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Changes in brain biogenic amines in cats performing a symmetrically reinforced go-nogo visual discrimination task
BEHAVIORAL AND NEURAL BIOLOGY 32, 133-147 (1981)
Changes in Brain Biogenic Amines in Cats Performing a Symmetrically Reinforced Go-Nogo Visual Discrimination Task A N N E KITSIKIS AND ANDRI~E G . ROBERGE i
Dkparternents de Physiologie et de Biochimie, Facultk de Mbdecine, Universit~ Laval, Quebec G1K 7P4, Canada Cats were trained on a symmetrically reinforced (SR) go-nogo visual discrimination task and sacrificed for biochemical assays on Day 5 of training, after reaching performance criterion or after a 6-day postcriterion period. Serotonin (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), and noradrenaline (NA) were measured in 13 brain structures. Dopamine (DA) was evaluated in the neostriatum and amygdala while tryptophan hydroxylase activity was measured in the brain stem raphe nuclei. A significant increase in 5-HT, 5-HIAA, and NA were observed in several brain structures on Day 5 and after cats reached performance criterion compared to untrained controls. Tryptophan hydroxylase activity decreased in the raphe nuclei in cats performing at criterion level and DA decreased in the amygdala and neostriatum. Fewer changes were observed on Day 5 than at performance criterion and several changes persisted after a 6-day postcriterion period without training. 5-HT and NA increases are discussed in terms of response suppression and reward behavior, respectively, and DA changes are related to motor organization on nogo trials. It is suggested that the biochemical changes observed are the result of training on the SR go-nogo task.
This work is part of an ongoing research project aimed at a better understanding of the role of biogenic amines in learning. Our main goals were first of all to determine how the acquisition of a behavioral task modifies the synthesis of serotonin (5-HT), noradrenaline (NA), and dopamine (DA) in the cat's brain and where these changes take place. Second, we wanted to evaluate biochemical changes at different stages of learning. The third goal was to investigate whether the biochemical changes observed and their anatomical localization vary as a function of the type of behavioral task cats are required to perform. The aim of the present research was to investigate whether the localized biochemical changes measured during or after the acquisition of a symmetrically i The authors gratefully acknowledge Gilles Huard's helpful technical assistance and Claude Roberge for the enzyme measurements. This research was supported by the Medical Research Council of Canada (MA-6590). 133 0163-1047/81/060133-15502.00/0 Copyright© 1981by AcademicPress, Inc. All rightsof reproductionin any formreserved.
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reinforced (SR) go-nogo visual discrimination task were different to those previously observed in cats performing an asymmetrically reinforced (AR) go-nogo task. A great deal of research has already been done at the Nencki Institute to identify the neuroanatomical substrates and behavioral differences of AR and SR go-nogo tasks in dogs (Dabrowska, 1972; Kowalska & Zielinski, 1976). We therefore know that the presence or absence of reinforcement on correct nogo trials changes the nature of a go-nogo task. The results presented here permit us to further differentiate AR and SR go-nogo tasks on a biochemical basis.
METHODS
Subjects. Twenty-six adult, male and female mongrel cats, weighing between 2.5 and 4 kg were used in these experiments and maintained at their initial weight _ 10%. They were housed in individual cages in a room with background music, constant temperature, and humidity. Their cages were cleaned by the same person throughout the training period. Cats were fed immediately after each training session or by 1000 hr in the morning in the case of untrained cats. They were maintained on a stable diet of cat food with water ad libitum. Sixteen animals were trained on a symmetrically reinforced go-nogo visual discrimination task and 10 animals served as untrained controls. One group of 6 trained and 4 untrained cats was sacrificed for biochemical assays on Day 5 of training. Another equivalent group was sacrificed on the third day of a postcriterion consolidation period and a third group of 4 trained and 2 untrained cats was killed 6 days (without training) after reaching criterion performance. Apparatus. The experimental setup consisted of a wooden box equipped with a one-way screen in front of which was placed a mobile tray with a centrally situated food well covered by a wooden block. A black cover represented the positive stimulus (S ÷) and a white cover the negative stimulus (S-). Training procedure. All animals underwent a 2-day shaping period during which they became habituated to the experimental box and learned to uncover the food well for reinforcement. During shaping, a naturalcolored wooden block was used and animals did not perform more than four complete trials before the onset of training. Training started the next day and continued for 5 days (group 1) or until a criterion performance of 90% correct responses on 2 consecutive days was reached. Learning was consolidated on the 3 following days before the second group of animals was sacrificed. Group 3 was killed 6 days after reaching criterion performance. They did not perform the task during the 6-day postcriterion period. Training was carried out in the morning between 0800 and 1200 hr and cats were trained in a different order each day so as to avoid interference of the circadian cycle in the training schedule. Fifty trials (25 go and
CHANGES IN BRAIN AMINES DURING LEARNING
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25 nogo) were given in a randomized order 5 days a week. On each trial the undirectional screen was lifted, exposing either a black (S÷) or a white (S-) wooden block covering the food well. During go trials animals were required to remove S+ within 5 sec in order to retrieve a small piece of meat. During nogo trials animals had to refrain from this response for 7 sec before being rewarded with a piece of meat by the experimenter. Intertrial intervals lasted 10 sec. Performance was measured by calculating response errors and response latencies of go and nogo trials separately. Biochemical assays. All animals were trained and sacrificed during the winter months in order to avoid seasonal biochemical variations. Cats were guillotined without anesthesia immediately after the last training session which always took place between 0800 and 1030 hr so as to avoid circadian fluctuations. One or two untrained controls were always sacrificed on the same morning as trained animals. The brain was rapidly removed and the prefontal cortex, neostriatum, septum, hypothalamus, thalamus, hippocampus, amygdala, mesencephalon, pons, medulla, and the respective raphe nuclei of the mesencephalon, pons, and medulla were dissected on ice, frozen on dry ice, and kept at -80°C until biochemical assays were performed. Amines measurement. Noradrenaline (NA), dopamine (DA), serotonin (5-HT), and 5-hydroxyindoleacetic acid (5-HIAA) were separated according to the method of Earley and Leonard (1978). DA and NA were oxidized according to Welch and Welch (1969), whereas the native fluorescence of 5-HT and 5-HIAA was read at 305 nm activation-535 nm emission in concentrated HCL containing 10% ascorbic acid, using an Aminco-Bowman spectrophotofluorimeter (American Instruments Co. Inc., Silver Springs, Md.). Recoveries of DA, NA, 5-HT, and 5-HIAA were 90 _+ 5, 117 + 6, 63 _+ 8, and 95 _+ 4%, respectively. All estimates were corrected for losses. Results are expressed in micrograms of amines or acid per gram of fresh tissue. 5-HT metabolism was studied more extensively, as former experiments had pointed to its involvement in the acquisition of specific behavioral tasks. However, were it not for material limitations NA and DA metabolism would also have been measured more completely. Tryptophan hydroxylase assay. Tryptophan hydroxylase activity was assayed using an isotopic modification (manuscript in preparation) of the technique described by Roberge and Poirier (1973). L-[Methylene-~4C] tryptophan and 5-[2-'4C]hydroxytryptamine binoxalate (5-HT) were used as labeled substrate and standard, respectively. The labeled 5-HT formed was measured with a Beckman LS-350 liquid scintillation counter (Beckman Instruments Inc., Irvine, Calif.). Recovery of 5-HT was 95 _+ 1%. Estimates were corrected for losses and results are expressed in picamoles of 5-HT formed per gram per hour.
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Protein determination. Total protein concentrations were determined according to the method of Bradford (1976), using bovine y-globulin as standard. Statistical analysis. Standard error of the mean and Student's t test (two-tailed) were calculated according to Lison (1958). Chemicals. Noradrenaline (DL-Arterenol, HCL), 5-hydroxyindole-3acetic acid, cyclohexyl-ammonium salt (5-HIAA), and DL-6-methyl5,6,7,8-tetrahydropterine dihydrochloride were purchased from Calbiochem, Los Angeles, Calif. Serotonin (5-hydroxytryptamine, HCL), iproniazid phosphate, dithiothreitol and Sephadex G-10 were obtained from Sigma Chemical Company, St. Louis, Missouri. Amberlite CG-50 type 11 200 mesh was obtained from BDH Chemicals, Ltd, Montreal. L-[Methylene-a4]tryptophan was purchased from Amersham Corporation, Oakville, Ontario, Canada. Columns (glass barrel ECONO-Columns, 0.7 × 4.0 cm i.d.) and bovine y-globulin were obtained from Bio-Rad Laboratories Ltd, Missisauga, Ontario, Canada. RESULTS
Behavioral Data Number o f errors. The learning curves of cats in groups 1, 2, and 3 are shown in Fig. 1. In Fig. 1A, the mean go and nogo error curves of six cats sacrificed on Day 5 are shown. Although cats made significantly (p < .001) more errors in nogo than in go trials during the whole 5-day period, on a daily basis, the difference was significant on the first 2 days of training only. In groups 2 and 3, three cats took much longer to reach criterion performance than the other cats. On account of these divergent results, the error curves of these cats were drawn separately and presented in parallel to the mean go and nogo error curves of the remaining cats in each group (Figs. 1B and C). The number of go and nogo errors were calculated for Days 1 to 5, Day 6 to criterion performance, and for the whole training period. In both groups, cats made significantly (p < .001) more nogo than go errors whatever the period. Moreover, nogo errors were significantly (p < .02, group 2; p < .001, group 3) more frequent in the first 5 days of training than between Day 6 and criterion performance. No difference was found for go errors. However the difference between go and nogo errors was not significant in all daily scores (Figs. IB and C). The performance of group 1 cats sacrificed on Day 5 did not differ from that of cats in group 2 and 3 during the first 5 days of training. The cats in group 2 reached criterion performance between Days 8 and 10 (mean 9.5), while the three cats in group 3 took between 13 and 15 days (mean 13.6). Of the three cats that took 21 to 22 days to reach criterion performance, two (one in group 2, one in group 3) were hyperactive.
