Physiology and Behavior, Vol. 13, pp. 693-696. Brain Research Publications Inc., 1974. Printed in the U.S.A.

BRIEF COMMUNICATION
Delayed Development of Amnestic Behavior Alter Hypoxia I

SUSAN J. SARA

Center for Experimental and Comparative Psychology, University of Louvain

Pellenberg, Belgium

(Received 12 September 1973)

SARA, S. J. Delayeddevelopment ofarnnesitc behaviorafter hypoxia. PHYSIOL. BEHAV. 13(5) 693--696, 1974. -Wistar
rats were trained in one trial to avoid a small dark compartment after which they were submitted to a 10 min hypoxia or
sham hypoxia. Different groups were tested at I, 3, 6 or 24 hr after treatment. All sham hypoxia groups avoided at all
testing times. Hypoxia groups avoided at 1 and 3 hr but not at 6 or 24 hr. The results are interpreted as evidence that
hypoxia interferes with memory retrieval and not with memory storage. It is suggested that hypoxia produces a behavioral
deficit by triggering an abnormal metabolic sequence of the biochemical substrates which are necessary for the retrieval
function.

Hypoxia Amnesia Memory Memory retrieval

STUDIES of the temporal course of amnesia induced by
cerebral protein inhibitors like cycloheximide have shown
that the response deficit due to such treatment is not seen
until 3 - 6 hr after training [2]. These results have been
taken as support for a two [2] or three [24] phase memory
storage process, the first being independent of the protein
synthesis process and probably supported by an ordered
pattern of reverberating neural activity [1 1]. The same
progressive decay, however, has been seen with electro-
convulsive shock treatment (ECS) as the amnestic agent
[5 ], thus putting into question the supposed mechanism of
the first phase. The temporal course in this study was
strikingly similar to that of the cycloheximide studies, but
the authors do not rule out the possibility, in spite of the
inclusion of an appropriate control experiment, that the
avoidance response seen in the first could be due to the
potentiation of the postictal effects of ECS by the training
manipulations. Albert [1 ] found a similar decay gradient
using cathodal polarization to disrupt memory. Spreading
depression (SD) produced performance decrement one hour
after training in the same experiment.
More recent studies have replicated the Geller and Jarvik
[5] data. McGaugh and Landfield's [12] data closely
resemble these results, if the ECS is given 20 sec after train-
ing, but an 8 sec delay between footshock (FS) and ECS
yields performance decrement event one hour after train-
ing. Miller and Springer [ 16] used ECS immediately follow-
ing FS and found failure to avoid at 30 min or one hr after
training, but there was good avoidance at 15 rain.
Since this delayed development of response decrement
has such an important significance for any model of
memory (see [15]), further study of these temporal rela-
tions seems to be appropriate, especially in view of the fact
that the phenomenon has not been observed in all labora-
tories. The present work examines the time course of
memory decay with hypoxia as the amnestic agent.
Hypoxia has been shown to produce retrograde amnesia
under diverse conditions [6, 8, 17, 26]. It produced a reli-
able but reversible retrograde amnesia in one trial passive
avoidance training [ 19], although the effect is limited to
certain parameters [ 18].
EXPERIMENT 1
Method
Animals. One-hundred nine male rats of the Wistar strain
were obtained from a commercial supplier. They were
housed 8 - 1 0 to a cage in the laboratory room for one week
before the start of the experiment. Animals had free access
to food and water at all times and weighed 1 5 0 - 2 0 0 g at
the time of the experiment.

The author recognizes the essential contributions made by M. David-Remacle and D. Lefevre especially in the discussion of the results. The
author would like also to thank Dr. C. Giurgea for his support and guidance. This research was supported by grants from "Fonds pour la
Recherche Fondamentale et Collective" and Union Chimique Beige (U.C.B.).

