Pergamon Press
Life Sciences, Vol . 24, pp . 1971-1978 Printed in the U .S .A .
EVIDENCE FCd2 THE EFFT7CT OF TRYPTOPHAN ON THE PATTERN C~ F LOOD COTiSOMPTI~i IN FREE FE©INa AND FOOD DEPRIVED RATS ' Colin J. Latham and Joha E. Blundell 3 BioPeychology Laboratories, Psychology Department, IInivereity of Leede, Leede LS2 9JT, IIK. (Received in final form April 9,
1979)
Summary The effects of injections of 50 mg/kg L-tryptophan upon meal size, meal frequency, rate of eating sad satiety ratios were measured in rats whose feeding behaviour was monitored continuously over 24-h periods. A number of precautions were taken to minimize the effects of novel or streeeful experimental procedures, to prevent the contamination of behaviour during periods of data collection and to maximise the detection of subtle effects on behaviour. Ia freely-feeding rate tryptophan brought about a significant diminution in the 24-h food intake and significantly reduced meal size . When food deprived rats were tested under similar circumstances tryptophan significantly reduced the size of the first large meal taken after the deprivation period and markedly extended the duration of the poet-meal interval . The conditions adopted is this study to improve the sensitivity of the behavioural assay for feeding have made possible the detection of certain small but clear effects of tryptophan on food consumption in rate . A number of experiments have shown that the pharmacological manipulation of eerotonin (5-HT) metabolism can lead to alterations in food intake (1) . Many of theee data do not provide direct evidence for a physiological role for 5-HT is the control of food consumption but merely demonstrate that alterations in food intake arise ae a consequence of experimental interventions believed to adjust the activity of aerotonergic systems. Additional evidence has led to various suggeetions concerning the possible functions of 5-HT in feeding processes.For example, it has been suggested that serotonin systems may contribute to the day and night control of satiation (2), may modulate other systems regulating body weight (3), may regulate the intake of protein (4) or the relative proportions of protein and carbohydrate (5) or may control specific parameters of feeding behaviour (6) . 1.
This work was carried out during the tenure of a Medical Research Council award to CJL, and was additionally supported by a research grant from Organoa.
2.
Part of this work has been presented at the Spring meeting of the British Pharmacological Society (lq]9) .
3.
Correspondence to JEB.
0024-3205/79/211971-0802 .00/0 Copyright (c) 1979 Pergamon Press Ltd
1972
Tryptophan and Feeding Behavior
Vol . 24, No . 21, 1979
One methodological weakness in the use of pharmacological tools to investigate the relationship between serotonin and behaviour is the tendency of certain chemical manipulations to exert a number of effects within a single neuro transmitter system (7) and to interfere with the activity of different neuronal systems (e .g . 8) . For example, although the immediate precursor of serotonin, 5-hydroxytryptophan has been shown to reduce food intake (9, 10), the convereion of this precursor to 5-HT in non-serotonergic neurons (11, 12) reduces its value as an experimental tool . However, the use of the precursor tryptophan Moreover, it does not appear to be hampered by this non-specific action (11) . has been shown that brain tryptophan and serotonin levels can be increased Although a weak following intra-peritoneal injections of tryptophan (13, 14) . effect of tryptophan loading on food intake has been observed (15-footnote), more recent studies in which tryptophan was administered to 18-h (16) and 24-h (17) food deprived rata failed to demonstrate a significant depression of food consumption during the first two hours after injection. However, one methodological weakness inherent in many tests for potential anoreotic properties of compounds concerns the advisability of measuring the weight of food consumed in a brief measurement period in rats subjected to food deprivation or to cyclic feeding regimes (6) . Although this general procedure has long been regarded as the standard device for screening anorexic agents, the tendency of food deprivation to alter brain neurochemistry (18), to introduce unusual behavioural responses into the animal's repertoire (19) and to undermine the notion of ecological validity (20) may lead to misleading inforThe methodological issue mation about the effect of drugs on feeding (21) . concerns the sensitivity and accuracy of the behavioural assay being used to An alternative to the standard assess potential changes in feeding behaviour. procedure involves the measurement of meal parameters and feeding profiles in non-deprived freely-feeding rats treated so as to minimise the contamination of feeding behaviour by competing activities . This procedure has been widely used in studies on the physiology of feeding (e .g . 22, 23) and has been shown to provide a sensitive technique for characterising the actions of different anorexic compounds (24, 25) and for detecting the effects of drugs normally concealed by less sensitive measuring devices (26) . Accordingly, the present investigation was designed to investigate the effect of tryptophan administration on meal parameters, food intake and feeding profiles in freely feeding rats allowed to eat with a minimum disturbance. In addition, as a check on previous studies, a second experiment examined the action of tryptophan in food deprived animals . Methods Animals and testin environment . The subjects were sixteen Lister strain male hooded rats 330- Og reared on a reversed 12/12 hour light-dark cycle . The animals were housed singly and throughout the course of the experiment all testing was carried out in these home cages. Each cage was placed in a temperature controlled, sound-attenuating ventilated experimental chamber in order to minimize any modification of the animal's behaviour due to disturbance is the laboratory environment . Food in the form of 45 mg precision pellets was available from a pellet-detecting eatometer located in each animal's home cage . Each eatometer consisted of a V-shaped trough in which a light source and photosensor were positioned at opposite ends of the base of the V . Normally a food pellet rested at the base of the V and occluded the photosensor. When the pellet was removed by the rat the light source activated the photosensor which Water in turn led to the delivery of a further pellet from a pellet dispenser. was continuously available from a water bottle attached to the cage wall oppoThe food contained 193ô digestible site to the wall containing the eatometer . protein and 51A~ digestible carbohydrate, and the pellets were eaten with negli-