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FIG. 1. (A) M e a n go and nogo errors _+SEM in daily 50-trial (25 go and 25 nogo) sessions in six cats trained for 5 days on a symmetrically reinforced (SR) g o - n o g o visual discrimination task. (B) Learning c u r v e s of cats trained to criterion p e r f o r m a n c e on a SR g o - n o g o visual discrimination task. M e a n go and nogo errors + S E M in daily 50-trial (25 go and 25 nogo) sessions are represented for four cats sacrificed at the end of a 3-day postcriterion consolidation period. Nogo error c u r v e s o f two cats of the same group that took m u c h longer to learn the task are presented. The few go errors made are not represented in this figure for t h e s e two cats. (C) M e a n go and nogo errors _+SEM of cats trained to criterion p e r f o r m a n c e and sacrificed 6 days (without training) after the 3-day consolidation period. Go and nogo errors of three cats. Nogo errors of one cat. *p < .05; **p < .01; ***p < .001.
Response latencies. R e s p o n s e latencies were calculated by measuring the interval that elapsed b e t w e e n the time the screen w a s lifted and the m o m e n t the cat's p a w touched the w o o d e n block covering the food well. Latencies were recorded by the experimenter, in seconds. R e s p o n s e latencies on correct go trials and incorrect nogo trials were compiled
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separately and the values obtained were expressed as the average percentage of responses at a given latency. When comparing go and nogo latencies, no significant difference was found for groups 2 and 3. If go trials are considered separately, short (< 1 sec) latencies were found to be significantly (p < .02) more frequent than 1 to 5 sec latencies in groups 2 and 3. No significant difference was found between long and short latencies in nogo trials for the same groups. In group 1, nogo latencies were longer (p < .05) than go latencies and significantly (p < .001) more nogo responses occurred between 1 and 7 sec than within 1 sec. No difference between long and short go latencies was observed.
Biochemical Results Variations in serotonin (5-HT) content in cats trained on a symmetrically reinforced (SR) go-nogo visual discrimination task are described in Table 1. After 5 days of training, a significant decrease was observed in frontal cortex (p < .05) and amygdala (p < .05) accompanied by a significant increase in hypothalamus (p < .05) and medulla (p < .05). In cats that reached criterion performance a generalized and significant increase in 5-HT content was demonstrated (p < .01 orp < .001) in 11 of the 13 structures used. 5-HT decreased significantly in the amygdala (p < .01) and remained unchanged in dorsal mesencephalon and medulla. In the postcriterion group sacrificed 6 days after reaching performance criterion, 5-HT content increased significantly in ventral mesencephalon (p < .001) and hippocampus (p < .01) and decreased in the amygdala. Variations in the 5-hydroxyindoleacetic acid (5-HIAA) content in cats trained on a SR go-nogo task are shown in Table 2. After 5 days of training, a significant increase was observed in five structures: hippocampus (19 < .01), dorsal mesencephalon (p < .05) mesencephalic raphe nuclei (p < .01), pons (p < .05), and pontine raphe nuclei (p < .05). 5-HIAA was normal in the eight remaining structures. At criterion level, a significant increase in the 5-HIAA content was noticed in neostriatum (p < .01), hippocampus Co < .01), thalamus (p < .01), dorsal mesencephalon (p < .05), and mesencephalic raphe nuclei (p < .05) and was found normal in the other eight structures. Following the postcriterion period, 5-HIAA increased significantly in neostriatum (p < .05), hypothalamus (p < .05), dorsal mesencephalon (t9 < .01), mesencephalic raphe nuclei (p < .05), and pontine raphe nuclei (p < .05) and was not affected in the remaining eight structures. In Table 3, variations in the noradrenaline (NA) content in cats trained on a SR go-nogo visual discrimination task are described. A significant increase in Day 5 trained cats was observed in hippocampus (p < .05), dorsal (p < .05), and ventral (p < .01) mesencephalon and a significant decrease in hypothalamus (p < .001). No significant change was noticed
Note. R e s u l t s a r e e x p r e s s e d parentheses. * p < .05. ** p < .01. *** p < .001.
Frontal cortex Neostriatum Amygdala Hippocampus Thalamus Hypothalamus Dorsal mesencephalon Ventral mesencephalon Mesencephalon raphe nuclei Pons Pons raphe nuclei Medulla Medulla raphe nuclei
Structures ± 0.09 ± 0.09 + 0.13 + 0.12 ___ 1.0 ± 0.13 ± 0.17 ± 0.15 ± 0.15 ± 0.16 ± 0.12 _+ 0 . 1 0 ± 0.13
in/zg of amines per g of tissue ± SEM.
0.97 1.07 1.13 0.87 0.96 1.05 1.24 1.11 1.34 1.10 0.89 0.88 1.00
Control (10) ± 0.08* _+ 0.11 _+ 0 . 0 6 * _+ 0 . 0 4 ± 0.34 ± 0.17" + 0.12 +_ 0 . 4 2 ± 0.20 ± 0.13 ± 0.08 ± 0.10" _+ 0 . 0 6
1.95 2.00 0.82 2.09 1.73 2.42 1.42 2.20 3.24 1.63 3.10 1.21 3.15
± ± ± ± + _+ ± ± ± ± ± ± _+
0.22 0.25 0.03* 0.26** 0.39 0.48 0.32 0.14"** 0.23 0.11 0.13 0.33 0.02 o f c a t s is in
1.28 1.40 0.86 1.57 1.34 1.57 1.57 1.93 1.59 1.24 1.11 1.38 1.28
Postcriterion (4)
T test. Number
± 0.32** ± 0.22*** _+ 0.04** ± 0.20*** ___ 0.22*** ± 0.63** ± 0.14 ± 0.34*** ± 0.43*** + 0.21"* _+ 0.44*** ± 0.07 ± 1.0"**
Criterion (6)
Visual Discrimination Task
Statistical analysis was done using Student's
0.69 1.00 0.90 0.72 1.43 1.50 1.47 1.10 1.63 1.2 0.86 1.28 0.81
Day 5 (6)
TABLE 1 V a r i a t i o n s in 5 - H T B r a i n C o n t e n t o f C a t s T r a i n e d o n a S y m m e t r i c a l l y R e i n f o r c e d G o - N o g o
Note. *p ** p *** p
1.02 1.00 0.77 1.04 1.06 1.20 1.01 1.09 1.11 1.02 0.91 0.92 1.12
_ 0.09 _+ 0.10 _+ 0.09 _+ 0.07 -+ 0.08 ± 0.06 -+ 0.07 _+ 0.08 ± 0.12 ± 0.10 ± 0.09 _+ 0.10 +_ 0.12
C ont rol s (10) 0.89 0.96 0.69 1.76 1.30 1.30 1.31 0.84 1.74 1.55 1.40 0.83 1.03
± 0.03 + 0.03 _+ 0.03 _+ 0.29** ± 0.22 _+ 0.09 _+ 0.15" +_ 0.06 _+ 0.17"* _+ 0.26* +_ 0.23* + 0.09 + 0.08
Day 5 (6) 1.35 1.71 0.69 1.54 1.62 1.38 0.75 I. 12 1.56 1.25 0.86 0.83 0.75
-+ 0.19 + 0.21"* -+ 0.05 _+ 0.16"* + 0.17"* +_ 0.16 ± 0.05* _+ 0.11 ± 0.20* _+ 0.20 _+ 0.15 + 0.07 ± 0.08
Cri t e ri on (6)
1.04 1.34 0.76 1.23 1.13 1.49 1.57 1.30 1.73 1.26 1.37 0.99 1.10
_ 0.18 _+ 0.10" _+ 0.05 _+ 0.20 ± 0.08 +_ 0.18" _+ 0.22** ± 0.21 _+ 0.25* ± 0.13 _+ 0.32* + 0.16 _+ 0.21
Postcriterion (4)
Results are e x p r e s s e d in/~g of acid pe r g of t is s ue _ S EM. S t a t i s t i c a l a n a l y s i s w a s done usi ng S t u d e n t ' s T test. N u m b e r of cats is in p a r e n t h e s e s . < .05. < .01. < .001.
