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Comparative studies with thieno- and benzodiazepines: Spatial delayed alternation behaviour in the monkey (Macaca mulatta)
Newrophammacology, Vol. 19. PP. 491 to 495 Pergamon Press Ltd 1980. Printed in Great Britain
COMPARATIVE STUDIES WITH THIENO- AND BENZODIAZEPINES: SPATIAL DELAYED ALTERNATION BEHAVIOUR IN THE MONKEY (MACACA MULATTA) A. N. NICHOLSONand CATHERINEM. WRIGHT Royal Air Force Institute of Aviation Medicine, Farnborough, Hampshire, England (Accepted
20 December
1974)
Summary-The effects of three benzodiazepines, temazepam, nordiazepam and clobazam, and two triazolo-~,4-thienodi~epines, brotizolam and WE 973, were studied on spatial delayed alternation in the monkey (Macaca muhtta).Experiments were carried out 1 hr after the intra~ritoneal injection of doses ranging from 0.5 to 3.0 mg. kg-’ body weight. No behavioural effects were observed with temazepam and clobazam. Nordiazepam reduced the number of correct responses, and the thienodiazepines reduced the number of correct responses, and increased total response time. In further experiments with doses ranging from 5.0 to 25.0 mg . kg- 1 body weight there was still no effect with clobazam, but temazepam reduced the number of correct responses and increased total response time. There would appear to be differential effects of diazepines on behaviour. The triazolo-1,4_thienodiazepines are particularly potent compared with the benzodiazepines, and the question arises whether they possess specific behavioural activity.
Previous studies from this laboratory have been concerned with the effects of benzodiazepines on behaviour in the monkey. With diazepam, delayed differentiation and spatial delayed alternation were impaired, though minor alterations in moiecular structure led to marked changes in effect. There was a loss of, or at least a considerable attenuation of, activity with hydroxylation, whereas activity was preserved, though modified by demethylation (Nicholson and Wright, 1973; Curry, Nicholson, Whelpton and Wright, 1977). It is difhcult to interpret these findings in terms of
effects in man (Brimblecombe, Nicholson and Wright, 1979), but, nevertheless, it is considered that the benzodiazepines have differential activity, and it is suggested that some benzodiazepines may have subtle, though as yet uncertain, effects on higher nervous function. To explore this possibility further, the studies have been extended to diazepines which have some pharmacological properties which are unusual for the group as a whole (Fig. 1). A l,S-benzodiazepine, clobazam, which possesses anti-anxiety activity, but has little effect on motor function (Fielding and
OH
Fig. 1. Structural formulae of the 1,4_benzodiazepines, (a) temazepam (3-hydroxydiazepam) and (b) nordiazepam (N-desmethyldiazepam), the 1,5-benzodiazepine, (c) clobazam, and the triazolo-1,4thienodiazepines, (d) WE 941 (brotizolam) and (e) WE 973, The formula for brotizolam is (2-bromo-4-(2-chlorophenyl~9-methyl-6H_-1,2,4-triazolo-[4,3-a]-l,4-diazepine. 491
492
A. N. NICHOLSON and CATHERINE M.
WRIGHT
Table 1. Change in number of correct responses 1 hr after drugs compared with placebo (means for five monkeys)
0.5
1.0
1.5
2.0
2.5
Nordiazepam
1.6
0.6
2.2 *
Temazepam
1.0
1.4
0.6
3.6 % 0.8
2.4 t 0.4
Clobazam Brotizolam
WE 973
-1.1 -0.4
-0.1 0.4
Dose (mg.kg-‘) 3.0 2.6 t 0.4
-0.1 3.0
-0.9 4.4
-0.7 5.6
-0.3 4.8
t
%
%
%
Z” % 6.7 % 5.3 % 8.5 F’ Least significant difference * P < 0.05; t P < 0.01; $P < 0.001 2.38 3.08 1.79
5.0
10.0
15.0
20.0
25.0
0.8
1.2
2.6
5.0
6.6
*
ll
0.1
0.5
0.1
0.9
n
1.7
% 8.7
Hoffman, 1979) has been studied, and also two 1,Cthienodiazepines which are particularly potent and possess some of the activity characteristic of the neuroleptics (Boehringer Ingelheim-Internal Report). METHODS
