Developmental exposure to chlorpyrifos alters reactivity to environmental and social cues in adolescent mice

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Toxicology and Applied Pharmacology 191 (2003) 189 –201

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Developmental exposure to chlorpyrifos alters reactivity to environmental and social cues in adolescent mice Laura Ricceri,a,* Nadja Markina,a,b Angela Valanzano,a Stefano Fortuna,c Maria Francesca Cometa,c Annarita Meneguz,c and Gemma Calamandreia b

a Section of Comparative Psychology, Laboratorio di Fisiopatologia, Istituto Superiore di Sanita`, Rome, Italy Department of Biology, Laboratory of Genetics and Physiology of Behavior, University of Moscow, Moscow, Russia c Section of Biochemistry, Laboratorio di Farmacologia, Istituto Superiore di Sanita`, Rome, Italy

Received 10 December 2002; accepted 1 May 2003

Abstract Neonatal mice were treated daily on postnatal days (pnds) 1 through 4 or 11 through 14 with the organophosphate pesticide chlorpyrifos (CPF), at doses (1 or 3 mg/kg) that do not evoke systemic toxicity. Brain acetylcholinesterase (AChE) activity was evaluated within 24 h from termination of treatments. Pups treated on pnds 1– 4 underwent ultrasonic vocalization tests (pnds 5, 8, and 11) and a homing test (orientation to home nest material, pnd 10). Pups in both treatment schedules were then assessed for locomotor activity (pnd 25), novelty-seeking response (pnd 35), social interactions with an unfamiliar conspecific (pnd 45), and passive avoidance learning (pnd 60). AChE activity was reduced by 25% after CPF 1– 4 but not after CPF 11–14 treatment. CPF selectively affected only the G4 (tetramer) molecular isoform of AChE. Behavioral analysis showed that early CPF treatment failed to affect neonatal behaviors. Locomotor activity on pnd 25 was increased in 11–14 CPF-treated mice at both doses, and CPF-treated animals in both treatment schedules were more active when exposed to environmental novelty in the novelty-seeking test. All CPF-treated mice displayed more agonistic responses, and such effect was more marked in male mice exposed to the low CPF dose on pnds 11–14. Passive avoidance learning was not affected by CPF. These data indicate that developmental exposure to CPF induces long-term behavioral alterations in the mouse species and support the involvement of neural systems in addition to the cholinergic system in the delayed behavioral toxicity of CPF. © 2003 Elsevier Inc. All rights reserved. Keywords: Chlorpyrifos; Acetylcholinesterase; Neonatal behavior; Novelty seeking; Social interaction; Behavioral teratology; Mouse

Introduction Chlorpyrifos (CPF) is a widely used organophosphorous pesticide (OP), which elicits toxicity by inhibiting acetylcholinesterase (AChE), the key molecule in the control of cholinergic transmission, in the central and peripheral nervous system. In recent years, CPF has replaced many other OP pesticides due to its relative safety and persistence. However, the potential health effects associated with human exposure to CPF are the subject of increasing concern. In

* Corresponding author. Section of Comparative Psychology, Lab. FOS, Istituto Superiore di Sanita`e`, V.le Regina Elena 299 Rome, Italy. Fax: ⫹39-06-4957821. E-mail address: [email protected] (L. Ricceri). 0041-008X/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0041-008X(03)00229-1

particular, it has been hypothesized that infants and children are exposed to CPF doses well above the established not observed effect level (NOEL), due to persistent accumulation of CPF on residential surfaces and toys after household application (Gurunathan et al., 1998). Recently, urinary biomarkers and their metabolites have been used to evaluate children exposure to different pesticides (Eskenazi et al., 1999; Qiao et al., 2001). These epidemiological data have indicated that the urinary concentrations of the primary CPF metabolite were up to two times higher in children than levels observed in comparable studies with adults. The great majority of available animal studies has been carried out in rats and indicates that CPF has higher systemic toxicity in neonates and weanlings than in adults (Pope et al., 1991; Zheng et al., 2000). CPF exposure in

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doses below the threshold for systemic toxicity exerts disruptive effects on neural cell development, with respect to DNA synthesis (Dam et al., 1998), gene transcription (Crumpton et al., 2000), cell differentiation (Roy et al., 1998), and synaptogenesis (Dam et al., 1999a,b). Furthermore, several rat studies have reported behavioral effects of CPF following similar subtoxic exposures. Short-term effects include delay in reflex development and alterations in locomotor activity in immature rats (Carr et al., 2001; Dam et al., 2000), and spatial learning deficits in weanling and adult rats (Jett et al., 2001; Levin et al., 2001). These effects appear to be gender selective and dependent on time of CPF administration. Most of the neurobehavioral adverse effects occur in the absence of significant AChE inhibition. AChE inhibition remains a reliable endpoint for acute CPF toxicity in adult animals (Dementi, 1999), but the same parameter might be misleading for detecting developmental effects on the brain following low-level CPF exposures (Pope et al., 1991). Developing animals have indeed a much faster rate of recovery of AChE activity compared to adults (Chakraborti et al., 1993). More than 80% of total brain AChE activity in adult rodents is due to the membrane-bound enzyme (Inestrosa et al., 1994; Lazar et al., 1984; Ochoa et al., 1982), and the remaining to the soluble intracellular portion. By contrast, during postnatal development (1–28 days) most of total brain AChE activity is due to the soluble portion and consequently, the AChE-soluble/membrane ratio is higher in pups than in adults (Bisso et al., 1988; Niemierko and Skangiel-Kramska, 1976; Wilson and Walker, 1974). Furthermore, AChE can be separated by sedimentation analysis into multiple molecular forms, catalytically equivalent within a given animal specie (Vigny et al., 1978). The monomeric globular (G1, 4S) and the tetrameric globular (G4, 10 S) are the predominant forms in mammalian brain. Each of these forms may be freely soluble or tightly bound within membrane (Schegg et al., 1992), and the relative proportion of the molecular forms of AChE changes in relationship to developmental stages of mammalian brain (Massoulie et al., 1993; Rieger and Vigny, 1976). In addition, membrane-bound G4 appears to be the physiologically critical form of AChE in CNS cholinergic neurons, whereas secreted soluble G4 may participate in important noncholinergic functions (Appleyard et al., 1988; Greenfield et al., 1980). So far, developmental studies in rats have used inhibition of total AChE as the main marker of CPF cholinotoxic effects, and CPF effects on the different AChE molecular forms have not been investigated. Considerable evidence also suggests that CPF could interfere with neurobehavioral development by acting on neurotransmitter systems in addition to the cholinergic system (Crumpton et al., 2000; Qiao et al., 2001; Slotkin et al., 2002). The analysis of a wider range of behavioral responses other than those traditionally thought to be cholinergic-mediated might therefore help to elucidate the mech-