Frontal cortex Neostriatum A m yg da ta Hippocampus T h alam u s Hypothalamus Dorsal M e s e n c e p h a l o n Ventral m e s e n c e p h a l o n M e s e n c e p h a l o n raphe nuclei Pons Pons raphe nuclei Medulla Medulla raphe nuclei
Structures
TABLE 2 Variations in 5 - H I A A C o n t e n t in t he Br ai n o f Cats T r a i n e d on a S y m m e t r i c a l l y R e i n f o r c e d G o - N o g o V i sua l D i s c r i m i n a t i o n T a s k
Note. *p ** p *** p
0.31 0.41 0.24 0,20 0.26 1.43 0.30 0.30 0.54 0.36 0.37 0.34 0.27
4- 0.05 + 0.03 4- 0.005 ___ 0.03 _+ 0.04 4- 0.07 4- 0.04 ± 0.05 ± 0.06 4- 0.04 ___ 0.03 4- 0.04 4- 0,04
Controls (10) 0.48 0.43 0.25 0.30 0.42 0.74 0.43 0.55 0,50 0.44 0.46 0.39 0.38
___ 0.03 ___ 0.03 4- 0.02 4- 0.02* 4- 0.03 _+ 0.16"** 4- 0.03* ± 0.07** - 0.001 4- 0.04 4- 0.03 4- 0.02 ± 0.02
Day 5 (6) 0.38 0.36 0.21 0.25 0.36 1.28 0.31 0.48 0.73 0.36 0.56 0.37 0.53
___ 0.07 ___ 0.05 4- 0.02 4- 0.06 ± 0.09 4- 0,30 4- 0.07 4- 0.07* 4-_ 0.05* _+ 0.05 ± 0.08* ___ 0.08 ± 0.10"*
Criterion (6)
0.22 4- 0.02 0.27 4- 0.04* 0.17 _ 0.009 0.23.4- 0.06 0.18 4- 0.01 0.93 4- 0.21"* 0.25 _+ 0.04 0.34 ___ 0.05 0,46 ± 0.05 0.31 ± 0.09 0.43 ± 0.06 0.31 _ 0.05 0.23 4- 0.04
Postcriterion (4)
Results are e x p r e s s e d i n / x g of N A per g of t i s s u e 4- S EM. S t a t i s t i c a l a na l ys i s w a s done usi ng S t u d e n t ' s T test. N u m b e r of cats is in p a r e n t h e s e s . < .05. < .01. < .001.
Frontal c o r t e x Neostriatum Amygdala Hippocampus T hal am us Hypothalamus Dorsal m e s e n c e p h a l o n Ventral m e s e n c e p h a l o n M e s e n c e p h a l o n raphe nuclei Pons Pons raphe nuclei Medulla Me du lla raphe nuclei
Structures
TABLE 3 Variations in Brain N A C o n t e n t in C at s T r a i n e d on a S y m m e t r i c a l l y R e i n f o r c e d G o - N o g o Visual D i s c r i m i n a t i o n T a s k
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TABLE 4 Variations in DA Content in the Brain of Cats Trained on a Symmetrically Reinforced G o - N o g o Visual Discrimination Task
Structures Neostriatum Amygdala
Controls (10)
Day 5 (6)
6.34 + 0.35 0.22 + 0.01
6.67 _+ 0.20 0.31 _+ 0.01
Criterion (6) 5.03 ___ 0.17" 0.12 _ 0.01"**
Postcriterion (4) 4.54 _+ 0.16"* 0.18 _+ 0.016"**
Note. Results are expressed in/zg of DA per g of tissue --- SEM. Statistical analysis was done using Student's T test. N u m b e r of cats is in parentheses. * p <0.05. ** p < 0.01. *** p < 0.001.
in the nine remaining structures. In cats that reached criterion level performance, a significant increase in 5-HIAA content was observed in ventral mesencephalon (p < .05) mesencephalic raphe nuclei (p < .05), pontine raphe nuclei (p < .05), and medullary raphe nuclei (p < .01) and was not affected in the nine remaining structures. After the postcriterion period, a significant decrease in NA content was observed in the neostriatum (p < .05) and hypothalamus (p < .01) only and was not changed in any of the other structures. Table 4 describes the concentrations of dopamine (DA) measured in neostriatum and amygdala in cats trained on a SR go-nogo visual discrimination task. A significant decrease was noticed at criterion level in neostriatum (p < .05) and amygdala (p < .001) and after the postcriterion period in neostriatum (p < .01) as well as amygdala Co < .001). In Table 5, variations in tryptophan hydroxylase activity measured in different raphe nuclei revealed a significant decrease in cats that reached criterion performance: mesencephalic raphe nuclei (p < .05), pontine raphe nuclei (p < .05), and medullary raphe nuclei (p < .01). No sigTABLE 5 Variations in Tryptophan Hydroxylase Activity in Raphe Nuclei of Cats Trained on a Symmetrically Reinforced G o - N o g o Visual Discrimination Task
Structures Mesencephalon Ports Medulla
Controls (10)
Day 5 (6)
70.2 ___ 2.5 70.7 -4- 2.6 70.0 ___ 1.7
65.5 _ 1.2 67.1 _+ 1.1 67.6 _+ 1.1
Criterion (6) 63.6 _ 1.1" 63.1 _+ 1.9" 64.6 __. 1.0"*
Postcriterion (4) 71.6 _+ 3.0 61.2 +_ 2.7 70.2 _+ 2.3
Note. Results are expressed in n mole • g-l. hr-i of 5-HT formed _+ SEM. Statistical analysis was done using Student's T test. • p < 0.05. • * p < 0.01.
CHANGES IN BRAIN AMINES DURING LEARNING
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nificant change was observed in cats trained for 5 days or those sacrificed 6 days after criterion performance.
