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Effects of benzodiazepines and naloxone on food intake and food preference in the rat
Appetite 1980, 1,215- 224
Effects of Benzodiazepines and Naloxone on Food Intake and Food Preference in the Rat SANDRA E. FILE Department of Pharma.cology, The School of Pharmacy, University of London
After four days of administration, chlord.iazepoxide (5 mg/ kg) increased the weight gain of rats on ad libitum food and water, whereas chlordiazepoxide (7'5 mg/kg) and lorazepam (0·25 and 0·5 mg/kg) decreased it. After a period of food deprivation, chlordiazepoxide (7'5 mg/kg) and lorazepam (0'5 mg/kg) increased food consumption after!, 2 and 6 h. In a food preference test chlordiazepoxide (5 mg/kg) enhanced consumption of chocolate chips, whereas lorazepam (0'25 and q'5 mg/kg) enhanced consumption of familiar chow. Chlordiazepoxide (5 and 7·5 mg/kg) enhanced consumption of unfamiliar chow, whether this was preferred or not, whereas lorazepam (0'25 and 0·5 mg/ kg) enhanced consumption of familiar chow. Naloxone (4 mg/kg) reduced food intake in the first! h after injection 'and reduced consumption of unfamiliar chow . Naloxone (2 and 4 mg/ kg) abolished consumption of chocolate chips in the food preference test.
Several authors have reported that both acute and chronic treatment with chlordiazepoxide increases the amount of food eaten by rats, following a period of deprivation (Iwahara & Iwasaki, 1969; Niki, 1965; Randall, Schallek, Heise, Keith & Bagdon, 1960). These effects have been found over the dose-range 5 to 50 mg/kg. Several benzodiazepines (after acute and chronic administration) have been found to increase the food intake in non-deprived rats (Sou brie, Kulkarni, Simon & Boissier, 1975; Wise & Dawson, 1974) and to enhance feeding elicited by hypothalamic stimulation (Watson, Short, Huenink & Hartman, 1980). Acute administ·ration of several benzodiazepines enhances the consumption of palatable foods, e.g. sweetened milk (Johnson, 1978; Margules & Stein, 1967; Poschel, 1971) and "free" food, as opposed to that obtained by lever-pressing (Tye, Nicholas & Morgan, 1975). In the experiments using sweetened milk this was both a palatable food and a novel food and it is therefore impossible to say from the results of these ~xperiments whether benzodiazepines enhance the consumption of novel food if this is not also palatable. The results from one experiment, however, suggest that the effects on consumption of novel food may be dose-dependent. Cooper and McClelland (1980) found that acute administration of 5 and 10 mg/ kg chlordiazepoxide increased the time that rats spent eating familiar food, but that at 15 mg/ kg the rats preferred novel food.
This work was supported by a Medical Research Council project grant and technical assistance was provided by L. Rooney. The video equipment was purchased with a grant from the Central Research Fund of London University. I am grateful to Endo Laboratories, Wyeth Laboratories and Roche Products for their generous gifts of drugs, and to Dr L. J. Herberg for the gift of diet Q. Requests for reprints should be sent to Sandra E. File, Department of Pharmacology, The School of Pharmacy, University of London. 29/ 39 Brunswick Square, London WCI N lAX. 0195- 6663/80/030215 + 10 $02'00/0
co 1980 Academic Press Inc. (London) Limited
216
s.
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Similarly, a low dose of diazepam (2·5 mg/kg) increased the time spent eating familiar food, whereas 5 mg/kg increased the time spent eating novel food (Cooper, Crummy & Skan, 1977). Contrasting effects of low and high doses of chlordiazepoxide have also been found with tail-pinch induced eating: 5 mg/kg reducing the latency to start eating and increasing the duration of eating and 20 mg/kg having the opposite effects (Robbins, Phillips & Sahakian, 1977). For a discussion of the dose-dependent effects of benzodiazepines on the selection between novel and familiar foods, see Cooper (1980). The present study was designed to investigate the effects of chronic (5 days) administration of low doses of chlordiazepoxide and lorazepam on food intake after deprivation and on food preference. So far the effects of benzodiazepines on food preference seem to have been investigated only after acute administration. Acutely, the benzodiazepines have a sedative action (see Greenblatt & Shader, 1977), whereas after a few days of administration there is tolerance to this effect, seen both in lever-pressing (Cook & Sepinwall, 1975; Stein & Berger, 1971) and in spontaneous motor activity and social interaction (File, 1979). Low doses of benzodiazepines were selected to be comparable with the doses used clinically. A five day pretreatment period was selected since tolerance to the sedative effects of low doses of benzodiazepines has been shown after this period (File, 1979, 1980). Thus after five days of treatment any benzodiazepine effects on feeding should not be secondary to a reduction in motor activity . In order to check the levels of motor activity following the dosing regime used in this study, spontaneous motor activity was measured in the test box used for the food preference test. After five days of administration benzodiizepines produce an anxiolytic profile in animal tests of anziety (e.g. Cook & Sepinwall, 1975; File & Hyde, 1978, 1979). It has been suggested that enhanced food consumption can be used to detect anxiolytic actions in benzodiazepines (Poschel, 1971), but it seems unlikely that all of the effects of benzodiazepines on feeding are secondary to an anxiolytic action (see Cooper, 1980). It is hoped that by in vestigating the effects of chronic benzodiazepines on several aspects of feeding that this study will help to illuminate whether these drugs do have any actions on feeding per se. For comparison, the effects of naloxone on food intake and preference were also investigated. Naloxone has been reported to reduce food intake in deprived rats (Brands, Thornhill, Hirst & Gowdey, 1979; Brown & Holtzman, 1979), having a greater effect in hypothalamically lesioned and obese rats than in normals (King, Castellanos, Kastin, Berzas, Mauk, Olson & Olson, 1979) and in genetically obese mice than normals (Margules, Moisset, Louis, Shibuya & Pert, 1978). It has also been found to reduce eating in obese patients suffering from the Prader-Willi syndrome (Kyriakides, Silverstone, Jeffcoate & Laurance, 1980). In non-deprived rats naloxone has been found to decrease consumption of palatable food (King et al., 1979). The doses of naloxone chosen for this study were within the range previously found to reduce food intake and the effects of these doses on motor activity were also measured . Naloxone has been reported to increase anxiety in man (Grevert & Goldstein, 1977) and in the social interaction test of anxiety it had effects opposite to those of the benzodiazepines (File, in press). The present study therefore explored whether the effects of naloxone and benzodiazepines on feeding were opposite in nature . METHODS
Animals Male hooded rats (from Olac Ltd., Bicester), weighing 250-300 g, were maintained on ad libitum food and water, except for overnight deprivation of food where specified.
