- Email: [email protected]
Neurobehavior effects in four strains of mice offspring exposed prenatally to alprazolam
Neurobehavior effects in four strains of mice offspring exposed prenatally to alprazolam William F. Rayburn, MD,a,b H. Dix Christensen, PhD,b,c Karen M. Gold, MD,b and Christina L. Gonzalez, BSb,c Albuquerque, NM, and Oklahoma City, Okla OBJECTIVE: This study was performed to determine whether prenatal exposure to alprazolam affects offspring behavior in different strains of mice. STUDY DESIGN: Eight to 11 gravid mice of the C3H/He, C57BL/6, A/J, and DBA/2 strains were given either an anxiolytic dose of alprazolam (0.32 mg/kg) or a placebo by gavage on day 18 of an anticipated 19- to 21-day gestation. Neurobehavior tasks were conducted to assess anxiety, learning and memory, and social interaction. Data were analyzed by analysis of variance or a Fisher exact probability test. RESULTS: Anxiety in alprazolam-exposed offspring was reduced in C3H/He (P < .05) and A/J (P < .05) newborn infants by separation vocalization but may be increased in the C3H/He adult strain on the plus maze task. Learning was slower among C57BL/6 mice exposed to alprazolam (P < .01), whereas memory was reduced in exposed A/J and DBA/2 offspring (P < .05). Alprazolam exposure was associated with more aggression among C3H/He and C57BL/6 male offspring (P < .01) and with less group activity by C57BL/6 offspring (P < .05). CONCLUSION: Altered behaviors in several mouse strains after prenatal exposure to alprazolam suggests a vulnerability of GABA-benozdiazepine receptor formation in fetal brain development. (Am J Obstet Gynecol 2002;187:968-72.)
Key words: Alprazolam, Xanax, prenatal exposure, mouse, behavior
Alprazolam, the most commonly taken benzodiazepine for anxiety and for premenstrual dysphoric disorder, is one of the most often prescribed drugs to women of reproductive age.1,2 Benzodiazepines exert a marked influence on the function of gamma-amino-butyric acid (GABA), the primary inhibitory neurotransmitter in the mammalian brain.3 GABAergic neurogenesis proceeds and correlates with the regional distribution of GABA/benzodiazepine receptors. Once pregnancy is diagnosed, prescribing alprazolam requires a careful balance between benefits to the mother and risks to the fetus. Alprazolam crosses the placenta easily because of its lipid solubility and its low molecular weight.4,5 Binding receptors in human fetal tissue have been identified by 12 weeks’ conceptual age.6,7 We were
From the Departments of Obstetrics and Gynecology, University of New Mexico Health Sciences Centera and the Department of Obstetrics and Gynecologyb and the College of Pharmacy,c University of Oklahoma Health Sciences Center. Supported by John W. Records Perinatal Research Fund. Presented at the Twenty-second Annual Meeting of the Society of Maternal-Fetal Medicine, New Orleans, La, January 14-19, 2002. Reprint requests: William F. Rayburn, MD, University of New Mexico, Department of Obstetrics and Gynecology, 2211 Lomas Blvd NE (ACC 4), Albuquerque, NM 87131. E-mail: [email protected] © 2002, Mosby, Inc. All rights reserved. 0002-9378/2002 $35.00 + 0 6/6/127133 doi:10.1067/mob.2002.127133
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unable to locate any studies about hazards to the human fetal brain. Animal investigations of prenatal insult with benzodiazepines have primarily involved the rodent.8 We reported anxiolytic efficacy without adverse effect using a 0.32-mg/kg dose of alprazolam in C57BL/6 and C3H/He mice and during certain tasks in A/J mice.9 In the same study, the maternal serum concentration of 47.3 ng/mL at 1 hour after dosing in C3H/He mice was comparable to effective concentrations of 20 to 40 ng/mL in humans treated for anxiety.10 The fetal brain concentration of alprazolam was 45.3 ng/g on gestational day 18. The objective of this investigation was to test the hypothesis that prenatal exposure to alprazolam affects the neurobehavior of several strains of mice offspring. By using a mouse model developed in our laboratory, effects from a relevant dose of alprazolam were sought in a randomized placebo-controlled manner. Material and methods This proposal was approved by our Animal Review Committee. Four strains of mice, C3H/He, C57BL/6, A/J, and DBA/2 (Jackson Laboratory, Bar Harbor, Me) were selected for differences in drug responsiveness and for having recombinant sets for any future genetic study.11 Each strain, used commonly in neurobehavior testing, has its own baseline of behaviors and neurobe-
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Table I. Performances on anxiety tasks by four mouse strains exposed prenatally either to alprazolam or to a placebo Mouse strain Tasks Separation vocalization (%) Alprazolam Placebo Elevated plus maze (No.) Arms entered (No.) Postnatal day 30 Alprazolam Placebo Postnatal day 60 Alprazolam Placebo Novelty exploration Arms entered (No.) Alprazolam Placebo Time per arm (s) Alprazolam Placebo
C3H/He
C57BL/6
23 ± 12* 81 ± 11
0 0
A/J
DBA/2
4 ± 4* 33 ± 15
15 ± 6 31 ± 15
2.6 ± 0.8 3.1 ± 0.8
2.3 ± 0.7 3.0 ± 0.8
0.6 ± 0.5 0.4 ± 0.3
1.3 ± 0.2 1.3 ± 0.2
0.3 ± 0.2* 0.9 ± 0.3
1.2 ± 0.3 1.0 ± 0.3
0.1 ± 0.1 0.1 ± 0.1
0.1 ± 0.1 0.1 ± 0.1
3.7 ± 0.5 5.5 ± 0.3
5.8 ± 0.2 5.4 ± 0.2
2.0 ± 0.4 1.4 ± 0.3
5.9 ± 0.2 5.8 ± 0.2
25 ± 3 24 ± 3
12 ± 1 12 ± 2
– –
21 ± 2 19 ± 1
Mean ± SEM. *P < .05 compared with corresponding placebo group in each strain.
