- Email: [email protected]
Two-way shuttlebox avoidance conditioning and brain NADH in rats
0031-9384/90 $3.00 + .OO
Physiology & Behavior, Vol. 48, pp. 515-517. e Pergamon F’ress plc, 1990. Printed in the U.S.A.
Two-Way Shuttlebox Avoidance Conditioning and Brain NADH in Rats ZHENG SHEN, GANG WANG AND SHU-ZHI LIN Department of Psychology, Peking University, Beijing 100871, P.R.C. Received 3 1 May 1989 MEN, Z., G. WANG AND S.-Z. LIN. Two-way shuttlebox avoidance conditioning and brain NALIH in rats. PHYSIOL BEHAV 4&I(4) 515-517, 1990.-The effects of amphetamine (1.5 mgkg) and caffeine (120 mg/kg, 75 mgikg) on shuttle behavior and on the concentrations of reduced nicotinamide adenine dinucleotide (NADH) of the brain were studied in 56 rats from the perspective of regional brain metabolism. Amphetamine potentiated the shuttle behavior including avoidance responses and response speed, while it increased NADH concentrations in the hippocampus. The low dose of caffeine increased avoidance responses. The high dose of caffeine inhibited avoidance responses, but it had no effect on NADH concentrations in discrete brain regions. Amphetamine
Caffeine
Shuttlebox
Avoidance conditioning
IT is well known that amphetamine is a psychomotor stimulant. Positron emission tomography has already revealed some new facts about the relationship between psychological processes and regional brain metabolism of glucose (3, 7, 8). We are trying to investigate the effect of amphetamine and caffeine on learning behavior from the perspective of regional brain glucose metabolism. It has been found that increased NADH in the brain of rats may be caused by increased jumping (9). Amphetamine strongly potentiated induced jumping behavior while it increased NADH concentrations in discrete brain regions. Caffeine increased NADH concentrations in discrete brain regions while it potentiated jumping during the first 3 minutes, but the effects of caffeine quickly disappeared (10). Amphetamine has been shown to increase two-way avoidance in the shuttlebox (2). Caffeine increased mouse activity (6). In the present experiment, we compare effects of amphetamine and caffeine on the conditioned avoidance responses of rats. Furthermore, we inquire into the relationship between the regional brain NADH and shuttle behavior in the rats.
Brain NADH
Hippocampus
each placed a short distance into a compartment from the doorway. The microcomputer detected the shuttle behavior so long as the rats passed through two beams. Both stimulation and response data
were recorded by an Apple II. Fifteen min following administration of the drug, the rats in the first three groups were put in the box for 25 min. The behavioral test was separated into two phases of 25 trials each, with an interphase interval of 5 min. The stimulus sequence of each trial is shown in Fig. 1. After a preparatory period of 10 set from the start of the trial, a light stimulus lasted 3 set, 2 set after the end of the light stimulus, an audio stimulus lasted 3 sec. Two seconds after the end of the audio stimulus, the electric current with 3-set duration was applied to the floor grid (foot shock) as the unconditioned stimulus. The electroshock was a series of electropulses (25 V, 50 Hz) including both sinusoid and rectangular waves. When the rat moved to the other part of the box in response to the light or audio stimulus before the application of the electric shock, a correct conditioned avoidance response was recorded. If the rat moved during the electroshock, it was regarded as an escape response. If the rat did not pass through the two beams during the trial, it was recorded as no response. Following the behavioral procedures, rats were quickly decapitated and their brains dissected. According to the method previously described (9,10), the brain was divided into six areas: cortex, hippocampus, caudate nucleus, diencephalon, cerebellum and brain stem; NADH concentrations of discrete brain structures were analyzed. The last group of rats was a normal control group decapitated and analyzed as the other groups except without the drug and behavior procedures.