BENZODIAZEPINES, NALOXONE AND FEEDING
217
They were housed in groups of six in a room maintained in an 11 h on: 13 h off light cycle, with lights on at 0700 h.
Drugs Chlordiazepoxide hydrochloride (Roche Products Ltd.) was dissolved in deionised water to concentrations of 2·5 mg/ml and 3·75 mg/m\. Lorazepam (Wyeth Laboratories) was dissolved in a solvent of2% benzyl alcohol, 18% polyethylene glycol and 80% propylene glycol, to a concentration of 4 mg/m\. It was then diluted further with deionised water to concentrations of 0·125 and 0·25 mg/m\. Naloxone hydrochloride (Endo Laboratories) was dissolved in saline to concentrations of 1 and 2 mg/m\. Control rats received injections of water, lorazepam vehicle or saline, respectively. Apparatus The food preference tests took place in a wooden box, 60 x 60 x 36 cm, in a light level of 10 scotopic lux. The animals were observed on video monitors in an adjacent room. Infrared cells in the walls of the box provided an automated measure of motor activity. Diet Except for half the groups of rats in Experiment 2 the rats were maintained on Heygates breeding diet. The alternative chow that was offered in the food preference test in Experiment 2 was No.3 expanded breeding diet (BP Nutrition UK Ltd.) and the "palatable" food offered was chocolate chips (Cadbury's Ltd.). Procedure The rats were randomly allocated (at least 6 per group) among the following groups: water, chlordiazepoxide (COP 5 and 7·5 mg/kg), vehicle, lorazepam (LOR 0·25 and 0·5 mg/kg) saline, naloxone (NAL 2 and 4 mg/kg). All the rats were weighed and received daily i.p. injections for five days before the feeding tests. Chlordiazepoxide and lorazepam were given chronically but naloxone was given only before the feeding tests; thus rats in this group received saline injections on the five previous days. In Experiment 1 the rats were housed singly on the afternoon of their fifth day of dosing and food deprived. The following morning each rat was injected and 30min later each was given a known weight of normal rat chow in the food hopper. The food, and any spillage, were reweighed after 30 min, 2 hand 6 h. The rats were then rehoused in groups of six and food once more removed. The following day 30 min after injection each rat was placed singly in the test box for 10 min. Two Petri dishes were in the centre of the box, one contained a known weight of normal rat chow, the other a known weight of chocolate chips. The amount eaten was determined by reweighing the food and spillage. Separate groups of rats were used for Experiment 2. Half of the COP and half of the LOR rats and their controls were' allocated to receive the normal diet (H) for four weeks before the feeding tests, the others received the expanded diet (Q) for four weeks. The rats allocated to the naloxone groups received diet H for four weeks. There were at least six rats in each subgroup. Five days before testing the rats were weighed and injected daily, as in Experiment 1. On the afternoon before testing the rats were food deprived. The following morning, 30 min after injection, each rat was placed for 10 min in the test box. There were two Petri dishes in the centre, one containing a known weight of diet H, and the other a known weight of diet Q. The time spent eating each type of diet as well as the amount
s.
