0361-9230/92$5.00 + .OO Copyright0 1992Pergamon Press Ltd.

Brain Research Bulletin,

Vol. 29, pp. 609-616, 1992 Printed in the USA. All rights reserved.

Eight-Arm Maze Performance, Neophobia, and Hippocampal Cholinergic Alterations After Prenatal Oxazepam in Mice GIOVANNI

LAVIOLA,’

CHAIM

G. PICK, ** JOSEPH

YANAI*

AND

ENRICO

ALLEVA

Section of Behavioral Pathophysiology, Laboratorio di Fisiopatologia di Organ0 e di Sistema, Istituto Superiore di Sanith, Roma, Italy *Department of Anatomy and Embryology, The Ross Laboratory for Studies in Neural Birth Defects, The Hebrew University-Hadassah Medical School, 91010 Jerusalem, Israel Received

4 June

199 1; Acckpted

7 January

1992

LAVIOLA, G., C. G. PICK, J. YANAI AND E. ALLEVA. Eight-arm mazeperformance, neophobia, and hippocampal cholinergic alterations after prenatal oxazepam in mice. BRAIN RES BULL 29(5) 609-616, 1992.-Outbred CD-1 mice were exposed to oxazepam (I 5 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. At adulthood, radial arm maze performance, activity-habituation test in an open-field arena (either single 15-min test or three 5-min sessions at 24-h intervals), approach to a novel stimulus object, and amphetamine or scopolamine effects thereon were assessed in male progeny. Overall, the oxazepam exposed (OX) mice were much less efficient in the radial arm maze task than the vehicle exposed (VEH) animals. Pre-test scopolamine injection, but not amphetamine, significantly impaired the arm maze performance of OX mice when compared with the corresponding VEHscopolamine animals. In separate nonleamed behavioral tasks, prenatal oxazepam did not affect either baseline activity levels in the open field or the response to the amphetamine and the scopolamine challenge, while it considerably increased the latency of first approach to a novel object and produced a deficit of habituation in the course of the subsequent exploratory period. Concomitant investigation at the neurochemical level showed that the adult OX animals had a significant increase in both B,, and in the affinity (Kd) of cholinergic muscarinic receptors in the hippocampal formation when compared to the vehicle-exposed controls. Prenatal henzodiazepine exposure Amphetamine Scopolamine

Eight-arm maze Activity-habituation test Choline& receptor binding Mouse

BOTH neuroanatomical and pharmacological evidence suggests a chronic inhibitory control by at least a portion of the CNS GABA-benzodiazepine (BDZ) systems over forebrain cholinergic projections to cortex (35). Cortical ACh release or turnover can be modified by acute treatment by BDZs or the GABA agonist muscimol(44). Sarter and Schneider (36) have shown high density BDZ binding sites in rat basal forebrain. Thus the substrates exist for GABAergic modulation of the cholinergic cortical projection, through activity at the GABA-BDZ receptor complex. The GABAergic mechanisms modulated by BDZ receptors are also widely expressed in the neural areas critically involved in gating of sensory input which may be responsible for selective attention, while memory processes have been shown to be modulated during acquisition by endogenous BDZ receptor ligands (21). Converging lines of evidence also suggest that damage to forebrain cholinergic projections are related to deficits in attention and memory in animals and humans (1 I). In rodents, both lesions to cholinergic systems and administration of cholinergic

Neophobia

Pain reactivity

antagonist drugs reliably impair performances in attention or memory tasks (see, e.g., 13). Prenatal BDZ exposure, because of the link between GABABDZ systems and specific cholinergic pathways (16,37), could alter, particularly during some critical stage of neurobehavioral development, the functional relationship between the two recognition sites. With regard to adult learning, the variable effects of prenatal benzodiazepine exposure are difficult to evaluate due to differences among experiments in organism, treatment and test variables. See ($17) and the Discussion of the present report. At this point, it could be useful to analyze further some of these aspects, and the present series of experiments was aimed at extending the evaluation of the behavioral alterations of adult male mice prenatally exposed to BDZ. The choice of the radial arm maze task, which has proven to be quite useful in the study of drug effects on spatial or “working memory” performance (25) and to represent a sensitive tool in behavioral toxicology and teratology studies (31) reflected the literature reports that

’ To whom requests for reprints should be addressed. ’ Current address: Cotzias Laboratory of Neuro-Oncology, Sloan-Kettering Memorial Cancer Center, Department of Neurology and Pharmacology, New York. NY 1002 1.

