Prolonged exposure to low doses of ozone: short- and long-term changes in behavioral performance in mice.

Two separate experiments were designed to assess the effects of ozone exposure on outbred CD-1 mice. In the first experiment, adult males were exposed continuously to O3 at 0, 0.3, or 0.6 ppm for 30 days and their behavior was assessed in a 5-min open-field test on exposure days 4 and 19 and on day 3 after the end of the exposure phase. In addition, mice performed a Morris water maze task from exposure day 24 to 28. In the second experiment, adult females were exposed from 30 days prior to the formation of breeding pairs until gestational day 17 to the same doses used in the first experiment. Litters were fostered at birth to untreated dams and neurobehavioral development of the offspring was investigated until adulthood. Specifically, somatic and sensorimotor development [postnatal day (PND) 2-20], homing performance (PND 12), motor activity (PND 21), passive avoidance (PND 22-23), water maze performances (PND 70-74), and response to a nociceptive stimulus (PND 100) were assessed. Results from both experiments confirm that exposure to O3 slightly but selectively affected neurobehavioral performance in rodents. Exposure to O3 did not grossly affect neurobehavioral development, whereas it consistently impaired reversal learning in the Morris water maze test in both prenatally and adult exposed mice. Moreover, longer latency to step-through in the first trial of the passive avoidance test and a decrease in wall rearing in the hot-plate test were recorded in O3 prenatally exposed mice. Except for the first open-field test, altered responses were observed only in animals exposed at the intermediate concentration of the gas. Adaptation and/or onset of compensatory mechanisms might be responsible for the lack of linear dose-response relationships.

Limited changes in handedness and morphine reactivity in CD-1 mice after pre- and postnatal ozone exposure.

Outbred CD 1 mice were either not exposed (control group) or exposed to ozone (O3) (0.3, 0.6, or 0.9 ppm), during foetal and neonatal life until the time of weaning (postnatal day (PND) 26). On PND 70 the subjects were tested for handedness using a paw preference task assessing both the animals' capability to reach a food pellet in a feeding tube and the individual preference for the use of one of the other forepaw. O3 exposure did not affect the animals' capability to learn the task but caused changes in handedness. Specifically, females exposed to the intermediate O3 concentration showed a reduced preference for the right paw than both their same-sex controls and 0.6 ppm males. On PND 100, mice underwent a hot plate test after IP treatment by either saline or morphine HCl (10 mg/kg). The results were generally in the direction of reduced drug sensitivity after exposure to the highest concentration. The evidence for this effect was more robust in the case of an organised avoidance response (wall-rearing) than in the case of a reflexive response (limb withdrawal); in the case of the former, latency data showed an effect on both males and females while frequency data showed an effect only in females. Overall, the O3 effects are suggestive of subtle CNS changes affecting mouse behavioural responses.

Behavioural and nociceptive response in male and female spiny mice (Acomyscahirinus) upon exposure to snake odour.

Predator cues (both mammalian odour or avian vocalizations) are known to elicit fear-associated responses in rodents, including analgesia. In previous studies it was reported that spiny mice fail to show fear responses when presented with the calls of an owl. In order to test the hypothesis that this species (living in semiarid and rocky areas) may react to stimuli coming from reptilian predators, 40 sexually mature spiny mice (20 males, 20 females) were individually exposed to a small cylinder containing either fresh sawdust or snake odour. Behavioural changes (5 min before and 15 min after odour exposure) as well as the subsequent performance in a hot-plate test (50±0.5°C) were assessed. Results indicate that exposure to the odour of a sympatric terrestrial predator affected both behavioural and physiological responses of spiny mice. Upon exposure to snake odour both sexes showed significant changes in the patterns of inactivity, sniffing, grooming, sniffing the stimulus object (SO), withdraw reaction and in the frequency of somersaults. However, males increased the frequency of rearing, sniffing the SO, decreasing grooming more than females. No analgesic effect of odour exposure emerged; however, males showed significantly shorter latencies and higher frequencies of hindpaw licking compared to females.

Increased number of mast cells in the central nervous system of adult male mice following chronic subordination stress.