The studies were carried out with 5 male monkeys mulatta) weighing between 8.2 and 12.4 (mean 10.4) kg, trained on a spatial delayed alternation task. The stimuli were 2 illuminated green discs displayed simultaneously on vertical panels spaced 12 cm apart with a food well in the midline below the panels. The animals were required to press the right and left hand panels alternately. With the initial presentation of both stimuli the animal pressed one of the panels to extinguish the light, and was rewarded with a food pellet. This response was not used in the analysis. With the succeeding 50 trials the correct response was to press the panel not rewarded in the previous trial. In the event of an error the trial was repeated until the correct response was made. The intertrial delay was 5 set, and each monkey was required to reach a level of performance of 40 correct out of 50 responses for 10 consecutive sessions (criterion) before experiments with drugs were commenced. The number of correct responses, the number of repeat errors, and the time taken to complete 50 trials were measured. (Macaca
Least significant difference 11P < 0.01; TP < 0.001 3.41 2.57 4.42
i;P < 0.05;
The tests were carried out 1 hr after an intraperitoneal injection of drug or drug vehicle (placebo control). The order of injection of drugs and of drug vehicle was randomized for each animal, and at least 4 days separated each injection. The drugs were 3-hydroxydiazepam (temazepam) and N-desmethyldiazepam (nordiazepam) as examples of the 1,4-benzodiazepines, clobazam as an example of the 1,5-benzodiazepines and WE 973 and WE 941 (brotizolam) as examples of the triazolo-thienodiazepines (see Fig. 1 for structural formulae). In the first experiment the doses were 0.5, 1.0, 1.5,2.0, 2.5 and 3.0mg. kg-’ body weight for all drugs. No behavioural effect was observed with temazepam and clobazam, and so a second experiment was carried out with the dose range 5.0, 10.0, 15.0, 20.0 and 25.0mg. kg-’ body weight. All drugs were dissolved in 5 ml polyethylene glycol. RESULTS
Analysis of performance was concerned with the change in the number of correct responses (excluding repeated responses if the initial response was incorrect), the number of repeat errors and the total response time, each compared with placebo. The measures were related to the performance of individual monkeys and to the dose level. Analysis of variance was the statistical method, and the results are given in Tables 1 and 2.
Table 2. Change in total response time (set) 1 hr after drugs compared with placebo (means for five monkeys)
0.5 Nordiazepam Temazenam
0.24 0.13
1.0 -0.19 -0.17
1.5 -0.14 0.02
2.0
2.5
-0.04 -0.10
-0.22 -0.19
Dose (mg kg- ‘) 3.0 0.02 -0.02
5.0 0.58**
10.0 0.14
15.0 0.37
8 Clobazam Brotizolam WE 973
0.06 -0.13
-0.09 0.50
*
-0.03 1.05
%
0.11 1.06
%
- 0.02 0.82
0.19 1.10
%
%
1.22 0.88 0.90 0.78 t % % % Least significant difference * P < 0.05; t P < 0.01; fPi0.001 0.61 0.79 0.46 0.35
-0.36
-0.19
-0.08
20.0
25.0
1.26
1.05
ll
ll
0.38
0.39
-0.04
** This value was due to an effect in one monkey.
Least significant difference p P < 0.05; 11P < 0.01; 7 P < 0.001 0.97 0.75 0.57
Diazepines and alternation behaviour
493
it is much less potent than diazepam in disturbing rotarod performance, and then only at doses which are markedly above those required to produce antiaggression and “anti-anxiety” effects. It is also difficult to establish impaired coordination with clobazam in man (Borland and Nicholson, 1975). In previous studies from this laboratory with a delayed differentiation task it was shown that hydroxylation of diazepam led to a loss of, or a considerable attenuation of, behavioural activity whereas with demethylation behavioural activity was preserved. Similar observations have been made with spatial delayed alternation. With nordiazepam the number of correct responses was reduced within the dose range 0.5-3.0 mg. kg- ‘, but there was no effect within this dose range with temazepam. Changes were observed with temazepam at much higher doses, but these were not used in the previous experiments. Differential effects on performance with nordiazepam and temazepam have also been observed in man. In dose ranges with hypnotic activity, temazepam has a pronounced effect on coordination, but it is difficult to establish such an effect with nordiazepam (Clarke and Nicholson, 1978). Though substitution of the benzo ring with a thieno group as with clotiazepam, would appear to have little effect in changing activity from that of diazepam as far as pharmacological tests in animals (Nakanishi, DISCUSSION Tsumagari, Takigawa, Shuto, Kenjo and Fukuda, The present studies have shown that the effects of 1972) and effects on sleep in man (Nakazawa, Kotorii, thieno- and benzodiazepines on spatial delayed