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Simulated marihuana smoking in the maternal and fetal guinea pig
Simulated marihuana smoking in the maternal and fetal guinea pig PAUL
R.
JOSEPH
SINGER, J.
SCIBETTA,
MORTIMER Rochester,
G. New
M.D. M.D.
ROSEN,
M.D
York
Maternal and fetal heart rate and brain wave (electroencephalogram [EEG]) changes were observed during simulated maternal marihuana smoking. The maternal heart rate change consisted of a delay in onset of an elevated heart rate, while the fetuses and newborn infants studied revealed a decrease in heart rate during the smoking period. The maternal EEG remained essentially unchanged during the smoking period. Following marihuana exposure, the maternal EEG changed to a pattern of low frequency and high-amplitude activity and in a few instances was similar to the “recovery spike-discharges” reported in other animal experiments with marihuana. The fetal EEG changed to a slow-frequency, high-voltage activity pattern during the smoking period. Following exposure to the marihuana, it changed to a lower voltage and higher frequency activity.
erty resides in several tetrahydrocannabinols, of which the major psychoactive compound is thought to be delta-9 tetrahydrocannabinol (dibenzopyran system) .* It has been estimated that between 12 and 20 million adolescents and young adults are using or have tried smoking Cannabis derivatives.3, 4 Uncertainty about the potential dangers of marihuana use prevails. Inasmuch as marihuana is reported to induce mental changes in the user,s analysis of its effects upon the central nervous system has been an appropriate target of medical research. More than 25 years ago, Wikler and Lloyd” described minimal and inconsistent scalp-recorded electroencephalogram (EEG) changes following marihuana smoking in 19 long-term narcotic addicts. More recently, EEG effects of marihuana or delta-9 tetrahydrocannabinol (THC) given to rabbits or rats was characterized by a generalized reduction in cortical EEG voltage and the appearance of bursts of high-voltage sharp waves.l-10 Rodin and associates,ll in a report on 10 medical students who had smoked marihuana for at least a year, found it im-
IN A co MPE N DIU M of medicines in 2737 B.C., the Chinese Emperor Shen Nung recorded some of the earliest observations on man’s use of marihuana.l The hemp plant, Cannabis satiua, has a history of use as a medicine, a source of fiber, and a drug in tribal religious ceremonies.’ Today, our principal interest in marihuana is its behavioral property for inducing euph0ria.l This prop-
From the Department Gynecology, Strong
of Obstetrics and Memorial Hospital.
Supported in part by a General Research Support Grant of the University of Rochester School of Medicine and Dentistry from the National Institutes of Health, a Graduate Research Training Grant in Reproductive Biology in Maternal Fetal Relationships (HDOOO15I I), and the 1. A. Hartford Foundation, Inc. 7;7c;ived Revised Accepted
for publication March March
December
28,
12, 1973. 26, 1973.
Reprint requests: Dr. Mortimer G. Rosen, Department of Obstetrics and Gynecology, Strong Memorial Hospital, 260 Crittenden Blvd., Rochester, New York 14642. 331
332
Singer,
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and
Rosen
Fig. 1. Diagram of experimental model: A and 13, Fetal EEG electrodes; C, fetal sewn in myometrium over fetus; D, ground wire placed beneath the maternal amphenol jack; F and G, maternal EEG electrodes; H, strain gauge to monitor respirations. Inset: Fetal electrode in detail.
possible by visual inspection to distinguish the premarihuana from postmarihuana EEG’s. There was a suggestion of more persistent alpha rhythm and slower frequency components after smoking. This was confirmed by power-density spectral analysis. Deliyannakis and colleagues12 reported minimal and inconsistent EEG findings in a study involving 27 admitted hashish-addicted soldiers. Heath’” reported minimal or no changes in E,EG’s recorded from scalp electrodes following marihuana smoking in a patient in whom electrodes had been implanted into numerous deep nuclear and surface areas of the brain. He did find that consistent EEG changes occurred in deep recordings from the septal region. These high-voltage slow waves were concomitant with the behavioral effects of marihuana smoking. Possible neurotoxic effects of marihuana smoking have been postulated by Kolansky and Moore14 who theorize that the psychic changes associated with Cannabis intoxication are the result of chemical (delta-9 THC) damage to the cerebral cortical cells. Furthermore, Campbell and co-worker@ have demonstrated cerebral atrophy by air encephalography in 10 individuals who had smoked marihuana for a period of 3 to 11 years.
ECG wire skin; E, maternal
A pertinent question for study concerns the possible effects of marihuana use during pregnancy. In this report, we will present observations on physiologic and behavioral variables following marihuana exposure in the maternal and fetal guinea pig. Methods Nine gravid guinea pigs were studied. Maturity of the fetus was estimated by gentle fetal vertex palpation through the maternal abdomen and by maternal symphyseal separation. In the maternal guinea pig, symphyseal separation occurs 48 to 72 hours prior to birth.l’; Maternal EEG electrode wires were implanted at least 24 hours prior to laparotomy. The guinea pigs were fasted overnight prior to the fetal operations. A flank incision overlying the palpable fetal skull was made after infiltrating the maternal abdominal wall with 2 C.C. of 0.25 per cent procaine hydrochloride. Light ether anesthesia was used when the peritoneum was opened. The fetal EEG electrodes consisted of 2 stainless steel needles attached to the ends of a metal spring (Fig. 1) . The details of the electrodes have been reported previ0us1y.‘~ The tips of the fetal electrode were pushed through the maternal myometrium and fetal skull into the brain in the central
Volume Number
Marihuana
117 3
Table I. Summary
‘Test tone 1.5, and
and fetal guinea
pig
333
of experiments Weight
Experiment NO. 1 2 3 (Day Pay 4 5 6 7 8 w
in maternal
(Grams)
Maternal
1)
No. of cigarettes smoked
Fetal
1,024 1,233 1,010
52, 60* 58, 65, 70, 82” 57, 61, 64”
1,018 1,193 989 1,102 1,008 837
40, 55*, 58” 42, 103, 40.
