Evaluation of a rapid technique for detecting minor tranquilizers

Nerrropharmarology, 1971,10,459-469 Pergamo~l Press. Printed

in Gt.Britain.

EVALUATION OF A RAPID TECHNIQUE FOR DETECTING MINOR TRANQUILIZERS C. ARON,P. SIMON,C. LAROUSSE and J. R. BOISSIER Unite de Recherches de Neuropsychopharmacologie 2, rue d’Alesia, Paris 14tme

de I’INSERM,

(Accepted 21 November 1970)

Summary-A technique, based on the inhibition by foot-shocks of a simple ongoing behaviour in mice (ambulation), is suggested for the preliminary screening of the minor tranquilizers. The effect of a large number of psychotropic drugs was studied: they include minor tranquilizers, hypnotics, anticonvulsants, antihistamines, neuroleptics, antidepressants, antiparkinsonism agents, stimulants, analgesics and others. The efficiency, specificity, reliability, simplicity and disadvantages of the method are discussed.

“MINOR TRANQUILIZERS", as distinguished from the “major tranquilizers” or “neuroleptics”, have been used in man to treat symptoms of anxiety and tension and of some psychoneurotic and psychosomatic disorders. They have also been used as an adjunct of neuroleptic medication, but have not been found to affect psychotic symptoms. At the present time, no satisfactory experimental method or design is available to produce, in animals, manifestations of anxiety similar to those observed in man. The techniques now existing have already been described in several reviews (COOK and KELLEHER, 1963; IRWIN, 1968; RANDALL and SCHALLEK, 1968; BOISSIERand SIMON,1969; GIURGEA,1969). Two main types of procedure have been used in attempts to detect the suspected “tranquilizing activity” of a new compound. The techniques belonging to the first type can easily be used for a fast preliminary screening: the association of sedative activity with anticonvulsant, anti-aggressive and muscle relaxant action is looked for; however, the relation between all these properties and the therapeutic effect is far from clear. The techniques belonging to the second type may possibly have more to do with the therapeutic effect. They are based on conditioned “emotional” reactive patterns, but the length of time involved in their application limits their use for the preliminary screening of new potentially anxiolytic agents. In a previous study (BOISSIER et al., 1968) the present authors suggested the use, in mice, of a rapid screening method involving the inhibition of a simple exploratory behaviour of a new environment by the application of an electric shock to the paws of the animal. Indeed, the minor tranquilizers studied were found to diminish this induced inhibition. The aim of the present study was to discover if this method could differentiate drugs with minor tranquilizing activity from all other psychotropic drugs. Because the druginduced disinhibition could result from a stimulating effect rather than from a decreased “anxiety”, we also investigated whether or not the behavioural disinhibition observed could be secondary to an increase in motor activity. 4.59 THE

460

C. ARON,P.SIMON, C. LAROUSSE and J. R. BOISSIER METHODS

The four-plates test Apparatus. The box was first used by SLOTNIK and JARVIK (1966) in their study of

septal mice. The chamber is made of an opaque plastic and has the shape of a parallelepiped rectangle (25 x 18 x 16 cm). The floor is covered with 4 rectangular metal plates (Il.3 x 7.7 cm) separated from one another by a gap of 4 mm. The plates are connected to a source of direct current and a 180 V difference of potential between 2 adjacent plates occurs for 0.5 set when the experimenter presses a switch. The voltage and duration parameters were selected in order to evoke a clear flight reaction in the control animals. Testing procedure. At the beginning of a test, a mouse was gently dropped onto a plate and allowed to explore the enclosure for 15 sec. Every time the mouse crossed from one plate to another, the experimenter pressed the switch and electrified the whole floor for 0.5 set; the mouse often crossed 2 or 3 plates; if it continued running, it received no new shock during the following 3 set; the number of times the apparatus was electrified was counted during the minute after the 15 set exploration. A parametric study was made to choose the values of the different variables (ARON, 1970). The activity box

The apparatus

based on photocells BOISSIERand SIMON (1965) was used.

