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Elicitation and modification of the acoustic startle reflex in animals prenatally exposed to cocaine
Neurotoxicology and Teratology, Vol. 13, pp. 541-546. © Pergamon Press plc, 1991. Printed in the U.S.A.
0892-0362/91 $3.00 + .00
Elicitation and Modification of the Acoustic Startle Reflex in Animals Prenatally Exposed to Cocaine J O H N A. F O S S 1 A N D E D W A R D P. R I L E Y *
*Department of Psychology, San Diego State University, San Diego, CA 92182 Received 26 July 1989 FOSS, J. A. AND E. P. RILEY. Elicitation and modification of the acoustic startle reflex in animals prenatally exposed to cocaine. NEUROTOXICOL TERATOL 13(5) 541-546, 1991.--In separate experiments, pregnant Long-Evans rats were administered cocaine orally (60 mg/kg/day) on gestation days 14-21, or subcutaneously (40 mg/kg/day) on gestational days 8-21. For each route of administration, a vehicle control group was pair-fed to the group administered cocaine and another control group was left untreated. Throughout pregnancy, the dams that received cocaine gained approximately 15% less weight than the untreated controls, but none of the dosage procedures affected the size of the fitters, or the weight and growth of the offspring. When the offspring reached adulthood, various assessments of reflex function were made using the acoustic startle reflex and prepulse inhibition of startle. There were no effects on startle habituation or reflex modification that could be attributed to the prenatal exposure to cocaine. Cocaine
Startle reaction
Pregnancy, animal
Prenatal exposure delayed effects
GENERAL METHOD
CLINICAL reports of neonates prenatally exposed to cocaine have described abnormal startle reactions, irritability, and unusual sensitivity to external stimuli (1,4). One component of this constellation, the startle reaction, can easily be measured in laboratory animals. Acute administration of cocaine in animals potentiates the acoustic startle reflex, and further analysis of this potentiation indicated that it is due to cocaine's action on dopamine-containing neurons (8). These catecholamine neurons begin to develop the functions necessary for chemical transmission during the last week of gestation in the rat (6), and the presence of cocaine at this point in development could produce permanent changes in these neurons or their contacts. Given the significance of dopamine-containing neurons to the modulation of the acoustic startle reflex, as well as the clinical reports of changes in the startle response of infants, the startle reflex could be a useful tool for the detection of persistent changes caused by prenatal exposure to cocaine. In a preliminary report, we found that oral administration of 60 mg/kg/day of cocaine to pregnant rats on gestation days 14-21 resulted in hyperreactivity, as measured by the acoustic startle response in 90-day-old, but not in 60-day-old offspring (12). The following experiments attempted to replicate and expand that preliminary finding. In addition to pregnant dams administered cocaine by gavage, a second set of rats that received cocaine by subcutaneous injection were examined. Unlike our preliminary study, a pair-feeding procedure was used, and various characteristics of the acoustic startle reflex were examined.
Breeding Nulliparous Long-Evans rats (Blue Spruce Farms, Altamont, NY) were adapted to the laboratory and its 12-h light/dark cycle for at least one week prior to mating. During that period, the animals were handled on three to five occasions. Females were placed individually with males overnight and the bedding checked the following morning for evidence of a sperm plug. Females in cages showing evidence of successful mating were transferred to a separate nursery and housed in clear plastic cages with hardwood bedding. The median weight of the animals entering the nursery [gestational day (GD) 1] was 273 g with a range of 237 g to 310 g. Parturition usually occurred during the night of GD 22. The day of birth was designated postnatal day (PD) 0, and litters were weighed and randomly culled to eight pups on PD 1, maintaining an equal number of males and females when possible. Pups were weaned on PD 21 and housed in metal hanging cages.
Prenatal Treatment Groups Two different procedures for administering cocaine were used in these experiments. However, in each case, as pregnant animals were identified they were assigned to one of three groups: one group (either C60 or C40) received cocaine; one control
~Requests for reprints should be addressed to John Foss, Argus Research Laboratories, 905 Sheehy Drive, Horsham, PA 19044.
541
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FOSS AND RILEY
group (PF) was administered the vehicle and pair-fed with the group receiving cocaine; and another control group (UT) was simply weighed along with the others. Differences in the route of administration, dosage, and duration of exposure defined two complete sets of animals. Oral administration. Rats in this set were obtained over a three-week period. Dams in the C60-G (G = gavage) group received 60 mg/kg of cocaine hydrochloride orally by gavage dally from GD 14 through GD 21. The drug was dissolved in distilled water and administered in a volume of 4.5 ml/kg. The dosage was the same as that used in the preliminary study and was chosen because it produced a slightly smaller weight gain in the surviving dams, but no change in the body weights or survival of their offspring. Based upon preliminary data, the administration period was limited to the one-hour period between 0930 and 1030. The PF-G group was pair-fed to the C60-G group and was intubated with distilled water. Throughout the dosage period, the food intake of each animal in the PF-G group was matched to a member of the C60-G group at least one day further along in gestation. The animals were fed the normal diet in meal form (Agway RMH3200) using metal feeders (Lab Products, H1306A) raised above the cage floor by a bracket. The meal was introduced as an alternative source of food along with the pelleted form of the diet after the rats were placed in the nursery and became the only food source after several days. During the dosing period, the food was removed at the time the animals were intubated and returned in the late afternoon when the cocainetreated rats became less active. The food allotment for the control animal was calculated by multiplying its weight by the ratio of food intake to body weight for the matched animal on the same gestational day. Subcutaneous administration. Rats in this set were obtained over a seven-week period. The dams in the C40-S (S = subcutaneous) group received 40 mg/kg of cocaine hydrochloride by subcutaneous injection on GD 8-GD 21. In other studies (14), that dosage produced plasma levels of over 1000 ng/ml without fatalities. The drug was dissolved in saline and given in a volume of 3 ml/kg; administration occurred between 1000 and 1100 hours daily. The PF-S group received the saline vehicle at the same time, and the food intake of these rats was matched to animals in the C40-S group during the dosage period.
