Pharmacology Biochemistry & Behavior, Vol. 3, pp. 775-780. Copyright © 1975 by ANKHO International Inc. All rights of reproduction in any form reserved. Printed in the U.S.A.
Changes in Brain Monoamine Metabolism and Carbon Dioxide Induced Amnesia in the Rat B. E. L E O N A R D A N D H. R I G T E R
Pharmacology Department, Scientific Development Group Organon, Oss, the Netherlands
(Received 16 O c t o b e r 1974) LEONARD, B. E. AND H. RIGTER. Changes in brain monoamine metabolism and carbon dioxide induced amnesia in the rat. PHARMAC. BIOCHEM. BEHAV. 3(5) 775-780, 1975. - The effect of treatment with carbon dioxide (CO2) on the performance of rats 24 hr after receiving a foot shock in a passive avoidance task was studied. Foot shock induced avoidance. Carbon dioxide produced retrograde amnesia for the foot shock induced avoidance response. Changes in brain monoamine metabolism were studied in groups of rats which had been treated with CO2, foot shock or foot shock + CO2. The rats were killed 24 hr after treatment. Changes mainly occurred in the brain stem and hippocampus. In the rats which had received foot shock alone, brain stem and hippocampal serotonin concentrations were raised. This rise was not observed when the foot shock was followed by CO 2 treatment. Furthermore, it was found that there was an increased release of noradrenaline in those rats subjected to foot shock alone but a decreased release of this amine in the group which received foot shock followed by CO2. It is suggested that the amnesic effect of CO2 parallels changes in brain serotonin and noradrenaline metabolism. Amnesia
Passive avoidance
Carbon dioxide
Hippocampus
THE m o s t c o m m o n paradigm for amnesia studies in rats involves (1) a training situation in w h i c h the animal acquires an aversive e x p e r i e n c e (f.e., a f o o t s h o c k ) , (2) followed by an amnesic t r e a t m e n t and (3) a retrieval test 24 hr later. Little is k n o w n a b o u t the role brain m o n o a m i n e s m a y play in amnesia. A n u m b e r o f investigators s t u d i e d the effects o f a f r e q u e n t l y used a m n e s i c agent, e l e c t r o c o n v u l sive shock, on brain m o n o a m i n e levels [8, 13, 14, 2 0 ] . However, these studies did n o t i n t e n d to correlate the changes in brain amine c o n c e n t r a t i o n s with amnesia. Thus, either the training e x p e r i e n c e was lacking [8, 13, 14] or the time interval was d i f f e r e n t f r o m the o n e used in amnesia studies [ 19,20]. We r e p o r t on the very first step in an investigation aimed at assessing possible associations b e t w e e n brain m o n o a m i n e m e t a b o l s i m and amnesia. C a r b o n dioxide (CO2), an effective amnesic agent [21, 22, 2 7 ] , was used t o p r o d u c e amnesia for a one-trial s t e p - t h r o u g h passive avoidance response. In E x p e r i m e n t 1, we d e m o n s t r a t e t h a t amnesia is p r e s e n t 24 hr a f t e r amnesic t r e a t m e n t . In E x p e r i m e n t 2, we f o l l o w e d the same paradigm but instead o f subjecting t h e animals t o a retrieval test, the rats were killed 24 h r a f t e r amnesic t r e a t m e n t and brain m o n o a m i n e levels were determined. In f u r t h e r studies [ 2 3 , 3 1 ] , we a t t e m p t to clarify the relationships b e t w e e n the changes in brain a m i n e c o n c e n t r a t i o n s and amnesia.