anisms underlying the behavioral toxicity of developmental CPF exposure. The present study consists of a longitudinal analysis of behavioral development of CD1 mice exposed to subtoxic CPF doses either on pnds 1 through 4 or 11 through 14, a treatment regimen extensively used in the rat (Slotkin et al., 2002). Our aim was threefold: 1. to assess CPF effects at different developmental stages (neonatal, weaning, adolescent, and adult stage) and on different behavioral endpoints, ranging from those reportedly sensitive to CPF action in the rat (locomotor activity, habituation, and cognitive performances) to those not strictly cholinergically regulated, such as (i) ultrasound emission and orientation to home nest material in the neonates and (ii) social/affiliative behaviors and novelty-seeking behavior in adolescent mice; 2. to measure CPF anticholinesterase effects on total, soluble, and membrane-bound AChE activities, providing more information on the CPF cholinotoxic effects during the developmental phase, and to analyze how the CPF inhibitory effects influence the G4 and G1 AChE isoforms in the soluble fractions, where G1 relative concentration is much higher in newborn than in young and adult animals; 3. to extend to the mouse species information on adverse CPF effects, up to now limited to the rat. The availability of genetically manipulated mice for OP detoxifying esterase (Shih et al., 1998) makes information on developmental CPF toxicity in the mouse relevant for future studies on individual susceptibility to pesticides’ toxic effects. CPF treatment effects were evaluated in both sexes, because previous data in rats show that behavioral effects of CPF are sexually dimorphic. In particular, behavioral patterns maturing at adolescence are markedly sexually dimorphic (Palanza et al., 2001; Terranova et al., 1993; Venerosi et al., 2001) and are thus extremely sensitive for revealing gender differences.

Methods Animals and treatment Male and female mice of an outbred Swiss-derived strain (CD-1), weighing 30 –35 and 25–27 g, respectively, were purchased from a commercial breeder (Charles River, Calco, Italy). Upon arrival at the laboratory, the animals were housed in an air-conditioned room (temperature 21 ⫾ 1°C, relative humidity 60 ⫾ 10%) with lights on from 8 AM to 8 PM. Adult virgin males and females were housed in same-sex pairs in 33 ⫻ 13 ⫻ 14-cm Plexiglas boxes with a metal top and sawdust as bedding. Pellet food (Enriched standard diet purchased from Mucedola, Settimo Milanese,

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Italy) and tap water were continuously available. After 3 weeks of acclimatization, breeding pairs were formed. Females were inspected daily for the presence of the vaginal plug (Pregnancy Day 0). The stud was removed 10 days after the discovery of the vaginal plug. Thirty-six litters were used and culled at birth to at least five males and five females, whenever possible. On the day of birth, pups were tattooed with blue ink on their backs for individual identification. CPF (Chem. Service, West Chester, PA) was dissolved in dimethyl sulfoxide to provide rapid and complete absorption (Whitney et al., 1995) and was injected subcutaneously in the nape of the neck in a volume of 3.3 ml/kg body weight; control animals received vehicle injections on the same schedule. Two CPF doses (1 or 3 mg/kg) were selected within the range of doses used in previous rat studies (a pilot study showed that the 5 mg/kg dose slightly affected body weight in mouse pups), and these produce neurochemical alterations in the developing brain without eliciting systemic toxicity, weight loss, or mortality (Slotkin et al., 2002). Both doses were administered in both treatment schedules (pnds 1– 4 or 11–14) to evidence potential dosedependent effects of CPF in developing mice (Fig. 1). Within each litter, all pups were treated and at least one male and one female were randomly assigned to vehicle (VEH), one male and one female to CPF 1 mg/kg (CPF1), and one male and one female to CPF 3 mg/kg (CPF3) treatment (split-litter design). All the pups from each litter underwent the entire series of behavioral tests, with the exception of the novelty-seeking test, where CPF1 animals were not assessed. Mice treated with CPF were monitored for signs of cholinergic intoxications (tremors, etc.) at 1 and 6 h following injection. Litters were weaned on pnd 23 and housed in cages containing littermates of the same sex. Additional animals were used to control for CPF effects at an age when AChE has attained the adult-like profile of activity and they were treated with CPF3 for 4 days from pnd 32 to 35. Biochemical assays Tissue preparation (pnds 0 –35 untreated animals; pnds 4, 14, 35 CPF-treated animals) A first aim of the biochemical analysis was to determine the developmental profile of both total and soluble AChE forms in the mouse species. To this aim, untreated male mice were sacrificed on pnd 0, 1, 5, 11, 14, 21, 28, or 35 (n ⫽ 10 in each age group). As for treated animals, analysis was performed 1, 4, or 24 h from the last VEH or CPF treatment. Treated and untreated mice were killed by decapitation, the whole brain minus cerebellum was rapidly removed, weighed, and separated in two groups as follows: Group 1: Preparation of total AChE (membrane-bound plus soluble). Tissues were homogenized in 20 vol of cold 0.038 M Tris-HCl buffer, pH 7.6, using a Braun Potter S for 3

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Fig. 1. Schematic diagram of study design. Pnds 1– 4 study: mice received subcutaneous injection of CPF (1 or 3 mg/kg) on pnds 1 through 4, and selected neonatal behavioral patterns were assessed in the first 2 postnatal weeks (ultrasonic vocalization profile on pnds 5,8, and 11, homing task on pnd 10). Afterward a locomotor activity test was carried out on pnd 25, a novelty seeking test on pnd 35, a social interaction test on pnd 45, and a passive avoidance learning task on pnd 60. In the pnds 11–14 study, mice were administered with CPF (1 or 3 mg/kg) on pnds 11 through 14, and then tested for locomotor activity on pnd 25, novelty seeking on pnd 35, social interaction test on pnd 45, and passive avoidance learning task on pnd 60.

