Development of GABAergic modulation of mouse locomotor activity and pain sensitivity after prenatal benzodiazepine exposure

Neurotoxicologyand Teratology, Vol.

14, 1-5, 1992

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Development of GAB Aergic Modulation of Mouse Locomotor Activity and Pain Sensitivity After Prenatal Benzodiazepine Exposure G I O V A N N I L A V I O L A , F L A V I A CHIARO'I*I'I A N D E N R I C O A L L E V A

Section of Behavioural Pathophysiology, Laboratorio di Fisiopatologia di Organo e di Sistema Istituto Superiore di Sanitd, Viale Regina Elena, 299, 1-00161 Roma, Italy R e c e i v e d 12 April 1991 LAVIOLA, G., F. CHIAROTTI AND E. ALLEVA. Development of GABAergic modulation of mouse locomotor activity and pain sensitivity after prenatal benzodiazepine exposure. NEUROTOXICOL TERATOL 14(1) 1-5, 1992.-Outbred CD-1 mice were exposed to oxazepam (15 mg/kg PO twice/day) on days 12-16 of fetal life, i.e., at a critical ontogenetic stage of Type II benzodiazepine (BDZ) receptor increase, and fostered at birth to untreated dams. Locomotor activity (single 30-min session in a Varimex apparatus), hot-plate responding, and muscimol (GABAa agonist) effects thereon [see normative data in (16)] were assessed on postnatal day 14, 21, or 28. Prenatal oxazepam did not affect the development of hot-plate responding and muscimol analgesia; however, it reduced activity on day 14 (as in previous studies) and modified the profile of muscimol effects at 21 days (time of fast appearance of an adult-like pattern of activity) and at 28 days. Specifically, oxazepam mice showed a faster recovery from the initial depression after 1 mg/kg of muscimol at the former age and a lack of rebound hyperactivity at the latter age. These effects might be explained either 1) by an accelerated development of GABAergic regulatory mechanisms, or 2) by the same monoaminergic system changes which can account for other effects of prenatal BDZ exposure (1,3). In any event, the dissociation phenomena found in the preseiat study strengthen the notion that GABAergic influences contribute to the modulation of locomotor activity and of pain reactivity by mechanisms which are at least in part separate from each other (16). Prenatal benzodiazepine exposure

Muscimol

Locomotor activity

SOME of the effects of prenatal benzodiazepine (BDZ) exposure on neurobehavioral development of laboratory animals have been studied extensively. For example, impairment of locomotor activity has been evidenced during the normal developmental stage of locomotor hyperactivity, which appears in immature rats and mice (when tested in social isolation) around the end of the 2nd postnatal week (16,17). This effect goes hand in hand with a reduction of the locomotor hyperactivity produced by amphetamine at the same age (3). These and other changes, which include a selective impairment of active avoidance at the young adult stage in the absence of passive avoidance deficits (1,3), appear to be related to specific alterations in norepinephrinecontaining neurons of the hypothalamus (3, 7, 25, 30). At the physiological level, Livezey and colleagues (18) have suggested that most of the behavioral anomalies observed in rats exposed prenatally to BDZs may be explained by an imbalance between forebrain inhibitory influences and brainstem arousal system(s). In this context, the increasing evidence for a role of GABAergic mechanisms in the modulation of behavioral arousal appears to have considerable significance [see Introduction in (16)]; specifically, pharmacological manipulations support the view of an inverse relationship between the rate or intensity of GABAergic transmission and behavioral arousal [(8,24); and studies using various types of GABA systems manipulations,