609

610

LAVIOLA

the hippocampus is a brain area thought to be important for maze spatial learning (30), and at the same time an area characterized by a high density of benzodiazepine receptors (20). In addition, in order to better characterize in mice the animal’s behavioral repertoire, additional measures in nonlearned tasks were used, which on the basis oftheir known selective sensitivity to anxiolytic drugs are indicative of abnormal arousal and/or anxiety. Namely, they were activity-habituation test (two schedules of exposure to an open-field apparatus), approach to a novel stimulus object (neophobia level), and pain reactivity (hot plate test). It was also deemed advisable to assess whether or not prenatal BDZ exposure can produce long-term changes in reactivity to selected drug challenges such as scopolamine (a cholinergic muscarinic receptor blocker) and amphetamine (a potent central stimulant which releases dopamine and norepinephrine at the neuronal level), which would be indicative of alterations in the subserving neurochemical systems (for this part of the experimental model see ref 5 and 24 for locomotor activity, and 6, 14. and 25, for the radial maze performance. The BDZ agent (oxazepam) and dosing schedule for prenatal treatment were the same as those used in several previous studies after the assessment of dose-response relations, doses were also selected on the basis of pharmacodynamic considerations and preliminary behavioral assessments (see particularly refs. 3. 5).

METHOD

Mice ofan outbred Swiss-derived strain (CD- I) weighing 2527 g were purchased from a commercial breeder (Charles River Italia. I-22050 Calco). Upon arrival at the laboratory, the animals were housed in an air conditioned room (temperature 2 I 1“C. relative humidity 60 IO%>) 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 X 27 X I5 cm Plexiglas boxes with sawdust as bedding and a metal top. Pellet food (Enriched standard diet purchased from Piccioni. I-25 100 Brescia) and water were continuously available. After 2-3 weeks, breeding pairs were formed and housed in 33 X 13 X 14 cm boxes. Oxazepam (Agrar. I00 195 Roma) was suspended in a 0.5% solution of sodium carboxymethylcellulose (Fluka AG, Switzerland) in water. Females were treated PO twice daily (between 9 a.m. and IO a.m. and between 7 p.m. and 8 p.m.) on pregnancy days 12-16. Dams received either oxazepam in a volume of 0.01 ml per g body weight or vehicle. Drug treatment was carried out with the help of a spot light, when needed, in the same room in which the animals were usually housed. These treatment and dosage schedules were chosen on the basis of previous multi-dose studies showing that. in this mouse strain, the 15 mg/kg dose of oxazepam (i) does not significantly affect dam reproductive performance or litter viability and (ii) produces only a temporary retardation in postnatal body weight gain and neurobehavioral development of neonates (3.5). The females were inspected twice daily at 9 a.m. and 8 p.m. for delivery (postnatal day 1). The stud was removed ten days after the finding of the plug. Litters to be used in Experiment I were reduced at birth to three males and three females (only male animals were subsequently used), while litters used in Experiment 2 were reduced to six males. At birth, pups were fostered to untreated dams of the same strain which had given birth to healthy litters within 24 h Mice were weaned on postnatal day 20.

ET AL.

All tests were performed in dim red light between 9.30 a.m. and 12 a.m., i.e.. during the initial hours ofthe dark period. The experimental designs were counterbalanced in order to equate the representation of various groups at different test times.

The data were analyzed using mixed-model analyses of variance (ANOVAs). These considered the litter random variable ( 10.12) nested under prenatal treatment (two levels), test type (this variable was considered in the case of open-field performance), and treatment before test (saline, amphetamine. or scopolamine). All ANOVAs with repeated measures also considered the within-subjects variable. Post-hoc comparisons within logical sets of means were performed by using the Tukey’s HSD test. The design of the experiments followed as closely as possible the methodological recommendations of the Collaborative Behavioral Teratology Study group (18) and the ethical recommendations of Bateson (7). EXPERIMENT

I

In the first experiment one set of prenatally VEH or OX mice was assessed when adult (7-8 week old) for performance in a radial eight-arm maze test for nine consecutive days and challenged on day IO with a single dose of scopolamine or amphetamine given IP 15-min before being introduced into the maze.