min. Aliquots (100 ␮l) of the homogenates were used immediately for enzymatic analyses. Group 2: Preparation of soluble AChE. The homogenates obtained as in group 1 were then centrifuged at 100,000 g for 1 h in a Centrikon T-1055 (Kontron) ultracentrifuge and supernatants used for enzymatic analyses. Enzymatic analysis AChE activity was determinated by a well-known and suitable spectrophotometric method (Ellman et al., 1961). Acetylthiocholine (AcThCh) 0.56 mM was used as substrate and incubation was in 0.05 M sodium phosphate buffer, pH 7.2, and lasted 30 min at 37°C (the absorbance was measured at ␭ ⫽ 412 nm). Specificity of the spectrophotometric method has been already described by using specific acetylcholinesterase (BW284C51) and pseudocholinesterase (iso-OMPA) inhibitors (0.01 mM) (Meneguz et al., 1992; Volpe et al., 1990; Buratti et al., 2002). Soluble AChE G1 and G4 molecular forms determination The molecular forms of AChE were separated by ultracentrifugation in sucrose gradient as previously described (Bisso et al., 1988). In brief, 150 ␮l of a fresh 100,000 g supernatant from group 2 was layered on 4.8 ml of 5–20% linear sucrose gradient and centrifuged at 38,000 rpm in a SW55B rotor in a Centrikon T-1055 (Kontron) ultracentrifuge for 18 h. In some experiments, catalase (11.3 S) and bovine serum albumin (4.3 S) were used as sedimentation standards. Seven-drop fractions were collected from the

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bottom of each centrifuge tube and AChE was determined in each fraction. The distribution profiles of individual molecular forms were evaluated. The G4 and G1 AChE molecular form inhibition was determinated by Ellman’s spectrophotometric method using acetylthiocholine as already described for the enzymatic analysis. All biochemical results were expressed as nanomoles of AcThCh hydrolyzed per minute per gram of tissue. Behavioral assessment Neonatal stage Ultrasonic vocalization (pnds 5, 8, 11). The ontogenetic pattern of ultrasound emission in rodents has proven to be a reliable diagnostic tool to detect changes in neurobehavioral development brought about by a variety of perinatal treatments (Adams, 1982; Branchi et al., 2001; Cagiano et al., 1988; Calamandrei et al. 1999). Within each litter, male and female pups assigned to the different treatments (N ⫽ 10 in each final VEH, CPF1, and CPF3 group) were assessed for ultrasonic vocalization during a 3-min test. Ultrasonic calls were recorded in a sound-attenuating chamber (Amplisilence, I-10070 Robassomero, Italy) during the dark period between 10:00 and 15:00 h. Single pups were removed from the litter and individually placed in a double-wall glass container (diameter, 5 cm; height, 10 cm). Two openings in the wall allowed water to be continuously pumped from a water bath into the double wall and, ultimately, to return to the pump. This maintained the surface at the same temperature (28 ⫾ 1°C). The number of ultrasonic calls emitted during the 3-min test was assessed listening to the audible frequencies output of a S-25 Bat Detector (UltraSound Advice, London, UK) tuned to a ⫾ 40 kHz range centered on 60 kHz according to the procedure described by Santucci et al. (1994). Homing (pnd 10). On pnd 10, male and female mice assigned to the different treatments (N ⫽ 10 in each final VEH, CPF1, and CPF3 group) were separated from the dam and kept for 30 min in an incubator (Elmed Ginevri 0GB 1000, Roma, Italy) at 28 ⫾ 1°C. Individual pups were then transferred to a Plexiglas arena (36 ⫻ 22.5 cm, walls 10 cm high) maintained at 28 ⫾ 1°C, with the floor subdivided by black lines in 12 quadrants. Wood shavings from the home cage were evenly spread under the wire-mesh floor on one side of the arena (14 ⫻ 22.5 cm, goal arena) and the pup was placed close to the wall on the opposite side. The time taken by the pup to place both forelimbs on the goal area was recorded (cutoff time 3 min). In addition, the pup’s overall activity was measured by counting the number of quadrants entered during the 3-min test period. Weaning stage: Locomotor activity (pnd 25) On pnd 25, mice were transferred to the experimental room and, after 20 min of acclimatization, they were individually introduced for 20 min into a clean Plexiglas arena

(55 ⫻ 55 ⫻ 50 cm) provided with two series of infrared beams (low and high level). Locomotion (horizontal movements, breaking of low-level infrared beams), Rearing (vertical movements, breaking of high-level infrared beams), and thigmotaxis (time and distance traveled immediately close to the walls, breaking of low-level infrared beams immediately close to the walls) were measured by an automated device (Acti-track system, Panlab, 08029 Barcelona, Spain). The test was carried out between 10:00 AM and 1:00 PM. (CPF 1– 4 group: VEH males ⫽ 17, VEH females ⫽ 13; CPF 1 males ⫽ 8, CPF 1 females ⫽ 14; CPF 3 males ⫽ 16, CPF 3 females ⫽ 10. CPF 11–14 group: VEH males ⫽ 18, VEH females ⫽ 12; CPF 1 males ⫽ 18, CPF 1 females ⫽ 15; CPF 3 males ⫽ 14, CPF 3 females ⫽ 12). Adolescent stage Novelty seeking (pnd 35) Apparatus. the experimental apparatus consisted of an opaque Plexiglas rectangular box with smooth walls, which was subdivided into two compartments (20 ⫻ 14 ⫻ 27 cm). The door between the two compartments, one black and one white, could be closed by means of a temporary partition. Each compartment was provided with four pairs of infrared photobeams, placed on the wall a few centimeters from the floor, 5.5 cm apart. Each beam interruption caused by the mouse was recorded by a computerized system. A custommade software extensively used to analyze these data (Adriani et al., 1998; Adriani and Laviola 2002) provided the following measures: (1) time spent in each compartment, (2) activity rate in each compartment (number of beam interruptions/second), (3) frequency of passages between the two compartments (number of passages/minute), and (4) latency (time between the opening of the partition and the first entrance in the novel compartment). The whole session was automatically subdivided into 5-min intervals. Procedure. The whole experimental schedule took a total of 4 days, each subject being tested between 9:30 AM and 12:30 PM. The white compartment of the apparatus was the familiar one. This procedure has been adopted since novelty preference has been shown to be independent from environmental cues provided (Bardo et al., 1988). Days 1, 2, and 3 (familiarization). Male and female mice were individually placed for 25 min in the white compartment of the apparatus. Activity rate was recorded only during Day 1. Day 4 (test of novelty preference). Animals were placed in the same white compartment explored in the previous days (familiar compartment). After 5 min, the partition separating the two compartments of the apparatus was opened, and mice were allowed to freely explore both compartments of the apparatus (the familiar white and the novel black one) for 20 min. Activity rates and time spent in each compart-