218
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consumed were recorded, because some of the data on food preference have been presented as the time spent eating (e.g. Cooper et al., 1977). The effects of five days pretreatment with chlordiazepoxide (5 and 7-5 mg/kg) and lorazepam (0·25 and 0·5 mg/kg) and acute doses of naloxone (2 and 4 mg/kg) on motor activity were measured in a separate group of rats. Each rat was placed singly in the wooden box for a 10 min trial 30 min after an i.p. injection of drug or vehicle control. Each drug group was tested against its own vehicle control and the rats were tested in an order randomised for drug treatment, between 0900 and 1200 h. After each trial any boluses were removed and the box was thoroughly wiped and dried. RESULTS
Table 1 shows the mean weight gain for each group of rats during the injection days before the feeding experiments, i.e. after three and four days of injections. Since there were no significant differences between any of the control scores they have been TABLE 1 Weight gain on ad libitum food and water for rats given daily control or benzodiazepine injections, and amount eaten after a period of food deprivation, following five days of benzodiazepine treatment and after an acute dose of naloxone
Wt gain (g) 3 days 4 days of injections Control
n= 16
CDP
n=6
(5 mg/kg) for 5 days CDP
n=6
(7·5 mg/kg) for 5 days LOR
n=7
(0·"25 mg/kg) for 5 days LOR
n=7
(0·5 mg/kg) for 5 days NAL
n=7
(2 mg/kg) acute NAL
n=7
(4 mg/kg) acute
Amount eaten (g) after 24 h deprivation 1h
2h
6h
14·74 ± 0·68
5·75 ± 0·28
8·37 ± 0·41
9·75 ± 1·0
18·0 ** ± 1·01
6·01 ± 0·36
8·86 ± 0-41
9·26 ± 0·48
5·38*** ± 0·73
12·02** ± 0·78
6·86* ± 0·51
10·10* ± 0·52
12·25* ± 0-47
6·86** ± 0·74
12·18** ± 0·84
6·2 ± 0·41
8·64 ± 0·93
10·25 ± 0·93
7·83* ± 0·65
13-48 ± 0·88
8·33* ± 1·12
10·14* ± 0·98
13-01 * ± 0·49
5·02 ± 0·63
8·57 ± 0·74
11·\7 ± 1·23
4·20* ± 0·34
8·25 ± 0·74
12·07 ± 1·36
9·52 ± 0·50 10·45 ± 0·69
Note: The scores are means±s.e.m. * p<0·U5; ** p
219
BENZODIAZEPINES, NALOXONE AND FEEDING
combined in Table 1, but statistical comparisons were made only between drug groups and their particular controls. It can be seen from Table 1 that all the rats showed some weight gain during the week before testing, as is usual for male hooded rats until they reach 450-500 g. The lower dose of chlordiazepoxide (5 mg/kg) significantly increased the weight gain by the fourth day, whereas the higher dose (7'5 mg/kg) resulted in significantly less weight gain after three and four injections, compared with the controls (see Table 1). Both doses of lorazepam reduced the weight gain (see Table 1). Thus it seems that in a free-feeding situation with only normal diet available the benzodiazepines (apart from 5 mg/kg COP) reduced weight gain, but that this effect occurred mainly during the first three days when the rats were still sedated after the drug injections. Table 1 also shows the amount of food consumed following overnight deprivation. The low doses of COP and LOR were without significant effects, but the higher doses increased food consumption at all the times tested. The low dose of naloxone was without significant effect, but at 4 mg/kg naloxone significantly reduced food consumption in the first 1h of the test. Thereafter the naloxone groups consumed more food, although not significantly so, than the controls. Table 2 shows the weights of chow and chocolate chips consumed in the food preference test. The low dose of chlordiazepoxide significantly increased the amount of TABLE 2 10-min test of food preference between lab chow and chocolate chips
Wt. of chow (mg) Control
n= 16
CDP
n=6
1040±260 890±260
Wt. of chocolate chips (mg) 190± 150 938± 350***
(5 mg/kg) for 5 days
CDP
n=6
1150±240
853 ± 350
(7'5 mg/kg) for 5 days LOR
n=7
2590±200**
49± 13
181O±200*
750±41O
(0'25 mg/kg) for 5 days LOR
n=7
(0'5 mg/kg) for 5 days NAL
n=7
495±270
0***
425 ± 260**
0***
(2 mg/kg) acute NAL
n=7
(4mg/kg) acute
Note: The scores are means ±s.e.m. *v < 0·05: **p
220
S. E. FILE
chocolate chips eaten, but the increase with the higher dose did not reach significance. In contrast, both doses of lorazepam increased consumption of the familiar chow. Naloxone (4 mg/kg) significantly reduced consumption of chow and both doses (2 and 4 mg/kg) completely abolished consumption of chocolate chips. It can be seen from Table 3 that chow H was preferred to chow Q, whether it was familiar or unfamiliar to the rats. Chlordiazepoxide (5 mg/kg) increased the time spent eating the unfamiliar diet H, and increased the amount consumed of both diets when these were unfamiliar. The higher dose of chlordiazepoxide had similar effects (see Table 3). These effects are in contrast to those found with lorazepam, where there was no consumption of the unfamiliar chow and a significant increase in consumption of familiar food, whether this was preferred or non-preferred. Naloxone (4 mg/kg) reduced the consumption of the unfamiliar chow. TABLE 3 Mean ± s.e .m. time spent eatingfamiliar and unfamiliar chow and mean amount consumed ± S.e.ln., during a JO-min preference test
Familiar chow Time (sec)
Control
n= 16
COP
n=7
5 days of (5 mg/kg) COP
n=7
5 days of (7'5mg/kg)
LOR n=8
5 days of (0'25 mg/kg)
LOR n=8
5 days of (0'5mg/kg)
NAL n=7
(2mg/kg)
NAL n=7 (4mg/kg)
* p <0'05;
Unfamiliar chow
Weight (mg)
Time (sec)
Diet H
Diet Q
Diet H
no
15·0
1090
160
75·5
30·7
1514
259
28·6
15·0
300
160
32·0
26·2
560
240
69·8
17·3
1187
167
238'2*
89·0
2430*
1003*
26·7
13-0
280
110
57·9
33·0
160
290
103·5
14·2
924
210
268'3**
146'5*
2130
816*
20·7
12·0
260
150
34·0
68-1
320
212
106·3
16·6
3600*
780*
0*
0*
0*
0*
22·1
14·7
290
270
112-8
17-3
4710*
670*
0*
0*
0*
0*
25-4
15·2
470
290
± ±
±
±
±
±
±
±
±
±
±
±
±
±
±
Diet Q Diet H
Weight (mg)
±
±
±
±
±
±
±
±
Diet Q Diet H
±
±
±
± ±
±
Diet Q
±
±
±
48·6
505
33·3
120
19·8
300
8·5
160
45·2
492
3'0*
11'0*
17·6
270
0·8
5·0
± ±
± ±
** P
±
±
±
±
BENZODIAZEPINES, NALOXONE AND FEEDING TABLE
M ell/1 (± s.e .m.) motor activity score durillg
221
4
10-mill period in the test box for rats receiving vehicle control or drug injections II
Chronic chlordiazepoxide" Control (water)
5 mg/kg
7·5 mg/kg
215·2±20·6
226·4±23-1
198·6± 18·9
Chronic lorazepam b Vehicle
0'25mg/kg
0'5mg/kg
232-4±20'2
191'2± 22-4
164·8 ± 26-4
Naloxone c Saline
2mg/kg
4mg/kg
220·1 ±25·2
212·0±23·8
175-4±28'6
a5 and 7·5 mg/kg 30 min before test and for 5 days previous. bOo 25 and O· 5 mg/kg 30 min before test and for 5 days previous. c2 and 4 mg/kg 30 min before test.