Noradrenaline
Serotonin
EXPERIMENT 1 ASSESSMENT OF CARBON DIOXIDE AS AN AMNESIC AGENT
Method Animals and Apparatus. F o r t y male rats o f an i n b r e d Wistar strain weighing 2 3 0 - 2 5 0 g were used. The animals were h o u s e d 10 per cage w i t h free access to f o o d and water. The animals were trained in a s t e p - t h r o u g h passive avoidance a p p a r a t u s o f the t y p e described b y Ader, et al. [ 1 ]. This consisted o f a 40 × 40 × 40 cm Lucite c h a m b e r w h i c h had a grid floor and black walls. The f r o n t wall was posit i o n e d at the edge o f a table and c o n n e c t e d to a 6 cm wide and 25 cm long r u n w a y w h i c h p r o j e c t e d over the floor. The r u n w a y was illuminated by m e a n s o f a 40 W lamp held 40 cm above it; the c h a m b e r was in darkness. When placed on the r u n w a y , a rat could e n t e r the d a r k e n e d c h a m b e r t h r o u g h a 6 × 6 cm o p e n i n g w h i c h could be closed b y means o f a h a n d o p e r a t e d guillotine d o o r . A s c r a m b l e d f o o t s h o c k (0.5 m A for 3 sec) could be delivered t h r o u g h the metal grid floor o f the c h a m b e r f r o m a 500 V AC source. Procedure. The rats were r a n d o m l y divided into 4 groups of 10 each. T h e y were given 3 pretraining trials on Day 1 o f the e x p e r i m e n t . A pretraining trial consisted o f placing the rat at the end o f the r u n w a y facing the e n t r a n c e o f the c h a m b e r ; the time t a k e n for the animal to e n t e r the cham775
776
LEONARD AND RIGTER
ber was r e c o r d e d a n d defined as t h e s t e p - t h r o u g h latency. U p o n e n t e r i n g t h e c h a m b e r , the d o o r was closed a n d t h e animal allowed to r e m a i n t h e r e for 10 sec. It was t h e n r e t u r n e d to its h o m e cage. T h e interval b e t w e e n t h e 3 pret r a i n i n g trials was a p p r o x i m a t e l y 2 hr. O n Day 2, t h e rats were s u b j e c t e d t o a single a c q u i s i t i o n trial. This was similar t o t h e p r e t r a i n i n g trials w i t h t h e e x c e p t i o n t h a t a f o o t s h o c k was given to 2 groups of animals 10 sec after t h e y had e n t e r e d t h e c h a m b e r (FS groups). T h e o t h e r 2 g r o u p s of rats did n o t receive f o o t s h o c k ( N o F S groups). I m m e d i ately on t e r m i n a t i n g the a c q u i s i t i o n trial, the rats were r e m o v e d f r o m the c h a m b e r a n d s u b j e c t e d to e i t h e r amnesic t r e a t m e n t (CO2 g r o u p s ) or s h a m amnesic t r e a t m e n t (NoCO2 groups). T h e amnesic t r e a t m e n t consisted in placing an animal in a closed c o n t a i n e r t h r o u g h w h i c h CO2 was passed u n t i l o x y g e n m e a s u r e m e n t s yielded zero readings [ 2 2 ] . The rats were r e m o v e d f r o m the b o x as s o o n as r e s p i r a t o r y arrest o c c u r r e d a n d were revived b y artificial r e s p i r a t i o n ; the time necessary for r e s p i r a t o r y arrest to o c c u r was 3 0 - - 3 5 sec. Rats receiving s h a m amnesic treatm e n t were placed in an air filled b o x for 35 sec. All animals were r e t u r n e d t o t h e i r h o m e cages at the e n d of t h e amnesic or sham a m n e s i c t r e a t m e n t . T h u s at t h e e n d of t h e acquisit i o n trial the 4 groups of rats h a d b e e n s u b j e c t e d to t h e following t r e a t m e n t s : NoFS-NoCO2 - n o f o o t shock, n o CO2 t r e a t m e n t ; NoFS-CO2 - n o f o o t s h o c k , b u t CO2 given; FS-CO2 foot s h o c k f o l l o w e d b y CO2 t r e a t m e n t ; FS-NoCOz f o o t s h o c k b u t n o CO2. T w e n t y - f o u r h o u r s after the a c q u i s i t i o n trial, t h e rats were s u b j e c t e d t o a single retrieval trial. T h e rats were placed at the e n d of the r u n w a y a n d t h e t i m e t a k e n for t h e m to e n t e r