Startle Reflex Apparatus The startle apparatus (San Diego Instruments) consisted of three separate chambers each containing an acrylic cylinder (17.5 cm long and 8.8 cm in diameter), that housed the animal. A transducer was coupled to this cylinder. A noise generator and tweeter in each chamber produced the stimuli, and a microcomputer controlled the timing and intensity of the stimuli and recorded the peak response from each chamber in the 75-ms period following a stimulus. Sound pressure levels were measured with a Bruel and Kjaer sound level meter (Model 2230 with the 0.5 inch 4155 micro~ phone) using the A-weighted scale with the integrating mode for background noise and the peak mode for eliciting stimuli. The ambient level of the chambers was 66 dB with the ventilation fans running. The eliciting stimuli were noise bursts, 15 ms in duration, with instantaneous rise-decay times. The measured peak levels were within 4 dB of the nominal values of 100, 115, or 130 dB. RESULTS Two of the 29 pregnant dams treated with 60 mg/kg cocaine died during the dosage period. These rats showed evidence of
TABLE 1 MATERNALANDOFFSPRINGCHARACTERISTICS: ORALADMINISTRATION Group LIT Number 10 Maternal Weight Gain (g) GD 14-21 71.5 (5.0) GD 1-21 144.7 (5.9) Number of Live 11.8 (0.6) Offspring (PD 1) Offspring Body Weight (g) Female PD 1 7.4 (0.2) PD 21 50.7 (1.4) Male PD 1 7.9 (0.1) PD 21 52.9 (1.5)
PF-G 11
C60-G 13
45.5 (3.4)* 120.2 (5.4)* 13.5 (0.5)
51.9 (2.2)* 124.5 (4.1)* 12.6 (0.5)
6.8 (0.2)* 49.5 (1.5)
7.2 (0.•) 50.9 (1.2)
7.2 (0.2)* 52.0 (1.7)
7.7 (0.2) 52.0 (1.3)
Data are the mean (SE). *p<0.05, one-sided, Dunnen's test with comparison to UT mean. seizures. Table 1 describes the characteristics of those dams with litters that were used for the behavioral experiments. The prenatal treatment was a significant factor for the weight gain of the dams during the dosage period, F(2,31)--13.8, p<0.001, and from GD 1 through GD 21, F(2,31)=6.22, p<0.01. Both the C60-G group and the PF-G group had smaller weight gains than the UT group. The number of offspring did not differ among the groups, and the weight of the C60-G offspring was not different from the offspring of untreated rats; however, the pups in the PF-G group weighed significantly less than animals in the untreated group on Day 1. These differences were not apparent at Day 21. Of the 49 pregnant dams subcutaneously injected with 40 mg/kg of cocaine daily from GD 8-21, only one rat died during the dosing period. The cocaine did produce inflammation and necrosis at the injection site, a phenomenon documented by others (2), so that the site of injection had to be moved daily. Table 2 describes the characteristics of those dams with litters used in the behavioral experiment. The prenatal treatment was again a significant factor in the weight gain during the dosage period, F(2,41)=13.7, p<0.001, and from GD 1 through GD 21, F(2,39) = 8.3, p<0.001, with the untreated group gaining more weight than both of the injected groups. There was no indication of differential emhryotoxicity or perinatal lethality between the groups. Body weights of the offspring, measured in the early postnatal period and one week after testing, were also not different. BEHAVIORAL STUDIES EXPERIMENT1: REFLEXELICITATION--ORALADMINISTRATION A simple test for changes in the elicitation of the acoustic startle reflex would examine responses to a single intensity of an eliciting stimulus. However, changes in reactivity may not be apparent at all intensities (3), so the addition of more conditions in the design can improve the sensitivity of the measure. In addition, the observation of an increase in response amplitude with stimulus intensity can verify the integrity of the basic reflex pathway.
PRENATAL COCAINE AND ACOUSTIC STARTLE
543
TABLE 2
UT PF-G C60-G
1200
MATERNAL AND OFFSPRING CHARACTERISTICS: SC ADMINISTRATION
Group UT
PF-S
Number 14 Maternal Weight Gain (g) GD 8-21 116.9 (4.6) GD 1-21 153.5 (5.5) Number of Live 12.1 (0.7) Offspring (PD 1) Offspring Body Weight (g) Female PD 1 7.0 PD 21 52.0 Male PD 1 7.3 PD 21 53.8
15 82.8 (6.2)* 125.5 (6.4)* 13.3 (0.4)
~"
lOOO
,oo
C40-S 15 84.4 (4.3)* 123.9 (5.0)* 12.7 (0.4)
i°
n-
(0.2) (1.0)
6.9 (0.1) 54.3 (1.0)
7.0 (0.1) 54.2 (1.3)
(0.2) (1.1)
7.3 (0.1) 56.6 (0.9)
7.3 (0.1) 56.6 (1.2)
Data are the mean (SE). *.o<0.05, one-sided, Dunnett's test with comparison to UT mean.
METHOD
Subjects A male and female were selected from 11 C60-G, 11 PF-G, and 10 UT litters. Half the litters from each prenatal treatment group were tested at 110 days and the other half at 130 days of age; the latter had been tested earlier in an habituation experiment described later in this paper.
Reflex Elicitation Design A rat was placed in a startle chamber and left undisturbed for 15 min. It then heard a series of six 115 dB stimuli. This adaptation condition was intended to isolate the large amplitude, but highly variable responses that characterize early trials. A series of 36 trials followed, in which each of three stimulus intensities (100, 115, and 130 dB) was presented 12 times in random order. The average intertrial interval was 30 s for both the adaptation series and the intensity series. The litters from each prenatal treatment group were divided among the different test chambers and that variable was used as a blocking factor in the analysis. Separate analyses were conducted for the initial adaptation trials and the subsequent intensity series. The scores used in the analysis were the averages of the peak responses for the first 6 trials and the 12 trials at each intensity. These scores were analyzed using the univariate procedures in the 4V program of the BMDP statistical package (9). Prenatal treatment and sex were between-subject factors; chamber was a blocking factor; and stimulus intensity was a within-subject factor. The probabilities for tests involving repeated measures were reduced by the Geisser/Greenhouse adjustment (15). Gabriel's (13) extension of Scheffe's test was used for post hoc analyses with p set at 0.05. RESULTS
Startle Response These data are presented in Fig. 1. Responses from males were larger than those of females [F(1,16)=9.96, p<0.01 for
A
115
100
115
130
Stimulus Sound Level (db) FIG. 1. Average startle responses (and SE) for the initial adaptation trials and 12 subsequent trials at each of three stimulus intensities for adult animals prenatally exposed to cocaine (C60-G) and their controls. The data are collapsed over two age groups with different experience.