METHOD Fifty-six rats weighing 200 + 25 g (SD) were divided randomly into 7 groups of eight. Saline (0.9%, 3 mvkg), amphetamine (d-amphetamine sulfate, Sigma, London, 1.5 mg/kg), and caffeine (Tianjin Med Corp., 120 mg/kg) were administered to the Fit three groups. According to previous findings, a dose of 120 mgikg caffeine provoked maximum jumping behavior. In the first experiment of the present study 120 mg/kg caffeine had no effect on shuttle behavior, so a second experiment was performed with this and a lower (75 mg/kg) dose. An automated system was used to evaluate the effects of the drugs on shuttle behavior. This system consisted of a personal computer (Apple II), a shuttlebox and its interface. The shuttlebox was divided into two equal parts (25 X 23 x23 cm). A wall separated the two parts and permitted the rats to go through an arch 11 cm in height and 8 cm in width. Two infrared beams were used,
RESULTS The behavioral results are shown in Table 1. The animals did not make an escape or avoidance response on the majority of trials, except the amphetamine group. The avoidance number for the rats injected with amphetamine (1.5 mg/kg) was greater than that for
515
516
SHEN, WANG AND LIN
-
./’
L i g h t timing(sec)
10
,/,’ _____---
3 & 8.
Amphetamine Caffeine Saline
..
Audio timing(sec)
15
** .’
[ Shock timing(sec)
P2 ESCAPE
1. The stimulus sequence of each trial.
the rats injected with saline. The increased number of avoidance responses of the amphetamine group was not accompanied by a decrease in escape responses. The average response time of the amphetamine group was shorter than that of the saline group. Both differences reached significant levels (pcO.05). Even though there were some differences in escape number, avoidance number, and average response time between the saline and caffeine group (120 mg/kg), the differences were not significant. The results show that the effects of amphetamine on shuttle behavior in rats are greater than those of caffeine. We compared the performance of the rats in phase 1 versus 2, and the results are shown in Fig. 2. The numbers of escape and avoidance responses in the second phase were greater than those in the first phase for amphetamine and saline but only the differences of the saline group were significant (p
.x’
/
0 L
Pl FIG.
.’
.’
Pl p2 AVOIDANCE
FIG. 2. Shuttle behavior; 8 rats/group. Amphetamine 1.5 mgikg, . -. - ; caffeine 120 mg/kg, ---; saline 0.9%. -. P,, phase 1; P,, phase 2. The differences between phase 1 and phase 2 were tested by the paired sample t-test, *p
The results of biochemical analysis of discrete brain structures are shown in Fig. 3. The NADH concentrations of the amphetamine group were the highest in the cortex, hippocampus, caudate nucleus and cerebellum; only the difference between amphetamine and saline groups in NADH concentration of the hippocampus was significant @<0.05). Caffeine (120 mg/kg) had no effects on concentrations of NADH in discrete brain structures (even in the brain stem the difference was not significant). There were no differences in NADH concentration between the normal group and the saline group. DISCUSSION
Others have found that a 1.5 mg/kg dose of amphetamine elicited the maximum locomotor response (5). The same dose of amphetamine increased rat’s jumping (9). In this experiment, the same dose of amphetamine increased avoidance responding and response speed. Although the models of animal behavior are different, amphetamine seems to have a similar effect on the locomotor, induced jump and conditioned avoidance response. According to other findings, amphetamine increased the number of avoidance responses of rats in the shuttlebox. But the “learning” was not genuinely enhanced by amphetamine, because the avoidance improvement disappeared when the drug was abruptly or gradually discontinued (2). In this experiment, am-
TABLE 1
TABLE 2
THE SHU’ITLE NUMBERS AND RESPONSE TIME IN THE DIFFERENT TREATMENTS (MEAN + SD.)
THE SHUTTLE NUMBERS AND RESPONSE TIME FOR THE DIFFERENT CAFFEINE DOSES (MEAN f SD.)