the d a r k e n e d c h a m b e r was r e c o r d e d . W h e n a rat failed to e n t e r w i t h i n 300 sec, it was r e m o v e d f r o m t h e r u n w a y a n d a l a t e n c y score o f 300 sec was assigned. The retrieval scores were divided i n t o 3 classes: (1) latencies of 0 - 1 0 sec; (2) latencies of 1 0 - 2 9 9 sec; (3) latencies o f 300 sec. Rats e n t e r i n g the c h a m b e r w i t h i n 10 sec were considered as n o t s h o w i n g passive a v o i d a n c e ; previous experim e n t s h a d s h o w n t h a t 10 sec r e p r e s e n t e d t h e m a x i m a l latency for rats w h i c h h a d n o t received a f o o t s h o c k at t h e
t i m e of the a c q u i s i t i o n trial. Rats e n t e r i n g t h e c h a m b e r w i t h i n 1 0 - 2 9 9 sec displayed i n c o m p l e t e passive avoidance while t h o s e t h a t failed to e n t e r w i t h i n 300 sec were regarded to show a c o m p l e t e passive a v o i d a n c e response. In the analysis of t h e results, the 3 classes received a weighting of 0, 1 a n d 2, respectively. T h e retrieval scores were analysed statistically using t h e Yates test [ 3 3 ] . F o r the analysis o f t h e o t h e r data the two-tailed M a n n W h i t n e y U test was e m p l o y e d , for c o m p a r i s o n s b e t w e e n groups, a n d t h e twotailed sign test, for c o m p a r i s o n s w i t h i n groups.
Results All groups of rats s h o w e d a r e d u c t i o n in their stept h r o u g h latencies during the course of t h e p r e t r a i n i n g trials. This was a p p a r e n t f r o m the significant differences b e t w e e n the latencies at t h e t i m e of t h e first p r e t r a i n i n g trial a n d those at t h e time of the acquisition trial (p = 0.001 for each group, sign test). T h e latencies at t h e t i m e of t h e acquisit i o n trial did n o t differ b e t w e e n t h e f o u r g r o u p s (Table 1). In the 2 N o F S groups, there was n o significant difference b e t w e e n the latencies at t h e retrieval a n d t h o s e at t h e acquisition trial ( p > 0 . 1 0 for b o t h groups). The latencies o f the N o F S groups at the test trial did n o t differ (Table 1). This suggests t h a t the CO2 t r e a t m e n t did n o t affect the stept h r o u g h response. T h e latencies of the four groups of animals at the time of the retrieval trial are s h o w n in Fig. 1. A d m i n i s t r a t i o n o f CO2 p r o d u c e d amnesia: the FS-CO2 group had significantly s h o r t e r latencies t h a n t h e FS-NoCO2 g r o u p (z = 4.13; p < 0 . 0 0 1 , Yates test). T h u s 8 o u t of 10 rats in the FS-CO2 g r o u p h a d a l a t e n c y similar to t h o s e o f the NoFS-NoCO2 group, while 9 o u t of 10 of the FS-NoCO2 g r o u p showed c o m p l e t e passive a v o i d a n c e a n d did n o t e n t e r t h e c h a m b e r . The latencies of FS-CO2 rats did n o t differ f r o m those of NoFS-NoCO2 or NoFS-CO2 rats (in b o t h cases: z = 1.49). On t h e o t h e r h a n d , the latencies of t h e FS-NoCO2 g r o u p were longer t h a n those of the N o F S groups (z = 4.37; p < 0 . 0 0 1 in b o t h cases).
TABLE 1 EFFECT OF PRETRAINING ON STEP-THROUGH LATENCIES OF GROUPS OF RATS
Trials Pretraining Group
t*
2*
3*
Acquisition*
Retrieval*
NoFS-NoCO~
9.3 ± 2.0
3.8 + 1.0
2.2 + 0.6
1.4 -+ 0.1
1.5 + 0.2
NoFS-CO 2
8.1 ± 1.7
3.9 ± 0.9
2.0 ± 0.4
1.1 + 0.3
1.6 ± 0.2
FS-CO 2
9.2 -+ 1.8
5.4 + 1.2
1.9 ± 0.4
1.5 ± 0.1
t
FS-NoCO 2
8.5 +- 1.5
5.2 + 1.2
1.3 ± 0.3
1.1 + 0.1
Step-through latencies (mean number of sec ± standard error of the mean) during pretraining trials and the acquisition trial. *Differences between groups within column are not significant (two-tailed Mann-Whitney U test). ~Latencies at the test trial are only given for groups which did not receive footshock at the acquisition trial. FS: footshock; NoFS: no footshock; CO:: COs-treatment; and NoCO2: no CO~-treatment.