havioral characteristics. The 25 adult female mice in each strain (100 total), received at 7 weeks of age, were housed at an American Association for the Accreditation of Laboratory Animal Care–approved facility. Maintenance of these animals has been described.12 For mating purposes, each female was placed in a cage with a randomly selected male of the same strain for a 2-hour exposure. Attempts at conception were undertaken for up to 5 consecutive days (one estrus cycle). Depending on the strain, we anticipated that 16 to 20 of the 25 mice would conceive. The day of conception, identified by the presence of a copulatory plug, was recorded as gestational day 0. Once gestation was confirmed by palpation, the females were housed individually in identical environments. Although binding sites are detected in rodents as early as gestational day 14, GABA/benzodiazepine receptors are not identified in all brain regions until gestational age 18.13 For this reason, the gravid mice were administered either the relevant dose of alprazolam (0.32 mg/kg) or a placebo on gestational day 18. Randomization in each strain either to the alprazolam group (n = 8-10) or to the placebo group (n = 10) was performed in a predetermined, computer-generated manner. Alprazolam was obtained from the commercially available product (Xanax, Pharmacia and Upjohn, Kalamazoo, Mich).14 Gavage was the route of drug delivery because alprazolam is taken orally by humans. Alprazolam was dissolved in propylene glycol, then diluted with water for a 3.2% propylene glycol formulation. The placebo consisted of the same formulation without alprazolam. Therapy was administered between 10 AM and 12 noon in a volume of 0.01 mL/g of body weight.
Beginning on gestational day 19, the dams were checked twice daily for the presence of litters. The day on which the litter was found at 8:30 AM was designated as being postnatal day 1. Maintenance of inbred mice strains precluded handling and weighing of the pups until postnatal day 4. The litter size was reduced to a maximum of six pups (3 females, 3 males) at postnatal day 4. Offspring in both exposure groups (alprazolam and placebo) undertook behavioral tasks at the same time during the prejuvenile, juvenile, and adult periods and under identical conditions. Several standard tasks were used to assess anxiety (separation vocalization, elevated plus maze, and novelty exploration), learning and memory (tube runway), and social interaction (social play and male aggression).15,16 The rationale and brief descriptions of these tasks and the times of testing during the life span are described in detail elsewhere.12,17,18 These tasks were similar to those that could be used in humans. The same field biologist (C. L. B.) observed the mice. We observed that these tasks were simple to conduct, nonpunishing, and quantitative. A litter was the unit of measurement. For there to be a noteworthy effect, we considered that a 50% difference in the mean task performance would be necessary between the alprazolam-exposed and the placebo-exposed groups in each strain. The data were reported as mean ± SEM or as a percentage in the litter. Intrastrain comparisons between offspring in the alprazolam-exposed and in the placebo-exposed groups were conducted with a 1-way analysis of variance and, in cases of nonparametric comparisons, with the Fisher exact probability test. InStat 2.05 GraphPad Software (GraphPad Software, Inc, San Diego,
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Table II. Performances on learning and memory tube runway tasks by 4 mice strains exposed prenatally either to alprazolam or to a placebo Mouse strain Tasks Learning (s) Postnatal day 35 Alprazolam Placebo Postnatal day 37 Alprazolam Placebo Postnatal day 39 Alprazolam Placebo Memory (s) Postnatal day 46 Alprazolam Placebo
C3H/He
C57BL/6
A/J
DBA/2
39.1 ± 8.1 42.3 ± 7.1
37.1 ± 7.7 34.3 ± 6.3
73.6 ± 4.7 62.4 ± 3.6
62.2 ± 8.4 61.7 ± 9.7
5.2 ± 0.6 5.3 ± 0.6
5.0 ± 0.4 4.7 ± 0.9
32.5 ± 7.5 22.3 ± 5.8
15.3 ± 6.0 10.3 ± 4.0
3.5 ± 0.3 3.6 ± 0.3
4.1 ± 0.3† 2.9 ± 0.5
12.0 ± 3.3 14.2 ± 4.2
8.3 ± 5.3 3.5 ± 0.7
5.5 ± 0.5 6.5 ± 0.8
5.5 ± 0.7 4.7 ± 0.7
44.3 ± 7.2* 25.1 ± 5.0
15.6 ± 5.5* 4.7 ± 0.7
Mean ± SE. *P < .05 compared with corresponding placebo group in each strain. †P < .01, compared with corresponding placebo group in each strain.