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ment were recorded throughout the entire session. Only VEH and CPF3 mice of both sexes underwent the test (CPF 1– 4 group: VEH males ⫽ 9, VEH females ⫽ 9; CPF 3 males ⫽ 9, CPF 3 females ⫽ 7. In the CPF 11–14 group VEH males ⫽ 9, VEH females ⫽ 8; CPF 3 males ⫽ 9, CPF 3 females ⫽ 8). Social interactions (pnd 45) On pnd 45, males and females assigned to the different treatment groups (CPF 1– 4 group: VEH males ⫽ 8, VEH females ⫽ 10; CPF 1 males ⫽ 10, CPF 1 females ⫽ 10; CPF 3 males ⫽ 8, CPF 3 females ⫽ 10. In the CPF 11–14 group VEH males ⫽ 11, VEH females ⫽ 10; CPF 1 males ⫽ 9, CPF 1 females ⫽ 9; CPF 3 males ⫽ 8, CPF 3 females ⫽ 8) underwent a 20-min social encounter with an untreated animal of the same sex and comparable age and body weight [design and procedures as in (Terranova et al., 1993)]. Immediately prior to the beginning of the encounter, they were marked for individual recognition with a nontoxic marker. The encounter took place between 10 AM and 3 PM in a test cage identical to the home cage supplied with clean sawdust bedding. Behavior was videotaped under red light. Recordings were scored by an observer blind to the treatment received by each pair. The data were recorded using a keyboard event recorder system connected to a computer for analysis (Observer, Noldus, Wageningen, NL). The behavioral responses scored and their classification in two main groups as described below (nonsocial and social) are based principally upon the ethological profile of mouse behavior described by Grant and Mackintosh (1963) and Van Oortmerssen (1971) [for previous use in our laboratory see (Terranova et al., 1993)]. Nonsocial behaviors Exploring—Moving around the cage, rearing, sniffing the air, the walls, or the sawdust. Inactive—Laying flat or standing still, with the eyes closed or opened in the total absence of movements. Digging—Digging in the sawdust, pushing and kicking it around using the snout and/or both the forepaws and hindpaws. Self-grooming—Wiping, licking, combing or scratching any part of own body. Social behaviors Investigative behaviors. Anogenital sniffing—Sniffing the anogenital region of the partner. Nose sniffing—Sniffing the head, or the snout of the partner. Body sniffing—Sniffing any other region of the body except for the tail. Follow— Following the partner around the cage, without any quick or sudden movement. Squire—Following the moving partner while maintaining a constant nose contact with its fur (mostly near the anogenital area). Mutual circle—Partners mutually sniffing each other’s anogenital region, while describing tight circles with their reciprocal following movements and maintaining close nose-anogenital contact.

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Affiliative behaviors. Social rest—Laying flat or standing still (with the eyes closed or opened) while maintaining close physical contact with the partner, which may be, in turn, either inactive or involved in mate-directed activity (i.e., grooming or sniffing the partner). Allogrooming— Grooming partner’s body. Soliciting behaviors. Push under—Pushing the snout or the whole anterior part of the body under the partner’s body, and then resting. Crawl over—Crawling over the partner’s back, crossing it transversally from one side to the other. Agonistic behavior (observed only in males). Attack— Rushing approach carried on over the back of the partner, often accompanied by biting attempts. Aggressive grooming—Allogrooming markedly intense and persistent, performed leaning on the partner’s back with the forepaws, and accompanied by gross movements of the head of the attacker and by vigorous pulling of the fur of the partner with a marked involvement of the teeth. Tail rattling—refers to a species-specific rattlesnake-like movement of the tail. Offensive Upright Posture—the mouse stands on its hind legs facing the opponent aggressively. Defensive postures—this item is comprehensive of two defensive elements: (i) the classical Defensive Upright Posture, the animal’s standing on its hindlimbs pushing the aggressive opponent with its forepaws, and (ii) a Submissive Posture, the animal lays on its back, with its head directed backwards flat against the cage floor. Adult stage Passive avoidance learning (pnd 60) The avoidance apparatus (Ugo Basile, Comerio, Italy) consisted of a tilting-floor Plexiglas cage, divided into two compartments (the start and the escape compartments, 18 ⫻ 9.5 ⫻ 16 cm each) by a sliding partition door. The start compartment was white and illuminated by a white light located on the top, whereas the escape compartment was black and maintained in the dark. The metallic grid floor (bars of 0.3 cm in diameter spaced 5 mm apart) was connected to a scrambling shocker set at 0.3 mA. Avoidance tests were performed between 9:30 AM and 12:30 PM, that is, during the initial portion of the dark period. The procedure consisted of two phases, acquisition and retention, which took place on two consecutive days. Acquisition phase. Mice were individually placed into the start compartment facing away from the doorway. The sliding door between the compartments was raised, and the mouse was allowed to cross into the dark chamber. When the mouse crossed (4-paw criterion) lowering the tilting floor, the door shut and a 3-s 0.4 mA foot shock was delivered to the grid floor. The trial ended when the mouse gave the step-through response or remained in the start compartment for 120 s, whichever event occurred first. At

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the end of each trial, mice were removed from the apparatus and left undisturbed for a 45-s intertrial interval. The acquisition phase ended either when the subject had remained in the start compartment for two consecutive trials (learning criterion), or after 10 trials ended by stepping through. If the learning criterion was achieved before the 10th trials, then all remaining trials (to a total of 10) were considered as 120-s latencies. Retention phase (24 h later). The procedure consisted of a single trial not punished by foot shock. The retention trial ended when the mouse either gave the step-through response or remained in the start compartment for 120 s. Latency to step through was used as an index of retention. To avoid confounding effects of estrous cycle on female’s learning performance (Frick and Berger-Sweeney, 2001), only males underwent passive avoidance test (CPF 1– 4 group: VEH ⫽ 9, CPF 1 ⫽ 9; CPF 3 ⫽ 8. In the CPF 11–14 group: VEH ⫽ 10; CPF 1 ⫽ 10; CPF 3 ⫽ 7).

Table 1 Brain AChE activity of CPF 1– 4, 11–14, and 31–35 treated mice CPF doses

1 mg/kg

3 mg/kg

Total AChE

Soluble AChE

Total AChE

Soluble AChE

pnd 4 pnd 14 pnd 35

80 ⫾ 3* 106 ⫾ 2 n.d

77 ⫾ 13 109 ⫾ 1 n.d

77 ⫾ 3* 105 ⫾ 2 105 ⫾ 1

74 ⫾ 5* 107 ⫾ 2 97 ⫾ 2

Data are mean percentage of control AChE activity (⫾ SE). * P ⬍ 0.01 t test vs control; nd, not determined.

repeated measures within animals. t tests with Bonferroni correction were applied to perform post hoc comparisons on ANOVA results. Biochemical data were analyzed by Student’s t test.

