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A signal detection analysis of centrally active drugs in mice
Life Sciences, Vol. 27, pp. 2633-2638 Printed in the U.S.A.
Pergamon Press
A SIGNAL DETECTION ANALYSIS OF CENTRALLY ACTIVE DRUGS IN MICE G. F. Gebhart, Arnold D. Sherman and Vahn A. Lewis Department of Pharmacology, University of lowa, lowa City, lowa 52242 SUMMARY A signal detection analysis procedure was employed to evaluate I0 drugs f o r analgetic efficacy using both a conventional analgesiometric test involving no t r a i n i n g (a t a i l f l i c k procedure) and a minimal-training s i t u a t i o n (a platform escape task). The signal detection parameters D' and CR were found to provide a more complete view of the e f f e c t s of the agents tested than did t r a d i t i o n a l threshold measurement. Morphine, ethanol and aspirin a l l s i g n i f i c a n t l y reduced D' in the t a i l f l i c k procedure while only morphine s i m i l a r l y reduced D' in the platform escape task. A major problem in evaluating the analgesia produced by a drug or t r e a t ment is that an attenuation in the response to noxious stimulation may be the r e s u l t of e i t h e r a reduction in the sensation of pain or a decreased a b i l i t y to respond to the noxious stimulation, or both. Sign~l detection theory (SDT) is a psychophysical procedure which has been applied to the analysis of the response to noxious stimulation in humans, in the evaluation of various drugs, acupuncture and verbal suggestion and placebo effects (cf. I ) . This procedure (as applied to nociception in humans) attempts to distinguish between the sensory experience of pain ( i . e . , the neurophysiological response) and the report of, or response to, pain ( i . e . , response bias), which are often not the same (2). The basic design of a SDT analysis assumes the form of a 2 X 2 contingency t a b l e , r e l a t i n g stimulus to response (2,3). Both stimulus and response are recorded as being present or absent; thus, there are four possible conditions: (a) stimulus and response ( ' h i t ' ) ; (b) stimulus, but no response ( ' m i s s ' ) ; (c) no stimulus and no response (correct r e j e c t i o n ) ; and (d) no stimulus, but a response ( f a l s e alarm). On a predetermined proportion of t r i a l s , the stimulus is presented and the presence or absence of a response is noted. On other t r i a l s , no stimulus (or a low level stimulus) is presented and the response to t h i s no signal or 'noise' is noted. A response to noise is a measure of the response bias of the subject while the a b i l i t y to detect and respond to a stimulus is a measure of the subject's s e n s i t i v i t y . Estimation of the subject's s e n s i t i v i t y and response bias in an SDT analysis separates the sensory and cognitive factors responsible for his response. The f i r s t SDT parameter is D' and is an estimate of the sensory d i s c r i m i n a b i l i t y between the no or low level stimulus and the stimulus present ( i . e . , signal) conditions. A low D' value indicates that these conditions are not very d i f f e r e n t and cannot be easily discriminated. A l t e r n a t i v e l y , low D' values also r e s u l t when the subject's sensory mechanisms have become 0024-3205/80/512633-06502.00/0 Copyright (c) 1980 Pergamon Press Ltd.
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i n s e n s i t i v e , thus making s t i m u l l which p h y s l c a l l y are d i f f e r e n t appear to be s i m i l a r . In terms of drug-induced analgesia, a low D' value would suggest that the agent is a f f e c t i n g the subject's s e n s i t i v i t y to pain rather than, or in addition to, his response to pain. The other SDT parameter, the response c r i t e r i o n (CR), estimates a subject's w i l l i n g n e s s to respond. A high c r i t e r i o n suggests a lack of w i l l ingness to respond while a low c r i t e r i o n suggests a high degree of responsiveness. This measure is s i m i l a r to a 'threshold' since i t describes a point at which the subject responds to s t i m u l a t i o n . Since both the D' and CR estimates vary independently, SDT can be used to evaluate whether s e n s i t i v i t y and/or responsiveness to noxious stimulation is altered by drugs. Rollman ( I ) has recently reviewed the theory of signal d e t e c t a b i l i t y , f u l l y explaining response patterns and the l i m i t a t i o n s of SDT, as well as pain studies employing SDT. The present study was designed to evaluate the a p p l i c a b i l i t y of the SDT method to the analysis of drug e f f e c t s in mice. We employed the conventional t a i l f l i c k analgesiometric procedure and a r a p i d l y acquired simple escape task to assess and catagorize the effects of various c e n t r a l l y active agents on animal behavior in response to noxious s t i m u l i . METHODS A t o t a l of 159 male CF-I mice, i n i t i a l l y weighing 30 gm each, were used. One hundred and four were employed in the t a i l f l i c k procedure; the remaining 55 were trained and tested in a grid shock platform-escape procedure. The following agents and doses (calculated as the free base) were evaluated: morphine s u l f a t e (3 and 5 mg/kg); naloxone hydrochloride (0.4 and 0.8 mg/kg); sodium pentobarbital (I0 and 20 mg/kg); ethanol (2.5 and 5 gm/kg); ketamine hydrochloride (4 and 8 mg/kg); chlordiazepoxide hydrochloride (25 and 50 mg/kg); chlorpromazine hydrochloride (2.5 and 5 mg/kg); a s p i r i n (20 and 40 mg/kg); sodium gamma-hydroxy-butyrate (125 and 250 mg/kg) and normal saline (I and 2 ml/kg). These dosages were selected on the basis of previous experience and i n t e r p o l a t i o n based on t h e i r known pharmacology. In the t a i l f l i c k procedure, control responses were obtained immediately p r i o r to i n t r a p e r i t o n e a l i n j e c t i o n of the agent to be evaluated. The mice, randomly divided into groups of 5 and assigned to a treatment group, were tested again beginning t h i r t y to f o r t y min a f t e r drug i n j e c t i o n . This time delay approximated the time of maximal e f f e c t of these agents. Each animal was exposed to four i n t e n s i t i e s of radiant heat applied to the t a i l and the responses were recorded. The next animal was then tested in a s i m i l a r manner so that each animal in a group of f i v e was rotated through the t e s t , being exposed to the four i n t e n s i t i e s of the stimulation in random order on each r o t a t i o n . This was continued u n t i l each animal had been through the t e s t i n g cycle a t o t a l of I0 times. The mice were tested at 60, 70, 80, and 90 v o l t s applied to the radiant Beat ~ource which corresponded, r e s e p e c t i v e l y , to temperatures of 30 , 43 , 57 , and 70vC when tested on a thermometer. In a l l cases, a response was recorded i f the t a i l was withdrawn s u f f i c i e n t l y to a c t i v a t e a l i g h t - s e n s i t i v e photocell w i t h i n 5 sec from the time of heat (lamp) onset. A l l withdrawals which activated a photocell c i r c u i t , whether a sharp r e f l e x i v e f l i c k of the t a i l or a slow voluntary movement, were considered as responses. I f no response occurred w i t h i n 5 sec, the heat source was turned o f f and a "no response" was recorded. Grid shock escape t r a i n i n g was carried out in the following manner. An animal was placed in a p l a s t i c chamber (15 cm wide X 30 cm long X 30 cm high) with a gridded f l o o r and foot shocks were delivered at an i n t e n s i t y of 0.2 mA
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u n t i l the animal escaped onto a raised platform (2.5 cm high) in the center of the box. A 2000 Hz tone was presented concurrently with every shock. Following an escape, the animal was returned to the grid f l o o r and presented with another shock-tone pair. This t r a i n i n g procedure was carried out for a total of 20 t r i a l s . I f within 5 sec of onset of the paired shock and tone the animal had not escaped onto the platform, both the shock and tone were turned o f f . A f t e r the t r a i n i n g period, any animal that f a i l e d to escape a l l subsequent shocks within 6 sec was removed from the experiment. Only one animal out of 55 was removed f o r f a i l u r e to a t t a i n this c r i t e r i o n . The schedule was then changed such that grid shock was presented on only 50% of the t r i a l s while the tone was presented on every t r i a l . This 50% grid shock/lO0% tone randomized paring was carried out for a total of 30 t r i a l s . The trained animals were then injected i n t r a p e r i t o n e a l l y with one dose level (Table 2) of the I0 agents l i s t e d above and tested 40 min l a t e r . Again, shocks were presented in 50% of 40 t r i a l s ; the tone was present on all t r i a l s . Any escape which placed a l l four feet on the platform within 6 sec was considered a response. Both shock and the tone were terminated a f t e r 6 sec i f there was no response. Using the t a i l f l i c k procedure, a classical threshold was calculated employing the responses to a l l four i n t e n s i t i e s used. The SDT parameters D' and CR were calculated by a comparison of the responses to the 70 and 80 v o l t levels of stimulation. All responses to the 70 v o l t (43°C) stimulus were considered false alarms and responses to the 80 v o l t stimulus (57oc) were considered h i t s . In the gridshock procedure, D' and CR were calculated between zero mA (tone only) and 0.2 mA (tone plus shock). Methods for calcul a t i o n of D' and CR are presented in several sources (2, 4). All s t a t i s t i c a l comparisons were made by means of a two-tailed Student's t t e s t , P < 0.05. RESULTS Data f o r the t a i l f l i c k test are presented in Table I. Threshold was s i g n i f i c a n t l y elevated by both doses of morphine, pentobarbital, ethanol, ketamine, chlorpromazine and gamma-hydroxy-butyrate in a dose-related fashion. Thus, a wide v a r i e t y of agents of d i f f e r i n g pharmacologic classes were able to s i g n i f i c a n t l y a l t e r the t a i l withdrawal response. Naloxone, chlordiazepoxide and a s p i r i n , however, did not increase (or decrease) the t a i l withdrawal threshold. The SDT withdrawal c r i t e r i o n (CR) was also s i g n i f i c a n t l y elevated by both doses of morphine, pentobarbital, ethanol, chlorpromazine, gammahydroxy-butyrate and the higher dose of ketamine (8 mg/kg). The c o r r e l a t i o n