BEHAVIORAL AND BIOCHEMICAL EFFECTS OF INTRACRANIAL INJECTION OF CYTOSINE ARABINOSIDE IN GOLDFISH* - PNAS
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BEHAVIORAL AND BIOCHEMICAL EFFECTS OF INTRACRANIAL
INJECTION OF CYTOSINE ARABINOSIDE IN GOLDFISH*
BY LUIGI CASOLA, RAMON LIM, t ROGER E. DAVIS, AND BERNARD W. AGRANOFF
MENTAL HEALTH RESEARCH INSTITUTE, UNIVERSITY OF MICHIGAN
Coinnmmunicated by J. L. Oncley, May 27, 1968
Previous studies have indicated that memory of a shock avoidance task in
the goldfish can be blocked by various antimetabolites injected before or shortly
after training trials. The intracranial injection of puromycin or acetoxycyclo-
heximide (AXM\), inhibitors of protein synthesis in the goldfish brain, or of
actinomycin D, a powerful inhibitor of DNA-dependent RNA synthesis, blocks
the formation of long-term memory." 2 These results implicate growth or
induction processes ill memory formation, but they do not rule out the possi-
bility that memory requires the formation of new cells in the brain. This possi-
bility might be tested with an inhibitor of DNA synthesis. Growth of the
cerebral cortex of rats raised in an "enriched environment" has been reported3
and might be interpreted to reflect hyperplasia. It has also been reported that
such rats show increased incorporation of H'-thymidine into glial nuclei.4 These
experiments suggest that glial proliferation is at least in part responsible for the
observed increase in cortical depth after exposure of rats to the enriched environ-
ment. Uptake of labeled thymidine into neurons of adult rats has been re-
ported, but proliferation of adult neurons remains to be demonstrated.5
The present report is of the effects of cytosine arabinoside (ara-C) on the
synthesis of DNA, RNA, and protein in brain, and on learning and retention of
avoidance responding. Ara-C inhibits selectively DNA synthesis in vivo6 and
in tissue cultures.7' 8 In addition, we have examined the effect of AXM on
DNA synthesis. Recent reports that antibiotic protein inhibitors impair DNA
synthesis in rat intestines' 10 suggested that AXM\I might block DNA synthesis
in goldfish brain.
Materials and Methods.-Goldfish weighing 8.5-11 gm. and 6-7 cm long were obtained
from Ozark Fisheries, Stoutland, Missouri. Prior to experiments, fish were kept in group
tanks in continuous light for 2-10 days. Fish were removed to individual, clear plastic
tanks 18-24 hr before the start of behavioral experiments and for 1 hr prior to chemical
studies. AXM was a gift of T. C. McBride (Pfizer and Co., Maywood, New Jersey),
and ara-C was kindly provided by the Upjohn Company (Kalamazoo, Michigan). Drugs,
dissolved in 0.15 M NaCl, and aqueous solutions of labeled thymidine and of uridine
were injected intracranially (IC) in a l-,gl volume."
DNA and RNA labeling: Groups of ten goldfish were given either H3-methyl-thymidine
(10 ttc, 14.5 c/mmole, New England Nuclear Corporation) or H3-5-uridine (5 gc, 8 c/
mmole, Schwarz BioResearch), and each group was killed at a different time after injec-
tion. In each case, brains from the groups of goldfish were blotted, combined, and
homogenized in 5 ml of cold water. An equal volume of 10% trichloroacetic acid (TCA)
was added to the homogenate; the resulting TCA-insoluble precipitate was washed and
extracted once with ethanol and twice with ether. The dry residue was suspended in 5
ml of 1 Al KOH and stirred at 370 for 2.5 hr.'2 The clear solution was neutralized with
perchloric acid and centrifuged at 2500 rpm for 5 min, and the radioactivity in 0.1 ml of
the RNA hydrolysate was measured in a scintillation counter. Radioactivity in DNA
was measured by counting 0.1 ml of a hot perchloric acid extract.'2 For these experi-
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138911IOCHEAIISTRY: CASOLA ET AL.