693
694
Apparatus. T h e t r a i n i n g cage was a t w o c o m p a r t m e n t
b o x c o n s t r u c t e d of a l u m i n i u m . T h e large c o m p a r t m e n t was
p a i n t e d w h i t e a n d h a d a gravel floor. It m e a s u r e d 4 0 × 4 0
× 4 0 cm a n d was c o n n e c t e d to t h e small c o m p a r t m e n t b y a
6 × 6 cm guillotine d o o r . T h e small c o m p a r t m e n t , mea-
suring 10 × 10 × 10 cm was p a i n t e d black. T h e f l o o r was
m a d e of m e t a l rods, 2.5 m m in w i d t h a n d 12 m m apart.
T h e f o o t s h o c k (FS) was delivered b y a F o r i n g e r s h o c k
scrambler. T h e s h o c k source was a 7 0 0 V, 50 Hz trans-
f o r m e r w h o s e o u t p u t c u r r e n t is limited b y a variable series
resistance. S h o c k level was 1 m A . T h e h y p o x i a e q u i p m e n t
was e x a c t l y as described b y Sara and Lefevre [ 1 8 ] . It
consisted of an airtight Plexiglas cage m e a s u r i n g 50 × 50 ×
30 cm. Pure n i t r o g e n was i n t r o d u c e d i n t o t h e cage and was
m i x e d w i t h the a t m o s p h e r e b y a v e n t i l a t o r a t t a c h e d to o n e
of t h e walls. A B e c k m a n n 7 7 7 analyser continuously
m e a s u r e d t h e p e r c e n t a g e o f o x y g e n in t h e a t m o s p h e r e in
t h e cage ( 3 . 5 - 4 % ) .
Experimental plan and procedure. T h e animals were
divided r a n d o m l y i n t o t w o groups, h y p o x i a ( H Y P ) and n o n
h y p o x i a ( N H Y P ) ; these g r o u p s were s u b d i v i d e d a c c o r d i n g
to testing time: 1 hr, 3 hr, 6 hr or 24 hr a f t e r training. T h u s
t h e r e were 8 separate groups: HYP-1, NHYP-1, HYP-3,
NHYP-3, HYP-6, NHYP-6, HYP-24, N H Y P - 2 4 .
T h e a n i m a l was placed in t h e large c o m p a r t m e n t in the
c o r n e r f a r t h e s t away f r o m t h e d o o r leading t o t h e small
c o m p a r t m e n t . T h e r e was a 3 rain free e x p l o r a t i o n p e r i o d
d u r i n g w h i c h t h e t o t a l t i m e s p e n t in each c o m p a r t m e n t was
n o t e d for each rat. A f t e r 3 m i n , the d o o r was lowered a n d
the f o o t s h o c k of o n e m A was a d m i n i s t e r e d for a 15 sec
period. T h e rat was a l m o s t always located in t h e s h o c k
c o m p a r t m e n t at t h e end o f t h e e x p l o r a t i o n p e r i o d ; in the
few e x c e p t i o n a l cases t h e e x p l o r a t i o n p e r i o d was e x t e n d e d
until t h e rat r e e n t e r e d t h e dark. In no case did this e x t e n -
tion exceed 10 sec. T h e rat was t h e n i n t r o d u c e d i n t o the
h y p o x i a b o x a n d m a i n t a i n e d at 3.5 4% 0 2 for l 0 m i n , or
placed in a Plexiglas b o x for 10 m i n at n o r m a l o x y g e n level.
T h e animals s p e n t t h e time b e t w e e n t r e a t m e n t and
testing in t h e g r o u p cage. At t h e a p p r o p r i a t e testing t i m e
the individual a n i m a l was placed in the a p p a r a t u s in the
same m a n n e r as t h e t r a i n i n g phase a n d t h e time s p e n t in
each c o m p a r t m e n t was r e c o r d e d , for a t o t a l of 180 sec.
Results
Animals s p e n t a t o t a l o f 82% of t h e t o t a l 180 sec p e r i o d
in t h e dark b e f o r e FS. A n analysis of variance s h o w e d n o
d i f f e r e n c e a m o n g all g r o u p s as t o t h e t o t a l time in t h e small
c o m p a r t m e n t b e f o r e training. T h e m e d i a n t i m e s p e n t in t h e
dark a f t e r FS for each g r o u p can be seen in Fig. 1.
Lack of n o r m a l d i s t r i b u t i o n o f t h e results i n d i c a t e d t h e
use o f n o n p a r a m e t r i c statistical analysis. HYP a n d N H Y P
groups were c o m p a r e d for e a c h testing time using a M a n n -