Vol . 24, No . 21, 1979
Tryptophan and Feeding Behavior
1973
Bible spillage . Data acquisition and analysis . The removal of a pellet from the trough of an eatometer activated the pellet dispenser associated with each eatometer, and also resulted in a discrete pulse being sent to a NOVA 840 mini-computer . A data acquisition programme determined the source and the exact time of arrival of each pulse and these data were recorded on a floppy disc system . At the end of 24 hours of continuous monitoring the disc was changed . This system enabled the analysis of feeding patterns for each 24 hour period to proceed without interfering with the continuous monitoring function of the computer . Data were analysed by a programme developed in our laboratories according to the following criteria . A meal was defined as the removal of 5 or more food pellets separated from any other number of 5 or more pellets by an interval of at least 15 minutes . In reality, this definition of a meal is quite conservative since it has been our experience of working with raw data from these animals under these conditions that meals are rarely less than 25 pellets (l .~.g) in size and are usually separated from each other by 30 minutes or more . In addition to determining the number of meals taken together with the duration (minutes) and size (grams) of each meal, the programme was designed to compute the rate of eating (g/min) within every meal . The rate was calculated from the median inter-pellet interval in order to avoid excessive influence from occasional very long intervals . Moreover, since the beginning and end of each meal were precisely defined, the pre- and post- meal intervals were obviously known and from these data the satiety ratio (size of the meal compared with the dvration of the post-meal interval) was computed . The criteria used in this study are consistent with those usually adopted by other researchers (27) . Procedure and design . Animale in the free-feeding experiment were habituated to the testing environment and allowed to feed from the eatometere for a period of 10 days . During this period animals were handled daily at the time at which injections were later to be given . In addition, for a further period of 3 days animals received an i .p . injection of 19i methylcellulose immediately before the onset of the dark phase of the light-dark cycle . These procedures were carried out in order to fully accustom animals to all aspects of the test procedure, prior to the onset of experimental treatments . This precaution was intended to ensure that any behavioural changes observed during the course of the experiment would be due to the treatments administered rather than to the introduction of novel or stressful procedures . At the end of this period of acclimatization any animals not displaying stable feeding patterns were removed from the experiment . Those animals which demonstrated stable temporal profiles of meal taking over several days entered the teat phase during which the experimental treatments were administered . Oa any test day animals received injections of a control solution of 1% methylcelluloae or 50mg/kg of 1-tryptophan as a fine suspension in the control solution. The i .p .,iajections were given just before the lights were turned off . Each animal served as its own control and received both treatments with 48 h intervening between successive tests . The order of presentation of the treatments was counterbalanced to avoid systematic ordering effects . Following each injection, the animals' patterns of feeding were continuously monitored for 24 hours and from the collected data the computer programme determined the number and sizes of all meals taken, the overall amount of food consumed, the rate of eating within meals and the distribution of eating between the light and dark phases of the 24 h cycle . Following the free-feeding experiment a second study vas carried out using a separate group of animals subjected to a 24 h period of food deprivation prior to each test session . These animals were exposed to a similar 10 day habituation period as the rats in the previous experiment . In addition, for a further week the rata were subjected to a cyclic food deprivation regime of 24h
1974
Tryptophan and Feeding Behavior
Vol . 24, No . 21, 1979
deprivation followed by 24 h free access to food . During this period the rata received injections of the control solution one hour before food was made avail -able at the beginning of the dark period. These procedures were implemented to allow the rats to become accustomed to the stress of the food deprivation and of the injection before the period of data collection . Results Free-feeding experiment . Table 1 shows that under the conditions employed in this study, 1-tryptophan in a dose of 50 mg/kg exerted a significant inhibitory effect on food intake measured over a 24 h period (t=3 .65, df=12, p< .01, 2-tailed test) . Further analysis of food consumption during the phases of the light dark cycle revealed that the inhibitory effect was limited to the dark period which followed immediately upon the administration of tryptophan (t !+ .04 df=12, p< .01, 2-tailed test) . In addition, examination of the data on feeding patterns set out in Table 1 indicates that tryptophan had no effect upon the number of meals taken by the rate but selectively reduced the sizes of the meals consumed during the dark period (t=3 .76, df--12, p< .01, 2-tailed teat) . TABLE 1 Effects of 1-tryptophan on Food Consumption and Meal Parameters in Free-feeding Rate
Measurement
Total food
Number of
Average meal
Rate of eat-
period
treatment
intake
meals (n)
size (g)
ing (g/min)
Whole
Control
26 .9 ± 0.8
13.0 ± 1.1
2.1 ± 0.2
0.41 ± 0.02
24 .3 ± 0.~ *
13.2 ± 0" 9
1.8 ± 0.2
0.40 ± 0.01
17 .6 ± 0.9
7.8 ± 0.6
2.3 ± 0.2
0.41 ± 0.02
15 .3 ± 0.7~
7.9 ± 0.6
1.9 ± 0.2~
o. 40
Control
9" 3 ± 1 .0
5.2 ± 0.9
1.8 ± 0.2
0.41 ± 0.02
Tryptophan
9.0 ± 0.7
5.3 ± 0.6
1.7 ± 0.4
0.40 ± 0.02
(0-24h) Dark . Phase (o-12h) Light Phase (12-24h)
Experimental
Tryptophan Control Tryptophan
(g)
± o. rn
Values in the body of the table are means ± S .E .M . (N--13) . "Different from control value at p t .01. tical testa.