DISCUSSION In this discussion, biochemical data will be considered first before trying to relate them to the test situation. Generally speaking, the present findings demonstrate a significant involvement of serotonergic (5-HT) pathways during the acquisition of the symmetrically reinforced (SR) go-nogo visual discrimination task accompanied by considerable interaction with dopaminergic (DA) and noradrenergic (NA) pathways. The high 5-HT content observed in several structures is reflected by the 5-HT to 5-HIAA ratio which is significantly higher in cats having reached performance criterion (1.88 _+ 0.07, p < 0.001) than in controls (1.04 _+ 0.01) or cats trained for 5 days only (0.99 + 0.04). Such a high 5-HT: 5-HIAA ratio indicates a low 5-HT turnover rate which is in agreement with the low tryptophan hydroxylase activity observed at criterion level. This low enzyme activity which occurs simultaneously with heightened 5-HT and 5-HIAA content might be explained by a biochemical feedback mechanism assumed by biogenic amines, as catechols have been reported to be potent inhibitors of tryptophan hydroxylase activity in beef pineal gland (Lovenberg, Jequier, & Sjoerdsma, 1967) and in cat brain (Roberge & Poirier, 1973). Although a low 5-HT metabolism is observed in most structures, the 5-HIAA increase in five structures may reflect a high 5-HT turnover rate particular to these areas. The above-mentioned increase in 5-HT and 5-HIAA levels in animals performing at criterion level may also have influenced the DA content in neostriatum and amygdala which was significantly decreased. We already know that the neostriatum is innervated by serotonergic neurones originating mainly in the dorsal raphe nuclei (B7; Geyer, Puerto, Dawsey, Knapp, Bullard, & Mandell, 1976; Ternaux, H6ry, Bourgoin, Adrien, Glowinski, & Hamon, 1977; Boulay, 1978) and that a fiber tract from the median raphe nuclei (B8) innervates the substantia-nigra-ventraltegmental area (Dray, Gonye, Oakley, & Tanner, 1976). Many reports have also indicated a functional interrelation between the nigrostriatal dopamine (DA) neurones and serotonergic fibers arising in the raphe nuclei (Kostowski, 1975; Roberge, Parent, & Boulay, 1976; Samanin & Garattini, 1975). It has also been found that an increase in DA content following the administration of L-DOPA with a decarboxylase inhibitor reduces neostriatal 5-HT (Roberge, 1979), while decreasing 5-HT, through a selective lesion in the dorsal raphe nuclei, increases DA in the amygdala (Roberge et al., 1976). The anatomical localization of biochemical changes observed in cats trained on a SR go-nogo task corresponds to well identified chemical pathways or to the site of origin of these pathways. An extensive review
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of these projections, mostly in rat, may be found in the numerous studies (Fallon, Koziell, & Moore, 1978; Fallon & Moore, 1978a, 1978b; Palkovits, Brownstein, & Saavedra, 1974; Palkovits, Saavedra, Jacobowitz, Kizer, Zaborsky, & Brownstein, 1977). When it comes to defining the role of specific anatomical structures in the performance of the SR go-nogo task or to relating changes in the levels of neurotransmitters (enzymes or metabolites) to precise physiological or behavioral functions, considerable obstacles are encountered. Nevertheless, we tend to consider that the biochemical changes observed reflect the SR go-nogo training situation at the time of sacrifice. A serious effort was made to control variables unrelated to the learning situation. Cats used for experiments were conditioned to their living quarters at least 3 weeks before experiments were initiated. Stable environmental conditions were maintained from the time cats reached our facilities until their sacrifice. This included an automatic lighting schedule, constant temperature and humidity, background music, and regular animal attendants. Seasonal biochemical variations were eliminated by training and sacrificing animals at the same time of the year. Circadian fluctuations were taken into account by training animals in the morning only on a daily rotating schedule and by sacrificing all groups of cats at the same time of day between 0800 and 1000 hr in the morning. Animals were also weighed regularly and maintained at their initial weight ___10%. We are aware that cage controls were not manipulated as much as trained cats and received no raw meat in their diet. However a previous study in our laboratory carried out in identical conditions (Everett & Roberge, submitted for publication) has shown that the biochemical assays of manipulated cats exposed daily to the same experimental box did not differ significantly from those of untrained, nonmanipulated cage controls. The fact that controls did not receive meat in their diet cannot be considered a determining factor in the differences observed as the biochemical assays of the postcriterion group of cats differed significantly from those of control cats and in the same way as trained cats, even though they had received no meat for 6 days preceding sacrifice. All cats in groups B and C reached criterion performance although learning did not occur at the same rate. Cats were therefore sacrificed when they reached the same level of performance, however long it took to reach that level. We suggest that when cats reach criterion performance a Certain biochemical state is attained which is independent of the length of the learning process. In support of this is the fact that the biochemical assays of the two slow-learning cats in group B and the one in group C did not differ significantly from those of the other cats. An attempt to relate biochemical changes to specific aspects of the SR go-nogo task requires a brief description of the task. Cats had to learn to discriminate between two stimuli that commanded either a motor re-
CHANGES IN BRAIN AMINES DURING LEARNING
145
sponse (go trial) or the suppression of the motor response (nogo trial) both of which were reinforced. Cats performed correctly on go trials from the beginning of training. Learning was therefore mainly related to nogo responses. For this reason we tend to consider the changes in neurotransmitter levels to be essentially due to nogo response training. The two main aspects of nogo performance to which biochemical changes may be related are the motor response suppression and the state of motivation including incentive and reward expectancy. The prevailing line of thought concerning 5-HT and NA implicates these two neurotransmitters in response inhibition (Stein & Wise, 1974; Warburton, 1977) and reward behavior (Stein, 1968), respectively. The decreased 5-HT metabolism activity in cats that performed at criterion level could therefore be related to response suppression on correct nogo trials. Although NA and 5-HT are thought to exert opposite effects on goal-directed behavior (Stein & Wise, 1974) this is not the case for cats performing at criterion level, in which these two neurotransmitters increased in parallel in the brainstem. This may be explained by the fact that the response suppression required of these cats was related to reward and not to punishment. Interestingly enough, 5-HT and NA behaved in an antagonistic manner in cats performing on an asymmetrically reinforced task in which correct nogo trials were not rewarded (Roberge & Kitsikis, submitted for publication) as well as in cats that did not learn the task after 1000 trials (unpublished data), and therefore received less reinforcement than the SR cats. They also exerted opposite effects in the hypothalamus of cats trained for 5 days only on the SR task. The decrease in NA in the neostriatum and hypothalamus at the end of the postcriterion period may be related to the absence of reward. If the 5-HT:NA ratio is calculated and compared to controls, a significant increase is observed in cats performing at criterion level (p < .001) and after the postcriterion period (p < .01) thus indicating a lower utilization of 5-HT than of NA. The selective decrease of 5-HT in the amygdala compared to the general increase in other structures is worth noting although we have no explanation for this finding except that it could have something to do with the motivational aspect of the performance. The DA decreases observed in the neostriatum and amygdala may well be related to some aspect of motor organization (Iversen & Koob, 1977). As DA content was not altered in cats unable to perform correct nogo responses after 1000 trials (unpublished data) we believe that DA changes in the present study to be due to response suppression on correct nogo trials, as "poor learner" cats performed on all trials as if they were go trials. It is interesting that biochemical changes in many anatomical structures were not present after 5 days of training and occurred when cats reached performance criterion. This gradual evolution of biochemical changes
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favors the view that they are related to learning processes and/or to improved performance that also progress with time. The biochemical results from the three cats that took longer to reach performance criterion did not differ from those of other cats, suggesting that it was the level of performance and not the length of training that determined the biochemical changes in our experiments. Altered neurotransmitter levels persist in some structures for at least 6 days after training, taking time to return to control levels. Besides the brain stem structures, biochemical changes occurred in frontal cortex, hippocampus, amygdala, neostriatum and hypothalamus, which are all structures that have been cited in the literature in relation to behavioral inhibition, learning, and motivation. It is not possible at the present time to attribute a specific role to each of these structures in the SR g o - n o g o task or to understand the complex circuits involved. The present experiments have shown that the biochemical changes observed in cats trained on a symmetrically reinforced g o - n o g o visual discrimination task are different to those obtained in cats trained on a similar task with asymmetrical reinforcement (Roberge & Kitsikis, submitted for publication). We consider these variations to be due to differences in the behavioral requirements of these tasks.
REFERENCES Boulay, M. (1978). Etude neurochimique de l'organisation topographique des voies s~rotoninergiques cbrbbrales chez le chat. Th~se de doctorat, Universit6 Laval. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72, 248-254. Dabrowska, J. (1972). On the mechanism ofgo-nogo symmetrically reinforced task in dogs. Acta Neurobiologae Experimentalis, 32, 345-349. Dray, A., Gonye, T. J., Oakley, N. R., & Tanner, T. (1976). Evidence for a raphe projection to the substantia nigra in rat. Brain Research, 113, 45-57. Earley, C. J., & Leonard, B. E. (1978). Isolation and assay of noradrenaline, dopamine, 5-hydroxytryptamine and several metabolites from brain tissue using disposable BioRad columns packed with Sephadex G-10. Journal of Pharmacological Methods 1, 67-69. Fallon, J. H., Koziell, D. A., & Moore, R. Y. (1978). Catecholamineinnervation of the basal forebrain II. Amygdala, suprarhinal cortex and entorhinal cortex. Journal of Comparative Neurology, 180, 509-531. Fallon, J. H., & Moore, R. Y. (1978). Catecholamine innervation of the basal forebrain. Ill. Olfactory bulb, anterior olfactory nuclei, olfactory tubercle and piriform cortex. Journal of Comparative Neurology, 180, 533-544. (a) FaUon, J. H., & Moore, R. Y. (1978). Catecholamine innervation of the basal forebrain. IV. Topography of the dopamine projection to the basal forebrain and neostriatum. Journal of Comparative Neurology, 180, 545-579. (b) Geyer, M. A., Puerto, A., Dawsey, W. J., Knapp, S., Bullard, W. P., & Mandell, A. J. (1976). Histologic and enzymatic studies of the mesolimbic area and mesostriatal serotoninergic pathways, Brain Research, 106, 241-256. Iversen, S. D., & Koob, G. F. (1977). Behavioral implications of dopaminergic neurons in