Results General toxicity and body weight

Statistical analysis Analyses of variance (ANOVAs) were performed on body weight, ultrasound vocalizations, and homing data. The model of such analysis (Chiarotti et al., 1987) included a within-litter treatment factor (2 levels); litter as block with respect to sex and repeated measures; sex as fixed-effect factor within litter; repeated measures as fixed factor within subjects. Postweaning behavioral data were analyzed considering the single animal as the statistical unit. In particular, for locomotor activity, treatment and sex were considered as grouping factors. As for Novelty-seeking and Social interactions data, treatment and sex were considered as grouping factors and time blocks as repeated measures within animals. However, aggressive behavior items (normally not expressed by adolescent females) were analyzed only in males. Finally, as for passive avoidance data, treatment was the grouping factor and time blocks were considered as

Fig. 2. Brain total and soluble AChE (nmol AcThCh hydrolized/min/g tissue) during postnatal development (pnds 0 –35) in CD-1 mice. Values are mean ⫾ SD (n ⫽ 10 in each age group).

The two CPF doses did not cause any overt signs of cholinergic intoxication at either age of treatment. This is not surprising since depression of brain AChE activity in the order of 50% is usually associated with cholinergic signs (Bignami et al., 1975) and a correlation between depression of brain AChE activity and an appearance of cholinergic signs has been questioned (Allen, 1990). Lack of systemic toxicity by CPF dosages administered in the present study was also suggested by comparable rates of body weight gain in CPF- and VEH-treated 1– 4 and 11–14 pups [main effect of CPF treatment, F(2,14) ⫽ 0.0, ns, respectively] Biochemical assays Age-related changes in total and soluble AChE (pnds 0 –35). Total AChE activity in CD-1 mouse brain progressively increased throughout postnatal development (Fig. 2) whereas soluble AChE activity remained almost unchanged from Postnatal Day 5 to 35. Soluble AChE contributed almost 50% to the overall brain AChE activity during the first 5 pnds, while from the second postnatal week onward the soluble AChE represented a minor component of the total AChE. Total and soluble AChE inhibition in CPF 1– 4, 11–14, and 32–35 pups (Table 1). CPF1 and CPF3 doses induced a significant but not dose-dependent inhibition of brain total and soluble AChE activity only on pnd 4, following daily sc treatments on pnds 1– 4 [CPF1: total AChE t ⫽ 8.95, P ⬍ 0.01 and soluble AChE t ⫽ 2.37, P ⫽ 0.09; CPF3: total AChE t ⫽ 4.58, P ⬍ 0.01 and soluble AchE P ⫽ 4.01, P ⬍ 0.01]. The AChE inhibition was transient as it was only observed after a 1-h interval from the last treatment and not after longer intervals (4 and 24 h, data not shown). In Fig.

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Neonatal stage: Neonatal behavior (ultrasonic vocalizations and homing) in CPF 1– 4 pups CPF treatment failed to affect the number of US vocalizations recorded on pnds 5, 8, and 11 [F(2,18) ⫽ 0.02, ns]. The ontogenetic profile of ultrasound production did not differ in the three treatment groups (Fig. 4, left graph). In both vehicle and CPF-treated pups, and regardless of sex, ultrasound emission rate decreases from pnd 5 to pnd 11, as previously shown in this strain of mice [F(2,18) ⫽ 43, P ⬍ 0.01] (Calamandrei et al., 1999). The two-way interaction between CPF dose and testing age was also not significant. Homing test performance on pnd 12 was also not affected by CPF treatment (Fig. 4, right graph): latency to reach the nest area, time spent in the nest area, and locomotor activity during the 5-min test did not differ in CPF-treated and control littermates [F(2,18) ⫽ 0.19, ns; F(2,18) ⫽ 0.20, ns; F(2,18) ⫽ 0.33, ns, respectively], despite that CPF3 pups tended to have shorter latencies to reach the nest area than the other groups. Weaning stage: Locomotor activity (pnd 25) CPF 1– 4. No effect of the treatment was found on distance traveled, frequency of rearing, and time spent in central and peripheral area of the arena [F(2,82) ⫽ 0.41, ns; F(2,82) ⫽ 1.38, ns;] (Fig. 5, left panel). Neither a significant main effect of sex nor an interaction between treatment and sex on any of the parameters considered were found.

Fig. 3. Typical sedimentation profiles of G4 (11.3S) and G1 (4.3S) brain AChE molecular forms obtained from soluble AChE fraction, in control and CPF-treated mice (3 mg/kg). Upper panel, CPF administered pnds 1– 4; middle panel, CPF administered pnds 11–14; lower panel, CPF administered pnds 32–35.

3 inhibition of soluble AChE G1 and G4 molecular forms in 1– 4, 11–14, and 32–35 VEH and CPF3 treated pups is reported. In CPF 1– 4 pups, sacrificed 1 h after the last treatment, an inhibition of the soluble G4 molecular form was observed, while the G1 form appeared essentially unaffected (Fig. 3A). By contrast, in CPF 11–14 and 32–35 pups, sedimentation profiles of both G1 and G4 molecular forms appeared unchanged when compared to control animals (Figs. 3B and C). Analysis carried out on the G4/G1 ratio confirmed these observations: this parameter was significantly reduced in CPF-treated animals only in the pnd 1– 4 group [(mean values ⫾ SE, n ⫽ 6) CPF 1– 4: VEH ⫽ 0.50 ⫾ 0.02, CPF3 ⫽ 0.35 ⫾ 0.01; t ⫽ 5.36, P ⬍ 0.01] and not in the other two age groups [CPF 11–14: VEH ⫽ 0.53 ⫾ 0.02, CPF3 ⫽ 0.53 ⫾ 0.01, ns; CPF 32–35: VEH ⫽ 0.43 ⫾ 0.01, CPF3 ⫽ 0.49 ⫾ 0.02, ns]

CPF 11–14. As for distance traveled, ANOVA showed that main effect of the treatment in the CPF 11–14 group just missed statistical significance [F(2,83) ⫽ 2.85, P ⫽ 0.06] (Fig. 5, right panel). Post hoc comparisons conducted on ANOVA results showed that CPF-treated animals (both doses) were more active than controls (post hoc t83 ⫽ ⫺2.27, Ps ⫽ 0.02). No effect of the treatment was found on rearing frequency [F(2,83) ⫽ 2.04, P ⫽ ns] and on time spent in the central and peripheral area of the arena. Neither

Fig. 4. Right panel: Ultrasonic vocalization profile of CPF treated pups on pnds 5, 8, and 11. Data are mean number of calls. Left panel: Homing test on pnd 10. Data are mean latency to reach the nest area of the experimental arena (left) and number of crossings (an index of locomotor activity) during the 5-min test (male and female data pooled). Vertical bars to the right of each graph indicate pooled SE. Male and female data were pooled, n ⫽ 20 in each group.