1390 Prtoc. N. A. S.
inents, xyleiie-dioxane-methyl cellosolve solvent (XDC)13 was used, and quenching was
determined by addition of an internal standard.
Protein labeling: H3-4,5-L-Leucine (10 Mc, 6 c/mmole, Schwarz BioResearch) was
injected intraperitoneally (IP) in 10 dul of 0.01 N HCL. Brains from groups of 30 fish
were pooled and homogenized in 15 ml of cold water. A 0.5-ml sample of the homogenate
was mixed with 5 ml of cold 10% TCA containing 2 mM L-leucine carrier. After cen-
trifugation, the supernatant was decanted, and the precipitate was suspended in 5 ml of
10% TCA and heated to 800 for 30 min. The mixture was again centrifuged, and 1 ml
of the combined supernatant fraction was counted in XDC. The precipitate was washed
with ethanol and ether. The dried powder was dissolved in 0.5 ml of 1 M Hyamine hy-
droxide and counted in 10 ml of toluene containing 5% ethanol, 0.5% 2,5-diphenyloxazole,
and 0.03% dimethyl 1,4-bis [2-(4-methyl-5-phenyloxazolyl) ]benzene.
Shock-avoidance training: The shuttlebox and training procedure designated task III
in a previous investigation was used.2 The shuttlebox consisted of a clear plastic tank
with a lid. The tank was separated into two equal compartments by an underwater
barrier. Above the barrier was a clear plastic gate hinged to the shuttlebox lid. The
fish had to push the gate to cross over the barrier. Each compartment had a pair
of stimulus electrodes on the walls adjacent to the barrier and a stimulus lamp outside the
end wall. Movement of fish over the barrier was registered by a photodetector which
was continuously illuminated by a narrow beam of light flanking the barrier in each
compartment. Ten shuttleboxes were operated simultaneously in a darkened room.
On day 1 of an experiment, fish were moved in home tanks to the experimental room,
placed in individual shuttleboxes for 5 min, and then given 20 trials at 1-min intervals.
At the start of a trial, the stimulus lamp outside the compartment containing the fish was
turned on. After 15 sec,14 a repetitive electrical shock was presented in the light com-
partment for an additional 20 sec. A trial automatically terminated after 35 see or when
the fish crossed over the barrier to the dark compartment. Fish could avoid the shock
by crossing the barrier during the first 15 see of a trial or could escape from it by crossing
during the last 20 sec. Avoidances and escapes were registered by a 20-pen Esterline-
Angus operations recorder. After the 25-min training session, fish were returned to home
tanks. Retention of avoidance responding was tested on day 4, or 72 hr after trial 20, by
giving the fish ten additional trials in a 15-min retraining session.
Retention was evaluated as the difference between the number of avoidances a fish
made in the ten retraining trials (A) and the score which was predicted (P) on the basis of
the fish's performance in the training session.2 We recorded six scores for individual fish:
avoidances and failures to escape in trials 1-10, 11-20, and 21-30. Fish who showed
five or more avoidances or more than five failures to escape in trials 1-10 were rejected.
Also omitted were fish who failed to escape more than five times in trials 21-30. The
remaining fish, approximately 90% of the total, fell into two classes. Class I fish showed
no avoidances in trials 1-20, while class II fish avoided at least once in trials 1-20. For
class I fish, the number of failures to escape in trials 1-10 and 11-20 were the two variables
used to obtain P. For class II fish, the total number of avoidances in trials 1-20 and
failures to escape in trials 1-10 were usvd. Weekly class I and II equations were ob-
tained from independent control fish who were trained during that week and the 2 weeks
before and after. Each equation was based on data from approximately 50 fish.
