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Psychosomatic digitalis-toxic arrhythmias in guinea pigs
Life Sciences, Vol . 22, pp . 2245-2250 Printed in the U .S .A .
Pergamon Press
PSYCHOSOMATIC DIGITALIS-TOXIC ARRHYTHMIAS IN GUINEA PIGS Benjamin H . Natelson, Normen A. Caginw, Kenneth Donner and Bruce E. Hamilton Department of Neurosciences, New Jersey Medical School and VA Hospital East Orange, NJ
07018
and 'Department of Medicine, Albert Einstein College of Medicine, Bronx, NY
10481
(Received in final form May 10, 1978) Summery Adult male guinea pigs were subjected daily to six 1 min tone light signals, each 5 min apart . For half of the animals, the signal was followed animalspshock wasnever delivered (signal, no-shock~gôup) .o Animals min~both groups were subjected to weekly injections of ouabain, a fast-acting digitalis glycoside . For the signal, shock group, significantly more potentiallyy lethal arrhythmias began during the signal than the min preceding it . Also, a significantly higher percentage of arrhythmies began during the signal periods for the signal, shock group when compered to the signal, no-shock group . These findings indicate that psychogenic factors may contribute to the development of digitalis-toxic arrhythmias. Digitalis, important in the treatment of heart disuse, is one of our mast commonly prescribed drugs. A major problem in usirg this drug has been the relatively narrow zone between therapeutic effectiveness and toxicity as evidenced by the onset of potentially lethal cardiac arrhythmias, which may occur despite doses and serum levels of digitalis not considered tonic (1, 2) . Factors other than overdosage thought to be responsible for precipitatirg arrhythmias include changes in concentration of hormones and ions 8a well as metabolic factors such es hypoxia (1). Although changes in autonomic nervous system tone have also been noted to affect individual responses to constant doses of cardiac glycosfdes (1), no data link this observation to the individual's interaction with his environment. In the experiments reported here, we show that environmental/psychologicel factors also can play a role in the ptbàiction o! digitalis toxicity. Specifically, we demonstrate that in guinea pigs given a fast acting digitalis preparation, ouabain, at doses that usually do not affect cardiac rhythm, a significant number of potentially lethal arrhythmias begin during a signal that had previously been paired with shock. Methods The subjects were 19 Hartley albino guinea pigs, weighing 570 - 890 g. Nine animals were subjected to s signalled shock paradigm ; nine animals served as signal, no-shock controls ; and an additional animal was initially in the signal, no-shock group but later served in the signal, shock group. On week-days, animals in both groups were placed into an experimental chamber prepared from a length of 4° diameter plastic pipe; the chamber had a grid floor and a panel containing 3 cue lights and a sonalert which faced the animal The chamber was kept in a sound attenuating booth. After electrocardiographic (ECG) leads were attached to the animals' shaved back, unülled space around the animal wan 0300-9653/78/0626-2245$02 .00/0 Copyright © 1978 Pergamon Press
2246
Psychosomatic Arrhythmias in Guinea Pige
Vol . 22, No . 2 4, 1978
packed with pieces of rubber foam to reduce the possibility oP movements which could affect the ECG tracing . The chamber was then closed and the animal left alone Por 10 - 15 min . Then, a 30-min conditioning session was started . During these sessions, the cue lights and sonalert were turned on for a 80 .75 sec signal period every 5 min. For animals in the signal, shock group only, constant current shock at 5 mA was delivered with a probability of 40% during the last 0 .75 sec of these signals to electrodes pressed firmly to the animals' rump. Shock was not programmed to follow every signal because such intermittently reinforced schedules have been shown to be more stressful (3) . On Fridays, animals were injected intraperitoneaüy with an assigned dose of ouabain, U .S .P ., 75 min prior to being put in the chamber. ~ Three animals in each group received weekly injections of 175 kg/kg; 4 no-shock and 5 shock animals received weekly injections of 210 Pg/kg; and 3 no~hodc and 2 shock animals received weekly injections of 225 !