Cocaine alters behavioral states in fetal sheep

Developmental Brain Research, 56 (1990) 41-45

41

Elsevier BRESD 51141

Cocaine alters behavioral states in fetal sheep D.J. Burchfield ~, E.M. Graham 2, R.M.

A b r a m s 1"2 a n d

K.J.

Gerhardt 3

Departments of ~Pediatrics, 2Obstetrics and Gynecology, and SCommunication Processes and Disorders, University of Florida, Gainesville, FL 32610 (U.S.A.) (Accepted 15 May 1990)

Key words: Cocaine; Fetus; Sleep; Behavioral state; Drug; Pregnancy

Intrauterine cocaine exposure causes subtle neurologic abnormalities in human newborn infants; however, the mechanism for these abnormalities is not known. To investigate whether cocaine alters fetal behavioral state, the electrocortical, electro-ocular and neck muscle electrical activity was monitored in 7 chronically instrumented fetal sheep before and during both saline and cocaine HCI infusions directly to the fetus, Saline infusion to the fetus had no effect on the percentage of time spent in rapid eye movement sleep compared to no infusion (37.5 + 11.6% vs 46.3 + 4.6%, mean _+ SD, P > 0.1). Cocaine infusion directly to the fetus had no effect on fetal arterial pOz, but did increase mean arterial pressure from 53.6 + 15 mmHg to 61.0 + 21 mmHg (P < 0.01). In addition, during cocaine infusion, the percentage of time spent in rapid eye movement sleep dropped to 3.9 + 5.1% (P < 0.0001) and the average duration of rapid eye movement epochs decreased from 10.1 + 3.0 rain precocaine infusion to 1.9 _+ 2.6 min during cocaine infusion (P < 0.02). The influence of cocaine was noted in a frequency analysis of the electrocorticogram. The amplitude of the energy centered at 1 Hz during cocaine infusion (73.8 _+ 4.0 dB) was greater than the amplitude during rapid eye movement sleep (65.5 _+ 4.7 dB) and less than the amplitude during non-rapid eye movement periods (79.9 +__4.5 dB) (P = 0.01). Cocaine appears to alter fetal behavioral state directly and this may play a role in the abnormal behavior in newborn infants exposed to cocaine in utero. INTRODUCTION

of c o c a i n e in p r e g n a n t s h e e p , fetal h y p o x i a and v o l u m e shifts f r o m m u l t i p l e b l o o d s a m p l i n g as well as a direct

C o c a i n e use by w o m e n of c h i l d b e a r i n g age has b e c o m e a major

public

in R E M sleep. In the p r e s e n t study we h y p o t h e s i z e that

of subtle

c o c a i n e has a direct effect on fetal b e h a v i o r a l states. To

n e u r o i o g i c a b n o r m a l i t i e s in n e w b o r n infants c o n t i n u e s to m o u n t 4"6"9"~5. T h e m e c h a n i s m for the irritability, a b n o r -

gases and b l o o d v o l u m e , factors which m a y h a v e b e e n

mal o r i e n t a t i o n r e s p o n s e s , v i g o r o u s sucking, a b n o r m a l

c o n f o u n d i n g v a r i a b l e s in the p r e v i o u s study.

cocaine

electroencephalogram,

issue ~6. E v i d e n c e

action of c o c a i n e w e r e possible causes of this t e r m i n a t i o n

implicating

intrauterine

health

exposure

and

as

other

a cause

neurologic

test the h y p o t h e s i s , we a t t e m p t e d to c o n t r o l for b l o o d

findings

s e e n in t h e s e c o c a i n e - e x p o s e d infants is not k n o w n . In s h e e p , m a t e r n a l c o c a i n e a d m i n i s t r a t i o n causes a d e c r e a s e in u t e r i n e b l o o d flow 17 and fetal h y p o x i a 5"29. If this is also true in h u m a n s , o x y g e n d e p r i v a t i o n m a y play a role in causing the n e u r o l o g i c s y n d r o m e s e e n in infants. H o w e v e r , b e c a u s e o f the t r a n s i e n t n a t u r e o f the n e u r o logical a b n o r m a l i t i e s , h y p o x i a - i n d u c e d brain d a m a g e is not likely to be the only cause. A d u l t h u m a n s w h o chronically abuse c o c a i n e s p e n d less of the sleep t i m e in rapid eye m o v e m e n t ( R E M ) sleep 21 - - a sleep state c o n s i d e r e d v e r y i m p o r t a n t in fetal and n e w b o r n life 2223"25. In an e a r l i e r study in s h e e p 5, we f o u n d that m a t e r n a l c o c a i n e a d m i n i s t r a t i o n during the b e g i n n i n g of fetal R E M sleep cycle was f o l l o w e d by a p r e m a t u r e t e r m i n a t i o n o f that sleep cycle. In that study d e s c r i b i n g the p h a r m a c o k i n e t i c s and p h a r m a c o - d y n a m i c s