Calif) and Statistix 4.1 Analytical Software (Analytic Software Co, Tallahassee, Fla) were used. A P < .05 was considered to be statistically significant. Results The duration of gestation varied between the four strains (C57BL/G: 19.7 days; C3H/HG: 20.1 days; DBA/2 days: 20.7; and AJ: 21.6 days). The gestational day of delivery did not differ between alprazolam and placebo exposure groups in each strain. The litter sizes, usually six to eight offspring, were not different between exposure groups. An equal proportion of male and female offspring was observed for each strain. Body weights at postnatal days 4 and 21 were indistinguishable between exposure groups in each strain. Results of performances on anxiety tasks are shown in Table I. Alprazolam-exposed pups in three strains were less likely than placebo-exposed pups to vocalize when separated from their mothers on postnatal day 6. Prenatal exposure to alprazolam did not have an impact on the number of open-arm entries on the elevated plus maze. The only exception was on postnatal day 60, when C3H/He adult mice that had been exposed to alprazolam exhibited less exploratory activity. Novelty exploration into the eight arms of the radial arm maze was similar between the two exposed groups in each strain. The average time in each arm was indistinguishable between exposure groups in each strain. The alprazolam-exposed A/J offspring did not explore, hindering interpretation of the elevated plus maze and of the novelty exploration tasks. Learning and memory was assessed by using the tube runway task. Table II displays results of tasks for the four strains. Learning to run the entire runway to acquire the
food pellets was rapid during the 5 consecutive days. Runway times for alprazolam-exposed C57BL/6J mice were less than the placebo group on days 4 and 5 of learning (postnatal days 38 and 39). A delay in performing the runway task at 7 days later (memory task) was evident in A/J and DBA/2 offspring exposed to alprazolam rather than to a placebo. Mice offspring exposed to alprazolam exhibited differences in social interaction (Table III). Alprazolam-exposed mice were less likely to congregate as a group while awake among the C3H/He and A/J strains and were less likely to sleep as a group among C57BL/6 and A/J strains. Compared with the placebo group, alprazolamexposed C3H/He offspring demonstrated a lower percentage of time trying to escape. Exposure to alprazolam affected wrestling among male offspring. Pairs of alprazolam-exposed male offspring in the C3H/He and C57BL/6 strains wrestled more than placebo-exposed mice (Table IV). This observation was present after food restriction on postnatal days 35 and 65 and after cage changing on postnatal day 65. Comment Alprazolam is prescribed commonly to women of reproductive age for anxiety and panic disorders. Reports about its safety during pregnancy are largely unavailable. A postmarketing evaluation in 542 instances of firsttrimester exposure to alprazolam revealed no evidence of an increase in spontaneous abortion or in congenital anomalies.19 Effects in the neonate after exposure to benzodiazepines shortly before birth include hypotonia and a poor suck.7,20 Prenatal doses of centrally acting drugs do not usually produce traditional toxicity signs (altered survival,
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Table III. Social interaction of four mouse strains exposed prenatally either to alprazolam or to a placebo Mouse strain Tasks Time all awake (%) Alprazolam Placebo Social play as a group (%) Postnatal day 17 Alprazolam Placebo Postnatal day 25 Alprazolam Placebo Escape attempts (%) Alprazolam Placebo Sleep as a group (%) Alprazolam Placebo
C3H/He
C57BL/6
A/J
DBA/2
45 ± 2* 56 ± 4
56 ± 6 53 ± 6
36 ± 3* 45 ± 3
26 ± 5 33 ± 3
48 ± 11 22 ± 8
28 ± 5* 59 ± 5
61 ± 10* 36 ± 7
29 ± 8 42 ± 13
33 ± 8 23 ± 6
10 ± 3* 22 ± 3
25 ± 3 32 ± 8
19 ± 6 19 ± 10
2.2 ± 0.3* 4.5 ± 0.8
3.1 ± 1.0 2.5 ± 0.9
2.3 ± 0.5 3.7 ± 0.7
4.4 ± 1.6 3.9 ± 0.6
83* 20
33* 89
13* 75
67 50
Mean ± SEM. *P < .05 compared with corresponding placebo group in each strain.