(DEWS,

1953),

which has been described by

Animals

Male mice of a Swiss strain (Charles Rivers), weighing from 18 to 25g, were used. Drug administration

All drugs were injected intraperitoneally 30 min before testing, except for reserpine, iproniazid and pargyline which were injected 24 hr before testing. Drugs were given in a water solution or in suspension with arabic gum, so that all mice received 0.5 ml of liquid for 20g of body weight. For each drug dose tested by the four-plates method, a group of 12 mice was compared to a group of 12 control animals injected with water or an arabic gum suspension. For the measures of activity, groups of 6 mice were used. The following drugs were tested: alimemazine, amitriptyline, hydrochloride, amobarbitone, dexamphetamine sulphate, aspirin, atropine sulphate, beclamide, benactyzine hydrochloride, butesamide, butobarbitone, caffeine, captodiamine, hydrochloride, chloral hydrate, chlorpromazine hydrochloride, clofenamide hydrochlordiazepoxide hydrochloride, bitartrate, dextropropoxyphene cresoxydiol, dextromoramide chloride, clorazepate, hydrochloride, diazepam, dibenzepine hydrochloride, diethazine hydrochloride, diphencloxazine hydrochloride, diphenhydramine, dispranol, emylcamate, eserine sahcylate, ethosuximide, N ethyl 3 piperidyl phenyl cyclopentyl (Ditran@), Auoresone, fluphenazine dichlorhydrate, haloperidol, hydroxyphenamate, hydroxyzine dichlorhydrate, imipramine hydrochloride, iproniazid, levomepromazine hydrochloride, lysergamide, mecloqualone, mephenoxalone, mephenytone, meprobamate, mesoridazine napadisylate, methaqualone, methocarbamol, a-methyldopa, methylpentynol carbamate, a-methyiparatyrosine hydrochloride, methysergide, metoclopramide, molindone, morphine hydrochloride, oxazepam, oxomemazine, DL parachloromethylamphetamine hydrobromide, parachlorophenylalanine, pargyhne, pemoline, pethidine hydrochloride, phenaglycodol, phenobarbitone, phensuximide, phenyl-acetylurea, phenytone, pimetixene, pimoside, prenylamine lactate, primidone,

Evaluation of a rapid technique for detectingminor tranquilizers

461

procyclidine hydrochloride, promethazine, propranolol hydrochloride, reserpine, scopolamine hydrobromide, sulpiride, tetrazepam, thalidomide, thioridazine hydrochloride, trihexyphenidyle hydrochloride, trimethadione, trimetosine, valnoctamide, yohimbine hydrochloride. RESULTS Control animals The first shock induced a flight reaction; the animal squeaked, ran across 1 or 2 plates and then froze in a corner of the box. It trembled, exhibited piloerection and urinated. After a few seconds, it slowly resumed its exploration, tentatively explored the adjacent plate edges with its paw or its nose, eventually placing its 2 fore-paws on one of the plates. At that moment, the plate was again electrified. An early or a late electrification, as related to the passage of the gap, totally changed the behaviour of the animals. If the floor was electrified exactly when the animal had 2 paws on one plate and 2 on the other, it induced the typical flight reaction. If it was electrified earlier, for example when the mouse had only 1 paw on the next plate, the animal retreated very fast and showed considerably fewer autonomic reactions (piloerection, urination, etc.). If it was electrified later, for example when only 1 paw was left on the first plate, the same happened: very rapid escape from the shock, very few autonomic reactions, Correct timing, therefore, seemed important. Within the first minute, the animal would normally take from 1 to 10 shocks, as shown in Fig. 1. The distribution of the results was close enough to the normal distribution for us to be able to compare the group of control mice to the group of treated ones by means of the t-test. We were interested only in evaluating the significance of the difference

__AI

2

Number FIG.