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groups activity was higher in the familiar compartment [F(1,30) ⫽ 75.88, P ⬍ 0.01]. However, a trend toward a selective activation in the novel compartment was displayed by CPF-treated females.

Figure 5. Locomotor activity test on pnd 25 (male and female data pooled). Data are mean distance traveled in a 20-min test (⫾SE) in CPF 1– 4 and CPF 11–14 groups. *A significant increase in locomotion in CPF 11–14 treated mice (P ⬍ 0.05).

a significant main effect of sex nor interactions on any of the parameters considered were found. Adolescent stage Novelty seeking (pnd 35–38) CPF 1– 4: Day 1. No main effect of the treatment was found on locomotor activity measured during the 25-min familiarization session on Day 1 in the white compartment [F(1,30) ⫽ 0.02, ns] (see Table 2). Day 4: Activity rate. The final test consisted of two phases: in the first one, before partition opening, the subject could only explore the familiar compartment; in the second one, after partition opening, the subject could freely move between the familiar and the novel compartment. Activity rate in the first phase did not differ between control and CPFtreated pups [F(1,30) ⫽ 1.73, ns P ⫽ 0.19]. Locomotor activity rates significantly increased in the second phase in all groups, and the effect was more marked in CPF-treated pups of both sexes [Partition opening effect F(1,30) ⫽ 295.4, P ⬍ 0.01; Partition opening treatment F(1,30) ⫽ 6.89, P ⫽ 0.01, Fig. 6]. When locomotor activity was analyzed in the white and black compartment separately, no significant treatment effect was evident. In both treatment

Day 4: Novelty seeking. No significant main effects of the CPF treatment were found on novelty preference. Both control and CPF-treated animals, regardless of sex, spent more time in the novel black compartment after partition opening [F(1,32) ⫽ 138.5, P ⬍ 0.01]. Latency to enter the novel compartment was also unaffected by the treatment [F(1,32) ⫽ 0.14, ns]. CPF 11–14: Day 1. No effect of the treatment was found on locomotor activity measured during the 25-min familiarization session on Day 1 in the white compartment [F(1,31) ⫽ 0.55, ns] (see Table 2). Day 4: Activity rate. Activity in the familiar compartment did not differ between control and CPF treated pups [F(1,31) ⫽ 0.04, ns]. Locomotor activity rates significantly increased in the second phase in all groups, and the effect was more marked in CPF-treated pups of both sexes [Partition opening effect F(1,31) ⫽ 295.7, P ⬍ 0.01; Treatment ⫻ 5-min blocks (4,120) ⫽ 2.28, P ⫽ 0.06, P ⬍ 0.01 after Tukey post hoc comparisons in the fourth 5-min block, see Fig. 6]. When locomotor activity was analyzed in the white and black compartment separately, no significant treatment effect was evident. In both treatment groups activity was higher in the familiar compartment [F(1,31) ⫽ 49.63, P ⬍ 0.01]. Again, a trend toward a selective activation in the novel compartment was displayed by CPFtreated females. Day 4: Novelty seeking. No significant main effects of the CPF treatment were found on novelty preference. Both control and CPF-treated animals, regardless of sex, spent more time in the novel black compartment after partition opening [F(1,33) ⫽ 63.8, P ⬍ 0.01]. Latency to enter the

Table 2 Novelty-seeking test (pnd 35) CPF 1–4

Day 1 Activity in the familiar compartment (number of counts) Day 4 Novelty preference (%) Latency to enter the novel compartment (s) Activity rate in the novel compartment Activity rate in the familiar compartment

CPF 11–14

Vehicle

CPF 3 mg/kg

Vehicle

CPF 3 mg/kg

1838 ⫾ 162

1826 ⫾ 187

2116 ⫾ 183

2207 ⫾ 134

65.2 ⫾ 1.6 14.2 ⫾ 4.3 3.0 ⫾ 0.8 4.0 ⫾ 0.8

65.0 ⫾ 2.0a 15.2 ⫾ 3.7 3.4 ⫾ 0.7 4.9 ⫾ 1.3b

61.8 ⫾ 3.0 31.7 ⫾ 7.9 3.1 ⫾ 0.4 4.1 ⫾ 1.0

66.4 ⫾ 1.6a 25.5 ⫾ 3.7 3.4 ⫾ 0.8 4.5 ⫾ 0.7b

Data are mean ⫾ SE. a All animals spent more time in the novel than in the familiar compartment, P ⬍ 0.01. b All animals were more active in the familiar than in the novel compartment, P ⬍ 0.01.

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Fig. 6. Novelty seeking test on pnd 35. Data are mean activity rates before and after free access to the novel compartment. The gray arrow indicates time of partition opening. **A significant increase in locomotion in CPF 1– 4 and CPF 11–14 treated mice (P ⬍ 0.01). Vertical bars to the right of each graph indicate pooled SE.

novel compartment was also unaffected by the treatment [F(1,33) ⫽ 0.49, ns]. Social interactions on pnd 45 CPF 1– 4. Among nonsocial responses, ANOVA showed a main effect of the treatment on frequency of self-grooming [VEH, 14.9 ⫾ 1.2; CPF1, 10.3 ⫾ 1.1; CPF3, 12.4 ⫾ 1.1 (mean values ⫾ SE); F(2,49) ⫽ 4.22, P ⬍ 0.05], with both doses of CPF significantly reducing self grooming (P’s ⬍ 0.01 after post hoc comparisons). Among social responses, no differences between VEH- and CPF-treated animals were evident in the two categories of investigative and affiliative responses either when considering the single items or when they were grouped, with main effect of the treatment on the category soliciting responses just missing statistical significance [frequency: VEH, 1.6 ⫾ 0.5; CPF1, 1.5 ⫾ 0.4; CPF3, 3.7 ⫾ 1.0; F(2,50) ⫽ 2.78, P ⫽ 0.07; VEH vs CPF3, P ⬍ 0.01 after post hoc comparisons]. Male agonistic behavior was significantly affected by CPF treatment as for frequency of aggressive grooming, increased in CPF1 mice [VEH, 2.3 ⫾ 0.9; CPF1, 7.7 ⫾ 2.2; CPF3, 16.5 ⫾ 1.9; F(2,25) ⫽ 2.96, P ⫽ 0.07, VEH vs CPF1 and VEH vs CPF3 P’s ⬍ 0.01 after post hoc comparisons]. In addition, when considering the category “agonistic responses” as a sum of the single items some treatmentinduced differences on temporal patterns of agonistic responses were evident (Fig. 7, upper graphs). Both frequency and duration of agonistic responses did not display variations throughout the 20-min session in the VEH group, whereas such responses were preferentially emitted in the initial phases of the session in CPF1-treated mice and in the last phases of the session in CPF3-treated mice [interaction Treatment ⫻ 5-min block, frequency F(6, 69) ⫽ 3.72, P ⬍ 0.01; duration, F(6,69) ⫽ 3.58, P ⬍ 0.01; VEH vs CPF1, P’s ⬍ 0.01 after post hoc comparisons in the first two blocks; VEH vs CPF3, P’s ⬍ 0.01 after post hoc comparisons in the last two blocks]. CPF 11–14. No treatment-induced differences were evident among nonsocial responses and among investigative and