alterOhshima, Horikawa and Tachibana, 1975) are connation differ, at least, with respect to dose. Within the cerned, the triazolo ring across the methyl group of dose range 0.5-3.0 mg . kg-l, there was no effect of the the diazepam molecule would appear to lead to 1,4_benzodiazepine, temazepam, and the 1,5-benzomarked changes in potency. Triazolam, a triazolo-1,4diazepine, clobazam, but the I+benzodiazepine, norbenzodiazepine, is a potent anti-convulsant with antidiazepam, and the thienodiazepines reduced the aggression, “anti-anxiety” and muscle relaxing number of correct responses and the thienodiazepines properties and increases drug-induced sleeping time increased response time. Within the dose range, more than diazepam (Rudzik, Hester, Tang, Straw 5.G25.0 mg. kg-‘, temazepam reduced the number of and Friis, 1973). It is also a potent hypnotic in man correct responses and increased response time, and (Nicholson and Stone, 1980). Pharmacological studies there was still no effect with clobazam. in animals also emphasise the activity of the triIt is difficult to be certain whether these effects azolo-l,4-thienodiazepines in anti-convulsant, “antimerely reflect the potency of the various compounds anxiety” and anti-aggression tests in animals, though or whether they reflect qualitative differences in ac- in many respects they differ from minor tranquillizers. tivity. The effect of the triazoIo-thienodiazepine, They are only weakly effective in inhibiting electrobrotizolam, compared with temazepam on behaviour shock convulsions in mice, and at very high doses in the monkey is certainly comparable with the rela- they are unable to produce coma or loss of righting tive hypnotic activity of these compounds in man reflexes even though sedation and ataxia are present (Nicholson and Stone, 1976 and unpublished observaat much lower doses. Additionally, they possess actions), though dissociation of psychomotor and be- tivity typical of neuroleptic drugs as brotizolam and havioural activity would appear to be a property WE 973 decrease locomotion and exploration in mice, specific to the l,S-benzodiazepine, clobazam. Characand brotizolam is effective in an active avoidance task teristically, benzo~i~epines possess anti-convul~nt in rats. In previous studies, the possibility that impaired and anti-aggression activity, prolong drug-induced performance on delayed differentiation with benzosleeping time and demonstrate “anti-anxiety” potendiazepines may imply behavioural disinhibition was tial as well as muscle relaxant properties (Randall, Scheckel and Pool, 1970; Barzaghi, Fournex and discussed. Such an effect with benzodiazepines has Mantegazza, 1973; De Angelis, Traversa and Vertua, also been discussed by Hasegawa, Ibuka and Iwahara 1974; Fielding and Hoffman, 1979) but with cloba- (1973) in the monkey, by Schallek, Kuehn and Kovacs zam there is a loss of muscular relaxant activitv. ~~,, and ~~~. (1972) in the cat and by Margules and Stein (1968),
There were no effects of clobazam on the number of correct responses or on total response time. Temazepam decreased the number of correct responses with 15.0 mg. kg-’ and above, and increased total response time with 20 mg . kg- 1 and above. An effect on total response time at the 5.0 mg. kg- ’ dose was due to a marked increase with one monkey only, and this effect was not observed with the same monkey at 10 and 15 mg . kg- ‘. Nordiazepam reduced the number of correct responses without an increase in response time within the lower dose range (1.5-3.0 mg . kg-‘), and the animals failed to respond with doses above 5.0 mg . kg- ‘. Brotizolam (1.5 mg . kg- 1 and above) reduced the number of correct responses and increased the total response time, while WE 973 decreased the number of correct responses with 0.5 mg.kg-’ and above, and increased the total response time with 1.5 mg. kg- ’ and above. Analysis of the number of repeat errors by the Fischer Exact Test showed a difference between the distribution of errors with brotizolam and WE 973 compared with nordiazepam, temazepam and clobazam. An additional statistical test showed that with brotizolam there was an increase in repeat errors at the higher doses (i.e. 2.0mg. kg-’ and above) and this was greater than with the lower doses (0.5-1.5 mg. kg-“) or with placebo (P < 0.05).