See text for full details of statis-
Tryptophan displayed little or no effect upon the rate of eating within meals. A more detailed examination of the action of tryptophan on meal size revealed that this treatment exerted an effect only during the first 4 h after administration . Table 2 indicates that when the data were partitioned into 2-h periods, tryptophan significantly reduced meal size during the first (t=3 .08, df--12, p < .Ol, 2-tailed test) and second (t=4 .54, df=12, p< .01, 2tailed test) 2-h periods but had no effect on meal size between 4 and 6 hours after administration . Tryptophan treatment produced no effect on the time
Vol . 24, No . 21, 1979
1975
Tryptophan ând Feeding Behavior
interval between injection and the start of the first meal . TABLE 2 Effect of 1-tryptophan on meal size in free-feeding rate during the first 6houra after administration
Measurement period (h)
Experimental treatment Control Tryptophan Values are means ± S .E .M .
0 - 2
2 - 4
4 - 6
2 .1 ± 0 .3
2 .3 ± 0 .3
2 .3 ± 0 .2
1 .4 ± 0 .2*
1 .7 ± 0 .2+
2 .5 ± 0 .3
(N=13) rt different from control at p< .O1
Deprivation experiment . Ia contrast to the results of the previous experiment, Table 3 shows that following a period of food deprivation tryptophan displayed no effect on food intake or meal parameters measured during the dark and TABLE Effects of 1-tryptophan on food intake and m eal~arameters in 24-h food-deprived rata .
Measurement period
Whole day (0 .24h) Dark Phase (o-12h) Light Phase (12-24h)
Experimental treatment
Food intake (g)
Number of meals (n)
Average meal size (g')
Rate of eating (g/min)
Control
31 .1 ± 1 .2
10.6 ± 0 .5
2 .9 ± 0 .3
0 .28±0 .02
Tryptophaa
30 .7 ± 1 .1
11 .3 ± 0 .5
2 .8 ± 0 .2
0 .31±0 .03
Control
26 .9 ± 1 .4
7 " 7 ± 0 .4
3 .5 ± 0.3
0 .27±0 .01
25 .6 ± 1 .1
8 .5 ± 0 .3
3 " 0 ± 0.4
0 .28±0 .04
4 .2 ± 0 .5
2 .8 ± 0 .5
1 .5 ± 0 .2
0.29±0 .02
5 .4 ± 0 .4
2 .8 ± 0 .4
1 .9 ± 0 .2
0.33±0 .02
Tryptophan Control Tryptophan
Values given are manna ± S .E .M . (N=7) light phaees or over the full 24-h period . However, is the carefully controlled conditions of thin study a abort-term effect of tryptophan on feeding wen detected . Following a period of food deprivation rats initially consume one very large meal and Table 4 shown that tryptophan produced a small but statistically significant reduction is the size of this first meal (t=2 .51, df=6, p~
1976
.05,
Tryptophan and Feeding Behavior
Vol . 24, No . 21, 1979
2-tailed teat) . TABLE 4 Effect of 1-tryptophan on the first meal following 24-h food deprivation
Meal parameters Experimental treatment
Control 1 9 Tryptophan
Meal size
(g)
11 .1 ± 0.8
0.52 ± 0.11
9.3 ± 0.8 *
Values given are means ± S.E .M .
Post-meal interval (h)
.04 ± 0.19*
Satiety ratio 21 .7 ± 3.2 .6 ± 0.9**
(N=7)
* significantly different from control at p < .05 or p c .01 (**), 2-tailed ttest .