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affiliative social responses. Among soliciting responses, ANOVA yielded a main effect of the treatment on the push-under response [frequency: VEH, 1.0 ⫾ 0.4; CPF1, 1.4 ⫾ 0.3; CPF3 2.7 ⫾ 0.8; F(2,50) ⫽ 3.57, P ⬍ 0.05, VEH vs CPF3, P ⬍ 0.01 after post hoc comparisons]. Male agonistic behavior was affected by CPF treatment: the category “agonistic responses” appeared significantly enhanced after both doses of CPF [main effect of Treatment: frequency F(2,25) ⫽ 2.94, P ⫽ 0.07; duration, F(6, 69) ⫽ 3.34, P ⫽ 0.05; P’s ⬍ 0.01 after post hoc comparisons, shown in Fig. 7, lower graphs]. Adult stage: Passive avoidance learning on pnd 60 CPF 1– 4 and 11–14: CPF treatment did not affect acquisition of the passive avoidance task. Both latencies to step through and number of trials to reach criterion did not differ between control and CPF-treated mice [CPF 1– 4: F(2,23) ⫽ 0.54, ns; F(2,23) ⫽ 0.83, ns respectively; CPF 11–14: F(2,24) ⫽ 0.09, ns; F(2,23) ⫽ 0.05, ns respectively]. No differences were evident in the 24-h retention performances [CPF 1– 4: F(2,23) ⫽ 0.34, ns; CPF 11–14: F(2,24) ⫽ 0.98, ns].

Discussion The results of this study indicate that developmental exposure to CPF at doses that do not cause systemic toxicity induces long-term behavioral alterations in the mouse. The

Fig. 7. Social interaction test on pnd 45. Data are mean total aggressive responses displayed by 45-day-old mice during a 20-min social interaction test. CPF 1– 4: Frequency of aggressive responses of CPF1 mice were significantly higher than Vehicle in the first two 5-min blocks; frequency of aggressive responses of CPF3 mice were significantly higher than Vehicle in the last two 5-min blocks. CPF 11–14: Both CPF1 and CPF3 mice displayed higher frequency and duration of aggressive responses throughout the 20-min test. Vertical bars to the right of each graph indicate pooled SE.

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behavioral changes reported here are mild and related to locomotor activity levels in a novel environment and to expression of social behavior. With the exception of hyperactivity recorded in the weaning phase (observed only in animals treated on Days 11–14), both periods of CPF exposure altered similar behavioral responses with respect to reaction to novel environmental and social cues. Finally, in accordance with rat data, the behavioral effects of CPF do not appear to be directly related to AChE inhibition. A significant AChE inhibition (about 25%) was found in mice treated with CPF on pnds 1– 4, 1 h but not 24 h from the last CPF administration. The percentage of inhibition is comparable to that reported for rats at the same age and for similar CPF doses (Dam et al., 2000; Slotkin et al., 2002), but the transience of the effects appears characteristic of the mouse species, and it is likely due to species-specific differences in AChE turnover and metabolism. Of note, such transient inhibition was found in both the membrane-bound and the soluble AChE fractions. These results rule out the hypothesis that CPF adverse effects might be underestimated in developmental studies when measuring the total AChE fraction only. No significant inhibition was found following CPF treatment on pnds 11–14 and on pnds 32–35, thus indicating an apparent selective vulnerability of the pnds 1– 4 mouse CNS to the anticholinesterase action of CPF. Nevertheless, it cannot be excluded that the rapid AChE activity increase by pnds 5 could “mask” a persisting moderate inhibitory effect. Previous rat studies evidenced a significant AChE inhibition also after CPF injections on pnds 11–14, but direct comparisons with the presents results should be avoided because the dose of CPF was higher than the one we administered to mice. On pnd 4 the anticholinesterase action of CPF targets primarily the G4 form, the one also affected by diisopropyl fluorophosphate exposure both at adulthood and in the first postnatal week (Meneguz et al., 1989, 1992). These results indicate that developmental CPF exerts its anticholinesterase action through the same mechanisms described for other OPs in the adult rodent brain rather than by age-specific mechanisms. Unfortunately, the amount of biological samples derived by brain pup homogenates was not sufficient to examine also the membrane-bound G4, that appears to have a critical function in the CNS cholinergic neurons (Schegg et al., 1992). At variance, secreted soluble G4 participates in noncholinergic functions, as shown by the release of soluble G4 from cerebellum and from dopaminergic neurons of substantia nigra after stimulation through mechanisms totally unrelated to cholinergic transmission (Appleyard et al., 1988; Greenfield et al., 1980). The overall reduction of soluble brain AChE activity in CPF3-treated 1– 4 pups is due almost exclusively to inhibition of this soluble G4 form. Thus, the biochemical data strongly support that evaluation of CPF neurotoxicity should not be based uniquely on AChE metabolism and turnover analysis. Furthermore, most of the behavioral effects were found following both CPF treatment schedules (pnds 1– 4 and pnds 11–14), whereas