Results.-DNA synthesis in goldfish brain: As shown in Figure 1, labeled
DNA was detected 30 minutes after injection of labeled thymidine. The
amount of radioactivity in DNA reached a maximum in one to two hours and
showed no appreciable decrease for two days. About 1 per cent of the labeled
precursor was incorporated into DNA. Figure 2A shows the amount of labeled
thymidine incorporated into DNA during a one-hour period at different times
after intracranial injection of ara-C. Radioactivity incorporated into DNA was
reduced to 5 per cent of the control value by one hour after the ara-C injection
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3.0
FIG. 1.-Time course of H3-
thymidine incorporation into 2.0
I0
goldfish brain after IC in-
jection. Solid line, DNA; E
dashed line, acid-soluble frac-
tion (see Materials and Meth- 1.0F
ode).
0 0 24 48
Hours after [3H]- Thymidine
and remained at this level for many hours. Recovery appeared complete in less
than 16 hours. The intracranial injection of AXM impaired thymidine in-
corporation into DNA within one hour, and the inhibition persisted for more than
48 hours (Fig. 2B). The low ratios reflect a decrease in thymidine incorporation
into DNA rather than a change in the levels of TCA-soluble radioactivity.
The persistence of label in the DNA seen in Figure 1 might reflect a slow turn-
over of brain DNA or the presence of a pool of radioactive thymidine supplying
continuous labeled precursor for DNA synthesis. The former possibility is
supported by experiments in which ara-C was injected one hour after the in-
jection of H3-thymidine. The ratio of radioactivity in DNA to that in the
TCA supernatant in animals killed at various times after injection of H3-thymi-
dine was no different than that in a control group (Fig. 3).
A B
~ ~ ~ ~ ~
o 0 20 4 0 4 4
I~~~
~~~
~~~
~~~ ~~~ ~~~ ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
0 1 2
Hours between drug andPHI -Thymidine
FIG. 2.-Inhibition of H3-thymidine incorporation into goldfish brain DNA by
IC injection of (A) ara-C, 100 ;&g, (B) AXM, 0.2 ,ug. Fish were killed 1 hr after
injection of H3-thymidine. Closed circle, ratio of a group which received the 113-
thymidine but no drug.
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0
e 0
F
9 FIG. 3.-Effect of ara-C on 2
loo prelabeled DNA. Solid line, /
ratio of H3-thymidine labeling
in DNA/TCA-soltble fractions
E s/shown in Fig. 1 with an addi-
tional 30-min point. In a sec-
ond experiment (dashed line),
O fish were given 100jug of ara-C
E
1 IC (arrow) 1 hr after H3-thymi-
Qfidine and were killed at the times
indicated.
5 10 15 24
Hours after [3H]- Thymidine
RNA and protein synthesis: H3-leucine and H3-uridine were administered
to different groups of fish one hour after they had received 100 /Ag of ara-C.
The radioactive precursors were also given to control groups that did not re-
ceive ara-C. The incorporation of radioactivity into protein during a 30-
minute period, into RNA during a 35-minute period, and that detected in the
corresponding TCA-soluble fractions appeared not to be inhibited by the ara-C
(Table 1).
Shock-avoidance behavior: Three groups of fish were given 100 ug of ara-C
at various times before and after the training session on day 1. One was in-
jected four hours pretrial, the others immediately (0 hours) and four hours post-
trial. The four-hour pretrial injection had no significant effect on avoidance
responding in the training session (F's less than 1) or in the retraining session
(Table 2). The 0- and 24-hour posttrial injections also had no significant effect
on retention tested on day 4 (A vs. P).
An injection of 0.2 jg of AX.M was administered to different groups 4 hours
pretrial and 24 hours posttrial. The results are presented in Table 2 with data
previously reported for fish injected immediately posttrial.2 The pretrial in-
jection resulted in a retention deficit on day 4, but the deficit was smaller (p
< 0.01) than that obtained with the zero-hour posttrial injection. The 24-hour
posttrial injection of AXM resulted in a positive retention score (p < 0.05).
That score, however, is not significantly different from the retention scores of the
group which received ara-C 24 hours posttrial and the scores of the two unin-
jected control groups (F < 1). The group which received the AXM 4 hours
pretrial showed more avoidances in trials 11-20 than the 0- and 24-hour post-
trial groups did (p < 0.01). A similar but nonsignificant increase in avoidance
TABLE 1. Effect of IC injection of 100 lg of ara-C on RNA and protein synthesis in goldfish
brain.