MONOAMINES AND AMNESIA
777
m ®
100.
class 1
Elmer s p e c t r o p h o t o f l u o r i m e t e r Model 2A. The statistical significance of the results was assessed using a S t u d e n t ' s t-test.
80-
D
o~
class 2
60 ~5
m
40
class 3
Results
The results are given in Figs. 2, 3 and 4. Neither C O : , nor foot shock or the c o m b i n a t i o n of b o t h treatments induced significant changes in amine metabolism in the cortex. In the mid-brain, foot shock caused an increase in tyrosine in the F S - C O : as well as the F S - N o C O : , group. N o n e of the other parameters was affected. CORTEX
20
MiD BRAIN
HIPPOCAMPUS
BRAIN STEM
TYROSINEI (~g/g } 30
no shock
no shock
shock
shock
no CO 2
CO 2
C02
no CO 2
treatment
FIG. l. Carbon dioxide induced amnesia for a passive avoidance response. The latency scores are divided into 3 classes: Class 1 indicates no avoidance; Class 2 incomplete avoidance; and Class 3 complete avoidance.
EXPERIMENT 2 C H A N G E S IN A M I N E M E T A B O L I S M
Method
F o u r groups of 20 rats were used. The e x p e r i m e n t was run in 5 r a n d o m i z e d blocks. Each b l o c k contained 16 rats, 4 of each group. T h e y were subjected to the pretraining and acquisition trials, as described above, and killed by decapitation 24 hr after the acquisition trial; they were n o t subjected to a retrieval trial before being killed. The brains were placed on ice and dissected within 3 rain into the cortex, mid-brain, brain stem and hippocampus. The olfactory bulbs, cerebellum and pineal gland were r e m o v e d . The brain stem was taken as the area posterior to the superior colliculi, i.e., colliculi, t e g m e n t u m , pons and medulla. The mid-brain comprised the striatum, h y p o t h a l a m u s , amygdala and septum. The h i p p o c a m p a l samples contained the area dentata and the subiculum in addition to the h i p p o c a m p u s proper dorsal to the rhinal sulcus. The h i p p o c a m p u s was studied separately, as a disturbance of its f u n c t i o n has been implicated in the causation of amnesia in man and animal [18,34]. After the dissection, the brain areas were frozen on solid carbon dioxide. Brain areas f r o m 4 rats were pooled for the biochemical determinations. The pooled brain areas were h o m o g e n i z e d in 12 ml of 0.01 N HC1 containing 1 ml of 10 percent sodium edate. A f t e r centrifugation at 800 G for 20 min, aliquots of the clear supernatant were r e m o v e d for the fluorimetric d e t e r m i n a t i o n of noradrenaline and d o p a m i n e [3,4], tyrosine [ 3 2 ] , normetanephrine by the m e t h o d of A n t o n and Sayre [5] as modified by Leonard and Tonge [ 1 5 ] , homovanillic acid [ 2 ] , t r y p t o p h a n [ 12] and gamma-amino-n-butyric acid (GABA) [30]. The pellet and the remainder of the supernatant fraction were extracted with b u t a n o l ; serotonin was determined by the m e t h o d of Snyder, et al. [26] and 5-hydroxyindoleacetic acid (5-HIAA) by the m e t h o d o f Giacolone and Valzelli as modified by Tonge and Leonard [29]. All fluorescence measurements were made using a Hitachi-Perkin
c
I~.g/g)
c02 co 2 s
c
co 2 c02
s
c c02 co 2
s
c
co2 c02
s
1
. . . . . . . . . . . . .