MATERIALS AND METHODS Seven pregnant ewes carrying singleton fetuses at 125 days gestation (145 days = term) were anesthetized with 2.5% halothane in oxygen and underwent a midline abdominal incision and a hysterotomy. Polyvinyl catheters (1.07 mm i.d.) were placed surgically via both fetal axillary arteries and a fetal vein. To record fetal behavioral states, electrodes were placed for detection of electrocortical (ECoG), electro-ocular (EOG), and neck muscle (EMG) activity5'23"25. The fetal scalp was incised and 1 mm holes were placed through the skull and ECoG electrodes soldered to stainless steel screws were placed on the dura. Next, EOG electrodes attached to screws were placed into holes drilled through the superior orbital ridge. The skin over the neck was also incised and a stainless steel wire electrode (Cooner Wire, Inc.) was sutured into a strap muscle to record neck EMG. Incisions were closed and the fetal head and foreleg were returned to the uterus. Sodium ampicillin, 500 rag, was placed into the uterine cavity and the peritoneum of the ewe before closing the incisions. Catheters and electrodes were tunnelled subcutaneously

Correspondence: D.J. Burchfield, Department of Pediatrics, Division of Neonatology, UF College of Medicine, Box J-296 JHMHC, Gainesville, FL 32610, U.S.A.

42 to the ewe's flank and stored in a pouch. Polyvinyl catheters (1.65 mm i.d.) were placed surgically into both femoral arteries and a femoral vein of the ewe. Ewes received penicillin and streptomycin daily t~or 3 days following surgery. Catheters were flushed daily with heparin, 1000 U/ml, to maintain patency. On the third postoperative day the ewes were placed standing in a cart in the laboratory. Fetal arterial blood pressure was measured and continuously recorded on a physiograph (Gould Instruments, Inc., Dayton, OH), and fetal arterial blood gases were determined using a standard commercial blood gas machine (Radiometer, Inc., Westlake, OH) to assess fetal well-being. Next, fetal ECoG, EOG, and neck EMG were amplified and filtered (band pass ranges: ECoG 0.3-30 Hz, EOG 0.3-30 Hz, EMG 30-300 Hz) and then recorded continuously on the physiograph and on FM tape (Bruel and Kjaer Instruments, Inc., Copenhagen; Model 7005) for at least 90 rain after the first behavioral state change. Behavioral state was determined by visual inspection of the recordings z5 and categorized as either REM (low voltage, high frequency ECoG with rapid electrical activity from the EOG, and inactivity from the neck EMG); non-REM (NREM) (high voltage, low frequency ECoG with relatively little EOG activity); or indeterminate (all other combinations). After this initial observation period with an equal number of REM and NREM epochs, an infusion of sterile saline was begun through the fetal intravenous catheter at 0.2 ml/min. This infusion continued for 90 rain while behavioral state recordings were continued. The next day, fetal behavioral states were observed for another 90 rain period with equal R E M and NREM epochs. Then, at the beginning of a REM epoch, cocaine hydrochioride 0.6 mg/min, as free base, was administered directly to the fetus intravenously for the next 90 min. Cocaine was dissolved in sterile saline so the administration rate was 0.2 ml/min, the same volume as received the prior day. Preliminary studies in our laboratory showed that this cocaine infusion rate led to a steady-state cocaine concentration in the fetal plasma within 10 rain. This concentration approximated the peak fetal concentration obtained after a moderate intravenous bolus of cocaine to the pregnant ewe 5. Several 1.5 ml arterial blood samples were obtained at various times during the infusion for measuring fetal cocaine concentration and arterial blood gases. Warmed saline was slowly infused over 30-60 s after blood removal to replace volume and maintain catheter patency. Care was taken to avoid rapid blood removal or volume replacement. In addition, fetal blood pressure was measured and continuously recorded using a Statham P23 Strain Gauge Transducer. The percentage of time in each behavioral state was determined for the 4 experimental conditions: presaline infusion, during saline infusion, precocaine infusion, and during cocaine infusion. In addition, the duration of each behavioral state epoch was averaged for each experimental condition. These data were analyzed by