RNA TCA Protein TCA
-- --(cpm X 10-4 per fish)
Control 1.68 18.5 0.698 1.75
Experimental 2.11 21.4 0.828 2.09
Each fish received either 10 sc of H3-leucine IP or 5 pc of H3-uridine IC.
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TABLE 2. Effect of ara-C and AXM on memory of avoidance training.
Training-- Retraining-
Trials Trials Trials Predicted Retention
1-10 11-20 Tim ke of 21-30 (A) (P) (A - P)
No. Mean SE Mean SE injec tion Mean SE Mean SE Mean SE
No injection
23 .70 .27 1.26 .50 None 5.74 .61 4.82 .36 +.92 .60
26 .96 .26 1.85 .46 None 5.54 .62 5.43 .27 +.11 .,;7
Saline
26 1.08 .2,5 2.65 .58 4 hr 6.69 .34 6.06 .33 +.63 .37
pretrial
10()0ug ara-C
24 1.12 .30 2.33 .57 4 hr 5,.83 .63 5.58 .30 +.26
pretrial
2)5 .52 .21 1.40 .46 0 hr 4.76 .59 4.94 .24 -.18 .48
posttrial
27 .85 .23 2.04 .50 24 hr 6.04 .35 5.37 .27 +.67 .51
posttrial
(0.2 jUg ANM
1. 32 .28 3.48 .71 4 hr 4.20 .49 5.33 .36 -1.13t .56
pretrial
24 .62 .26 1.50 .55 0 hr 1.88 .48 4.80 .28 -2.93* .3.5
posttrial
27 .63 .20 1.37 .40 24 hr 5.93 .46 5.03 .25 +.90t .41
posttrial
* p < 0.01.
t p < 0.05.
in trials 11-20 was exhibited by the fish given ara-C four hours pretrial and by
fish given a 10-,Ml injection of the saline solution. Analysis of variance reveals
that the differences in responding in trials 11-20 among the groups injected
pretrial with AXM, ara-C, or saline are not significant. The enhancement of
avoidance responding by the pretrial injection of AXM, as well as by the 24-
hour posttrial injection, thus appears to be a response to handling and the IC
injection and not to the drug.
Discussion.-H3-thymidine has proved valuable not only for examining the
synthesis of DNA in vivo but also for determining autoradiographically the
location of the cells which synthesize DNA and for investigating whether cell
replication occurs during the period allowed for the incorporation of the pre-
cursor.'5 Studies in adult mammals have demonstrated radioactivity in glial
cell nuclei; this suggests that mitotic activity persists in the adult brain.'6 We
found a rapid incorporation of radioactive thymidine into goldfish brain. Auto-
radiograms from pilot studies in goldfish (Fig. 4) further indicate that the DNA
precursor is incorporated into cells deep in the brain after either an IC or an IP
injection. It is not known whether the labeled cells are glia or neurons or
whether the radioactive thvmidine was incorporated in preparation for mitosis.
MA/itosis may not be a necessary consequence of DNA synthesis. '7
Ara-C is an antibiotic known to inhibit both DNA synthesis and mitosis.6
It has been postulated that ara-C acts by blocking the conversion of cytosine
ribonucleotide to deoxyribonucleotide.18 Recent studies have indicated, how-
ever, that ara-C suppresses the synthesis of DNA by inhibiting DNA polymer-
ase.'9 The 100-1g dose of ara-C given to goldfish rapidly inhibited synthesis of
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DNA but not of RNA or protein. The inhibition of DNA synthesis by AXM
in goldfish brain agrees with the reported effects of cycloheximide and puromycin
in rat intestinal mucosa.9' 10 While AXM additionally suppresses protein syn-
TECTUM ltA
X
CERE BE LLUM
I
MOLECULAR LYR.
* I
GRANULAR LYR.