Table IV. Percent of male pairs that wrestled after prenatal exposure either to alprazolam or to a placebo Mouse strain Tasks Food restriction Postnatal day 35 Alprazolam Placebo Postnatal day 65 Alprazolam Placebo Cage change Alprazolam Placebo
C3H/He
C57BL/6
A/J
DBA/2
85* 44
33† 0
0 0
0 0
78† 25
50† 0
50 50
88 83
86† 0
50† 0
0 0
33 33
*P < .05 compared with corresponding placebo group in each strain. †P < .01 compared with corresponding placebo group in each strain.
growth, and morphology) on offspring but may be linked with select functional deficits, impairments, or behavioral alterations. A preliminary finding in our laboratory indicated that C3H/He mice exposed prenatally to alprazolam exhibited less anxiety, frustration during an anticipation task, and caution in novel situations compared with a placebo control group. The current prospective investigation was undertaken to better determine whether these altered behaviors were present in other mouse strains and during a greater variety of tasks. The four mouse strains used in these studies provided a range of potential responses from which drug-host interactions could be explored. Findings from this study uncovered many alterations in behavior among the alprazolam-exposed mice in the different strains. Certain strains of mice exhibited approachavoidance responses when undergoing anxiety-provoking or social interaction tasks. Separation vocalization, an early sign of anxiety, was less apparent in the exposed
C3H/He and A/J strains. Subtle differences in social interaction were observed in juveniles and adults. The C57BL/6 offspring, after in utero exposure to alprazolam, were more likely to function independently, rather than as a group while awake or asleep. Pairs of C3H/He and C57BL/6 male mice that had been exposed to alprazolam were likely to wrestle for more prolonged periods. Memory difficulties in exposed A/J and DBA/2 offspring suggest a vulnerability to receptor development for recalling integrated responses. Dosing at gestational day 18 in the current mouse study was selected as a time when benzodiazepine receptors are present in all brain regions and when an exposure to alprazolam is beyond the first trimester in a human. This exposure period would theoretically correspond to short-term use of an anxiolytic agent either during a stressful period or during a panic attack, rather than chronic therapy, which would less likely be prescribed during pregnancy.
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Differences in performances of certain tasks suggest that a specific inhibitory function is removed with shortterm prenatal exposure to a relevant dose of alprazolam.21 Altered reactivity of neurons to stress is most probably due to benzodiazepine neurotransmitter dysfunction, presumably on GABA neuronal inhibition. A role of GABA or a GABA/benzodiazepine receptor complex is involved in the organization of integrated stress responses. Either a dampened or an exaggerated behavior effect from alprazolam could be affected by compensatory changes in other systems. The fetal mouse brain could be vulnerable at multiple neural receptor sites that are critical to the expression of organized stress responses.22 Interstrain differences in fetal development would lead to questions about observed variations in response to alprazolam. With the use of birth as a time marker, the C57BL/6 pups lagged developmentally behind the C3H/He and A/J offspring. In contrast, the DBA/2 pups were more advanced. Because alprazolam was administered on gestation day 18, could variations in strain maturation affect a region of the C57BL/6 mouse brain and less or none in the other strains? Earlier dosing, such as on gestation day 17, may elicit a more vigorous response. For the DBA/2 and A/J mice, did the longer gestation period affect timing of brain development, thereby missing a vulnerable period from alprazolam exposure? Alprazolam may influence the expression of the GABAA receptor subtype in a specific brain region (cerebellum, cortex, corpus striatum, and thalamus). Are differences in any task performance due to interstrain variations in receptor molecular determinants? Observations from our study should provide additional information to the scientist who is interested in understanding more about postnatal effects of GABA-modulating drugs on the early developing brain. These findings may promote a deeper interest in understanding alterations in neural mechanisms underlying the organization of integrated responses. Strategies described here using this less-advanced mammal cannot provide a precise replica of the human situation. From a clinical perspective, a phenomenon observed from these investigations provides, at least, a beginning reference. Our conclusions, despite extrapolation variations between mammalian species, may be used in counseling women who ask for alprazolam during pregnancy. These subtle alterations in behavior indicate areas of potential concern in developing children. Reported experience in human subjects is now necessary to make more definitive statements about safety.