RESULTS

Cocaine infusion of 0.6 mg/min led to a steady-state plasma cocaine concentration within 10 min of beginning the infusion (Fig. 1). Unlike the fetal hypoxia seen after maternal cocaine administration 5, arterial blood gases did not change significantly during continuous direct infusion of cocaine to the fetus. Fig. 2 graphically compares fetal

40

pO2 In FETAL SHEEP DURING DIRECT COCAINE INFUSION

30 ¸

T

T

,

T

torr 20 10

0 -10

mean ÷ / -

10 20

0

30

40

50

60

70

sd

BO 90

TI.E ( IH)

40-

30-

COCAINE CONCENTRATION DURING CONTINUOUS FETAL INFUSION

500. 550500. 450. 400

analysis of variance and Tukey A comparison. A real time frequency analyzer (mode[ 2123. Bruel and Kjaer Instr., Copenhagen) was used offiine to analyze the taped ECo(i signals. Selected for analysis were 5 min periods during a period ot REM and NREM in the precocaine infusion and during a 5 rain period 70 min after the continuous infusion of cocaine had begun. Thirty successive 10 s samples of ECoG were subjected to 1/3 octave band filtering centered at 1 Hz and 20 Hz. The constant percentage bandwidth filters have a slope of 18 dB/octave. Dynamic range exceeds 80 dB and internal noise levels are less than 0.1 ,uV. The amplitudes in one-third octave bands centered at I Hz and 2() Hz were expressed in relative decibels. Standard deviation of measurements for the 10 s periods was 2.5 dB. Bands at 1 Hz and 20 Hz were chosen because they best delineated REM and NREM behavioral states, although similar trends in the low and high frequency spectra could be seen using 4 Hz and 16 Hz bands, respectively. The mean relative dB levels at each of the frequencies for each 5 rain interval were compared by analysis of variance and Tukey A comparison.

pO2 IN FETAL SHEEP AFTER MATERNAL ADMINISTRATION OF COCAINE

T

torr 20-

ng/ml 3 5 0 . 300 250. 200.

150. I O0

~

1

1

kiln + / - SO

50,

Oi 0

mean + / -

10-

I 20

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40

I

60 TIME (kilN)

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I

80

1O0

Fig. 1. Fetal plasma cocaine concentration (mean + S.D.) during direct infusion of 0.6 mg/min of cocaine HCI.

0 -10

-5

0

5

10

15

20

25

sd

30

TIME (WIN) Fig. 2. Fetal arterial p O 2 (mean _+ S.D.) during direct infusion of cocaine HC1 (top panel) and after maternal administration of 2 mg/kg of cocaine HC1 (bottom panel) from a prior study.

43

PRE-COCAINE

REM

DURING COCAINE INFUSION

NREM

0 0

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TIME (rain) Fig. 3. Strip chart recording of fetal E C O G (top), E O G (middle), and neck muscle E M G ( b o t t o m ) during a 20 min p e r i o d b e f o r e cocaine infusion to the fetus (left) and a 20 min period during cocaine administration (right). N o t e that the E C O G a p p e a r s to be of an intermediate voltage c o m p a r e d to R E M and N R E M , the lack of E O G activity c o m p a r e d to during R E M , and the repetitive bursts of neck activity.

paO2 during continuous fetal cocaine infusion (upper panel) to fetal paO2 after maternal administration of 2 mg/kg of cocaine from a previous study (lower panel) 5. Mean arterial pressure increased from 53.6 + 15 mmHg to 61.0 + 21 mmHg (mean + S.D., P < 0.01) by 5 min and remained elevated. Fig. 3 demonstrates the strip chart recording from one fetus. Normal cycling of behavioral states is seen in the precocaine infusion period. During cocaine administration one notes an intermediate ECoG signal, little eye

100, 80.

r--i REM mm NREM ~ INDETERMINATE

movement as detected by EOG and repetitive bursts of neck muscle activity. The percentage of time the fetuses spent in REM, NREM, and indeterminate behavioral states was unaffected by saline administration (Fig. 4). The percentage of time spent in REM was 37.5 + 11.6% before saline infusion, 46.3 _+ 4.6% during saline infusion and 37.0 + 11.0% before cocaine infusion (P > 0.1). These percentages correspond well to previously published reports on fetal behavioral states in sheep 1'23'2s. In addition, the fraction of sleep time spent in NREM and indeterminate