+g/kg. Because placing the digitalized animal in the chamber occasionally elicited transient arrhythmias, we did not start the session until we had recorded 3 consecutive, normal rhythm strips, 5 min apart (iP abnormal rhythm strips persisted beyond 30 min, the animal was returned to its home cage) . During the 30-min conditioning session which followed, ECGs were repeatedly taken for 2 min periods: the minute before every signal and the minute during every signal . For the signal, shock group, shock was programmed to follow only the fifth signal . Electrocardiographic traces were examined for arrhythmias by BHN and NAC . Both had to agree that an arrhythmia existed for it to be counted . Since arrhythmias occasionally were sustained, data were limited to the time when the first abnormal beat was seen - during the minute prior to the signal or during the minute of the signal . Because arrhythmias developing after shock delivery could have occurred from the physical stimulus itself, any arrhythmias seen in either group after the fifth signal were discarded Prom analysis . Thus, on the ouabain-injection day, any arrhythmias that were counted were related only to the experimental paradigm and not to shock . When listed, means are followed by standard errors. Tests for significance within a group were based on the t test for related measures ; tests for significance between the 2 groups were determined by Student's t test . All tests were one-tailed and a significance level of .05 was chosen . Results A similar number of ouabain-injection experiments was done on animals in the signal, no-shock group (2 .3 ± 0 .5) and in the signal, shock group (2 .9 ± 0.7). Varying numbers of experiments were performed on each animal because of death by intercurrent infection or from what appeared to be sensitization to ouabain ; in 4 animals in the no-shock group end 8 in the shock group, the dose of ouabain which an the first injection day had produced, at most, transient arrhythmias when placed fn the chamber, produced death on some subsequent injection day shortly before or after being placed in the chamber. We do not know the reason for this apparent sensitization to ouabain . Our hypothesis in initiating these studies was that the probability of arrhythmogenesis would be greater during a signal previously paired with shock than before it. We also reasoned that in animals never subjected to shock, arrhythmogenesis would not relate to signal . The data matched our expectations: in signal, shock animals, arrhythmias began significantly more often during the 60 sec signal period than during the preceeding 60 sec (1 .1 + 0 .3 end 0.4 + 0 .2, respectively; p < .025, Fig. 1) ; in signal, no-shock animals, arrhythmic episodes weré noted with similar rarity both before and during the signal (0 .3 + 0.2 and 0 .3 + 0 .2, respectively). The probability of arrhythmogenesis occurring anytime in é session incréased monotonically with increasing ouabain doses in the signal, shock group (33%, 48%, 82%, respectively) but not in the signal, ra-shock group (17%, 38%, 22%, respectively) . Because dosage and number of ouabain injections were not statistfcaüy different for
Vol . 22, No . 24, 1978
Psychosomatic Arrhythmias is Guinea Piga
2247
the 2 groups, comparisons between them can be made . In order to compare the frequency of arrhythmias across the 2 getups, data were standardized for each animal by conversion
100 N
a r~ ~ o=
eo
W ~ 60 O ~ Q Q Z = W ~ 40
~3
W tA a Z O W N
20
0 Before Signal
During Signal
NO SHOCK
Before Signal
During Slgnal
SHOCK
FIG . 1 Percentage of total sessions in which arrhythmias developed for signal, no-shock and signal, shock groups . Each line represents 1 animal's data . Numbers in parentheses refer to the actual number of sessions in which errhythmias were seen for each animal . Five animals in the no-shock group and 3 animals in the shock group developed no arrhythmias . Significantly more arrhythmias developed durUg the signal for just the signal, shock group . This group also had a significantly greater percentage of arrhythmias beginning during the signal period than the no-shock Group "
2248
Psychosomatic Arrhythmias in Guinea Pige
Vol . 2 2, No . 24, 1978
to percentage of total experiments showing arrhythmias. As expected, there was no significant difference between frequency oP arrhythmias for the 2 groups prior to the slgnel, but a significant difference in frequency of arrhythmias between the 2 groups was found during the signal (no-shock = 8 .7 ± 8 .8% ; shock = 34 .8 ± 12 .3% ; p < 0 .05 ; Fig. 1) .
FIG. 2 Electrocardiographic record from a guinea pig which had previously had signal paired with shock. On the day this record was made, shock was not delivered prior to the onset of this arrhythmia. The record is normal, until the start of the signal (arrow in B) . Slowing then occurs which is followed by some single ventricular premature contractions and then short runs of ventricular tachycardia . Record is continuous through the cessation of the signal in G, and then alternate 12 sec strips are displayed. Trace has returned to normal by M .