The test procedure in the radial eight-arm maze is described in detail elsewhere (32). Briefly, a week before starting of the experiment, mice were put on a regimen of water deprivation that consisted of the administration of water for 30 min once a day. After a week, the mice were introduced individually into the maze for IO min of habituation without reinforcement of water. In the following 9 days of the test, each arm of the maze was baited with water drops of 50 ~1 as a reinforcement. Unlike the habituation day (day 0), the animals were left in the maze until they had either entered all the 8 arms or until they had made 16 entries, whichever occurred first. Thus, it became possible to determine: (a) the number of correct entries in the first eight attempts (defined as choice of arms containing baits); (b) the number of trials needed to enter all arms (the maximum allowed was 16 entries). On day IO, the trained animals were challenged either with scopolamine and amphetamine. One mouse from each litter was weighed and randomly assigned to each of the following treatments given IP I5 min before testing in a volume of 0.0 I ml/g body weight: saline solution (NaCI 0.9%); scopolamine hydrocloride 2 mg/kg; d-amphetamine sulphate I mg/kg (SIGMA Chemical Company, St Louis, MO, USA). Drug dosages were chosen according to the literature data (see, e.g., 25, and to our previous experience. Upon injection, the animals were immediately returned to their respective home cages. Because the animals, while exploring the maze, take spatial cues from the extra-maze environment, the maze orientation with regard to the visual cues on the walls of the room remained constant. After each trial, the fecal boli and urine deposits were removed and the floor was cleaned with detergent.

FUNCTION

AFTER PRENATAL EXPERIMENT

611

OXAZEPAM 2

Open-Jield Behavior To exclude any relationship between maze-learning performance and altered locomotor activity profile, open-field behavior was also assessed independently in another set of adult animals, which underwent the-same prenatal exposure as those of Experiment 1. The use of two schedules of open-field test and drug administration was aimed at assessing changes in habituation profiles (within- vs. between-session) and/or in acute drug response, which would be indicative of alterations in the subserving neurochemical systems [for this part of the experimental model see (24)j. The apparatus was an open-field arena [described in detail elsewhere (2)] made of black Plexiglas, with a light green bottom subdivided by black lines into 7 X 7 cm squares. Crossings of square limits with both forepaws were recorded by a counter mechanically activated by the experimenter. A test started by placing the animal at the center of the arena. Immediately after each test the apparatus was thoroughly cleaned by cotton pads wetted with 96% ethanol. One mouse from each litter was weighed and randomly assigned to each of the following treatments, given IP 15 min before testing in a volume of 0.0 1 ml/g body weight: saline solution (NaCl 0.9%); scopolamine hydrocloride 2 mg/kg; d-amphetamine sulphate I mg/kg (SIGMA Chemical Company, St Louis, MO). Drug dosages were chosen according to literature data and to our previous experience (2,24). Upon injection, the animals were immediately returned to their respective home cages. All observations were performed by an experimenter blind to the assignment of animals to different groups. The use of a single dose is not a serious problem in the case of scopolamine, because the effects on activity do not show a marked variation until a much higher (toxic) dosage level. By contrast, amphetamine effects are biphasic [hyperactivity at low doses and stereotypy at higher doses, particularly following repeated treatment (24). Therefore, the I mg/kg dose was chosen somewhat arbitrarily to minimize both the chance of missing a hyperactivity response and the risk of triggering stereotypy. Three subjects from each litter were given either a single I 5-min test, while the others were treated repeatedly before each ofthe three S-min tests on successive days. Approach to II Novel Stimulus Object The test procedure is described in detail elsewhere ( 1). Briefly, imm~iately after the end of the single extended ( f 5-min) version of the activity test a white glass ball (diameter 2 cm) was introduced by the experimenter into the box. The ball was gently placed in the cage corner opposite to the one where the animal took refuge when the experimenter’s hand approached. Several precautions (plastic gloves, alcohol washing of the ball, etc.) were taken to minimize the introduction via the stimulus object of human or mouse odor stimuli. The latency to make a first approach to the object was used as the end-point (cutoff time was 200 s). The number of approaches during the subsequent 3-min period was also recorded. All observations were performed by an experimenter blind to the assignment of animals to different groups.