2)
42, 57” 58, 60, 65*, 60 75* !I,, Y59* 109* .58. 60”
Smoke
1 3 ‘) 2 2 2 3 3 1 3
78
exposure
(minutes)
7.0 --. 0 ‘)3 14.0 15.6 15.3 14.0 23.0 2’2.0 6.0 22.6
fetus. cigarette 3 hours.
smoking
immediately
followed
by next
in all
regions, analogous to a biparietal location. The depth of electrode penetration was between 1 and 2 mm. beyond the inner table of the sk~11.l~ The metal spring remained external to the uterus and held the electrodes in position without gross loss of amniotic fluidsI During the operation, the uterus and abdominal contents were kept warm with saline-soaked sponges (temperature maintained at 37 to 38’ C.). All the electrode wires were connected to a jack placed in the skin prior to closure of the abdomen (Fig. 1). Three to four hours following operation, the now alert maternal pig was placed in the smoking machine, and the jacks were connected to a polygraph for recording. The recording consisted of maternal and fetal EEG, electrocardiogram (ECG) , and maternal respiratory movement (Fig. 1) . All marihuana was administered in the form of 500 mg. cigarettes made with a hand-operated machine. The marihuana used in these experiments was supplied by the National Institutes of Mental Health and contained 1.4 per cent of delta-9 THC by weight. I8 Good-quality marihuana cigarettes are said to contain 500 mg. of Cannabis sativa with an average delta-9 THC content of 1 per cent.? Experiments on the effects of marihuana smoking in human subjects followed the procedure of allowing the subject 8 to 12 minutes to smoke each cigarette, subjects taking long puffs, inhaling deeply, and main-
animals
except
No.
9. In
this
animal,
cigarette
smoking
at 0,
taining inspiration for 20 seconds.lg Therefore, the cycle designed for the maternal guinea pig was selected to be analogous to the human act of smoking marihuana. A smoking apparatus was designed especially for the guinea pig and was used for all experiments. This apparatus consisted of a clear plastic container, approximately twice the volume of the maternal guinea pig, equipped with solenoid-operated portholes regulating smoke and air entry into the chamber. Smoking consisted of a repeated, 3 stage, 30 second cycle, as follows: ( 1) By means of a vacuum pump and cam, a puff of smoke was drawn from the lighted cigarette into the animal compartment (3 seconds) ; (2) the compartment was then closed off and the smoke remained within the compartment ( 12 seconds) ; (3) the compartment was then evacuated by the vacuum and the smoke replaced by fresh air ( 15 seconds) . At the end of this interval, the cycle began again with another puff of smoke. Prior to beginning the simulated smoking experience, the animal was placed in the smoking apparatus with all conditions simulating the actual smoking period except there was absence of the smoke. Recordings of both maternal and fetal EEG’s and ECG’s were obtained for a period ranging from 10 to 134 minutes. This recording supplemented a recording of maternal EEG’s taken one day prior to the laparotomy. At the end of this
334
Singer,
Scibetta,
and
Rosen
I
1
0
5
IO
b
20
25
20
MINUTES Fig. 2. A comparison marihuana cigarette tion of heart rate.
of maternal (Exp. No. 8).
and Note
fetal heart rate changes after the decrease in fetal heart rate during
smoking maternal
of one eleva-
Table II. Maternal heart rate changesassociatedwith marihuana smoking Experiment No.
No.
*No
further labor
cigarettes
smoked
2 3 (Day (Day 4 7 8 9 tin
of
Baseline
3 3
1)
2)
200-210 200-2 10 260-280 190-210 170-230 180-220 240-260
2 2 3 1 3
recording during
past 96 minutes.
Heart with
Heart
rate
returned
to base-line
rate Peak smoking 240 230 300 220 258 250 260
values
the next
Time (minutes) before return to base line 96+* 42 24 18 64 20 5t
day.
smoking.
presmoking period, the animal was exposed to smoke with one cigarette at a time. The maternal pig was exposed to each cigarette for not less than 6 minutes and not more than 10 minutes. The total duration of exposure to smoke can be seenin Table I. Results
Maternal heart rate changes. During the smoking of the marihuana cigarettes, a progressive increase in heart rate is generally noted (Table II). It begins 2 to 6 minutes after exposure to the first cigarette and lasts from 18 to more than 96 minutes. Because of the very wide range of base-line heart rates, it is difficult to establish a direct relationship between the magnitude of the increased heart rate and the number of cigarettes smoked.
Fetal heart rate (FHR) changes. FHR depression occurred during the period in which the mother was exposed to marihuana smoke and was herself experiencing an increasedheart rate (Fig. 2). With each additional exposure to a marihuana cigarette, the FHR progressively declined, as can be seen in Fig, 3. This figure compares fetal heart rate changes on 2 consecutive days. On each day, 2 marihuana cigarettes were smoked. It can be seen that the heart rate depression was more pronounced during the second cigarette. In 5 animals, the heart rate returned toward the baseline within 8 minutes following exposure to the last cigarette (Table III). The sixth fetus (Experiment No. 4) experienced prolonged bradycardia for one hour following smoking, before the heart rate returned to pre-existing levels. Also of
Volume Number
117 3
Marihuana
and fetal guinea
DAY2
DAYI
280-
in maternal
pig
335
-280‘
240 -
-240
1, 200% L 160-
-200
k
3
- 160 ypTm
Fig. 3. Fetal heart rate changes after 2 marihuana No. 3). Note more pronounced heart rate depression
Table
III.
Fetal heart
rate changes
associated Heart
Experiment NO. 2 3 (Day (Day 4 7 8 9
Table
Experiment No.
Weight (Gm.1
5
55 65 58 75
6 *Returned
to base-line
heart
rate
during
Minutes before return to control
110 170 110 170 120 100 80
heart rate changes associated
latter
part
(Exp.
7 5 60 1 2 8
with marihuana
“Recurrent bradycardia” (heart rate during this period) 160-l 10 240-180 240-190 190-160 190- 70 220-110 190- 80
smoking
Control
Change with smoking
Minutes before return to control
280-360 270-340 270-360 246-282
270 200 180 318
2.0 4.0 1.0 0*
of smoking
interest is the recurrent bradycardia seen in all of the fetuses. This delayed drop in the FHR usually occurred 8 to 40 minutes foIlowing the period of smoking, with the time of recurrence apparently related neither to the number of cigarettes smoked by the mother nor the maternal heart rate at that time. Neonatal heart rate changes. Heart rate changes associated with marihuana cigarette smoking were studied in 4 newborn animals within 9 hours of their birth (TabIe IV). The variation in control heart rates made determination of changes during smoking, as well as possible recurrent bradycardia or
days
smoking
rate
160-180 210-230 240-258 180-200 160-230 198-230 180-220
2)
IV. Neonatal
with marihuana
Maximum deceleration with smoking
Control 1)
cigarettes on 2 consecutive during the second cigarette.
period.
tachycardia, difficult. Three of the newborn animals responded to the smoking period with a deceleration similar to that seen in the previously described fetuses. The fourth and largest newborn animal initially responded to the marihuana smoking with an increased heart rate as did the maternal guinea pigs. In 2 of the newborn animals (58 and 75 grams), a recurrent bradycardia was noted (Fig. 4). Respiration. Maternal respiratory movements were monitored throughout each experiment. The respiratory rate during the control period prior to the smoking ranged between 50 and 80 breaths per minute. Dur-
336
Singer,
Scibetta,
and
Rosen
x0330-
-360 -330
NO270-
-300 -270
240210& 1803 ISOQ mg 3303002702402lOIBO-
NEWBORN 5Bgns
1
-240
I CKjHAI 1
CIC 1 * HHH
-210 - 180 - 150 - 120 -330 -300 -270
J
3
Do'*O 'i0.x'
NEWBORN 75gms.