It can be seen from Table 4 that after five days of pretreatment neither dose of chlordiazepoxide or lorazepam had a significant sedative effect, and nor did naloxone (2 and 4 mg/kg) reduce motor activity significantly. It therefore seems unlikely that the drug effects on feeding were secondary to drug-induced sedation. DISCUSSION
With ad libitum food and water the effect of three to four days of benzodiazepine injections was to reduce the amount of weight gain, except for chlordiazepoxide (5 mg/kg) which enhanced weight gain. This effect is most likely due to the initial sedative effects of the benzodiazepines, which reduce motor activity and social interaction (File, 1979, 1980). However, there is rapid tolerance to these sedative effects and the doses used in the present experiment were no longer sedative after five days of dosing. After a period of food deprivation the chronic administration of the benzodiazepines led to an increase in food consumption at the higher doses. This increase was detectable even 6 h after injection. This extends to lorazepam the effects previously found with chlordiazepoxide (Iwahara & Iwasaki, 1969; Niki, 1965: Randall et aI., 1960). However, the effects of lorazepam and chlordiazepoxide on consumption of palatable food were in contrast; low doses of chlordiazepoxide enhanced consumption of the palatable, novel food (chocolate chips) whereas both doses of lorazepam enhanced consumption of the familiar chow. In this respect the effects of lorazepam seem to be different from those of other benzodiazepine derivatives. The effects of chlordiazepoxide to enhance consumption of novel food were not restricted to novel, palatable foods. Both doses increased the consumption of unfamiliar chow, even when this was the less preferred food. Once more the effects of
222
S. E. FILE
lorazepam were different; both doses enhanced consumption of the familiar chow and abolished consumption of the unfamiliar one. Whilst lorazepam acted like chlordiazepoxide to enhance food consumption in the home-cage, after a period of food deprivation, it had contrasting effects on the consumption of novel versus familiar foods. The acute effects of these two benzodiazepine derivatives are similar in causing sedation, but with chronic (five days) administration some differences have emerged. Lorazepam (0'25 and 0·5 mg/kg) produces a less striking anxiolytic profile in the social interaction test (File, 1980), than does chlordiazepoxide. It is therefore possible that the differences relating to the selection of novel food could be a reflection of differences in anxiolytic action. However, not all the effects of the two benzodiazepines on feeding can be attributed to an anxiolytic action; certainly the effects of chlordiazepoxide (7'5 mg/kg) to enhance food consumption in the home-cage, as well as to enhance consumption of unfamiliar chow in the test box, cannot be attributed solely to an anxiolytic action and hence a reduction in neophobia. Chlordiazepoxide and lorazepam also differ in that the former has several active metabolites whereas the latter has none (Greenblatt & Shader, 1977). Studies on the effects of chlordiazepoxide metabolites on feeding are needed in order to assess whether the differences reported in this study are due to the action of a metabolite rather than to that of the parent compound. The differences between the two benzodiazepines found ih this study suggest that any weight gain in patients (Greenblatt & Shader, 1977) might be due to rather different behavioural patterns: an iqcreased consumption of novel and palatable foods in the case of chlordiazepoxide, as opposed to an increased intake of familiar foods in the case of lorazepam. The effects of naloxone to reduce food intake following deprivation were in agreement with previous reports (Brands et aI., 1979; Frenk & Rogers, 1979; Holtzman, 1974; Margules et al., 1978), including the short duration of its action, followed by a rebound increase in eating (King et al., 1979). This suggests that naloxone may not be a useful appetite suppressant as there might be an increased food intake after the drug wore off. King et al. (1979) found that naloxone also reduced the consumption of unfamiliar chow. Naloxone (1,2 and 4 mg/kg) reduces exploratory head-dipping in rats (File, in press), and naloxone also reduces activity in a novel open field (Green et aI., 1979; Rodgers & Deacon, 1979). Therefore, the reduced response to unfamiliar food may be part of a general pattern of decreased responding to novel stimuli. In its effects on the consumption of novel food naloxone resembled lorazepam, and it is this action of naloxone that might be of greatest use clinically to reduce food intake in obese patients. REFERENCES
Brands, B., Thornhill, J. A., Hirst, M., & Gowdey, C. W. Suppression of food intake and body weight gain by naloxone. Life Sciences, 1979, 24, 1773-1778. Brown, D. R., & Holtzman, S. G. Suppression of deprivation-induced food and water intake in rats and mice by naloxone. Pharmacology, Biochemistry and Behavior, 1979, 11, 567-574. Cook, L., & Sepinwall, J. Behavioral analysis of the effects and mechanisms of action of benzodiazepines. In E. Costa & P. Greengard (Eds.), Mechanism of Action of Benzodiazepines. Pp. 1-28. New York: Raven Press, 1975. :=ooper, S. J. Benzodiazepines as appetite-enhancing compounds. Appetite, 1980,1,7-19. Cooper, S. J., Crummy, Y. M. T., & Skan, A. Changes in food choice towards familiar and unfamiliar foods following benzodiazepine administration. Experimental Brain Research, 1977,28, R12-13. Cooper, S. J., & McClelland, A. Effects of chlordiazepoxide, food familiarisation, and prior shock experience on food choice in rats. PharmacologJ', Biochemistry and Behavior, 1980,12,23-28.
