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Prenatal exposure to benzodiazepine—I. Prenatal exposure to lorazepam in mice alters open-field activity and GABAA receptor function
Neuropharmocology Vol. 30, No. 1, pp. 53-58, Printed in Great Britain. All rights reserved
1991
0028-3908/91$3.00+ 0.00 Copyright 0 1991Pergamon Press plc
PRENATAL EXPOSURE TO BENZODIAZEPINE-I. PRENATAL EXPOSURE TO LORAZEPAM IN MICE ALTERS OPEN-FIELD ACTIVITY AND GABA, RECEPTOR FUNCTION S. CHESLEY,M. LUMPKIN, A. SCHATZKI, W. R. GALPERN, D. J. GREENBLATT,R. I. SHADERand L. G. MILLER* From the Division of Clinical Pharmacology, Departments of Psychiatry and Pharmacology, Tufts University School of Medicine and New England Medical Center, Boston (Accepted 17 July 1990)
Summary-Prenatal exposure to benzodiazepines may lead to developmental abnormalities in humans and animals. To assess the behavioral and neurochemical effects of such exposure, pregnant mice were treated with lorazepam, 2 mg/kg/day, from days 13-20 of gestation, and open-field activity was assessed in offspring at 3 and 6 weeks of age and the function of GABA, receptors at 6 weeks of age. Activity was increased in mice exposed to lorazepam, compared to untreated or vehicle-treated controls at 3 weeks, but was unchanged at 6 weeks. Muscimol-stimulated uptake of chloride was decreased in lorazepamtreated mice, compared to controls, with a decrease in maximum uptake but no change in the EC,, for muscimol. Concentrations of lorazepam in maternal plasma and brain showed a similar brain:plasma ratio as previously reported and concentrations in fetal brain were about 50% of maternal levels. Lorazepam persisted for 48 hours after birth in dams but not in the offspring. These results indicate persistent behavioral and neurochemical alterations after prenatal exposure to lorazepam. This model may be useful in assessing other effects of prenatal exposure to benzodiazepine. Key words-lorazepam,
prenatal exposure, GABA, chloride, benzodiazepine.
Benzodiazepines are the most commonly prescribed psychoactive drugs and among the most widely prescribed of any class of drugs (Greenblatt, Abernethy and Shader, 1983). In addition, both survey and anecdotal evidence indicate that benzodiazepines are sub,ject to abuse, often in combination with other drugs of abuse (Woods, Katz and Winger, 1987). Thus, a large number of women of childbearing age are exposed to benzodiazepines. As such, it is likely that. a substantial number of pregnant women are exposed, although precise data from the U.S. are not available. Data from a Swedish study indicate chronic use of benzodiazepines in 0.5-l% of pregnancies and this proportion may be greater in the U.S. (Laegreid, Olegard, Wahlstrom and Conradi, 1987). Initial reports of the teratogenecity of benzodiazepines were not confirmed (Safra and Oakley, 197 5; Rosenberg, Mitchell, Parsels, Pahayan, Louik and Shapiro, 1983). Subsequently, several reports indicated a “withdrawal” syndrome, occurring in infants exposed to benzodiazepines in utero, although follow-up data on these children are not available (Kanto, 1982; Scanlon, 1975). Recently, Laegreid et al. (1987) reported 7 cases of a “benzodiazepine
embryopathy” characterized by dysmorphism, growth restriction, and psychomotor and mental retardation. Although this series was not populationbased or controlled, it again raises the possibility of serious effects of prenatal exposure to benzodiazepines in humans. A substantial literature in animals indicates behavioral abnormalities in animals exposed to benzodiazepines prenatally (for reviews, see Kellogg, 1988; Tucker, 1985). In general, motor activity and exploratory behavior are reduced in exposed newborn animals and reflex development and operant responses may be altered in adult animals exposed in utero. A modest literature also exists concerning the neurochemical effects of prenatal exposure to benzodiazepines, although results concerning the binding of benzodiazepines are conflicting (e.g. Massotti, Alleva, Baalazs and Guidotti, 1980; Livezey, Marczynski and Isaac, 1986). In a chick model, alterations in binding and function at the y-aminobutyric acid (GABAA) receptor chloride channel were previously reported in late embryos and adult chickens exposed in ouo (Miller, Weill, Roy and Gaver, 1988b, Miller, Roy, Weill and Lopez, 1989). To assess the behavioral and neurochemical effects of prenatal exposure to benzodiazepines in a mammalian model, mice were exposed prenatally to lorazepam and open-field activity and GABA, receptor function were evaluated.
*To whom correspondence should be addressed at: New England Medical Center, 171 Harrison Ave. Boston, MA 02111, U.S.A. NP 3011-n
53
s.
54
CHFSLEY et
of age. Open-field activity was determined for a period of 5 min in a Digiscan activity monitor (Omnitech, Columbus, Ohio). The testing of groups was performed in random order.
METHODS
Materials
Female mice, 7-10 weeks of age, were obtained from Charles River Laboratories (Wilmington, Massachusetts) and maintained on a 12-hr light-dark cycle with food and water ad libitum. Estrus was assessed by vaginal smears and pregnancy was determined by weighing and palpation. Osmotic pumps were obtained from Alza (Palo Alto, California). Lorazepam was a gift from Wyeth Laboratories (Philadelphia, Pennsylvania) and [3”C11], specific activity 12.5 mCi/g, was obtained from New England Nuclear (Boston, Massachusetts). All other reagents were obtained from standard commercial sources.