Pain reactivity

Mouse development

see (16, 21, 28, 29) and below]. Direct evidence on the effects of early BDZ exposure on the behavioral functions of GABAergic mechanisms is not available. However, in one of our previous studies mice treated with oxazepam on days 12-16 of pregnancy failed to show at 28 days the hyperactivity which is normally induced by a high morphine dose (2). This may constitute indirect evidence of a change in GABAergic function, since suppression of GABAergic influences has been indicated as one of the mechanisms mediating opiate hyperactivity (9). The available neurochemical evidence also points to the potential interest of studies aimed at assessing the effects of early BDZ exposure on GABAergic behavioral functions. On the one hand, it is not precisely known at which point in ontogenesis the linkage between the BDZ receptors (BZR) and the GABA recognition sites is established (11, 15, 22, 27). On the other hand, BDZ Type II binding sites, which are coupled to GABAa receptors and/or chloride ionophores, show already at birth an adult-like concentration level (i.e., their proliferation is mostly prenatal), while Type I binding sites, which are not coupled to GABA receptors, proliferate primarily during the first two weeks of postnatal life (13). Therefore, it is conceivable that occupancy of the BZR during the last part of the prenatal stage (third week of pregnancy) could alter the functional relationship be-

Part of the data reported here were presented at the "2nd Meeting of the European Behavioural Pharmacology Society" (Athens, Greece 1988).

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LAVIOLA, CHIAROTTI AND ALLEVA

tween the two recognition sites (15) although the direct biochemical evidence on this point is still inadequate [(15, 22, 26, 27); see the Discussion section]. The present study was aimed at extending previous evaluations of prenatal oxazepam effects to the parallel development of two GABAergic modulatory influences which have recently been assessed in our laboratory (16); namely, that on locomotor activity (in a Varimex apparatus) and that on pain reactivity (hot plate test). The attention was focussed on three stages of development. At the preweaning (late previsual) phase (testing on day 14) pain responses are well developed, the locomotor tendency is already quite strong (but still unchecked by habituation processes), and the sensitivity to muscimol depression and analgesia is maximal. At two successive postweaning stages (testing on day 21 and day 28, respectively), the animals show an adultlike activity pattern (including habituation) and sensitivity to muscimol depression and analgesia is progressively reduced. Moreover, around the end of the fourth postnatal week the animal is in a transitional phase in which muscimol depression is followed by a marked rebound hyperactivity not observed at either 3 or 5 weeks [see (16) and the Discussion below]. METHOD

Animals and Breeding Mice of an outbred Swiss-derived strain (CD-1) weighing 25-27 g were purchased from a commercial breeder (Charles River Italia, 1-22050 Calco). Upon arrival at the laboratory the animals were housed in an air-conditioned room (temperature 21--_ I°C, relative humidity 60± 10%) with lights on from 9:30 p.m. to 9:30 a.m. Males and nulliparous females were housed separately in groups of 8-10 in 42 × 27 × 15 cm Plexiglas boxes with sawdust as bedding and a metal top. Pellet food (Enriched Standard Diet purchased from Piccioni, 1-25100 Brescia) and water were continuously available. After 2-3 weeks, breeding pairs were formed and housed in 33 × 13 × 14 cm boxes. Oxazepam (Agrar, 1-00195 Roma) was suspended in a 0.5% solution of Sodium Carboxymethylcellulose (Fluka AG, Switzerland) in water. Females were treated PO twice daily (between 9 and I0 a.m. and between 7 and 8 p.m.) on pregnancy days 12-16. Dams received either oxazepam (15 mg/kg per dose) in a volume of 0.01 ml per g body weight or vehicle. This treatment was chosen on the basis of previous multidose studies showing that, in this mouse strain, the 15 mg/kg dose of oxazepam given twice daily 1) does not significantly affect dam reproductive performance or litter viability and 2) produces only a temporary retardation in postnatal body weight gain and neurobehavioral development of neonates (3). The females were inspected twice dally at 9 a.m. and 8 p.m. for the presence of a vaginal plug (pregnancy day 0) and for delivery (postnatal day 1). The stud was removed ten days after the finding of the plug. All litters were reduced at birth to four males and four females and fostered to untreated dams of the same strain which had given birth to healthy litters within 24 h. Except during test sessions, mothers remained with the pups until weaning, which took place on day 20 of age. When weaned, mice were separated according to sex with no more than four animals per cage.