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Watson, P. J., Short, M. A., Huenink, G. L., & Hartman, D. F. Diazepam effects on hypothalamically elicited drinking and eating. Physiology and Behavior, 1980,24, 39-44. Wise, R. A., & Dawson, V. Diazepam-induced eating and lever pressing for food in sated rats. J oumal of Comparative and Physiological Psychology, 1974, 86, 930-941.
Received 9 June, 1980; revision 14 July, 1980
Effects of Benzodiazepines and Naloxone on Food Intake and Food Preference in the Rat SANDRA E. FILE Department of Pharma.cology, The School of Pharmacy, University of London
After four days of administration, chlord.iazepoxide (5 mg/ kg) increased the weight gain of rats on ad libitum food and water, whereas chlordiazepoxide (7'5 mg/kg) and lorazepam (0·25 and 0·5 mg/kg) decreased it. After a period of food deprivation, chlordiazepoxide (7'5 mg/kg) and lorazepam (0'5 mg/kg) increased food consumption after!, 2 and 6 h. In a food preference test chlordiazepoxide (5 mg/kg) enhanced consumption of chocolate chips, whereas lorazepam (0'25 and q'5 mg/kg) enhanced consumption of familiar chow. Chlordiazepoxide (5 and 7·5 mg/kg) enhanced consumption of unfamiliar chow, whether this was preferred or not, whereas lorazepam (0'25 and 0·5 mg/ kg) enhanced consumption of familiar chow. Naloxone (4 mg/kg) reduced food intake in the first! h after injection 'and reduced consumption of unfamiliar chow . Naloxone (2 and 4 mg/ kg) abolished consumption of chocolate chips in the food preference test.
Several authors have reported that both acute and chronic treatment with chlordiazepoxide increases the amount of food eaten by rats, following a period of deprivation (Iwahara & Iwasaki, 1969; Niki, 1965; Randall, Schallek, Heise, Keith & Bagdon, 1960). These effects have been found over the dose-range 5 to 50 mg/kg. Several benzodiazepines (after acute and chronic administration) have been found to increase the food intake in non-deprived rats (Sou brie, Kulkarni, Simon & Boissier, 1975; Wise & Dawson, 1974) and to enhance feeding elicited by hypothalamic stimulation (Watson, Short, Huenink & Hartman, 1980). Acute administ·ration of several benzodiazepines enhances the consumption of palatable foods, e.g. sweetened milk (Johnson, 1978; Margules & Stein, 1967; Poschel, 1971) and "free" food, as opposed to that obtained by lever-pressing (Tye, Nicholas & Morgan, 1975). In the experiments using sweetened milk this was both a palatable food and a novel food and it is therefore impossible to say from the results of these ~xperiments whether benzodiazepines enhance the consumption of novel food if this is not also palatable. The results from one experiment, however, suggest that the effects on consumption of novel food may be dose-dependent. Cooper and McClelland (1980) found that acute administration of 5 and 10 mg/ kg chlordiazepoxide increased the time that rats spent eating familiar food, but that at 15 mg/ kg the rats preferred novel food.
This work was supported by a Medical Research Council project grant and technical assistance was provided by L. Rooney. The video equipment was purchased with a grant from the Central Research Fund of London University. I am grateful to Endo Laboratories, Wyeth Laboratories and Roche Products for their generous gifts of drugs, and to Dr L. J. Herberg for the gift of diet Q. Requests for reprints should be sent to Sandra E. File, Department of Pharmacology, The School of Pharmacy, University of London. 29/ 39 Brunswick Square, London WCI N lAX. 0195- 6663/80/030215 + 10 $02'00/0
co 1980 Academic Press Inc. (London) Limited
216
s.
E. FILE
Similarly, a low dose of diazepam (2·5 mg/kg) increased the time spent eating familiar food, whereas 5 mg/kg increased the time spent eating novel food (Cooper, Crummy & Skan, 1977). Contrasting effects of low and high doses of chlordiazepoxide have also been found with tail-pinch induced eating: 5 mg/kg reducing the latency to start eating and increasing the duration of eating and 20 mg/kg having the opposite effects (Robbins, Phillips & Sahakian, 1977). For a discussion of the dose-dependent effects of benzodiazepines on the selection between novel and familiar foods, see Cooper (1980). The present study was designed to investigate the effects of chronic (5 days) administration of low doses of chlordiazepoxide and lorazepam on food intake after deprivation and on food preference. So far the effects of benzodiazepines on food preference seem to have been investigated only after acute administration. Acutely, the benzodiazepines have a sedative action (see Greenblatt & Shader, 1977), whereas after a few days of administration there is tolerance to this effect, seen both in lever-pressing (Cook & Sepinwall, 1975; Stein & Berger, 1971) and in spontaneous motor activity and social interaction (File, 1979). Low doses of benzodiazepines were selected to be comparable with the doses used clinically. A five day pretreatment period was selected since tolerance to the sedative effects of low doses of benzodiazepines has been shown after this period (File, 1979, 1980). Thus after five days of treatment any benzodiazepine effects on feeding should not be secondary to a reduction in motor activity . In order to check the levels of motor activity following the dosing regime used in this study, spontaneous motor activity was measured in the test box used for the food preference test. After five days of administration benzodiizepines produce an anxiolytic profile in animal tests of anziety (e.g. Cook & Sepinwall, 1975; File & Hyde, 1978, 1979). It has been suggested that enhanced food consumption can be used to detect anxiolytic actions in benzodiazepines (Poschel, 1971), but it seems unlikely that all of the effects of benzodiazepines on feeding are secondary to an anxiolytic action (see Cooper, 1980). It is hoped that by in vestigating the effects of chronic benzodiazepines on several aspects of feeding that this study will help to illuminate whether these drugs do have any actions on feeding per se. For comparison, the effects of naloxone on food intake and preference were also investigated. Naloxone has been reported to reduce food intake in deprived rats (Brands, Thornhill, Hirst & Gowdey, 1979; Brown & Holtzman, 1979), having a greater effect in hypothalamically lesioned and obese rats than in normals (King, Castellanos, Kastin, Berzas, Mauk, Olson & Olson, 1979) and in genetically obese mice than normals (Margules, Moisset, Louis, Shibuya & Pert, 1978). It has also been found to reduce eating in obese patients suffering from the Prader-Willi syndrome (Kyriakides, Silverstone, Jeffcoate & Laurance, 1980). In non-deprived rats naloxone has been found to decrease consumption of palatable food (King et al., 1979). The doses of naloxone chosen for this study were within the range previously found to reduce food intake and the effects of these doses on motor activity were also measured . Naloxone has been reported to increase anxiety in man (Grevert & Goldstein, 1977) and in the social interaction test of anxiety it had effects opposite to those of the benzodiazepines (File, in press). The present study therefore explored whether the effects of naloxone and benzodiazepines on feeding were opposite in nature . METHODS
Animals Male hooded rats (from Olac Ltd., Bicester), weighing 250-300 g, were maintained on ad libitum food and water, except for overnight deprivation of food where specified.