i

3

4

of shocks(N)

received

1. Distributionof resultsfor 600control animals.

462

C. ARON, P. SIMON,C. LAROUSSE and J. R. BOISSIER

between the increase in the number of punished crossings from one plate to another of treated mice and that of the controls. The activity measures were used only as a qualitative index of stimulation or sedation in a different environment. Pre-treated mice

For the treated mice, the number of punished crossings was increased by 2 groups of drugs. In the first group were various types of psychotropic drugs which all increased motor activity: caffeine, cocaine, dexamphetamine, DL parachloromethylamphetamine, phenobarbitone, trihexyphenidyle (Table 1). In the other group were substances which did not increase motor activity: firstly, drugs which clinicians know as minor tranquilizers : benactyzine, chlordiazepoxide, diazepam, emylcamate, hydroxyphenamate, meprobamate, oxazepam, phenaglycodol and trimetosine (Table 2) ; and secondly, 2 hypnotics (butobarbitone and chloral hydrate), 2 anticonvulsants (mephenytone and phenacetylurea), 2 antihistamines (diphenhydramine and promethazine), 1 antidepressant (amitriptyline), 1 central anticholinergic (Ditran@) (Table 3). Among the drugs which did not increase the number of punished crossings were some drugs reputed as minor tranquilizers (Table 4), most of the anticonvulsants, antihistamines, drugs used in parkinsonism, and antidepressants, and all the neuroleptics and the central analgesics which we have studied. Also among them were: lysergamide and methysergide, antiserotonin agents, and parachlorophenylalanine, an inhibitor of serotonin synthesis; a-methyl-dopa, metoclopramide and sulpiride which have some relation to the neuroleptics, prenylamine, a sedative drug, propranolol, a /3-adrenergic-blocking agent, yohimbine an a-adrenergic-blocking agent, a-methylparatyrosine, an inhibitor of tyrosine hydroxylase and finally eserine (Table 3). DISCUSSION

The first point of significance is the route of administration of the drugs and the interval chosen between the injection and the testing. We decided to keep these 2 parameters constant so as to maintain our usual conditions for preliminary screening. Probably, this fact alone accounts for some of the negative results and for differences within a given class of drugs. Another handicap may have been the similarity of the punished behaviour to the locomotor reaction induced by the punishment itself: punished behaviour was the crossing of a gap, at a normal or a slow pace, and the locomotor reaction induced by the punishment was a flight during which the mouse ran, squeaked and would, as a rule, cross l-3 gaps, unpunished. What are the factors or drug actions which might affect the behaviour of mice in the test situation? If their behaviour were primarily controlled by 2 main sets of motivation -on the one hand, the desire to move or to explore the new environment and on the other, the fear of moving, of crossing the gaps, caused by the foot-shocks-it is evident that certain drug actions should affect the results of the test. It is to be expected that the number of shocks received in any 1 min would increase if the overall drive to move were increased, if the sensitivity to the shock were decreased, or if the short-term memory of the animal were modified. It could not be inferred with certainty from this test that a drug known to have any of these actions had a tranquilizing action. For example, phenobarbitone has been used by clinicians at low doses as a minor tranquilizer. It does increase the number of punished crossings in the test, but it does so at a dose which increases spontaneous activity as well. Therefore, in a preliminary screening, we might have attributed

463

Evaluation of a rapid technique for detecting minor tranquilizers TABLE

1. DRUGS WHICH INCREASED BOTH PUNISHED BEHAVIOLJRIN THE TEST AND AMBULATION [Number of punished crossings, expressed as a percentage of controls (10 mice per group)]

-

Doses (mg/kg i.p.) 30 min before the test 0.5

1

2

4

8

16

32

120

118 (144) 131

141* (216) 150* (119) 109

175* (310) 264% (174) 147 (120)

214* (445)

272* (516)

Pemoline

130 (161) 146* (90) 111

(1::)

(Z)

Cocaine

81 (1::)

(KZ;

(37899

&

Caffeine

121

117 (131)

133 (273)

153 (267)

178* (154)

187* (157)

153* (246) 185* (178)

200* (152)

146* (135)

0.12

0.25

o-Amphetamine Parachloromethylamphetamine

118

Trihexyphenidyl

117 (112)

167* (124)

177* (135)

_-__ *The difference from the controls is statistically significant (P < 0.05). Figures in parentheses refer to motor activity, expressed as a percentage of controls.