REFERENCES
1. Brawman-Mintzer O. Pharmacologic treatment of generalized anxiety disorder. Psychiatr Clin North Am 2001;24:119-35.
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2. Evans S, Haney M, Levin F, Foltin R, Fishman M. Mood and performance changes in women with premenstrual dysphoric disorder: acute effects of alprazolam. Neuropsychopharm 1998;19: 499-516. 3. Yourn AB, Chu D. Distribution of GABAA and GABAB receptors in mammalian brain. Drug Dev Res 1990;21:161-7. 4. Mandelli M, Morselli PL, Nordio S, Pardi G, Principi N, Sereni F, et al. Placental transfer of diazepam and its disposition in the newborn. Clin Pharmacol Ther 1975;17:564-72. 5. Garzone PD, Kroboth PD. Pharmacokinetics of the newer benzodiazepines. Clin Pharmacokinet 1989;16:336-64. 6. Aaltonen L, Erkkola R, Kanto J. Benzodiazepine receptors in the human fetus. Biol Neonate 1983;44:54-7. 7. Brooksbank BW, Atkinson DJ, Balazs R. Biochemical development of the human brain, III: benzodiazepine receptors, free gamma-aminobutyrate (GABA) and other amino acids. J Neurosci Res 1982;8:581-94. 8. Kellogg CK. Benzodiazepines and the developing central nervous system: laboratory findings and clinical implications. In: Zagon IS, Slotkin TA, editors. Maternal substance abuse and the developing nervous system. New York: Academic Press; 1992. p. 283-321. 9. Coleman F, Christensen H, Kupiec T, Gonzalez C, Rayburn W. A therapeutic dose of alprazolam (Xanax) and its effect on perinatal outcomes of inbred mice. J Matern Fetal Invest 1996;6: 110-8. 10. Benet LZ, Oie S, Schwartz. Design and optimization of dosage regimens; pharmacokinetic data: alprazolam. In: Hardman JG, Limbird LE, Molinoff PB, Ruddon RW, Goodman, Gilman, editors. The pharmacological basis of therapeutics. 9th ed. New York: McGraw-Hill; 1996. p. 1713. 11. Green MC, Witham BA. Handbook on genetically standardized Jax mice. 4th ed. Bar Harbor (ME): Jackson Laboratory; 1991. 12. Rayburn W, Christensen H, Gonzalez C. A placebo-controlled comparison between betamethasone and dexamethasone for fetal maturation: differences on neurobehavior in mice offspring. Am J Obstet Gynecol 1997;176:842-51. 13. Lauder JM, Han VKM, Henderson P, Verdoorn T, Towle AC. Prenatal ontogeny of the GABAergic system in the rat brain: an immunocytochemical study. Neuroscience 1986;19:465-93. 14. Xanax. In: Physicians’ desk reference. 52th ed. Montvale (NJ): Medical Economics; 1998. p. 2294-8. 15. Irvin S. Comprehensive observational assessment: a systematic, quantitative procedure for assessing the behavioral and physiologic state of the mouse. J Psychopharmacol 1968;13:222-57. 16. McClearn G. Selected use of the mouse in behavioral research. In: Foster H, Small J, Fox J, editors. The mouse in biomedical research, volume 4. Experimental biology and oncology. New York: Academic Press; 1982. p. 37-49. 17. Stanton ME. Workshop on the qualitative and quantitative comparability of human and animal developmental neurotoxicity, work group I report: comparability of measures of developmental neurotoxicity in humans and laboratory animals. Neurotoxicol Teratol 1990;12:261-7. 18. Bignami G, Alleva E, Chiarotti F, Laviola G. Selective changes in mouse behavioral development after prenatal benzodiazepine exposure: a progress report. Neuro Psychopharmacol Biol Psychiat 1992;16:587-604. 19. St. Clair SM, Schirmer RG. First trimester exposure to alprazolam. Obstet Gynecol 1992;80:843-6. 20. Barry WS, St. Clair SM. Exposure to benzodiazepines in utero. Lancet 1987;1:436-7. 21. Kellogg CK, Primus RJ, Bitran D. Sexually dimorphic influences of prenatal exposure to diazepam on behavioral responses to environmental challenge and on gamma-aminobutyric acid (GABA) stimulated uptake in the brain. J Pharmacol Exp Ther 1991;256:259-65. 22. Wang JB, Kofuii P, Fernando JC, Moss SJ, Huganir RL, Burt DR. The alpha 1, alpha 2, and alpha 3 subunits of GABAA receptors: comparison in seizure-prone and -resistant mice and during development. J Mol Neurosci 1992;3:177-84.
Key words: Alprazolam, Xanax, prenatal exposure, mouse, behavior
Alprazolam, the most commonly taken benzodiazepine for anxiety and for premenstrual dysphoric disorder, is one of the most often prescribed drugs to women of reproductive age.1,2 Benzodiazepines exert a marked influence on the function of gamma-amino-butyric acid (GABA), the primary inhibitory neurotransmitter in the mammalian brain.3 GABAergic neurogenesis proceeds and correlates with the regional distribution of GABA/benzodiazepine receptors. Once pregnancy is diagnosed, prescribing alprazolam requires a careful balance between benefits to the mother and risks to the fetus. Alprazolam crosses the placenta easily because of its lipid solubility and its low molecular weight.4,5 Binding receptors in human fetal tissue have been identified by 12 weeks’ conceptual age.6,7 We were