BENZODIAZEPINES, NALOXONE AND FEEDING
217
They were housed in groups of six in a room maintained in an 11 h on: 13 h off light cycle, with lights on at 0700 h.
Drugs Chlordiazepoxide hydrochloride (Roche Products Ltd.) was dissolved in deionised water to concentrations of 2·5 mg/ml and 3·75 mg/m\. Lorazepam (Wyeth Laboratories) was dissolved in a solvent of2% benzyl alcohol, 18% polyethylene glycol and 80% propylene glycol, to a concentration of 4 mg/m\. It was then diluted further with deionised water to concentrations of 0·125 and 0·25 mg/m\. Naloxone hydrochloride (Endo Laboratories) was dissolved in saline to concentrations of 1 and 2 mg/m\. Control rats received injections of water, lorazepam vehicle or saline, respectively. Apparatus The food preference tests took place in a wooden box, 60 x 60 x 36 cm, in a light level of 10 scotopic lux. The animals were observed on video monitors in an adjacent room. Infrared cells in the walls of the box provided an automated measure of motor activity. Diet Except for half the groups of rats in Experiment 2 the rats were maintained on Heygates breeding diet. The alternative chow that was offered in the food preference test in Experiment 2 was No.3 expanded breeding diet (BP Nutrition UK Ltd.) and the "palatable" food offered was chocolate chips (Cadbury's Ltd.). Procedure The rats were randomly allocated (at least 6 per group) among the following groups: water, chlordiazepoxide (COP 5 and 7·5 mg/kg), vehicle, lorazepam (LOR 0·25 and 0·5 mg/kg) saline, naloxone (NAL 2 and 4 mg/kg). All the rats were weighed and received daily i.p. injections for five days before the feeding tests. Chlordiazepoxide and lorazepam were given chronically but naloxone was given only before the feeding tests; thus rats in this group received saline injections on the five previous days. In Experiment 1 the rats were housed singly on the afternoon of their fifth day of dosing and food deprived. The following morning each rat was injected and 30min later each was given a known weight of normal rat chow in the food hopper. The food, and any spillage, were reweighed after 30 min, 2 hand 6 h. The rats were then rehoused in groups of six and food once more removed. The following day 30 min after injection each rat was placed singly in the test box for 10 min. Two Petri dishes were in the centre of the box, one contained a known weight of normal rat chow, the other a known weight of chocolate chips. The amount eaten was determined by reweighing the food and spillage. Separate groups of rats were used for Experiment 2. Half of the COP and half of the LOR rats and their controls were' allocated to receive the normal diet (H) for four weeks before the feeding tests, the others received the expanded diet (Q) for four weeks. The rats allocated to the naloxone groups received diet H for four weeks. There were at least six rats in each subgroup. Five days before testing the rats were weighed and injected daily, as in Experiment 1. On the afternoon before testing the rats were food deprived. The following morning, 30 min after injection, each rat was placed for 10 min in the test box. There were two Petri dishes in the centre, one containing a known weight of diet H, and the other a known weight of diet Q. The time spent eating each type of diet as well as the amount
s.