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the mesolimbic system. In E. Costa & G. L. Gessa (Eds.), Advances in Biochemical Pharmacology, Vol 16, pp. 209-214. New York: Raven Press. Kotowski, W. (1975). Interactions between serotoninergic and catecholaminergic systems in the brain. Polish Journal of Pharmacology and Pharmacy, 27, 15-24. Kowalska, D., & Zielinski, K. (1976). Asymmetrical versus symmetrical go-nogo avoidance differentiation in dogs. Aeta Nervosa Superior (Praha) 18, 1-2. Lison, L. (1958). Statistique Appliqube ~ la Biologie Expkrimentale. Paris: Gauthier Villars. Lovenberg, W., Jequier, E., & Sjoerdsma, A. (1967). Tryptophan hydroxylation: Measurement in pineal gland, brainstem and carcinoid tumor. Science 155, 217-219. Palkovits, M., Brownstein, M., & Saavedra, J. M. (1974). Serotonin content of the brain stem nuclei in the rat. Brain Research, 80, 237-249. Palkovits, M., Saavedra, J. M., Jacobowitz, D. M., Kizer, J. S., Brownstein, M. J., & Ztiborszky, L. (1977). Serotonergic innervation of the forebrain: Effect of lesions on serotonin and tryptophan hydroxylase levels. Brain Research, 130, 121-134. Poschel, B. P. H., & Ninteman, F. W. (1975). Role of norepinephrine, dopamine and serotonin in reward mechanisms. In E. F. Domino & J. M. Davis (Eds.), Neurotransmitter Balances Regulating Behavior, pp. 57-73. Ann Arbor, Mich.: NPP Books. Roberge, A. G. (1979). A morphological and biochemical dissociation between dopamine and serotonin in cat brain. In E. Usdin, I. J. Kopin, & J. Barchas, (Eds.), Cateeholamines: Basic and Clinical Frontiers, Vol. 2, pp. I110-1112. New York: Pergamon. Roberge, A. G., Parent, A., & Boulay, M. (1976). Drmonstration d'une relation inversement proportionnelle entre la dopamine et la serotonine dans certaines structures crrrbrales: Aspects neurochimique et morphologique. Journal of Neurochemist~, 26, 591-595. Roberge, A. G., & Poirier, L. J. (1973). Effect of chronically administered L-DOPA on DOPA/5 HTP decarboxylase and tyrosine and tryptophan hydroxylases in cat brain. Journal of Neural Transmission, 34, 171-185. Samanin, R., & Garattini, S. (1975). The serotoninergic system in the brain and its possible functional connections with other aminergic systems. Life Sciences, 17, 1201-1210. Stein, L. (1968). Chemistry of reward and punishment. In D. H. Efron, J. O. Cole, J. Levine, & J. R. Wittenborn (Eds.), Psychopharmacology. A Review of Progress 19571967. pp 105-135. Washington, D.C.: Public Health Service Publication No. 1836, U.S. Government Printing Office. Stein, L., & Wise, C. D. (1974). Serotonin and behavioral inhibition. In E. Costa, G. L, Gessa, & M. Sandier (Eds.), Advances in Psychopharmaeology, Vol. 11, pp 281-291. New York: Raven Press. Ternaux, J. P., Hrry, F., Bourgoin, S., Adrien, J., Glowinski, J., & Hamon, N. (1977). The topographical distribution of serotoninergic terminals in the neostriatum of the rat and the caudate nucleus of the cat. Brain Research, 121, 311-326. Waldmeier, P. C., & Delini-Stula, A. A. (1979). Serotonin-dopamine interactions in the nigrostriatal system. European Journal of Pharmacology, 15, 363-373. Warburton, D. M. (1977). Stimulus selection and behavioral inhibition. In L. L. Iversen, S. D. Iversen, & S. H. Snyder (Eds.), Handbook ofPsychopharmacology, pp 385-431. New York: Plenum. Welch, A. S., & Welch, B. L. (1969). Solvent extraction method for simultaneous determination of norepinephrine, dopamine, serotonin and 5-hydroxyindoleacetic acid in a single mouse brain. Analytical Biochemistry 30, 161-179.
Changes in Brain Biogenic Amines in Cats Performing a Symmetrically Reinforced Go-Nogo Visual Discrimination Task A N N E KITSIKIS AND ANDRI~E G . ROBERGE i
Dkparternents de Physiologie et de Biochimie, Facultk de Mbdecine, Universit~ Laval, Quebec G1K 7P4, Canada Cats were trained on a symmetrically reinforced (SR) go-nogo visual discrimination task and sacrificed for biochemical assays on Day 5 of training, after reaching performance criterion or after a 6-day postcriterion period. Serotonin (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), and noradrenaline (NA) were measured in 13 brain structures. Dopamine (DA) was evaluated in the neostriatum and amygdala while tryptophan hydroxylase activity was measured in the brain stem raphe nuclei. A significant increase in 5-HT, 5-HIAA, and NA were observed in several brain structures on Day 5 and after cats reached performance criterion compared to untrained controls. Tryptophan hydroxylase activity decreased in the raphe nuclei in cats performing at criterion level and DA decreased in the amygdala and neostriatum. Fewer changes were observed on Day 5 than at performance criterion and several changes persisted after a 6-day postcriterion period without training. 5-HT and NA increases are discussed in terms of response suppression and reward behavior, respectively, and DA changes are related to motor organization on nogo trials. It is suggested that the biochemical changes observed are the result of training on the SR go-nogo task.
This work is part of an ongoing research project aimed at a better understanding of the role of biogenic amines in learning. Our main goals were first of all to determine how the acquisition of a behavioral task modifies the synthesis of serotonin (5-HT), noradrenaline (NA), and dopamine (DA) in the cat's brain and where these changes take place. Second, we wanted to evaluate biochemical changes at different stages of learning. The third goal was to investigate whether the biochemical changes observed and their anatomical localization vary as a function of the type of behavioral task cats are required to perform. The aim of the present research was to investigate whether the localized biochemical changes measured during or after the acquisition of a symmetrically i The authors gratefully acknowledge Gilles Huard's helpful technical assistance and Claude Roberge for the enzyme measurements. This research was supported by the Medical Research Council of Canada (MA-6590). 133 0163-1047/81/060133-15502.00/0 Copyright© 1981by AcademicPress, Inc. All rightsof reproductionin any formreserved.
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reinforced (SR) go-nogo visual discrimination task were different to those previously observed in cats performing an asymmetrically reinforced (AR) go-nogo task. A great deal of research has already been done at the Nencki Institute to identify the neuroanatomical substrates and behavioral differences of AR and SR go-nogo tasks in dogs (Dabrowska, 1972; Kowalska & Zielinski, 1976). We therefore know that the presence or absence of reinforcement on correct nogo trials changes the nature of a go-nogo task. The results presented here permit us to further differentiate AR and SR go-nogo tasks on a biochemical basis.
METHODS
Subjects. Twenty-six adult, male and female mongrel cats, weighing between 2.5 and 4 kg were used in these experiments and maintained at their initial weight _ 10%. They were housed in individual cages in a room with background music, constant temperature, and humidity. Their cages were cleaned by the same person throughout the training period. Cats were fed immediately after each training session or by 1000 hr in the morning in the case of untrained cats. They were maintained on a stable diet of cat food with water ad libitum. Sixteen animals were trained on a symmetrically reinforced go-nogo visual discrimination task and 10 animals served as untrained controls. One group of 6 trained and 4 untrained cats was sacrificed for biochemical assays on Day 5 of training. Another equivalent group was sacrificed on the third day of a postcriterion consolidation period and a third group of 4 trained and 2 untrained cats was killed 6 days (without training) after reaching criterion performance. Apparatus. The experimental setup consisted of a wooden box equipped with a one-way screen in front of which was placed a mobile tray with a centrally situated food well covered by a wooden block. A black cover represented the positive stimulus (S ÷) and a white cover the negative stimulus (S-). Training procedure. All animals underwent a 2-day shaping period during which they became habituated to the experimental box and learned to uncover the food well for reinforcement. During shaping, a naturalcolored wooden block was used and animals did not perform more than four complete trials before the onset of training. Training started the next day and continued for 5 days (group 1) or until a criterion performance of 90% correct responses on 2 consecutive days was reached. Learning was consolidated on the 3 following days before the second group of animals was sacrificed. Group 3 was killed 6 days after reaching criterion performance. They did not perform the task during the 6-day postcriterion period. Training was carried out in the morning between 0800 and 1200 hr and cats were trained in a different order each day so as to avoid interference of the circadian cycle in the training schedule. Fifty trials (25 go and
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25 nogo) were given in a randomized order 5 days a week. On each trial the undirectional screen was lifted, exposing either a black (S÷) or a white (S-) wooden block covering the food well. During go trials animals were required to remove S+ within 5 sec in order to retrieve a small piece of meat. During nogo trials animals had to refrain from this response for 7 sec before being rewarded with a piece of meat by the experimenter. Intertrial intervals lasted 10 sec. Performance was measured by calculating response errors and response latencies of go and nogo trials separately. Biochemical assays. All animals were trained and sacrificed during the winter months in order to avoid seasonal biochemical variations. Cats were guillotined without anesthesia immediately after the last training session which always took place between 0800 and 1030 hr so as to avoid circadian fluctuations. One or two untrained controls were always sacrificed on the same morning as trained animals. The brain was rapidly removed and the prefontal cortex, neostriatum, septum, hypothalamus, thalamus, hippocampus, amygdala, mesencephalon, pons, medulla, and the respective raphe nuclei of the mesencephalon, pons, and medulla were dissected on ice, frozen on dry ice, and kept at -80°C until biochemical assays were performed. Amines measurement. Noradrenaline (NA), dopamine (DA), serotonin (5-HT), and 5-hydroxyindoleacetic acid (5-HIAA) were separated according to the method of Earley and Leonard (1978). DA and NA were oxidized according to Welch and Welch (1969), whereas the native fluorescence of 5-HT and 5-HIAA was read at 305 nm activation-535 nm emission in concentrated HCL containing 10% ascorbic acid, using an Aminco-Bowman spectrophotofluorimeter (American Instruments Co. Inc., Silver Springs, Md.). Recoveries of DA, NA, 5-HT, and 5-HIAA were 90 _+ 5, 117 + 6, 63 _+ 8, and 95 _+ 4%, respectively. All estimates were corrected for losses. Results are expressed in micrograms of amines or acid per gram of fresh tissue. 5-HT metabolism was studied more extensively, as former experiments had pointed to its involvement in the acquisition of specific behavioral tasks. However, were it not for material limitations NA and DA metabolism would also have been measured more completely. Tryptophan hydroxylase assay. Tryptophan hydroxylase activity was assayed using an isotopic modification (manuscript in preparation) of the technique described by Roberge and Poirier (1973). L-[Methylene-~4C] tryptophan and 5-[2-'4C]hydroxytryptamine binoxalate (5-HT) were used as labeled substrate and standard, respectively. The labeled 5-HT formed was measured with a Beckman LS-350 liquid scintillation counter (Beckman Instruments Inc., Irvine, Calif.). Recovery of 5-HT was 95 _+ 1%. Estimates were corrected for losses and results are expressed in picamoles of 5-HT formed per gram per hour.