Item Escape numbers Avoidance numbers Response time
Saline n=8
Caffeine n= 8 (120 mgkg)
Amphetamine n=8 (1.5 mg/kg)
12.25 ” 11.03
16.38 k 7.93
21.25 r 4.11
2.88 t 2.30
1.63 k 2.45
11.63 t 9.21*
12.11 + 0.55
12.25 f 0.54
11.26 ? 0.85*
*p
Item Escape numbers Avoidance numbers Response time (set)
Saline n=8
Low Dose n = 8 (75 mg/kg)
High Dose n=8 (120 mgkg)
18.00 + 9.70
18.00 2 6.00
14.40 k 9.21
2.58 2 2.00 12.03 k 0.43
6.25 k 5.83** 11.73 -r- 0.68
0.20 k 0.45* 12.55 t 0.31
*p
517
SHUTTLE BEHAVIOR AND NADH Normal
20
group
Amphetamine
Caffeine
amphetamine group, although the saline group does show an
FIG. 3. NADH concentrations in 6 brain structures; 8 rats/group. Fr, frontal cortex; Hi, hippocampus; Ca, caudate nucleus; Ce, cerebellum; St, brain stem; Di, diencephalon. Only the NADH level in the hippocampus of the amphetamine-treated group differed from the controls, *p
increase in avoidance responding. On the other hand, the amphetamine group performs avoidance responses at a higher rate in the first phase. It is difficult to assess effects of amphetamine on learning process in the experiment. The hippocampus may play an important role in learning. Bilateral temporal lobectomy (including the hippocampus) produces anterograde amnesia in humans (1). In rats, the protein content of the hippocampus increases during a learning task (4). In our experiment, the delayed conditioning procedure. raised the NADH concentration in the hippocampus in the amphetamine group. It is possible that amphetamine increased the level of NADH in the majority of brain structures, and especially in the hippocampus, and improved the utilization of glucose at the same time. There is a linear increase in activity with caffeine doses ranging from 25 to 150 mg/kg in mice (6). In a previous experiment, caffeine (120 mg/kg) caused a transient change in jumps and brain NADH. It potentiated jumps and increased brain NADH for a short period (3 min), but the effects of caffeine quickly disappeared (10). In this experiment, the low dose of caffeine facilitated the leaming performance, increasing the number of the avoidance responses; the high dose of caffeine prevented learning, while it did not obviously increase NADH concentrations in discrete brain regions. Possibly the caffeine raised NADH levels but then they returned to normal by the time they were measured after the behavioral test.
phetamine increased the response speed and the avoidance. It seems to have a facilitating action on the learning process but there are contrary facts in the experiment. There are no significant differences between the first phase and the second phase of the
Supported by the National Natural Foundation of China. We thank Editor-in-Chief M. J. Wayner for his helpful advice and revising the early drafts of this manuscript.
18
2 0
I
Fr
Hi
St
Di
BRAIN STFlUCTURE
ACKNOWLEDGEMENTS
REFERENCES 1. Carlson, N. R. Physiology of behavior. 3rd ed. Boston: Allyn and Bacon Inc.; 1986:54-57. 2. Driscoll, P. Roman high-avoidance and low-avoidance rats: Present status of the Swiss sublines, RAH/verh and RLA/verh and effects of amphetamine on shuttlebox performance. Behav. Genet. 16:355-364; 1986. Frackowiak, R. S.; Pozzilli, J. C.; Legg, N. J. Regional cerebral oxygen supply utilization in dementia. Brain 104:753-778; 1981. Hyden, H.; Ronannbark, L. Distribution of S-100 and 14-3-2 proteins of neuronal cell membranes. J. Neurol. Sci. 39:241-252; 1978. Iversen, S. D.; Iversen, L. L. Behavioral pharmacology. New York: Oxford University Press; 1981: 154. Knoll, J. Motimeter a new sensitive apparatus for the quantitative
7.
8. 9. 10.
measurement of hypermobility caused by psychostimulants. Arch. Int. Pharmacodyn. Ther. 130:141-147; 1961. Mazziotta, J. C.; Phelps, M. E.; Kuhl, D. E.; Packwood, J. Tomographic mapping of human cerebral metabolism: auditory stimulation. Neurology 32:921-937; 1982. Phelps, M. E. Positron computed tomography studies of cerebral glucose metabolism in nuclear medicine. Semin. Nucl. Med. 11: 32-49; 1981. Shen, 2.; Lin, S. 2. Brain NADH and jumping behavior in rat. Life Sci. 37:731-738; 1985. Shen, Z.; Lin, S. Z. Effects of amphetamine and caffeine on jumping behavior and brain NADH in rats. Chung Kuo Yao Li Hsueh bao 8:97-100; 1987.