-240 - 210 -180 - I50 L I20
Fig. 4. Newborn heart rate changes after exposure to 3 marihuana cigarettes Note recurrence of heart rate depression 20 minutes or more following smoking. ing the smoking period, the respirations varied with each portion of the smoking cycle. In that portion of the cycle in which smoke filled the chamber, respirations were shallow and the rate decreased to 20 to 30 breaths per minute. During the expulsion of the smoke and its replacement with fresh air, 2 different patterns were seen. The guinea pig would either breathe very deeply at a rate of 25 to 35 breaths per minute or breathe quite rapidly (80 to 90 breaths per minute) at a normal depth of respiration. One of these patterns continued until smoke began to fill the chamber, signaling the onset of the next cycle, and the animal would again revert to the shallow, slow respirations. The guinea pig resumed pre-existing breathing patterns within 2 to 4 minutes following the last cigarette. No further variations in respiration were observed during the remainder of each experiment. Observed behavior. The behavior of the maternal guinea pig and newborn animal was noted during each experiment. The most striking change in behavior was seen during and immediately following the smoking period. Throughout the control period, the animals were active. During smoking, the mothers and newborn animals were noticeably disturbed by the smoke, as demonstrated by their attempts to turn around. Following
(Exp. No. 6)
the smoking period, the animals changed their behavior and became relatively immobile. They would remain practically motionless and unreactive to stimuli which normally would disturb them, such as door opening or dropping an instrument on the floor. This persisted for 90 to 120 minutes. Quantitation of this effect with increasing numbers of cigarettes ~~3s not possible because of our desire not to stimulate the animals in order to check their level of awareness but merely to observe their spontaneous movement. All of thr animals demonstrated the “sedated period” for variable durations. Electroencephalograms. Maternal EEG. Prior to the smoking period, the maternal EEG displayed a spectrum of activity, varying from continuous lower voltage fast activity (Fig. 5, A) to somewhat higher voltage activity with slower wave frequencies (Fig. 6, A 1. During marihuana smoking, the EEG pattern was similar to the low-voltage fast activity [Figs. 5 and 6, B). This low-voltage fast activity was replaced 15 to 30 minutes post smoking with higher voltages and lower frequency patterns as illustrated in Figs. 5 and 6, C. This pattern of slower frequency, higher voltage activity lasted for variable periods of time. It reappeared up to 24 hours after smoking and
Volume Number
117
Marihuana
3
in maternal
Fig. 5. Maternal EEG changes associated with marihuana smoking recording with continuous lower voltage fast activity. B, During similarity to A. C, Two hours and forty-two minutes post smoking voltage “recovery spike-discharges,”
and
at times,
a very high-voltage EEG. of the presmoking fetal EEG is seen in Fig. 7, A. During the period of exposure to the marihuana smoke, fetal EEG activity demonstrated a trend toward increased voltage of 10 cycles per second wave forms (Fig. 7, B). Over a time
Fetal EEG. An example
guinea
pig
337
(Exp. No. 3). A, Control the smoking period with with few-frequency, high-
Fig. 6. Maternal EEG changes associated with marihuana smoking (Exp. recording with higher voltage, slower wave forms. B, During smoking voltage, fast wave forms (see also Fig. 7, A and B). C, Forty-eight minutes slow-frequency, high-voltage activity.
showed,
fetal
No. 2). A, Control period with lower post smoking; note
span of 15 to 30 minutes following the last cigarette, the EEG activity showed increasing frequency and decreasing voltage (Fig. 7, C). This pattern of low-voltage, fast activity lasted for variable periods of time, generally disappearing within 50 to 90 minutes after exposure to the marihuana.
338
Singer,
Scibetta,
and
Rosen
Fig. 7. Fetal EEG changes associated with maternal marihuana Control recording. B, During smoking period with trend toward per second wave forms. C. Thirty-four minutes post smoking. voltage, faster activity pattern.
Comment The results of this study demonstrate maternal and fetal heart rate changes associated with maternal marihuana smoking in the pregnant guinea pig. The maternal heart rate change described (i.e., the delayed onset of elevated heart rate) is consistent with that of other studies of cardiovascular changes associated with marihuana smoking.1g-27 The very brief delay (2 to 6 minutes) in onset may be indicative of rapid absorption through the lung. Its duration of effect (18 to 96 minutes, with the heart rate used as the indicator) is again consistent with that of other marihuana studies.?“, *I The fetuses and newborn animals studied generally revealed a decrease in heart rate during and, in a few instances, continuing after the marihuana smoking period. During this period, the magnitude of the deceleration would increase as the animal was exposed to additional marihuana cigarettes. It may be postulated that the fetal heart rate changes were caused by a drop in maternal blood pressure. However, this has been an inconsistent finding in other marihuana studies.Q w z4 The decelerations seen at various time intervals after the smoking period (i.e., the “recurrent bradycardias”) may also be drug related. Human studies
smoking
(Exp.
No.
3).