1~9/g ~ 300
200
0
FIG. 2. Effect of treatment with foot shock and/or CO 2 at the time of the acquisition trial on the concentration of tyrosine, tryptophan and gamma-aminobutyric acid (GABA) in different brain areas at the time of the retrieval trial. C: control group receiving neither foot shock nor CO2; CO2: group subjected to CO 2 treatment alone; S: group subjected to foot shock alone; S+CO2: group subjected to foot shock followed by CO 2 treatment.* The significance of the difference between the control group and the experimental groups shown by p
Changes in Brain Monoamine Metabolism and Carbon Dioxide Induced Amnesia in the Rat B. E. L E O N A R D A N D H. R I G T E R
Pharmacology Department, Scientific Development Group Organon, Oss, the Netherlands
(Received 16 O c t o b e r 1974) LEONARD, B. E. AND H. RIGTER. Changes in brain monoamine metabolism and carbon dioxide induced amnesia in the rat. PHARMAC. BIOCHEM. BEHAV. 3(5) 775-780, 1975. - The effect of treatment with carbon dioxide (CO2) on the performance of rats 24 hr after receiving a foot shock in a passive avoidance task was studied. Foot shock induced avoidance. Carbon dioxide produced retrograde amnesia for the foot shock induced avoidance response. Changes in brain monoamine metabolism were studied in groups of rats which had been treated with CO2, foot shock or foot shock + CO2. The rats were killed 24 hr after treatment. Changes mainly occurred in the brain stem and hippocampus. In the rats which had received foot shock alone, brain stem and hippocampal serotonin concentrations were raised. This rise was not observed when the foot shock was followed by CO 2 treatment. Furthermore, it was found that there was an increased release of noradrenaline in those rats subjected to foot shock alone but a decreased release of this amine in the group which received foot shock followed by CO2. It is suggested that the amnesic effect of CO2 parallels changes in brain serotonin and noradrenaline metabolism. Amnesia
Passive avoidance
Carbon dioxide
Hippocampus
THE m o s t c o m m o n paradigm for amnesia studies in rats involves (1) a training situation in w h i c h the animal acquires an aversive e x p e r i e n c e (f.e., a f o o t s h o c k ) , (2) followed by an amnesic t r e a t m e n t and (3) a retrieval test 24 hr later. Little is k n o w n a b o u t the role brain m o n o a m i n e s m a y play in amnesia. A n u m b e r o f investigators s t u d i e d the effects o f a f r e q u e n t l y used a m n e s i c agent, e l e c t r o c o n v u l sive shock, on brain m o n o a m i n e levels [8, 13, 14, 2 0 ] . However, these studies did n o t i n t e n d to correlate the changes in brain amine c o n c e n t r a t i o n s with amnesia. Thus, either the training e x p e r i e n c e was lacking [8, 13, 14] or the time interval was d i f f e r e n t f r o m the o n e used in amnesia studies [ 19,20]. We r e p o r t on the very first step in an investigation aimed at assessing possible associations b e t w e e n brain m o n o a m i n e m e t a b o l s i m and amnesia. C a r b o n dioxide (CO2), an effective amnesic agent [21, 22, 2 7 ] , was used t o p r o d u c e amnesia for a one-trial s t e p - t h r o u g h passive avoidance response. In E x p e r i m e n t 1, we d e m o n s t r a t e t h a t amnesia is p r e s e n t 24 hr a f t e r amnesic t r e a t m e n t . In E x p e r i m e n t 2, we f o l l o w e d the same paradigm but instead o f subjecting t h e animals t o a retrieval test, the rats were killed 24 h r a f t e r amnesic t r e a t m e n t and brain m o n o a m i n e levels were determined. In f u r t h e r studies [ 2 3 , 3 1 ] , we a t t e m p t to clarify the relationships b e t w e e n the changes in brain a m i n e c o n c e n t r a t i o n s and amnesia.