Neurochemical testing
In three experiments, muscimol-stimulated uptake of chloride was determined in the cortex at 6 weeks of age. Uptake was assessed as previously described (Miller et al., 1988b; Miller et al., 1989). Briefly, cortical synaptoneurosomes were prepared as described. After centrifugation, the tissue was resuspended gently in assay buffer (145 mM NaCl, 6 mM KCl, 1 mM MgCl, and 10 mM HEPES) and incubated for 10min at 30°C. To 100~1 of membranes was added 100~1 of a solution containing [3”Cll] (0.2pCi/ml of assay buffer), with or without muscimol (final concentration, l-50 p M). Samples were vortexed immediately and after 6 set the incubation was terminated by addition of 3 ml ice-cold assay buffer containing picrotoxin, 6 PM and filtration on Whatman GFjC filters, using a Brandel M24 harvester. Filters were counted by conventional scintillation spectrometry. The amount of [36Cl-] bound to the filters in the absence of muscimol was subtracted from all values to eliminate non-GABA-related uptake. Three to five determinations were performed in duplicate or triplicate for each litter.
Administration of drug
At day 13 after mating, osmotic pumps containing lorazepam, 2 mg/kg/day, were implanted subcutaneously under brief ether anesthesia. Control mice received PEG 400 vehicle alone. The weight of the animal was assumed to be 40 g, based on mean weight in pregnancy in previous experiments. The pumps were removed under brief ether anesthesia on day 20. Fostering
Within 16 hr of delivery, the litters were culled to 8-10 mice and distributed as shown in Figure 1. Litters from all three exposure groups, control, vehicle and lorazepam-treated, were distributed to dams from the three exposure groups, resulting in 9 combinations of prenatal exposure and fostering. Mice were weaned at 3 weeks and housed in singlesex cages, 4-S per cage.
Benzodiazepine concentrations
In a separate experiment, dams were sacrificed at day 6 of administration of lorazepam and concentrations of lorazepam in plasma and cortex were determined in dams and in fetal forebrain. Similar determinations were performed two days after birth. Concentrations of lorazepam were determined by gas-liquid chromatography, as previously described (Greenblatt, Franke and Shader, 1978).
Behavioral testing
The mice were weighed weekly and, in three experiments, underwent open-field testing at 3 and 6 weeks 3 Weeks
Maternal •J
controt
Vehicle PUP exposure
Lorozepom
al.
exposure
6 Weeks
Control
Control
Vehicle
Lorazepam
PUP exposure
Fig. 1. Effects of prenatal exposure to lorazepam on open-field activity. Mice underwent a 5 min open-field trial at the designated ages. Pups were exposed to lorazepam or vehicle through the dam. There were no differences in mice with differing maternal exposures but similar exposure of pup at either time. At 3 weeks, lorazepam-exposed pups had increased activity compared to the other groups but no differences were observed at 6 weeks. Results are by litter, mean + SEM, n = 3 for each combination of pup and maternal exposure, n = 8-10 per litter.
.,,_ ._ ..,, Ill
Prenatal lorazepam exposure
55
40
Analysis of data
r
Data were analyzed by litter and within litters by sex. Comparisons between groups were performed using analysis of variance with Dunnett’s comparison. For uptake of chloride, the EC,, was determined by fitting a sigmoidal function, as previously described (Miller, Greenblatt, Paul and Shader, 1987a).
&
e
n
..’
,...“’
30
,:’
z \ d ;
..’
..’
20
,.;.p.
d
E
Behavioral testing
At 3 weeks of age, three separate litters each of unexposed (control) mice and vehicle-exposed mice had similar motor activity (Fig. 1). However, motor activity was significantly increased in three litters of mice exposed to lorazepam. Within exposure groups, there were no differences among the fostering conditions. Activity was similar in male and female mice (data not shown). At 6 weeks of age, activity was similar in all three exposure groups. There were no differences among the fostering conditions, and activity was similar in male and female mice. Nexrochemical testing
At 6 weeks of age, muscimol-stimulated uptake of chloride at 50 PM muscimol was similar in three separate litters, each of unexposed (control) and vehicle-exposed mice (Fig. 2). Uptake of chloride was significantly decreased in three litters of mice exposed to lorazepam. Within exposure groups, there were no differences among the fostering conditions. Represen-
Maternol
T
q
exposure
,Y~---------d
H R
.F
PUP exposure
/
b
RESULTS
:
10
z
s
-)
I
0
... ... . .
Control Vehicle
--A-
Lorozepam
-
I
I
I
I
IO
I
20
30
40
50
Muscimol
[~Ml
Fig. 3. Effects of prenatal exposure to lorazepam on muscimol-stimulated uptake of chloride. Pups were exposed to lorazepam or vehicle through the dam. Uptake was performed at 6 weeks of age. Data are from a representative experiment for mice reared by untreated control dams. Results are for a litter of 8 mice per group, means of n = 3-5 determinations for each group.
tative muscimol-stimulated uptake curves for chloride, for litters exposed to each treatment but reared by untreated dams, are presented in Figure 3. The EC,, for muscimol-stimulated uptake of chloride was unchanged among treatment groups and fostering conditions (Table 1). Uptake of chloride, independent of GABA, was not different among the treatment conditions. In one experiment, uptake was compared in control-fostered mice of each treatment group, which had undergone behavioral testing, compared to those without prior testing. No differences were observed in tested compared to untested animals among any exposure group.
control
Concentrations of lorazepam
Lorazepam was present in maternal plasma and cortex after 6 days of administration and the brain : plasma ratios were similar to those previously reported (Miller, Greenblatt, Barnhill and Shader, 1988a; Table 2). Fetal forebrain also contained substantial concentrations of lorazepam, approximately one-half of that present in maternal cortex. Two days after birth, lorazepam was undetectable in maternal plasma but small concentrations remained in Vehicle PUP
Lorozepam
exposure
Fig. 2. Effects of prenatal exposure to lorazepam on muscimol-stimulated uptake of chloride. Pups were exposed to lorazepam or vehicle through the dam. Uptake was performed at 6 weeks of age. There were no differences in uptake at 50pM muscimol, with differing maternal exposures but similar exposure of litters, but uptake was decreased for all pups exposed to lorazepam, compared to (control groups (P < 0.05). Results are by litter, mean k SEM, n = 3 for each combination of pup and maternal exposure, n = 8-10 per litter.
