid floor. A Hunter timer was used to time the 5-sec interval
between placing the animal in the maze and the onset of shock. The
Hunter and a Standard timer that was used to measure the time required
to reach.the.goal box were activated simultaneously by a foot switch.
The shock equipment and timers were turned off by a single hand switch.
A stop watch was used to measure the time the animals were retained in

the goal box and the intertrial interval.

Procedure. CXM was prepared as previously described and injected
at a dose of 3.0 mg/kg. Thirty minutes after injection, the rats were
placed in the.maze.with the house lights on. They were allowed to
explore.the.stem and goal boxes for 2 min. During that time grey card-
board inserts covered the rear walls of both goal boxes. The house
lights were off during training and testing. The rats were trained to
avoid or escape shock by going to the brighter arm of the maze. For
each trial an animal was placed in the stem of the maze and the shock
timer was simultaneously activated. The animal could avoid shock by
entering-the correct goal box within 5 sec or escape shock by entering
the box after 5 sec. Shock continued until the animal entered the
correct box or for.a maximum of approximately 45 sec. A response was
counted as.correct if the animal entered the brighter arm of the maze
without first entering the dark arm. After a correct response, the

rat was retained in the maze for 10 sec and after an intertrial interval

23

of approximately 1 min, the next trial began. Trials continued until
the animal had reached a criterion of 5 correct responses in 6 trials
or to a maximum of 25 trials. Retention was tested 6 hr later by
retraining to the same criterion.

The primary method of analysis for acquisition was the number of
trials required to reach the training criterion. Retention was measured
by the percentage of savings on trials to criterion in testing as
compared to training. A savings percentage was computed for each
animal by the equation given by Barondes and Cohen (1967a) where per-
cent savings - [(I-C) - (R-C))/(I—C)] x 100. I and R are the number
of trials required to reach criterion in training and testing,
respectively. C is the criterion number of trials; in this case,

C - 5. By the method of computation used here, an animal that reached
criterion within the first five trials of testing was assigned a

savings score of 100%. A score of zero savings was assigned to

animals that required as many or more trials to reach the criterion

in testing as in training. A score of 25 was used for animals that

did not reach the training criterion in 25 trials while animals that

died within 72 hours after the experiment were excluded from the analysis

(Table 2).

Results

The drug-treated group in Experiment 3 required significantly more
trials to reach the training criterion than did their saline control;
however, this was not replicated in Experiment 4 (Table 3). Neither
experiment resulted in significant differences between the groups on
the percent savings measure of retention (Mann-Whitney U Test, p>.20).

In Experiment 3, the distribution of savings for the control group was

24

Table 3. Mean trials to criterion during training for CXM-treated and
control rats of Experiments 3 and 4

 

 

 

Experiment Mean trials to criterion

Number Saline Drug df t p
3 10.91 15.42 21 -2.29 <.05
4 13.81 10.40 19 1.50 >.05

 

dichotomous.in that all animals had a score of 0 or 100% while in
Experiment 4 a more variable range of scores was obtained. The mean
percent savings for each group is shown in Table 4. A within-group

comparison of trials to criterion in training and testing indicated

Table 4. Mean percent savings on trials to criterion for CXM-treated
and control rats of Experiments 3 and 4

 

 

 

Experiment Mean percent savings
Number Saline Drug
3 72.7(14.1)a 64.3(9.2)
4 61.0(10.8) 52.5(15.6)

 

f3( ) ‘ one standard error.

that the saline groups of both experiments and the CXM group of Experi-
ment 3 learned and retained the discrimination, while the drug treated
group in Experiment 4 failed to show a significant reduction in trials

between training and testing (Table 5).

25

Table 5. Within-group comparisons on trials to criterion in training
and testing (Experiments 3 and 4)

 

 

Training Testing

Group mean mean df t p
Exp. 3

Saline 10.91 6.18 10 2.82 <.02

Drug 15.42 8.25 11 4.32 <.01
Exp. 4

Saline 13.81 8.36 10 3.02 <.02

Drug 10.40 8.30 9 1.07 >020

 

Experiment 5
Subcutaneous CXM Treatments in Rats Trained a Fixed Number of Trials

 

The evidence that the effect of CXM on retention may be blocked
by overtraining raised the question of whether a lesser degree of train-
ing might result in amnesia for rats injected subcutaneously with CXM.
The mean trials to criterion of the saline groups of Experiments 3 and
4 was used as a basis for determining the number of trials used in this
experiment which employed a fixed-trial procedure. Three groups that
received fewer trials than this mean of 12.36 were trained and tested
in Experiment 5. In preliminary experiments increasing the time the
animal was retained in the goal box resulted in more rapid acquisition
of a brightness discrimination. In the current eXperiment, goal box
times of 10 and 30 sec were compared for rats treated with CXM and their

saline controls.

26

Method

§§, Male SpraguerDawley rats (Spartan Research Animal) that
weighed between 244 and 307 g were housed 10 or 11 rats to a cage in
metal cages (21 x 18 x 12-1/2"). Other conditions of maintenance were
the same as in Experiments 3 and 4. Thirty rats were treated with CXM
(3.0 mg/kg) and 30 were used as saline-injected controls, and 10 animals

in each of these groups were trained 2, 5, or 12 trials.

Eguipgen . The Y-maze, shock equipment, and timers were the same
as used in the experiments described above. To eliminate the possibility
that the rats were using the noise of the high intensity lamp as a cue,
the lamps were replaced by a 15 watt (115 v. ac) bulb. This was mounted
on a magnet and could be moved from goal box to goal box where it was
attached to a plate on the rear wall. As before, a black cardboard

insert covered the rear wall of the incorrect goal box.