between the threshold (as determined by the classical procedure) and CR was 0.93. The sensory discriminative measure D' was s i g n i f i c a n t l y depressed to below control levels by ethanol (2.5 and 5 mg/kg), chlorpromazine (5 mg/kg), aspirin (20 and 40 mg/kg), and morphine (3 and 6 mg/kg). Naloxone, however, produced a s i g n i f i c a n t elevation of D' above control levels. Control animals averaged I0.0 + 0 hits and 2.0 + 0.3 false alarms in the t a i l f l i c k t e s t . The data f o r the platform escape are presented in Table 2. All agents except naloxone, chlordiazepoxide, and. a s p i r i n elevated the response c r i t e r i o n , CR. Only morphine, however, s i g n i f i c a n t l y depressed D'. Control animals averaged 20.0 + 0 hits and 4.7 + 0.2 false alarms. DISCUSSION Signal detection analysis has provided some new information about drug e f f e c t s in the standard t a i l f l i c k analgesiometric t e s t and in a foot shock platform-escape procedure. The CR estimates p a r a l l e l closely those of t r a d i tional threshold measurements, but the D' data provide other, independent information. Traditional threshold measurements are nonspecific tests for
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TABLE I DRUG EFFECTS ON TAIL FLICK RESPONSES Drug
Dose a
Saline
Threshold b
1 ml/kg 2 ml/kg Morphine 3 5 Naloxone 0.4 0.8 Pent6barbital I0 20 Ethanol 2.5 gm/kg 5 gm/kg Ketamine 4 8 Chlordi25 azepoxide 50 Chlorproma2.5 zine 5 Aspirin 20 40 Gamma125 hydroxy~50 butyrate a b c + +
69.88 70.14 +81.04 +85.95 72.47 70.24 ~73.76 +76.14 +77.18 ~90.14 +75.00 +78.32 71.90 70.76 #73.81 +78.30 72.20 73.41 ÷75.38 +81.40
+ ¥ ¥ ¥ ¥ ¥ ¥ ¥ ¥ ¥ ¥ ¥ ¥ ¥ 7 ¥ ¥ ¥ ¥ ¥ -
1.35 1.17 1.40 1.35 1.63 1.13 1.61 1.03 1.80 1.74 1,02 1.00 1.05 1.12 0.87 1.00 1.58 0.76 0.65 0.72
CR c 70.67 70.90 +85.00 +88.32 73.45 7~04 +78.24 ÷86.24 ÷81.67 +97.60 74.22 +79.76 71.42 72.11 +78.04 +83.45 72.45 72.15 +75.00 #83.40
D' c
+ 0.63 ¥ 0.50 T 2.15 T 3.05 ¥ 1.14 ¥ 0.76 ¥ 0.64 ¥ 0.51 ¥4.95 ¥ 1.54 ¥ 1.48 ¥ 1.06 T 0.47 ¥ 1.07 ¥0.51 ¥ 0.87 ¥ 0.92 ¥ 0.86 ¥ 1.26 ¥ 1.07 -
N
4.05 + 0.24 3.84 T 0.30 # I . 0 0 T 0.40 +1.02 ¥ 0.41 +5.97 7 0.83 +5.20 ¥ 0.56 3.17 ¥ 1.31 3.00 ¥ 0.97 ¢2.50 ¥ 0 . 5 2 #2.32 ¥ 0.59 3.83 ¥ 0.71 3.97 ¥ 0.54 4 . i 6 ¥ 0.27 4 . 0 1 T 0.40 3.54 ¥ 0.40 +2.32 T 0.42 ¢2.52 ¥ 0.41 +2.41 ¥ 0.50 4.12 ¥ 1.57 3.89 ¥ 1.23 -
7 5 7 6 5 4 5 4 6 4 5 5 5 4 5 7 5 5 5 5
In mg/kg unless otherwise s p e c i f i e d . Expressed as mean + SE of v o l t a g e applied to the lamp. C a l c u l a t e d on the basis of values recorded between 70 and 80 v o l t s . I n d i c a t e s a s i g n i f i c a n t increase above c o n t r o l , P < 0.05. I n d i c a t e s a s i g n i f i c a n t decrease below c o n t r o l , P <-0.05.
TABLE I I DRUG EFFECTS ON GRID SHOCK ESCAPE RESPONSES a Drug Saline Morphine Naloxone Pentobarbital Ethanol Ketamine Chlordiazepoxide Chlorpomazine Aspirin Gamma-hydroxy-butyrate a See legend, Table I .
Dose 1 ml/kg 5 0.4 I0 2.5 gm/kg 4 25 5 20 120
CR 0.I0 +0.33 0.13 +0.38 ÷0.35 +0.26 0.09 +0.44 0.I0 +0.27
+ + + + + + + + + +
C' 0.44 0.05 0.II 0.02 0.03 0.II 0.03 0.06 0.II 0.08
2.37 +1.46 2.32 2.25 2.31 2.34 2.41 2.21 2.54 2.32
+ + + + + + + + + +
N 0.08 0.02 0.I0 0.04 0.15 0.II 0.I0 0.I0 0.42 0,01
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analgesia and can be greatly influenced by drugs having primarily general depressant properties (e.g. pentobarbital, ethanol, e t c . : see Table I ) . However, drug effects r e f l e c t e d by D' are interpreted to be i n d i c a t i v e of analgetic e f f e c t s . Aspirin and morphine had r e l a t i v e l y greater effects on D' than on CR compared to the other drugs tested in the t a i l f l i c k t e s t , but aspirin did not a l t e r e i t h e r the threshold or withdrawal c r i t e r i a , CR. This suggests that a s p i r i n ' s sole analgetic e f f e c t is to decrease the d i s c r i m i n a b i l i t y between non-noxious and noxious s t i m u l i . Aspirin does not have a marked central depressant action and the f a i l u r e of aspirin to change CR or threshold is consistant with such a hypothesis. This is not to suggest that analgesia could not also be produced by a change in threshold, but does indicate that such a change in threshold is neither necessary nor s u f f i c i e n t . Ethanol and chlorpromazine also s i g n i f i c a n t l y depressed D' in the t a i l f l i c k test but were less e f f e c t i v e than morphine, although both agents s i g n i f i c a n t l y elevated the withdrawal c r i t e r i o n as well. Ethanol has long been recognized to have analgetic properties and this has been confirmed in this t e s t . Chlorpromazine, on the other hand, is generally considered not to have analgetic propert i e s , but d i f f e r e n t i a t i o n between antipsychotic t r a n q u i l i z e r s and analgetic agents in analgesiometric tests has been d i f f i c u l t . Naloxone, the 'pure' narcotic antagonist, produced an unexpected s i g n i f i c a n t increase in d i s c r i m i n a b i l i t y in the t a i l f l i c k t e s t . I t would be of considerable i n t e r e s t to determine whether t h i s change is related to naloxone's narcotic antagonist properties ( e . g . , of endogenous opioid peptides) or is a nonspecific e f f e c t .