W h i t n e y U test. T h e r e was a significant d i f f e r e n c e b e t w e e n
H Y P 2 4 a n d N H Y P 2 4 (U = 26, p < 0 . 0 0 2 ) i n d i c a t i n g t h a t
h y p o x i a does, u n d e r t h e p r e s e n t e x p e r i m e n t a l c o n d i t i o n s ,
p r e v e n t e x p r e s s i o n of an a v o i d a n c e r e s p o n s e at 24 hr. T h e r e
was also a d i f f e r e n c e b e t w e e n the g r o u p s H Y P 6 and N H Y P 6
(U = 124, p = 0.01). A t 1 a n d 3 h r after t r e a t m e n t , g r o u p s
HYP1 a n d NHYP1 and groups HYP3 and N H Y P 3 ;
t h e r e were n o differences. A d d i t i o n a l c o m p a r i s o n s were
m a d e b e t w e e n t h e HYP1 and H Y P 2 4 and also b e t w e e n
HYP1 and H Y P 6 ; in b o t h cases t h e d i f f e r e n c e was signifi-
cant (U = 14, p < 0 . 0 2 a n d U = 16, p < 0 . 0 0 2 , respectively).
T h e r e were n o d i f f e r e n c e s b e t w e e n NHYP1 a n d N H Y P 2 4 or
SARA
180
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m
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,<
a
z
b.I
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I 3 6 24
TiME (IN HRS) A F T E R TRAINING
FIG. 1. Median total time in the dark (shock) compartment after
training, for each of the eight hours with interquartrile range.
Shaded bars are the NHYP groups. Note that only the HYP6 and
HYP24 groups display a lack of avoidance of the small compart-
ment. The differences among the NHYP are not significant. The
number of animals in each group is indicated in parentheses.
b e t w e e n NHYP1 a n d NHYP6. T h u s a v o i d a n c e b e h a v i o r was
seen for all N H Y P g r o u p s at all testing times. F u r t h e r m o r e ,
at 1 hr and 3 hr, the HYP a n i m a l s avoid as well as t h e
c o n t r o l animals. Failure t o avoid a p p e a r s b e t w e e n 3 and 6
hr after t r e a t m e n t .
EXPERIMENT 2
T h e d i f f e r e n c e s seen at 6 hr a n d 24 hr b e t w e e n t h e
g r o u p HYP a n d g r o u p N H Y P could c o n c e i v a b l y be due to
c o n s e q u e n c e s n o t related to m e m o r y . A d i m i n u t i o n of loco-
m o t o r activity favors a r e t e n t i o n score in a passive avoid-
ance task, so it is necessary t o insure t h a t i n t e r a c t i o n o f FS
a n d HYP does n o t i n d u c e e i t h e r a decrease in l o c o m o t i o n
up t o 3 hr a f t e r t r e a t m e n t , or an increase in activity begin-
ning at 6 h r after. E i t h e r of these f a c t o r s could a c c o u n t for
the decay g r a d i e n t f o u n d in E x p e r i m e n t 1. I n d e e d , t h e r e is
evidence t h a t t h e r e is a l o w e r activity level in mice u p t o 3
hr, b u t n o t 24 hr o f a FS g r o u p c o m p a r e d to a FS-ECS
g r o u p (A. Geller, personal c o m m u n i c a t i o n ) . F u r t h e r m o r e ,
c y c l o h e x i m i d e t r e a t e d mice also show decrease in activity
up to 3 hr p o s t t r e a t m e n t a f t e r an initial increase lasting less
t h a n 1 hr [ 2 5 ] , H o w e v e r , an a t t e m p t has b e e n m a d e to
dissociate t h e effects of c y c l o h e x i m i d e o n m e m o r y from its
effects o n activity [ 2 2 ] . Nevertheless, the possibility
r e m a i n s t h a t c e r t a i n s y s t e m i c effects of i n t e r a c t i o n b e t w e e n
t r a i n i n g a n d a m n e s t i c t r e a t m e n t could a c c o u n t , in part, for
the r e t e n t i o n score s h o r t l y a f t e r t r e a t m e n t or for t h e for-
DELAYED AMNESIA AFTER HYPOXIA
getting at longer intervals. Since the decay curve in most
studies closely resembles the activity curve of the FS-ECS
or the FS cycloheximide injected animals, it is essential to
eliminate this possible artifact as an explanation of the