Procedure. After injection and habituation as previously described,
the rats were trained 2, 5, or 12 trials to avoid or escape shock by
running to the brighter side of the maze. The first trial that the
animal entered the incorrect goal box and was shocked was counted as
trial 1. Within each group half the animals were retained in the goal
box for 10.sec and half for 30 sec after each trial. The intertrial
interval was approximately 1 min. Six hours after the end of training,
12 additional trials were given to test for retention. The results were
analyzed in terms of percent correct responses for all 12 test trials
and for the first and last 6 trials separately. In this and the experi-
ments that follow, if more than one experimenter was used the conditions

were counterbalanced for experimenters.

27

Results

Neither CXM.nor the time the rats were retained in the goal box had
any effect on test performance (Figure 2). The analysis of variance
in percent correct responses in 12 test trials indicated that increasing
the number of training trials improved test performances but there was
no interaction of this with either of the other two variables (Table 6).
When the first and second halves of testing were analyzed separately,
only the second half showed a significant effect of the number of train-

ing trials (F a 5.94, df = 2,48, p<.01).

Table 6. Analysis of variance for percent correct responses (test
trials 1-10, Experiment 5)

 

 

Source Mean Sq. df F

A (goal box time) 205.35 1 1.05

B (no. of trials) 889.40 2 4.53a
C (CXM) 18.15 1 .09

AB 24.80 2 °l3

AC .15 1 .00

BC 72.80 2 .37

ABC 31.40 2 .16

Error 196.44 48

 

aF 05 (2,48) = 3.19.

28

 

100 —-
808——
60 —‘
Percent
Correct
Responses
40 .-
20 ——
011111:

 

 

2 4 6 8 10 12
Number of training trials
Figure 2. Percent correct responses in 12 test trials for Experi-

ment 5. Rats were trained 2, 5, or 12 trials 30 min after injections
of 0.9% saline ——-——-—-or CXM ----- and were tested 6 hr later.

29

Experiments 6 and 7
Retention After Intracerebral CXM Treatments in Rats

 

In the only experiments to show memory impairments in rats after
treatment with one of the glutamaride derivatives, intracerebral injec-
tions of AXM were used (Daniels, 1971; 1972; Serota, 1971; Serota 6t
aZ., 1972). As rats treated subcutaneously with CXM had normal reten-
tion, the possibility that the route of administration was critical was
investigated by administering the agent intracerebrally. No information
as to the dose requirement for inhibition of protein synthesis via
intracerebral injections of CXM in rats was available; however, with
AXM twice the amount of drug required to inhibit brain protein synthesis
by 95% in the mouse produced comparable inhibition in the rat brain
(Serota, 1971). The amount of CXM used in Experiments 6 and 7 is twice
the amount shown by Barondes (1970a) to produce 95% inhibition in the

mouse (400 vs 200 pg).
Method

§§, Twenty-two, male, Sprague-Dawley rats (Spartan Research
Animal) were distributed between groups as shown in Table 7. They were
housed in group cages (3 to 4 per cage) and maintained as previously

described except during Experiment 6 a 24-hr light cycle was in effect.

Equipment. Except for different goal box stimuli, the equipment
used was the same as described above. In Experiment 6 the correct goal
box was illuminated by an 8 v. dc, .25 amp bulb. The incorrect goal
box was denoted by the absence of this stimulus, i.e., no black inserts
covered the back wall of the goal box as in previous experiments. The

stimulus light in Experiment 7 was a 15 watt (115 v. ac) candelabra bulb.

30

Table 7. Summary of animals used and their mean body weights for
Experiments 6 and 7

 

 

 

 

Experiment 6 Experiment 7
Category Saline CXM Saline CXM
Number trained 5 aa 5 6
No. that met training 5 4 3 6
criterion
No. with zero savings 0 1 3 6
Mean body wt. (g) 329 321

 

aTwo animals were discarded because they were too ill to complete
the experiment.

Injectionprocedure. For injection of CXM, the animals were anesthe-
tized with methoxyflurane (Metofane, Pitman-Moore). They were placed
in a KOpf stereotaxic holder for rats and the head was adjusted so that
the midline between bregma and lambda was in a horizontal plane. The
scalp was opened along the midline with a cold cautery instrument and
deflected laterally with retractors. Holes were placed bilaterally in
the skull at coordinates 3.5 mm anterior to the interaural line and 3.0
mm lateral to the midline. A 30 ga needle was lowered into the brain to
a depth of 4.0 mm below the skull surface. A polyethylene tube connected
this needle to a 100 pl syringe. The experimental animals were injected
with 200 pg of CXM prepared in 0.9% saline solution in each hemisphere.
In Experiment 6 the concentration was 200 ug/lO pl and injections of
10 ul were made into each hemisphere 5 hr before training. In Experi-
ment 7 the concentration was 100 ug/lO p1 and injections of 20 p1 were
placed in each side 4 hr before training. Control rats received injec-

tions of 0.9% saline solution in volumes equivalent to those of the

31

experimental animals. After surgery and injection, the animals were

kept in a heated recovery cage until they began to move about (1 to 2 hr).

Behavioral procedure. Procedures were essentially the same as in

 

Experiments 3 and 4 except that in EXperiment 6 the animals were trained
to a criterion of five correct avoidances in six trials. With this cri-
terion the animal had to enter the correct goal box within 5 sec without
first entering the incorrect box. In Experiment 7, the criterion was
again five correct responses in six trials, i.e., correct escape responses
were also counted. The animals were trained to a maximum of 50 (Experi-
ment 6) or 20 (Experiment 7) trials and those not meeting the training

criterion were assigned a score equal to the maximum number of trials.

Results

Intracerebral injections of CXM had no effect on acquisition of
the brightness discrimination (p>.20). While the absolute value of the
savings scores for the drug-treated groups was somewhat smaller than for
controls, the two groups were not significantly different from each other
(Mann-Whitney U test, p>.10) in retention of avoidance or escape respond-
ing (Table 8).