A. N. NICHOLSON and CATHEKINE M. WRIGHT
494
Heise, Laughlin and Keller (1970) Wedeking (1969) and Iwahara (1977) in the rat. However, recently, response perseveration rather than disinhibition has been proposed as the behavioural basis of the changes observed with benzodiazepines (Dantzer, 1977). The interpretation of the effect of benzodiazepines on behaviour remains uncertain, and similar studies with brain lesions suggest that other factors such as the spatial element of the task may be involved (Mishkin, 1964; Iversen and Mishkin, 1970). Behavioural studies with brain lesions also raise the question whether the integrity of certain forebrain systems may be impaired by benzodiazepines. Impairments in learning and retention of spatial delayed alternation have been observed in the monkey with lesions of the frontal lobes and with lesions of deep temporal lobe structures (Orbach, Milner and Rasmussen, 1960; Divac, Rosvold and Szwarcbart, 1967; Mishkin, Vest, Waxler and Rosvold, 1969) while response perseveration has been seen after similar lesions in tests which involve extinction, differentiation, and discrimination reversals (Butter, Mishkin and Rosvold, 1963; Mishkin, 1964; Butters and Rosvoid, 1968; Mishkin et rd., 1969; Butter, 1969; Iversen and Mishkin. 1970). Care must be exercised in comparing the behavioural effects of drugs and of brain lesions, but the importance of frontal and temporal pathways for the integrity of complex behaviour is well established, and the effects of some benzodiazepines may well involve such higher nervous pathways. It is difficult to be certain whether the present observations in the monkey relate to such higher nervous function in man, but the observations on spatial delayed alternation may suggest subtle, though as yet uncertain, effects on human behaviour, and that such effects may be more likely with the triazolo-1,4-thienodiazepines than with the 1,4- and 15benzodiazepines.
REFERENCES
Barzaghi, F., Fournex, R. and Mantegazza,
P. (1973). Pharmacological and toxicological properties of clobazam (l-phenyl-5-methyl-8-chloro-l,2,4,5-tetrahydro-2,4diketo-3H-l$benzodiazepine), a new psychotherapeutic agent. Arzneimittel-Forsch. (Drug Res.) 23: 683-686. Borland, R. G. and Nicholson, A. N. (1975). Immediate effects on human performance of a 1,5_benzodiazepine (clobazam) compared with the 1,4_benzodiazepines chlordiazepoxide hydrochloride and diazepam. Br. J. clin. Pharmac. 2: 215 -221. Brimblecombe. R., Nicholson, A. N. and Wright, C. M. (1979). Adverse central effects of drugs in man: Predictive value of behavioural studies in rodents and primates Pharmac,. Thrr. 5: 455-460.
Butter, C. M. (1969). Perseveration in extinction and in discrimination reversal tasks following selective frontal ablations in Macaca mulatta. Physiol. Behau. 4: 163-171. Butter, C. M., Mishkin, M. and Rosvold, H. E. (1963). Conditioning and extinction of a food-reward response after selective ablations of frontal cortex in rhesus monkeys. Expl Nrurol. 7: 65-75. Butters, N. and Rosvold. H. E. (1968). Effect of caudate and septal nuclei lesions on resistance to extinction and
delayed alternation. J. camp. physiol. Psycho/. 65: 397403. Clarke, C. H. and Nicholson, A. N. (1978). Immediate and residual effects in man of the metabolites of diazepam. Br. J. clin. Pharmac. 6: 325-331. Curry, S. H., Nicholson. A. N., Whelpton, R. and Wright, C. M. (1977). Behavioural and pharmacokinetic studies in the monkey (Macaca mulatta) with diazepam, nordiazepam and related 1,4-benzodiazepines. Br. J. Pharmac. 61: 3255330. De Angelis. L.. Traversa, U. and Vertua, R. (1974). Comparative evaluation of the central nervous system activity of diazepam and its metabolites (demethyl-diazepam). methyl-oxazepam and oxazepam. Pharmac. Res. Comman. 6: 61.-75.
