INVOLVEMENT OF SEROTONIN AND CATE~HOLAM~NE METABOLISM IN CATS TRAINED TO PERFORM A DELAYED RESPONSE TASK L. VACHCIN and ANDR&E G. ROBEROE” Dtpartement
de Biochimie, FacultC de Muecine,
UniversitC Lava& Q&bee, Canada, GIK 7P4
Abstract-The effects of the a~uisitjon of a delayed response task on the central ~t~holamine and serotonin metabolism were investigate in normal, undru~ed cats. The training (11 ~n~~utive days) produced an increased serotonin content in the pirifo~ lobe, me~n~phalon without the raphe nuclei and medulla without the raphe nuclei, whereas the 5-hydrox~ndolea~tic acid ievel was increased in the piriform lobe and mesencephalon without the raphe nucIei, but decreased in the pons without the rapbe nuclei. The noradrenaline content was decreased by the training in the frontal cortex and piriform lobe, whereas the dopamine concentration was increased in the piriform lobe. Tryptophan hydroxylase activity was increased in the piriform lobe, in the mesencephalon without the raphe nuclei and in the raphe nuclei of the mesencephalon, pons and medulla whereas training did not produce any significant effect on the dopamine B-hydroxylase activity. These results indicate the involvement of an active biochemical mechanism fulfilled by the serotoninergic mesolimbic system in association with the noradrenergic pontocorticolimbic system and the fimbic dopaminer~~ system. They also suggest that these well localized biochemical changes reflect processes involved in the performana: criterion of the delayed response task.
changes take pface during and after the acquisition of a DR task and (3) to anaIyse these biocbemicaf changes in relation to the cognitive, motivational and motor mecha&sms mobilized by learning a DR task. colo$ical treatments (ROBERGE,Rau & BOISVERT, A preliminary report was presented to the Society 1978; KITSIKIS & ROBERGE, 1973a,b; KITSIKIS, for Neuroscience at Atlanta, Georgia in 1979. ROBERGE& FRENETTE,1972; ROBEROE,BOISVERT& EVERETT,1980) have attempted to define the neuroanatomical and neurochemical substrates involved in EXPERIMENTAL PROCEDURES the performance of a delayed r&ponse (DR) task in cats. Our interest in the DR task is related to those aspects of ~roto~iner~c and ~techolaminer~c Fourteen ad& mongrel female cats, weighing metabo~sm possibly involved in the performance of 3.0 f 0.2 kg, were housed in individual cages in a room with background music, constant temperature and humidthis task. We have already been able to demonstrate ity and adapted to these en~ronmenta~ conditions for 10 (ROBERGEet at, 1980) that an a~umui~tion of serotonin in the CNS foflowing the administration of days after they were examined by a veterinarian. Cages 5-hydroxy+-tryptophan impaired the performance of were cleaned by the same person throughout the training period. They were fed Dr. Ballard’s cat food and maincats trained on a DR task without affecting the cognitained at 90-95x of their initial weight, with water ud libitive aspects of the task and that cats unable to learn turn. Trained animals were fed immediately after the daily the DR task within loo0 trials revealed a low testing session by the experimentor, according to the dopamine content in the neostriatum @&SIKIS & amount of meat consumed during this period, or at 10.00 h
approaches, using lesions &WAC, 1968, 1973; LAWICICA & Kmcwm, 1961; WARREN,W.4aRENd;: AKERT, 19621, electrical stirn~i~ti~n BISON, 1977; UNGHER,APPEL& SIRIANU,1966) and pharma-
DIFFERENT
ROBEWE, 1973b).
The purpose of this study is therefore (I) to investigate whether serotonin (5HT) and ~te~hol~ine
me~bo~sm is rn~ifi~ as a consequence of learning a DR task in normal, undrugged cats; (2) to identify different neuro~atomi~~ sites at which biochemica1 ’ Address reprint request to: And&e G. Roberge, Dtpartement de Biochimie, FacultC de MCdecine, UniversitC Lava], Qutbec, Canada, GlK 7P4. Abbreviations: DR, delayed response; !XHAA, Shydroxyindoleacetic acid; SHT. serotonin; NA, noradrenaline. 189
in the morning in the case of untrained cats. Eight cats were trained on a defayed response (DR) task and the six others served as controls. This experiment was done during summer time. Delayed response task. The experimental set-up consisted of a wooden box equipped with a glass screen permitting animals to see a tray with two food weib carved 11 cm apart. While the light was on, a piece of meat was placed in one of the food wells and then both wells were covered with identical wooden blocks and the light turned off. At the end of the delay period, the light was automatically turned on by a device connected to a chronometer, the screen was lifted and the cat allowed to respond.