Table I. Effects of prenatal exposure to lorazepam on EC% for muscimol-stimulated uutake of chloride in cortex Maternal exaosure Pup exposure
Control
Vehicle
Lorazepam
Control Vehicle Lorazepam
3.0 f 0.5 4.5 * 1.0 3.5 _+ 1.2
3.8 f 0.8 3.5 * 1.3 3.7 * 1.3
4.0 * 1.1 3.0 5 0.6 3.9 f 0.8
Data are in [PM] muscimol. Uptake was performed at 6 weeks of age. Data were fitted to the function y = xp + b and the EC, determined. Results are by litter, mean f SEM, n = 3, each litter n = 8-10 mice, 3-5 determinations per litter. There were no significant differences.
S. CHEsLEY et al.
56
Table 2. Concentrations of lorazepam in dam, fetus and offspring after administration bv osmotic ourno Gestational day 19
Postpartum day 2
46 f I 118+ 16
0 11.7 f 5.2
57 * 7
undetectable
DlW?l
Plasma cortex Fern
(forebrain) fforebrain)
-
Offsorinr
Concentrations of lorazepam were determined by gas-liquid chromatography. Results are ng/ml for plasma, rig/g for brain. Results are mean f SEM, n = 5 for dams, n = 9 for fetus and offspring; - = not measured.
maternal cortex. Lorazepam was undetectable in the forebrain of newborn. Postnatal development
Weights of lorazepam-exposed pups were decreased, compared to control and vehicle-treated mice for the first 1-2 weeks, regardless of fostering but from 3 to 6 weeks, the weights were similar among all exposure and fostering groups (data not shown). There were no differences in weights between male and female mice. No gross dysmorphism was observed in any group and litters were of similar size in each group. DISCUSSION
A number of previous studies have addressed behavioral and neurochemical effects of prenatal exposure to benzodiazepines. Initial studies indicated that reflex development and the startle reflex in particular, was altered in newborn animals exposed to benzodiazepines prenatally (Kellogg, Tervo, Ison and Paris, 1980). Subsequent results extended these findings to other aspects of behavior and in some cases to mature animals. Additional forms of behavior affected in newborns include motor activity and exploratory behavior and discriminant responses (Gai and Grimm, 1982; Kellogg, Ison and Miller, 1983; Simmons, Miller and Kellogg, 1984; Alleva, Leviola, Tirelli and Bignami, 1985). In mature animals, operant responses, avoidance and stress responses and the startle reflex may be altered by prenatal exposure to benzodiazepines (Alleva and Bignami, 1986; Gavish, Avnimelech-Gigus, Feldon and Myslobodsky, 1985; Livezey, Radulovacki, Isaac and Marczynski, 1985). Several studies have investigated effects of prenatal exposure to benzodiazepines on the binding of benzodiazepines in brain. However, the results of these studies are conflicting, including no change or decreases in binding and varying regional specificity. These differences may be due, in part, to differences in choice of drug and dose and in part to variations in preparation of membranes and assay conditions (Miller, Greenblatt and Shader, 1987b). Most previous behavioral and neurochemical studies of prenatal administration of benzodiazepines are affected by limitations in study design, which, in
turn, limit the conclusions that can be drawn. These limitations fall into the major categories of choice and administration of drug, controls for prenatal and postnatal effects and analysis of data. With regard to choice of drug, diazepam was used in almost all previous studies but it is not ideal, due to the active metabolites, desmethyldiazepam and oxazepam. Also, the prolonged half-life of desmethyldiazepam may complicate assessment of the true exposure interval. In only one previous study were the concentrations of drug determined to ensure delivery of drug to the dam and fetus. These data are critical to confirming exposure to drug in individual studies and to comparing studies with different exposure protocols. With regard to controls, several studies did not include vehicle-treated controls and thus, effects may not be specific. Although the majority of studies included controls, many did not control for maternal fostering effects, thus allowing rearing of the offspring by dams exposed to benzodiazepines. In view of the multiple effects of benzodiazepines and the demonstrated importance of fostering on development (Coyle, Wayner and Singer, 1976), the results of these studies may be confounded by alterations in fostering. Finally, most studies compared results in individual animals across litters. Even assuming adequate fostering controls, developmental effects, specific to each litter, might overwhelm or alter putative effects of drug. Thus, the appropriate unit of analysis is the litter and results should be compared between litters rather than individuals. In the present study, an attempt was made to circumvent these limitations by using a benzodiazepine, lorazepam, with an intermediate half-life and no active metabolites. In addition, concentrations were determined in maternal plasma and brain and in fetal brain, to ensure delivery of drug to the dam and fetus and to assess persistence of the drug after birth. The regimen chosen, involving administration of drug by osmotic pumps, produced maternal brain:plasma ratios similar to those reported in young male mice and produced concentrations in fetal brain approximately 50% of those in dams. Small, but detectable, maternal concentrations of lorazepam persisted at 48 hr after birth but lorazepam was not detectable in offspring at this time. These data confirm the delivery of lorazepam by osmotic pump, as well as the importance of cross-fostering, due to persistent maternal concentrations of drug. It should be noted that other methods of delivery of drug, such as intermittent administration, might produce differing concentration patterns of the drug and perhaps differing behavioral and neurochemical effects. With regard to rearing, a complicated protocol, using all combinations of untreated, vehicle-treated and drug-treated dams and offspring was employed. The results indicated no differences among different rearing conditions within exposure groups. Therefore,