Experiment 8
Retention after CXM Treatments in Swiss-Webster Mice

 

This experiment was an attempt to replicate Barondes and Cohen's
(1968a) results in a strain of mice different from the one they used.
In their experiment, CXM (120 mg/kg) resulted in significant memory
impairments 6‘hr or 1 or 7 days after training to a criterion of five
correct responses in six trials (Table 1); however, in the current

experiment only the 6-hr retention interval was studied.

32

Table 8. Median trials to training criterion and mean percent savings
(Experiments 6 and 7)

 

 

 

 

Experiment Median trials to criterion Mean percent savings
Number Saline CXM Saline CXM
6 35.0 23.5 84.8 48.5
7 20.0 13.5 91.3 62.8
Method

§§, The Ss were Swiss-Webster albino mice (Spartan Research
Animal). They were housed in groups in clear plastic shoe box cages
which were located on open shelves where food and water were available
at all times. Approximately 1 week prior to the start of the experiment,
a light-dark cycle of 12 hr for each condition was initiated. Before
that time the animals had been kept in the light 24 hr a day. Six mice
were injected with CXM and six were saline controls. At the time of

injection their weights ranged from 35 to 46 g.

Equipment. The training and testing apparatus was a T-maze con-
structed from 1/4" clear plexiglass. The stem was 10-1/2 x 3" and the
arms or horizontal part of the T were 2-1/2 x 9". The rear walls of the
arms were lined with white contact paper while the remaining walls were
lined in grey. A black cardboard insert was available to alternately
cover the left or right half of the rear wall. This denoted the incor-
rect side of the maze. The correct side was illuminated by a high
intensity lamp (Everready, Model EV-202, 12 v bulb) which was centered
behind the rear wall. The head of the lamp was turned from side to side

as the position of the correct goal box changed. The floor was

33

constructed from 1/16" stainless steel rods spaced 1/4" apart. In the
horizontal part of the T, the rods spanned the width of both goal boxes
making it necessary to manually terminate shock upon a correct response.
The shock equipment described above was used to deliver the .4-.5 ma

foot shock.

Procedure. CXM (120 mg/kg) was dissolved in 0.9% saline and
injected subcutaneously in a volume of 12 ml/kg body weight. Control
mice received an equivalent volume of 0.9% saline solution. Thirty min
after injection, the mice were given a 2—min habituation period in the
T-maze. During this time they could freely explore the stem and both
goal boxes. House lights remained on for habituation but were off during
training and testing .

Following habituation the mice were trained to avoid or escape
shock by running to the brighter side of the maze. A trial began when
the mouse was lifted by the tail and placed in the stem of the maze and
the shock timer was simultaneously activated. The animal could avoid
shock entirely by entering the correct goal box within 5 sec after he
was placed in the maze. After the 5—sec interval, shock came on and
stayed on until the animal entered the correct goal box or until approxi-
mately 45 sec had elapsed. After entering the correct box, the animal
was retained there for 10 sec by a plexiglass insert which was slipped
over the goal box opening. After each trial, the mouse was removed and
placed in a holding cage for an intertrial interval of approximately
1 min. A trial was counted as correct if the animal entered the brighter
arm of the maze without first entering the dark arm. It was not required
that the shock be avoided for the trial to be correct. Training con—
tinued until the.mouse had reached a criterion of five correct responses

in six trials or to a maximum of 30 trials.

34

Retention of this training was tested 6 hr later. Mice were
retrained until they achieved the above criterion. In some cases
retraining was terminated when it was evident that the animal had
zero savings. In this and even more so in the experiments that follow
it was fairly common to have mice that did not meet the criterion within
the number of trials specified for the experiment. A training score
was derived.by projecting from performance at the time the maximum
number of trials was reached. The score used was the trial at which

the mouse could have first met the criterion.

Results

The median trials to criterion for saline and CXM groups were
respectively 12.5 and 9.0. The Mann—Whitney U test indicated that the
two groups were not significantly different in trials to criterion
during training (p>.20). When tested 6 hr after training, four saline
and five drug treated mice failed to show any savings. 0f the three
mice that had savings, two were animals that did not reach the training
criterion and the third had required 26 trials to meet the criterion.
From these results, it was apparent without statistical analysis that
there was no difference between the two groups in retention of the
discrimination. It was also apparent that neither group had learned
the discrimination or at least did not remember it during the retention
test .

EXperiments 9 and 10
Retention after CXM Treatments in Charles River Swiss Albino Mice

 

 

There is a well established literature showing strain differences
in the learning ability and susceptibility of mice to drugs (Bovet et aZ.,

1969, for review). The two experiments that follow were to rule out the

35

possibility that strain differences could account for the failure to
replicate the results of Barondes and Cohen (1968a). Mice obtained

from their supplier were used in these (and subsequent) experiments.
The methods in the two experiments:were identical except that in

Experiment 10, training was extended beyond the specified criterion.

Method

§§, Male, Swiss albino mice (Charles River Breeding Co.) that
weighed between 20 and 41 g were housed and maintained as previously
described except that the 12-hr light-dark cycle was used throughout.
On 5 days preceding training, the mice were removed from their cages
and handled for approximately 2 min each. In Experiment 9, five mice
were treated with CXM and five injected with saline. In Experiment

10, five were injected with the drug and six with saline.

Equipment. For these experiments, a grey, plexiglass divider
was installed between the two arms of the T—maze. This prevented the
mice from crossing directly to the correct side after an incorrect
response and also made the two goal boxes more distinct by reducing

light scatter into the darker (incorrect) arm.

Procedure. Injection and behavioral procedures were essentially
the same as those described above. In Experiment 9, training was
continued to a criterion of five correct responses in six trials to a
maximum of 22 trials, while in Experiment 10 training was continued
eight trials beyond this criterion or for 22 trials. The additional
eight trials were ignored for the analysis, i.e., trials to criterion
and percent savings were based on the point at which criterion was first
met. The added trials were simply an attempt to improve savings by

extending training.