190
L.
'VhCIfON and As%& 6. ROBE%&
Training procedure. Cats first underwent a shaping period of 2-3 days during which they were adapted to the experimental set-up and learned to uncover the food well in order to retrieve a piece of meat (2 g). This period was followed by DR training which consisted of 48 trials a day on 11 consecutive days. Animals were then killed irrespective of their level of performance. Each session included 24 trials without a delay, interspersed by 24 trials with delays, using a titration technique. Delays were progressively increased by 2 s after two consecutive correct responses until animals reached a 30s delay, and then by 5 s up to the maximal delay of 60s If animals made 2 consecutive errors, delays were decreased by 2 or 5 s. Perseverance, defined as 10 successive responses at the same food well, was corrected by reinforcing the other food well until 2 correct responses were performed in a block of 10 successive trials, Trials, with or without delay, and the side of the reinforced food well were presented in a randomized sequence. defined to avoid the same situation in more than 3 successive trials. Biochemical
assays
Animals were killed without anesthesia 24 h after the last training session, using a guillotine specially designed for this purpose. All cats were killed between 14.00 h and 16.00 h. canning and ending with a control. The other controls being intercalated with trained animals. The prefrontal and frontal cortex, neostriatum, septum, hypothalamus, thalamus, hippocampus, piriform lobe (amygdala), mesencephalon without the raphe nuclei, ports without the raphe nuclei, medulla without the raphe nuclei and mesencephalon, pons and medulla raphe nuclei, were respectively dissected on ice and kept frozen at -80°C untif biochemical assays were performed. Amines and add were determined by structures including controls and trained animals done on the same day. Amine determinations. Dopamine, noradrenaline (NA), serotonin (5HT) and 5-hydroxyindoleacetic acid (S-HIAA) were determined according to the method of PARLEY& I.,EONARD (19X3), using Bio-Rad cofumns packed with gef filtration, Sephadex G-10. Native fluorescence of 5-HT and S-NIAA was read at 305 nm activation 535 nm emission in IO mg% ascorbic acid-concentrated HCl. Dopamine and NA were oxidized according to EARLEY& LEONARD(1978) and to WELCH & WELCH techniques (196% respectively. Amines and S-HIAA were measured using an AmincoBowman s~ctroptiotofiuori~eter (American instruments Co. Inc., Silver Spring, Maryland. U.S.A.). All estimates were corrected for losses. Recoveries were 69 rt 5% 77 f 4x, 103 k 4% and 81 + 6% for S-HI, 5-HIAA, NA and dopamine, respectively. Results are expressed in pg of amines or acid/g of fresh tissue. Tr~~~o~~~~~ydroxj~~ase iassay. Tryptopban hydroxyfase activity was measured according to the method described by ROBBRGE & Potar~~ (1973) and modified in an isotopic method (manuscript in preparation) to use t-[methylene-14C]tryptophan as substrate and 5.[2m”C]hydroxy’tryptamine binoxalate (serotonin, 5-HT) as standard. Labelled 5-HT was measured using a Beckman LS-350 liquid scintilIation counter peckman Instruments Tue., Irvine, California, U.S.A.). Estimates were corrected for losses. Recovery of 5-HT was 93 i I%. Results are expressed in pmol of 5-WT formed/mg of protein/min. Dopamine fi-hydroxylaw assay. Dopamine /I-hydroxylase activity was photometrically measured according to Kato, Kuzuya & Nagatsu (t 974). Octopamine formed was deter-