Dantzer, R. (1977). Behavioural effects of benzodiazepines. A review. Biohehuc. Rec. I: 71-86. Divac, I., Rosvold, H. E. and Szwarcbart, M. K. (1967). Behavioural effects of selective ablation of the caudate nucleus. J. romp. physiol. Psycho/. 63: 184190. Fielding, S. and Hoffman, 1. (1979). Pharmacology of antianxiety drugs with special reference to clobazam. Br. J. clin. Pharmac. 7: 7-15s. Hasegawa, Y., Ibuka, N. and Iwahara, S. (1973). Effects of chlordiazepoxide upon successive red-green discrimination responses in Japanese monkeys, Macaca fuscata. Psychopharmucologia 30: 89-94. Heise, G. A.. Laughlin, N. and Keller, C. (1970). A behavioural and pharmacological analysis of reinforcement withdrawal. Psychopharmacologia 16: 345-368. Iversen, S. and Mishkin, M. (1970). Perseverative interference in monkeys following selective lesions of the inferior prefrontal convexity. E.xpl Brain Res. II: 376386. Iwahara. S. (1977). Contributions from the animal laboratorydrug and response inhibition. Mod. Prohl. Pharmacopsych. 12: 59 76. Margules, D. L. and Stein, L. (1968). Increase of “antianxiety” activity and tolerance of behavioural depression during chronic administration of oxazepam. Psychopharmacologia 13: 74-80. Mishkin, M. (1964). Perseveration of central sets after frontal lesions in monkey. In: The Frontal Granular Cortex and Behauiour (Warren. J. M. and Akert. K.. Eds).I,.& no. 219-241. McGraw-Hill, New York. Mishkin, M.. Vest, B., Waxler, M. and Rosvold, H. E. (1969). A re-examination of the effects of frontal lesions on object alternation. Neuropsychologia 7: 357-363. Nakanishi, M., Tsumagari, T.. Takigawa, Y., Shuto, S., Kenjo, T. and Fukuda, T. (1972). Studies on psychotropic drugs. XIX : Psychopharmacological studies of l-methyl-5-O-chlorophenyl-7-ethyl-l,2-dihydro-3Hthieno[2,3-e][l,4]diazkpin-Z-one (Y-6047). Ar&wimittrlForsch. (Druq Rrs.) 22: 1905 1914. Nakazawa, Y.,‘. Kotorii, M., Ohshima, M., Horikawa, S. and Tachibana, H. (1975). Effects of thienodiazepine derivatives on human sleep as compared to those of benzodiazepine derivatives. Psychopharmacologia 44: 165-171. Nicholson, A. N. and Stone, B. M. (1976). Effect of a metabolite of diazepam, 3-hydroxydiazepam (temazePam), on sleep in man. Br. J. clin. Pharmac. 3: 543-550. Nicholson. A. N. and Stone, B. M. (1980). Activity of the hypnotics, flunitrazepam and triazolam, in man. Br. J. clin. Pharmac. In press. Nicholson, A. N. and Wright, C. M. (1973). Inhibitory and disinhibitory effects of nitrazepam. diazepam and flurazepam hydrochloride on delayed matching behaviour in monkeys (Macaw muluttu). Neuropharmacologp 13: 9 199926. Orbach. J., Milner, B. and Rasmussen, T. (1960). Learning and retention in monkeys after amygdala-hippocampus resection. Archs Neural. 3: 23&251. Randall, L. 0.. Scheckel. C. L. and Pool, W. (1970). Phar-
Diazepines
and alternation
macology of medazepam and metabolites. Archs inc. Pharmacodyn. 185: 135-148. Rudzik, A. D., Hester, J. B., Tang, A. H., Straw, R. N. and Friis, W. (1973). Triazolobenzodiazepines, a new class of central nervous system depressant compounds. In: The Benzodiazepines (Garattini, S., Mussini, E. and Randall, L. O., Eds), pp. 285-297. Raven Press, New York.
behaviour
495
Schallek, W., Kuehn, A. and Kovacs, J. (1972). Effects of chlordiazepoxide hydrochloride on discrimination responses and sleep cycles in cats. Neuropharmacology 11: 69-79. Wedeking, P. W. (1969). Disinhibition effect of chlordiazepoxide. Psvchon. Sci. 15: 232-233.
COMPARATIVE STUDIES WITH THIENO- AND BENZODIAZEPINES: SPATIAL DELAYED ALTERNATION BEHAVIOUR IN THE MONKEY (MACACA MULATTA) A. N. NICHOLSONand CATHERINEM. WRIGHT Royal Air Force Institute of Aviation Medicine, Farnborough, Hampshire, England (Accepted
20 December
1974)
Summary-The effects of three benzodiazepines, temazepam, nordiazepam and clobazam, and two triazolo-~,4-thienodi~epines, brotizolam and WE 973, were studied on spatial delayed alternation in the monkey (Macaca muhtta).Experiments were carried out 1 hr after the intra~ritoneal injection of doses ranging from 0.5 to 3.0 mg. kg-’ body weight. No behavioural effects were observed with temazepam and clobazam. Nordiazepam reduced the number of correct responses, and the thienodiazepines reduced the number of correct responses, and increased total response time. In further experiments with doses ranging from 5.0 to 25.0 mg . kg- 1 body weight there was still no effect with clobazam, but temazepam reduced the number of correct responses and increased total response time. There would appear to be differential effects of diazepines on behaviour. The triazolo-1,4_thienodiazepines are particularly potent compared with the benzodiazepines, and the question arises whether they possess specific behavioural activity.