Vol . 22, No . 24, 1978
Psychosomatic Arrhythmias in guinea Pigs
2249
The two groups differed in other ways when the ECGs from the signalled period were First, 7 of the 11 arrhythmias noted in the group for which signal was compared . associated with shock began during the first presentation of the signal ; in contrast, all 3 arrhythmias noted in the no-shock group began during either the fourth or fifth signal In addition to this, the arrhythmias that developed during the signal period for the shock group were more sustained than those appearing in the no-shock group . This is seen by the fact that following log transformation of data because of heterogeneity oP variance (4), significantly more abnormal beats per arrhythmic episode were seen in the signal, shock group than in the signal, no-shock group (p < 0 .025). The arrhythmias that developed during the signal for the group in which the signal had beén a cue for shock were varied and striking : first degree A-V block (operationally defined as a PA interval 50916 longer than that seen prior to the delivery of the first signal), Wenekebach phenomenon, sinus arrest, junetional tachycardia, ventricular escape beats, ventricular ventricular tachycardia and idioventricular pre-mature contractions, bradycardia . Figure 2 is an example of one such arrhythmia elicited during a signal period. Discussion Since a significant number of these arrhythmias was provoked only by a sonalert and lights, it is apparent that they are psychosomatic in origin. It ie clear that the signal by itself (see data for signal, no-shock group) was not capable of precipitating a significant number of arrhythmias. Classically, pairing signal with shock is thought to produce a state of anxiety or fear during the signal by Pavlovien conditioning. These experiments do not elucidate the mental state of the test animal but indicate that the amount of arousal produced by confrontation of sonalert and cue lights, never paired with shock, is inadequate to produce a significant number of arrhythmias in digitalized animals. However, the degree of arousal produced by confrontation with a signal that had been associated with shock in the pest did produce a significant number of digitalis-toxic errhythmias . Regardless whether the mechanism for these arrhythmias is due to a state of marked undifferentiated arousal or to the specific emotion of fear, the fact that an environmental stimulus can produce errhythmias in a digitalized animal indicates modulation by the central nervous system. Earlier work has shown that similar arrhythmias can be elicited by the combination of sub-toxic doses of ouabain plus posterior hypothalamic stimulation (5), can be delayed by cervical spinal cord section (8), and are accompanied by dramatic increases in directly measured vagal and sympathetic neural outflow (7) . Thus, activation of the autonomic nervous system is important In the genesis of the arrhythmias. The bradycardia that develops during a pre~hock signal (Fig . 2 and ref . 8) plus the bradyarrhythmias we noted would suggest that the vague is the important component of the autonomic nervous system in the pathogenesis of the arrhythmias shown here. Previous experiments have shown that psychologically stressful factors can reduce the threshold for electrically induced ventricular fibrillation in normal dogs and can produce frank arrhythmias for a few days following experimental myocardial infarction (9 11). To these important demonstrations of the role of environmental factors in producing cardiac rhythm dysfunction, we now provide a model of psychosomatic digitalis-toxic arrhythmias which has the advantage of being reproducible over time . The existence of this model thus suggests a reason, other than metabolic, why individual responses to constant doses of cardiac glycosides can vary dramatically. Acknowledgements This study was made possible by grants from the Foundation of the College of Medicine and Dentistry of New Jersey and the American Heart Association, Bergen County and Union County Chapters. BHN is a recipient of a VA Career Development Award. The authors acknowledge the secretarial aide of L. Fallone .
2250
Psychosomatic Arrhythmias in Guinea Pige
Vol . 22, No . 24, 1978
References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 .
T.W . 3MITH, Am . J. Med. 58 470-476 (1975) . J.A . INGELF~~OLDMAN, New En . J. Med. 294 887-870 (1978) . W.L . 3AWREY, J. Com~Physiol. Psycho B.J . WINER, Sta~~sficai rrm pici~es in ~erimental Design pp. 397-401 McGraw Hil1, NY (1871) . D.E . EVANS and R.A. GILLIS, J. Pharm. E~ . Ther . 185 577-596 (1975) . B. LEVITT, N.A . CAGIN, J. , . ~1'. MITTAG and A. RAINFS, J. Pharm. E~ . Ther . 185 24-28 (1973). , . E~Y.J . SOHN, B. LEVITT and F.G . STANDAERT, J. Pharm. E~ " Thera . 183 154-1B8 (1972). ~. .~E~D end T.J . TEYLER, J. Com . Ph sioL Ps choL 70 242-253 (1970). B. LOWN, R. VERRIER and R. CORBA , c ence - 8 ~973). R. CORBALAN, R. VERRIER end B. LOWN, ~GPePd10I. 34 892-898 (1974). J.E . BKINNER, J.T . LIE and M.L. ENTMAN, rc. 7-(1975).