Physiology & Behavior, Vol. 48, pp. 515-517. e Pergamon F’ress plc, 1990. Printed in the U.S.A.
Two-Way Shuttlebox Avoidance Conditioning and Brain NADH in Rats ZHENG SHEN, GANG WANG AND SHU-ZHI LIN Department of Psychology, Peking University, Beijing 100871, P.R.C. Received 3 1 May 1989 MEN, Z., G. WANG AND S.-Z. LIN. Two-way shuttlebox avoidance conditioning and brain NALIH in rats. PHYSIOL BEHAV 4&I(4) 515-517, 1990.-The effects of amphetamine (1.5 mgkg) and caffeine (120 mg/kg, 75 mgikg) on shuttle behavior and on the concentrations of reduced nicotinamide adenine dinucleotide (NADH) of the brain were studied in 56 rats from the perspective of regional brain metabolism. Amphetamine potentiated the shuttle behavior including avoidance responses and response speed, while it increased NADH concentrations in the hippocampus. The low dose of caffeine increased avoidance responses. The high dose of caffeine inhibited avoidance responses, but it had no effect on NADH concentrations in discrete brain regions. Amphetamine
Caffeine
Shuttlebox
Avoidance conditioning
IT is well known that amphetamine is a psychomotor stimulant. Positron emission tomography has already revealed some new facts about the relationship between psychological processes and regional brain metabolism of glucose (3, 7, 8). We are trying to investigate the effect of amphetamine and caffeine on learning behavior from the perspective of regional brain glucose metabolism. It has been found that increased NADH in the brain of rats may be caused by increased jumping (9). Amphetamine strongly potentiated induced jumping behavior while it increased NADH concentrations in discrete brain regions. Caffeine increased NADH concentrations in discrete brain regions while it potentiated jumping during the first 3 minutes, but the effects of caffeine quickly disappeared (10). Amphetamine has been shown to increase two-way avoidance in the shuttlebox (2). Caffeine increased mouse activity (6). In the present experiment, we compare effects of amphetamine and caffeine on the conditioned avoidance responses of rats. Furthermore, we inquire into the relationship between the regional brain NADH and shuttle behavior in the rats.
Brain NADH
Hippocampus
each placed a short distance into a compartment from the doorway. The microcomputer detected the shuttle behavior so long as the rats passed through two beams. Both stimulation and response data
were recorded by an Apple II. Fifteen min following administration of the drug, the rats in the first three groups were put in the box for 25 min. The behavioral test was separated into two phases of 25 trials each, with an interphase interval of 5 min. The stimulus sequence of each trial is shown in Fig. 1. After a preparatory period of 10 set from the start of the trial, a light stimulus lasted 3 set, 2 set after the end of the light stimulus, an audio stimulus lasted 3 sec. Two seconds after the end of the audio stimulus, the electric current with 3-set duration was applied to the floor grid (foot shock) as the unconditioned stimulus. The electroshock was a series of electropulses (25 V, 50 Hz) including both sinusoid and rectangular waves. When the rat moved to the other part of the box in response to the light or audio stimulus before the application of the electric shock, a correct conditioned avoidance response was recorded. If the rat moved during the electroshock, it was regarded as an escape response. If the rat did not pass through the two beams during the trial, it was recorded as no response. Following the behavioral procedures, rats were quickly decapitated and their brains dissected. According to the method previously described (9,10), the brain was divided into six areas: cortex, hippocampus, caudate nucleus, diencephalon, cerebellum and brain stem; NADH concentrations of discrete brain structures were analyzed. The last group of rats was a normal control group decapitated and analyzed as the other groups except without the drug and behavior procedures.