TABLE 2. MINOR TRANQUILIZERS WHICH INCREASED THE PUNISHED BEHAVIOUR

[Number of punished crossings. exuressed as a percentage of controls (10 mice per group)] Doses (mg/kg i.p.) 30 min before the test 0.5 Chlordiazepoxide

1 104

2

Oxazepam

97

124 (81) 198% (90) 116

Benactyzine

99

188*

Diazepam (1::)

,:ZY;

4

8

16

32

115 (56) 205* (49) 195* (91) 210* (97)

146* (26) 50

160* (13)

100

162* (58) 261* (104)

150* (37) 199* (102)

Emylcamate

137 (59)

Hydroxyphenamate Meprobamate

100

Phenaglycodol

143

Trimetosine

89

113

130 (62)

150* (58)

64

128

256

201* (101) 134 (91) 148* (128) 163

215* (76) 194* (106)

183

140

63 (47)

(%; 178* (65) 83

133 (10) 70

*The difference from the controls is statistically significant (P < 0.05). Figures in parentheses refer to motor activity, expressed as percentage of controls.

C. ARON, P. SIMON,C. LAROUSSEand 5. R. BOIWER

464

TABLE3. EFFECTOF VARIOUSPSYCHOTROPIC DRUGS:PART I [Number of punished crossings, expressed as a percentage of the controls (10 mice per group)] Doses (mg/kg i.p.) 30 min before the test 0.125

0.25

0.5

1

2

Hypnotics Phenobarbitone Butobarbitone

4

8

16

32

64

100

106

108

96

220*

242*

160’ (139) 251*

(146)

(67) 100 59 95 129

(86)

‘;;I

68 95 123

87 104 125

147 129

147 114

147 142

95

Amobarbitone Mecloqualone Methaqualone Chloral

100 67

77 99

Anticonvulsants Phenytone Mephenytoine

126

Primidone Trimethadione Phensuximide Ethosuximide Beclamide Phenylacetylurea

127 106 83

Antihistamines Alimemazine Diphenhydramine

75

Oxomemazine Pimetixene Promethazine

Neuroleptics Chlorpromazine Fluphenazine Levopromazine Thioridazine Mesoridazine Haloperidol t Pimozide Reserpine Reserpine (during 5 days) Sulpiride Molindone

100

100 94

82

96

75

130 90

94 78 88

50 95 125

90 56 87

100

84 108

(101) 103

(120) 121

73

125

80 157* (81)

100 (66)

91 84 141 (30)

74 55

39

61

75

46 74 82

67 67

125 102

90 33

72

77

76

73

63

(Z)

*The difference from controls is statistically significant (P < 0.05). t Haloperidol was shown inactive down to 0.03 mg/kg. Figures in parentheses refer to motor activity, expressed as a percentage of controls,

147 186* (102) 108 99

127

108 155*

84

104 169* (88)

89

96 166*

256

127 (61)

90 62 121 100

106

128

92

240* (96)

167 152* (105) 102

110 96 85 123 157* (129)

80 97 36 175* 243* (86)

Evaluation

of a rapid technique for detecting minor tranquilizers

465

TABLE 3. EFFECT OF VARIOUS PSYCHOTROPIC DRUGS:PART II [Number of punished crossings, expressed as a percentage of the controls (10 mice per group)1

Doses (mg/kg i.p.) 30 min before the test 0.125

0.25

0.5

1

Antidepressants Pargyline Iproniazid Dibenzepine Imipramine Clomipramine Amitriptyline