Apparatus and Procedures Five litters from each of two original groups of 15 litters (prenatal OX or vehicle, VEH) were randomly assigned to testing at three different postnatal ages (day 14, 21, or 28). On each testing day the pups were weighed at the nearest 0.01 g on a

PK-300 Mettler Balance set for automatic compensation of variation due to movements during weighing. One male and one female from each litter were randomly assigned to one of four treatment conditions (injected IP, in a volume of 0.01 mug body weight): saline solution (NaC1 0.9%) and three doses of muscimol (Sigma Chemical Company, St. Louis, MO). Fourteen-dayold mice received a 0.1, 0.3, or 0.5 mg/kg dose, while 21-, or 28-day-old mice received a 0.1, 0.5, or 1.0 mg/kg dose. Drug dosages were scaled according to the literature data concerning drug sensitivity at different developmental stages [see, e.g., (20, 28, 29)] and to our previous experience which included the demonstration of muscimol bioavailability in the CNS after systemic treatment on postnatal days 7-35 (16). Upon injection, the animals were immediately returned to their respective home boxes (dams were not removed). Fifteen min later, pups were introduced individually in a clean box of the same type as the home cage. The box was placed on a Varimex Activity Meter apparatus (four units, Columbus Instruments, OH), set at a standard level. Only the horizontal sensor systems were used, and the recording session lasted 30 min. Immediately after the activity session, individual pups were placed on a hot-plate apparatus (Model-D837 Socrel, Basile 1-21025 Comerio) set at 55 ---0.5°C. Paw-licking or jumping was used as the end-point in determining the response latency (cutoff time 60 s). All tests were performed in dim red light between 9:30 and 12 a.m., i.e., during the initial hours of the dark period. The experimental designs were counterbalanced in order to equate the representation of various groups at different test times. In the case of activity tests, the designs were also counterbalanced for assignment of animals to different Varimex units. The design of the experiments followed as closely as possible the methodological recommendations of the Collaborative Behavioral Teratology Study group (13) and the ethical recommendations of Bateson (4).

Design and Statistical Analysis The data were first analyzed by mixed-model ANOVAs considering all variables, namely, prenatal exposure (two levels), the litter random variable (5,6), sex, testing age (14, 21, or 28 days), and pretest treatment (saline and low, intermediate, and high muscimol dose). Since the latter variable introduced a bias (due to the different dose ranges used at different ages), separate mixed model ANOVAs were subsequently performed for each developmental age. All ANOVAs on activity data also considered the within-subject (repeated measures) variable (five successive within-session blocks, of 6 min each). Post hoc comparisons within logical sets of means were performed using the Tukey's HSD test. RESULTS The data on activity (Fig. 1) showed the expected overall increase with age, F(2,24) = 32.17, p<0.001. Moreover, withinsession trends varied, consisting of a progressive increase of activity on day 14 after a low initial level, and of a substantial activity decrease (within-sessi~)n habituation) from day 21 onwards [age × blocks, F(8,24)= 16.41, p<0.001]. Muscimol effects were mainly in the direction of response depression but varied considerably as a joint function of age of testing, drug dosage, and blocks, F(24,24)=4.84, p<0.001. ANOVAs performed separately for each age showed drug depression on ages 14 and 21 [drug × blocks, F(12,96)=2.54 and 2.51, p<0.005]. Specifically, on day 14 the animals were al-

PRENATAL O X A Z E P A M AND ONTOGENY OF MUSCIMOL EFFECTS

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FIG. 1. Activity (Varimex counts) of immature mice prenatally exposed to oxazepam or vehicle and injected IP 15 min before testing with either saline or different muscimol doses adjusted for age (see the Method section). Data from the five males and five females in each final group were pooled in the absence of main effect and interactions of the sex variable (N= 10 for each final group). (+-S.E.M. indicated by vertical bar to the right, calculated on the basis of the appropriate error mean square in the ANOVA.)