phenomen. One approach to control for systemic effects of
FS-amnestic treatment is to include a noncontingent foot-
shock group (NCFS) which receives a footshock outside of
the apparatus and then the amnestic treatment, after which
the animal is tested at the appropriate time in the experi-
mental apparatus [13,16]. It is necessary to control for
exposure to the apparatus, so that animals are introduced
to the FS or NCFS environment before the training session.
Such a maneuver assumes that the two events are totally
independent, which may not be the case.
Experiment 2 addresses itself to the question of whether
the results found in Experiment 1 could be due to a differ-
ential influence on spontaneous locomotor activity of FS
alone and the interaction of FS-HYP. Treatment of both
groups during training and testing session is identical,
except for the level of oxygen after FS, thereby eliminating
possible differences introduced by pretraining manipula-
tions. The testing phase of the experiment involved a
measurement of activity for 3 rain.
Me t h o d
Animals. Thirty-six rats were purchased from the same
source as in Experiment 1. They had the same weight and
were housed under the same conditions as in Experiment 1.
Apparatus. The training cage and hypoxia equipment
were the same as used in Experiment 1. The activity cage
was constructed of tubular Plexiglas, 27 cm in dia. and
27 cm high. Four pairs of photo electric cells measured
horizontal and vertical movements. Each interruption signal
was amplified and automatically recorded with a Sodeco
electronic counter. An automatic timer limited the record-
ing period to 3 min. The entire apparatus, with the excep-
tion of the counter and timer, was enclosed by a wooden
box.
Procedure. The rats were divided into three groups HYP
and NHYP and then subdivided according to testing time as
in Experiment 1. There were 6 groups of 6 rats, HYP1,
NHYP1, HYP6, NHYP6, HYP24 and NHYP24. The proce-
dure at the training time was exactly as it was in Experi-
ment 1. The rat was placed in the corner of the large
compartment and allowed to freely explore for 3 min; the
door was lowered at the end of this period and the animal
received FS for 15 sec, after which it was removed and
placed in the hypoxia cage at 3.5% 02 and maintained at
this level for 10 min. The NHYP treatment groups received
the same training and then were isolated in a Plexiglas cage
for 10 min. After treatment, the animals were returned to
the living cages until the appropriate testing time. The test
consisted of placing the rat individually in the activity cage
and registering the horizontal and vertical movements for 3
min.
Results
The horizontal and vertical activity for each group and
each time can be seen in Table 1. An analysis of variance
showed no difference among groups (F = 1.4, dr= 45).
DISCUSSION
The results support the findings in other laboratories
695
TABLE 1
HORIZONTAL AND VERTICAL ACTIVITY FOR EACH GROUP
Mean total
Group n displacements
HYPI 6 35
NHYP1 6 24
HYP6 6 22
NHYP6 6 16
HYP24 6 25
NHYP24 6 27
using other amnestic agents that the performance of a
passive avoidance task [5, 12, 13] or a discriminated
o p e r a n t avoidance [24] deteriorates over time after
amnestic treatment; Experiment 2 indicates that the change
is not due to a modification of spontaneous locomotor
activity. Unlike cycloheximide and FS-ECS, FS-HYP does
not produce changes in locomotor activity at the times
tested.
Many investigators take the decay of memory gradient as
evidence for a dual or multiprocess storage mechanism [ 1,
12, 24, 27] i.e. that a short term memory (STM) persists
for an extended period and that the amnestic agent pre-
vents permanent storage of long term memory (LTM).