the adaptation series and F(1,39)= 14.18, p<0.001 for the intensity series], but the effects did not interact with prenatal exposure. The older rats with previous experience in the testing situation also responded less than the younger, naive rats IF(l,16) = 5.05, p < 0 . 0 5 for the adaptation series and F ( 1 , 3 9 ) = 6 . 9 6 , p<0.025 for the intensity series], but again the effects did not interact with prenatal exposure. The effect of prenatal treatment was statistically significant for the intensity series, F(2,39)= 3.45, p<0.05, but not for the adaptation trials (p>0.35). However, none of the paired comparisons were statistically significant. These data differed from those obtained in our preliminary experiment (12) in that the C60-G group did not demonstrate elevated-startle responses; instead, both the C60-G and PF-G groups had smaller responses. EXPERIMENT2: REFLEXELIC1TATION-SUBCUTANEOUSADMINISTRATION The failure to observe an increase in the amplitude of the startle response following prenatal exposure prompted us to assess the subcutaneous route of exposure. In this experiment, dams were administered 40 mg/kg subcutaneously between GD 8 and GD 21 to increase the exposure of the fetuses to cocaine without increasing maternal toxicity. METHOD
Subjects A male and female were selected from 15 C40-S, 15 PF-S, and 14 UT litters. The rats were between 80 and 82 days of age at the start of testing.
Reflex Elicitation Design The experimental design was similar to that described above with two alterations. Only the 115 and 130 dB stimuli were presented, the former in both the adaptation series and the intensity series. In addition, the rats were tested for three sessions and were placed in a different chamber for each session.
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FOSS AND RILEY
,--
[11]UT PF-S == C 4 0 - 8
1200 1000
±
1000 Session 1 ¢0 tO CL mr .N o5 E O Z
0 A 115
115
Session 2
8OO
6OO
•
--
UT
400 "4 ,'Y"'"..
2OO
130
Stimulus Sound Level (clb) 0
FIG. 2. Average startle response (and SE) for the initial adaptation trials and 12 subsequent trials at two stimulus intensities for adult animals prenatally exposed to cocaine (C40-S) and their controls.
RESULTS
Responses declined over the three test sessions for the intensity series, F(2,164)= 167.42, p<0.001, but not for the adaptation series (p>0.80), but this factor, as well as gender, did not interact with prenatal exposure (see Fig. 2). Although the group exposed to cocaine did respond slightly more in the adaptation and intensity series, the effect of prenatal treatment was not significant for either the adaptation series (p>0.35) or the intensity series (p>0.30). EXPERIMENT
3: R E F L E X
HABITUATION
Apparent changes in reactivity may be due to a change in the subject's response to repeated stimuli; for example, an enhancement of the habituation process could lower the overall score for reactivity. However, the reflex elicitation design, with a separate adaptation series, variable intensities and intertrial intervals, and the different levels of experience in the animals, did not provide a straightforward means of examining habituation, so the following experiment was conducted. METHOD
Subjects A male and female were selected for testing at 115 days of age from 11 C60-G, 11 PF-G, and 10 UT litters. The rats had no previous experience in the acoustic startle apparatus.
i
i
i
i
~
i
~
1
5
10
15
1
5
10
Block
(10 trials)
i__
15
FIG. 3. Habituation of the acoustic startle response within a session and across two daily sessions of 150 trials for animals prenatally exposed to cocaine (C60-G). The data are the averages of normalized responses which in turn were computed from the averages of responses in 10 trial blocks.
by the grand sum for the animal and then multiplying by 10000. This procedure corrected the scores for variations in reactivity between individual rats, but retained the differences between blocks of trials and sessions which were the focus of this experimeut. Responses declined between sessions, F ( I , 6 0 ) = 14.65, p<0.001, and there was a more rapid decline to asymptote in the second session, F(14,840)=4.41, p<0.001. However, the interactions of these effects with prenatal treatment were not statisticaUy significant, so there was no evidence that habituation was affected by prenatal exposure to cocaine (see Fig. 3). EXPERIMENT
4: REFLEX
MODIFICATION
The reflex link between an eliciting stimulus and the startle response can be modulated by concurrent stimuli (16). The effect of the concurrent stimuli is often a reduction in response amplitude and has been labelled "prepulse inhibition." Reflex modification can be used to assess an animal's sensitivity to stimuli, and was employed in this study to examine whether the hypersensitivity reported in the human infant could be detected in adult rats exposed prenatally to cocaine. METHOD
Reflex Habituation Design
Subjects
A rat was placed in a test chamber and left undisturbed for 30 rain. It then heard a series of 150 eliciting stimuli, separated by 20-s intervals, at an intensity of 115 dB. The session was repeated in the same chamber on the following day. The averages of responses in 10 trial blocks were the basic scores for the analysis.
A male and female rat between 147 and 148 days of age were selected for testing from 11 C60-G, 11 PF-G, and 10 UT litters. The rats had been tested in previous experiments described in this paper.
RESULTS
Startle Response Habituation The scores for the 30 blocks of trials were in-st summed to examine overall group differences, which were not significant (p>0.40). The data were then normalized by dividing each score
Reflex Modification Design A rat was placed in the apparatus and left undisturbed for 15 min. It then received a series of six adaptation trials with 115 dB eliciting stimuli. These were followed by series of 36 trials divided between three conditions: a 115 dB eliciting stimulus alone (baseline) or 60 ms following 60 dB and 70 dB bursts of noise 20 ms in duration. The trials were separated by an aver-
PRENATAL COCAINE AND ACOUSTIC STARTLE
545
1000
and larger effects of the prepulse, gender did not interact with prenatal treatment.