In addition tryptophan brought about a marked elongation of the interval between the end of the first meal and the initiation of the second meal (t-2 .50, df=6, pc .05, 2-tailed teat) . In turn, computation of the satiety ratio Prom meal size and poet-meal interval showed that tryptophan exerted a considerable effect on this parameter (t=3 .78, df--6, pc .01, 2-tailed test) . Discussion These experiments have demonstrated that injections of 1-tryptophan can be shown to have an effect upon the feeding behaviour of rata . The effect of this compound on food intake ie not massive but it definitely existé and can be rev ealed by a carefully conducted behavioural teat . In considering the detection of the effects of drugs upon food intake, two methodological issues are worth mentioning . First, food consumption is a manifestation of behaviour that may be easily disrupted by the introduction of novel or stressful procedures such as injections or handling, or by activities going on outside of the animal's cage but within the laboratory environment . Under such circumstances feeding behaviour may be disturbed to such an extent as to mask the effect of any additional experimental treatment . Second, in drug experiments food intake is often measured following a period of food deprivation during which time the animal may be in a highly reactive state and may show anomolous reactions toward any experimental intrusion . Although the nature of the interaction between these two seta of conditions is not known, it can be argued that the overall affect may be to magnify the effect on food intake of a powerful drug and to mask the effect of a weakly acting compound . These comments are intended to draw attention to the sensitivity of the behavioural assays commonly employed for the assessment of the effects of . drugs on food intake . Since great care is usually taken to ensure that biochemical assays are sensitive and free from contaminating influences in order to detect subtle effects of drugs on brain tissues, it seems appropriate that equal care should be exercised in the construction of behavioural assays . In the present study a number of procedures were adopted to habituate animals to all features of the experimental regime and to prevent the contamination of feeding behaviour by environmental disturbances . These
Vol . 24, No . 21, 1979
Tryptophan and Feeding Behavior
1977
procedures were intended to maximise the sensitivity of this behavioural assay. IInder the conditions of this study, tryptophan displayed an effect on food intake for 12-hour and 24-hour periods in free-feeding rate but no such effects were detected in the intakes of rats subjected to food deprivation. This sugg eets that the free-feeding animal constitutes a more-sensitive preparation for the detection of weak drug effects than does a deprived rat . In addition to the effect of tryptophan on the weight of food consumed, the first experiment further reveals that tryptophan administration curtailed eating by reducing the size of meals rather than by reducing meal frequency. This finding ie consistent with the effect brought about by other agents believed to influence serotoain metabolism including fenYluramine (24, 25, 28), ~2G 6582 (6) and 5-hydroxy-tryptophan (Blundell, Ciotti and Latham - in preparation), and is in keeping with the idea that pharmacological manipulation of serotonin systems leads to caasistent effects on the pattern of food consumption. Data from the first experiment indicated that the action of tryptophan was limited to about the first four hours after administration . For this reason, a close analysis was carried out of the early profile of feeding following food deprivation. Although previous studies have failed to observe an effect of tryptophan on food intake during this particular time period, the present experiment showed that tryptophan reduced the size of the first very large meal consumed after deprivation and also prolonged the post-meal interval . Taken together, these two effects contributed to as increased satiety ratio indicating that tryptophan administration increased the capacity of the meal to postpone further food consumption. It seems likely that the detection of these effects was made possible through the improved sensitivity of the behavioural assay compared with previous techniques . Although these findings do not provide any direct evidence on a possible functional role of serotonin in the control of feeding behaviour, they have contributed to the fund of information which sow exists on the relationship be tween fluctuations in food intake and adjustments in aerotonin metabolism (29) . Consideration should be given to the question of whether the effects of tryptophan administration on meal size and satiety ratio xere brought about by alterâtions in the concentration of brain or peripheral stores of serotonin and, in turn, by action upon central systems influencing food intake or peripheral proces~es concerned with gastric emptying or gut motility . At the moment the mechanisms through which acute tryptophan administration leads to subtle adjustments in meal parameters are not known. Acknowledgements : The authors are indebted to Peter Higginbotham for the development of the computer programmes used for data ânalysis . We are grateful to Mavis Walton for help with the preparation of the manuscript . References 1. 2. 3. 4. 5. 6. 7. 8.