218
E. FILE
consumed were recorded, because some of the data on food preference have been presented as the time spent eating (e.g. Cooper et al., 1977). The effects of five days pretreatment with chlordiazepoxide (5 and 7-5 mg/kg) and lorazepam (0·25 and 0·5 mg/kg) and acute doses of naloxone (2 and 4 mg/kg) on motor activity were measured in a separate group of rats. Each rat was placed singly in the wooden box for a 10 min trial 30 min after an i.p. injection of drug or vehicle control. Each drug group was tested against its own vehicle control and the rats were tested in an order randomised for drug treatment, between 0900 and 1200 h. After each trial any boluses were removed and the box was thoroughly wiped and dried. RESULTS
Table 1 shows the mean weight gain for each group of rats during the injection days before the feeding experiments, i.e. after three and four days of injections. Since there were no significant differences between any of the control scores they have been TABLE 1 Weight gain on ad libitum food and water for rats given daily control or benzodiazepine injections, and amount eaten after a period of food deprivation, following five days of benzodiazepine treatment and after an acute dose of naloxone
Wt gain (g) 3 days 4 days of injections Control
n= 16
CDP
n=6
(5 mg/kg) for 5 days CDP
n=6
(7·5 mg/kg) for 5 days LOR
n=7
(0·"25 mg/kg) for 5 days LOR
n=7
(0·5 mg/kg) for 5 days NAL
n=7
(2 mg/kg) acute NAL
n=7
(4 mg/kg) acute
Amount eaten (g) after 24 h deprivation 1h
2h
6h
14·74 ± 0·68
5·75 ± 0·28
8·37 ± 0·41
9·75 ± 1·0
18·0 ** ± 1·01
6·01 ± 0·36
8·86 ± 0-41
9·26 ± 0·48
5·38*** ± 0·73
12·02** ± 0·78
6·86* ± 0·51
10·10* ± 0·52
12·25* ± 0-47
6·86** ± 0·74
12·18** ± 0·84
6·2 ± 0·41
8·64 ± 0·93
10·25 ± 0·93
7·83* ± 0·65
13-48 ± 0·88
8·33* ± 1·12
10·14* ± 0·98
13-01 * ± 0·49
5·02 ± 0·63
8·57 ± 0·74
11·\7 ± 1·23
4·20* ± 0·34
8·25 ± 0·74
12·07 ± 1·36
9·52 ± 0·50 10·45 ± 0·69
Note: The scores are means±s.e.m. * p<0·U5; ** p
219
BENZODIAZEPINES, NALOXONE AND FEEDING
combined in Table 1, but statistical comparisons were made only between drug groups and their particular controls. It can be seen from Table 1 that all the rats showed some weight gain during the week before testing, as is usual for male hooded rats until they reach 450-500 g. The lower dose of chlordiazepoxide (5 mg/kg) significantly increased the weight gain by the fourth day, whereas the higher dose (7'5 mg/kg) resulted in significantly less weight gain after three and four injections, compared with the controls (see Table 1). Both doses of lorazepam reduced the weight gain (see Table 1). Thus it seems that in a free-feeding situation with only normal diet available the benzodiazepines (apart from 5 mg/kg COP) reduced weight gain, but that this effect occurred mainly during the first three days when the rats were still sedated after the drug injections. Table 1 also shows the amount of food consumed following overnight deprivation. The low doses of COP and LOR were without significant effects, but the higher doses increased food consumption at all the times tested. The low dose of naloxone was without significant effect, but at 4 mg/kg naloxone significantly reduced food consumption in the first 1h of the test. Thereafter the naloxone groups consumed more food, although not significantly so, than the controls. Table 2 shows the weights of chow and chocolate chips consumed in the food preference test. The low dose of chlordiazepoxide significantly increased the amount of TABLE 2 10-min test of food preference between lab chow and chocolate chips
Wt. of chow (mg) Control
n= 16
CDP
n=6
1040±260 890±260
Wt. of chocolate chips (mg) 190± 150 938± 350***
(5 mg/kg) for 5 days
CDP
n=6
1150±240
853 ± 350
(7'5 mg/kg) for 5 days LOR
n=7
2590±200**
49± 13
181O±200*
750±41O
(0'25 mg/kg) for 5 days LOR
n=7
(0'5 mg/kg) for 5 days NAL
n=7
495±270
0***
425 ± 260**
0***
(2 mg/kg) acute NAL
n=7
(4mg/kg) acute
Note: The scores are means ±s.e.m. *v < 0·05: **p
220
S. E. FILE
chocolate chips eaten, but the increase with the higher dose did not reach significance. In contrast, both doses of lorazepam increased consumption of the familiar chow. Naloxone (4 mg/kg) significantly reduced consumption of chow and both doses (2 and 4 mg/kg) completely abolished consumption of chocolate chips. It can be seen from Table 3 that chow H was preferred to chow Q, whether it was familiar or unfamiliar to the rats. Chlordiazepoxide (5 mg/kg) increased the time spent eating the unfamiliar diet H, and increased the amount consumed of both diets when these were unfamiliar. The higher dose of chlordiazepoxide had similar effects (see Table 3). These effects are in contrast to those found with lorazepam, where there was no consumption of the unfamiliar chow and a significant increase in consumption of familiar food, whether this was preferred or non-preferred. Naloxone (4 mg/kg) reduced the consumption of the unfamiliar chow. TABLE 3 Mean ± s.e .m. time spent eatingfamiliar and unfamiliar chow and mean amount consumed ± S.e.ln., during a JO-min preference test
Familiar chow Time (sec)
Control
n= 16
COP
n=7
5 days of (5 mg/kg) COP
n=7
5 days of (7'5mg/kg)
LOR n=8
5 days of (0'25 mg/kg)
LOR n=8
5 days of (0'5mg/kg)
NAL n=7
(2mg/kg)
NAL n=7 (4mg/kg)
* p <0'05;
Unfamiliar chow
Weight (mg)
Time (sec)
Diet H
Diet Q
Diet H
no
15·0
1090
160
75·5
30·7
1514
259
28·6
15·0
300
160
32·0
26·2
560
240
69·8
17·3
1187
167
238'2*
89·0
2430*
1003*
26·7
13-0
280
110
57·9
33·0
160
290
103·5
14·2
924
210
268'3**
146'5*
2130
816*
20·7
12·0
260
150
34·0
68-1
320
212
106·3
16·6
3600*
780*
0*
0*
0*
0*
22·1
14·7
290
270
112-8
17-3
4710*
670*
0*
0*
0*
0*
25-4
15·2
470
290
± ±
±
±
±
±
±
±
±
±
±
±
±
±
±
Diet Q Diet H
Weight (mg)
±
±
±
±
±
±
±
±
Diet Q Diet H
±
±
±
± ±
±
Diet Q
±
±
±
48·6
505
33·3
120
19·8
300
8·5
160
45·2
492
3'0*
11'0*
17·6
270
0·8
5·0
± ±
± ±
** P
±
±
±
±
BENZODIAZEPINES, NALOXONE AND FEEDING TABLE
M ell/1 (± s.e .m.) motor activity score durillg
221
4
10-mill period in the test box for rats receiving vehicle control or drug injections II
Chronic chlordiazepoxide" Control (water)
5 mg/kg
7·5 mg/kg
215·2±20·6
226·4±23-1
198·6± 18·9
Chronic lorazepam b Vehicle
0'25mg/kg
0'5mg/kg
232-4±20'2
191'2± 22-4
164·8 ± 26-4
Naloxone c Saline
2mg/kg
4mg/kg
220·1 ±25·2
212·0±23·8
175-4±28'6
a5 and 7·5 mg/kg 30 min before test and for 5 days previous. bOo 25 and O· 5 mg/kg 30 min before test and for 5 days previous. c2 and 4 mg/kg 30 min before test.