Pentobarbital, ketamine and gamma-hydroxy-butyrate all produced similar effects in the t a i l f l i c k test. Winters et a l . , (5) have suggested that there are two classes of anesthetic agents: cataleptic anesthetics, whose actions are characterized by an activated EEG, including gamma-hydroxy-butyrate, ketamine, phencyclidine, and trichlorethylene; and the depressant anesthetics, which characteristically depress the EEG at anesthetic doses, such as halothane and the bartiturates. In this study, anesthetics of both classes increased the withdrawal threshold and CR, but did not exhibit significant effects on D' at non-anesthetic doses. In the grid shock platform-escape t e s t , the CR was elevated by morphine, ethanol, chlorpromazine and several anesthetics, indicating f u r t h e r that response bias is sensitive to a v a r i e t y of c e n t r a l l y acting agents. Only morphine, however, produced a s i g n i f i c a n t decrease in D' at the doses studied. The D' as estimated in the t a i l f l i c k t e s t was influenced by a greater v a r i e t y of drugs than in the grid shock escape task. However, a number of experimental variables could account for such differences. Only one dose per agent was employed in the grid shock procedure and i t is possible that higher doses of the other agents could also have altered D'. Each type of drug appeared to present a p a r t i c u l a r p r o f i l e in these tests and p o t e n t i a l l y these properties may be useful in f u r t h e r c l a s s i f y i n g drug groups. However, at this time i t is not clear why the same drugs altered responses in the t a i l f l i c k t e s t to a greater extent than in the grid shock t e s t . I t may be because the t a i l f l i c k test represents a s p i n a l l y mediated r e f l e x whereas the grid shock t e s t represents a behavior integrated at higher CNS levels. Methodologically, the application of SDT in these tests suffers from some limitations. I t would have been desirable to be able to present more t r i a l s in the t a i l f l i c k t e s t , but the danger of i n j u r i n g the animals' t a i l s limited the number of tests that could be tolerated. Increasing the number of t r i a l s
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is important since the r e l i a b i l i t y of D' and CR are d i r e c t l y related to the accuracy of the estimate of the response p r o b a b i l i t i e s . In addition, the ideal SDT experiment accumulates data and portrays i t as the recelver operating c h a r a c t e r i s t i c (ROC), p l o t t i n g false alarms on the abcissa and hits on the ordinate. The ROC function should have a slope of I , indicating equal variances f o r the two d i s t r i b u t i o n s ; D' can thus be calculated from the graph directly. The estimation of D' is based on the assumption of equal variances and the r e l a t i v e l y small sample of responses we collected do not allow D' to be determined for each subject and then averaged across subjects. The variances f o r false alarms and hits among control animals in the two procedures at least suggests Intra-experimental r e l i a b i l i t y . In spite of these drawbacks, the d i s c r i m i n a b i l i t y in the t a i l f l i c k procedure was s i g n i f i c a n t l y higher than that for the grid shock ( t a i l f l i c k D'=4.05 for controls; grid shock D'=2.37 for c o n t r o l s ) . I t is possible, however, that D' in the t a i l f l l c k t e s t is spuriously high because of the small test sample size. Nonetheless, the t a l l f l i c k test results were informative about the r e l a t i v e properties of the drugs used in this study and moreover indicate that SDT analysis c l e a r l y d i f f e r e n t i a t e d effects of analgetics from those of anesthetics. On a methodological basis, the grid shock procedure should be the better method because there was a true no signal condition and a larger sample of responses were obtained. Paradoxically, this method appeared to be less s e n s i t i v e , and the animals had poorer d i s c r i m i n a b i l i t y scores. However, an analgetic agent per se should be the only agent among those we tested (at the doses employed) which would depress pain s e n s i t i v i t y ( i . e . , reduce D' for noxious stimulation) and this was true in the grid shock procedure. Thus, the grid shock escape paradigm may represent the more appropriate procedure of the two employed in this experiment. Acknowledgement: Naloxone was generously provided by Endo Laboratories, Garden City, N. J. Supported by USPHS Grant NS12114 to G.F.G. Dr. Sherman's current address is: The Department of Psychiatry, University of Iowa; Dr. Lewis is c u r r e n t l y at The Department of Pharmacology, University of Texas, Dental Branch, Houston, Texas. REFERENCES I.
ROLLMAN, G. B., Pain 3:
187-212 (1977).
2.
GREEN, D. M. and SWETS, J. A.: (Wiley, New York) (1966).
3.
SWETS, J. A., ed.: Signal Detection and Recognition by Human Observers. John Wiley and Sons, Inc. New York, 1964.
4.
CLARK, W. C. Anesthesiology, 4_00, 272-287, (1974).
5.
WINTERS, W. D., FERRAN-ALLADO, GUZMAN, T., FLORES, G. and ALCARAZ, M. Neuropharmacology I I , 303-315, 1972.