55 30

§

['---I REM i NREM ~ INDETERMINATE

25

60'

Ji~

PRE-SAUNE

PRE-COCAINE COCAINE

Fig. 4. P e r c e n t time in the different behavioral states u n d e r the 4 e x p e r i m e n t a l conditions. Values are m e a n s + S.D. *P < 0.04301 c o m p a r e d to o t h e r R E M periods. §P < 0.002 c o m p a r e d to o t h e r i n d e t e r m i n a t e periods.

§

TIME

L PRE-SALINE SAUNE PRE-COCAINE COCAINE Fig. 5. A v e r a g e d u r a t i o n of each b e h a v i o r a l state. Values are m e a n s + S . D . *P < 0.02 c o m p a r e d to o t h e r R E M periods. §P < 0.006 c o m p a r e d to o t h e r i n d e t e r m i n a t e periods.

44

100-

IHz BIB 20 Hz

" "io bJ

_]_

80.

"8

5

> i-

5

L,d no

60'

40

REM

i Ii

NREM

COCAINE

Fig. 6. Power in relative dB at 1 Hz (open bars) and 20 Hz (filled bars) (mean + S.D.) during REM, NREM and exactly 70 min into the cocaine infusion. *P = 0.01 compared to its accompanying20 Hz signals. ~P = 0.01 compared to other 1 Hz signals.

were unaltered by saline and were similar to previously published reports 1'23'25. During cocaine infusion, the percentage of time that the fetus spent in REM sleep dropped significantly to 3.9 + 5.1% (P < 0.0001 compared to the 3 other observation periods) (Fig. 4). Not only was the total percentage of time spent in REM diminished, but the average duration of REM epochs was also shortened from 10.1 + 3.0 min precocaine infusion to 1.9 + 2.6 min during cocaine infusion (P < 0.02) (Fig. 5). This attenuation of REM sleep was accompanied by a significant increase in the percentage of time and duration of the indeterminate behavioral states (Figs. 4, 5). Cocaine infusion altered the spectral amplitude in the 1 Hz frequency band of the ECoG in the fetus. As seen in Fig. 6, the amplitude at 20 Hz was similar during REM, NREM and the indeterminate periods. However, whereas the mean amplitude at the 1 Hz frequency band was 65.5 + 4.7 dB during REM and 79.9 + 4.5 dB during NREM, the spectral amplitude during cocaine infusion was 73.8 + 4.0 dB, a value intermediate and statistically different than the values during REM and NREM (P = 0.01 v s REM and P = 0.01 v s NREM).

DISCUSSION In this study REM sleep in the fetal lamb was prematurely terminated by cocaine infusion and it appears that this is a direct effect of the drug on the fetal brain. During the experiment, care was taken to avoid unnecessary noise, disturbance of the ewe, rapid blood sampling and catheter flushing, and other procedures that conceivably could alter fetal behavioral state. Direct administration of cocaine to the fetus did not cause significant hypoxemia as has been reported after infusion into a maternal vein5'29.