8oo
I
6°° i
11:
DISCUSSION
lot
m m eeo-a
0 A
Beseline
60 db 70 db PrepuCe
CondUon FIG. 4. Modification of tlie acoustic startle response by preliminary stimuli ("Prepulses") of 60 and 70 dB for adult rats prenatally exposed to cocaine (cr0-G) and their controls. Data are the averages of responses in the adaptation trials and the subsequent conditions with only the eliciting stimulus ("Baseline") or with the eliciting stimulus preceded by a noise burst with a sound level of 60 or 70 dB.
age of 30 s. The basic scores were the averages of the responses in each condition, with the differences between the baseline condition and the two prepulse conditions used for the analysis of reflex modification. Animals from the treatment groups were distributed to the different chambers and this variable was used as a blocking factor. RESULTS The scores for the adaptation and baseline trials (Fig. 4) were first examined for differences in reactivity. In both conditions, the C60-G and PF-G groups had lower responses than the UT animals, although these effects were not significant for the adaptation series (/7>0.25), they were significant for the baseline condition, F(2,40) = 3.38, p < 0 . 0 5 . The differences between the baseline score and each of the prepulse conditions were also smaller for the gavage groups, and were reflected in the significant effect of prenatal treatment, F(2,40)=4.61, p<0.05. Although gender was a significant factor, with males demonstrating larger responses in both the adaptation and baseline conditions
The startle response would appear to be an appropriate behavior for examining the consequences of prenatal exposure to cocaine. Clinical studies have reported larger startle reactions in infants exposed to cocaine prenatally (1), and adult animals treated acutely with cocaine show facilitated startle responses (8). In addition, there is an abundant literature which shows that alterations in neurolransmitter processes can modulate the startle reaction (7). The experiments reported in this study, however, do not point to a strong or even consistent effect of prenatal cocaine exposure on subsequent startle reactions in adults. Startle reactions were not consistently affected by the prenatal exposure, showing increases in one experiment and decreases or no effect in others. The results are not due to intrinsic unreliability in the measure since characteristics of the reflex--the increase in amplitude with increases in stimulus intensity, the habituation of responses within and across sessions, and the reduction in amplitude in conditions with prepulse stimuli--were all obvious in these experiments. We had also thought that the hypersensitivity reported in clinical descriptions of exposed infants might be detected in animal models using a reflex modification design. Again, the resuits did not support our conjecture. If there was any effect, it was that the extraneous stimuli, or "prepulses," were less effective in the intubated groups. The dosages of cocaine and routes of administration used in these experiments have also been used in other laboratories (10,14). Cocaine produced modest maternal toxicity among the surviving dams. There was a significant decrease in the amount of weight gained from GD I through GD 21, approximately 14% for the oral dosage and 19% for the subcutaneous dosage. There were no signs of increased embryotoxicity or perinatal death, and the weights of the offspring were unaffected. These results agree with other studies that have found that cocaine has a low potential for producing morphological teratogenesis (5,11). In conclusion, these experiments do not support the hypothesis that prenatal cocaine exposure to moderate levels of cocaine in the rat will produce persistent changes in the acoustic startle reflex or its modulation by concurrent stimuli. ACKNOWLEDGEMENTS The authors want to thank Rebecca Butt and Wendy Gagnon for their technical assistance throughout this study. This work was supported in part by grant DA04275 to E.P.R. from NIDA and a Research Scientist Development Award to E.P.R. from NIAAA (AA00077). These experiments were conducted at SUNY-Albany.
REFERENCES
1. Anday, E. K.; Cohen, M. E.; Kelly, N. E.; Le'itner, D. S. Effect of in-utero cocaine exposure on startle and its modification. Dev. Pharmacol. Ther. 12:137-145; 1989. 2. Brnckner, J. V.; Jiang, W. D.; Ho, B. T.; Levy, B. M. Histopathological evaluation of cocaine-induced skin lesions ha the rat. J. Cutan. Pathol. 9:83-95; 1982. 3. Campo, A.; del Cerro, M.; Foss, J. A.; Ison, J. R.; Orr, J. L.; Warren, P. H.; Monjan, A. A. Impairment in auditory and visual function follows periuatal viral infection in the rat. Int. J. Neurosei. 27:85-90; 1985. 4. Chasnoff, I. J.; Burns, K. A.; Bums, W. T. Cocaine use in pregnancy: Perinatal morbidity and mortality. Neurotoxicol. Teratol. 9:291-293; 1987. 5. Church, M. W.; Dintcheff, B. A.; Gessner, P. K. Dose-dependent consequences of cocaine on pregnancy outcome in the Long-Evans rat. Neumtoxicol. Teratol. 10:51-58; 1988.
6. Coyle, J. T. Biochemical aspects of neurotransmission in the developing brain. Int. Rev. Neurobiol. 20:65-103; 1977. 7. Davis, M. The mammalian startle response. In: Eaton, R. C., ed. Neural mechanisms of startle behavior. New York: Plenum Press; 1984:287-351. 8. Davis, M. Cocaine: Excitatory effects on sensorimotor reactivity measured with acoustic startle. Psychopharmaeology (Berlin) 86:3136; 1985. 9. Dixon, W. J. BMDP statistical software manual. Berkeley: University of California Press; 1985. 10. Dow-Edward, D.; Fico, T. A.; Osman, M; Gamagaris, Z. Comparison of oral and subcutaneous routes of cocaine administration on behavior, plasma drug concentration, and toxicity in female rats. Pharmacol. Biochem. Behav. 33:167-173; 1989. 11. Fantel, A. G.; MacPhail, B. J. The teratogenicity of cocaine. Neurotoxicol. Teratol. 26:17-19; 1982.