METHOD Fifty-six rats weighing 200 + 25 g (SD) were divided randomly into 7 groups of eight. Saline (0.9%, 3 mvkg), amphetamine (d-amphetamine sulfate, Sigma, London, 1.5 mg/kg), and caffeine (Tianjin Med Corp., 120 mg/kg) were administered to the Fit three groups. According to previous findings, a dose of 120 mgikg caffeine provoked maximum jumping behavior. In the first experiment of the present study 120 mg/kg caffeine had no effect on shuttle behavior, so a second experiment was performed with this and a lower (75 mg/kg) dose. An automated system was used to evaluate the effects of the drugs on shuttle behavior. This system consisted of a personal computer (Apple II), a shuttlebox and its interface. The shuttlebox was divided into two equal parts (25 X 23 x23 cm). A wall separated the two parts and permitted the rats to go through an arch 11 cm in height and 8 cm in width. Two infrared beams were used,
RESULTS The behavioral results are shown in Table 1. The animals did not make an escape or avoidance response on the majority of trials, except the amphetamine group. The avoidance number for the rats injected with amphetamine (1.5 mg/kg) was greater than that for
515
516
SHEN, WANG AND LIN
-
./’
L i g h t timing(sec)
10
,/,’ _____---
3 & 8.
Amphetamine Caffeine Saline
..
Audio timing(sec)
15
** .’
[ Shock timing(sec)
P2 ESCAPE
1. The stimulus sequence of each trial.
the rats injected with saline. The increased number of avoidance responses of the amphetamine group was not accompanied by a decrease in escape responses. The average response time of the amphetamine group was shorter than that of the saline group. Both differences reached significant levels (pcO.05). Even though there were some differences in escape number, avoidance number, and average response time between the saline and caffeine group (120 mg/kg), the differences were not significant. The results show that the effects of amphetamine on shuttle behavior in rats are greater than those of caffeine. We compared the performance of the rats in phase 1 versus 2, and the results are shown in Fig. 2. The numbers of escape and avoidance responses in the second phase were greater than those in the first phase for amphetamine and saline but only the differences of the saline group were significant (p
.x’
/
0 L
Pl FIG.
.’
.’
Pl p2 AVOIDANCE
FIG. 2. Shuttle behavior; 8 rats/group. Amphetamine 1.5 mgikg, . -. - ; caffeine 120 mg/kg, ---; saline 0.9%. -. P,, phase 1; P,, phase 2. The differences between phase 1 and phase 2 were tested by the paired sample t-test, *p
The results of biochemical analysis of discrete brain structures are shown in Fig. 3. The NADH concentrations of the amphetamine group were the highest in the cortex, hippocampus, caudate nucleus and cerebellum; only the difference between amphetamine and saline groups in NADH concentration of the hippocampus was significant @<0.05). Caffeine (120 mg/kg) had no effects on concentrations of NADH in discrete brain structures (even in the brain stem the difference was not significant). There were no differences in NADH concentration between the normal group and the saline group. DISCUSSION
Others have found that a 1.5 mg/kg dose of amphetamine elicited the maximum locomotor response (5). The same dose of amphetamine increased rat’s jumping (9). In this experiment, the same dose of amphetamine increased avoidance responding and response speed. Although the models of animal behavior are different, amphetamine seems to have a similar effect on the locomotor, induced jump and conditioned avoidance response. According to other findings, amphetamine increased the number of avoidance responses of rats in the shuttlebox. But the “learning” was not genuinely enhanced by amphetamine, because the avoidance improvement disappeared when the drug was abruptly or gradually discontinued (2). In this experiment, am-
TABLE 1
TABLE 2
THE SHU’ITLE NUMBERS AND RESPONSE TIME IN THE DIFFERENT TREATMENTS (MEAN + SD.)
THE SHUTTLE NUMBERS AND RESPONSE TIME FOR THE DIFFERENT CAFFEINE DOSES (MEAN f SD.)