n- T
Pergamon Press
PSYCHOSOMATIC DIGITALIS-TOXIC ARRHYTHMIAS IN GUINEA PIGS Benjamin H . Natelson, Normen A. Caginw, Kenneth Donner and Bruce E. Hamilton Department of Neurosciences, New Jersey Medical School and VA Hospital East Orange, NJ
07018
and 'Department of Medicine, Albert Einstein College of Medicine, Bronx, NY
10481
(Received in final form May 10, 1978) Summery Adult male guinea pigs were subjected daily to six 1 min tone light signals, each 5 min apart . For half of the animals, the signal was followed animalspshock wasnever delivered (signal, no-shock~gôup) .o Animals min~both groups were subjected to weekly injections of ouabain, a fast-acting digitalis glycoside . For the signal, shock group, significantly more potentiallyy lethal arrhythmias began during the signal than the min preceding it . Also, a significantly higher percentage of arrhythmies began during the signal periods for the signal, shock group when compered to the signal, no-shock group . These findings indicate that psychogenic factors may contribute to the development of digitalis-toxic arrhythmias. Digitalis, important in the treatment of heart disuse, is one of our mast commonly prescribed drugs. A major problem in usirg this drug has been the relatively narrow zone between therapeutic effectiveness and toxicity as evidenced by the onset of potentially lethal cardiac arrhythmias, which may occur despite doses and serum levels of digitalis not considered tonic (1, 2) . Factors other than overdosage thought to be responsible for precipitatirg arrhythmias include changes in concentration of hormones and ions 8a well as metabolic factors such es hypoxia (1). Although changes in autonomic nervous system tone have also been noted to affect individual responses to constant doses of cardiac glycosfdes (1), no data link this observation to the individual's interaction with his environment. In the experiments reported here, we show that environmental/psychologicel factors also can play a role in the ptbàiction o! digitalis toxicity. Specifically, we demonstrate that in guinea pigs given a fast acting digitalis preparation, ouabain, at doses that usually do not affect cardiac rhythm, a significant number of potentially lethal arrhythmias begin during a signal that had previously been paired with shock. Methods The subjects were 19 Hartley albino guinea pigs, weighing 570 - 890 g. Nine animals were subjected to s signalled shock paradigm ; nine animals served as signal, no-shock controls ; and an additional animal was initially in the signal, no-shock group but later served in the signal, shock group. On week-days, animals in both groups were placed into an experimental chamber prepared from a length of 4° diameter plastic pipe; the chamber had a grid floor and a panel containing 3 cue lights and a sonalert which faced the animal The chamber was kept in a sound attenuating booth. After electrocardiographic (ECG) leads were attached to the animals' shaved back, unülled space around the animal wan 0300-9653/78/0626-2245$02 .00/0 Copyright © 1978 Pergamon Press
2246
Psychosomatic Arrhythmias in Guinea Pige
Vol . 22, No . 2 4, 1978
packed with pieces of rubber foam to reduce the possibility oP movements which could affect the ECG tracing . The chamber was then closed and the animal left alone Por 10 - 15 min . Then, a 30-min conditioning session was started . During these sessions, the cue lights and sonalert were turned on for a 80 .75 sec signal period every 5 min. For animals in the signal, shock group only, constant current shock at 5 mA was delivered with a probability of 40% during the last 0 .75 sec of these signals to electrodes pressed firmly to the animals' rump. Shock was not programmed to follow every signal because such intermittently reinforced schedules have been shown to be more stressful (3) . On Fridays, animals were injected intraperitoneaüy with an assigned dose of ouabain, U .S .P ., 75 min prior to being put in the chamber. ~ Three animals in each group received weekly injections of 175 kg/kg; 4 no-shock and 5 shock animals received weekly injections of 210 Pg/kg; and 3 no~hodc and 2 shock animals received weekly injections of 225 !