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12. Foss, J. A.; Riley, E. P. Behavioral evaluation of animals exposed prenatally to cocaine. Teratology 37:517; 1988. 13. Gabriel, K. R. A procedure for testing the homogeneity of all sets of means in analysis of variance. Biometrics 20:459-477; 1964. 14. Spear, L. P.; Frambes, N. A.; Kirstein, C. L. Fetal and maternal brain and plasma levels of cocaine and benzoyleegonine following chronic subcutaneous administration of cocaine during gestation in
FOSS AND RILEY
rats. Psychopharmacology (Berlin) 97:427-431; 1989. 15. Vitaliano, P. P.; Prinz, P.; Vitiello, M. V.; Olshan, A.; Roehrs, T. A. On the use of repeated measures designs in psychopharmacology. Psychopharmacology (Berlin) 72:247-249; 1981. 16. Wecker, J. R.; Ison, J. R.; Foss, J. A. Reflex modification as a test for sensory function. Neurotoxicol. Teratol. 7:733-738; 1985.
0892-0362/91 $3.00 + .00
Elicitation and Modification of the Acoustic Startle Reflex in Animals Prenatally Exposed to Cocaine J O H N A. F O S S 1 A N D E D W A R D P. R I L E Y *
*Department of Psychology, San Diego State University, San Diego, CA 92182 Received 26 July 1989 FOSS, J. A. AND E. P. RILEY. Elicitation and modification of the acoustic startle reflex in animals prenatally exposed to cocaine. NEUROTOXICOL TERATOL 13(5) 541-546, 1991.--In separate experiments, pregnant Long-Evans rats were administered cocaine orally (60 mg/kg/day) on gestation days 14-21, or subcutaneously (40 mg/kg/day) on gestational days 8-21. For each route of administration, a vehicle control group was pair-fed to the group administered cocaine and another control group was left untreated. Throughout pregnancy, the dams that received cocaine gained approximately 15% less weight than the untreated controls, but none of the dosage procedures affected the size of the fitters, or the weight and growth of the offspring. When the offspring reached adulthood, various assessments of reflex function were made using the acoustic startle reflex and prepulse inhibition of startle. There were no effects on startle habituation or reflex modification that could be attributed to the prenatal exposure to cocaine. Cocaine
Startle reaction
Pregnancy, animal
Prenatal exposure delayed effects
GENERAL METHOD
CLINICAL reports of neonates prenatally exposed to cocaine have described abnormal startle reactions, irritability, and unusual sensitivity to external stimuli (1,4). One component of this constellation, the startle reaction, can easily be measured in laboratory animals. Acute administration of cocaine in animals potentiates the acoustic startle reflex, and further analysis of this potentiation indicated that it is due to cocaine's action on dopamine-containing neurons (8). These catecholamine neurons begin to develop the functions necessary for chemical transmission during the last week of gestation in the rat (6), and the presence of cocaine at this point in development could produce permanent changes in these neurons or their contacts. Given the significance of dopamine-containing neurons to the modulation of the acoustic startle reflex, as well as the clinical reports of changes in the startle response of infants, the startle reflex could be a useful tool for the detection of persistent changes caused by prenatal exposure to cocaine. In a preliminary report, we found that oral administration of 60 mg/kg/day of cocaine to pregnant rats on gestation days 14-21 resulted in hyperreactivity, as measured by the acoustic startle response in 90-day-old, but not in 60-day-old offspring (12). The following experiments attempted to replicate and expand that preliminary finding. In addition to pregnant dams administered cocaine by gavage, a second set of rats that received cocaine by subcutaneous injection were examined. Unlike our preliminary study, a pair-feeding procedure was used, and various characteristics of the acoustic startle reflex were examined.
Breeding Nulliparous Long-Evans rats (Blue Spruce Farms, Altamont, NY) were adapted to the laboratory and its 12-h light/dark cycle for at least one week prior to mating. During that period, the animals were handled on three to five occasions. Females were placed individually with males overnight and the bedding checked the following morning for evidence of a sperm plug. Females in cages showing evidence of successful mating were transferred to a separate nursery and housed in clear plastic cages with hardwood bedding. The median weight of the animals entering the nursery [gestational day (GD) 1] was 273 g with a range of 237 g to 310 g. Parturition usually occurred during the night of GD 22. The day of birth was designated postnatal day (PD) 0, and litters were weighed and randomly culled to eight pups on PD 1, maintaining an equal number of males and females when possible. Pups were weaned on PD 21 and housed in metal hanging cages.
Prenatal Treatment Groups Two different procedures for administering cocaine were used in these experiments. However, in each case, as pregnant animals were identified they were assigned to one of three groups: one group (either C60 or C40) received cocaine; one control
~Requests for reprints should be addressed to John Foss, Argus Research Laboratories, 905 Sheehy Drive, Horsham, PA 19044.
541
542
FOSS AND RILEY
group (PF) was administered the vehicle and pair-fed with the group receiving cocaine; and another control group (UT) was simply weighed along with the others. Differences in the route of administration, dosage, and duration of exposure defined two complete sets of animals. Oral administration. Rats in this set were obtained over a three-week period. Dams in the C60-G (G = gavage) group received 60 mg/kg of cocaine hydrochloride orally by gavage dally from GD 14 through GD 21. The drug was dissolved in distilled water and administered in a volume of 4.5 ml/kg. The dosage was the same as that used in the preliminary study and was chosen because it produced a slightly smaller weight gain in the surviving dams, but no change in the body weights or survival of their offspring. Based upon preliminary data, the administration period was limited to the one-hour period between 0930 and 1030. The PF-G group was pair-fed to the C60-G group and was intubated with distilled water. Throughout the dosage period, the food intake of each animal in the PF-G group was matched to a member of the C60-G group at least one day further along in gestation. The animals were fed the normal diet in meal form (Agway RMH3200) using metal feeders (Lab Products, H1306A) raised above the cage floor by a bracket. The meal was introduced as an alternative source of food along with the pelleted form of the diet after the rats were placed in the nursery and became the only food source after several days. During the dosing period, the food was removed at the time the animals were intubated and returned in the late afternoon when the cocainetreated rats became less active. The food allotment for the control animal was calculated by multiplying its weight by the ratio of food intake to body weight for the matched animal on the same gestational day. Subcutaneous administration. Rats in this set were obtained over a seven-week period. The dams in the C40-S (S = subcutaneous) group received 40 mg/kg of cocaine hydrochloride by subcutaneous injection on GD 8-GD 21. In other studies (14), that dosage produced plasma levels of over 1000 ng/ml without fatalities. The drug was dissolved in saline and given in a volume of 3 ml/kg; administration occurred between 1000 and 1100 hours daily. The PF-S group received the saline vehicle at the same time, and the food intake of these rats was matched to animals in the C40-S group during the dosage period.