AchE inhibition was found only immediately after the pnd 1– 4 exposure. Such discrepancy suggests, in line with developmental CPF studies in rats (Dam et al., 1999b; Slotkin et al., 2002), that neurotransmitter systems other than the cholinergic system, are very likely targeted by neonatal CPF exposure. The mouse neonatal behaviors analyzed in the present study, related to the pup’s response to isolation from the nest environment and modulated by several neurochemical systems (see Bignami, 1996; Branchi et al., 2001; Kehoe et al., 2001), are not affected by early CPF treatment. Gross effects of CPF on mouse sensorimotor coordination can be ruled out, since pup locomotor competences assessed during the homing test are unaffected by CPF; we cannot exclude, however, that maturation of single sensorimotor reflexes are targeted by CPF also in the mouse species, in accordance with that reported in neonate female rats by Dam et al. (2000). Locomotor activity in the weaning stage is enhanced in both sexes but only after later treatment (pnds 11–14). These findings are in full agreement with the decreased habituation rate (increase of locomotor activity) observed in rats of 4/8 postnatal weeks receiving CPF on pnds 11– 14 (Levin et al., 2001). Other rat data, however, indicated a decrease in locomotor activity and rearing behavior in adolescent males following treatment with CPF 1 mg/kg on pnds 1– 4 (Dam et al., 2000). Despite the direction of the effect and the period of CPF exposure for this parameter in mice and rats appearing to differ, as a whole these data confirm that locomotor activity is a sensitive endpoint for the toxic effects of low repeated CPF doses in rodents. As concerns adolescent behaviors, CPF treatment partially affects a typical age-specific response, such as the novelty-seeking behavior, that reflects the high levels of behavioral arousal of periadolescent rats and mice. This behavioral pattern is significantly associated with reward processes and related dopaminergic brain areas (Dellu et al., 1996; Laviola and Adriani, 1998). The alterations found in this test, which are comparable in both treatment groups, for both CPF doses, and in the two sexes, did not involve the novelty preferences per se. Both CPF and vehicle-treated mice also displayed activity rates lower in the novel than in the familiar compartment, indicating appropriate risk assessment when exposed to novelty. In accordance with activity data on pnd 25, the effects of CPF again are related to locomotor activity levels and emerged only when animals had free access to the novel compartment, suggesting that the test challenge uncovered subtle differences in reaction to novelty. Similarly, the alterations in locomotor activity previously reported in rat studies have been recorded in a novel experimental context (Levin et al., 2001). Finally, as already observed in rats, significant sexual differences did not emerge in this context, without considering a trend for CPF females to be more active than CPF males in the novel compartment. The social behavior repertoire analyzed on Day 45 indi-

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cated that CPF treatment at the higher dose increased the expression of soliciting responses in both treatment groups and in both sexes. This trend could suggest either a higher propensity to interact with a social mate or enhanced arousal in responding to social cues. The latter hypothesis seems to be supported by the increase of agonistic items in CPFtreated males. When considering the aggressive items as a whole, CPF mice treated on pnds 11–14 displayed more agonistic responses than vehicle mice, and the low CPF dose was the most effective. The presence of aggressive components in male social behavior at a stage when the affiliative/social components of the interaction should be prevalent might suggest an alteration in the normal development of social behavior patterns (Terranova et al., 1993; Venerosi et al., 2001). Further studies are warranted to characterize possible effects of CPF treatment on social behavior at adulthood, when male intraspecific agonistic behavior has a more relevant functional role in the ecoethological needs of the mouse species (Alleva, 1993). Finally, adult performance in an inhibitory avoidance task in mice was not altered by neonatal CPF treatment. This learning task, that is sensitive to alteration of cholinergic functions in rodents (Bammer, 1982; Calamandrei et al., 1996), requires the animals to withhold a spontaneous locomotor response (step-through). Interestingly both control and CPF-treated mice successfully inhibited the stepthrough response, thus indicating that the increased activity levels shown by CPF-treated animals in the previous tests did not bias the avoidance performance. Possible effects of neonatal CPF on shock sensitivity thresholds, that could mask associative learning alterations in treated mice, cannot be dismissed on the basis of our results. As a whole, we cannot exclude the possibility that the use of other learning paradigms, focused on spatial orientation, could provide evidence for CPF effects on mouse learning too (for rat data see Levin et al., 2001; Jett et al., 2001). CPF was thought initially to interfere with only cholinergic developmental markers (Dementi, 1999; Pope et al., 1991). However, effects on noradrenergic and dopaminergic function have been recently reported, based on measurements of transmitter turnover and response to drug challenges (Slotkin et al., 2002). Our present findings in the mouse species support the view that subtoxic CPF exposures can produce neurobehavioral effects long after termination of exposure, likely due to functional alterations in multiple CNS regions regulated by different neurotransmitters. As a whole, the hyperactivity found in 11–14 CPFtreated mice in a standard test of open field on pnd 25 and in both treatment groups (1– 4 and 11–14) in the more challenging novelty-seeking task could be interpreted as the behavioral output of a reduced cholinergic inhibitory tone induced by the CPF treatment in the first 2 postnatal weeks, a critical period for development of basal forebrain cholinergic function and late hippocampus synaptogenesis (Berger-Sweeney and Hohmann, 1997; Calamandrei et al.,

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1996; Semba and Fibiger, 1988). However, dopaminergic mechanisms are also strongly implicated in novelty-induced motor activity in rodents (Hooks and Kalivas, 1995). The effects on social/affiliative behavior are more difficult to interpret: of note is the convergence of effects, as both treatment schedules tend to affect similar behavioral items within the context of social behavior, namely increasing “soliciting” responses in both sexes and enhancing aggressive components in males. The involvement of other neural systems (primarily catecholaminergic and GABAergic) can be hypothesized; as an example, social/investigative responses in rodents are extremely sensitive to agents affecting GABAergic (Laviola et al., 1991; Piret et al., 1991) and catecholaminergic neurotransmission (Kudryavtseva, 2000; Miczek et al., 1994). It must be pointed out that direct CPF toxic effects on cholinergic neurotransmission in early phases of postnatal development may in turn influence the functional maturation of GABAergic and/or catecholaminergic system (Berger-Sweeney and Hohmann, 1997). In conclusion, CPF appears to affect mouse neonatal brain development through multiple mechanisms, including interference with basal processes controlling cell replication and differentiation as well as specific effects on cholinergic system similar to effects in rats (Crumpton et al., 2000; Pope, 1999; Slotkin, 1999; Slotkin et al., 2002). The analysis of a wider range of behavioral and neurochemical endpoints than previously considered may help to elucidate the CNS targets for the delayed behavioral toxicity of CPF.

Acknowledgments This work was supported by ISS Project 1105/RI 20012002 and by the joint Italy-Russia collaborative research project n.N73⬘Med3 “Developmental role of central cholinergic system: evaluation of pre and perinatal treatments targeting the basal forebrain.” The authors acknowledge Paola Lorenzini for skillful technical assistance in biochemical determinations, Maria Puopolo for expert statistical advice, Maria Luisa Scattoni for skillful support in passive avoidance data collection, and Joanne Berger-Sweeney for critical comments on the manuscript.

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