+g/kg. Because placing the digitalized animal in the chamber occasionally elicited transient arrhythmias, we did not start the session until we had recorded 3 consecutive, normal rhythm strips, 5 min apart (iP abnormal rhythm strips persisted beyond 30 min, the animal was returned to its home cage) . During the 30-min conditioning session which followed, ECGs were repeatedly taken for 2 min periods: the minute before every signal and the minute during every signal . For the signal, shock group, shock was programmed to follow only the fifth signal . Electrocardiographic traces were examined for arrhythmias by BHN and NAC . Both had to agree that an arrhythmia existed for it to be counted . Since arrhythmias occasionally were sustained, data were limited to the time when the first abnormal beat was seen - during the minute prior to the signal or during the minute of the signal . Because arrhythmias developing after shock delivery could have occurred from the physical stimulus itself, any arrhythmias seen in either group after the fifth signal were discarded Prom analysis . Thus, on the ouabain-injection day, any arrhythmias that were counted were related only to the experimental paradigm and not to shock . When listed, means are followed by standard errors. Tests for significance within a group were based on the t test for related measures ; tests for significance between the 2 groups were determined by Student's t test . All tests were one-tailed and a significance level of .05 was chosen . Results A similar number of ouabain-injection experiments was done on animals in the signal, no-shock group (2 .3 ± 0 .5) and in the signal, shock group (2 .9 ± 0.7). Varying numbers of experiments were performed on each animal because of death by intercurrent infection or from what appeared to be sensitization to ouabain ; in 4 animals in the no-shock group end 8 in the shock group, the dose of ouabain which an the first injection day had produced, at most, transient arrhythmias when placed fn the chamber, produced death on some subsequent injection day shortly before or after being placed in the chamber. We do not know the reason for this apparent sensitization to ouabain . Our hypothesis in initiating these studies was that the probability of arrhythmogenesis would be greater during a signal previously paired with shock than before it. We also reasoned that in animals never subjected to shock, arrhythmogenesis would not relate to signal . The data matched our expectations: in signal, shock animals, arrhythmias began significantly more often during the 60 sec signal period than during the preceeding 60 sec (1 .1 + 0 .3 end 0.4 + 0 .2, respectively; p < .025, Fig. 1) ; in signal, no-shock animals, arrhythmic episodes weré noted with similar rarity both before and during the signal (0 .3 + 0.2 and 0 .3 + 0 .2, respectively). The probability of arrhythmogenesis occurring anytime in é session incréased monotonically with increasing ouabain doses in the signal, shock group (33%, 48%, 82%, respectively) but not in the signal, ra-shock group (17%, 38%, 22%, respectively) . Because dosage and number of ouabain injections were not statistfcaüy different for
Vol . 22, No . 24, 1978
Psychosomatic Arrhythmias is Guinea Piga
2247
the 2 groups, comparisons between them can be made . In order to compare the frequency of arrhythmias across the 2 getups, data were standardized for each animal by conversion
100 N
a r~ ~ o=
eo
W ~ 60 O ~ Q Q Z = W ~ 40
~3
W tA a Z O W N
20
0 Before Signal
During Signal
NO SHOCK
Before Signal
During Slgnal
SHOCK
FIG . 1 Percentage of total sessions in which arrhythmias developed for signal, no-shock and signal, shock groups . Each line represents 1 animal's data . Numbers in parentheses refer to the actual number of sessions in which errhythmias were seen for each animal . Five animals in the no-shock group and 3 animals in the shock group developed no arrhythmias . Significantly more arrhythmias developed durUg the signal for just the signal, shock group . This group also had a significantly greater percentage of arrhythmias beginning during the signal period than the no-shock Group "
2248
Psychosomatic Arrhythmias in Guinea Pige
Vol . 2 2, No . 24, 1978
to percentage of total experiments showing arrhythmias. As expected, there was no significant difference between frequency oP arrhythmias for the 2 groups prior to the slgnel, but a significant difference in frequency of arrhythmias between the 2 groups was found during the signal (no-shock = 8 .7 ± 8 .8% ; shock = 34 .8 ± 12 .3% ; p < 0 .05 ; Fig. 1) .
FIG. 2 Electrocardiographic record from a guinea pig which had previously had signal paired with shock. On the day this record was made, shock was not delivered prior to the onset of this arrhythmia. The record is normal, until the start of the signal (arrow in B) . Slowing then occurs which is followed by some single ventricular premature contractions and then short runs of ventricular tachycardia . Record is continuous through the cessation of the signal in G, and then alternate 12 sec strips are displayed. Trace has returned to normal by M .