Startle Reflex Apparatus The startle apparatus (San Diego Instruments) consisted of three separate chambers each containing an acrylic cylinder (17.5 cm long and 8.8 cm in diameter), that housed the animal. A transducer was coupled to this cylinder. A noise generator and tweeter in each chamber produced the stimuli, and a microcomputer controlled the timing and intensity of the stimuli and recorded the peak response from each chamber in the 75-ms period following a stimulus. Sound pressure levels were measured with a Bruel and Kjaer sound level meter (Model 2230 with the 0.5 inch 4155 micro~ phone) using the A-weighted scale with the integrating mode for background noise and the peak mode for eliciting stimuli. The ambient level of the chambers was 66 dB with the ventilation fans running. The eliciting stimuli were noise bursts, 15 ms in duration, with instantaneous rise-decay times. The measured peak levels were within 4 dB of the nominal values of 100, 115, or 130 dB. RESULTS Two of the 29 pregnant dams treated with 60 mg/kg cocaine died during the dosage period. These rats showed evidence of
TABLE 1 MATERNALANDOFFSPRINGCHARACTERISTICS: ORALADMINISTRATION Group LIT Number 10 Maternal Weight Gain (g) GD 14-21 71.5 (5.0) GD 1-21 144.7 (5.9) Number of Live 11.8 (0.6) Offspring (PD 1) Offspring Body Weight (g) Female PD 1 7.4 (0.2) PD 21 50.7 (1.4) Male PD 1 7.9 (0.1) PD 21 52.9 (1.5)
PF-G 11
C60-G 13
45.5 (3.4)* 120.2 (5.4)* 13.5 (0.5)
51.9 (2.2)* 124.5 (4.1)* 12.6 (0.5)
6.8 (0.2)* 49.5 (1.5)
7.2 (0.•) 50.9 (1.2)
7.2 (0.2)* 52.0 (1.7)
7.7 (0.2) 52.0 (1.3)
Data are the mean (SE). *p<0.05, one-sided, Dunnen's test with comparison to UT mean. seizures. Table 1 describes the characteristics of those dams with litters that were used for the behavioral experiments. The prenatal treatment was a significant factor for the weight gain of the dams during the dosage period, F(2,31)--13.8, p<0.001, and from GD 1 through GD 21, F(2,31)=6.22, p<0.01. Both the C60-G group and the PF-G group had smaller weight gains than the UT group. The number of offspring did not differ among the groups, and the weight of the C60-G offspring was not different from the offspring of untreated rats; however, the pups in the PF-G group weighed significantly less than animals in the untreated group on Day 1. These differences were not apparent at Day 21. Of the 49 pregnant dams subcutaneously injected with 40 mg/kg of cocaine daily from GD 8-21, only one rat died during the dosing period. The cocaine did produce inflammation and necrosis at the injection site, a phenomenon documented by others (2), so that the site of injection had to be moved daily. Table 2 describes the characteristics of those dams with litters used in the behavioral experiment. The prenatal treatment was again a significant factor in the weight gain during the dosage period, F(2,41)=13.7, p<0.001, and from GD 1 through GD 21, F(2,39) = 8.3, p<0.001, with the untreated group gaining more weight than both of the injected groups. There was no indication of differential emhryotoxicity or perinatal lethality between the groups. Body weights of the offspring, measured in the early postnatal period and one week after testing, were also not different. BEHAVIORAL STUDIES EXPERIMENT1: REFLEXELICITATION--ORALADMINISTRATION A simple test for changes in the elicitation of the acoustic startle reflex would examine responses to a single intensity of an eliciting stimulus. However, changes in reactivity may not be apparent at all intensities (3), so the addition of more conditions in the design can improve the sensitivity of the measure. In addition, the observation of an increase in response amplitude with stimulus intensity can verify the integrity of the basic reflex pathway.
PRENATAL COCAINE AND ACOUSTIC STARTLE
543
TABLE 2
UT PF-G C60-G
1200
MATERNAL AND OFFSPRING CHARACTERISTICS: SC ADMINISTRATION
Group UT
PF-S
Number 14 Maternal Weight Gain (g) GD 8-21 116.9 (4.6) GD 1-21 153.5 (5.5) Number of Live 12.1 (0.7) Offspring (PD 1) Offspring Body Weight (g) Female PD 1 7.0 PD 21 52.0 Male PD 1 7.3 PD 21 53.8
15 82.8 (6.2)* 125.5 (6.4)* 13.3 (0.4)
~"
lOOO
,oo
C40-S 15 84.4 (4.3)* 123.9 (5.0)* 12.7 (0.4)
i°
n-
(0.2) (1.0)
6.9 (0.1) 54.3 (1.0)
7.0 (0.1) 54.2 (1.3)
(0.2) (1.1)
7.3 (0.1) 56.6 (0.9)
7.3 (0.1) 56.6 (1.2)
Data are the mean (SE). *.o<0.05, one-sided, Dunnett's test with comparison to UT mean.
METHOD
Subjects A male and female were selected from 11 C60-G, 11 PF-G, and 10 UT litters. Half the litters from each prenatal treatment group were tested at 110 days and the other half at 130 days of age; the latter had been tested earlier in an habituation experiment described later in this paper.