Deutsch [3], on the other hand, suggests that perhaps
memory is a single process and the amnestic treatment
accelerates forgetting, the decay of memory curve being an
accelerated forgetting curve. However, experiments showing
recovery of memory (eg. [14, 16, 19]) demand that for-
getting, at least after amnestic treatment, be considered as
retrieval failure (see [ 1 5 - 2 3 ] ).
Perhaps a certain amount of storage of information
occurs during learning but the significant aspect of
memory, that which produces an adaptive behavior, is the
integration of this information into existing functional
systems (see [18]). These systems undergo a constant
reorganization as a result of ongoing experience, especially
retrieval activities. Amnestic treatments prevent this inte-
grative activity from occuring after training, but this does
not exclude the possibility that such activity could be
triggered again at a later time, perhaps by a reminder event,
(see [20] ) thus explaining the recovery from amnesia after
ECS or hypoxia. It is also possible that this activity does
continue for some time after amnestic treatment and that
the biochemical substates necessary to sustain such activity
are gradually altered over time as a result of an abnormal
metabolic sequence triggered by the amnestic agent. This
would, of course, account for the decay gradient. When the
biochemical state returns to normal, the possibility for
further organization of the memory and hence its eventual
retrieval also returns. Reminder events such as the training
environment, increase the probability of such integrative
activity occurring. (Note that this position is different from
the "state dependent" explanation of retrograde amnesia
(eg. [4]) in that the adaptive behavior can be expressed
696
w i t h o u t r e i n s t a t i n g t h e a p p r o p r i a t e physiological state of
the o r g a n i s m [20] .)
T h e r e is s o m e physiological e v i d e n c e t h a t m e m o r y exists
in a p r i m i t i v e f o r m at t h e reticular and t h e n at the t h a l a m i c
level d u r i n g t h e early stages o f a c q u i s i t i o n , b e f o r e an adap-
tive b e h a v i o r a l r e s p o n s e can be observed [ 7 ] . J o h n [10]
has also suggested t h a t m e m o r y storage can o c c u r w i t h o u t
the i n f o r m a t i o n being available in a f o r m w h i c h has b e h a v -
ioral c o n s e q u e n c e s . F u r t h e r m o r e , Hine and P a o l i n o [9] a n d
Schoel a n d A g r a n o f f [21] have d e m o n s t r a t e d t h a t animals
w h i c h b e h a v e " a m n e s t i c a l l y " a f t e r ECS t r e a t m e n t r e t a i n
a u t o n o m i c responses. Perhaps t h e cortical i n t e g r a t i o n
necessary for an o r g a n i z e d skeletal r e s p o n s e ( a v o i d a n c e ) is
p r e v e n t e d or i n t e r r u p t e d b y t h e a m n e s t i c t r e a t m e n t , while
primitive s u b c o r t i c a l traces are f o r m e d . T h e d e c a y of
m e m o r y a f t e r a m n e s t i c t r e a t m e n t could be e x p l a i n e d b y
t h e fact t h a t s u b c o r t i c a l l y s t o r e d m e m o r y a t t r i b u t e s can
m e d i a t e an adaptive r e s p o n s e for a limited period after
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SARA
learning, b u t require f u r t h e r o r g a n i z a t i o n and i n t e g r a t i o n in
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tion specific to a p a r t i c u l a r p r i m a r y m e m o r y trace as o t h e r s
have suggested [ t 5 ] ; retrieval in this m o d e l is c o n s i d e r e d as
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cortex. T h e r e is increasing evidence t h a t events w h i c h o c c u r
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