Anti-parkinsonism Atropine Scopolamine Diethazine Procyclidine

4

8

16

105 83

9.5

100

113

125

122 140

80

100 103

89

126 94

55 98 113 147* (87)

65 98 112 159* (76)

107

87

86

58

85 125

110 134

118 132 118

63

31

123 131

63 105

83

55

132

151 102

81 132

110

105

50 96

89

149”

32

64

128

256

119

105 88 70

117 105

110 97

101

72

133

114

93

97

85 109 110

114 135

110 119

Analgesics Aspirin Dextromoramide Dextropropoxyphene Morphine Pethidine

Various other drugs Lysergamide Ditran” Methysergide Yohimbine Eserine a-Methyldopa Prenylamine Metoclopramide Propranolol Parachlorophenylalanine (per 0s) a-Methylp-tyrosine

2

60

172* 67 64

92 56 130

86 51 130

128 102

114

134 104 95

466

C. ARON, P. SIMON,C. LAROUSSEand J. R. BOISSIER TABLE4. MINOR TRANQUILIZERS WHICH DID NOTSIGNIFICANTLY INCREASE THE PUNISHED BEHAVIOUR [Number of punished crossings, expressed as a percentage of controls (10 mice per group)] Doses (mg/kg i.p.) 30 min before the test 0.5

Butesamide Captodiamine Clofenamide Clorazepate Cresoxydiol Diphencloxazine Dispranol Fluoresone Hydroxyzine Mephenoxalone Methocarbamol Methylpentynol Tetrazepam Thalidomide Valnoctamide

87

1

2

4

8

12

123

80

79

82

75 89

92 120

60 62

48 14

62

68

52 78

1;: 98

92

112

81 120

97 104

16

256

32

64

128

58 45

61 45

59

85

81

60

78 135 79

82 130

109 85

75

86 141 104 126 61

95 90 85

140 93

150 93

75 69 140 68

103

80 78 102

512

85 81

109 -

--

its effect on the test to overall stimulation. This raises a problem of interpretation: if a minor tranquilizer is found to have slightly stimulating effects, the result may be taken for those given by a stimulant and the minor tranquilizing property overlooked. This ambiguity is likely to be classified by more elaborate tests (based on Conditioned Emotional Response or conflict). We would discard without hesitation, in view of the results of motility measures which reflect their well known stimulating action, caffeine, cocaine, dexamphetamine, DL parachloromethylamphetamine, phenobarbitone and trihexyphenidyl. For all the other drugs which were shown to affect the test positively, since they are neither known as analgesics nor as affecting memory in man, we would tend to postulate that they do, in fact, possess an anxiolytic activity. It appears, however, that not all drugs known as minor tranquilizers and not all hypnotics, do increase the number of punished crossings. Perhaps, therefore, they do not really have an anxiolytic action. It is possible that the test does not detect those drugs which are anxiolytic at sedative doses. This is made all the more likely by the fact that these drugs were selected by pharmacologists from a large number of substances producing sedation. Whether a tranquilizer should be a sedative is a matter for discussion; a therapeutic dose of a minor tranquilizer is not necessarily a sedative one. It has been shown in rats that prolonged treatment will attenuate oxazepam-induced sedation but not its “disinhibitory or therapeutic effect” (MARGULES and STEIN, 1968). That mephenytone and phenacetylurea have minor tranquilizing properties is possible but is of no consequence since these drugs are too toxic to be used as minor tranquilizers. Two antihistamines out of 5 increased punished crossings, thus indicating anxiolytic action. This seems to confirm a recent study by RAULT (1969) who could not find a central property common to all antihistaminic drugs. Amitriptyline has been singled out by the test among all the antidepressants studied. Some clinicians view it as the antidepressant that can be given to depressed patients with a lesser risk of suicide. The result of this study suggests that part of its therapeutic efficacy might be related to its anxiolytic action.