Reflex Elicitation Design A rat was placed in a startle chamber and left undisturbed for 15 min. It then heard a series of six 115 dB stimuli. This adaptation condition was intended to isolate the large amplitude, but highly variable responses that characterize early trials. A series of 36 trials followed, in which each of three stimulus intensities (100, 115, and 130 dB) was presented 12 times in random order. The average intertrial interval was 30 s for both the adaptation series and the intensity series. The litters from each prenatal treatment group were divided among the different test chambers and that variable was used as a blocking factor in the analysis. Separate analyses were conducted for the initial adaptation trials and the subsequent intensity series. The scores used in the analysis were the averages of the peak responses for the first 6 trials and the 12 trials at each intensity. These scores were analyzed using the univariate procedures in the 4V program of the BMDP statistical package (9). Prenatal treatment and sex were between-subject factors; chamber was a blocking factor; and stimulus intensity was a within-subject factor. The probabilities for tests involving repeated measures were reduced by the Geisser/Greenhouse adjustment (15). Gabriel's (13) extension of Scheffe's test was used for post hoc analyses with p set at 0.05. RESULTS
Startle Response These data are presented in Fig. 1. Responses from males were larger than those of females [F(1,16)=9.96, p<0.01 for
A
115
100
115
130
Stimulus Sound Level (db) FIG. 1. Average startle responses (and SE) for the initial adaptation trials and 12 subsequent trials at each of three stimulus intensities for adult animals prenatally exposed to cocaine (C60-G) and their controls. The data are collapsed over two age groups with different experience.
the adaptation series and F(1,39)= 14.18, p<0.001 for the intensity series], but the effects did not interact with prenatal exposure. The older rats with previous experience in the testing situation also responded less than the younger, naive rats IF(l,16) = 5.05, p < 0 . 0 5 for the adaptation series and F ( 1 , 3 9 ) = 6 . 9 6 , p<0.025 for the intensity series], but again the effects did not interact with prenatal exposure. The effect of prenatal treatment was statistically significant for the intensity series, F(2,39)= 3.45, p<0.05, but not for the adaptation trials (p>0.35). However, none of the paired comparisons were statistically significant. These data differed from those obtained in our preliminary experiment (12) in that the C60-G group did not demonstrate elevated-startle responses; instead, both the C60-G and PF-G groups had smaller responses. EXPERIMENT2: REFLEXELIC1TATION-SUBCUTANEOUSADMINISTRATION The failure to observe an increase in the amplitude of the startle response following prenatal exposure prompted us to assess the subcutaneous route of exposure. In this experiment, dams were administered 40 mg/kg subcutaneously between GD 8 and GD 21 to increase the exposure of the fetuses to cocaine without increasing maternal toxicity. METHOD
Subjects A male and female were selected from 15 C40-S, 15 PF-S, and 14 UT litters. The rats were between 80 and 82 days of age at the start of testing.
Reflex Elicitation Design The experimental design was similar to that described above with two alterations. Only the 115 and 130 dB stimuli were presented, the former in both the adaptation series and the intensity series. In addition, the rats were tested for three sessions and were placed in a different chamber for each session.
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FOSS AND RILEY
,--
[11]UT PF-S == C 4 0 - 8
1200 1000
±
1000 Session 1 ¢0 tO CL mr .N o5 E O Z
0 A 115
115
Session 2
8OO
6OO
•
--
UT
400 "4 ,'Y"'"..
2OO
130
Stimulus Sound Level (clb) 0
FIG. 2. Average startle response (and SE) for the initial adaptation trials and 12 subsequent trials at two stimulus intensities for adult animals prenatally exposed to cocaine (C40-S) and their controls.
RESULTS
Responses declined over the three test sessions for the intensity series, F(2,164)= 167.42, p<0.001, but not for the adaptation series (p>0.80), but this factor, as well as gender, did not interact with prenatal exposure (see Fig. 2). Although the group exposed to cocaine did respond slightly more in the adaptation and intensity series, the effect of prenatal treatment was not significant for either the adaptation series (p>0.35) or the intensity series (p>0.30). EXPERIMENT
3: R E F L E X
HABITUATION
Apparent changes in reactivity may be due to a change in the subject's response to repeated stimuli; for example, an enhancement of the habituation process could lower the overall score for reactivity. However, the reflex elicitation design, with a separate adaptation series, variable intensities and intertrial intervals, and the different levels of experience in the animals, did not provide a straightforward means of examining habituation, so the following experiment was conducted. METHOD
Subjects A male and female were selected for testing at 115 days of age from 11 C60-G, 11 PF-G, and 10 UT litters. The rats had no previous experience in the acoustic startle apparatus.
i
i
i
i
~
i
~
1
5
10
15
1
5
10
Block
(10 trials)
i__
15
FIG. 3. Habituation of the acoustic startle response within a session and across two daily sessions of 150 trials for animals prenatally exposed to cocaine (C60-G). The data are the averages of normalized responses which in turn were computed from the averages of responses in 10 trial blocks.
by the grand sum for the animal and then multiplying by 10000. This procedure corrected the scores for variations in reactivity between individual rats, but retained the differences between blocks of trials and sessions which were the focus of this experimeut. Responses declined between sessions, F ( I , 6 0 ) = 14.65, p<0.001, and there was a more rapid decline to asymptote in the second session, F(14,840)=4.41, p<0.001. However, the interactions of these effects with prenatal treatment were not statisticaUy significant, so there was no evidence that habituation was affected by prenatal exposure to cocaine (see Fig. 3). EXPERIMENT
4: REFLEX
MODIFICATION
The reflex link between an eliciting stimulus and the startle response can be modulated by concurrent stimuli (16). The effect of the concurrent stimuli is often a reduction in response amplitude and has been labelled "prepulse inhibition." Reflex modification can be used to assess an animal's sensitivity to stimuli, and was employed in this study to examine whether the hypersensitivity reported in the human infant could be detected in adult rats exposed prenatally to cocaine. METHOD
Reflex Habituation Design
Subjects
A rat was placed in a test chamber and left undisturbed for 30 rain. It then heard a series of 150 eliciting stimuli, separated by 20-s intervals, at an intensity of 115 dB. The session was repeated in the same chamber on the following day. The averages of responses in 10 trial blocks were the basic scores for the analysis.
A male and female rat between 147 and 148 days of age were selected for testing from 11 C60-G, 11 PF-G, and 10 UT litters. The rats had been tested in previous experiments described in this paper.