36

Results

Neither experiment resulted in differences between the CXM and
control mice in training trials to criterion (p>.20) with most mice in
both groups rapidly reaching the criterion (Table 9). There was a good

proportion of animals in both experiments that had zero savings on

Table 9. Distribution of mice used in Experiments 9 and 10 and their
median trials to criterion in training

 

 

 

 

Experiment 9 Experiment 10
Category Saline CXM Saline CXM
No. trained 5 5 6 6
No. that met 5 5 5 4
training criterion
No. with zero savings 3 5 3 3
Median trials to 7 7 7 15.5

criterion

 

trials to criterion (Table 9) and again no differences between CXM and
control mice on the savings measure of retention (p>.20) (Table 10).
Animals in Experiment 10 (where training was more extensive) tended to
have greater savings; however, the increase in savings between experi-
ments was not significant (.05<p<.10).

Experiment 11.
Retention in CXM-Treated Mice: Day vs Night Training

 

The results of Experiments 9 and 10 indicated that a good propor-
tion of the mice were rapidly reaching criterion during training yet
when tested 6 hr later, they gave little indication of having learned

the discrimination. One possible explanation for such a result is that

37

Table 10. Mean percent savings for Experiments 9 and 10

 

7 : 1 _ Percent savipgs
Experiment Saline CXM

 

9 23.4 0
10 33.0 38.3

 

they were starting the experiment with a pretraining preference for the
brighter side of the maze. An effort was made to reduce the light
preference by housing some of the animals in a dimmer environment prior
to starting the experiment. The effect of training in the light part

of the light-dark cycle as opposed to the dark was investigated so as to
rule out the possibility that a tendency to train at night could account
for the poor acquisition and a lack of CXM effect on retention in the

earlier experiments.

Method

§§, Male, Swiss albino mice (Charles River Breeding Co.) that

weighed between 27 and 40 g were housed four per cage in plastic shoe
box cages. For approximately half the animals, these cages were located
on open shelves where the ambient light for the light part of the 12-hr
light-dark cycle was about 9 to 10 footcandles (fc). The shoe box cages
for the remaining mice were located in metal cages which reduced the
ambient light to approximately 5 fc. The mice were maintained under
these conditions for 10 to 14 days prior to training. 0f the animals
maintained under each condition approximately half were treated with

CXM 30 min prior to training and half were injected with saline. In

38
each of these subgroups, half the animals were trained during the light
part of the light-dark cycle (9:00 a.m. to 9:00 p.mu) and half during
the dark part of the cycle (9:00 p.m. to 9:00 a.m.). The distribution

of animals in the various subgroups is shown in Table 11.

Equipment. In a further effort to make the correct and incorrect
goal boxes more distinctive and to facilitate learning, the previously
grey side.walls of the goal boxes in the Tdmaze were lined with white
paper such as that which covered the rear walls. Black inserts that
covered both the rear and side walls were used to denote the incorrect
box. A high intensity lamp of the type used in the previous experiments
was located on each side of the maze. These were directed so that the
light entered primarily through the side wall of the goal box with
only the light on the correct side being on. To make the start of a
trial more discrete, the stem.was divided into a start box and runway

by the addition of a guillotine door.

Procedure. The injection and habituation procedures were the same
as in earlier experiments. The training procedures were essentially
the same; however, instead of starting the trial when the mouse was
drapped by the tail into the maze, the mice were grasped in the experi—
menter's hand and placed in the start box so that they were facing
approximately 180° away from the goal boxes. A couple of seconds later
the start-box door was lifted and the shock timer was activated. Shock
onset followed 5 sec later and shock continued until the mouse entered
the correct goal.box or for a maximum.of approximately 45 sec. The same
criterion (five out of six) that had been used in prior experiments
was used here, but in this experiment the criterion run of six trials

could begin no earlier than the first trial on which the mouse made an

39

 

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40

incorrect response and was shocked. This eliminated the possibility of
the mouse making.four or five of the criterion responses before he had
been shocked as had happened in earlier experiments. Training was con-
tinued until criterion was met or to a maximum of 22 trials. Testing
or retraining took place 6 hr later. The mice were retrained to the
same criterion. During retraining all responses, including any pre-
shock responses, were potentially includable in the criterion run.
All mice were retrained at least 10 trials to permit a comparison on
the percentage of correct responses independent of training performance.
Composite groups obtained by pooling across two of the three
treatment variables at a time were used for analyzing training trials
to criterion and percent savings (Mann-Whitney U test). Retention
was also analyzed in terms of percent correct responses in test trials

1-10 by an analysis of variance.

Results

Neither CXM, nor the time.of training, nor the brightness of the
maintenance environment affected training.performance (p>.20 for all
comparisons, Table 12). Retention was similarly unaffected by these
variables (p§.05 in all cases) whether measured by percent savings
(Table 12) or percent correct responses (Figure 3, Table 13). The only
variable to approach significance (p<.10) was the comparison between
day and night training with respect to percent savings. Animals trained
at night tended to have higher savings scores. Table 11 indicates that
most animals met the criterion (in some cases in as few as six trials)

and yet there was again a good proportion of animals with zero savings.

41

 

 

 

 

 

 

 

 

 

 

 

 

 

80"‘
(F...
60— ‘—’
~——+ 7“
Percent 40"'
Correct
Responses
20"'
0
2.981 2m 8.3101 8,810.4
Brighter Environment Dimmer Environment

Figure 3. Percent correct responses for 10 test trials 6 hr after
mice were trained to a criterion of five correct responses in six trials.
Saline (S) or CXM (120 mg/kg) was administered 30 min before training.

PM - trained between 9:00 p.m. and 9:00 a.m., AM - trained between
9:00 a.m. and 9:00 p.m., Brighter Environment - maintenance environment =
9 to 10 fc; Dimmer Environment - maintenance environment = 5 fc.