Evaluation of a rapid technique for detecting minor tranquilizers

461

No neuroleptic, at any dose, gave positive results. However, at low doses, some neuroleptics-for example fluphenazine (RICKELSet al., 1968)-are sometimes used instead of benzodiazepines and shown to have the same action on some types of patients. This test, like all so far known except that of VOGELet al. (1969), has the disadvantage of not discriminating between those neuroleptics which possess an anxiolytic property and those which do not. Morphine, in contrast to what could be expected of an “analgesic”, did not increase the number of punished crossings and even decreased it. Similar results have been found before (BARRY and MILLER, 1962) in conflict situations involving foot-shock punishment. There is no simple satisfactory explanation. Yohimbine significantly decreased the number of punished crossings. It has been reported to induce anxiety (LANG and GERSHON, 1963) and thus it is not surprising that it acts in a manner opposite to that of the minor tranquilizers. On the whole, what is the value of the test? Does it have any of the virtues which JANSSEN(1964) asks the ideal screening test? “High eficiency, high speed, simplicity”? This technique is simple, does not require expensive equipment nor unusual animals. Three doses of a drug can be studied within 1 hr, even by an untrained experimenter. It might be asked why the test is not automatized; the reason is that we have not found the perfect system that would shock the animal exactly when-and only when-it is actually crossing a gap. “Reproducibility” ? The behaviour of the control animals is very reliable and for the standard-drugs like diazepam, chlorpromazine, imipramine, we have been able to repeatedly reproduce the results. “SpeciJicity, a given drug effect being characteristic of a well-defined class of chemicals and indicative of a spec@c mode of action”? The question arises here of how precisely

the group of the minor tranquilizers is defined. In the introduction, we defined them by their clinical activity but this raises the problem of the methodology and interpretation of clinical studies. What is studied is difficult to quantify, and criteria of anxiety, emotion or neurotic symptoms have often been arbitrarily chosen. Besides, even though many studies have been made in this field, they often lack a control group of subjects and this makes them almost meaningless. Therefore, we did not think it possible to take into account a great number of the drugs classified as minor tranquilizers to prove (or disprove) that the test does indeed screen out all the minor tranquilizers known at the present time. The well known benzodiazepines, diazepam and chlordiazepoxide, are generally recognized by most clinicians to possess therapeutic activity and they have always given positive results in this test. Clearly the minor tranquilizers were not the only drugs which gave positive results in the test. Stimulating drugs also increased punished responses, but the 2 groups could easily be differentiated by simple observation, or by measuring motor activity. There is not enough evidence to prove that there is only one specific mode of action for all the non-stimulating drugs which gave positive results in the test. “Adequate design, adequate data processing,

statistical analysis and symbolization” ?

Randomization can easily be-and must be-complete, and the experimenter should work in a blind manner. We found that, under those conditions, the subjective bias could almost be eliminated, since a change of experimenter did not bring about different results. The statistical analysis of the results presents no difficulty. “Good correlation with other tests . . . in man”? We can only speculate on this, even if