J .E. BLUI~ELL, Int. J. Obesity , _1, 15-42 (1970 . B .G . HOEBEL, . Ann. Rev. Pharmacol . Toxicol . ~, 605-621 (19']7) . D .V . COSCINA, In : Anorexia Nervosa, ed . R .A . Vigersl~y, 9'7-107, Raven Press, New York (1977 . D .V .M . ASHLEY and G.H . ANDERSON . J. Nutr . 1~, 1412-1421 (19'J5) . J.D . FERNSTROM and R .J . WQRTMAN. In : Advances in P cho harmacolo Vol. ll, Serotonin : New Vistas, ads. E. Costa and M. Sandier, 133-1 2, Raven Press, New York 197 J.E . BLIINDELL and C.J . " LATHAM . In : Central Mechanisms of Anorexic s ads. S. Garretini and R. Samanin, 83-109, Raven Press, New York 19'J A.J . MANDELL, Ann. Rev. Pharmacol. Toxicol. 18, 461-493 (1978) . S .H . SNYDER, E .G . ~n~nN and M .J . ICOHAR . In : Serotonin and Behaviour
1978
Tryptophan and Feeding Behavior
Vol . 24, No . 21, 1979
ede . J . Barchae and E. Usdin, 9'7-108, Academic Preen, New York (1973) . 9 . D . JOYCE and N . MROSOVSRY, Psychopharmacologie ~, 417-423 (1964) . 10 . J .E . BLUNDELL and M .B . LESB~I, J . Pharm . Pharmacol . 27, 31-37 (1975) . 11 . A .T .B . MOIR and D . F]CCLESTON, J . Neurochem . ~, 1093-1108 (1968) . 12 . D .V . COSCINA, J .J . WARSH, D .D . GODSE and H .C . STANCER, Rea . Comet. Chem . Pathol . Pharmacol . ~, 617-620 (1974) . 13 . D . ECCLESTON, G .W . ASHCRC~T and T .B .B . CRAWFORD, J . Neurochem . _12, 493-503 " (1965) . 14 . J .D . FERNSTRCM and R .J . WURTMAN, Science 1~, 149-152 (1971) . 15 . J .D . FERNSTR~I and R .J . WURTMAN, Science 17 , 414-416 (1972) " 16 . A .M . HARREIT and L . MCSHARRY, J . Pharm. Pharmaco1 .27, 889-895 (1975) . 17 . S .B . WEINBrrRGER, S . KNAPP and A .J . MANDELL, Life Sci . _22, 1595-1602 (1978) . 18 . J . PEREZ-CRUET, A . TAGLIAMONTE, P . TAGLIAMONTE and G .L . GESSA, Life Sci . 11, 31-39 (1972) . 19 . G . MQRAH, Psychol . Bull . 82, 543-557 (1975) . 20. G .H . COLLIER, E. HIRSCH andP .H . HAMr "T N, Phyeiol . Behav . 9, 705-716 (1972) . 21 . J .E . BLUI~ELL and C .J . LATHAM . In : Chemical Influences on Behaviour, eds. S . Cooper and K. Brown, 201-254, Academic Press, London 1979 . 22 . C .P . RICHTER, Quart . Rev . Biol . 2, 3d7-343 (1927) " 23. J . LE MAGNEN and S . TALL~i, J . PY~yeiol . (Parie ) 60, 143-154 (1968) . 28, 47124 . J .E . BLUP~ELL, C .J . LATHAM and M .B . LESFIFM, J . Pharmac . Pharmacol . _ 477 (1976) . 25 . R .F . DAMES, Some neurochemical and physiological factors controlling free feeding patterns in the rat . Unpublished Ph .D . thesis McCÜll University (1876) . 26 . J . E . BLUNDELL, E . TCI+BROS, P .J . ROGERS and C .J . LATHAM, Prog . Neuropharma col . (19']9) - in preen . of Feedi Control, ed . 27 . .PANKSEPP, J In : Hun r Model~ Co utable Theo D .A . Booth, 144-1 Academic Press, London 19'75 . 28 . J .E . BLUNDELL and M .B . LESBIIi . In : Recent Advances in Obeeit Research, ed . A . Howard, 368-371 . Newman, London 1975 . 29 . J . E . BLUNDELL, In : Serotonin in Health and Disease, Vol . 5 . Ed . W .B .Eesman Spectrum, New fork 1979 .
Life Sciences, Vol . 24, pp . 1971-1978 Printed in the U .S .A .
EVIDENCE FCd2 THE EFFT7CT OF TRYPTOPHAN ON THE PATTERN C~ F LOOD COTiSOMPTI~i IN FREE FE©INa AND FOOD DEPRIVED RATS ' Colin J. Latham and Joha E. Blundell 3 BioPeychology Laboratories, Psychology Department, IInivereity of Leede, Leede LS2 9JT, IIK. (Received in final form April 9,
1979)
Summary The effects of injections of 50 mg/kg L-tryptophan upon meal size, meal frequency, rate of eating sad satiety ratios were measured in rats whose feeding behaviour was monitored continuously over 24-h periods. A number of precautions were taken to minimize the effects of novel or streeeful experimental procedures, to prevent the contamination of behaviour during periods of data collection and to maximise the detection of subtle effects on behaviour. Ia freely-feeding rate tryptophan brought about a significant diminution in the 24-h food intake and significantly reduced meal size . When food deprived rats were tested under similar circumstances tryptophan significantly reduced the size of the first large meal taken after the deprivation period and markedly extended the duration of the poet-meal interval . The conditions adopted is this study to improve the sensitivity of the behavioural assay for feeding have made possible the detection of certain small but clear effects of tryptophan on food consumption in rate . A number of experiments have shown that the pharmacological manipulation of eerotonin (5-HT) metabolism can lead to alterations in food intake (1) . Many of theee data do not provide direct evidence for a physiological role for 5-HT is the control of food consumption but merely demonstrate that alterations in food intake arise ae a consequence of experimental interventions believed to adjust the activity of aerotonergic systems. Additional evidence has led to various suggeetions concerning the possible functions of 5-HT in feeding processes.For example, it has been suggested that serotonin systems may contribute to the day and night control of satiation (2), may modulate other systems regulating body weight (3), may regulate the intake of protein (4) or the relative proportions of protein and carbohydrate (5) or may control specific parameters of feeding behaviour (6) . 1.
This work was carried out during the tenure of a Medical Research Council award to CJL, and was additionally supported by a research grant from Organoa.
2.
Part of this work has been presented at the Spring meeting of the British Pharmacological Society (lq]9) .
3.
Correspondence to JEB.