Signal Detection Theory and Psychophysics.
Pergamon Press
A SIGNAL DETECTION ANALYSIS OF CENTRALLY ACTIVE DRUGS IN MICE G. F. Gebhart, Arnold D. Sherman and Vahn A. Lewis Department of Pharmacology, University of lowa, lowa City, lowa 52242 SUMMARY A signal detection analysis procedure was employed to evaluate I0 drugs f o r analgetic efficacy using both a conventional analgesiometric test involving no t r a i n i n g (a t a i l f l i c k procedure) and a minimal-training s i t u a t i o n (a platform escape task). The signal detection parameters D' and CR were found to provide a more complete view of the e f f e c t s of the agents tested than did t r a d i t i o n a l threshold measurement. Morphine, ethanol and aspirin a l l s i g n i f i c a n t l y reduced D' in the t a i l f l i c k procedure while only morphine s i m i l a r l y reduced D' in the platform escape task. A major problem in evaluating the analgesia produced by a drug or t r e a t ment is that an attenuation in the response to noxious stimulation may be the r e s u l t of e i t h e r a reduction in the sensation of pain or a decreased a b i l i t y to respond to the noxious stimulation, or both. Sign~l detection theory (SDT) is a psychophysical procedure which has been applied to the analysis of the response to noxious stimulation in humans, in the evaluation of various drugs, acupuncture and verbal suggestion and placebo effects (cf. I ) . This procedure (as applied to nociception in humans) attempts to distinguish between the sensory experience of pain ( i . e . , the neurophysiological response) and the report of, or response to, pain ( i . e . , response bias), which are often not the same (2). The basic design of a SDT analysis assumes the form of a 2 X 2 contingency t a b l e , r e l a t i n g stimulus to response (2,3). Both stimulus and response are recorded as being present or absent; thus, there are four possible conditions: (a) stimulus and response ( ' h i t ' ) ; (b) stimulus, but no response ( ' m i s s ' ) ; (c) no stimulus and no response (correct r e j e c t i o n ) ; and (d) no stimulus, but a response ( f a l s e alarm). On a predetermined proportion of t r i a l s , the stimulus is presented and the presence or absence of a response is noted. On other t r i a l s , no stimulus (or a low level stimulus) is presented and the response to t h i s no signal or 'noise' is noted. A response to noise is a measure of the response bias of the subject while the a b i l i t y to detect and respond to a stimulus is a measure of the subject's s e n s i t i v i t y . Estimation of the subject's s e n s i t i v i t y and response bias in an SDT analysis separates the sensory and cognitive factors responsible for his response. The f i r s t SDT parameter is D' and is an estimate of the sensory d i s c r i m i n a b i l i t y between the no or low level stimulus and the stimulus present ( i . e . , signal) conditions. A low D' value indicates that these conditions are not very d i f f e r e n t and cannot be easily discriminated. A l t e r n a t i v e l y , low D' values also r e s u l t when the subject's sensory mechanisms have become 0024-3205/80/512633-06502.00/0 Copyright (c) 1980 Pergamon Press Ltd.
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i n s e n s i t i v e , thus making s t i m u l l which p h y s l c a l l y are d i f f e r e n t appear to be s i m i l a r . In terms of drug-induced analgesia, a low D' value would suggest that the agent is a f f e c t i n g the subject's s e n s i t i v i t y to pain rather than, or in addition to, his response to pain. The other SDT parameter, the response c r i t e r i o n (CR), estimates a subject's w i l l i n g n e s s to respond. A high c r i t e r i o n suggests a lack of w i l l ingness to respond while a low c r i t e r i o n suggests a high degree of responsiveness. This measure is s i m i l a r to a 'threshold' since i t describes a point at which the subject responds to s t i m u l a t i o n . Since both the D' and CR estimates vary independently, SDT can be used to evaluate whether s e n s i t i v i t y and/or responsiveness to noxious stimulation is altered by drugs. Rollman ( I ) has recently reviewed the theory of signal d e t e c t a b i l i t y , f u l l y explaining response patterns and the l i m i t a t i o n s of SDT, as well as pain studies employing SDT. The present study was designed to evaluate the a p p l i c a b i l i t y of the SDT method to the analysis of drug e f f e c t s in mice. We employed the conventional t a i l f l i c k analgesiometric procedure and a r a p i d l y acquired simple escape task to assess and catagorize the effects of various c e n t r a l l y active agents on animal behavior in response to noxious s t i m u l i . METHODS A t o t a l of 159 male CF-I mice, i n i t i a l l y weighing 30 gm each, were used. One hundred and four were employed in the t a i l f l i c k procedure; the remaining 55 were trained and tested in a grid shock platform-escape procedure. The following agents and doses (calculated as the free base) were evaluated: morphine s u l f a t e (3 and 5 mg/kg); naloxone hydrochloride (0.4 and 0.8 mg/kg); sodium pentobarbital (I0 and 20 mg/kg); ethanol (2.5 and 5 