mined using a Gilford 300-N Micro-Ssmplc speL.trophotameter (Gilford Instrument Laboratories Inc. 0herhn. Ohio. U.S.A.). Estimales were corrected for losses Rccovery of octopamine was X3 f 6”:,. Results arc exprc~scd in pm01 of octopamine formedimg of protein:min. Protrin ~~r~~~~tiaf~ojls. Totat protein con~o~ratl~~n~ were determined according to ERAI~I~~IT (I%%). uwg bovine gamma globulin as standard. Slarisrical analysis. Non-parametric analyses (Friedman two-way analysis of variance. Wilcoxon test1 were used as described by SIEGEL(19561.Standard error of the mean and Student’s r-test were calculated according to Ltso~ t t 9581. Chemicals Noradrenaline (rrt-arterenol. HCI), S-hydroxy indole-!acetic acid, cyclohexyl-ammonium salt (S-HIAA) and IIL-6methyl-5.6.7.8tetrahydropterine dihydroxychloride were from Calbiochem, Los Angeles, California, USA. Serotonin (5-hydroxytryptamine Hfl), ut-octopamine HCI. fumaric acid (sodium salt), pargyline HCI, tysaminc HC’l. iproniazid phosphate, dithiothreitoi, Sephadex G-10 and Dowex 50-W-X4 were purchased from Sigma Chemical Co.. St-Louis. Missouri, U.S.A. .V-ethylmaleimide and Amberlite CC-50 (II) type II 200 mesh were obtained from RDH Chemicals Ltd.. Montreal, Quebec. Canada. L-[methylene-l~C]tryptophan was from Amersham Corporation. Oakvitle, Ontario, Canada. 5-[2-‘cCfhydtoxy tryptamine binoxalace was purchased from New England Nuclear. Lachine, Montreal, Quebec. Canada. Columns [glass barrel ECONQ=columns, id. 7 x 40 mm) and hovinc gamma globulin were obtained from Bio-Rad Laboratories Ltd.. Missisauga. Ontario, Canada. RESULTS Behavioral
data
The
daily performance (percent of correct responses) and response latencies (s) of c&s perforthy: in 0 s delay triais are represented in Fig, I. Cats maintained a performance of 97% correct responses DELAYor 0 #et
1 2 3 4 s 6 r 8 SlOttmVB
Fro. 1. Performance (% correct responses) and response latencies (s) of cats performing a delayed response task at 0 s delay on 1I consecutive days.
Delayed response effect on cat biogenic amine synthesis INTERPOSED
DELAYS
T
0
’ 1
’ 2
’ 3
’ 4
’ 5
’ 6
’ 7
’ 6
* 9
’ 10
’ 11 DAYS
3
4
5
6
7
S
91011DAYS
1.5 oz
0
12
FIG. 2. Performance (mean delays) and response latencies (s) of cats performing a delayed response task with interposed delays on 11 consecutive days.
throughout the experiment and only slight, non-significant variations were observed during the I1 days of training Response later&es, however, decreased (P < 0.01) from 1.6 s on the first day to 0.6 s on the last day of training Although the variation in response latencies was not significant during the first 5 days, they were significantly decreased during the last 6 days of training (P < 0.02). The performance (mean delay) and response latenties (s) of cats in interposed delay trials is shown in Fig. 2. On account of the titration technique, the mean delay is considered a better measure of performance than the number of correct responses. Although the performance on delay trials was found to improve (P < 0.01) during the whole 11 days of training, it is clear that the main increase in mean delays occurred within the first 5 days, jumping from 4.5 to :7 s. Following the fifth day, performance reached a plateau at a mean of 18.04 f 0.34 s. In fact, a Friedman two-way analysis of variance, done on the first 5 days, showed a significant improvement in the performance of cats (P < 0.001). The same analysis, done on the last 6 days, showed no significant change in performance. On the other hand, response latencies in trials with delays decreased progressively and significantly (P < 0.05) during the experiment, varying between 3.3 and 1.7 s. In this case, the improvement was found significant neither during the first 5 days nor during the last 6 days of training taken separately. Higher response latencies (Wilcoxon, P < 0.01) observed in interposed delay trials than in OS delay trials were noted throughout the experiment. Biochemical data
The effect of the ll-day DR training period on serotonin 5-hydroxyindoleacetic acid (5-HT), (5-HIAA) and noradrenaline (NA) content in 13 CNS
191