Prenatal lorazepam exposure this cumbersome study design could be replaced by a more simple protocol in future studies, such as rearing of each exposure group by an untreated dam. In addition, no differences between males and female in motor activity or GABA, receptor function were found, so that it is reasonable to include all offspring in the analyses. Finally, the results were evaluated by litter rather than by individual, obviating possible confounding by effects of litter. Although it is possible that exposure to lorazepam altered lactation or feeding, at 6 weeks no differences in the weight of pups exposed to lorazepam or reared by lorazepamexposed dams were found. Thus, it is unlikely that the results are explained by nutritional effects. Results of open-field activity, in the present study, indicate that offspring exposed to lorazepam had increased activity at three weeks of age, compared to controls. By 6 weeks of age, the activity was similar to control groups. The period between 2 and 3 weeks of’ age appeared to be critical for motor development in rodents (Kelley, Cador and Stinus, 1989). Thus, the results may indicate an age-specific effect of prenatal administration of Iorazepam on motor development. Alternatively, since the mice were tested twice, results at 6 weeks may represent learning effects, with a possible differential between exposed and unexposed mice. Subsequent studies with testing of animals at 6 weeks only could resolve this issue. Neurochemical results indicated a decrease in GABA, receptor function in mature mice, exposed to lorazepam prenatally. A decrement was observed in maximal uptake, but not in the EC, for muscimol, suggesting an overall decrease in function rather than a change in sensitivity to GABA. This alteration may bc due to a decrease in number of GABA, receptors, as previously reported for the benzodiazepine site as noted above, although an alteration in the coupling ofGABA to the chloride channel cannot be excluded. Studies of ligand binding, as well as of gene expression for GABA, receptor subunits, may be useful in addressing this issue. The model reported here may be useful for assessing various aspects of prenatal exposure to benzodiazepines, including effects of intermittent administration of drug, exposure during specific periods of gestation and different benzodiazepines. In addition, the model should facilitate studies concerning t1.e mechanism of effects of benzodiazepines, thus contributing to the understanding of the modulation of GABA, receptor structure and function. A:knowledgements-The
authors thank Fred Lopez, Lisie Flschman, and Jack Heller for assistance, Dr Michael Thompson for use of facilities, Dr Klaus Miczek for discussions and MS Feroline Laughlin for preparation of the manuscript. Supported in part by Grants DA-05258, MH-34223, and AG-00106 from the U.S. Public Health Service, and by a grant from the March of Dimes Birth Defects Foundation, Dr Miller is the recipient of a Faculty Development Award in Clinical Pharmacology from the Pharmaceutical Manufacturers Association Foundation.
51 REFERENCES
Alleva E. and Bignami G. Prenatal benzodiazepine effectsin mice: Postnatal behavioral development, response to drug challenges, and adult discrimination learning. Neurotoxicologv 7: 303-318 (1986). Alleva E., Laviola G., Tirelii E. and Bignami G. Short-, medium-, and long-term effects of prenatal oxazepam on neuro~havioral development of mice. ~~~c~o~~~rrnacology 87: 434-441 (1985). Coyle I., Wayner M. J. and Singer G. Behavioral teratogenesis: a critical evaluation. Phurmac. Biochem. Bekzu. 4: 191-200 (1976). Gai N. and Grimm V. E. The effect of prenatal exposure to diazepam on aspects of postnatal development and behavior in rats. Psychopharmacology 78: 225-229 (1982).
Gavish M., Avnimelech-Gigus N., Feldon J. and Myslobodsky M. Prenatal chlordiazepoxide effects on metrazol seizures and benzodiazepine receptors density in adult albino rats. Life Sci. 36: 1693-1698 (1985). Greenblatt D. J., Abernethy D. R. and Shader R. I. Current status of benzodiazepines. N. Engi. f. Med. 309: 410-416 (1983). Greenblatt D. J., Franke K. and Shader R. I. Analysis of lorazepam and its glucuronide metabolite by electroncapture gas-liquid chromatography: Use in pharmacokinetic studies of lorazepam. J. Chromar. 146: 311-329 (1978). Kanto J. H. Use of benzodiazepines during pregnancy, labor, and lactation. Drugs 23: 354-380 (1982). Kelley A. G., Cador M. and Stinus L. Exploration and its measurement. In: Neuromethods, Vol. 13 (Boulton A. A., Baker G. B. and Greenshaw A. J., Eds), pp. 95-143. Clifton, New Jersey (1989). Kellogg C. K. Effect of benzodiazepines in neonates. Prog. Brain Res. 73: 207-228 (1988). Kellogg C., Ison J. R. and Miller R. K. Prenatal diazepam exposure: effects on auditory temporal resolution in rats. ~~ychophurmQcoiogy 79: 332-337 (1983).
Kellogg C., Tervo D., Ison J. and Paris T. Prenatal exposure to diazepam alters behavioral deveIopment in rats. Science 20% 205-207 (1980). Laegreid L., Olegard R., Wahlstriim J. and Conradi N. Abnormalities in children exposed to benzodiazepines in utero. Lance1 1: 108-109 (1987). Lauer J. A., Adams P. M. and Johnson K. M. Perinatal diazepam exposure: behavioral and neurochemical consequences. Neurotoxic. Teratol. 9: 213-219 (1987). Livezey G. T., Marczynski T. J. and Isaac L. Prenatal diazepam: chronic anxiety and deficits in brain receptors in mature rat progeny. Neurobehao. Toxic. Teratol. 8: 425-432 (1986).