0024-3205/79/211971-0802 .00/0 Copyright (c) 1979 Pergamon Press Ltd
1972
Tryptophan and Feeding Behavior
Vol . 24, No . 21, 1979
One methodological weakness in the use of pharmacological tools to investigate the relationship between serotonin and behaviour is the tendency of certain chemical manipulations to exert a number of effects within a single neuro transmitter system (7) and to interfere with the activity of different neuronal systems (e .g . 8) . For example, although the immediate precursor of serotonin, 5-hydroxytryptophan has been shown to reduce food intake (9, 10), the convereion of this precursor to 5-HT in non-serotonergic neurons (11, 12) reduces its value as an experimental tool . However, the use of the precursor tryptophan Moreover, it does not appear to be hampered by this non-specific action (11) . has been shown that brain tryptophan and serotonin levels can be increased Although a weak following intra-peritoneal injections of tryptophan (13, 14) . effect of tryptophan loading on food intake has been observed (15-footnote), more recent studies in which tryptophan was administered to 18-h (16) and 24-h (17) food deprived rata failed to demonstrate a significant depression of food consumption during the first two hours after injection. However, one methodological weakness inherent in many tests for potential anoreotic properties of compounds concerns the advisability of measuring the weight of food consumed in a brief measurement period in rats subjected to food deprivation or to cyclic feeding regimes (6) . Although this general procedure has long been regarded as the standard device for screening anorexic agents, the tendency of food deprivation to alter brain neurochemistry (18), to introduce unusual behavioural responses into the animal's repertoire (19) and to undermine the notion of ecological validity (20) may lead to misleading inforThe methodological issue mation about the effect of drugs on feeding (21) . concerns the sensitivity and accuracy of the behavioural assay being used to An alternative to the standard assess potential changes in feeding behaviour. procedure involves the measurement of meal parameters and feeding profiles in non-deprived freely-feeding rats treated so as to minimise the contamination of feeding behaviour by competing activities . This procedure has been widely used in studies on the physiology of feeding (e .g . 22, 23) and has been shown to provide a sensitive technique for characterising the actions of different anorexic compounds (24, 25) and for detecting the effects of drugs normally concealed by less sensitive measuring devices (26) . Accordingly, the present investigation was designed to investigate the effect of tryptophan administration on meal parameters, food intake and feeding profiles in freely feeding rats allowed to eat with a minimum disturbance. In addition, as a check on previous studies, a second experiment examined the action of tryptophan in food deprived animals . Methods Animals and testin environment . The subjects were sixteen Lister strain male hooded rats 330- Og reared on a reversed 12/12 hour light-dark cycle . The animals were housed singly and throughout the course of the experiment all testing was carried out in these home cages. Each cage was placed in a temperature controlled, sound-attenuating ventilated experimental chamber in order to minimize any modification of the animal's behaviour due to disturbance is the laboratory environment . Food in the form of 45 mg precision pellets was available from a pellet-detecting eatometer located in each animal's home cage . Each eatometer consisted of a V-shaped trough in which a light source and photosensor were positioned at opposite ends of the base of the V . Normally a food pellet rested at the base of the V and occluded the photosensor. When the pellet was removed by the rat the light source activated the photosensor which Water in turn led to the delivery of a further pellet from a pellet dispenser. was continuously available from a water bottle attached to the cage wall oppoThe food contained 193ô digestible site to the wall containing the eatometer . protein and 51A~ digestible carbohydrate, and the pellets were eaten with negli-
Vol . 24, No . 21, 1979
Tryptophan and Feeding Behavior
1973
Bible spillage . Data acquisition and analysis . The removal of a pellet from the trough of an eatometer activated the pellet dispenser associated with each eatometer, and also resulted in a discrete pulse being sent to a NOVA 840 mini-computer . A data acquisition programme determined the source and the exact time of arrival of each pulse and these data were recorded on a floppy disc system . At the end of 24 hours of continuous monitoring the disc was changed . This system enabled the analysis of feeding patterns for each 24 hour period to proceed without interfering with the continuous monitoring function of the computer . Data were analysed by a programme developed in our laboratories according to the following criteria . A meal was defined as the removal of 5 or more food pellets separated from any other number of 5 or more pellets by an interval of at least 15 minutes . In reality, this definition of a meal is quite conservative since it has been our experience of working with raw data from these animals under these conditions that meals are rarely less than 25 pellets (l .~.g) in size and are usually separated from each other by 30 minutes or more . In addition to determining the number of meals taken together with the duration (minutes) and size (grams) of each meal, the programme was designed to compute the rate of eating (g/min) within every meal . The rate was calculated from the median inter-pellet interval in order to avoid excessive influence from occasional very long intervals . Moreover, since the beginning and end of each meal were precisely defined, the pre- and post- meal intervals were obviously known and from these data the satiety ratio (size of the meal compared with the dvration of the post-meal interval) was computed . The criteria used in this study are consistent with those usually adopted by other researchers (27) . Procedure and design . Animale in the free-feeding experiment were habituated to the testing environment and allowed to feed from the eatometere for a period of 10 days . During this period animals were handled daily at the time at which injections were later to be given . In addition, for a further period of 3 days animals received an i .p . injection of 19i methylcellulose immediately before the onset of the dark phase of the light-dark cycle . These procedures were carried out in order to fully accustom animals to all aspects of the