42

Table 12. 'Median trials to criterion and mean percent savings for
composite groups of Experiment 11

 

Median training trials

 

Groups to criterion . Mean percent savings
Saline 10.5 35.8
CXM 12.5 35.6
Brighter env. 15.0 40.0
Dimmer env. 10.0 30.9
Trained 9 p.m.-9 a.m. 15.0 45.6
Trained 9 a.m.-9 p.m. 10.0 25.8

 

Experiment 12
CXM Treatments in Mice Trained in a Black-White Discrimination

 

The results of Experiment 11 indicated that the preference for the
brighter side of the maze persisted despite manipulation of the amount
of light in the maintenance environment. This being the case, training
to the brighter side of the maze was in effect training an already
preferred response. In Experiment 12, mice were trained to avoid or
escape shock by running to the darker arm of the maze. To eliminate the
possibility that the preference demonstrated in Experiments 9-11 was
a heat preference rather than a light preference, the stimulus light
previously used was discarded. In this experiment, the mice were trained
to discriminate between the black and white goal boxes, both of which
were illuminated from overhead by the room light. This experiment also
included a group that received a larger dose of CXM (150 mg/kg) than

had been employed in the earlier experiments.

43

Method

Twenty-three mice (Charles River Breeding Co.) that weighed between
34 and 49 g were distributed as shown in Table 13), housed in group
cages, and maintained under the conditions previously described. Injec-
tions were prepared in 0.9% saline and.administered in a volume of 10
mllkg body weight at doses of 0, 120, or 150 mg/kg approximately 30 min
before habituation. The mice were trained in the T—maze used in prior
experiments; however, the stimulus light was not employed. To make the
two goal boxes more distinctive, all four walls of the goal boxes were
covered in white. The black cardboard inserts that marked the correct
goal box were extended so that they covered the four walls. Rectangles
of white or black construction paper were used to cover the plexiglass

floor beneath the grid bars of the goal boxes.

' Table 13. Distribution of mice used in Experiment 12 and their median
trials to criterion

 

 

CXM CXM
Category Saline 120 mg/kg 150 mg/kg
No. trained 9 7 7
No. that met training 4 3 3
criterion
Median trials to 22 22 24
criterion
No. with zero savings8 3 2 0

 

aThese values do not include three animals that did not meet
criterion during training or testing.

44

The basic model for this experiment was the same as those pre-
viously described; however, several procedural changes were made. The
time the animals were retained in the goal box was increased from 10
to 30 sec, while the intertrial interval was decreased to 30 sec.
Training was again to a criterion of five correct responses in six
trials but was terminated if the animal did not reach criterion in 20
trials. Testing took place 24 hr, rather than 6 hr, after training
and all animals were given 20 test trials. Analysis was in terms of
training trials to criterion, percent savings, and percent correct

responses during the test trials.

Results

There was no significant effect of CXM on trials to criterion
during training (Kruskall—Wallis analysis of variance.in ranks, Siegel,
1956, p>.30). Once again, despite the inclusion of a group that
received a larger dose of CXM than had previously been used, the drug
also failed to result in a significant impairment of retention (p>.20).
In fact, the ordering of the means was exactly opposite of what would be
expected if an impairment obtained (Figure 4). Percent correct
responses was similarly unaffected with F rations less than 1 occurring
regardless of whether the trials analyzed were trials 1-10, 11-20, or
the total of 20 trials. The Wilcoxon nonparametric test for repeated
measures (Siegel, 1956) indicated that neither the saline group nor the
group treated with 120 mg/kg CXM showed a significant reduction in
trials to criterion between training and testing (p>.05), while for the
group that received 150 mg/kg this reduction was significant at the .05
level. This would indicate that the group that received the larger
dose of the drug was the only group to learn and retain the

discrimination.

45

 

 

 

 

 

 

 

 

60 {-
(6)
(7) """’
40L-
Percent
Savings ._£Zl_w
20 "'
0
0 120 150

CXM (mg/k8)

Figure 4. Percent savings on trials to criterion for mice trained
to a criterion of five correct responses in six trials after treatment
with 0, 120, or 150 mg/kg CXM. They were trained 30 min after injec-
tion and tested 24 hr later.‘ The numbers in parentheses are the nUmber
of animals in each group.

DISCUSSION OF BEHAVIORAL EXPERIMENTS

The results of the experiments reported above can be summarized as
follows: (1) The major and most consistent outcome was that subcutaneous
treatments with CXM had no effect on retention of brightness discrimi-
nated avoidance training in rats and mice; (2) Rats treated intracere-
brally with CXM had lower savings scores than their controls, but this
difference too fell short of statistical significance; (3) There was
never any effect of CXM on acquisition of discrimination by mice; in
rats, in only one of four experiments was there a statistically signifi-
cant impairment of acquisition related to CXM administration; (4) Results
that are secondary to the purpose of this investigation were as follows:
a) Comparing intervals of 10 and 30 sec., the time rats were retained
in the goal box did not affect brightness-discriminated avoidance; b)
Decreasing the brightness of the maintenance environment for approxi-
mately 10 days before training had no effect on brightness-discriminated
avoidance in mice; c) Training at night tended to result in greater
savings scores for mice, but this was not a significant effect.

Is Inhibition of Protein Synthesis the Effective
Mechanism of Drug;Induced Amnesia?

Despite inhibition of brain protein synthesis by more than 90%,
rats treated with CXM showed normal retention whether trained to cri-
terion or for a fixed number of trials. Such evidence is contradictory
to the hypothesis that CXM impairs retention because it inhibits brain
protein synthesis. Other mechanisms of action such as induction of

46

47
abnormal electrical activity in the brain (Cohen and Barondes, 1967) or
the blocking of adrenergic sites by peptidyl puromycin (Roberts, Flexner,
and Flexner, 1970) have been suggested as a basis for puromycin's
effect on retention. While these specific mechanisms are not charac-
teristic of CXM, the evidence that CXM can produce close to maximum
inhibition of protein synthesis without impairing retention suggests
that inhibition of protein synthesis may not be the critical variable

in CXMrinduced amnesia.