Livezey G. T., Radulovacki M., Isaac L. and Marczynski T. J. Prenatal exposure to diazepam results in enduring reductions in brain receptors and deep slow wave sleep. Bruin Res. 334: 361-365 (1985).
Massotti M., Alleva F. R., Baalazs T. and Guidotti A. GABA and the benzodiazepine receptors in the offspring of dams receiving diazepam: ontogenetic studies. Neuropharmacology 19: 951-956 (1980).
Miller L. G., Greenblatt D. J., Paul S. M. and Shader R. I. Benzodiazepine receptor occupancy in uivo: correlation with brain concentrations and pharmacodynamic actions. J. Pharmac. exp. Ther. 240: 516522 (1987a). Miller L. G.. Greenblatt D. J. and Shader R. I. Benzodiazepine receptor binding: influence of physiologic and pharmacologic factors. Biopharm. Drug Disp. 8: 103-l 14 (I987bf. Miller L.. G., Greenblatt D. J., Bamhill J. G. and Shader R. I. Chronic benzodiazenine administ~tion: I. Tolerance is associated with do~nregulation of ~nzodi~pine receptor binding and y-aminobutyric acid, receptor
58
S. CHFSLEY
function. J. Pharmac. exp. Ther. 246: 170-176 (1988a). Miller L. G., Roy R. B., Weill C. L. and Lopez F. Prenatal lorazepam exposure alters GABA, binding and function in adult animals. Brain Res. Bull. 23: 171-174 (1989).
Miller L. G., Weill C. L. Roy R. B. and Gaver A. Lorazepam administration during embryonic development alters GABA, receptor binding and function. Deu. Brain Res. 44: 241-246 (1988b). Rosenberg L., Mitchell A. A., Parsels I. L., Pahayan H., Louik C. and Shapiro S. Lack of relation of oral clefts to diazepam use in pregnancy. N. Engl. J. Med. 309: 1282-1285 (1983).
et d.
Safra M. J., Oakley G. P. Jr Association between cleft lip and prenatal exposure to diazepam. Lancer 2: 478480 (1975). Scanlon J. W. Effect of benzodiazepines in neonates. N. Engl. J. Med. 292: 649650,
(1975).
Simmons R. D., Miller R. K. and Kellogg C. K. Prenatal exposure to diazepam alters central and peripheral responses to stress in adult rat offspring. Brain Res. 307: 3946 (1984). Tucker J. C. Benzodiazepines and the developing rat: a critical review. Neurosci. Biobehau. Rev. 9: 101-111 (1985). Woods J. H., Katz J. L. and Winger G. Abuse liability of benzodiazepines. Pharmac. Rev. 39: 251419 (1987).
1991
0028-3908/91$3.00+ 0.00 Copyright 0 1991Pergamon Press plc
PRENATAL EXPOSURE TO BENZODIAZEPINE-I. PRENATAL EXPOSURE TO LORAZEPAM IN MICE ALTERS OPEN-FIELD ACTIVITY AND GABA, RECEPTOR FUNCTION S. CHESLEY,M. LUMPKIN, A. SCHATZKI, W. R. GALPERN, D. J. GREENBLATT,R. I. SHADERand L. G. MILLER* From the Division of Clinical Pharmacology, Departments of Psychiatry and Pharmacology, Tufts University School of Medicine and New England Medical Center, Boston (Accepted 17 July 1990)
Summary-Prenatal exposure to benzodiazepines may lead to developmental abnormalities in humans and animals. To assess the behavioral and neurochemical effects of such exposure, pregnant mice were treated with lorazepam, 2 mg/kg/day, from days 13-20 of gestation, and open-field activity was assessed in offspring at 3 and 6 weeks of age and the function of GABA, receptors at 6 weeks of age. Activity was increased in mice exposed to lorazepam, compared to untreated or vehicle-treated controls at 3 weeks, but was unchanged at 6 weeks. Muscimol-stimulated uptake of chloride was decreased in lorazepamtreated mice, compared to controls, with a decrease in maximum uptake but no change in the EC,, for muscimol. Concentrations of lorazepam in maternal plasma and brain showed a similar brain:plasma ratio as previously reported and concentrations in fetal brain were about 50% of maternal levels. Lorazepam persisted for 48 hours after birth in dams but not in the offspring. These results indicate persistent behavioral and neurochemical alterations after prenatal exposure to lorazepam. This model may be useful in assessing other effects of prenatal exposure to benzodiazepine. Key words-lorazepam,
prenatal exposure, GABA, chloride, benzodiazepine.