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Protein determination. Total protein concentrations were determined according to the method of Bradford (1976), using bovine y-globulin as standard. Statistical analysis. Standard error of the mean and Student's t test (two-tailed) were calculated according to Lison (1958). Chemicals. Noradrenaline (DL-Arterenol, HCL), 5-hydroxyindole-3acetic acid, cyclohexyl-ammonium salt (5-HIAA), and DL-6-methyl5,6,7,8-tetrahydropterine dihydrochloride were purchased from Calbiochem, Los Angeles, Calif. Serotonin (5-hydroxytryptamine, HCL), iproniazid phosphate, dithiothreitol and Sephadex G-10 were obtained from Sigma Chemical Company, St. Louis, Missouri. Amberlite CG-50 type 11 200 mesh was obtained from BDH Chemicals, Ltd, Montreal. L-[Methylene-a4]tryptophan was purchased from Amersham Corporation, Oakville, Ontario, Canada. Columns (glass barrel ECONO-Columns, 0.7 × 4.0 cm i.d.) and bovine y-globulin were obtained from Bio-Rad Laboratories Ltd, Missisauga, Ontario, Canada. RESULTS
Behavioral Data Number o f errors. The learning curves of cats in groups 1, 2, and 3 are shown in Fig. 1. In Fig. 1A, the mean go and nogo error curves of six cats sacrificed on Day 5 are shown. Although cats made significantly (p < .001) more errors in nogo than in go trials during the whole 5-day period, on a daily basis, the difference was significant on the first 2 days of training only. In groups 2 and 3, three cats took much longer to reach criterion performance than the other cats. On account of these divergent results, the error curves of these cats were drawn separately and presented in parallel to the mean go and nogo error curves of the remaining cats in each group (Figs. 1B and C). The number of go and nogo errors were calculated for Days 1 to 5, Day 6 to criterion performance, and for the whole training period. In both groups, cats made significantly (p < .001) more nogo than go errors whatever the period. Moreover, nogo errors were significantly (p < .02, group 2; p < .001, group 3) more frequent in the first 5 days of training than between Day 6 and criterion performance. No difference was found for go errors. However the difference between go and nogo errors was not significant in all daily scores (Figs. IB and C). The performance of group 1 cats sacrificed on Day 5 did not differ from that of cats in group 2 and 3 during the first 5 days of training. The cats in group 2 reached criterion performance between Days 8 and 10 (mean 9.5), while the three cats in group 3 took between 13 and 15 days (mean 13.6). Of the three cats that took 21 to 22 days to reach criterion performance, two (one in group 2, one in group 3) were hyperactive.
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FIG. 1. (A) M e a n go and nogo errors _+SEM in daily 50-trial (25 go and 25 nogo) sessions in six cats trained for 5 days on a symmetrically reinforced (SR) g o - n o g o visual discrimination task. (B) Learning c u r v e s of cats trained to criterion p e r f o r m a n c e on a SR g o - n o g o visual discrimination task. M e a n go and nogo errors + S E M in daily 50-trial (25 go and 25 nogo) sessions are represented for four cats sacrificed at the end of a 3-day postcriterion consolidation period. Nogo error c u r v e s o f two cats of the same group that took m u c h longer to learn the task are presented. The few go errors made are not represented in this figure for t h e s e two cats. (C) M e a n go and nogo errors _+SEM of cats trained to criterion p e r f o r m a n c e and sacrificed 6 days (without training) after the 3-day consolidation period. Go and nogo errors of three cats. Nogo errors of one cat. *p < .05; **p < .01; ***p < .001.
Response latencies. R e s p o n s e latencies were calculated by measuring the interval that elapsed b e t w e e n the time the screen w a s lifted and the m o m e n t the cat's p a w touched the w o o d e n block covering the food well. Latencies were recorded by the experimenter, in seconds. R e s p o n s e latencies on correct go trials and incorrect nogo trials were compiled
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KITSIKISAND ROBERGE
separately and the values obtained were expressed as the average percentage of responses at a given latency. When comparing go and nogo latencies, no significant difference was found for groups 2 and 3. If go trials are considered separately, short (< 1 sec) latencies were found to be significantly (p < .02) more frequent than 1 to 5 sec latencies in groups 2 and 3. No significant difference was found between long and short latencies in nogo trials for the same groups. In group 1, nogo latencies were longer (p < .05) than go latencies and significantly (p < .001) more nogo responses occurred between 1 and 7 sec than within 1 sec. No difference between long and short go latencies was observed.
Biochemical Results Variations in serotonin (5-HT) content in cats trained on a symmetrically reinforced (SR) go-nogo visual discrimination task are described in Table 1. After 5 days of training, a significant decrease was observed in frontal cortex (p < .05) and amygdala (p < .05) accompanied by a significant increase in hypothalamus (p < .05) and medulla (p < .05). In cats that reached criterion performance a generalized and significant increase in 5-HT content was demonstrated (p < .01 orp < .001) in 11 of the 13 structures used. 5-HT decreased significantly in the amygdala (p < .01) and remained unchanged in dorsal mesencephalon and medulla. In the postcriterion group sacrificed 6 days after reaching performance criterion, 5-HT content increased significantly in ventral mesencephalon (p < .001) and hippocampus (p < .01) and decreased in the amygdala. Variations in the 5-hydroxyindoleacetic acid (5-HIAA) content in cats trained on a SR go-nogo task are shown in Table 2. After 5 days of training, a significant increase was observed in five structures: hippocampus (19 < .01), dorsal mesencephalon (p < .05) mesencephalic raphe nuclei (p < .01), pons (p < .05), and pontine raphe nuclei (p < .05). 5-HIAA was normal in the eight remaining structures. At criterion level, a significant increase in the 5-HIAA content was noticed in neostriatum (p < .01), hippocampus Co < .01), thalamus (p < .01), dorsal mesencephalon (p < .05), and mesencephalic raphe nuclei (p < .05) and was found normal in the other eight structures. Following the postcriterion period, 5-HIAA increased significantly in neostriatum (p < .05), hypothalamus (p < .05), dorsal mesencephalon (t9 < .01), mesencephalic raphe nuclei (p < .05), and pontine raphe nuclei (p < .05) and was not affected in the remaining eight structures. In Table 3, variations in the noradrenaline (NA) content in cats trained on a SR go-nogo visual discrimination task are described. A significant increase in Day 5 trained cats was observed in hippocampus (p < .05), dorsal (p < .05), and ventral (p < .01) mesencephalon and a significant decrease in hypothalamus (p < .001). No significant change was noticed
Note. R e s u l t s a r e e x p r e s s e d parentheses. * p < .05. ** p < .01. *** p < .001.
Frontal cortex Neostriatum Amygdala Hippocampus Thalamus Hypothalamus Dorsal mesencephalon Ventral mesencephalon Mesencephalon raphe nuclei Pons Pons raphe nuclei Medulla Medulla raphe nuclei
Structures ± 0.09 ± 0.09 + 0.13 + 0.12 ___ 1.0 ± 0.13 ± 0.17 ± 0.15 ± 0.15 ± 0.16 ± 0.12 _+ 0 . 1 0 ± 0.13
in/zg of amines per g of tissue ± SEM.