Retention by Rats After CXM Treatments

In Experiments 3 and 4 where rats were treated subcutaneously with
CXM, the treated and control groups differed by less than 10% savings
in trials to criterion. This difference should be contrasted with the
results of the experiments listed. in Table l where, in all cases, the
control group's savings exceeded those of treated animals by at least
35% and in some cases by almost 70%.

Reported failures to induce amnesia with AXM or CXM may be cate-
gorized into two major classes: (1) those related to the degree and
duration of inhibition of protein synthesis and (2) those which involved
a manipulation or procedure which presumably did not affect this inhi-
bition yet blocked or attenuated drug-induced amnesia. It is unlikely
that the lack of amnesia in Experiments 3-5 can be attributed to inade-
quate inhibition of protein synthesis. The biochemical experiments
indicated that 2.4 mg/kg doses of CXM resulted in 93.6% calculated
inhibition of brain protein synthesis for the period 30 to 60 min after
injection. The subcutaneous dose used in all behavioral experiments
with rats was 3.0 mg/kg. This dose was selected after pilot studies

indicated that 2.4 mg/kg did not affect retention and that larger doses

48
were lethal for a major proportion of the animals. It seems reasonable
to assume that calculated inhibition of at least 93.6% would be achieved
with 3.0 mg/kg CXM, and this is well within the range of the 90—95%
inhibition reportedly required to induce amnesia (Barondes, 1970a).

It would be advantageous to have information concerning the duration
of inhibition of protein synthesis in rats that were treated with CXM.

In mice there is a rather drastic drop in CXMFinduced inhibition of
protein synthesis between 60 and 90 mdn after injection (Barondes,
1970). In the experiments reported here, the average time per training
trial was approximately 1-1/2 min, Thus, when an animal required more
than 20 trials to reach.the.training criterion, training occasionally
exceeded the 30-min interval for which inhibition of protein synthesis
was measured. With the present data, it cannot be stated that greater
than 90% inhibition of protein synthesis was maintained throughout train-
ing; however, there is evidence that contradicts this as an explanation
for the lack of CXMrinduced amnesia in rats. This comes from Experiment
5 in.which animals were trained 2, 5, or 12 trials. Training for these
animals was confined to much shorter interval and yet no memory impair-
ments were observed. Thus, it seems probable that the failure to induce
amnesia by subcutaneous injections of CXM in rats cannot be attributed
to inadequate inhibition.of protein synthesis nor to an insufficient
duration of this inhibition.

When.similarly designed investigations result in drastically dif-
ferent results, the final designation of the variables responsible for
the differences is not possible without experiments in which the variables
in question are controlled and differential effects are shown. 0n the
other hand, it is possible to rule out (as an explanation for the dif-

ferent results) any variables which have been held constant in the

49
experiments which produced different results. The training and testing
parameters selected for Experiments 3 and 4 were based on those of
Barondes and Cohen (1968b) but differed in that a Y—maze rather than a
T—maze was used. A comparison of their methods (see Table l and the
discussion preceding it) and the methods used here will show that the
two designs shared many similarities. If control group performance may
be taken as evidence for equivalence of procedures, there is. strong
indication that the procedures were equivalent despite the use of a
different training apparatus. Their control groups required an average
of 13 to 16 trials to attain the training criterion of five correct
responses in six trials and six hours later had savings of 72 to 77%.
The control groups for Experiments 3 and 4 required an average of
12.36 trials to reach the same criterion and after six hours showed
savings of 67%. This would seem to indicate that the procedures were
sufficiently alike to rule out.differences.in the training and testing
methods as an explanation for failure to produce their mouse results
in rats, i.e., for the failure to show CXMrinduced amnesia.

Variables that interact with CXM to counteract its effect on
retention were discussed above. Two of these (overtraining and shock
parameters) were explored in the context of the present discussion.

Overtraining can be ruled out as an explanation of the failure to
show CXMéinduced amnesia with subcutaneous injections in rats- Such
an explanation is contradicted by Experiment 5 where training as limited
as two trials failed to reveal amnesia for drug treated animals.

Considering the shock parameters, intensity of shock can also be
ruled out. The level used in these experiments was the same as that in
four of five of the experiments in Table l (.5 ma) and the fifth experi-

ment listed there used an even more intense shook. Shock of long

50
duration blotks.CXMrinduced amnesia (Flood et al., 1972). In maze
experiments, the duration of shock typically varies across.trials
depending on how long the animal takes to reach the correct goal box.
In the experiments reported here, the animals were removed if they had
not entered the.correct.goal.box within approximately 45 sec after the
start of the trial. For a post hoc determination of whether shock
duration had affected percent savings for the CXM-treated rats, percent
savings for the treated groups of Experiments 3 and 4 were tested.for
correlation with the number of shocks longer than 10 sec received during
training. For Experiment 3, the resulting coefficient was a small
positive number while in Experiment 4 the coefficient was negative, and
in neither case was the correlation.statistically significant (p>.10).

While it is still possible that some variation in the procedures
for training and testing may have accounted for the difference in the
results obtained here and those of.other investigators, comparisons of
the reported methods and; consideration of variables known to interact
with CXM to reduce amnesia have not suggested the nature of this
variation.

It was previously mentioned that CXM-induced amnesia has been
reported for mice but not rats and that AXM-induced amnesia has been
observed in rats and mice (Barondes and Cohen, 1968a; Daniels, 1971;
Serota, 1971). The.investigators that used rats both used intracerebral
injections of AXM and strains of rats that differed from each other and
from the one used here. In this context, a third set of alternatives
should be examined in.connection with the failure to show.CXM-induced
amnesia in rats. These include a) strain or species differences in the

effects of the protein inhibitors, b) differential effects of AXM vs

 

 

 

I‘ll- Illl. '1
‘l‘ll'lul

51
CXM, and c) differential effects related to the route of administration
of the drug.

In comnection with the first alternative, evidence.that-there are
species differences.in some of the effects of CXM was noted above, i.e.,
there were large.differences in the dose requirements for lethality and
for inhibition of protein synthesis between rats and mice. However, it
was also suggested that the degree inhibition obtained with sun mg/kg
of CXM was comparable to that other investigators have found to be
effective in the induction of amnesia. If species differences.are to
be accepted as explanation of the current data, then it must be assumed
that the.difference.is related to some other reaction.to CXMN. With
reference to strain.differences, Randt et al. (1971) reporteduthat
different strains of mice performed differently in passive avoidance
training and that there was an.interaction between the strain variable
and the effect of CXM on retention. The possibility of strain or species
differences cannot be excluded with the information at hand.