B
FIG. 4.-Tracings of H3-thymidine autoradiograms of 8-EL transverse sections of goldfish
brain. (A) Midbrain section from a goldfish killed 3 hr after an IC injection of 80 Mc of H3-
thymidine (6.7 c/mmole) in 8 pl of saline. (B) Section of hindbrain of a goldfish who received
an IP injection of 333 pc of H3-thymidine in 100 ,4 of saline 3 hr prior to being killed. The
brains were fixed in formalin and embedded in paraffin. Brain sections were dipped in Kodak
NTB-3 emulsion, dried, stored at - 15° for 4 weeks, and then developed and lightly stained
with chromalum gallocyanin. The dots illustrate the location of the most conspicuously
labeled cells.
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thesis in the goldfish brain,20 it has no immediate effect on the synthesis of
RNA.21
The lack of interference with avoidance responding by ara-C suggests that
the formation of memory does not depend on DNA synthesis. It is possible
that the prolonged inhibition of DNA synthesis obtained with AXM is con-
tributory to the amnesic effects of AXM. But to obtain a memory loss with a
posttrial injection of AXM, the injection must be given within one hour follow-
ing the last trial.2 This suggests that the memory processes susceptible to dis-
ruption by AXM are completed within an hour or two following training. In
addition, the difference between the retention deficits produced by the four-hour
pretrial and the zero-hour posttrial injections of AXM indicates that those
effects of AXM on brain metabolism which are most disruptive to memory
formation occur within a few hours following the injection. The metabolic
effects of AXM four hours after injection did not appear to disturb avoidance
responding during the training session (Table 2). This is compatible with the
proposal, originally based on results obtained with puromycin,22 that memory
during training is a temporary or short-term form mediated by processes other
than protein and DNA synthesis. The resistance of memory (during the train-
ing session) to puromycin and AXM further implies that these agents interfere
specifically with memory fixation, or the formation of long-term memory. The
sum of our investigations with antibiotic antimetabolites in goldfish implicate
the synthesis of protein and possibly of RNA in the formation of long-term
memory. Models of memory based on DNA synthesis or cell replication are not
supported.
*
This study was supported by grants from the National Science Foundation and the Na-
tional Institute of Mental Health.
t Special research fellow, National Institute of Mental Health.
1 Agranoff, B. W., R. E. Davis, and J. J. Brink, Brain Res., 1, 303 (1966).
2 Agranoff, B. W., R. E. Davis, L. Casola, and R. Lim, Science, 158, 1600 (1967).
3 Diamond, M. C., J. Comp. Neurol., 131, 357 (1967).
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5 Altman, J., Anat. Record, 145, 573 (1963).
6 Cohen, S. S., Progr. Nucleic Acid Res., 5, 1 (1966).
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8 Kaplan, A. S., M. Brown, and T. Ben-Porat, Mol. Pharmacol., 4, 131 (1968).
9 Verbin, R. S., and E. Farber, J. Cell Biol., 35, 649 (1967).
10 Estensen, R. D., and R. Baserga, J. Cell Biol., 30, 13 (1966).
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12 Santen, R. J., and B. W. Agranoff, Biochim. Biophys. Acta, 72, 251 (1963).
13 Bruno, G. A., and J. E. Christian, Anal. Chem., 33, 1216 (1961).
14 This interval was erroneously described in ref. 2 as 20 sec.
15 Taylor, J. H., P. S. Woods, and W. L. Hughes, these PROCEEDINGS, 43, 122 (1957).
16 Hommes, 0. R., and C. P. LeBlond, J. Comp. Neurol., 129, 269 (1967).
'7 Pelc, S. R., J. Cell Biol., 22, 21 (1964).
18 Chu, M. Y., and G. A. Fischer, Biochem. Pharmacol., 11, 423 (1962).
19 Furth, J. J., and S. S. Cohen, Cancer Res., 27, 1528 (1967).
20 Brink, J. J., R. E. Davis, and B. W. Agranoff, J. Neurochem., 13, 889 (1966).
21 Casola, L., and B. W. Agranoff, in Proceedings of the First International Meeting of the
International Society for Neurochemistry (1967), p. 4.
22 Davis, R. E., and B. W. Agranoff, these PROCEEDINGS, 55, 555 (1966).
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