RESULTS
Startle Response Habituation The scores for the 30 blocks of trials were in-st summed to examine overall group differences, which were not significant (p>0.40). The data were then normalized by dividing each score
Reflex Modification Design A rat was placed in the apparatus and left undisturbed for 15 min. It then received a series of six adaptation trials with 115 dB eliciting stimuli. These were followed by series of 36 trials divided between three conditions: a 115 dB eliciting stimulus alone (baseline) or 60 ms following 60 dB and 70 dB bursts of noise 20 ms in duration. The trials were separated by an aver-
PRENATAL COCAINE AND ACOUSTIC STARTLE
545
1000
and larger effects of the prepulse, gender did not interact with prenatal treatment.
8oo
I
6°° i
11:
DISCUSSION
lot
m m eeo-a
0 A
Beseline
60 db 70 db PrepuCe
CondUon FIG. 4. Modification of tlie acoustic startle response by preliminary stimuli ("Prepulses") of 60 and 70 dB for adult rats prenatally exposed to cocaine (cr0-G) and their controls. Data are the averages of responses in the adaptation trials and the subsequent conditions with only the eliciting stimulus ("Baseline") or with the eliciting stimulus preceded by a noise burst with a sound level of 60 or 70 dB.
age of 30 s. The basic scores were the averages of the responses in each condition, with the differences between the baseline condition and the two prepulse conditions used for the analysis of reflex modification. Animals from the treatment groups were distributed to the different chambers and this variable was used as a blocking factor. RESULTS The scores for the adaptation and baseline trials (Fig. 4) were first examined for differences in reactivity. In both conditions, the C60-G and PF-G groups had lower responses than the UT animals, although these effects were not significant for the adaptation series (/7>0.25), they were significant for the baseline condition, F(2,40) = 3.38, p < 0 . 0 5 . The differences between the baseline score and each of the prepulse conditions were also smaller for the gavage groups, and were reflected in the significant effect of prenatal treatment, F(2,40)=4.61, p<0.05. Although gender was a significant factor, with males demonstrating larger responses in both the adaptation and baseline conditions
The startle response would appear to be an appropriate behavior for examining the consequences of prenatal exposure to cocaine. Clinical studies have reported larger startle reactions in infants exposed to cocaine prenatally (1), and adult animals treated acutely with cocaine show facilitated startle responses (8). In addition, there is an abundant literature which shows that alterations in neurolransmitter processes can modulate the startle reaction (7). The experiments reported in this study, however, do not point to a strong or even consistent effect of prenatal cocaine exposure on subsequent startle reactions in adults. Startle reactions were not consistently affected by the prenatal exposure, showing increases in one experiment and decreases or no effect in others. The results are not due to intrinsic unreliability in the measure since characteristics of the reflex--the increase in amplitude with increases in stimulus intensity, the habituation of responses within and across sessions, and the reduction in amplitude in conditions with prepulse stimuli--were all obvious in these experiments. We had also thought that the hypersensitivity reported in clinical descriptions of exposed infants might be detected in animal models using a reflex modification design. Again, the resuits did not support our conjecture. If there was any effect, it was that the extraneous stimuli, or "prepulses," were less effective in the intubated groups. The dosages of cocaine and routes of administration used in these experiments have also been used in other laboratories (10,14). Cocaine produced modest maternal toxicity among the surviving dams. There was a significant decrease in the amount of weight gained from GD I through GD 21, approximately 14% for the oral dosage and 19% for the subcutaneous dosage. There were no signs of increased embryotoxicity or perinatal death, and the weights of the offspring were unaffected. These results agree with other studies that have found that cocaine has a low potential for producing morphological teratogenesis (5,11). In conclusion, these experiments do not support the hypothesis that prenatal cocaine exposure to moderate levels of cocaine in the rat will produce persistent changes in the acoustic startle reflex or its modulation by concurrent stimuli. ACKNOWLEDGEMENTS The authors want to thank Rebecca Butt and Wendy Gagnon for their technical assistance throughout this study. This work was supported in part by grant DA04275 to E.P.R. from NIDA and a Research Scientist Development Award to E.P.R. from NIAAA (AA00077). These experiments were conducted at SUNY-Albany.
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6. Coyle, J. T. Biochemical aspects of neurotransmission in the developing brain. Int. Rev. Neurobiol. 20:65-103; 1977. 7. Davis, M. The mammalian startle response. In: Eaton, R. C., ed. Neural mechanisms of startle behavior. New York: Plenum Press; 1984:287-351. 8. Davis, M. Cocaine: Excitatory effects on sensorimotor reactivity measured with acoustic startle. Psychopharmaeology (Berlin) 86:3136; 1985. 9. Dixon, W. J. BMDP statistical software manual. Berkeley: University of California Press; 1985. 10. Dow-Edward, D.; Fico, T. A.; Osman, M; Gamagaris, Z. Comparison of oral and subcutaneous routes of cocaine administration on behavior, plasma drug concentration, and toxicity in female rats. Pharmacol. Biochem. Behav. 33:167-173; 1989. 11. Fantel, A. G.; MacPhail, B. J. The teratogenicity of cocaine. Neurotoxicol. Teratol. 26:17-19; 1982.
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12. Foss, J. A.; Riley, E. P. Behavioral evaluation of animals exposed prenatally to cocaine. Teratology 37:517; 1988. 13. Gabriel, K. R. A procedure for testing the homogeneity of all sets of means in analysis of variance. Biometrics 20:459-477; 1964. 14. Spear, L. P.; Frambes, N. A.; Kirstein, C. L. Fetal and maternal brain and plasma levels of cocaine and benzoyleegonine following chronic subcutaneous administration of cocaine during gestation in
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rats. Psychopharmacology (Berlin) 97:427-431; 1989. 15. Vitaliano, P. P.; Prinz, P.; Vitiello, M. V.; Olshan, A.; Roehrs, T. A. On the use of repeated measures designs in psychopharmacology. Psychopharmacology (Berlin) 72:247-249; 1981. 16. Wecker, J. R.; Ison, J. R.; Foss, J. A. Reflex modification as a test for sensory function. Neurotoxicol. Teratol. 7:733-738; 1985.