Both the second.and third alternatives would seem to be contradicted
by evidence related to the induction of amnesia in mice by AXM and CXM.
Considering the alternative related to differential effects of AXM and
CXM in mice, when the dose levels were such that the two agents resulted
in equivalent inhibition of protein synthesis, they had equivalent
effects on retention (Barondes, 1970b; Barondes and Cohen, 1968a, 1968b).
Evidence contrary to the alternative related to route of administration
is that.both subcutaneous (Barondes and Cohen, 1968a) and intracerebral
(Siegel et a1. , 1971) treatments with CXM impaired retention in mice as
did.administration of AXM via either route (Barondes and Cohen, 1968b;

Cohen and Barondes, 1968b).

52

Therefore, in order for either the second or third alternative to
be accepted as the correct hypothesis, an interaction between the
species (rat vs mouse) and one of the other variables (nature of the
agent or route of administration) would be required. There is some
indication of an interaction between species and route of administra-
tion with respect to the protein inhibiting properties of these drugs.
In rats 40 ug of AXM injected intracerebrally (Serota, 1971) resulted
in inhibition of protein synthesis that was equivalent to that produced
by 20 ug treatments in mice (Barondes and Cohen, 1967b). This 2:1 ratio,
which is approximately equal to the ratio of the weights of the brain
cortices for the two species (Daniels, 1971), should be contrasted with
the much greater difference between rats and mice in their suscepti-
bility to the protein inhibiting.pr0perties of CXM administered subcu—
taneously (a ratio of about 40:1 in terms of mg/kg required to inhibit
protein synthesis by more than 90% (Barondes, 1970b; Experiment 2 above).
In Experiments 6 and 7 where rats were treated intracerebrally with
CXM, differences between the saline and CXM groups on percent savings
were between 30 and 35%. This is approximately the same degree of
impairment observed by Cohen and Barondes (1968b) for mice treated with
AXM and should be contrasted with the 10% saline-CXM difference obtained
when subcutaneous injections of CXM were used (Experiments 3 and 4).
As no data are available to show what degree of inhibition of brain
protein synthesis resulted from the intracerebral injections of CXM
in Experiments 6 and 7, it cannot be ruled out that these treatments
produced larger absolute differences than subcutaneous injections
because they produced a greater degree of inhibition. 0n the other
hand, an equally tenable hypothesis is that the critical.variab1e that

was affected by the route of administration was some other aspect of

53
CXM's effect in rats which was absent when the agent was administered

subcutaneously.

Retention in Mice After CXM Treatments

 

Subcutaneous injections of CXM also failed to impair.retention in
mice. During the course of Experiments 8-12, there were nine saline-
CXM comparisons on percent savings. Of these there were only three in
which the magnitude of percent savings for the CXM group was lower than
that of the control group. When percent savings was analyzed for each
experiment, these analyses indicated that there was no effect of CXM
treatments on retention. In two experiments a second measure of reten-
tion was also considered. In Experiment 11, this was the percent
correct responses in the first 10 test trials, while in Experiment 12
the percent correct responses in test trials 1-11, 11-20, or 1-20 were
also considered. None of these comparisons resulted in significant
differences between the groups treated with CXM and those injected with
saline.

The failure to.show an impairment of retention with subcutaneous
injections of CXM in mice is consistent with the data reported above
for rats but is of course in conflict with the evidence of other
investigators (Barondes and Cohen, 1968b; Siegel at al., 1971). It is
possible that some common factor in the experiments reported here for
rats and mice worked against the induction of amnesia by subcutaneous
injections of CXM; however, the results of the two series of experiments
were sufficiently different to suggest that this was not the case.
Where the control rats of Experiments 3 and 4 had acquisition and
savings scores comparable to those reported byBarondes and Cohen

(1968b) for mice, the control mice trained in Experiments 8-12 typically

54
had much smaller savings. The mean value for savings on trials to
criterion ranged from.approximate1y 17% up to 64% in the saline control
groups; however, only two.of these groups had savings in excess of 35%.
Savings values for the mice treated with CXM were comparable. For mice
trained to the light.(Experiments 8—11), 51% of the control mice and
61% of those treated with CXM had zero savings six hours after.training.
From these data, it is evident that a good percentage of the animals
had failed to learn the discrimination even though they met the train-
ing criterion. As this was the case, the absence of a CXM effect on
retention is not necessarily contrary to the evidence of other.investi-
gators. With a baseline as low as that. obtained with control mice, it
would be difficult to demonstrate a significant reduction in savings.
This was most clearly indicated by Experiment 9 in which all CXM?
treated mice had zero savings, yet this was not different from the
' control group savings of approximately 23%. These experiments point up
one of the problems inherent with the use of a low criterion.of learning,
i.e., they illustrate a condition under which an animal may readily
meet the.criterion without having learned the discrimination. In this
case, this appeared to be due for a pretraining preference for-the
brighter side of the maze. The experiments also illustrate problems
related to the use of a percent savings measure of retention. This
method which is designed to control for individual differences in
learning assumes a positive correlation between training and testing
performance for untreated groups. However, in the experiments described
above, animals that required fewer trials to reach training criterion
were more likely to have zero or a low percentage of savings. This is
partly inherent in the.method of computation. Animals that require

only a few training trials to reach criterion have a more limited

55
distribution of savings.scores available to them. For example, an
animal that takes six trials in training to reach the criterion of five
correct responses in six trials has only two possible savings scores.
If he meets the test criterion.in.five trials, he has 100% savings but
with one error during testing‘his savings are reduced to zero. The
percent savings measure of retention may be more efficient when a
stricter criterion is used for training but for experiments in which
training is to be very limited, it would seem that another measure of
retention.should'be,sought.