Rapid eye movement sleep, or dream sleep, is theorized to be important in normal cerebral maturation and development 22. The brain is metabolically active during this behavioral state 1'2°, and this increased metabolic rate is accompanied by an increase in cerebral blood flowz 19,10. Roffwarg et al. zz hypothesized that REM sleep provided intense stimulation to the central nervous system during a time when the developing brain receives very little external stimulation; that is, during prenatal and newborn periods. Early studies indicate that functional stimulation potentiates structural growth in the nervous system3'z2'28, and this stimulation may precede myelinization14. Thus, drugs interfering with REM sleep might affect some attributes of cerebral growth function. Cocaine is lipid soluble and rapidly crosses the placenta after maternal administration 5,s. Other drugs have been shown to cross the placenta and alter fetal behavioral state 25-z7. However, in contrast to the indeterminate state observed with cocaine administration, methadone and morphine caused arousal in fetal sheep 24"~6. This behavioral state consists of a desynchronized, low voltage ECoG, often indistinguishable from the ECoG pattern of REM sleep. In the present study, the strip chart recording of the ECoG signal during cocaine infusions appeared to be distinctly different from either REM or NREM periods. However, during fetal cocaine infusion repetitive neck contractions could have introduced movement artifacts which may have potentially contaminated the ECoG signal. The amplitude in the low frequency band (1 Hz) during cocaine infusion was intermediate to the amplitude during REM cycles and NREM cycles. In humans, cocaine can also cause increased electroencephalographic amplitude 1° and increased beta activityx~, which may relate to seizure development. Clinical evidence in human newborns supports a 'fetal cocaine exposure' syndrome consisting of irritability, jitteriness and excessive crying4,6'15. We find it interesting that anxiety and irritability are common in adult volunteers deprived of REM sleep 7'12, and recognize that a cause and effect relationship may exist between REM deprivation through cocaine exposure and 'fetal cocaine exposure syndrome'. The mechanism for cocaine's curtailment of REM sleep may be through its known inhibitory properties on the locus ceruleus TM, lesions of which produce decreased REM sleep time 13. In addition, the clinical syndrome in newborn humans may be related to the development of a physical dependence on cocaine, a phenomena described by Szeto et at. 26 in fetal sheep with chronic methadone exposure, but not investigated in the present study. We conclude that cocaine acts directly on the fetal

45 brain to a t t e n u a t e R E M sleep, a behavioral state which is p r o b a b l y crucial for normal central nervous system maturation. REFERENCES 1 Abrams, R.M., Hutchison, A.A., Jay, T.M., Sokoloff, L. and Kennedy, C., Local cerebral glucose utilization non-selectively elevated in rapid eye movement sleep of the fetus, Dev. Brain Res., 40 (1988) 65-70. 2 Abrams, R.M., Post, D.J., Burchfield, D.J., Gomez, K.J., Hutchison, A.A. and Conlon, M., Local cerebral blood flow increased in rapid eye movement sleep in fetal sheep, Am. J. Obstet. Gynecol., 162 (1990) 278-281. 3 Bennett, E.L., Diamond, M.C., Krech, D. and Rosenzweig, M.R., Chemical and anatomic plasticity of brain, Science, 146 (1964) 610-619. 4 Bingol, N., Fuchs, M., Diaz, W., Stone, R.X. and Gromish, D.S., Teratogenicity of cocaine in humans, J. Pediatr., 111 (1987) 571-578. 5 Burchfield, D.J., Abrams, R.M., Miller, R., Braun, S. and DeVane, C.L., Pharmacokinetics and pharmacodynamics of cocaine in pregnant sheep, Ped. Res., 23(4) (1988) 256A. 6 Chasnoff, I.J., Burns, W.J., Scholl, S.H. and Burns, K.A., Cocaine use in pregnancy, N. Engl. J. Med., 313 (1985) 666-669. 7 Dement, W., The effect of dream deprivation, Science, 131 (1960) 1705-1707. 8 DeVane, C.L., Simpkins, J.W., Miller, R.L. and Braun, S., Tissue distribution of cocaine in the pregnant rat, Life Sci., 45 (1989) 1271-1276. 9 Doberczak, T.M., Shanzer, S., Senie, R.T. and Kandall, S.R., Neonatal neurologic and electroencephalographic effects of intrauterine cocaine exposure, J. Pediatr., 113 (1988) 354-358. 10 Herning, R.I., Hooker, W.D. and Jones, R.T., Cocaine effects on electroencephalographic cognitive event-related potentials and performance, Electroencephalogr. Clin. Neurophysiol., 66 (1987) 34-42. 11 Herning, R.I., Jones, R.T., Hooker, W.D. et al., Cocaine increases EEG beta: a replication and extension of Hans Berger's historic experiments, Electroencephalogr. Clin. Neurophysiol., 60 (1985) 470-477. 12 Hartman, E., Antidepressants and sleep: clinical and theoretical implications. In A. Kales (Ed.), Sleep-Physiology and Pathology -- A Symposium, Lippencott, Philadelphia, PA, 1969, pp. 308-316. 13 Jouvet, M. and Delorme, E, Locus ceruleus et sommeil paradoxal, C. R. Soc. Biol., 159 (1965) 895-899. 14 Langworthy, O.R., Development of behavior patterns and

Acknowledgements. The authors wish to acknowledge C. Lindsey DeVane, Pharm.D. and Ronald L. Miller for performing plasma cocaine measurements. Supported by NIDA Grant DA 05170.

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