Noradrenaline
Serotonin
EXPERIMENT 1 ASSESSMENT OF CARBON DIOXIDE AS AN AMNESIC AGENT
Method Animals and Apparatus. F o r t y male rats o f an i n b r e d Wistar strain weighing 2 3 0 - 2 5 0 g were used. The animals were h o u s e d 10 per cage w i t h free access to f o o d and water. The animals were trained in a s t e p - t h r o u g h passive avoidance a p p a r a t u s o f the t y p e described b y Ader, et al. [ 1 ]. This consisted o f a 40 × 40 × 40 cm Lucite c h a m b e r w h i c h had a grid floor and black walls. The f r o n t wall was posit i o n e d at the edge o f a table and c o n n e c t e d to a 6 cm wide and 25 cm long r u n w a y w h i c h p r o j e c t e d over the floor. The r u n w a y was illuminated by m e a n s o f a 40 W lamp held 40 cm above it; the c h a m b e r was in darkness. When placed on the r u n w a y , a rat could e n t e r the d a r k e n e d c h a m b e r t h r o u g h a 6 × 6 cm o p e n i n g w h i c h could be closed b y means o f a h a n d o p e r a t e d guillotine d o o r . A s c r a m b l e d f o o t s h o c k (0.5 m A for 3 sec) could be delivered t h r o u g h the metal grid floor o f the c h a m b e r f r o m a 500 V AC source. Procedure. The rats were r a n d o m l y divided into 4 groups of 10 each. T h e y were given 3 pretraining trials on Day 1 o f the e x p e r i m e n t . A pretraining trial consisted o f placing the rat at the end o f the r u n w a y facing the e n t r a n c e o f the c h a m b e r ; the time t a k e n for the animal to e n t e r the cham775
776
LEONARD AND RIGTER
ber was r e c o r d e d a n d defined as t h e s t e p - t h r o u g h latency. U p o n e n t e r i n g t h e c h a m b e r , the d o o r was closed a n d t h e animal allowed to r e m a i n t h e r e for 10 sec. It was t h e n r e t u r n e d to its h o m e cage. T h e interval b e t w e e n t h e 3 pret r a i n i n g trials was a p p r o x i m a t e l y 2 hr. O n Day 2, t h e rats were s u b j e c t e d t o a single a c q u i s i t i o n trial. This was similar t o t h e p r e t r a i n i n g trials w i t h t h e e x c e p t i o n t h a t a f o o t s h o c k was given to 2 groups of animals 10 sec after t h e y had e n t e r e d t h e c h a m b e r (FS groups). T h e o t h e r 2 g r o u p s of rats did n o t receive f o o t s h o c k ( N o F S groups). I m m e d i ately on t e r m i n a t i n g the a c q u i s i t i o n trial, the rats were r e m o v e d f r o m the c h a m b e r a n d s u b j e c t e d to e i t h e r amnesic t r e a t m e n t (CO2 g r o u p s ) or s h a m amnesic t r e a t m e n t (NoCO2 groups). T h e amnesic t r e a t m e n t consisted in placing an animal in a closed c o n t a i n e r t h r o u g h w h i c h CO2 was passed u n t i l o x y g e n m e a s u r e m e n t s yielded zero readings [ 2 2 ] . The rats were r e m o v e d f r o m the b o x as s o o n as r e s p i r a t o r y arrest o c c u r r e d a n d were revived b y artificial r e s p i r a t i o n ; the time necessary for r e s p i r a t o r y arrest to o c c u r was 3 0 - - 3 5 sec. Rats receiving s h a m amnesic treatm e n t were placed in an air filled b o x for 35 sec. All animals were r e t u r n e d t o t h e i r h o m e cages at the e n d of t h e amnesic or sham a m n e s i c t r e a t m e n t . T h u s at t h e e n d of t h e acquisit i o n trial the 4 groups of rats h a d b e e n s u b j e c t e d to t h e following t r e a t m e n t s : NoFS-NoCO2 - n o f o o t shock, n o CO2 t r e a t m e n t ; NoFS-CO2 - n o f o o t s h o c k , b u t CO2 given; FS-CO2 foot s h o c k f o l l o w e d b y CO2 t r e a t m e n t ; FS-NoCOz f o o t s h o c k b u t n o CO2. T w e n t y - f o u r h o u r s after the a c q u i s i t i o n trial, t h e rats were s u b j e c t e d t o a single retrieval trial. T h e rats were placed at the e n d of the r u n w a y a n d t h e t i m e t a k e n for t h e m to e n t e r the d a r k e n e d c h a m b e r was r e c o r d e d . W h e n a rat failed to e n t e r w i t h i n 300 sec, it was r e m o v e d f r o m t h e r u n w a y a n d a l a t e n c y score o f 300 sec was assigned. The retrieval scores were divided i n t o 3 classes: (1) latencies of 0 - 1 0 sec; (2) latencies of 1 0 - 2 9 9 sec; (3) latencies o f 300 sec. Rats e n t e r i n g the c h a m b e r w i t h i n 10 sec were considered as n o t s h o w i n g passive a v o i d a n c e ; previous experim e n t s h a d s h o w n t h a t 10 sec r e p r e s e n t e d t h e m a x i m a l latency for rats w h i c h h a d n o t received a f o o t s h o c k at t h e
t i m e of the a c q u i s i t i o n trial. Rats e n t e r i n g t h e c h a m b e r w i t h i n 1 0 - 2 9 9 sec displayed i n c o m p l e t e passive avoidance while t h o s e t h a t failed to e n t e r w i t h i n 300 sec were regarded to show a c o m p l e t e passive a v o i d a n c e response. In the analysis of t h e results, the 3 classes received a weighting of 0, 1 a n d 2, respectively. T h e retrieval scores were analysed statistically using t h e Yates test [ 3 3 ] . F o r the analysis o f t h e o t h e r data the two-tailed M a n n W h i t n e y U test was e m p l o y e d , for c o m p a r i s o n s b e t w e e n groups, a n d t h e twotailed sign test, for c o m p a r i s o n s w i t h i n groups.