The hot-plate test, a standard procedure, was used to assess the animal’s sensitivity to noxious stimuli: Immediately after the activity session. individual mice were placed on a hot-plate

apparatus (Model-D837 Socrel, Basile I-2 1025 Comerio) set at 55 + 0.5”C. Mice paw-licking or jumping was used as the endpoint in determining the response latency (cutoff time was 100 s). ~~scur~n~c Receptor Binding Immediately after completion of experiments I and 2, the mature 8-g-week-old animals were sacrificed by decapitation. The time from the moment of capturing the animal to decapitation was less than 4 s. The brains were rapidly removed and the hippocampal formation was dissected, immediately frozen and kept at -80°C for subsequent assay. 3Hquinuciidinyl benzilate (46 Ci mmol) was obtained from Amersham (England). Brilliant blue G and atropine sulphate were obtained from Sigma (Israel). All salts and buffers were Analar grade. The saturation binding experiment was performed according to Yamamura and Snyder (40). To assay specific binding, 50 ~1 of homogenized supernatant was allowed to incubate for 60 min at 25°C in 0.45, 1.0,2.0,4.0,6.0,8.0 nM 3H-QNB. All reaction vials contained the appropriate amount of 3H-QNB plus 0.05M Na phosphate buffer, pH = 7.4, in order to reach 2 ml of the desired concentrations. Reactions at each concentration were performed as above in triplicate to determine nonspecific binding; the identical experiment was performed in triplicate but with the addition of 10-5 M atropine. After 1 h of incubation, the reaction vials were passed through a Whatman GF/B 25 mm fiberglass filter positioned over a vacuum. This filter was then washed 3 times with 3 ml of the same ice-cold phosphate buffer. The filters were then transferred to 5 ml scintillation vials and 5 ml of 60% toluene/40% LUMAX were added as a scintillation cocktail. The samples were then counted in a Packard Tri Carb Liquid Scintillation Spectrometer. Results were plotted according to Scatchard (38) to obtain Kd and Bmax values and were expressed in pmol/mg protein. Protein was measured by the procedure of Bradford (9). RESULTS

Experiment I (ARM-MAZE) As shown in Fig. 1 the g-day profiles of maze learning of the prenatally OX and VEH mice run separate and consistently parallel because the first few days onwards. The performance of the OX group consisted of a stable response level throughout all nine trials, thus allowing a consistent separation from the profile of prenatal VEH group, which was characterized by a day by day increased efficiency in the maze task. In particular, (see Fig. 1, left) statistical analysis yielded a signi~cant effect of repeated measures, F(8, 48) = 4.14, p < 0.00 1, and of prenatal exposure, F( 1, 6) = 12.69, p < 0.01, with the OX mice giving as a total a lower number of correct entries during the first eight attempts. The higher level interaction between the above variables was not significant. In the same direction there were marked differences between prenatally exposed groups in the number of attempts needed to enter ail arms (see Fig. 1, right). In particular, statistical analysis yielded a significant effect of prenatal exposure, F( 1, 6) = 9.23, p < 0.02, and of repeated measures, F(8, 48) = 3.77, p < 0.001, with VEH mice much more efficient than the other group. The higher level interaction between the above variables was not significant. Drug Challenge on Day 10 The data from the test on day ten with or without amphetamine scopolamine challenge are reported in Fig 2. The differ-

612

LAVIOLA

ET

AL.

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FIG. I. Eight-arm maze performances of undrugged adult mice receiving prenatal oxazepam (OX) or vehicle (VEH). (A) show the number of correct entries in the first eight attempts, (B) shows the number of entries needed to visit all 8 arms (maximum possible 16). The bars on the right hand in each square indicate whole-session averages for each group (SEM). *p < 0.05, **p < 0.01 (N = 24).

ences due to prenatal treatment were essentially in the same direction as reported above. OX mice (see Fig. 2, left) appeared to have needed a lower number of correct entries during the first eight attempts, while a main effect of prenatal exposure just missed statistical significance. F( I. 6) = 5.22, p < 0.06. Acute drug treatment before testing apparently did not affect the performance and the same was in the case of the higher level interaction between this variable and prenatal exposure. Nevertheless. an accurate inspection of results and appropriate multiple comparisons seemed to be justified in such a situation [see chapter 9 in (43)]: they revealed that pretest scopolamine but not amphetamine. significantly decreased the number of correct entries by OX mice when compared with the better performance of their respective VEH controls (post hots, p < 0.05).