Previous studies from this laboratory have been concerned with the effects of benzodiazepines on behaviour in the monkey. With diazepam, delayed differentiation and spatial delayed alternation were impaired, though minor alterations in moiecular structure led to marked changes in effect. There was a loss of, or at least a considerable attenuation of, activity with hydroxylation, whereas activity was preserved, though modified by demethylation (Nicholson and Wright, 1973; Curry, Nicholson, Whelpton and Wright, 1977). It is difhcult to interpret these findings in terms of
effects in man (Brimblecombe, Nicholson and Wright, 1979), but, nevertheless, it is considered that the benzodiazepines have differential activity, and it is suggested that some benzodiazepines may have subtle, though as yet uncertain, effects on higher nervous function. To explore this possibility further, the studies have been extended to diazepines which have some pharmacological properties which are unusual for the group as a whole (Fig. 1). A l,S-benzodiazepine, clobazam, which possesses anti-anxiety activity, but has little effect on motor function (Fielding and
OH
Fig. 1. Structural formulae of the 1,4_benzodiazepines, (a) temazepam (3-hydroxydiazepam) and (b) nordiazepam (N-desmethyldiazepam), the 1,5-benzodiazepine, (c) clobazam, and the triazolo-1,4thienodiazepines, (d) WE 941 (brotizolam) and (e) WE 973, The formula for brotizolam is (2-bromo-4-(2-chlorophenyl~9-methyl-6H_-1,2,4-triazolo-[4,3-a]-l,4-diazepine. 491
492
A. N. NICHOLSON and CATHERINE M.
WRIGHT
Table 1. Change in number of correct responses 1 hr after drugs compared with placebo (means for five monkeys)
0.5
1.0
1.5
2.0
2.5
Nordiazepam
1.6
0.6
2.2 *
Temazepam
1.0
1.4
0.6
3.6 % 0.8
2.4 t 0.4
Clobazam Brotizolam
WE 973
-1.1 -0.4
-0.1 0.4
Dose (mg.kg-‘) 3.0 2.6 t 0.4
-0.1 3.0
-0.9 4.4
-0.7 5.6
-0.3 4.8
t
%
%
%
Z” % 6.7 % 5.3 % 8.5 F’ Least significant difference * P < 0.05; t P < 0.01; $P < 0.001 2.38 3.08 1.79
5.0
10.0
15.0
20.0
25.0
0.8
1.2
2.6
5.0
6.6
*
ll
0.1
0.5
0.1
0.9
n
1.7
% 8.7
Hoffman, 1979) has been studied, and also two 1,Cthienodiazepines which are particularly potent and possess some of the activity characteristic of the neuroleptics (Boehringer Ingelheim-Internal Report). METHODS
The studies were carried out with 5 male monkeys mulatta) weighing between 8.2 and 12.4 (mean 10.4) kg, trained on a spatial delayed alternation task. The stimuli were 2 illuminated green discs displayed simultaneously on vertical panels spaced 12 cm apart with a food well in the midline below the panels. The animals were required to press the right and left hand panels alternately. With the initial presentation of both stimuli the animal pressed one of the panels to extinguish the light, and was rewarded with a food pellet. This response was not used in the analysis. With the succeeding 50 trials the correct response was to press the panel not rewarded in the previous trial. In the event of an error the trial was repeated until the correct response was made. The intertrial delay was 5 set, and each monkey was required to reach a level of performance of 40 correct out of 50 responses for 10 consecutive sessions (criterion) before experiments with drugs were commenced. The number of correct responses, the number of repeat errors, and the time taken to complete 50 trials were measured. (Macaca
Least significant difference 11P < 0.01; TP < 0.001 3.41 2.57 4.42
i;P < 0.05;
The tests were carried out 1 hr after an intraperitoneal injection of drug or drug vehicle (placebo control). The order of injection of drugs and of drug vehicle was randomized for each animal, and at least 4 days separated each injection. The drugs were 3-hydroxydiazepam (temazepam) and N-desmethyldiazepam (nordiazepam) as examples of the 1,4-benzodiazepines, clobazam as an example of the 1,5-benzodiazepines and WE 973 and WE 941 (brotizolam) as examples of the triazolo-thienodiazepines (see Fig. 1 for structural formulae). In the first experiment the doses were 0.5, 1.0, 1.5,2.0, 2.5 and 3.0mg. kg-’ body weight for all drugs. No behavioural effect was observed with temazepam and clobazam, and so a second experiment was carried out with the dose range 5.0, 10.0, 15.0, 20.0 and 25.0mg. kg-’ body weight. All drugs were dissolved in 5 ml polyethylene glycol. RESULTS
Analysis of performance was concerned with the change in the number of correct responses (excluding repeated responses if the initial response was incorrect), the number of repeat errors and the total response time, each compared with placebo. The measures were related to the performance of individual monkeys and to the dose level. Analysis of variance was the statistical method, and the results are given in Tables 1 and 2.