A,
irlcreased voltage of 10 cycles Note
trend
toward
lower
dealing with the clinical effects of marihuana smoking have noted a variability in performance of tasks during marihuana intoxication, which may be related to the fact that the subjects seemed to go “in and out” of their drug-affected state.?O This sporadic nature of drug effect could explain the recurrence of a fetal heart rate deceleration sometime after the smoking period. Maternal respiration was apparently unaffected by the marihuana except during the actual smoking period. During this time, the guinea pig responded to marihuana smoke by decreasing its respiratory rate and volume while exposed to the smoke and then breathing more deeply or more rapidly during the fresh air portion of the cycle. These changes may be attributed to the irritant nature of smoke on the guinea pig’s respiratory tract, as has been well described in human studies following hashish smoking.“p Because of the relative brevity of this altered pattern of respiration and the rapid (2 to 4 minutes) reversal to a normal respiratory pattern, it is unlikely that any significant change in pH, PO?, or Pco? occurred. No further respiratory changes were seen following the actual exposure to the smoke, again consistent with a number of other studies.‘, I91 ‘a Sedation following marihuana smoking is
Volume Number
117 3
also a well-recognized response to the drug. z”, 2913o This behavioral change following marihuana is suggestive evidence for absorption of a quantity of the drug through the lungs during the smoking period. Visual analysis of the EEG’s of maternal, fetal, and newborn guinea pigs was less clear. It is believed, however, that the visual defurther investigation. scriptions warrant Some of the animals showed more change in the EEG following exposure to marihuana than others. The maternal EEG during the smoking period tended to show an electrical activity pattern quite similar to that of control recordings, with a gradual transition following exposure to the marihuana toward a pattern of low-frequency, high-voltage activity. In a few of the maternal EEG’s, this “recovery pattern” demonstrated, at times, very high-voltage, spikelike waves of 10 to 14 cycles per second. These so-called recovery spike-discharges *have also been reported in other marihuana animal experiments.7-10 It has been postulated that these bursts in the EEG represent a drug-induced increased excitability of the neurons.? Studies involving human subjects have demonstrated a variable electroencephalographic response to marihuana or hashish smoking.@, 11-13,2o Reports of these EEG changes in human subjects have ranged from a somewhat more persistent alpha rhythmI to long-lasting alpha blocking, as well as amplitude decrease
Marihuana
3. 4. 5. 6. 7.
a. 9. 10.
Grinspoon, L.: Sci. Am. 221: 17, 1969. Pillard, R. C.: N. Engl. J. Med. 283: 294, 1970. Goddard, J. L.: Life, October, 1969. National Association of Blue Shield Plans: Drug Abuse, The Chemical Cop-Out, 1969. Kolansky, H., and Moore, W. T.: J. A. M. A. 216: 486, 1971. Wikler, A., and Lloyd, B. J., Jr.: Fed. Proc. 4: 141, 1945. Bose, B. C., Saifi, A. Q., and Bhagwat, A. W.: Arch. Int. Pharmacodyn. Ther. 147: 285, 1964. Lipparini, F., Carolis, A. S. D., and Longo, V. G.: Physiol. Behavior 4: 527, 1969. Masur, J., and Khazan, N.: Life Sci. 9: 1275, 1970. Pirch, J. H., Cohn, R. A., Barnes, P. R.,
and
fetal
guinea
pig
339
and intermittent disorganization of the tracing.12 Of interest is the similar EEG response to marihuana of high-voltage sharp waves as recorded from surface electrodes in animal studies7-10 and as recently reported in one human study recording the EEG from deep nuclear regions.13 The fetal EEG response to marihuana smoking was quite distinct from the maternal changes discussed above. During the smoking period, there was a trend toward a slowfrequency, high-voltage pattern of activity, toward lower with a gradual transition voltage and higher frequency activity following exposure to the marihuana. Of importance, however, is the fact that changes in the fetal heart rate and brain wave were demonstrated and were consistent in time with marihuana smoking. Since blood studies of marihuana concentration and other metabolites, as well as respiratory gas studies, were not available, the information recorded here must be accepted as observation, that and fetal physiologic and is, maternal behavioral parameters were recorded to have changed during and after the smoking of marihuana and similar changes were not present during a period of observation prior to the onset of the smoking protocol. Appreciation McFarland for Mrs. Margaret Braun for their
REFERENCES 1. 2.
in maternal
is expressed to Mrs. Lynne T. her continuous assistance and to Steinbrecher and Mr. Leonard technical advice.
and Barratt, E. S.: Neuropharmacology 11: 231, 1972. 11. 12.
13. 14. 15.
16. 17. 18.
Rodin, E. A., Domino, E. F., and Porzak, J. P.: J. A. M. A. 213: 1300, 1970. Deliyannakis, E., Panagopoulos, C., and Huott, A. D.: Clin. Electroencephalogr. 1: 128, 1970. Heath, R. G.: Arch. Gen. Psychiatry 26: 577, 1972. Kolansky, H., and Moore, W. T.: J. A. M. A. 222: 35, 1972. Campbell, A. M. G., Evans, M., Thompson, M. J.: Lancet 2: J. L. G., and Williams, 1219, 1971. Scibetta, J. J., and Rosen, M. G.: Obstet. Gynecol. 33: 830, 1969. Rosen, M. G., and McLaughlin, A.: Exp. Neurol. 16: 181, 1966. National Institutes of Mental Health. De-
340
19. 20. 21. 22.
23.
24.
Singer,
Scibetta,
and
Rosen
partment of Health. Education, and Welfare. Cannabis satixa Mexican, female, 1969. Weil, A. T., Zinberg, N. E., and Nelsen, J, M.: Science 162: 1234, 1968. Hollister, L. E.: Science 172: 21, 1971. Johnson, S., and Domino, E. F.: Clin. Pharmacol. Ther. 12: 762, 1971. Renault, P. F., Schuster, C. R., Heinrirh, R., and Freeman, D. X.: Science 174: 589, 1971. Williams, E. G., Himmelsbach, C. K., Wikler, A., Ruble, D. C., and Lloyd, B. J., Jr.: Public Health Rep. 61: 1059, 1946. Hollister, L. E., Richards, R. K., and Gillespie, H. K.: Clin. Pharmacol. Ther. 9: 783, 1968.
2:).
26. 27.
28.
29. DO.
Manno. J. E Kiphngerz C;. I:., Haine. b. t;.. Benrtett, I F.. and Fc)trrey, R. II.: Clili. Pharmacol. Ther. 11: 808, 1970. Iabcll, H., and Jasinski. D. R.: Paychopharmacologia 14: 11.5, 1969. Mayor’s Committee on Marihuana: The Marihuana Problem in the City of New York, Lancaster. Pennsylvania. 19-14, Jacques Cattell Pres>. Tennant, Jr., F. S., Preble, M.? Prendergast, P.: ,J. ‘4. M. A. 216: 1965, T. J., and \‘rntry. 1971. Geber, W. F.. and Schramm. L. C.: .4rch. Int. Pharmacodyn. Ther. 177: 224, 1969. Hockman, C. H., Perrin, R. G., and Kalant, H.: Science 172: 968. 1971.