Results All groups of rats s h o w e d a r e d u c t i o n in their stept h r o u g h latencies during the course of t h e p r e t r a i n i n g trials. This was a p p a r e n t f r o m the significant differences b e t w e e n the latencies at t h e t i m e of t h e first p r e t r a i n i n g trial a n d those at t h e time of the acquisition trial (p = 0.001 for each group, sign test). T h e latencies at t h e t i m e of t h e acquisit i o n trial did n o t differ b e t w e e n t h e f o u r g r o u p s (Table 1). In the 2 N o F S groups, there was n o significant difference b e t w e e n the latencies at t h e retrieval a n d t h o s e at t h e acquisition trial ( p > 0 . 1 0 for b o t h groups). The latencies o f the N o F S groups at the test trial did n o t differ (Table 1). This suggests t h a t the CO2 t r e a t m e n t did n o t affect the stept h r o u g h response. T h e latencies of the four groups of animals at the time of the retrieval trial are s h o w n in Fig. 1. A d m i n i s t r a t i o n o f CO2 p r o d u c e d amnesia: the FS-CO2 group had significantly s h o r t e r latencies t h a n t h e FS-NoCO2 g r o u p (z = 4.13; p < 0 . 0 0 1 , Yates test). T h u s 8 o u t of 10 rats in the FS-CO2 g r o u p h a d a l a t e n c y similar to t h o s e o f the NoFS-NoCO2 group, while 9 o u t of 10 of the FS-NoCO2 g r o u p showed c o m p l e t e passive a v o i d a n c e a n d did n o t e n t e r t h e c h a m b e r . The latencies of FS-CO2 rats did n o t differ f r o m those of NoFS-NoCO2 or NoFS-CO2 rats (in b o t h cases: z = 1.49). On t h e o t h e r h a n d , the latencies of t h e FS-NoCO2 g r o u p were longer t h a n those of the N o F S groups (z = 4.37; p < 0 . 0 0 1 in b o t h cases).
TABLE 1 EFFECT OF PRETRAINING ON STEP-THROUGH LATENCIES OF GROUPS OF RATS
Trials Pretraining Group
t*
2*
3*
Acquisition*
Retrieval*
NoFS-NoCO~
9.3 ± 2.0
3.8 + 1.0
2.2 + 0.6
1.4 -+ 0.1
1.5 + 0.2
NoFS-CO 2
8.1 ± 1.7
3.9 ± 0.9
2.0 ± 0.4
1.1 + 0.3
1.6 ± 0.2
FS-CO 2
9.2 -+ 1.8
5.4 + 1.2
1.9 ± 0.4
1.5 ± 0.1
t
FS-NoCO 2
8.5 +- 1.5
5.2 + 1.2
1.3 ± 0.3
1.1 + 0.1
Step-through latencies (mean number of sec ± standard error of the mean) during pretraining trials and the acquisition trial. *Differences between groups within column are not significant (two-tailed Mann-Whitney U test). ~Latencies at the test trial are only given for groups which did not receive footshock at the acquisition trial. FS: footshock; NoFS: no footshock; CO:: COs-treatment; and NoCO2: no CO~-treatment.