gm/kg); ketamine hydrochloride (4 and 8 mg/kg); chlordiazepoxide hydrochloride (25 and 50 mg/kg); chlorpromazine hydrochloride (2.5 and 5 mg/kg); a s p i r i n (20 and 40 mg/kg); sodium gamma-hydroxy-butyrate (125 and 250 mg/kg) and normal saline (I and 2 ml/kg). These dosages were selected on the basis of previous experience and i n t e r p o l a t i o n based on t h e i r known pharmacology. In the t a i l f l i c k procedure, control responses were obtained immediately p r i o r to i n t r a p e r i t o n e a l i n j e c t i o n of the agent to be evaluated. The mice, randomly divided into groups of 5 and assigned to a treatment group, were tested again beginning t h i r t y to f o r t y min a f t e r drug i n j e c t i o n . This time delay approximated the time of maximal e f f e c t of these agents. Each animal was exposed to four i n t e n s i t i e s of radiant heat applied to the t a i l and the responses were recorded. The next animal was then tested in a s i m i l a r manner so that each animal in a group of f i v e was rotated through the t e s t , being exposed to the four i n t e n s i t i e s of the stimulation in random order on each r o t a t i o n . This was continued u n t i l each animal had been through the t e s t i n g cycle a t o t a l of I0 times. The mice were tested at 60, 70, 80, and 90 v o l t s applied to the radiant Beat ~ource which corresponded, r e s e p e c t i v e l y , to temperatures of 30 , 43 , 57 , and 70vC when tested on a thermometer. In a l l cases, a response was recorded i f the t a i l was withdrawn s u f f i c i e n t l y to a c t i v a t e a l i g h t - s e n s i t i v e photocell w i t h i n 5 sec from the time of heat (lamp) onset. A l l withdrawals which activated a photocell c i r c u i t , whether a sharp r e f l e x i v e f l i c k of the t a i l or a slow voluntary movement, were considered as responses. I f no response occurred w i t h i n 5 sec, the heat source was turned o f f and a "no response" was recorded. Grid shock escape t r a i n i n g was carried out in the following manner. An animal was placed in a p l a s t i c chamber (15 cm wide X 30 cm long X 30 cm high) with a gridded f l o o r and foot shocks were delivered at an i n t e n s i t y of 0.2 mA
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u n t i l the animal escaped onto a raised platform (2.5 cm high) in the center of the box. A 2000 Hz tone was presented concurrently with every shock. Following an escape, the animal was returned to the grid f l o o r and presented with another shock-tone pair. This t r a i n i n g procedure was carried out for a total of 20 t r i a l s . I f within 5 sec of onset of the paired shock and tone the animal had not escaped onto the platform, both the shock and tone were turned o f f . A f t e r the t r a i n i n g period, any animal that f a i l e d to escape a l l subsequent shocks within 6 sec was removed from the experiment. Only one animal out of 55 was removed f o r f a i l u r e to a t t a i n this c r i t e r i o n . The schedule was then changed such that grid shock was presented on only 50% of the t r i a l s while the tone was presented on every t r i a l . This 50% grid shock/lO0% tone randomized paring was carried out for a total of 30 t r i a l s . The trained animals were then injected i n t r a p e r i t o n e a l l y with one dose level (Table 2) of the I0 agents l i s t e d above and tested 40 min l a t e r . Again, shocks were presented in 50% of 40 t r i a l s ; the tone was present on all t r i a l s . Any escape which placed a l l four feet on the platform within 6 sec was considered a response. Both shock and the tone were terminated a f t e r 6 sec i f there was no response. Using the t a i l f l i c k procedure, a classical threshold was calculated employing the responses to a l l four i n t e n s i t i e s used. The SDT parameters D' and CR were calculated by a comparison of the responses to the 70 and 80 v o l t levels of stimulation. All responses to the 70 v o l t (43°C) stimulus were considered false alarms and responses to the 80 v o l t stimulus (57oc) were considered h i t s . In the gridshock procedure, D' and CR were calculated between zero mA (tone only) and 0.2 mA (tone plus shock). Methods for calcul a t i o n of D' and CR are presented in several sources (2, 4). All s t a t i s t i c a l comparisons were made by means of a two-tailed Student's t t e s t , P < 0.05. RESULTS Data f o r the t a i l f l i c k test are presented in Table I. Threshold was s i g n i f i c a n t l y elevated by both doses of morphine, pentobarbital, ethanol, ketamine, chlorpromazine and gamma-hydroxy-butyrate in a dose-related fashion. Thus, a wide v a r i e t y of agents of d i f f e r i n g pharmacologic classes were able to s i g n i f i c a n t l y a l t e r the t a i l withdrawal response. Naloxone, chlordiazepoxide and a s p i r i n , however, did not increase (or decrease) the t a i l withdrawal threshold. The SDT withdrawal c r i t e r i o n (CR) was also s i g n i f i c a n t l y elevated by both doses of morphine, pentobarbital, ethanol, chlorpromazine, gammahydroxy-butyrate and the higher dose of ketamine (8 mg/kg). The c o r r e l a t i o n between the threshold (as determined by the