SALINE

As concerns the second parameter (see Fig. 2, right), in general to have needed a higher number of attempts to enter all arms, F( 1, 6) = 8.27, p < 0.05 and acute drug treatment also affected the performance, F(2, 12) = 3.39, p < 0.05. Moreover, the latter variable significantly interacted with prenatal exposure. F‘(2. 12) = 5.94. p < 0.0 I. An accurate inspection of data and appropriate multiple comparisons revealed that in absence of significant amphetamine effects, OX animals pretest treated with scopolamine appeared to have needed a significantly higher number of attempts to enter all arms than both the respective saline controls and VEH-scopolamine mice @ < 0.0 I). Remark that pretest saline injection (on day 10) seemed to improve the performance of OX-treated mice leading them to give a similar level to prenatal controls. (This would perhaps reflect OX mice appeared

* Ll!dlii

AMPHET. PRENATAL

SALINE

SCOPOL.

EXPOSURE

0

VEHICLE

m

AMPHET.

SCOPOL.

OXAZEPAM

FIG. 2. Mean eight-arm maze performances (SEM) on testing day 10 of adult OX or VEH mice injected IP I5 min before testing with saline, amphetamine (1 mg/kg). or scopolamine (2 mg/kg). Animals were the same as in Fig. 1. *p < 0.05. **p < 0.0 1. (N = 8 in each final group).

FUNCTION

AFTER

PRENATAL

TABLE

Pre-test Injection Saline Amphetamine Scopolamine

the single extended session and the repeated brief tests. The ANOVA showed a significant effect oftreatment before testing, F(2, 24) = 35.75, p < 0.001, as well as an interaction between this variable and repeated measures, F(4, 48) = 9.18, p < 0.001, although all the interactions between each of these variables and prenatal exposure were not statistically significant.

1

SingleExtended Open-fieldTest Prenatal Exposure

613

OXAZEPAM

Brief Repeated

VEH

ox

VEH

ox

199.1 (k33.2) 353.6 (* 107.4) 457.8 (*I 10.7)

218.1 (k31.4) 386.6 (k93.9) 443.6 (k98.3)

181.6 (k21.6) 315.5 (k78.6) 370.5 (+101.6)

189.5 (k26.5) 356.9 (k78.1) 357.4 (k86.9)

Approach to a Novel Object The data on the latency to approach a novel stimulus object and acute drug reactivity obtained at the end of the single extended activity session of Experiment 2, are reported in Fig. 3 (left). Both amphetamine and scopolamine markedly enhanced the latency of animals to explore and to approach a novel object, F(2, 24) = 9.24, p < 0.001. Moreover, a significant interaction between prenatal exposure and treatment before testing appeared in the ANOVA, F(2, 24) = 4.97, p < 0.01. Exploration of data and successive multiple comparisons revealed that a significant difference was limited to the saline groups. In fact, OX-saline mice exhibited considerably longer times than VEH-saline animals (p < 0.0 1). When considering the frequency of approaches in the subsequent 3-min period (see Fig 3, right), VEH animals showed a high initial response level followed by marked decreases as the session progressed (habituation profile). Morover, particularly in the first part of the session, a marked reduction in the number of approaches was measured following amphetamine and scopolamine challenge (p < 0.0 1). OX-saline mice showed a very low level of approaches at the starting of the test, which was followed by a progressive increase in the course of the session, while the initial attenuation of the response to both amphetamine and scopolamine may be an artifact due to the low level of OX-saline group (“floor effect”). These drugs were able to reduce the number of approaches at the end of the session in front of the higher level of respective controls. The ANOVA showed an interaction between prenatal treatment and repeated measures, F(2, 24) = 5.40, p < 0.01,

Values are means (?SEM) of crossings in the two schedules of openfield test (see Method section) by OX or VEH adult mice injected IP 15 min before testing with saline, amphetamine (I mg/kg) or scopolamine (2 mg/kg). Indicated are whole-session averages (single-extended test) and total average level of 3 sessions (brief-repeated test) (n = 8 in each final group).

a nonspecific effect of a more stressful handling level of arousal of the animal.)

on the general

Experiment 2 (Open-Field) The data on open-field activity and acute drug reactivity obtained in Experiment 2 are reported in Table 1. All animals showed a typical activity profile for adult outbred CD-l adult mice (for sake of brevity, data are here shown as whole-session averages), consisting of a higher initial level followed by a decrease in the course of the session (habituation), which was obtained either in the single-extended and in the repeated-brief tests (within- and between-session). Moreover, marked activity increases were produced both by amphetamine and by scopolamine. These drug effects did not seem to be different between

T

T

PRENATAL EXPOSURE 0

VEH.

m

ox.