R.
JOSEPH
SINGER, J.
SCIBETTA,
MORTIMER Rochester,
G. New
M.D. M.D.
ROSEN,
M.D
York
Maternal and fetal heart rate and brain wave (electroencephalogram [EEG]) changes were observed during simulated maternal marihuana smoking. The maternal heart rate change consisted of a delay in onset of an elevated heart rate, while the fetuses and newborn infants studied revealed a decrease in heart rate during the smoking period. The maternal EEG remained essentially unchanged during the smoking period. Following marihuana exposure, the maternal EEG changed to a pattern of low frequency and high-amplitude activity and in a few instances was similar to the “recovery spike-discharges” reported in other animal experiments with marihuana. The fetal EEG changed to a slow-frequency, high-voltage activity pattern during the smoking period. Following exposure to the marihuana, it changed to a lower voltage and higher frequency activity.
erty resides in several tetrahydrocannabinols, of which the major psychoactive compound is thought to be delta-9 tetrahydrocannabinol (dibenzopyran system) .* It has been estimated that between 12 and 20 million adolescents and young adults are using or have tried smoking Cannabis derivatives.3, 4 Uncertainty about the potential dangers of marihuana use prevails. Inasmuch as marihuana is reported to induce mental changes in the user,s analysis of its effects upon the central nervous system has been an appropriate target of medical research. More than 25 years ago, Wikler and Lloyd” described minimal and inconsistent scalp-recorded electroencephalogram (EEG) changes following marihuana smoking in 19 long-term narcotic addicts. More recently, EEG effects of marihuana or delta-9 tetrahydrocannabinol (THC) given to rabbits or rats was characterized by a generalized reduction in cortical EEG voltage and the appearance of bursts of high-voltage sharp waves.l-10 Rodin and associates,ll in a report on 10 medical students who had smoked marihuana for at least a year, found it im-
IN A co MPE N DIU M of medicines in 2737 B.C., the Chinese Emperor Shen Nung recorded some of the earliest observations on man’s use of marihuana.l The hemp plant, Cannabis satiua, has a history of use as a medicine, a source of fiber, and a drug in tribal religious ceremonies.’ Today, our principal interest in marihuana is its behavioral property for inducing euph0ria.l This prop-
From the Department Gynecology, Strong
of Obstetrics and Memorial Hospital.
Supported in part by a General Research Support Grant of the University of Rochester School of Medicine and Dentistry from the National Institutes of Health, a Graduate Research Training Grant in Reproductive Biology in Maternal Fetal Relationships (HDOOO15I I), and the 1. A. Hartford Foundation, Inc. 7;7c;ived Revised Accepted
for publication March March
December
28,
12, 1973. 26, 1973.
Reprint requests: Dr. Mortimer G. Rosen, Department of Obstetrics and Gynecology, Strong Memorial Hospital, 260 Crittenden Blvd., Rochester, New York 14642. 331
332
Singer,
Scibetta,
and
Rosen
Fig. 1. Diagram of experimental model: A and 13, Fetal EEG electrodes; C, fetal sewn in myometrium over fetus; D, ground wire placed beneath the maternal amphenol jack; F and G, maternal EEG electrodes; H, strain gauge to monitor respirations. Inset: Fetal electrode in detail.
possible by visual inspection to distinguish the premarihuana from postmarihuana EEG’s. There was a suggestion of more persistent alpha rhythm and slower frequency components after smoking. This was confirmed by power-density spectral analysis. Deliyannakis and colleagues12 reported minimal and inconsistent EEG findings in a study involving 27 admitted hashish-addicted soldiers. Heath’” reported minimal or no changes in E,EG’s recorded from scalp electrodes following marihuana smoking in a patient in whom electrodes had been implanted into numerous deep nuclear and surface areas of the brain. He did find that consistent EEG changes occurred in deep recordings from the septal region. These high-voltage slow waves were concomitant with the behavioral effects of marihuana smoking. Possible neurotoxic effects of marihuana smoking have been postulated by Kolansky and Moore14 who theorize that the psychic changes associated with Cannabis intoxication are the result of chemical (delta-9 THC) damage to the cerebral cortical cells. Furthermore, Campbell and co-worker@ have demonstrated cerebral atrophy by air encephalography in 10 individuals who had smoked marihuana for a period of 3 to 11 years.
ECG wire skin; E, maternal
A pertinent question for study concerns the possible effects of marihuana use during pregnancy. In this report, we will present observations on physiologic and behavioral variables following marihuana exposure in the maternal and fetal guinea pig. Methods Nine gravid guinea pigs were studied. Maturity of the fetus was estimated by gentle fetal vertex palpation through the maternal abdomen and by maternal symphyseal separation. In the maternal guinea pig, symphyseal separation occurs 48 to 72 hours prior to birth.l’; Maternal EEG electrode wires were implanted at least 24 hours prior to laparotomy. The guinea pigs were fasted overnight prior to the fetal operations. A flank incision overlying the palpable fetal skull was made after infiltrating the maternal abdominal wall with 2 C.C. of 0.25 per cent procaine hydrochloride. Light ether anesthesia was used when the peritoneum was opened. The fetal EEG electrodes consisted of 2 stainless steel needles attached to the ends of a metal spring (Fig. 1) . The details of the electrodes have been reported previ0us1y.‘~ The tips of the fetal electrode were pushed through the maternal myometrium and fetal skull into the brain in the central
Volume Number
Marihuana
117 3
Table I. Summary
‘Test tone 1.5, and
and fetal guinea
pig
333
of experiments Weight
Experiment NO. 1 2 3 (Day Pay 4 5 6 7 8 w
in maternal
(Grams)
Maternal
1)
No. of cigarettes smoked
Fetal
1,024 1,233 1,010
52, 60* 58, 65, 70, 82” 57, 61, 64”
1,018 1,193 989 1,102 1,008 837
40, 55*, 58” 42, 103, 40.