Item Escape numbers Avoidance numbers Response time
Saline n=8
Caffeine n= 8 (120 mgkg)
Amphetamine n=8 (1.5 mg/kg)
12.25 ” 11.03
16.38 k 7.93
21.25 r 4.11
2.88 t 2.30
1.63 k 2.45
11.63 t 9.21*
12.11 + 0.55
12.25 f 0.54
11.26 ? 0.85*
*p
Item Escape numbers Avoidance numbers Response time (set)
Saline n=8
Low Dose n = 8 (75 mg/kg)
High Dose n=8 (120 mgkg)
18.00 + 9.70
18.00 2 6.00
14.40 k 9.21
2.58 2 2.00 12.03 k 0.43
6.25 k 5.83** 11.73 -r- 0.68
0.20 k 0.45* 12.55 t 0.31
*p
517
SHUTTLE BEHAVIOR AND NADH Normal
20
group
Amphetamine
Caffeine
amphetamine group, although the saline group does show an
FIG. 3. NADH concentrations in 6 brain structures; 8 rats/group. Fr, frontal cortex; Hi, hippocampus; Ca, caudate nucleus; Ce, cerebellum; St, brain stem; Di, diencephalon. Only the NADH level in the hippocampus of the amphetamine-treated group differed from the controls, *p
increase in avoidance responding. On the other hand, the amphetamine group performs avoidance responses at a higher rate in the first phase. It is difficult to assess effects of amphetamine on learning process in the experiment. The hippocampus may play an important role in learning. Bilateral temporal lobectomy (including the hippocampus) produces anterograde amnesia in humans (1). In rats, the protein content of the hippocampus increases during a learning task (4). In our experiment, the delayed conditioning procedure. raised the NADH concentration in the hippocampus in the amphetamine group. It is possible that amphetamine increased the level of NADH in the majority of brain structures, and especially in the hippocampus, and improved the utilization of glucose at the same time. There is a linear increase in activity with caffeine doses ranging from 25 to 150 mg/kg in mice (6). In a previous experiment, caffeine (120 mg/kg) caused a transient change in jumps and brain NADH. It potentiated jumps and increased brain NADH for a short period (3 min), but the effects of caffeine quickly disappeared (10). In this experiment, the low dose of caffeine facilitated the leaming performance, increasing the number of the avoidance responses; the high dose of caffeine prevented learning, while it did not obviously increase NADH concentrations in discrete brain regions. Possibly the caffeine raised NADH levels but then they returned to normal by the time they were measured after the behavioral test.
phetamine increased the response speed and the avoidance. It seems to have a facilitating action on the learning process but there are contrary facts in the experiment. There are no significant differences between the first phase and the second phase of the
Supported by the National Natural Foundation of China. We thank Editor-in-Chief M. J. Wayner for his helpful advice and revising the early drafts of this manuscript.
18
2 0
I
Fr
Hi
St
Di
BRAIN STFlUCTURE
ACKNOWLEDGEMENTS
REFERENCES 1. Carlson, N. R. Physiology of behavior. 3rd ed. Boston: Allyn and Bacon Inc.; 1986:54-57. 2. Driscoll, P. Roman high-avoidance and low-avoidance rats: Present status of the Swiss sublines, RAH/verh and RLA/verh and effects of amphetamine on shuttlebox performance. Behav. Genet. 16:355-364; 1986. Frackowiak, R. S.; Pozzilli, J. C.; Legg, N. J. Regional cerebral oxygen supply utilization in dementia. Brain 104:753-778; 1981. Hyden, H.; Ronannbark, L. Distribution of S-100 and 14-3-2 proteins of neuronal cell membranes. J. Neurol. Sci. 39:241-252; 1978. Iversen, S. D.; Iversen, L. L. Behavioral pharmacology. New York: Oxford University Press; 1981: 154. Knoll, J. Motimeter a new sensitive apparatus for the quantitative
7.
8. 9. 10.
measurement of hypermobility caused by psychostimulants. Arch. Int. Pharmacodyn. Ther. 130:141-147; 1961. Mazziotta, J. C.; Phelps, M. E.; Kuhl, D. E.; Packwood, J. Tomographic mapping of human cerebral metabolism: auditory stimulation. Neurology 32:921-937; 1982. Phelps, M. E. Positron computed tomography studies of cerebral glucose metabolism in nuclear medicine. Semin. Nucl. Med. 11: 32-49; 1981. Shen, 2.; Lin, S. 2. Brain NADH and jumping behavior in rat. Life Sci. 37:731-738; 1985. Shen, Z.; Lin, S. Z. Effects of amphetamine and caffeine on jumping behavior and brain NADH in rats. Chung Kuo Yao Li Hsueh bao 8:97-100; 1987.