From the Departments of Obstetrics and Gynecology, University of New Mexico Health Sciences Centera and the Department of Obstetrics and Gynecologyb and the College of Pharmacy,c University of Oklahoma Health Sciences Center. Supported by John W. Records Perinatal Research Fund. Presented at the Twenty-second Annual Meeting of the Society of Maternal-Fetal Medicine, New Orleans, La, January 14-19, 2002. Reprint requests: William F. Rayburn, MD, University of New Mexico, Department of Obstetrics and Gynecology, 2211 Lomas Blvd NE (ACC 4), Albuquerque, NM 87131. E-mail: [email protected] © 2002, Mosby, Inc. All rights reserved. 0002-9378/2002 $35.00 + 0 6/6/127133 doi:10.1067/mob.2002.127133
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unable to locate any studies about hazards to the human fetal brain. Animal investigations of prenatal insult with benzodiazepines have primarily involved the rodent.8 We reported anxiolytic efficacy without adverse effect using a 0.32-mg/kg dose of alprazolam in C57BL/6 and C3H/He mice and during certain tasks in A/J mice.9 In the same study, the maternal serum concentration of 47.3 ng/mL at 1 hour after dosing in C3H/He mice was comparable to effective concentrations of 20 to 40 ng/mL in humans treated for anxiety.10 The fetal brain concentration of alprazolam was 45.3 ng/g on gestational day 18. The objective of this investigation was to test the hypothesis that prenatal exposure to alprazolam affects the neurobehavior of several strains of mice offspring. By using a mouse model developed in our laboratory, effects from a relevant dose of alprazolam were sought in a randomized placebo-controlled manner. Material and methods This proposal was approved by our Animal Review Committee. Four strains of mice, C3H/He, C57BL/6, A/J, and DBA/2 (Jackson Laboratory, Bar Harbor, Me) were selected for differences in drug responsiveness and for having recombinant sets for any future genetic study.11 Each strain, used commonly in neurobehavior testing, has its own baseline of behaviors and neurobe-
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Table I. Performances on anxiety tasks by four mouse strains exposed prenatally either to alprazolam or to a placebo Mouse strain Tasks Separation vocalization (%) Alprazolam Placebo Elevated plus maze (No.) Arms entered (No.) Postnatal day 30 Alprazolam Placebo Postnatal day 60 Alprazolam Placebo Novelty exploration Arms entered (No.) Alprazolam Placebo Time per arm (s) Alprazolam Placebo
C3H/He
C57BL/6
23 ± 12* 81 ± 11
0 0
A/J
DBA/2
4 ± 4* 33 ± 15
15 ± 6 31 ± 15
2.6 ± 0.8 3.1 ± 0.8
2.3 ± 0.7 3.0 ± 0.8
0.6 ± 0.5 0.4 ± 0.3
1.3 ± 0.2 1.3 ± 0.2
0.3 ± 0.2* 0.9 ± 0.3
1.2 ± 0.3 1.0 ± 0.3
0.1 ± 0.1 0.1 ± 0.1
0.1 ± 0.1 0.1 ± 0.1
3.7 ± 0.5 5.5 ± 0.3
5.8 ± 0.2 5.4 ± 0.2
2.0 ± 0.4 1.4 ± 0.3
5.9 ± 0.2 5.8 ± 0.2
25 ± 3 24 ± 3
12 ± 1 12 ± 2
– –
21 ± 2 19 ± 1
Mean ± SEM. *P < .05 compared with corresponding placebo group in each strain.
havioral characteristics. The 25 adult female mice in each strain (100 total), received at 7 weeks of age, were housed at an American Association for the Accreditation of Laboratory Animal Care–approved facility. Maintenance of these animals has been described.12 For mating purposes, each female was placed in a cage with a randomly selected male of the same strain for a 2-hour exposure. Attempts at conception were undertaken for up to 5 consecutive days (one estrus cycle). Depending on the strain, we anticipated that 16 to 20 of the 25 mice would conceive. The day of conception, identified by the presence of a copulatory plug, was recorded as gestational day 0. Once gestation was confirmed by palpation, the females were housed individually in identical environments. Although binding sites are detected in rodents as early as gestational day 14, GABA/benzodiazepine receptors are not identified in all brain regions until gestational age 18.13 For this reason, the gravid mice were administered either the relevant dose of alprazolam (0.32 mg/kg) or a placebo on gestational day 18. Randomization in each strain either to the alprazolam group (n = 8-10) or to the placebo group (n = 10) was performed in a predetermined, computer-generated manner. Alprazolam was obtained from the commercially available product (Xanax, Pharmacia and Upjohn, Kalamazoo, Mich).14 Gavage was the route of drug delivery because alprazolam is taken orally by humans. Alprazolam was dissolved in propylene glycol, then diluted with water for a 3.2% propylene glycol formulation. The placebo consisted of the same formulation without alprazolam. Therapy was administered between 10 AM and 12 noon in a volume of 0.01 mL/g of body weight.