structures is described in Table 1 and compared to control values obtained in untrained cats. In trained animals, a significant increase in 5-HT content in the amygdala (P < 0.05), mesencephalon without raphe nuclei (P < 0.01) and medulla without raphe nuclei (P < 0.01) was found. The 5-HIAA level also increased in the amygdala (P c 0.05), mesencephalon without raphe nuclei (P < 0.01) but decreased in the pons without raphe nuclei (P < 0.001). 5-HT and 5-HIAA content was not affected by training in the prefrontal and frontal cortex, neostriatum, thalamus, hypothalamus, hippocampus, mesencephalon raphe nuclei, pons raphe nuclei and medulla raphe nuclei. The NA content was si~ifi~tly decreased in the frontal cortex (P < 0.05) and amygdala (P < 0.01) of trained cats but not in any of the other structures studied (Table 1). On the other hand, dopamine /?-hydroxylase activity (expressed in pmol of octopamine formed/mg of protein/min) in the frontal cortex (12.32 rt: 0.65). thalamus (12.60 f 1.07) hippocampus (8.19 It: 0.66), amygdala (9.89 f 0.68) mesencephalon without raphe nuclei (7.84 f 0.44) and pons without raphe nuclei (9.67 + 0.64) was unaffected by training. Dopamine content was determined in two structures: the piriform lobe (amygdala) in which a significant increase (P < 0.05) was found (1.77 f 0.26 and 2.56 + 0.27 pg of tissue & S.E.M. in controls and trained cats, respectively) and the neostriatum in which no significant was observed change (18.88 + 1.33 fig/g of tissue + SE.M.). In Table 2, tryptophan hydroxylase activity is shown for control and trained animals. Training produced an increased activity in the piriform lobe (amygdala, P < O.OOl),mesencephalon without raphe nuclei (P < O.Ol), mesencephalon raphe nuclei (P < O.OS),pons raphe nuclei (P < 0.05) and medulla raphe nuclei (P < 0.05). No significant change was found in the septum, thalamus, hippocampus, pons without raphe nuclei and medulla without raphe nuclei. DISCUSSION The main findings of the present study are that the acquisition and performance of a delayed response (DR) task during 11 days in cats induced per se specific and localized biochemical changes in the CNS. Serotoninergic metabolism was particularly affected as shown by an acceleration of S-HT synthesis in the mesolimbic system. Such binominal disturbances in 5-HT metabolism were accompanied by a decreased noradrenaline (NA) content in the frontal cortex and piriform lobe and an increased dopamine level in the piriform lobe. In the present study, the analysis of the DR problem presented by FLETCHER(1965) was adopted. He defines the task as “an intratrial performance task in which the correct instrumental response is the overt terminal response of an orienting-response chain initiated totally, accurately and immediately by an observing response made at the beginning of each trial”
L. VACHON and ANDR~E G. RORERGE
192 TABLE
1. EFFECTS OF DELAYED RESPONSE TASK ON
SEROTONIN, IN
SW”l”ml Controls
Tramed cat\
(6)
(71
structurest Prefrontal cortex Frontal cortex Neostriatum Thakanus Hypothalamus Hippocampus Piriform lobe (amygdala) Mesencephalon (without raphe nuclei) Pons (without raphe nuclei) Medulla (without raphe nuclei) Raphe nuclei (mesencephalon) Raphe nuclei (pas) Raphe nuclei (medulla)
1.69 1.59 2 23 2.25 3.05 I .90 1.98 2.10
* i i_ k * k f +
5-HYDROXYINDOLEACETIC
ACID
AND
NORADRENALINI!
COYTtN1
CAT BRAIN
5.Hydroxylndoleacetlc acnd CO”tr”ls Trained cats 17)
(61
0.30 0.26 0.21 0 36 0.48 0.25 0.24 0.13
19o+O.15 I 57 f 0.24 2.16 k 0.28 2 55 i 0.23 3 34 + 0.24 2.15 f 021 2.64 t 0.20’ 297 i 022”
1.13 i 091 * 1.04 i 1.42 i_ 1.7X + 1.64 & 1.26 k 1.69 i:
0.28 0.14 0.16 0.25 0.16 0.23 0.18 0.25
I32 I 14 1.20 I47 2.25 I.52 1.96 2 56
+ 0.16 + 0.30 i 0.12 i 0.10 IO.23 ir 0.13 i 0 25’ i 0 12”
(‘O!liTOll; lh) 1.26 2.09 0 69 1.69 4.45 0.52 131 0.69
horddrcnalmr Trained cat\ 1.’