468

C. ARON, P. SIMON,C. LAROUSSEand J. R. BOISSIER

we change the requirement to “relationship of what is observed in the test situation to those clinical effects which are looked for”. It could be asked whether the effect of the minor tranquilizers on the punished crossings does not, in fact, reflect a side effect of these drugs which has nothing to do either with anxiety or with any of the symptoms against which the drugs are used. It would be difficult to say that we have here a model of a conflictual situation, in which the control animals react in an ill-adapted way, but in which the animals treated with minor tranquilizers react in a much more adapted manner; there is no evidence of this. But it can be said that in this test situation, the animals are motivated by 2 antagonistic drives, as suggested earlier. Under the influence of the minor tranquilizers, for example diazepam, what happens seems like an underestimation of the negative reinforcement (or “risk”) or an overestimation of the positive reinforcement (or “reward”): such an effect might be therapeutic if underestimation of rewards and overestimation of risks is an important factor in human neuroses. In the everyday life of a well-adapted person, however, such an effect would clearly be undesirable at times. One would, for example, assume from this that a driver under the influence of a minor tranquilizer could be a dangerous driver. To conclude: what place does this test have among those already in use ? Since existing tests are unspecific or long, and since this test is simple, reproducible, fast and reasonably efficient and specific, it would appear to be useful in a preliminary screening as one of a battery of tests. A drug active in this test should be studied further, with the longer and probably more specific tests based on conditioned emotional responses or conflict. Prolonged systematic use of this test by several experimenters should indicate its value. REFERENCES ARON, C. (1970). Dun test pharmacologique utilisable pour la selection de tranquillisants. These de Medecine, Paris. BARRY, H. and MILLER, N. E. (1962). Effects of drugs on approach-avoidance conflict tested repeatedly by means of a “telescope alley”. J. camp. physiol. Psychol. 55: 201-210. B~ISSIER,J. R. and SIMON,P. (1965). Action de la cafeine sur la motilite spontanQ de la souris. Archs int. Pharmacodyn. Thk. 158: 212-221. BOISSDER, J. R. and SIMON,P. (1969). Evaluation of experimental technique in the psychopharmacology of emotion. Ann. N. Y. Acad. Sci. 159: 898-914. BOISSIER,J. R., SIMON,P. and ARON, C. (1968). A new method for the rapid screening of minor tranquilizers in mice. Eur. J. Pharmac. 4: 145-151. &oK, L. and KELLEHER,R. T. (1963). Effects of drugs on behavior. A. Rev. Pharmac. 3: 205-222. DEWS, P. B. (1953). The measurement of the influence of drugs on voluntary activity in mice. Br. J. Pharmac. Chemother. 8: 4U8. GIURGEA,C. (1969). Pharmacological criteria for the classification of tranquilizers. In: The Present Status of Psychotropic Drugs (CERLETTI,A., Ed.), pp. 154-167. Ext. Med. Foun., Basle. IRWIN, S. (1968). Anti-neurotics: Practical pharmacology of the sedative-hypnotics and minor tranquilizers. In: Psychopharmacology, u Review of Progress, 1957-1967 (EFRON, D. H., Ed.), pp. 185-204. Public Health Service Publications, Washington. JANSSEN,P. A. J. (1964). Screening tests and prediction from animal to man. In: Animal Behavior and Drug Action (STEINBERG,H., Ed.), pp. 264-268. Churchill, London. LANG, W. J. and GERSHON,S. (1963). Effects of psychoactive drugs on yohimbine-induced responses in conscious dogs-a proposed screening procedure for anti-anxiety agents. Archs int. Pharmacodyn. ThPr. 142: 457-472. MARGULES,D. L. and STEIN,L. (1968). Increase of antianxiety activity and tolerance of behavioral depression during chronic administration of oxazepam. Psychopharmacologic 13: 74-80. RANDALL,L. 0. and SCHALLEK,W. (1968). Pharmacologic activity of certain benzodiazepines. In: Pyschopharmacology, a Review ofProgress, 1957-1967 (EFRON,D. H., Ed.), pp. 153-184. Public Health Service Publications, Washington.

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RAULT, B. (1969). Contribution a I’btude de l’action psycholeptique experimentale des anti-histaminiques. Thtse de pharmacie, Paris. RICKELS, K., RAAB, E., GORDON, P. E., LAQUER,K. G., DE SILVERIO,R. V. and HESBACHER,P. (1968). Differential effects of chlordiazepoxide and fluphenazine in two anxious patient populations. Psychopharmacologiu 12: 181-192. SLQTNIK,B. M. and JARVIK, M. E. (1966). Deficits in passive avoidance and fear conditioning in mice with septal lesions. Science, N. Y. 154: 1207-1208. VOGEL,J. R., BEER,B. and CU)DY, D. E. (1969). A simple and reliable conflict procedure for the evaluation of minor tranquilizers. Phurmacologist 11: 246.