test procedure, prior to the onset of experimental treatments . This precaution was intended to ensure that any behavioural changes observed during the course of the experiment would be due to the treatments administered rather than to the introduction of novel or stressful procedures . At the end of this period of acclimatization any animals not displaying stable feeding patterns were removed from the experiment . Those animals which demonstrated stable temporal profiles of meal taking over several days entered the teat phase during which the experimental treatments were administered . Oa any test day animals received injections of a control solution of 1% methylcelluloae or 50mg/kg of 1-tryptophan as a fine suspension in the control solution. The i .p .,iajections were given just before the lights were turned off . Each animal served as its own control and received both treatments with 48 h intervening between successive tests . The order of presentation of the treatments was counterbalanced to avoid systematic ordering effects . Following each injection, the animals' patterns of feeding were continuously monitored for 24 hours and from the collected data the computer programme determined the number and sizes of all meals taken, the overall amount of food consumed, the rate of eating within meals and the distribution of eating between the light and dark phases of the 24 h cycle . Following the free-feeding experiment a second study vas carried out using a separate group of animals subjected to a 24 h period of food deprivation prior to each test session . These animals were exposed to a similar 10 day habituation period as the rats in the previous experiment . In addition, for a further week the rata were subjected to a cyclic food deprivation regime of 24h
1974
Tryptophan and Feeding Behavior
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deprivation followed by 24 h free access to food . During this period the rata received injections of the control solution one hour before food was made avail -able at the beginning of the dark period. These procedures were implemented to allow the rats to become accustomed to the stress of the food deprivation and of the injection before the period of data collection . Results Free-feeding experiment . Table 1 shows that under the conditions employed in this study, 1-tryptophan in a dose of 50 mg/kg exerted a significant inhibitory effect on food intake measured over a 24 h period (t=3 .65, df=12, p< .01, 2-tailed test) . Further analysis of food consumption during the phases of the light dark cycle revealed that the inhibitory effect was limited to the dark period which followed immediately upon the administration of tryptophan (t !+ .04 df=12, p< .01, 2-tailed test) . In addition, examination of the data on feeding patterns set out in Table 1 indicates that tryptophan had no effect upon the number of meals taken by the rate but selectively reduced the sizes of the meals consumed during the dark period (t=3 .76, df--12, p< .01, 2-tailed teat) . TABLE 1 Effects of 1-tryptophan on Food Consumption and Meal Parameters in Free-feeding Rate
Measurement
Total food
Number of
Average meal
Rate of eat-
period
treatment
intake
meals (n)
size (g)
ing (g/min)
Whole
Control
26 .9 ± 0.8
13.0 ± 1.1
2.1 ± 0.2
0.41 ± 0.02
24 .3 ± 0.~ *
13.2 ± 0" 9
1.8 ± 0.2
0.40 ± 0.01
17 .6 ± 0.9
7.8 ± 0.6
2.3 ± 0.2
0.41 ± 0.02
15 .3 ± 0.7~
7.9 ± 0.6
1.9 ± 0.2~
o. 40
Control
9" 3 ± 1 .0
5.2 ± 0.9
1.8 ± 0.2
0.41 ± 0.02
Tryptophan
9.0 ± 0.7
5.3 ± 0.6
1.7 ± 0.4
0.40 ± 0.02
(0-24h) Dark . Phase (o-12h) Light Phase (12-24h)
Experimental
Tryptophan Control Tryptophan
(g)
± o. rn
Values in the body of the table are means ± S .E .M . (N--13) . "Different from control value at p t .01. tical testa.
See text for full details of statis-
Tryptophan displayed little or no effect upon the rate of eating within meals. A more detailed examination of the action of tryptophan on meal size revealed that this treatment exerted an effect only during the first 4 h after administration . Table 2 indicates that when the data were partitioned into 2-h periods, tryptophan significantly reduced meal size during the first (t=3 .08, df--12, p < .Ol, 2-tailed test) and second (t=4 .54, df=12, p< .01, 2tailed test) 2-h periods but had no effect on meal size between 4 and 6 hours after administration . Tryptophan treatment produced no effect on the time
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Tryptophan ând Feeding Behavior
interval between injection and the start of the first meal . TABLE 2 Effect of 1-tryptophan on meal size in free-feeding rate during the first 6houra after administration
Measurement period (h)
Experimental treatment Control Tryptophan Values are means ± S .E .M .
0 - 2
2 - 4
4 - 6
2 .1 ± 0 .3
2 .3 ± 0 .3
2 .3 ± 0 .2
1 .4 ± 0 .2*
1 .7 ± 0 .2+
2 .5 ± 0 .3
(N=13) rt different from control at p< .O1
Deprivation experiment . Ia contrast to the results of the previous experiment, Table 3 shows that following a period of food deprivation tryptophan displayed no effect on food intake or meal parameters measured during the dark and TABLE Effects of 1-tryptophan on food intake and m eal~arameters in 24-h food-deprived rata .
Measurement period
Whole day (0 .24h) Dark Phase (o-12h) Light Phase (12-24h)
Experimental treatment
Food intake (g)
Number of meals (n)
Average meal size (g')
Rate of eating (g/min)
Control
31 .1 ± 1 .2
10.6 ± 0 .5
2 .9 ± 0 .3
0 .28±0 .02
Tryptophaa
30 .7 ± 1 .1
11 .3 ± 0 .5
2 .8 ± 0 .2
0 .31±0 .03
Control
26 .9 ± 1 .4
7 " 7 ± 0 .4
3 .5 ± 0.3
0 .27±0 .01
25 .6 ± 1 .1
8 .5 ± 0 .3
3 " 0 ± 0.4
0 .28±0 .04
4 .2 ± 0 .5
2 .8 ± 0 .5
1 .5 ± 0 .2
0.29±0 .02
5 .4 ± 0 .4
2 .8 ± 0 .4
1 .9 ± 0 .2
0.33±0 .02
Tryptophan Control Tryptophan
Values given are manna ± S .E .M . (N=7) light phaees or over the full 24-h period . However, is the carefully controlled conditions of thin study a abort-term effect of tryptophan on feeding wen detected . Following a period of food deprivation rats initially consume one very large meal and Table 4 shown that tryptophan produced a small but statistically significant reduction is the size of this first meal (t=2 .51, df=6, p~
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.05,
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Vol . 24, No . 21, 1979
2-tailed teat) . TABLE 4 Effect of 1-tryptophan on the first meal following 24-h food deprivation
Meal parameters Experimental treatment
Control 1 9 Tryptophan
Meal size
(g)
11 .1 ± 0.8
0.52 ± 0.11
9.3 ± 0.8 *
Values given are means ± S.E .M .
Post-meal interval (h)
.04 ± 0.19*
Satiety ratio 21 .7 ± 3.2 .6 ± 0.9**
(N=7)
* significantly different from control at p < .05 or p c .01 (**), 2-tailed ttest .