Table 2. Change in total response time (set) 1 hr after drugs compared with placebo (means for five monkeys)
0.5 Nordiazepam Temazenam
0.24 0.13
1.0 -0.19 -0.17
1.5 -0.14 0.02
2.0
2.5
-0.04 -0.10
-0.22 -0.19
Dose (mg kg- ‘) 3.0 0.02 -0.02
5.0 0.58**
10.0 0.14
15.0 0.37
8 Clobazam Brotizolam WE 973
0.06 -0.13
-0.09 0.50
*
-0.03 1.05
%
0.11 1.06
%
- 0.02 0.82
0.19 1.10
%
%
1.22 0.88 0.90 0.78 t % % % Least significant difference * P < 0.05; t P < 0.01; fPi0.001 0.61 0.79 0.46 0.35
-0.36
-0.19
-0.08
20.0
25.0
1.26
1.05
ll
ll
0.38
0.39
-0.04
** This value was due to an effect in one monkey.
Least significant difference p P < 0.05; 11P < 0.01; 7 P < 0.001 0.97 0.75 0.57
Diazepines and alternation behaviour
493
it is much less potent than diazepam in disturbing rotarod performance, and then only at doses which are markedly above those required to produce antiaggression and “anti-anxiety” effects. It is also difficult to establish impaired coordination with clobazam in man (Borland and Nicholson, 1975). In previous studies from this laboratory with a delayed differentiation task it was shown that hydroxylation of diazepam led to a loss of, or a considerable attenuation of, behavioural activity whereas with demethylation behavioural activity was preserved. Similar observations have been made with spatial delayed alternation. With nordiazepam the number of correct responses was reduced within the dose range 0.5-3.0 mg. kg- ‘, but there was no effect within this dose range with temazepam. Changes were observed with temazepam at much higher doses, but these were not used in the previous experiments. Differential effects on performance with nordiazepam and temazepam have also been observed in man. In dose ranges with hypnotic activity, temazepam has a pronounced effect on coordination, but it is difficult to establish such an effect with nordiazepam (Clarke and Nicholson, 1978). Though substitution of the benzo ring with a thieno group as with clotiazepam, would appear to have little effect in changing activity from that of diazepam as far as pharmacological tests in animals (Nakanishi, DISCUSSION Tsumagari, Takigawa, Shuto, Kenjo and Fukuda, The present studies have shown that the effects of 1972) and effects on sleep in man (Nakazawa, Kotorii, thieno- and benzodiazepines on spatial delayed alterOhshima, Horikawa and Tachibana, 1975) are connation differ, at least, with respect to dose. Within the cerned, the triazolo ring across the methyl group of dose range 0.5-3.0 mg . kg-l, there was no effect of the the diazepam molecule would appear to lead to 1,4_benzodiazepine, temazepam, and the 1,5-benzomarked changes in potency. Triazolam, a triazolo-1,4diazepine, clobazam, but the I+benzodiazepine, norbenzodiazepine, is a potent anti-convulsant with antidiazepam, and the thienodiazepines reduced the aggression, “anti-anxiety” and muscle relaxing number of correct responses and the thienodiazepines properties and increases drug-induced sleeping time increased response time. Within the dose range, more than diazepam (Rudzik, Hester, Tang, Straw 5.G25.0 mg. kg-‘, temazepam reduced the number of and Friis, 1973). It is also a potent hypnotic in man correct responses and increased response time, and (Nicholson and Stone, 1980). Pharmacological studies there was still no effect with clobazam. in animals also emphasise the activity of the triIt is difficult to be certain whether these effects azolo-l,4-thienodiazepines in anti-convulsant, “antimerely reflect the potency of the various compounds anxiety” and anti-aggression tests in animals, though or whether they reflect qualitative differences in ac- in many respects they differ from minor tranquillizers. tivity. The effect of the triazoIo-thienodiazepine, They are only weakly effective in inhibiting electrobrotizolam, compared with temazepam on behaviour shock convulsions in mice, and at very high doses in the monkey is certainly comparable with the rela- they are unable to produce coma or loss of righting tive hypnotic activity of these compounds in man reflexes even though sedation and ataxia are present (Nicholson and Stone, 1976 and unpublished observaat much lower doses. Additionally, they possess actions), though dissociation of psychomotor and be- tivity typical of neuroleptic drugs as brotizolam and havioural activity would appear to be a property WE 973 decrease locomotion and exploration in mice, specific to the l,S-benzodiazepine, clobazam. Characand