Beginning on gestational day 19, the dams were checked twice daily for the presence of litters. The day on which the litter was found at 8:30 AM was designated as being postnatal day 1. Maintenance of inbred mice strains precluded handling and weighing of the pups until postnatal day 4. The litter size was reduced to a maximum of six pups (3 females, 3 males) at postnatal day 4. Offspring in both exposure groups (alprazolam and placebo) undertook behavioral tasks at the same time during the prejuvenile, juvenile, and adult periods and under identical conditions. Several standard tasks were used to assess anxiety (separation vocalization, elevated plus maze, and novelty exploration), learning and memory (tube runway), and social interaction (social play and male aggression).15,16 The rationale and brief descriptions of these tasks and the times of testing during the life span are described in detail elsewhere.12,17,18 These tasks were similar to those that could be used in humans. The same field biologist (C. L. B.) observed the mice. We observed that these tasks were simple to conduct, nonpunishing, and quantitative. A litter was the unit of measurement. For there to be a noteworthy effect, we considered that a 50% difference in the mean task performance would be necessary between the alprazolam-exposed and the placebo-exposed groups in each strain. The data were reported as mean ± SEM or as a percentage in the litter. Intrastrain comparisons between offspring in the alprazolam-exposed and in the placebo-exposed groups were conducted with a 1-way analysis of variance and, in cases of nonparametric comparisons, with the Fisher exact probability test. InStat 2.05 GraphPad Software (GraphPad Software, Inc, San Diego,
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Table II. Performances on learning and memory tube runway tasks by 4 mice strains exposed prenatally either to alprazolam or to a placebo Mouse strain Tasks Learning (s) Postnatal day 35 Alprazolam Placebo Postnatal day 37 Alprazolam Placebo Postnatal day 39 Alprazolam Placebo Memory (s) Postnatal day 46 Alprazolam Placebo
C3H/He
C57BL/6
A/J
DBA/2
39.1 ± 8.1 42.3 ± 7.1
37.1 ± 7.7 34.3 ± 6.3
73.6 ± 4.7 62.4 ± 3.6
62.2 ± 8.4 61.7 ± 9.7
5.2 ± 0.6 5.3 ± 0.6
5.0 ± 0.4 4.7 ± 0.9
32.5 ± 7.5 22.3 ± 5.8
15.3 ± 6.0 10.3 ± 4.0
3.5 ± 0.3 3.6 ± 0.3
4.1 ± 0.3† 2.9 ± 0.5
12.0 ± 3.3 14.2 ± 4.2
8.3 ± 5.3 3.5 ± 0.7
5.5 ± 0.5 6.5 ± 0.8
5.5 ± 0.7 4.7 ± 0.7
44.3 ± 7.2* 25.1 ± 5.0
15.6 ± 5.5* 4.7 ± 0.7
Mean ± SE. *P < .05 compared with corresponding placebo group in each strain. †P < .01, compared with corresponding placebo group in each strain.
Calif) and Statistix 4.1 Analytical Software (Analytic Software Co, Tallahassee, Fla) were used. A P < .05 was considered to be statistically significant. Results The duration of gestation varied between the four strains (C57BL/G: 19.7 days; C3H/HG: 20.1 days; DBA/2 days: 20.7; and AJ: 21.6 days). The gestational day of delivery did not differ between alprazolam and placebo exposure groups in each strain. The litter sizes, usually six to eight offspring, were not different between exposure groups. An equal proportion of male and female offspring was observed for each strain. Body weights at postnatal days 4 and 21 were indistinguishable between exposure groups in each strain. Results of performances on anxiety tasks are shown in Table I. Alprazolam-exposed pups in three strains were less likely than placebo-exposed pups to vocalize when separated from their mothers on postnatal day 6. Prenatal exposure to alprazolam did not have an impact on the number of open-arm entries on the elevated plus maze. The only exception was on postnatal day 60, when C3H/He adult mice that had been exposed to alprazolam exhibited less exploratory activity. Novelty exploration into the eight arms of the radial arm maze was similar between the two exposed groups in each strain. The average time in each arm was indistinguishable between exposure groups in each strain. The alprazolam-exposed A/J offspring did not explore, hindering interpretation of the elevated plus maze and of the novelty exploration tasks. Learning and memory was assessed by using the tube runway task. Table II displays results of tasks for the four strains. Learning to run the entire runway to acquire the
food pellets was rapid during the 5 consecutive days. Runway times for alprazolam-exposed C57BL/6J mice were less than the placebo group on days 4 and 5 of learning (postnatal days 38 and 39). A delay in performing the runway task at 7 days later (memory task) was evident in A/J and DBA/2 offspring exposed to alprazolam rather than to a placebo. Mice offspring exposed to alprazolam exhibited differences in social interaction (Table III). Alprazolam-exposed mice were less likely to congregate as a group while awake among the C3H/He and A/J strains and were less likely to sleep as a group among C57BL/6 and A/J strains. Compared with the placebo group, alprazolamexposed C3H/He offspring demonstrated a lower percentage of time trying to escape. Exposure to alprazolam affected wrestling among male offspring. Pairs of alprazolam-exposed male offspring in the C3H/He and C57BL/6 strains wrestled more than placebo-exposed mice (Table IV). This observation was present after food restriction on postnatal days 35 and 65 and after cage changing on postnatal day 65. Comment Alprazolam is prescribed commonly to women of reproductive age for anxiety and panic disorders. Reports about its safety during pregnancy are largely unavailable. A postmarketing evaluation in 542 instances of firsttrimester exposure to alprazolam revealed no evidence of an increase in spontaneous abortion or in congenital anomalies.19 Effects in the neonate after exposure to benzodiazepines shortly before birth include hypotonia and a poor suck.7,20 Prenatal doses of centrally acting drugs do not usually produce traditional toxicity signs (altered survival,
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Table III. Social interaction of four mouse strains exposed prenatally either to alprazolam or to a placebo Mouse strain Tasks Time all awake (%) Alprazolam Placebo Social play as a group (%) Postnatal day 17 Alprazolam Placebo Postnatal day 25 Alprazolam Placebo Escape attempts (%) Alprazolam Placebo Sleep as a group (%) Alprazolam Placebo
C3H/He
C57BL/6
A/J
DBA/2
45 ± 2* 56 ± 4
56 ± 6 53 ± 6
36 ± 3* 45 ± 3
26 ± 5 33 ± 3
48 ± 11 22 ± 8
28 ± 5* 59 ± 5
61 ± 10* 36 ± 7
29 ± 8 42 ± 13
33 ± 8 23 ± 6
10 ± 3* 22 ± 3
25 ± 3 32 ± 8
19 ± 6 19 ± 10
2.2 ± 0.3* 4.5 ± 0.8
3.1 ± 1.0 2.5 ± 0.9
2.3 ± 0.5 3.7 ± 0.7
4.4 ± 1.6 3.9 ± 0.6
83* 20
33* 89
13* 75
67 50
Mean ± SEM. *P < .05 compared with corresponding placebo group in each strain.