It can be seen from Table 4 that after five days of pretreatment neither dose of chlordiazepoxide or lorazepam had a significant sedative effect, and nor did naloxone (2 and 4 mg/kg) reduce motor activity significantly. It therefore seems unlikely that the drug effects on feeding were secondary to drug-induced sedation. DISCUSSION
With ad libitum food and water the effect of three to four days of benzodiazepine injections was to reduce the amount of weight gain, except for chlordiazepoxide (5 mg/kg) which enhanced weight gain. This effect is most likely due to the initial sedative effects of the benzodiazepines, which reduce motor activity and social interaction (File, 1979, 1980). However, there is rapid tolerance to these sedative effects and the doses used in the present experiment were no longer sedative after five days of dosing. After a period of food deprivation the chronic administration of the benzodiazepines led to an increase in food consumption at the higher doses. This increase was detectable even 6 h after injection. This extends to lorazepam the effects previously found with chlordiazepoxide (Iwahara & Iwasaki, 1969; Niki, 1965: Randall et aI., 1960). However, the effects of lorazepam and chlordiazepoxide on consumption of palatable food were in contrast; low doses of chlordiazepoxide enhanced consumption of the palatable, novel food (chocolate chips) whereas both doses of lorazepam enhanced consumption of the familiar chow. In this respect the effects of lorazepam seem to be different from those of other benzodiazepine derivatives. The effects of chlordiazepoxide to enhance consumption of novel food were not restricted to novel, palatable foods. Both doses increased the consumption of unfamiliar chow, even when this was the less preferred food. Once more the effects of
222
S. E. FILE
lorazepam were different; both doses enhanced consumption of the familiar chow and abolished consumption of the unfamiliar one. Whilst lorazepam acted like chlordiazepoxide to enhance food consumption in the home-cage, after a period of food deprivation, it had contrasting effects on the consumption of novel versus familiar foods. The acute effects of these two benzodiazepine derivatives are similar in causing sedation, but with chronic (five days) administration some differences have emerged. Lorazepam (0'25 and 0·5 mg/kg) produces a less striking anxiolytic profile in the social interaction test (File, 1980), than does chlordiazepoxide. It is therefore possible that the differences relating to the selection of novel food could be a reflection of differences in anxiolytic action. However, not all the effects of the two benzodiazepines on feeding can be attributed to an anxiolytic action; certainly the effects of chlordiazepoxide (7'5 mg/kg) to enhance food consumption in the home-cage, as well as to enhance consumption of unfamiliar chow in the test box, cannot be attributed solely to an anxiolytic action and hence a reduction in neophobia. Chlordiazepoxide and lorazepam also differ in that the former has several active metabolites whereas the latter has none (Greenblatt & Shader, 1977). Studies on the effects of chlordiazepoxide metabolites on feeding are needed in order to assess whether the differences reported in this study are due to the action of a metabolite rather than to that of the parent compound. The differences between the two benzodiazepines found ih this study suggest that any weight gain in patients (Greenblatt & Shader, 1977) might be due to rather different behavioural patterns: an iqcreased consumption of novel and palatable foods in the case of chlordiazepoxide, as opposed to an increased intake of familiar foods in the case of lorazepam. The effects of naloxone to reduce food intake following deprivation were in agreement with previous reports (Brands et aI., 1979; Frenk & Rogers, 1979; Holtzman, 1974; Margules et al., 1978), including the short duration of its action, followed by a rebound increase in eating (King et al., 1979). This suggests that naloxone may not be a useful appetite suppressant as there might be an increased food intake after the drug wore off. King et al. (1979) found that naloxone also reduced the consumption of unfamiliar chow. Naloxone (1,2 and 4 mg/kg) reduces exploratory head-dipping in rats (File, in press), and naloxone also reduces activity in a novel open field (Green et aI., 1979; Rodgers & Deacon, 1979). Therefore, the reduced response to unfamiliar food may be part of a general pattern of decreased responding to novel stimuli. In its effects on the consumption of novel food naloxone resembled lorazepam, and it is this action of naloxone that might be of greatest use clinically to reduce food intake in obese patients. REFERENCES
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Watson, P. J., Short, M. A., Huenink, G. L., & Hartman, D. F. Diazepam effects on hypothalamically elicited drinking and eating. Physiology and Behavior, 1980,24, 39-44. Wise, R. A., & Dawson, V. Diazepam-induced eating and lever pressing for food in sated rats. J oumal of Comparative and Physiological Psychology, 1974, 86, 930-941.
Received 9 June, 1980; revision 14 July, 1980