ready significantly affected at the intermediate (0.3 mg/kg) dose (post hocs in the last three blocks, p < 0 . 0 1 ) . On day 21, muscimol depression occurred throughout the session in prenatal controis after 0.5 or 1 mg/kg. On day 28, a more complex profile of drug effects emerged [drug × blocks, F ( 1 2 , 9 6 ) = 9 . 1 8 , p < 0 . 0 0 1 ] . The lower and the intermediate doses were ineffective, while a biphasic within-session trend appeared at the higher (1 mg/kg) dose; namely, animals showed rebound hyperactivity after the initial depression (p<0.05 vs. saline controls in the last two blocks). As in previous work using either an open-field test (4) or the same automated device of the present study (2), prenatal oxazepam reduced activity at 14 days [prenatal treatment × blocks, F(4,32) = 3.43, p < 0 . 0 1 ] . Such lowering of the baseline did not prevent muscimol from exerting depressant effects similar to those found in prenatal controls (ps<0.05 or less in post hocs comparing the 0.3 and 0.5 mg/kg groups with the corresponding saline group). By contrast, the muscimol profile was substan-

FIG. 2. Hot-plate responding of immature mice prenatally exposed to oxazepam or vehicle and injected IP with either saline or different muscimol doses adjusted for age (see the Method section). Data from the five males and five females in each final group were pooled in the absence of main effect and interactions of the sex variable (N= 10 for each final group). (---S.E.M. indicated by vertical bar to the right, calculated on the basis of the appropriate error mean square in the ANOVA.) Data refer to the same animals of Fig. 1.

tially modified at 21 and 28 days. At the former age, the depression after 1 mg/kg was initially the same in the oxazepam and the control groups, but only the oxazepam mice subsequently recovered and were as active as the prenatal control-no muscimol animals at the end of the session. At 28 days, the oxazepam animals failed to show rebound hyperactivity after depression by 1 mg/kg of muscimol. The interactions between prenatal treatment, pretest treatment and blocks missed overall statistical significance, F(12,160)= 1.25 and 1.42, p < 0 . 2 : 0.1, for 21- and 28-day groups, respectively; appropriate betweengroups comparisons [performed according to the rationale given in (31), pp. 187-189] revealed that in the last two 6-min blocks the oxazepam mice treated with 1 mg/kg of muscimol were significantly more active or less active than the corresponding prenatal control muscimol treated animals at 21 and 28 days, respectively. The results of the hot-plate tests are reported in Fig. 2. Overall, these data point to monotonic trends of both pain reactivity and response to muscimol, with the longest response latency and the more pronounced analgesic effects of the drug occurring at 14 days [in the overall ANOVA, age × drug: F(6,72) = 9.17, p < 0.001; in post hoc comparisons, p < 0 , 0 1 for

4

LAVIOLA, CHIAROTTI AND ALLEVA

the differences between the 14-day-old mice and the other ages]. More specifically, ANOVAs performed separately for each age showed that muscimol given on day 14 produced a marked analgesia, F(3,21)= 52.09, p<0.001, which was evident already at the intermediate (0.3 mg/kg) dose (p<0.05). On day 21, the lowest dose producing a significant change in response latency was 0.5 mg/kg (p<0.01), while on day 28 the drug was effective only at the highest (1.0 mg/kg) dose (p<0.01). These behavioral profiles were not affected by prenatal oxazepam exposure. DISCUSSION The effects of age and muscimol on activity and pain reactivity of control mice found in the present experiment replicated the results of a previous study [for a brief summary see the introduction; for details and discussion see (16)]. Pain reactivity and muscimol analgesia were not modified by prenatal oxazepam. If only these data were considered, one might be tempted to conclude against the occurrence of functionally important changes in GABA regulatory mechanisms. Analgesia produced by systemic administration of GABA agonists is not prevented by naloxone pretreatment, apparently ruling out an involvement of endogenous opioid systems, but seems to be due to a disinhibitory effect on some CNS cholinergic regulatory mechanism (12). Therefore, the present negative data are in agreement with some of our previous results which apparently exclude substantial functional repercussions of any cholinergic system changes that may occur after prenatal BDZ exposure (1,3). The data on activity, however, suggest that prenatal BDZ exposure produces some significant changes in GABAergic mechanisms which modulate arousal or otherwise contribute to the control of motor responding. At 21 days, depression by the highest muscimol dose (1 mg/kg) was followed in the oxazepam mice by a fast recovery which contrasted with the extended response suppression in the prenatal controls. Moreover, oxazepam mice failed to show the rebound hyperactivity which normally occurs at 28 days after the initial depression by the same dose and disappears within the following few days [see tests at 28 and 35 days in (16)]. Some of the features in this profile, particularly the remarkable recovery at 21 days from the depression initially produced by the highest (1 mg/kg) muscimol dose but not from the depression produced by the intermediate (0.5 mg/kg) dose, suggest that prenatal BDZ exposure may accelerate the maturation processes which are responsible for successive modifications of the GABAergic agonist's effects. This hypothesis, however, is questioned by the fact that the muscimol profile in prenatal oxazepam mice at 21 days of age fell short of an exact replica of the drug effects in prenatal controls at 28 days, missing the significant elevation above the activity levels of untreated animals (rebound hyperactivity). Therefore, it remains to be ascertained whether or not such a replica can be obtained by extending the tests to intermediate ages. From a different viewpoint, one should consider the possibility that the prevention by prenatal oxazepam of both muscimol