Table IV. Percent of male pairs that wrestled after prenatal exposure either to alprazolam or to a placebo Mouse strain Tasks Food restriction Postnatal day 35 Alprazolam Placebo Postnatal day 65 Alprazolam Placebo Cage change Alprazolam Placebo
C3H/He
C57BL/6
A/J
DBA/2
85* 44
33† 0
0 0
0 0
78† 25
50† 0
50 50
88 83
86† 0
50† 0
0 0
33 33
*P < .05 compared with corresponding placebo group in each strain. †P < .01 compared with corresponding placebo group in each strain.
growth, and morphology) on offspring but may be linked with select functional deficits, impairments, or behavioral alterations. A preliminary finding in our laboratory indicated that C3H/He mice exposed prenatally to alprazolam exhibited less anxiety, frustration during an anticipation task, and caution in novel situations compared with a placebo control group. The current prospective investigation was undertaken to better determine whether these altered behaviors were present in other mouse strains and during a greater variety of tasks. The four mouse strains used in these studies provided a range of potential responses from which drug-host interactions could be explored. Findings from this study uncovered many alterations in behavior among the alprazolam-exposed mice in the different strains. Certain strains of mice exhibited approachavoidance responses when undergoing anxiety-provoking or social interaction tasks. Separation vocalization, an early sign of anxiety, was less apparent in the exposed
C3H/He and A/J strains. Subtle differences in social interaction were observed in juveniles and adults. The C57BL/6 offspring, after in utero exposure to alprazolam, were more likely to function independently, rather than as a group while awake or asleep. Pairs of C3H/He and C57BL/6 male mice that had been exposed to alprazolam were likely to wrestle for more prolonged periods. Memory difficulties in exposed A/J and DBA/2 offspring suggest a vulnerability to receptor development for recalling integrated responses. Dosing at gestational day 18 in the current mouse study was selected as a time when benzodiazepine receptors are present in all brain regions and when an exposure to alprazolam is beyond the first trimester in a human. This exposure period would theoretically correspond to short-term use of an anxiolytic agent either during a stressful period or during a panic attack, rather than chronic therapy, which would less likely be prescribed during pregnancy.
972 Rayburn et al
Differences in performances of certain tasks suggest that a specific inhibitory function is removed with shortterm prenatal exposure to a relevant dose of alprazolam.21 Altered reactivity of neurons to stress is most probably due to benzodiazepine neurotransmitter dysfunction, presumably on GABA neuronal inhibition. A role of GABA or a GABA/benzodiazepine receptor complex is involved in the organization of integrated stress responses. Either a dampened or an exaggerated behavior effect from alprazolam could be affected by compensatory changes in other systems. The fetal mouse brain could be vulnerable at multiple neural receptor sites that are critical to the expression of organized stress responses.22 Interstrain differences in fetal development would lead to questions about observed variations in response to alprazolam. With the use of birth as a time marker, the C57BL/6 pups lagged developmentally behind the C3H/He and A/J offspring. In contrast, the DBA/2 pups were more advanced. Because alprazolam was administered on gestation day 18, could variations in strain maturation affect a region of the C57BL/6 mouse brain and less or none in the other strains? Earlier dosing, such as on gestation day 17, may elicit a more vigorous response. For the DBA/2 and A/J mice, did the longer gestation period affect timing of brain development, thereby missing a vulnerable period from alprazolam exposure? Alprazolam may influence the expression of the GABAA receptor subtype in a specific brain region (cerebellum, cortex, corpus striatum, and thalamus). Are differences in any task performance due to interstrain variations in receptor molecular determinants? Observations from our study should provide additional information to the scientist who is interested in understanding more about postnatal effects of GABA-modulating drugs on the early developing brain. These findings may promote a deeper interest in understanding alterations in neural mechanisms underlying the organization of integrated responses. Strategies described here using this less-advanced mammal cannot provide a precise replica of the human situation. From a clinical perspective, a phenomenon observed from these investigations provides, at least, a beginning reference. Our conclusions, despite extrapolation variations between mammalian species, may be used in counseling women who ask for alprazolam during pregnancy. These subtle alterations in behavior indicate areas of potential concern in developing children. Reported experience in human subjects is now necessary to make more definitive statements about safety.
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