Benzodiazepines are the most commonly prescribed psychoactive drugs and among the most widely prescribed of any class of drugs (Greenblatt, Abernethy and Shader, 1983). In addition, both survey and anecdotal evidence indicate that benzodiazepines are sub,ject to abuse, often in combination with other drugs of abuse (Woods, Katz and Winger, 1987). Thus, a large number of women of childbearing age are exposed to benzodiazepines. As such, it is likely that. a substantial number of pregnant women are exposed, although precise data from the U.S. are not available. Data from a Swedish study indicate chronic use of benzodiazepines in 0.5-l% of pregnancies and this proportion may be greater in the U.S. (Laegreid, Olegard, Wahlstrom and Conradi, 1987). Initial reports of the teratogenecity of benzodiazepines were not confirmed (Safra and Oakley, 197 5; Rosenberg, Mitchell, Parsels, Pahayan, Louik and Shapiro, 1983). Subsequently, several reports indicated a “withdrawal” syndrome, occurring in infants exposed to benzodiazepines in utero, although follow-up data on these children are not available (Kanto, 1982; Scanlon, 1975). Recently, Laegreid et al. (1987) reported 7 cases of a “benzodiazepine
embryopathy” characterized by dysmorphism, growth restriction, and psychomotor and mental retardation. Although this series was not populationbased or controlled, it again raises the possibility of serious effects of prenatal exposure to benzodiazepines in humans. A substantial literature in animals indicates behavioral abnormalities in animals exposed to benzodiazepines prenatally (for reviews, see Kellogg, 1988; Tucker, 1985). In general, motor activity and exploratory behavior are reduced in exposed newborn animals and reflex development and operant responses may be altered in adult animals exposed in utero. A modest literature also exists concerning the neurochemical effects of prenatal exposure to benzodiazepines, although results concerning the binding of benzodiazepines are conflicting (e.g. Massotti, Alleva, Baalazs and Guidotti, 1980; Livezey, Marczynski and Isaac, 1986). In a chick model, alterations in binding and function at the y-aminobutyric acid (GABAA) receptor chloride channel were previously reported in late embryos and adult chickens exposed in ouo (Miller, Weill, Roy and Gaver, 1988b, Miller, Roy, Weill and Lopez, 1989). To assess the behavioral and neurochemical effects of prenatal exposure to benzodiazepines in a mammalian model, mice were exposed prenatally to lorazepam and open-field activity and GABA, receptor function were evaluated.
*To whom correspondence should be addressed at: New England Medical Center, 171 Harrison Ave. Boston, MA 02111, U.S.A. NP 3011-n
53
s.
54
CHFSLEY et
of age. Open-field activity was determined for a period of 5 min in a Digiscan activity monitor (Omnitech, Columbus, Ohio). The testing of groups was performed in random order.
METHODS
Materials
Female mice, 7-10 weeks of age, were obtained from Charles River Laboratories (Wilmington, Massachusetts) and maintained on a 12-hr light-dark cycle with food and water ad libitum. Estrus was assessed by vaginal smears and pregnancy was determined by weighing and palpation. Osmotic pumps were obtained from Alza (Palo Alto, California). Lorazepam was a gift from Wyeth Laboratories (Philadelphia, Pennsylvania) and [3”C11], specific activity 12.5 mCi/g, was obtained from New England Nuclear (Boston, Massachusetts). All other reagents were obtained from standard commercial sources.
Neurochemical testing
In three experiments, muscimol-stimulated uptake of chloride was determined in the cortex at 6 weeks of age. Uptake was assessed as previously described (Miller et al., 1988b; Miller et al., 1989). Briefly, cortical synaptoneurosomes were prepared as described. After centrifugation, the tissue was resuspended gently in assay buffer (145 mM NaCl, 6 mM KCl, 1 mM MgCl, and 10 mM HEPES) and incubated for 10min at 30°C. To 100~1 of membranes was added 100~1 of a solution containing [3”Cll] (0.2pCi/ml of assay buffer), with or without muscimol (final concentration, l-50 p M). Samples were vortexed immediately and after 6 set the incubation was terminated by addition of 3 ml ice-cold assay buffer containing picrotoxin, 6 PM and filtration on Whatman GFjC filters, using a Brandel M24 harvester. Filters were counted by conventional scintillation spectrometry. The amount of [36Cl-] bound to the filters in the absence of muscimol was subtracted from all values to eliminate non-GABA-related uptake. Three to five determinations were performed in duplicate or triplicate for each litter.
Administration of drug
At day 13 after mating, osmotic pumps containing lorazepam, 2 mg/kg/day, were implanted subcutaneously under brief ether anesthesia. Control mice received PEG 400 vehicle alone. The weight of the animal was assumed to be 40 g, based on mean weight in pregnancy in previous experiments. The pumps were removed under brief ether anesthesia on day 20. Fostering
Within 16 hr of delivery, the litters were culled to 8-10 mice and distributed as shown in Figure 1. Litters from all three exposure groups, control, vehicle and lorazepam-treated, were distributed to dams from the three exposure groups, resulting in 9 combinations of prenatal exposure and fostering. Mice were weaned at 3 weeks and housed in singlesex cages, 4-S per cage.
Benzodiazepine concentrations
In a separate experiment, dams were sacrificed at day 6 of administration of lorazepam and concentrations of lorazepam in plasma and cortex were determined in dams and in fetal forebrain. Similar determinations were performed two days after birth. Concentrations of lorazepam were determined by gas-liquid chromatography, as previously described (Greenblatt, Franke and Shader, 1978).
Behavioral testing
The mice were weighed weekly and, in three experiments, underwent open-field testing at 3 and 6 weeks 3 Weeks
Maternal •J
controt
Vehicle PUP exposure
Lorozepom
al.
exposure
6 Weeks
Control
Control
Vehicle
Lorazepam
PUP exposure
Fig. 1. Effects of prenatal exposure to lorazepam on open-field activity. Mice underwent a 5 min open-field trial at the designated ages. Pups were exposed to lorazepam or vehicle through the dam. There were no differences in mice with differing maternal exposures but similar exposure of pup at either time. At 3 weeks, lorazepam-exposed pups had increased activity compared to the other groups but no differences were observed at 6 weeks. Results are by litter, mean + SEM, n = 3 for each combination of pup and maternal exposure, n = 8-10 per litter.
.,,_ ._ ..,, Ill
Prenatal lorazepam exposure
55
40
Analysis of data
r
Data were analyzed by litter and within litters by sex. Comparisons between groups were performed using analysis of variance with Dunnett’s comparison. For uptake of chloride, the EC,, was determined by fitting a sigmoidal function, as previously described (Miller, Greenblatt, Paul and Shader, 1987a).