In addition tryptophan brought about a marked elongation of the interval between the end of the first meal and the initiation of the second meal (t-2 .50, df=6, pc .05, 2-tailed teat) . In turn, computation of the satiety ratio Prom meal size and poet-meal interval showed that tryptophan exerted a considerable effect on this parameter (t=3 .78, df--6, pc .01, 2-tailed test) . Discussion These experiments have demonstrated that injections of 1-tryptophan can be shown to have an effect upon the feeding behaviour of rata . The effect of this compound on food intake ie not massive but it definitely existé and can be rev ealed by a carefully conducted behavioural teat . In considering the detection of the effects of drugs upon food intake, two methodological issues are worth mentioning . First, food consumption is a manifestation of behaviour that may be easily disrupted by the introduction of novel or stressful procedures such as injections or handling, or by activities going on outside of the animal's cage but within the laboratory environment . Under such circumstances feeding behaviour may be disturbed to such an extent as to mask the effect of any additional experimental treatment . Second, in drug experiments food intake is often measured following a period of food deprivation during which time the animal may be in a highly reactive state and may show anomolous reactions toward any experimental intrusion . Although the nature of the interaction between these two seta of conditions is not known, it can be argued that the overall affect may be to magnify the effect on food intake of a powerful drug and to mask the effect of a weakly acting compound . These comments are intended to draw attention to the sensitivity of the behavioural assays commonly employed for the assessment of the effects of . drugs on food intake . Since great care is usually taken to ensure that biochemical assays are sensitive and free from contaminating influences in order to detect subtle effects of drugs on brain tissues, it seems appropriate that equal care should be exercised in the construction of behavioural assays . In the present study a number of procedures were adopted to habituate animals to all features of the experimental regime and to prevent the contamination of feeding behaviour by environmental disturbances . These
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Tryptophan and Feeding Behavior
1977
procedures were intended to maximise the sensitivity of this behavioural assay. IInder the conditions of this study, tryptophan displayed an effect on food intake for 12-hour and 24-hour periods in free-feeding rate but no such effects were detected in the intakes of rats subjected to food deprivation. This sugg eets that the free-feeding animal constitutes a more-sensitive preparation for the detection of weak drug effects than does a deprived rat . In addition to the effect of tryptophan on the weight of food consumed, the first experiment further reveals that tryptophan administration curtailed eating by reducing the size of meals rather than by reducing meal frequency. This finding ie consistent with the effect brought about by other agents believed to influence serotoain metabolism including fenYluramine (24, 25, 28), ~2G 6582 (6) and 5-hydroxy-tryptophan (Blundell, Ciotti and Latham - in preparation), and is in keeping with the idea that pharmacological manipulation of serotonin systems leads to caasistent effects on the pattern of food consumption. Data from the first experiment indicated that the action of tryptophan was limited to about the first four hours after administration . For this reason, a close analysis was carried out of the early profile of feeding following food deprivation. Although previous studies have failed to observe an effect of tryptophan on food intake during this particular time period, the present experiment showed that tryptophan reduced the size of the first very large meal consumed after deprivation and also prolonged the post-meal interval . Taken together, these two effects contributed to as increased satiety ratio indicating that tryptophan administration increased the capacity of the meal to postpone further food consumption. It seems likely that the detection of these effects was made possible through the improved sensitivity of the behavioural assay compared with previous techniques . Although these findings do not provide any direct evidence on a possible functional role of serotonin in the control of feeding behaviour, they have contributed to the fund of information which sow exists on the relationship be tween fluctuations in food intake and adjustments in aerotonin metabolism (29) . Consideration should be given to the question of whether the effects of tryptophan administration on meal size and satiety ratio xere brought about by alterâtions in the concentration of brain or peripheral stores of serotonin and, in turn, by action upon central systems influencing food intake or peripheral proces~es concerned with gastric emptying or gut motility . At the moment the mechanisms through which acute tryptophan administration leads to subtle adjustments in meal parameters are not known. Acknowledgements : The authors are indebted to Peter Higginbotham for the development of the computer programmes used for data ânalysis . We are grateful to Mavis Walton for help with the preparation of the manuscript . References 1. 2. 3. 4. 5. 6. 7. 8.
J .E. BLUI~ELL, Int. J. Obesity , _1, 15-42 (1970 . B .G . HOEBEL, . Ann. Rev. Pharmacol . Toxicol . ~, 605-621 (19']7) . D .V . COSCINA, In : Anorexia Nervosa, ed . R .A . Vigersl~y, 9'7-107, Raven Press, New York (1977 . D .V .M . ASHLEY and G.H . ANDERSON . J. Nutr . 1~, 1412-1421 (19'J5) . J.D . FERNSTROM and R .J . WQRTMAN. In : Advances in P cho harmacolo Vol. ll, Serotonin : New Vistas, ads. E. Costa and M. Sandier, 133-1 2, Raven Press, New York 197 J.E . BLIINDELL and C.J . " LATHAM . In : Central Mechanisms of Anorexic s ads. S. Garretini and R. Samanin, 83-109, Raven Press, New York 19'J A.J . MANDELL, Ann. Rev. Pharmacol. Toxicol. 18, 461-493 (1978) . S .H . SNYDER, E .G . ~n~nN and M .J . ICOHAR . In : Serotonin and Behaviour
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