Vol . 22, No . 24, 1978
Psychosomatic Arrhythmias in guinea Pigs
2249
The two groups differed in other ways when the ECGs from the signalled period were First, 7 of the 11 arrhythmias noted in the group for which signal was compared . associated with shock began during the first presentation of the signal ; in contrast, all 3 arrhythmias noted in the no-shock group began during either the fourth or fifth signal In addition to this, the arrhythmias that developed during the signal period for the shock group were more sustained than those appearing in the no-shock group . This is seen by the fact that following log transformation of data because of heterogeneity oP variance (4), significantly more abnormal beats per arrhythmic episode were seen in the signal, shock group than in the signal, no-shock group (p < 0 .025). The arrhythmias that developed during the signal for the group in which the signal had beén a cue for shock were varied and striking : first degree A-V block (operationally defined as a PA interval 50916 longer than that seen prior to the delivery of the first signal), Wenekebach phenomenon, sinus arrest, junetional tachycardia, ventricular escape beats, ventricular ventricular tachycardia and idioventricular pre-mature contractions, bradycardia . Figure 2 is an example of one such arrhythmia elicited during a signal period. Discussion Since a significant number of these arrhythmias was provoked only by a sonalert and lights, it is apparent that they are psychosomatic in origin. It ie clear that the signal by itself (see data for signal, no-shock group) was not capable of precipitating a significant number of arrhythmias. Classically, pairing signal with shock is thought to produce a state of anxiety or fear during the signal by Pavlovien conditioning. These experiments do not elucidate the mental state of the test animal but indicate that the amount of arousal produced by confrontation of sonalert and cue lights, never paired with shock, is inadequate to produce a significant number of arrhythmias in digitalized animals. However, the degree of arousal produced by confrontation with a signal that had been associated with shock in the pest did produce a significant number of digitalis-toxic errhythmias . Regardless whether the mechanism for these arrhythmias is due to a state of marked undifferentiated arousal or to the specific emotion of fear, the fact that an environmental stimulus can produce errhythmias in a digitalized animal indicates modulation by the central nervous system. Earlier work has shown that similar arrhythmias can be elicited by the combination of sub-toxic doses of ouabain plus posterior hypothalamic stimulation (5), can be delayed by cervical spinal cord section (8), and are accompanied by dramatic increases in directly measured vagal and sympathetic neural outflow (7) . Thus, activation of the autonomic nervous system is important In the genesis of the arrhythmias. The bradycardia that develops during a pre~hock signal (Fig . 2 and ref . 8) plus the bradyarrhythmias we noted would suggest that the vague is the important component of the autonomic nervous system in the pathogenesis of the arrhythmias shown here. Previous experiments have shown that psychologically stressful factors can reduce the threshold for electrically induced ventricular fibrillation in normal dogs and can produce frank arrhythmias for a few days following experimental myocardial infarction (9 11). To these important demonstrations of the role of environmental factors in producing cardiac rhythm dysfunction, we now provide a model of psychosomatic digitalis-toxic arrhythmias which has the advantage of being reproducible over time . The existence of this model thus suggests a reason, other than metabolic, why individual responses to constant doses of cardiac glycosides can vary dramatically. Acknowledgements This study was made possible by grants from the Foundation of the College of Medicine and Dentistry of New Jersey and the American Heart Association, Bergen County and Union County Chapters. BHN is a recipient of a VA Career Development Award. The authors acknowledge the secretarial aide of L. Fallone .
2250
Psychosomatic Arrhythmias in Guinea Pige
Vol . 22, No . 24, 1978
References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 .
T.W . 3MITH, Am . J. Med. 58 470-476 (1975) . J.A . INGELF~~OLDMAN, New En . J. Med. 294 887-870 (1978) . W.L . 3AWREY, J. Com~Physiol. Psycho B.J . WINER, Sta~~sficai rrm pici~es in ~erimental Design pp. 397-401 McGraw Hil1, NY (1871) . D.E . EVANS and R.A. GILLIS, J. Pharm. E~ . Ther . 185 577-596 (1975) . B. LEVITT, N.A . CAGIN, J. , . ~1'. MITTAG and A. RAINFS, J. Pharm. E~ . Ther . 185 24-28 (1973). , . E~Y.J . SOHN, B. LEVITT and F.G . STANDAERT, J. Pharm. E~ " Thera . 183 154-1B8 (1972). ~. .~E~D end T.J . TEYLER, J. Com . Ph sioL Ps choL 70 242-253 (1970). B. LOWN, R. VERRIER and R. CORBA , c ence - 8 ~973). R. CORBALAN, R. VERRIER end B. LOWN, ~GPePd10I. 34 892-898 (1974). J.E . BKINNER, J.T . LIE and M.L. ENTMAN, rc. 7-(1975).
n- T