Furthermore, these results suggest that in addition to considera-
tion of overtraining, designs which are intended to show drugrinduced
amnesia.must be such that the animals are not undertrained.in the
acquisition phase of the experiment. In.the current design, more
extended training was not attempted due to the necessity of confining
training to the period when the protein inhibitor was most effective.

In the experiments where light was the positive stimulus, the
problem of-a low criterion.of training was compounded by apparent pre-
training preference of the mice for the brighter side of the maze. This
preference was evaluated by testing the hypothesis that light and dark
responses were equally probable during the first two trials of.training
in Experiment 11.“ For trial one the hypothesis was rejected for the
saline animals (p - .01) but not for those treated with CXM (p a .12).
For trial 2 the hypothesis was.rejected for the treated mice (p = .02)
but not for the control mice (p a .17). In both cases the rejection
was based on more animals choosing light, and this was also the case
for the tests which resulted in the higher probability values, which

were suggestive if not statistically significant.

56

This preference was a strong and very reliable phenomenon that
persisted despite the manipulation of the light in the maintenance
environment and despite the change to dark as the positive stimulus in
Experiment 12. In an experiment now in progress, it has been observed
that for the first.10 trials of trainingpmice trained to white respond
correctly approximately 60% of the time while those trained to black
make about 35% correct responses. This experiment (as was Experiment
12) is being carried out without a stimulus lamp or bulb. This eliminates
the possibility that the preference could be attributed to either heat
or noises from the stimulus light. The only other alternative to the
assumption that the mice were demonstrating a light preference is that
they found something aversive in the odor of the inserts used to cover
the walls of the darker goal box. This possibility has not been
excluded at the present time.

It has been suggested that the results of experiments in.which
animals were trained to the light were not necessarily contradictory
because of the low baseline of retention. There is evidence that this
was not the only factor which prevented the demonstration of CXMrinduced
amnesia. In Experiment 12, mice were trained to enter the darker goal
box in order to escape shock. These animals required more trials to
reach the training.criterion than groups which had been trained to
light in the previous experiments. Both groups that had been treated
with CXM.(120 or 150 mg/kg) had larger savings scores than the control
group (see Figure 6). While there was no significant drug effect on
the analysis of variance, it was surprising that the only group to show
a significant reduction in trials to criterion between training and
testing was the group that had received 150 mg/kg CXM. To date there

has been no attempt to replicate this finding and, as it is an isolated

57

case that does not fit with the existing evidence, no interpretation
seems indicated at this time.
Brightness.Discrimination Training
After CXM Treatment

None of the experiments with mice resulted in a significant train-
ing effect of CXM treatment, nor were rats treated intracerebrally with
CXM different from their controls. In contrast to these results and to
the evidence from experiments. presented in Table 1, subcutaneous injec-
tions impaired acquisition of the brightness discrimination in Experiment
3. While the observation of a CXM effect on acquisition is not without
precedent (Squire and Barondes, 1972b), it is not clear whether the
effect observed here.was real or whether it should be attributed to an
unfortunate distribution of animals between the two groups assigned by
random selection. In Experiment 4, there was a reversal of the drug
effect on acquisition which approached statistical significance. The
procedures of Experiment 4 were exactly the same as those in Experiment
3, except that the animals were slightly younger, weighed less, and
had been in the laboratory for a shorter time prior to the experiment.
There were more deaths of CXM-treated animals in Experiment 4 than in
Experiment 3 where the drugrtreated rats required more trials to reach
the training criterion. It was considered that the lower trials to
criterion for CXM animals in Experiment 4 might have been due to having
deleted from the analysis more of the animals that suffered intense
effects of the drug. This was not the case, however, as including
animals that died increased the mean trials to criterion for the CXM-

treated group of Experiment 3 and decreased this value in Experiment 4.

58

Effect of Secondm. Variables on
Discriminated Avoidance

The effect of the variables that were investigated secondarily to
the CXM treatment warrants only a brief discussion. It was not sur-
prising that' manipulation of time in the goal box, the brightness of
the maintenance enVironment, and the time of training all failed to
show significant effects. as the levels used were selected as- a possible
means of facilitating avoidance training to keep within the time limits
of the action of CXM and were not purported to be a meaningful test of
these variables in, and. of themselves. For example, in case of. time in
the goal box, Denny (1973) has shown that increasing goal box time
facilitated training. but differences on the order of 120 sec were

required to show a significant improvement.

SUMMARY

Rats and mice were treated subcutaneously with CXM in doses suf—
ficient to inhibit protein synthesis by more than 90%. Thirty min
later they were trained in brightness—discriminated shock avoidance
in a Y— or T-maze. Other rats were treated intracerebrally with CXM
and trained 4 or 5 hr after injection. Six or 24 hr after training,
the performance of rats and mice that had been treated with CXM was
compared with that of saline-injected mice that were similarly trained.
No consistent differences were observed between animals treated subcu-
taneously and their controls. The magnitude of difference between
treated and control rats was greater when CXM was administered intra-
cerebrally, but this too fell short of statistical significance. As
the failure to show CXM-induced amnesia.was contrary to the existing
literature, possible explanations were explored. It was suggested
that the failure to produce amnesia in rats may have been due to strain
and/or species differences in susceptibility to some undetermined
action of CXM which was not necessarily related to inhibition of pro-
tein synthesis. For mice the lack of effect appeared to be due to a
failure of both CXMFtreated and control mice to learn the discrimina-

tion during initial training.

59

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