brotizolam is effective in an active avoidance task teristically, benzo~i~epines possess anti-convul~nt in rats. In previous studies, the possibility that impaired and anti-aggression activity, prolong drug-induced performance on delayed differentiation with benzosleeping time and demonstrate “anti-anxiety” potendiazepines may imply behavioural disinhibition was tial as well as muscle relaxant properties (Randall, Scheckel and Pool, 1970; Barzaghi, Fournex and discussed. Such an effect with benzodiazepines has Mantegazza, 1973; De Angelis, Traversa and Vertua, also been discussed by Hasegawa, Ibuka and Iwahara 1974; Fielding and Hoffman, 1979) but with cloba- (1973) in the monkey, by Schallek, Kuehn and Kovacs zam there is a loss of muscular relaxant activitv. ~~,, and ~~~. (1972) in the cat and by Margules and Stein (1968),
There were no effects of clobazam on the number of correct responses or on total response time. Temazepam decreased the number of correct responses with 15.0 mg. kg-’ and above, and increased total response time with 20 mg . kg- 1 and above. An effect on total response time at the 5.0 mg. kg- ’ dose was due to a marked increase with one monkey only, and this effect was not observed with the same monkey at 10 and 15 mg . kg- ‘. Nordiazepam reduced the number of correct responses without an increase in response time within the lower dose range (1.5-3.0 mg . kg-‘), and the animals failed to respond with doses above 5.0 mg . kg- ‘. Brotizolam (1.5 mg . kg- 1 and above) reduced the number of correct responses and increased the total response time, while WE 973 decreased the number of correct responses with 0.5 mg.kg-’ and above, and increased the total response time with 1.5 mg. kg- ’ and above. Analysis of the number of repeat errors by the Fischer Exact Test showed a difference between the distribution of errors with brotizolam and WE 973 compared with nordiazepam, temazepam and clobazam. An additional statistical test showed that with brotizolam there was an increase in repeat errors at the higher doses (i.e. 2.0mg. kg-’ and above) and this was greater than with the lower doses (0.5-1.5 mg. kg-“) or with placebo (P < 0.05).
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Heise, Laughlin and Keller (1970) Wedeking (1969) and Iwahara (1977) in the rat. However, recently, response perseveration rather than disinhibition has been proposed as the behavioural basis of the changes observed with benzodiazepines (Dantzer, 1977). The interpretation of the effect of benzodiazepines on behaviour remains uncertain, and similar studies with brain lesions suggest that other factors such as the spatial element of the task may be involved (Mishkin, 1964; Iversen and Mishkin, 1970). Behavioural studies with brain lesions also raise the question whether the integrity of certain forebrain systems may be impaired by benzodiazepines. Impairments in learning and retention of spatial delayed alternation have been observed in the monkey with lesions of the frontal lobes and with lesions of deep temporal lobe structures (Orbach, Milner and Rasmussen, 1960; Divac, Rosvold and Szwarcbart, 1967; Mishkin, Vest, Waxler and Rosvold, 1969) while response perseveration has been seen after similar lesions in tests which involve extinction, differentiation, and discrimination reversals (Butter, Mishkin and Rosvold, 1963; Mishkin, 1964; Butters and Rosvoid, 1968; Mishkin et rd., 1969; Butter, 1969; Iversen and Mishkin. 1970). Care must be exercised in comparing the behavioural effects of drugs and of brain lesions, but the importance of frontal and temporal pathways for the integrity of complex behaviour is well established, and the effects of some benzodiazepines may well involve such higher nervous pathways. It is difficult to be certain whether the present observations in the monkey relate to such higher nervous function in man, but the observations on spatial delayed alternation may suggest subtle, though as yet uncertain, effects on human behaviour, and that such effects may be more likely with the triazolo-1,4-thienodiazepines than with the 1,4- and 15benzodiazepines.
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Schallek, W., Kuehn, A. and Kovacs, J. (1972). Effects of chlordiazepoxide hydrochloride on discrimination responses and sleep cycles in cats. Neuropharmacology 11: 69-79. Wedeking, P. W. (1969). Disinhibition effect of chlordiazepoxide. Psvchon. Sci. 15: 232-233.