I
i 0.15 ri_0.36 It 0.20 k 0.26 + 0.87 * 0.1 I * 0 17 + 0 06
I 29 I IY 0.64 1.61 4.00 0.57 0 74 0.59
~ (I.22 - 022’ f 0 I II +: OIX : 0.66 i- 0.08 + 0.09** f 0 05
1.31 c 0.28
I 24 i 0.34
0.88 f 0 I2
0 37 i 003***
0 54 2 0 Oh
047 : 0.06
0.79 * 0.05
1.48 + 0.17**
1.26 f 028
134 *01x
0.47 * 0.05
0.50 :.
3.43 fc 0.32
3.60 ir 0.29
2.10 + 0.32
2.18 * 0.21
0.91 + 0.16
IO4 3 0.13
1.51 t 0.25 1.07 + 0.10
1.26 i 0.27 1.22 + 0.10
I.09 * 0.19 1.76 f 030
0.78 * 0.06 2.00 + 0.27
0.90 + 0. I2 0.34 * 0.05
0 76 2 0.1 0.28 t 0 04
0 05
I
t Results are expressed in fig of amine or acid/g of fresh tissue. Statistical analysis was done using Student’s t-test. Number of animals is in bra&&s. * P < 0.05. ** P < 0.01. *** P < 0.001.
and considers it as a performance problem consisting of 4 phases: a baiting, a covering, a delay and a response phase. The initial and most important events are the observing response elicited by the placement of the bait followed by the orienting response chain, overt or covert, initiated by the observing response. Maintaining the orienting response chain during the delay is crucial for the successful instrumental response at the end of the delay. The analytical problem is to specify the conditions under which a response once initiated is maintained within a given trial. The only learning that occurs with practice is the fixation of an adequate orienting-response chain which will terminate in a correct response. In the light of the above remarks, cats in the present experiment, by performing at increasingly longer delays and responding at shorter latencies, improved their observation and orienting-response. In order to maintain the integrity of the observing and orienting responses, cats must eliminate competing response tendencies which interfere with performance, distractibility being one of the critical factors in performance decrement. Successful performance, therefore, depends on decreased distractibility and narrowed attention. Inasmuch as competing responses must be eliminated to perform the correct instrumental response, it seems plausible to attribute improved performance to some kind of ‘inhibitory’ mechanism permitting attention to be focussed on relevant stimuli. Considering that S-HT metabolism increased during training and that S-HT has been reported to play a role in behavioral ‘inhibition’ (ESSMAN,1974; MORGANE & STERN,1978; GRAFF8c SILVEIRA FKHO, 1978; ROBERGEet a!., 1980) we are tempted to associate this biochemical change with improved performance. As improved behavioral inhibition does not necessarily
imply central chemical inhibition thus the biochemical variations observed will be discussed in terms of changes in synthesis, turnover rate and concentrations of neurotransmitters rather than by inferring central inhibitory or excitatory mechanisms. The fact that the parallel increase in (1) S-HT and S-HIAA content in the mesolimbic system, resulting in a 5-HT:5-HIAA ratio of equivalent magnitude in both controls and trained cats and (2) tryptophan hydroxylase activity in the serotoninergic cell bodies of the raphe nuclei as well as in nerve terminals (piriform lobe and mesencephalon) indicates a general stimulation of 5-HT synthesis which seemingly reflects an active mechanism. The enhancements of 5-HT synthesis observed in trained animals are interrelated with a decreased NA content in the frontal cortex and piriform lobe (amygdala) and a higher dopamine content in the piriform lobe. As decreases or increases in brain amine content are generally explained as changes in turnover rate TABLE
2.