0.97 1.07 1.13 0.87 0.96 1.05 1.24 1.11 1.34 1.10 0.89 0.88 1.00
Control (10) ± 0.08* _+ 0.11 _+ 0 . 0 6 * _+ 0 . 0 4 ± 0.34 ± 0.17" + 0.12 +_ 0 . 4 2 ± 0.20 ± 0.13 ± 0.08 ± 0.10" _+ 0 . 0 6
1.95 2.00 0.82 2.09 1.73 2.42 1.42 2.20 3.24 1.63 3.10 1.21 3.15
± ± ± ± + _+ ± ± ± ± ± ± _+
0.22 0.25 0.03* 0.26** 0.39 0.48 0.32 0.14"** 0.23 0.11 0.13 0.33 0.02 o f c a t s is in
1.28 1.40 0.86 1.57 1.34 1.57 1.57 1.93 1.59 1.24 1.11 1.38 1.28
Postcriterion (4)
T test. Number
± 0.32** ± 0.22*** _+ 0.04** ± 0.20*** ___ 0.22*** ± 0.63** ± 0.14 ± 0.34*** ± 0.43*** + 0.21"* _+ 0.44*** ± 0.07 ± 1.0"**
Criterion (6)
Visual Discrimination Task
Statistical analysis was done using Student's
0.69 1.00 0.90 0.72 1.43 1.50 1.47 1.10 1.63 1.2 0.86 1.28 0.81
Day 5 (6)
TABLE 1 V a r i a t i o n s in 5 - H T B r a i n C o n t e n t o f C a t s T r a i n e d o n a S y m m e t r i c a l l y R e i n f o r c e d G o - N o g o
Note. *p ** p *** p
1.02 1.00 0.77 1.04 1.06 1.20 1.01 1.09 1.11 1.02 0.91 0.92 1.12
_ 0.09 _+ 0.10 _+ 0.09 _+ 0.07 -+ 0.08 ± 0.06 -+ 0.07 _+ 0.08 ± 0.12 ± 0.10 ± 0.09 _+ 0.10 +_ 0.12
C ont rol s (10) 0.89 0.96 0.69 1.76 1.30 1.30 1.31 0.84 1.74 1.55 1.40 0.83 1.03
± 0.03 + 0.03 _+ 0.03 _+ 0.29** ± 0.22 _+ 0.09 _+ 0.15" +_ 0.06 _+ 0.17"* _+ 0.26* +_ 0.23* + 0.09 + 0.08
Day 5 (6) 1.35 1.71 0.69 1.54 1.62 1.38 0.75 I. 12 1.56 1.25 0.86 0.83 0.75
-+ 0.19 + 0.21"* -+ 0.05 _+ 0.16"* + 0.17"* +_ 0.16 ± 0.05* _+ 0.11 ± 0.20* _+ 0.20 _+ 0.15 + 0.07 ± 0.08
Cri t e ri on (6)
1.04 1.34 0.76 1.23 1.13 1.49 1.57 1.30 1.73 1.26 1.37 0.99 1.10
_ 0.18 _+ 0.10" _+ 0.05 _+ 0.20 ± 0.08 +_ 0.18" _+ 0.22** ± 0.21 _+ 0.25* ± 0.13 _+ 0.32* + 0.16 _+ 0.21
Postcriterion (4)
Results are e x p r e s s e d in/~g of acid pe r g of t is s ue _ S EM. S t a t i s t i c a l a n a l y s i s w a s done usi ng S t u d e n t ' s T test. N u m b e r of cats is in p a r e n t h e s e s . < .05. < .01. < .001.
Frontal cortex Neostriatum A m yg da ta Hippocampus T h alam u s Hypothalamus Dorsal M e s e n c e p h a l o n Ventral m e s e n c e p h a l o n M e s e n c e p h a l o n raphe nuclei Pons Pons raphe nuclei Medulla Medulla raphe nuclei
Structures
TABLE 2 Variations in 5 - H I A A C o n t e n t in t he Br ai n o f Cats T r a i n e d on a S y m m e t r i c a l l y R e i n f o r c e d G o - N o g o V i sua l D i s c r i m i n a t i o n T a s k
Note. *p ** p *** p
0.31 0.41 0.24 0,20 0.26 1.43 0.30 0.30 0.54 0.36 0.37 0.34 0.27
4- 0.05 + 0.03 4- 0.005 ___ 0.03 _+ 0.04 4- 0.07 4- 0.04 ± 0.05 ± 0.06 4- 0.04 ___ 0.03 4- 0.04 4- 0,04
Controls (10) 0.48 0.43 0.25 0.30 0.42 0.74 0.43 0.55 0,50 0.44 0.46 0.39 0.38
___ 0.03 ___ 0.03 4- 0.02 4- 0.02* 4- 0.03 _+ 0.16"** 4- 0.03* ± 0.07** - 0.001 4- 0.04 4- 0.03 4- 0.02 ± 0.02
Day 5 (6) 0.38 0.36 0.21 0.25 0.36 1.28 0.31 0.48 0.73 0.36 0.56 0.37 0.53
___ 0.07 ___ 0.05 4- 0.02 4- 0.06 ± 0.09 4- 0,30 4- 0.07 4- 0.07* 4-_ 0.05* _+ 0.05 ± 0.08* ___ 0.08 ± 0.10"*
Criterion (6)
0.22 4- 0.02 0.27 4- 0.04* 0.17 _ 0.009 0.23.4- 0.06 0.18 4- 0.01 0.93 4- 0.21"* 0.25 _+ 0.04 0.34 ___ 0.05 0,46 ± 0.05 0.31 ± 0.09 0.43 ± 0.06 0.31 _ 0.05 0.23 4- 0.04
Postcriterion (4)
Results are e x p r e s s e d i n / x g of N A per g of t i s s u e 4- S EM. S t a t i s t i c a l a na l ys i s w a s done usi ng S t u d e n t ' s T test. N u m b e r of cats is in p a r e n t h e s e s . < .05. < .01. < .001.
Frontal c o r t e x Neostriatum Amygdala Hippocampus T hal am us Hypothalamus Dorsal m e s e n c e p h a l o n Ventral m e s e n c e p h a l o n M e s e n c e p h a l o n raphe nuclei Pons Pons raphe nuclei Medulla Me du lla raphe nuclei
Structures
TABLE 3 Variations in Brain N A C o n t e n t in C at s T r a i n e d on a S y m m e t r i c a l l y R e i n f o r c e d G o - N o g o Visual D i s c r i m i n a t i o n T a s k
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TABLE 4 Variations in DA Content in the Brain of Cats Trained on a Symmetrically Reinforced G o - N o g o Visual Discrimination Task
Structures Neostriatum Amygdala
Controls (10)
Day 5 (6)
6.34 + 0.35 0.22 + 0.01
6.67 _+ 0.20 0.31 _+ 0.01
Criterion (6) 5.03 ___ 0.17" 0.12 _ 0.01"**
Postcriterion (4) 4.54 _+ 0.16"* 0.18 _+ 0.016"**
Note. Results are expressed in/zg of DA per g of tissue --- SEM. Statistical analysis was done using Student's T test. N u m b e r of cats is in parentheses. * p <0.05. ** p < 0.01. *** p < 0.001.
in the nine remaining structures. In cats that reached criterion level performance, a significant increase in 5-HIAA content was observed in ventral mesencephalon (p < .05) mesencephalic raphe nuclei (p < .05), pontine raphe nuclei (p < .05), and medullary raphe nuclei (p < .01) and was not affected in the nine remaining structures. After the postcriterion period, a significant decrease in NA content was observed in the neostriatum (p < .05) and hypothalamus (p < .01) only and was not changed in any of the other structures. Table 4 describes the concentrations of dopamine (DA) measured in neostriatum and amygdala in cats trained on a SR go-nogo visual discrimination task. A significant decrease was noticed at criterion level in neostriatum (p < .05) and amygdala (p < .001) and after the postcriterion period in neostriatum (p < .01) as well as amygdala Co < .001). In Table 5, variations in tryptophan hydroxylase activity measured in different raphe nuclei revealed a significant decrease in cats that reached criterion performance: mesencephalic raphe nuclei (p < .05), pontine raphe nuclei (p < .05), and medullary raphe nuclei (p < .01). No sigTABLE 5 Variations in Tryptophan Hydroxylase Activity in Raphe Nuclei of Cats Trained on a Symmetrically Reinforced G o - N o g o Visual Discrimination Task
Structures Mesencephalon Ports Medulla
Controls (10)
Day 5 (6)
70.2 ___ 2.5 70.7 -4- 2.6 70.0 ___ 1.7
65.5 _ 1.2 67.1 _+ 1.1 67.6 _+ 1.1
Criterion (6) 63.6 _ 1.1" 63.1 _+ 1.9" 64.6 __. 1.0"*
Postcriterion (4) 71.6 _+ 3.0 61.2 +_ 2.7 70.2 _+ 2.3
Note. Results are expressed in n mole • g-l. hr-i of 5-HT formed _+ SEM. Statistical analysis was done using Student's T test. • p < 0.05. • * p < 0.01.