B

PRENATAL EXPOSURE

I

VEHICLE

OXAZEPAM

0--a

.-.

6---a

._.

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SALINE AMPHET.

(lmqlkq

(2tnglkg)

SALINE

AMPHET. SCOPOL. 1- min blocks

PRE-TEST

INJECTION

SALINE

AMPHETAMINE PRE-TEST

1

scoiiii

SCOPOLAMINE

INJECTION

FIG. 3. Mean latency (SEM) of first approach to a novel object, assessed at the end of the single extended open-field session in adult OX or VEH mice (A), and mean frequency of approaches to the novel object during a 3-min test (B), injected IP 30 min before testing with saline, amphetamine (1 mg/kg) or scopolamine (2 mg/kg). Animals are the same as in Table 2. *p < 0.05, **p < 0.01 (N = 8 in each final group).

614

LAVIOLA

and a main effect of treatment before testing, F(2, 24) =3.89, p < 0.05, as well as an interaction between these variables, F(4. 48) = 3.48, p < 0.01. In fact, appropriate multiple comparisons yielded significant differences (p < 0.05 or less) between each of the saline groups and the amphetamine and scopolamine groups which did not differ from each other. Moreover, the OX-saline group differed significantly from the VEH-saline group (p < 0.05 or less) on the first and the third block.

Hot-Plate Trst No significant differences due to prenatal exposure were evident in response to noxious stimuli with VEH and OX mice showing a lick latency 5.88 (+0.65) and of 7.24 (+1.48), respectively. Mxscurinic Receptor Binding As shown in Table 2, significant differences @ c 0.01) due to prenatal exposure were found in the number of cholinergic muscarinic receptors in the hippocampal formation as defined by the Scatchard analysis (Bmax). Prenatal oxazepam significantly increased also the affinity of muscarinic receptors, as reflected by the mean dissociation constants (I(d), when compared with prenatal controls @ < 0.01). Remark that both groups of prenatally exposed animals include between others data derived from the analysis of brains from mice pretest injected with saline. scopolamine, or amphetamine. DISCUSSlON

The results reported above confirm and extend those of previous studies which were carried out using mainly rats and diazepam treatment. Marczynski and colleagues (27) reported that adult male rats prenatally exposed to diazepam, when tested in the radial arm maze, showed lack of spontaneous exploratory activity and poor spatial “working memory.” Similarly, we reported here a learning deficit in the radial maze of adult mice prenatally exposed to oxazepam. On the other hand, the present data are in apparent contrast with those of another study using mice and developmental diazepam exposure. Benton and colleagues (8) found in mice that pre- but not postnatal administration of diazepam improved adult learning in the radial-arm maze. Strain or test variables, but especially differences in statistical methodology (10) can easily account for these discrepancies. The present results also extend to an appetitive task previous findings showing long-term effects of prenatal exposure of mice to oxazepam on active avoidance learning (5). In fact, the number ofcorrect responses within each trial throughout the 9-day experiment clearly indicates that choice accuracy is significantly lower in the OX group. The eight-arm maze is a behavioral task apparently dependent on the integrity of the septohippocampal cholinergic pathways (4 I ). A reduction in choline uptake in the frontal cortex but not in the hippocampus was previously reported for adult rats exposed to oxazepam during development (15). We observed that prenatal exposure to oxazepam induced long-term changes in density and affinity of the cholinergic muscarinic receptors in the hippocampal formation. More important, the marked scopolamine impairment of maze performance in the prenatal oxazepam group also suggested functional alterations in choline& muscarinic regulatory mechanisms. Following administration of a muscarinic cholinergic antagonist such as scopolamine, the long-term effects of early oxazepam exposure on the GABA/ BDZ receptor complex appear enhanced, thus leaving room to

TABLE Prenatal Exposure

Vehicle Oxazepam

ET AL.