EFFECTS OF DELAYED RESPONSE TASK ON TRYPTO-
PHAN
HYDROXYLASE
Septum Thalamus Hippocampus Pwllorm lobe (amygdalnl Mesenaphalon (tithou! raphe nuclei) Pans (without raphc nuckl) Medulln (without nphc nuckl) Raphe nuclei (mesenaphalon) Raphe nuclei (ponsl Raphe nuclei (medulla)
ACTIVITY
IN
CAT
BRAIN
I61 2 I9 I JI 201 2 .?I
+ 007 t 0.11 .? n.O8 2 0.10 f 0.02
I 54 * “OX 2 02 _+0 I I 14RfOo6 2.52 * 0@5*** 271 +nO4**
204 I 68 2 29 2YB 2.47
2017 t 0.05 + 0.15 + 0.13 * 0.14
2 08 I.56 2.6s 337 2.80
* 0 09 f 0.09 * 0.06’ f0Ov * 0.w
t Activity is expressed in pmol of serotonin formed/mg of protein/min. Statistical analysis was done using Student’s z-test. Number of animals is in brackets. l P < 0.05. **P < 0.01. ***P < 0.001.
Delayed response effect on cat biogenic amine synthesis COSTA, NEFFT, TWANG & NOAI, 19693, the present results thus suggest that the DR training produced a high NA turnover rate in the frontal cortex and amygdala and a low dopamine turnover rate in the frontal cortex and amygdala and a low dopamine turnover rate in the amygdala. Although these biochemical changes occurred at the same time as learning, we are presently unable to relate these specific changes to definite stages or cognitive components of the DR task. Several authors, using other experimental approaches, consider the prefontal cortex @WAC, 1973; WILCOR, 1977;LAWICKA& KONORSKI,1961; WARREN et al., 1962), the striatum (DIVAC, 1968; UNGHERet al., 1966), the subthalamic area (ADEY, WALTER& LINDSLEY,1962) and the cingulum (KORItXZ & ONIANI, 1972) necessary to an efficient performance of a DR task. Although we observed no biochemical changes in the prefrontal cortex and the neostriatum, we are quite aware that some other neurotr~smitters might be respon~ble for the involvement of these structures in a DR task.
(LIN,
The present findings suggest that in normal undrugged cats, learning a DR task involves an active serotoninergic mechanism fulfilled by the serotoninergic mesolimbic system in association with the noradrenergic pontocorticolimbic system and the dopam~ner~~ hmbic system, respectively. Al~ou~ all the neuroanatomical and mono~i~er~c systems taking part in a DR situation are not yet entirely known, the present study gives ad~tional info~ation concerning the effects of acquisition of a DR task on
193
some metabolic events which have been found different from those observed in successive discrimination (J. EVEREIT & A. 0, ROBERGE,unpublished observations) and in ‘go-nogo’ reinforced visual discrimination (A. KIISIKIS & A. G. ROBERGE,~pub~shed ob~rvations). Further inv~tiga~ons are however needed to associate these neurochemical changes to specific aspects of learning prose. For the moment the ~ha~oral i~ibition blatting attention to be focussed on relevant stimuli might be associated to the stimulated S-HT metabolism, whereas the fixation of an adequate orienting response chain might be related to the catecholaminergic metabolism. We are tempted to consider the changes observed to be related to the training undergone by our cats and not to any unspecific variable such as manipulation since in a recent study (J. Evnnnrr & A. G. ROBERGE, unpubIjshed ob~rva~~s) have shown that biochemical measurements in manipulated, untrained cats, placed daily in the ex~~mental box did not differ from those of untrained non-m~ipulat~ cage controls. Moreover, in another experiment, the biochemical assays of cats unable to learn a ‘go-nogo’ visual dis~i~nati~~ task after performing more than 1000 trials differed from those of trained cats, untrained manipulated cats and untrained non-manipulated cats (unpublished data).
~~~ow~e~~e~ents-~is
work was supported by a grant
(MA-6590) of the Medical Research Council of Canada. The technical assistance of C. ROsERoE and G. HUARD is gratefully acknowledged. We also thank the members of the Biom~~ne Center for their helpful assistance and Dr ANNE Kmsms who revised the manuscript.
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1l-121. (Accepted
2
1 September
1980)