rebound activity at 28 days and of morphine hyperactivity at the same age (2) be due to the same functional change; in fact, the suppression of GABAergic influences is deemed to play a substantial role in the production of activity enhancement by morphine (9). The available data, however, cannot discriminate between two possible types of changes, either in GABAergic reactivity per se or in other mechanisms which are activated by GABAergic hyperstimulation, apparently in an age-dependent fashion. [Specifically, a phenomenon such as muscimol rebound hyperactivity at 28 days might be due either to a predominance of the agonist's action at (a) site(s) where it can produce mainly stimulant effects, as in the case of dopaminergic activation by intraA10 administration of muscimol (10), or by an activation of compensatory mechanisms which can counteract the depressant effect.] In fact, the hypothesis that non-GABAergic mechanisms are affected is the only one compatible with a unitary explanation of a wide variety of prenatal BDZ effects. This applies to the hypoactivity and hyposensitivity to amphetamine at the end of the second postnatal week (3), to the modification of the activity responses to muscimol (present study) and morphine (2) at the end of the fourth postnatal week, and to the selective impairment of active avoidance at the young adult stage (1,3); all these effects could be functional consequences of those changes in monoaminergic functions which are well documented by neurochemical studies, such as the reduction of hypothalamic NE level and turnover (11,25). The monoaminergic hypothesis, however, is apparently inadequate to account for the accelerated recovery from muscimol depression found in prenatal oxazepam mice at 21 days. On the other hand, the biochemical data on GABAergic functions after BDZ exposure are still too limited to either support, or vice versa discount, the hypothesis of a role of GABAergic system changes. For example, diazepam at a high dosage level reduced the muscimol stimulation of 3H-diazepam binding to neural membranes in the postnatal period (19). By contrast, diazepam at a lower dosage level enhanced the GABA stimulation of 3nflunitrazepam binding at 90 days (11). Another study using a still lower dose of prenatal diazepam failed to show changes in 3H-muscimol binding to high affinity GABA receptors, while glutamic acid decarboxylase activity was enhanced (23). The latter effect, however, occurred only in the first postnatal week; therefore, it appears to be of little value for explaining changes in the response to muscimol at 21 and 28 days. In conclusion, it appears that both a unitary (monoaminergic) and a dual (monoaminergic and GABAergic) working hypothesis must be considered when designing further studies of the subtle effects of prenatal BDZ exposure on behavioral developmeat. ACKNOWLEDGEMENTS This research was supported as part of the Sub-project on Behavioral Pathophysiology (Project on Non-Infectious Pathology) of the Istituto Superiore di SanitY, and by grant No. 89.04677.CT04 from the Consiglio Nazionale delle Ricerche. We acknowledge Dr. Giorgio Bignami for critically reading successive versions of the manuscript.

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