classical procedure) and CR was 0.93. The sensory discriminative measure D' was s i g n i f i c a n t l y depressed to below control levels by ethanol (2.5 and 5 mg/kg), chlorpromazine (5 mg/kg), aspirin (20 and 40 mg/kg), and morphine (3 and 6 mg/kg). Naloxone, however, produced a s i g n i f i c a n t elevation of D' above control levels. Control animals averaged I0.0 + 0 hits and 2.0 + 0.3 false alarms in the t a i l f l i c k t e s t . The data f o r the platform escape are presented in Table 2. All agents except naloxone, chlordiazepoxide, and. a s p i r i n elevated the response c r i t e r i o n , CR. Only morphine, however, s i g n i f i c a n t l y depressed D'. Control animals averaged 20.0 + 0 hits and 4.7 + 0.2 false alarms. DISCUSSION Signal detection analysis has provided some new information about drug e f f e c t s in the standard t a i l f l i c k analgesiometric t e s t and in a foot shock platform-escape procedure. The CR estimates p a r a l l e l closely those of t r a d i tional threshold measurements, but the D' data provide other, independent information. Traditional threshold measurements are nonspecific tests for
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TABLE I DRUG EFFECTS ON TAIL FLICK RESPONSES Drug
Dose a
Saline
Threshold b
1 ml/kg 2 ml/kg Morphine 3 5 Naloxone 0.4 0.8 Pent6barbital I0 20 Ethanol 2.5 gm/kg 5 gm/kg Ketamine 4 8 Chlordi25 azepoxide 50 Chlorproma2.5 zine 5 Aspirin 20 40 Gamma125 hydroxy~50 butyrate a b c + +
69.88 70.14 +81.04 +85.95 72.47 70.24 ~73.76 +76.14 +77.18 ~90.14 +75.00 +78.32 71.90 70.76 #73.81 +78.30 72.20 73.41 ÷75.38 +81.40
+ ¥ ¥ ¥ ¥ ¥ ¥ ¥ ¥ ¥ ¥ ¥ ¥ ¥ 7 ¥ ¥ ¥ ¥ ¥ -
1.35 1.17 1.40 1.35 1.63 1.13 1.61 1.03 1.80 1.74 1,02 1.00 1.05 1.12 0.87 1.00 1.58 0.76 0.65 0.72
CR c 70.67 70.90 +85.00 +88.32 73.45 7~04 +78.24 ÷86.24 ÷81.67 +97.60 74.22 +79.76 71.42 72.11 +78.04 +83.45 72.45 72.15 +75.00 #83.40
D' c
+ 0.63 ¥ 0.50 T 2.15 T 3.05 ¥ 1.14 ¥ 0.76 ¥ 0.64 ¥ 0.51 ¥4.95 ¥ 1.54 ¥ 1.48 ¥ 1.06 T 0.47 ¥ 1.07 ¥0.51 ¥ 0.87 ¥ 0.92 ¥ 0.86 ¥ 1.26 ¥ 1.07 -
N
4.05 + 0.24 3.84 T 0.30 # I . 0 0 T 0.40 +1.02 ¥ 0.41 +5.97 7 0.83 +5.20 ¥ 0.56 3.17 ¥ 1.31 3.00 ¥ 0.97 ¢2.50 ¥ 0 . 5 2 #2.32 ¥ 0.59 3.83 ¥ 0.71 3.97 ¥ 0.54 4 . i 6 ¥ 0.27 4 . 0 1 T 0.40 3.54 ¥ 0.40 +2.32 T 0.42 ¢2.52 ¥ 0.41 +2.41 ¥ 0.50 4.12 ¥ 1.57 3.89 ¥ 1.23 -
7 5 7 6 5 4 5 4 6 4 5 5 5 4 5 7 5 5 5 5
In mg/kg unless otherwise s p e c i f i e d . Expressed as mean + SE of v o l t a g e applied to the lamp. C a l c u l a t e d on the basis of values recorded between 70 and 80 v o l t s . I n d i c a t e s a s i g n i f i c a n t increase above c o n t r o l , P < 0.05. I n d i c a t e s a s i g n i f i c a n t decrease below c o n t r o l , P <-0.05.
TABLE I I DRUG EFFECTS ON GRID SHOCK ESCAPE RESPONSES a Drug Saline Morphine Naloxone Pentobarbital Ethanol Ketamine Chlordiazepoxide Chlorpomazine Aspirin Gamma-hydroxy-butyrate a See legend, Table I .
Dose 1 ml/kg 5 0.4 I0 2.5 gm/kg 4 25 5 20 120
CR 0.I0 +0.33 0.13 +0.38 ÷0.35 +0.26 0.09 +0.44 0.I0 +0.27
+ + + + + + + + + +
C' 0.44 0.05 0.II 0.02 0.03 0.II 0.03 0.06 0.II 0.08
2.37 +1.46 2.32 2.25 2.31 2.34 2.41 2.21 2.54 2.32
+ + + + + + + + + +
N 0.08 0.02 0.I0 0.04 0.15 0.II 0.I0 0.I0 0.42 0,01
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analgesia and can be greatly influenced by drugs having primarily general depressant properties (e.g. pentobarbital, ethanol, e t c . : see Table I ) . However, drug effects r e f l e c t e d by D' are interpreted to be i n d i c a t i v e of analgetic e f f e c t s . Aspirin and morphine had r e l a t i v e l y greater effects on D' than on CR compared to the other drugs tested in the t a i l f l i c k t e s t , but aspirin did not a l t e r e i t h e r the threshold or withdrawal c r i t e r i a , CR. This suggests that a s p i r i n ' s sole analgetic e f f e c t is to decrease the d i s c r i m i n a b i l i t y between non-noxious and noxious s t i m u l i . Aspirin does not have a marked central depressant action and the f a i l u r e of aspirin to change CR or threshold is consistant with such a hypothesis. This is not to suggest that analgesia could not also be produced by a change in threshold, but does indicate that such a change in threshold is neither necessary nor s u f f i c i e n t . Ethanol and chlorpromazine also s i g n i f i c a n t l y depressed D' in the t a i l f l i c k test but were less e f f e c t i v e than morphine, although both agents s i g n i f i c a n t l y elevated the withdrawal c r i t e r i o n as well. Ethanol has long been recognized to have analgetic properties and this has been confirmed in this t e s t . Chlorpromazine, on the other hand, is generally considered not to have analgetic propert i e s , but d i f f e r e n t i a t i o n between antipsychotic t r a n q u i l i z e r s and analgetic agents in analgesiometric tests has been d i f f i c u l t . Naloxone, the 'pure' narcotic antagonist, produced an unexpected s i g n i f i c a n t increase in d i s c r i m i n a b i l i t y in the t a i l f l i c k t e s t . I t would be of considerable i n t e r e s t to determine whether t h i s change is related to naloxone's narcotic antagonist properties ( e . g . , of endogenous opioid peptides) or is a nonspecific e f f e c t .