2)
42, 57” 58, 60, 65*, 60 75* !I,, Y59* 109* .58. 60”
Smoke
1 3 ‘) 2 2 2 3 3 1 3
78
exposure
(minutes)
7.0 --. 0 ‘)3 14.0 15.6 15.3 14.0 23.0 2’2.0 6.0 22.6
fetus. cigarette 3 hours.
smoking
immediately
followed
by next
in all
regions, analogous to a biparietal location. The depth of electrode penetration was between 1 and 2 mm. beyond the inner table of the sk~11.l~ The metal spring remained external to the uterus and held the electrodes in position without gross loss of amniotic fluidsI During the operation, the uterus and abdominal contents were kept warm with saline-soaked sponges (temperature maintained at 37 to 38’ C.). All the electrode wires were connected to a jack placed in the skin prior to closure of the abdomen (Fig. 1). Three to four hours following operation, the now alert maternal pig was placed in the smoking machine, and the jacks were connected to a polygraph for recording. The recording consisted of maternal and fetal EEG, electrocardiogram (ECG) , and maternal respiratory movement (Fig. 1) . All marihuana was administered in the form of 500 mg. cigarettes made with a hand-operated machine. The marihuana used in these experiments was supplied by the National Institutes of Mental Health and contained 1.4 per cent of delta-9 THC by weight. I8 Good-quality marihuana cigarettes are said to contain 500 mg. of Cannabis sativa with an average delta-9 THC content of 1 per cent.? Experiments on the effects of marihuana smoking in human subjects followed the procedure of allowing the subject 8 to 12 minutes to smoke each cigarette, subjects taking long puffs, inhaling deeply, and main-
animals
except
No.
9. In
this
animal,
cigarette
smoking
at 0,
taining inspiration for 20 seconds.lg Therefore, the cycle designed for the maternal guinea pig was selected to be analogous to the human act of smoking marihuana. A smoking apparatus was designed especially for the guinea pig and was used for all experiments. This apparatus consisted of a clear plastic container, approximately twice the volume of the maternal guinea pig, equipped with solenoid-operated portholes regulating smoke and air entry into the chamber. Smoking consisted of a repeated, 3 stage, 30 second cycle, as follows: ( 1) By means of a vacuum pump and cam, a puff of smoke was drawn from the lighted cigarette into the animal compartment (3 seconds) ; (2) the compartment was then closed off and the smoke remained within the compartment ( 12 seconds) ; (3) the compartment was then evacuated by the vacuum and the smoke replaced by fresh air ( 15 seconds) . At the end of this interval, the cycle began again with another puff of smoke. Prior to beginning the simulated smoking experience, the animal was placed in the smoking apparatus with all conditions simulating the actual smoking period except there was absence of the smoke. Recordings of both maternal and fetal EEG’s and ECG’s were obtained for a period ranging from 10 to 134 minutes. This recording supplemented a recording of maternal EEG’s taken one day prior to the laparotomy. At the end of this
334
Singer,
Scibetta,
and
Rosen
I
1
0
5
IO
b
20
25
20
MINUTES Fig. 2. A comparison marihuana cigarette tion of heart rate.
of maternal (Exp. No. 8).
and Note
fetal heart rate changes after the decrease in fetal heart rate during
smoking maternal
of one eleva-
Table II. Maternal heart rate changesassociatedwith marihuana smoking Experiment No.
No.
*No
further labor
cigarettes
smoked
2 3 (Day (Day 4 7 8 9 tin
of
Baseline
3 3
1)
2)
200-210 200-2 10 260-280 190-210 170-230 180-220 240-260
2 2 3 1 3
recording during
past 96 minutes.
Heart with
Heart
rate
returned
to base-line
rate Peak smoking 240 230 300 220 258 250 260
values
the next
Time (minutes) before return to base line 96+* 42 24 18 64 20 5t
day.
smoking.
presmoking period, the animal was exposed to smoke with one cigarette at a time. The maternal pig was exposed to each cigarette for not less than 6 minutes and not more than 10 minutes. The total duration of exposure to smoke can be seenin Table I. Results
Maternal heart rate changes. During the smoking of the marihuana cigarettes, a progressive increase in heart rate is generally noted (Table II). It begins 2 to 6 minutes after exposure to the first cigarette and lasts from 18 to more than 96 minutes. Because of the very wide range of base-line heart rates, it is difficult to establish a direct relationship between the magnitude of the increased heart rate and the number of cigarettes smoked.
Fetal heart rate (FHR) changes. FHR depression occurred during the period in which the mother was exposed to marihuana smoke and was herself experiencing an increasedheart rate (Fig. 2). With each additional exposure to a marihuana cigarette, the FHR progressively declined, as can be seen in Fig, 3. This figure compares fetal heart rate changes on 2 consecutive days. On each day, 2 marihuana cigarettes were smoked. It can be seen that the heart rate depression was more pronounced during the second cigarette. In 5 animals, the heart rate returned toward the baseline within 8 minutes following exposure to the last cigarette (Table III). The sixth fetus (Experiment No. 4) experienced prolonged bradycardia for one hour following smoking, before the heart rate returned to pre-existing levels. Also of
Volume Number
117 3
Marihuana
and fetal guinea
DAY2
DAYI
280-
in maternal
pig
335
-280‘
240 -
-240
1, 200% L 160-
-200
k
3
- 160 ypTm
Fig. 3. Fetal heart rate changes after 2 marihuana No. 3). Note more pronounced heart rate depression
Table
III.
Fetal heart
rate changes
associated Heart
Experiment NO. 2 3 (Day (Day 4 7 8 9
Table
Experiment No.
Weight (Gm.1
5
55 65 58 75
6 *Returned
to base-line
heart
rate
during
Minutes before return to control
110 170 110 170 120 100 80
heart rate changes associated
latter
part
(Exp.
7 5 60 1 2 8
with marihuana
“Recurrent bradycardia” (heart rate during this period) 160-l 10 240-180 240-190 190-160 190- 70 220-110 190- 80
smoking
Control
Change with smoking
Minutes before return to control
280-360 270-340 270-360 246-282
270 200 180 318
2.0 4.0 1.0 0*
of smoking
interest is the recurrent bradycardia seen in all of the fetuses. This delayed drop in the FHR usually occurred 8 to 40 minutes foIlowing the period of smoking, with the time of recurrence apparently related neither to the number of cigarettes smoked by the mother nor the maternal heart rate at that time. Neonatal heart rate changes. Heart rate changes associated with marihuana cigarette smoking were studied in 4 newborn animals within 9 hours of their birth (TabIe IV). The variation in control heart rates made determination of changes during smoking, as well as possible recurrent bradycardia or
days
smoking
rate
160-180 210-230 240-258 180-200 160-230 198-230 180-220
2)
IV. Neonatal
with marihuana
Maximum deceleration with smoking
Control 1)
cigarettes on 2 consecutive during the second cigarette.
period.
tachycardia, difficult. Three of the newborn animals responded to the smoking period with a deceleration similar to that seen in the previously described fetuses. The fourth and largest newborn animal initially responded to the marihuana smoking with an increased heart rate as did the maternal guinea pigs. In 2 of the newborn animals (58 and 75 grams), a recurrent bradycardia was noted (Fig. 4). Respiration. Maternal respiratory movements were monitored throughout each experiment. The respiratory rate during the control period prior to the smoking ranged between 50 and 80 breaths per minute. Dur-
336
Singer,
Scibetta,
and
Rosen
x0330-
-360 -330
NO270-
-300 -270
240210& 1803 ISOQ mg 3303002702402lOIBO-
NEWBORN 5Bgns
1
-240
I CKjHAI 1
CIC 1 * HHH
-210 - 180 - 150 - 120 -330 -300 -270
J
3
Do'*O 'i0.x'
NEWBORN 75gms.