&
e
n
..’
,...“’
30
,:’
z \ d ;
..’
..’
20
,.;.p.
d
E
Behavioral testing
At 3 weeks of age, three separate litters each of unexposed (control) mice and vehicle-exposed mice had similar motor activity (Fig. 1). However, motor activity was significantly increased in three litters of mice exposed to lorazepam. Within exposure groups, there were no differences among the fostering conditions. Activity was similar in male and female mice (data not shown). At 6 weeks of age, activity was similar in all three exposure groups. There were no differences among the fostering conditions, and activity was similar in male and female mice. Nexrochemical testing
At 6 weeks of age, muscimol-stimulated uptake of chloride at 50 PM muscimol was similar in three separate litters, each of unexposed (control) and vehicle-exposed mice (Fig. 2). Uptake of chloride was significantly decreased in three litters of mice exposed to lorazepam. Within exposure groups, there were no differences among the fostering conditions. Represen-
Maternol
T
q
exposure
,Y~---------d
H R
.F
PUP exposure
/
b
RESULTS
:
10
z
s
-)
I
0
... ... . .
Control Vehicle
--A-
Lorozepam
-
I
I
I
I
IO
I
20
30
40
50
Muscimol
[~Ml
Fig. 3. Effects of prenatal exposure to lorazepam on muscimol-stimulated uptake of chloride. Pups were exposed to lorazepam or vehicle through the dam. Uptake was performed at 6 weeks of age. Data are from a representative experiment for mice reared by untreated control dams. Results are for a litter of 8 mice per group, means of n = 3-5 determinations for each group.
tative muscimol-stimulated uptake curves for chloride, for litters exposed to each treatment but reared by untreated dams, are presented in Figure 3. The EC,, for muscimol-stimulated uptake of chloride was unchanged among treatment groups and fostering conditions (Table 1). Uptake of chloride, independent of GABA, was not different among the treatment conditions. In one experiment, uptake was compared in control-fostered mice of each treatment group, which had undergone behavioral testing, compared to those without prior testing. No differences were observed in tested compared to untested animals among any exposure group.
control
Concentrations of lorazepam
Lorazepam was present in maternal plasma and cortex after 6 days of administration and the brain : plasma ratios were similar to those previously reported (Miller, Greenblatt, Barnhill and Shader, 1988a; Table 2). Fetal forebrain also contained substantial concentrations of lorazepam, approximately one-half of that present in maternal cortex. Two days after birth, lorazepam was undetectable in maternal plasma but small concentrations remained in Vehicle PUP
Lorozepam
exposure
Fig. 2. Effects of prenatal exposure to lorazepam on muscimol-stimulated uptake of chloride. Pups were exposed to lorazepam or vehicle through the dam. Uptake was performed at 6 weeks of age. There were no differences in uptake at 50pM muscimol, with differing maternal exposures but similar exposure of litters, but uptake was decreased for all pups exposed to lorazepam, compared to (control groups (P < 0.05). Results are by litter, mean k SEM, n = 3 for each combination of pup and maternal exposure, n = 8-10 per litter.
Table I. Effects of prenatal exposure to lorazepam on EC% for muscimol-stimulated uutake of chloride in cortex Maternal exaosure Pup exposure
Control
Vehicle
Lorazepam
Control Vehicle Lorazepam
3.0 f 0.5 4.5 * 1.0 3.5 _+ 1.2
3.8 f 0.8 3.5 * 1.3 3.7 * 1.3
4.0 * 1.1 3.0 5 0.6 3.9 f 0.8
Data are in [PM] muscimol. Uptake was performed at 6 weeks of age. Data were fitted to the function y = xp + b and the EC, determined. Results are by litter, mean f SEM, n = 3, each litter n = 8-10 mice, 3-5 determinations per litter. There were no significant differences.
S. CHEsLEY et al.
56
Table 2. Concentrations of lorazepam in dam, fetus and offspring after administration bv osmotic ourno Gestational day 19
Postpartum day 2
46 f I 118+ 16
0 11.7 f 5.2
57 * 7
undetectable
DlW?l
Plasma cortex Fern
(forebrain) fforebrain)
-
Offsorinr
Concentrations of lorazepam were determined by gas-liquid chromatography. Results are ng/ml for plasma, rig/g for brain. Results are mean f SEM, n = 5 for dams, n = 9 for fetus and offspring; - = not measured.