Pentobarbital, ketamine and gamma-hydroxy-butyrate all produced similar effects in the t a i l f l i c k test. Winters et a l . , (5) have suggested that there are two classes of anesthetic agents: cataleptic anesthetics, whose actions are characterized by an activated EEG, including gamma-hydroxy-butyrate, ketamine, phencyclidine, and trichlorethylene; and the depressant anesthetics, which characteristically depress the EEG at anesthetic doses, such as halothane and the bartiturates. In this study, anesthetics of both classes increased the withdrawal threshold and CR, but did not exhibit significant effects on D' at non-anesthetic doses. In the grid shock platform-escape t e s t , the CR was elevated by morphine, ethanol, chlorpromazine and several anesthetics, indicating f u r t h e r that response bias is sensitive to a v a r i e t y of c e n t r a l l y acting agents. Only morphine, however, produced a s i g n i f i c a n t decrease in D' at the doses studied. The D' as estimated in the t a i l f l i c k t e s t was influenced by a greater v a r i e t y of drugs than in the grid shock escape task. However, a number of experimental variables could account for such differences. Only one dose per agent was employed in the grid shock procedure and i t is possible that higher doses of the other agents could also have altered D'. Each type of drug appeared to present a p a r t i c u l a r p r o f i l e in these tests and p o t e n t i a l l y these properties may be useful in f u r t h e r c l a s s i f y i n g drug groups. However, at this time i t is not clear why the same drugs altered responses in the t a i l f l i c k t e s t to a greater extent than in the grid shock t e s t . I t may be because the t a i l f l i c k test represents a s p i n a l l y mediated r e f l e x whereas the grid shock t e s t represents a behavior integrated at higher CNS levels. Methodologically, the application of SDT in these tests suffers from some limitations. I t would have been desirable to be able to present more t r i a l s in the t a i l f l i c k t e s t , but the danger of i n j u r i n g the animals' t a i l s limited the number of tests that could be tolerated. Increasing the number of t r i a l s
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is important since the r e l i a b i l i t y of D' and CR are d i r e c t l y related to the accuracy of the estimate of the response p r o b a b i l i t i e s . In addition, the ideal SDT experiment accumulates data and portrays i t as the recelver operating c h a r a c t e r i s t i c (ROC), p l o t t i n g false alarms on the abcissa and hits on the ordinate. The ROC function should have a slope of I , indicating equal variances f o r the two d i s t r i b u t i o n s ; D' can thus be calculated from the graph directly. The estimation of D' is based on the assumption of equal variances and the r e l a t i v e l y small sample of responses we collected do not allow D' to be determined for each subject and then averaged across subjects. The variances f o r false alarms and hits among control animals in the two procedures at least suggests Intra-experimental r e l i a b i l i t y . In spite of these drawbacks, the d i s c r i m i n a b i l i t y in the t a i l f l i c k procedure was s i g n i f i c a n t l y higher than that for the grid shock ( t a i l f l i c k D'=4.05 for controls; grid shock D'=2.37 for c o n t r o l s ) . I t is possible, however, that D' in the t a i l f l l c k t e s t is spuriously high because of the small test sample size. Nonetheless, the t a l l f l i c k test results were informative about the r e l a t i v e properties of the drugs used in this study and moreover indicate that SDT analysis c l e a r l y d i f f e r e n t i a t e d effects of analgetics from those of anesthetics. On a methodological basis, the grid shock procedure should be the better method because there was a true no signal condition and a larger sample of responses were obtained. Paradoxically, this method appeared to be less s e n s i t i v e , and the animals had poorer d i s c r i m i n a b i l i t y scores. However, an analgetic agent per se should be the only agent among those we tested (at the doses employed) which would depress pain s e n s i t i v i t y ( i . e . , reduce D' for noxious stimulation) and this was true in the grid shock procedure. Thus, the grid shock escape paradigm may represent the more appropriate procedure of the two employed in this experiment. Acknowledgement: Naloxone was generously provided by Endo Laboratories, Garden City, N. J. Supported by USPHS Grant NS12114 to G.F.G. Dr. Sherman's current address is: The Department of Psychiatry, University of Iowa; Dr. Lewis is c u r r e n t l y at The Department of Pharmacology, University of Texas, Dental Branch, Houston, Texas. REFERENCES I.
ROLLMAN, G. B., Pain 3:
187-212 (1977).
2.
GREEN, D. M. and SWETS, J. A.: (Wiley, New York) (1966).
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SWETS, J. A., ed.: Signal Detection and Recognition by Human Observers. John Wiley and Sons, Inc. New York, 1964.
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CLARK, W. C. Anesthesiology, 4_00, 272-287, (1974).
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WINTERS, W. D., FERRAN-ALLADO, GUZMAN, T., FLORES, G. and ALCARAZ, M. Neuropharmacology I I , 303-315, 1972.
Signal Detection Theory and Psychophysics.