2

Bmax

0. I 10t- 0.04 0.134 * 0.04*

I(d

1.73 & 0.10 2.36 f 0.08*

The calculated muscarinic receptors B,, and I(d in the hippocampus of prenatal VEH or OX adult mice. B,, is expressed in pmol/mg protein and I(d is expressed in nM (Mean + SEM). Numbers are pmol/mg protein (n = 24 animals: 9 Scatchard plots for each group). * p < 0.00 I for the differences between prenatal VEH and OX animals.

an exaggerated behavioral response (i.e., performance deficit). This interaction between scopolamine and early oxazepam in the radial maze task is in agreement with the observation of Side1 and colleagues (39). They reported a potentiation by baclofen (a GABA agonist) of scopolamine-induced performance deficits in the eight-arm maze in adult rats, suggesting a pharmacological interaction between GABA and cholinergic systems (see also Introduction). So, early oxazepam exposure because of the link between BDZ and GABA systems may have disrupted in some way the normal patterning of the processes existing between forebrain GABAergic and cholinergic systems. The specificity of the proactive oxazepam effects on maze learning performance reported in the present study is further confirmed by the absence of changes in locomotor activity, as measured in both schedules of exposure to the open-field test (no effect of prenatal exposure on baseline activity-habituation profiles) or in response to the modulatory effects of amphetamine and scopolamine. This apparently denies that the reduced efficiency in the radial maze task can be ascribed to altered motor skills or reduced exploratory behavior of OX mice. Long-term alterations on active avoidance learning at the adult stage caused by prenatal oxazepam exposure were previously reported by our group. Similarly, these effects were dissociated from nonspecific changes in activity levels (5). The two responses have also been dissociated in a study comparing the performances of different mouse strains. In fact. although the level of spontaneous alternation in a maze was comparable among the strains, the mice exhibited different levels of locomotor activity in response to amphetamine and scopolamine (6). It has been suggested that the hypermotihty exhibited by animals receiving scopolamine contributes to the decrease in performance of the maze task by increasing perseverative and circling behaviors (14). Spontaneous motor activity has been widely utilized as a simple task measuring exploratory behavior in a nonspatial, nonwater reinforced environment (33). However, in the present study we report that amphetamine administration. at a relatively low dose (I mg/kg), induced hyperactivity in both schedules of open-field test but failed to affect significantly the performance in the eight-arm maze. This finding further supports the hypothesis that the increased performance deficit seen with scopolamine cannot be easily explained by a nonspecific change in motor activity in the radial-arm maze. The data on the latency of first approach to a novel stimulus object and on the frequency of explorative contacts revealed that adult OX mice showed a behavioral profile characterized both by increased neophobia and by a deficit of habituation. as revealed by the perseverative approaching/explorative behavior toward the novel stimuls object. Similarly, Marcinski and colleagues (27) reported in adult rats prenatally exposed to diazepam

FUNCTION

AFTER PRENATAL

615

OXAZEPAM

a reduced control over emotional responses, when challenged by a novel and “intimidating” environmental stimuli. Pain reactivity was not modified in oxazepam mice, as already observed in a series of previous ontogenetic studies (4,22). The failure to find any difference due to early BDZ exposure is intriguing in view of the wide literature involving the activity of the central GABA-BDZ receptor complex in the modulation of pain perception (29,34,42). As concerns the mechanisms by which long-term BDZ effects may be produced, the data extend previous models based on the finding of specific changes in sensory functions (18) or arousal-attention phenomena (17,26-28). Therefore, the production of reduced maze learning and the impaired coping with a novel stimulus by early BDZ exposure could involve impairment at the level of processing of sensory information (17) and/

or a modification of the functional value of various stimuli which contribute to the modulation of different behavioral responses (23). The present findings indicating significant changes in cholinergic parameters upon prenatal BDZ exposure (especially the increased Bmax), suggest that at least part of the behavioral alterations are due to central cholinergic functions. ACKNOWLEDGEMENTS This research was supported as part of the Sub-project on Behavioral Pathophysiology (Project on Non-infectious Pathology) of the Istituto Superiore di Sanita, by grant No. 90.04056.CT04 fr;m the Consiglio Nazionale delle Ricerche. bv USPHS arant DA-6670. and bv a grant from The Israeli Anti Drug’Authority_We thank Flavia Chiarotti for expert statistical advice. C.G. Pick was supported by a joint exchange program between the Italian CNR and the Israeli NCRD.

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