-240 - 210 -180 - I50 L I20
Fig. 4. Newborn heart rate changes after exposure to 3 marihuana cigarettes Note recurrence of heart rate depression 20 minutes or more following smoking. ing the smoking period, the respirations varied with each portion of the smoking cycle. In that portion of the cycle in which smoke filled the chamber, respirations were shallow and the rate decreased to 20 to 30 breaths per minute. During the expulsion of the smoke and its replacement with fresh air, 2 different patterns were seen. The guinea pig would either breathe very deeply at a rate of 25 to 35 breaths per minute or breathe quite rapidly (80 to 90 breaths per minute) at a normal depth of respiration. One of these patterns continued until smoke began to fill the chamber, signaling the onset of the next cycle, and the animal would again revert to the shallow, slow respirations. The guinea pig resumed pre-existing breathing patterns within 2 to 4 minutes following the last cigarette. No further variations in respiration were observed during the remainder of each experiment. Observed behavior. The behavior of the maternal guinea pig and newborn animal was noted during each experiment. The most striking change in behavior was seen during and immediately following the smoking period. Throughout the control period, the animals were active. During smoking, the mothers and newborn animals were noticeably disturbed by the smoke, as demonstrated by their attempts to turn around. Following
(Exp. No. 6)
the smoking period, the animals changed their behavior and became relatively immobile. They would remain practically motionless and unreactive to stimuli which normally would disturb them, such as door opening or dropping an instrument on the floor. This persisted for 90 to 120 minutes. Quantitation of this effect with increasing numbers of cigarettes ~~3s not possible because of our desire not to stimulate the animals in order to check their level of awareness but merely to observe their spontaneous movement. All of thr animals demonstrated the “sedated period” for variable durations. Electroencephalograms. Maternal EEG. Prior to the smoking period, the maternal EEG displayed a spectrum of activity, varying from continuous lower voltage fast activity (Fig. 5, A) to somewhat higher voltage activity with slower wave frequencies (Fig. 6, A 1. During marihuana smoking, the EEG pattern was similar to the low-voltage fast activity [Figs. 5 and 6, B). This low-voltage fast activity was replaced 15 to 30 minutes post smoking with higher voltages and lower frequency patterns as illustrated in Figs. 5 and 6, C. This pattern of slower frequency, higher voltage activity lasted for variable periods of time. It reappeared up to 24 hours after smoking and
Volume Number
117
Marihuana
3
in maternal
Fig. 5. Maternal EEG changes associated with marihuana smoking recording with continuous lower voltage fast activity. B, During similarity to A. C, Two hours and forty-two minutes post smoking voltage “recovery spike-discharges,”
and
at times,
a very high-voltage EEG. of the presmoking fetal EEG is seen in Fig. 7, A. During the period of exposure to the marihuana smoke, fetal EEG activity demonstrated a trend toward increased voltage of 10 cycles per second wave forms (Fig. 7, B). Over a time
Fetal EEG. An example
guinea
pig
337
(Exp. No. 3). A, Control the smoking period with with few-frequency, high-
Fig. 6. Maternal EEG changes associated with marihuana smoking (Exp. recording with higher voltage, slower wave forms. B, During smoking voltage, fast wave forms (see also Fig. 7, A and B). C, Forty-eight minutes slow-frequency, high-voltage activity.
showed,
fetal
No. 2). A, Control period with lower post smoking; note
span of 15 to 30 minutes following the last cigarette, the EEG activity showed increasing frequency and decreasing voltage (Fig. 7, C). This pattern of low-voltage, fast activity lasted for variable periods of time, generally disappearing within 50 to 90 minutes after exposure to the marihuana.
338
Singer,
Scibetta,
and
Rosen
Fig. 7. Fetal EEG changes associated with maternal marihuana Control recording. B, During smoking period with trend toward per second wave forms. C. Thirty-four minutes post smoking. voltage, faster activity pattern.
Comment The results of this study demonstrate maternal and fetal heart rate changes associated with maternal marihuana smoking in the pregnant guinea pig. The maternal heart rate change described (i.e., the delayed onset of elevated heart rate) is consistent with that of other studies of cardiovascular changes associated with marihuana smoking.1g-27 The very brief delay (2 to 6 minutes) in onset may be indicative of rapid absorption through the lung. Its duration of effect (18 to 96 minutes, with the heart rate used as the indicator) is again consistent with that of other marihuana studies.?“, *I The fetuses and newborn animals studied generally revealed a decrease in heart rate during and, in a few instances, continuing after the marihuana smoking period. During this period, the magnitude of the deceleration would increase as the animal was exposed to additional marihuana cigarettes. It may be postulated that the fetal heart rate changes were caused by a drop in maternal blood pressure. However, this has been an inconsistent finding in other marihuana studies.Q w z4 The decelerations seen at various time intervals after the smoking period (i.e., the “recurrent bradycardias”) may also be drug related. Human studies
smoking
(Exp.
No.
3).