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nificant change was observed in cats trained for 5 days or those sacrificed 6 days after criterion performance.
DISCUSSION In this discussion, biochemical data will be considered first before trying to relate them to the test situation. Generally speaking, the present findings demonstrate a significant involvement of serotonergic (5-HT) pathways during the acquisition of the symmetrically reinforced (SR) go-nogo visual discrimination task accompanied by considerable interaction with dopaminergic (DA) and noradrenergic (NA) pathways. The high 5-HT content observed in several structures is reflected by the 5-HT to 5-HIAA ratio which is significantly higher in cats having reached performance criterion (1.88 _+ 0.07, p < 0.001) than in controls (1.04 _+ 0.01) or cats trained for 5 days only (0.99 + 0.04). Such a high 5-HT: 5-HIAA ratio indicates a low 5-HT turnover rate which is in agreement with the low tryptophan hydroxylase activity observed at criterion level. This low enzyme activity which occurs simultaneously with heightened 5-HT and 5-HIAA content might be explained by a biochemical feedback mechanism assumed by biogenic amines, as catechols have been reported to be potent inhibitors of tryptophan hydroxylase activity in beef pineal gland (Lovenberg, Jequier, & Sjoerdsma, 1967) and in cat brain (Roberge & Poirier, 1973). Although a low 5-HT metabolism is observed in most structures, the 5-HIAA increase in five structures may reflect a high 5-HT turnover rate particular to these areas. The above-mentioned increase in 5-HT and 5-HIAA levels in animals performing at criterion level may also have influenced the DA content in neostriatum and amygdala which was significantly decreased. We already know that the neostriatum is innervated by serotonergic neurones originating mainly in the dorsal raphe nuclei (B7; Geyer, Puerto, Dawsey, Knapp, Bullard, & Mandell, 1976; Ternaux, H6ry, Bourgoin, Adrien, Glowinski, & Hamon, 1977; Boulay, 1978) and that a fiber tract from the median raphe nuclei (B8) innervates the substantia-nigra-ventraltegmental area (Dray, Gonye, Oakley, & Tanner, 1976). Many reports have also indicated a functional interrelation between the nigrostriatal dopamine (DA) neurones and serotonergic fibers arising in the raphe nuclei (Kostowski, 1975; Roberge, Parent, & Boulay, 1976; Samanin & Garattini, 1975). It has also been found that an increase in DA content following the administration of L-DOPA with a decarboxylase inhibitor reduces neostriatal 5-HT (Roberge, 1979), while decreasing 5-HT, through a selective lesion in the dorsal raphe nuclei, increases DA in the amygdala (Roberge et al., 1976). The anatomical localization of biochemical changes observed in cats trained on a SR go-nogo task corresponds to well identified chemical pathways or to the site of origin of these pathways. An extensive review
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of these projections, mostly in rat, may be found in the numerous studies (Fallon, Koziell, & Moore, 1978; Fallon & Moore, 1978a, 1978b; Palkovits, Brownstein, & Saavedra, 1974; Palkovits, Saavedra, Jacobowitz, Kizer, Zaborsky, & Brownstein, 1977). When it comes to defining the role of specific anatomical structures in the performance of the SR go-nogo task or to relating changes in the levels of neurotransmitters (enzymes or metabolites) to precise physiological or behavioral functions, considerable obstacles are encountered. Nevertheless, we tend to consider that the biochemical changes observed reflect the SR go-nogo training situation at the time of sacrifice. A serious effort was made to control variables unrelated to the learning situation. Cats used for experiments were conditioned to their living quarters at least 3 weeks before experiments were initiated. Stable environmental conditions were maintained from the time cats reached our facilities until their sacrifice. This included an automatic lighting schedule, constant temperature and humidity, background music, and regular animal attendants. Seasonal biochemical variations were eliminated by training and sacrificing animals at the same time of the year. Circadian fluctuations were taken into account by training animals in the morning only on a daily rotating schedule and by sacrificing all groups of cats at the same time of day between 0800 and 1000 hr in the morning. Animals were also weighed regularly and maintained at their initial weight ___10%. We are aware that cage controls were not manipulated as much as trained cats and received no raw meat in their diet. However a previous study in our laboratory carried out in identical conditions (Everett & Roberge, submitted for publication) has shown that the biochemical assays of manipulated cats exposed daily to the same experimental box did not differ significantly from those of untrained, nonmanipulated cage controls. The fact that controls did not receive meat in their diet cannot be considered a determining factor in the differences observed as the biochemical assays of the postcriterion group of cats differed significantly from those of control cats and in the same way as trained cats, even though they had received no meat for 6 days preceding sacrifice. All cats in groups B and C reached criterion performance although learning did not occur at the same rate. Cats were therefore sacrificed when they reached the same level of performance, however long it took to reach that level. We suggest that when cats reach criterion performance a Certain biochemical state is attained which is independent of the length of the learning process. In support of this is the fact that the biochemical assays of the two slow-learning cats in group B and the one in group C did not differ significantly from those of the other cats. An attempt to relate biochemical changes to specific aspects of the SR go-nogo task requires a brief description of the task. Cats had to learn to discriminate between two stimuli that commanded either a motor re-
CHANGES IN BRAIN AMINES DURING LEARNING
145
sponse (go trial) or the suppression of the motor response (nogo trial) both of which were reinforced. Cats performed correctly on go trials from the beginning of training. Learning was therefore mainly related to nogo responses. For this reason we tend to consider the changes in neurotransmitter levels to be essentially due to nogo response training. The two main aspects of nogo performance to which biochemical changes may be related are the motor response suppression and the state of motivation including incentive and reward expectancy. The prevailing line of thought concerning 5-HT and NA implicates these two neurotransmitters in response inhibition (Stein & Wise, 1974; Warburton, 1977) and reward behavior (Stein, 1968), respectively. The decreased 5-HT metabolism activity in cats that performed at criterion level could therefore be related to response suppression on correct nogo trials. Although NA and 5-HT are thought to exert opposite effects on goal-directed behavior (Stein & Wise, 1974) this is not the case for cats performing at criterion level, in which these two neurotransmitters increased in parallel in the brainstem. This may be explained by the fact that the response suppression required of these cats was related to reward and not to punishment. Interestingly enough, 5-HT and NA behaved in an antagonistic manner in cats performing on an asymmetrically reinforced task in which correct nogo trials were not rewarded (Roberge & Kitsikis, submitted for publication) as well as in cats that did not learn the task after 1000 trials (unpublished data), and therefore received less reinforcement than the SR cats. They also exerted opposite effects in the hypothalamus of cats trained for 5 days only on the SR task. The decrease in NA in the neostriatum and hypothalamus at the end of the postcriterion period may be related to the absence of reward. If the 5-HT:NA ratio is calculated and compared to controls, a significant increase is observed in cats performing at criterion level (p < .001) and after the postcriterion period (p < .01) thus indicating a lower utilization of 5-HT than of NA. The selective decrease of 5-HT in the amygdala compared to the general increase in other structures is worth noting although we have no explanation for this finding except that it could have something to do with the motivational aspect of the performance. The DA decreases observed in the neostriatum and amygdala may well be related to some aspect of motor organization (Iversen & Koob, 1977). As DA content was not altered in cats unable to perform correct nogo responses after 1000 trials (unpublished data) we believe that DA changes in the present study to be due to response suppression on correct nogo trials, as "poor learner" cats performed on all trials as if they were go trials. It is interesting that biochemical changes in many anatomical structures were not present after 5 days of training and occurred when cats reached performance criterion. This gradual evolution of biochemical changes
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favors the view that they are related to learning processes and/or to improved performance that also progress with time. The biochemical results from the three cats that took longer to reach performance criterion did not differ from those of other cats, suggesting that it was the level of performance and not the length of training that determined the biochemical changes in our experiments. Altered neurotransmitter levels persist in some structures for at least 6 days after training, taking time to return to control levels. Besides the brain stem structures, biochemical changes occurred in frontal cortex, hippocampus, amygdala, neostriatum and hypothalamus, which are all structures that have been cited in the literature in relation to behavioral inhibition, learning, and motivation. It is not possible at the present time to attribute a specific role to each of these structures in the SR g o - n o g o task or to understand the complex circuits involved. The present experiments have shown that the biochemical changes observed in cats trained on a symmetrically reinforced g o - n o g o visual discrimination task are different to those obtained in cats trained on a similar task with asymmetrical reinforcement (Roberge & Kitsikis, submitted for publication). We consider these variations to be due to differences in the behavioral requirements of these tasks.
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