maternal cortex. Lorazepam was undetectable in the forebrain of newborn. Postnatal development
Weights of lorazepam-exposed pups were decreased, compared to control and vehicle-treated mice for the first 1-2 weeks, regardless of fostering but from 3 to 6 weeks, the weights were similar among all exposure and fostering groups (data not shown). There were no differences in weights between male and female mice. No gross dysmorphism was observed in any group and litters were of similar size in each group. DISCUSSION
A number of previous studies have addressed behavioral and neurochemical effects of prenatal exposure to benzodiazepines. Initial studies indicated that reflex development and the startle reflex in particular, was altered in newborn animals exposed to benzodiazepines prenatally (Kellogg, Tervo, Ison and Paris, 1980). Subsequent results extended these findings to other aspects of behavior and in some cases to mature animals. Additional forms of behavior affected in newborns include motor activity and exploratory behavior and discriminant responses (Gai and Grimm, 1982; Kellogg, Ison and Miller, 1983; Simmons, Miller and Kellogg, 1984; Alleva, Leviola, Tirelli and Bignami, 1985). In mature animals, operant responses, avoidance and stress responses and the startle reflex may be altered by prenatal exposure to benzodiazepines (Alleva and Bignami, 1986; Gavish, Avnimelech-Gigus, Feldon and Myslobodsky, 1985; Livezey, Radulovacki, Isaac and Marczynski, 1985). Several studies have investigated effects of prenatal exposure to benzodiazepines on the binding of benzodiazepines in brain. However, the results of these studies are conflicting, including no change or decreases in binding and varying regional specificity. These differences may be due, in part, to differences in choice of drug and dose and in part to variations in preparation of membranes and assay conditions (Miller, Greenblatt and Shader, 1987b). Most previous behavioral and neurochemical studies of prenatal administration of benzodiazepines are affected by limitations in study design, which, in
turn, limit the conclusions that can be drawn. These limitations fall into the major categories of choice and administration of drug, controls for prenatal and postnatal effects and analysis of data. With regard to choice of drug, diazepam was used in almost all previous studies but it is not ideal, due to the active metabolites, desmethyldiazepam and oxazepam. Also, the prolonged half-life of desmethyldiazepam may complicate assessment of the true exposure interval. In only one previous study were the concentrations of drug determined to ensure delivery of drug to the dam and fetus. These data are critical to confirming exposure to drug in individual studies and to comparing studies with different exposure protocols. With regard to controls, several studies did not include vehicle-treated controls and thus, effects may not be specific. Although the majority of studies included controls, many did not control for maternal fostering effects, thus allowing rearing of the offspring by dams exposed to benzodiazepines. In view of the multiple effects of benzodiazepines and the demonstrated importance of fostering on development (Coyle, Wayner and Singer, 1976), the results of these studies may be confounded by alterations in fostering. Finally, most studies compared results in individual animals across litters. Even assuming adequate fostering controls, developmental effects, specific to each litter, might overwhelm or alter putative effects of drug. Thus, the appropriate unit of analysis is the litter and results should be compared between litters rather than individuals. In the present study, an attempt was made to circumvent these limitations by using a benzodiazepine, lorazepam, with an intermediate half-life and no active metabolites. In addition, concentrations were determined in maternal plasma and brain and in fetal brain, to ensure delivery of drug to the dam and fetus and to assess persistence of the drug after birth. The regimen chosen, involving administration of drug by osmotic pumps, produced maternal brain:plasma ratios similar to those reported in young male mice and produced concentrations in fetal brain approximately 50% of those in dams. Small, but detectable, maternal concentrations of lorazepam persisted at 48 hr after birth but lorazepam was not detectable in offspring at this time. These data confirm the delivery of lorazepam by osmotic pump, as well as the importance of cross-fostering, due to persistent maternal concentrations of drug. It should be noted that other methods of delivery of drug, such as intermittent administration, might produce differing concentration patterns of the drug and perhaps differing behavioral and neurochemical effects. With regard to rearing, a complicated protocol, using all combinations of untreated, vehicle-treated and drug-treated dams and offspring was employed. The results indicated no differences among different rearing conditions within exposure groups. Therefore,
Prenatal lorazepam exposure this cumbersome study design could be replaced by a more simple protocol in future studies, such as rearing of each exposure group by an untreated dam. In addition, no differences between males and female in motor activity or GABA, receptor function were found, so that it is reasonable to include all offspring in the analyses. Finally, the results were evaluated by litter rather than by individual, obviating possible confounding by effects of litter. Although it is possible that exposure to lorazepam altered lactation or feeding, at 6 weeks no differences in the weight of pups exposed to lorazepam or reared by lorazepamexposed dams were found. Thus, it is unlikely that the results are explained by nutritional effects. Results of open-field activity, in the present study, indicate that offspring exposed to lorazepam had increased activity at three weeks of age, compared to controls. By 6 weeks of age, the activity was similar to control groups. The period between 2 and 3 weeks of’ age appeared to be critical for motor development in rodents (Kelley, Cador and Stinus, 1989). Thus, the results may indicate an age-specific effect of prenatal administration of Iorazepam on motor development. Alternatively, since the mice were tested twice, results at 6 weeks may represent learning effects, with a possible differential between exposed and unexposed mice. Subsequent studies with testing of animals at 6 weeks only could resolve this issue. Neurochemical results indicated a decrease in GABA, receptor function in mature mice, exposed to lorazepam prenatally. A decrement was observed in maximal uptake, but not in the EC, for muscimol, suggesting an overall decrease in function rather than a change in sensitivity to GABA. This alteration may bc due to a decrease in number of GABA, receptors, as previously reported for the benzodiazepine site as noted above, although an alteration in the coupling ofGABA to the chloride channel cannot be excluded. Studies of ligand binding, as well as of gene expression for GABA, receptor subunits, may be useful in addressing this issue. The model reported here may be useful for assessing various aspects of prenatal exposure to benzodiazepines, including effects of intermittent administration of drug, exposure during specific periods of gestation and different benzodiazepines. In addition, the model should facilitate studies concerning t1.e mechanism of effects of benzodiazepines, thus contributing to the understanding of the modulation of GABA, receptor structure and function. A:knowledgements-The
authors thank Fred Lopez, Lisie Flschman, and Jack Heller for assistance, Dr Michael Thompson for use of facilities, Dr Klaus Miczek for discussions and MS Feroline Laughlin for preparation of the manuscript. Supported in part by Grants DA-05258, MH-34223, and AG-00106 from the U.S. Public Health Service, and by a grant from the March of Dimes Birth Defects Foundation, Dr Miller is the recipient of a Faculty Development Award in Clinical Pharmacology from the Pharmaceutical Manufacturers Association Foundation.
51 REFERENCES
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