A,
irlcreased voltage of 10 cycles Note
trend
toward
lower
dealing with the clinical effects of marihuana smoking have noted a variability in performance of tasks during marihuana intoxication, which may be related to the fact that the subjects seemed to go “in and out” of their drug-affected state.?O This sporadic nature of drug effect could explain the recurrence of a fetal heart rate deceleration sometime after the smoking period. Maternal respiration was apparently unaffected by the marihuana except during the actual smoking period. During this time, the guinea pig responded to marihuana smoke by decreasing its respiratory rate and volume while exposed to the smoke and then breathing more deeply or more rapidly during the fresh air portion of the cycle. These changes may be attributed to the irritant nature of smoke on the guinea pig’s respiratory tract, as has been well described in human studies following hashish smoking.“p Because of the relative brevity of this altered pattern of respiration and the rapid (2 to 4 minutes) reversal to a normal respiratory pattern, it is unlikely that any significant change in pH, PO?, or Pco? occurred. No further respiratory changes were seen following the actual exposure to the smoke, again consistent with a number of other studies.‘, I91 ‘a Sedation following marihuana smoking is
Volume Number
117 3
also a well-recognized response to the drug. z”, 2913o This behavioral change following marihuana is suggestive evidence for absorption of a quantity of the drug through the lungs during the smoking period. Visual analysis of the EEG’s of maternal, fetal, and newborn guinea pigs was less clear. It is believed, however, that the visual defurther investigation. scriptions warrant Some of the animals showed more change in the EEG following exposure to marihuana than others. The maternal EEG during the smoking period tended to show an electrical activity pattern quite similar to that of control recordings, with a gradual transition following exposure to the marihuana toward a pattern of low-frequency, high-voltage activity. In a few of the maternal EEG’s, this “recovery pattern” demonstrated, at times, very high-voltage, spikelike waves of 10 to 14 cycles per second. These so-called recovery spike-discharges *have also been reported in other marihuana animal experiments.7-10 It has been postulated that these bursts in the EEG represent a drug-induced increased excitability of the neurons.? Studies involving human subjects have demonstrated a variable electroencephalographic response to marihuana or hashish smoking.@, 11-13,2o Reports of these EEG changes in human subjects have ranged from a somewhat more persistent alpha rhythmI to long-lasting alpha blocking, as well as amplitude decrease
Marihuana
3. 4. 5. 6. 7.
a. 9. 10.
Grinspoon, L.: Sci. Am. 221: 17, 1969. Pillard, R. C.: N. Engl. J. Med. 283: 294, 1970. Goddard, J. L.: Life, October, 1969. National Association of Blue Shield Plans: Drug Abuse, The Chemical Cop-Out, 1969. Kolansky, H., and Moore, W. T.: J. A. M. A. 216: 486, 1971. Wikler, A., and Lloyd, B. J., Jr.: Fed. Proc. 4: 141, 1945. Bose, B. C., Saifi, A. Q., and Bhagwat, A. W.: Arch. Int. Pharmacodyn. Ther. 147: 285, 1964. Lipparini, F., Carolis, A. S. D., and Longo, V. G.: Physiol. Behavior 4: 527, 1969. Masur, J., and Khazan, N.: Life Sci. 9: 1275, 1970. Pirch, J. H., Cohn, R. A., Barnes, P. R.,
and
fetal
guinea
pig
339
and intermittent disorganization of the tracing.12 Of interest is the similar EEG response to marihuana of high-voltage sharp waves as recorded from surface electrodes in animal studies7-10 and as recently reported in one human study recording the EEG from deep nuclear regions.13 The fetal EEG response to marihuana smoking was quite distinct from the maternal changes discussed above. During the smoking period, there was a trend toward a slowfrequency, high-voltage pattern of activity, toward lower with a gradual transition voltage and higher frequency activity following exposure to the marihuana. Of importance, however, is the fact that changes in the fetal heart rate and brain wave were demonstrated and were consistent in time with marihuana smoking. Since blood studies of marihuana concentration and other metabolites, as well as respiratory gas studies, were not available, the information recorded here must be accepted as observation, that and fetal physiologic and is, maternal behavioral parameters were recorded to have changed during and after the smoking of marihuana and similar changes were not present during a period of observation prior to the onset of the smoking protocol. Appreciation McFarland for Mrs. Margaret Braun for their
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in maternal
is expressed to Mrs. Lynne T. her continuous assistance and to Steinbrecher and Mr. Leonard technical advice.
and Barratt, E. S.: Neuropharmacology 11: 231, 1972. 11. 12.
13. 14. 15.
16. 17. 18.
Rodin, E. A., Domino, E. F., and Porzak, J. P.: J. A. M. A. 213: 1300, 1970. Deliyannakis, E., Panagopoulos, C., and Huott, A. D.: Clin. Electroencephalogr. 1: 128, 1970. Heath, R. G.: Arch. Gen. Psychiatry 26: 577, 1972. Kolansky, H., and Moore, W. T.: J. A. M. A. 222: 35, 1972. Campbell, A. M. G., Evans, M., Thompson, M. J.: Lancet 2: J. L. G., and Williams, 1219, 1971. Scibetta, J. J., and Rosen, M. G.: Obstet. Gynecol. 33: 830, 1969. Rosen, M. G., and McLaughlin, A.: Exp. Neurol. 16: 181, 1966. National Institutes of Mental Health. De-
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19. 20. 21. 22.
23.
24.
Singer,
Scibetta,
and
Rosen
partment of Health. Education, and Welfare. Cannabis satixa Mexican, female, 1969. Weil, A. T., Zinberg, N. E., and Nelsen, J, M.: Science 162: 1234, 1968. Hollister, L. E.: Science 172: 21, 1971. Johnson, S., and Domino, E. F.: Clin. Pharmacol. Ther. 12: 762, 1971. Renault, P. F., Schuster, C. R., Heinrirh, R., and Freeman, D. X.: Science 174: 589, 1971. Williams, E. G., Himmelsbach, C. K., Wikler, A., Ruble, D. C., and Lloyd, B. J., Jr.: Public Health Rep. 61: 1059, 1946. Hollister, L. E., Richards, R. K., and Gillespie, H. K.: Clin. Pharmacol. Ther. 9: 783, 1968.
2:).
26. 27.
28.
29. DO.
Manno. J. E Kiphngerz C;. I:., Haine. b. t;.. Benrtett, I F.. and Fc)trrey, R. II.: Clili. Pharmacol. Ther. 11: 808, 1970. Iabcll, H., and Jasinski. D. R.: Paychopharmacologia 14: 11.5, 1969. Mayor’s Committee on Marihuana: The Marihuana Problem in the City of New York, Lancaster. Pennsylvania. 19-14, Jacques Cattell Pres>. Tennant, Jr., F. S., Preble, M.? Prendergast, P.: ,J. ‘4. M. A. 216: 1965, T. J., and \‘rntry. 1971. Geber, W. F.. and Schramm. L. C.: .4rch. Int. Pharmacodyn. Ther. 177: 224, 1969. Hockman, C. H., Perrin, R. G., and Kalant, H.: Science 172: 968. 1971.