MONOAMINES AND AMNESIA
777
m ®
100.
class 1
Elmer s p e c t r o p h o t o f l u o r i m e t e r Model 2A. The statistical significance of the results was assessed using a S t u d e n t ' s t-test.
80-
D
o~
class 2
60 ~5
m
40
class 3
Results
The results are given in Figs. 2, 3 and 4. Neither C O : , nor foot shock or the c o m b i n a t i o n of b o t h treatments induced significant changes in amine metabolism in the cortex. In the mid-brain, foot shock caused an increase in tyrosine in the F S - C O : as well as the F S - N o C O : , group. N o n e of the other parameters was affected. CORTEX
20
MiD BRAIN
HIPPOCAMPUS
BRAIN STEM
TYROSINEI (~g/g } 30
no shock
no shock
shock
shock
no CO 2
CO 2
C02
no CO 2
treatment
FIG. l. Carbon dioxide induced amnesia for a passive avoidance response. The latency scores are divided into 3 classes: Class 1 indicates no avoidance; Class 2 incomplete avoidance; and Class 3 complete avoidance.
EXPERIMENT 2 C H A N G E S IN A M I N E M E T A B O L I S M
Method
F o u r groups of 20 rats were used. The e x p e r i m e n t was run in 5 r a n d o m i z e d blocks. Each b l o c k contained 16 rats, 4 of each group. T h e y were subjected to the pretraining and acquisition trials, as described above, and killed by decapitation 24 hr after the acquisition trial; they were n o t subjected to a retrieval trial before being killed. The brains were placed on ice and dissected within 3 rain into the cortex, mid-brain, brain stem and hippocampus. The olfactory bulbs, cerebellum and pineal gland were r e m o v e d . The brain stem was taken as the area posterior to the superior colliculi, i.e., colliculi, t e g m e n t u m , pons and medulla. The mid-brain comprised the striatum, h y p o t h a l a m u s , amygdala and septum. The h i p p o c a m p a l samples contained the area dentata and the subiculum in addition to the h i p p o c a m p u s proper dorsal to the rhinal sulcus. The h i p p o c a m p u s was studied separately, as a disturbance of its f u n c t i o n has been implicated in the causation of amnesia in man and animal [18,34]. After the dissection, the brain areas were frozen on solid carbon dioxide. Brain areas f r o m 4 rats were pooled for the biochemical determinations. The pooled brain areas were h o m o g e n i z e d in 12 ml of 0.01 N HC1 containing 1 ml of 10 percent sodium edate. A f t e r centrifugation at 800 G for 20 min, aliquots of the clear supernatant were r e m o v e d for the fluorimetric d e t e r m i n a t i o n of noradrenaline and d o p a m i n e [3,4], tyrosine [ 3 2 ] , normetanephrine by the m e t h o d of A n t o n and Sayre [5] as modified by Leonard and Tonge [ 1 5 ] , homovanillic acid [ 2 ] , t r y p t o p h a n [ 12] and gamma-amino-n-butyric acid (GABA) [30]. The pellet and the remainder of the supernatant fraction were extracted with b u t a n o l ; serotonin was determined by the m e t h o d of Snyder, et al. [26] and 5-hydroxyindoleacetic acid (5-HIAA) by the m e t h o d o f Giacolone and Valzelli as modified by Tonge and Leonard [29]. All fluorescence measurements were made using a Hitachi-Perkin
c
I~.g/g)
c02 co 2 s
c
co 2 c02
s
c c02 co 2
s
c
co2 c02
s
1
. . . . . . . . . . . . .
1~9/g ~ 300
200
0
FIG. 2. Effect of treatment with foot shock and/or CO 2 at the time of the acquisition trial on the concentration of tyrosine, tryptophan and gamma-aminobutyric acid (GABA) in different brain areas at the time of the retrieval trial. C: control group receiving neither foot shock nor CO2; CO2: group subjected to CO 2 treatment alone; S: group subjected to foot shock alone; S+CO2: group subjected to foot shock followed by CO 2 treatment.* The significance of the difference between the control group and the experimental groups shown by p