Mast cells (MCs) are amine-storing cells with heterogeneous histological, biochemical, and functional properties. They are found in connective tissue as well as in the peripheral and central nervous system (CNS) of many mammalian species. In this study we investigated whether the distribution of MCs in the CNS of adult male CD-1 mice was modified following repeated defeat stress. Experimental subjects underwent a 3-week period of fighting encounters with a highly aggressive resident. On the test day they were divided into three groups: (a) paired with the resident for 20 min; (b) placed in a cage containing the soiled bedding of the resident for 20 min; (c) placed in a cage with clean sawdust for 20 min. Results show that previous defeat stress increases the number of MCs in the thalamus, habenula, and hypothalamus of subjects exposed to a fighting opponent or to a clean cage, compared to subjects placed in a cage with the bedding of the opponent or to a group-housed, nondefeated control. These results, together with previous reports in birds and rodents, suggest that MCs have a wider role than previously expected and might be involved in the behavioral response to highly relevant psychosocial stimuli.

Behavioral effects of peripheral interleukin-1 administration in adult CD-1 mice: specific inhibition of the offensive components of intermale agonistic behavior.

Peripheral administration of interleukin-1beta (IL-1beta) in rodents reduces exploratory behavior in a novel environment while decreasing social investigation of a juvenile conspecific. In this study we wanted to test the effects of peripherally administered IL-1beta on another aspect of the mouse social repertoire, namely intraspecific fighting towards an adult male intruder. In the first experiment, sickness behavior induced by IL-1beta (1 microg/mouse) in adult CD-1 mice was assessed by direct observation of behavioral changes following placement into a novel environment. Three hours after injection, subjects were individually introduced for 20 min in a cage with clean sawdust and a number of behavioral items recorded. Blood samples were collected at the end of the testing session. Body temperature was measured right before, 1 h and 3.5 h following injection. In IL-1beta treated mice, exploration (assessed by measuring duration and frequency of Wall Rearing and Rearing behaviors) was nearly totally suppressed, while duration and frequency of behaviors such as Grooming, Bar Holding, and Digging were also markedly reduced. Administration of IL-1beta significantly elevated CORT secretion above basal levels and, as previously reported for mice, induced hypothermia (about 2 degrees C). In the second experiment, we assessed mice receiving IL-1beta (0.25; 0.5 or 1 microg/mouse or saline solution) in a social context. Three hours after injection, subjects were placed into a neutral cage for 20 min with a non-injected adult male conspecific and aggressive behavior scored. Overall, IL-1beta administration affected the social repertoire of treated mice in a dose-dependent fashion. Specifically, agonistic components of aggressive behavior were nearly totally suppressed, while the defensive elements, such as Upright Defensive posture, Upright Submissive posture, Crouching, or Flee were not affected by IL-1beta. Overall these data support the notion that sickness behavior induced by IL-1beta administration represents an organized behavioral strategy and is not an aspecific response to an illness-type of condition.

A description of the ontogeny of mouse agonistic behavior.

The development of agonistic behavior was characterized in outbred Swiss CD-1 male Mus domesticus. At weaning (postnatal day [PND] 21), mice were housed either individually or as male pairs. Social encounters were carried out between dyads of initially unfamiliar same-age and same-housing subjects every 3rd day, from PND 23 to 47. The majority of both offensive and defensive elements had their onset around PND 29. Overall, their expression increased around puberty (i.e., on PND 35), which also represented the peak of an inverted U-shaped profile for the frequency of the "ambivalent" tail rattling behavior. A stability of dominance-submission relationships over development appeared, and early short latencies to display either the first crouched posture (subordinate) or the first attack (dominant) turned out to be possible predictors of adult social status. Ongoing individual housing was associated with a greater expression and an earlier onset of fighting behavior.

Behavioural characterization of interleukin-6 overexpressing or deficient mice during agonistic encounters.

Interleukin-6 (IL-6) is a cytokine released by activated immune cells which has been shown to affect brain function. In this experiment aggressive and affiliative behaviour exhibited during agonistic encounters by transgenic male mice either not expressing (IL-6 -/-) or overexpressing (NSE-hIL-6) IL-6 in the central nervous system was investigated. All subjects were isolated for 24 days before the aggressive encounter and were 52 days old at the time of testing. Subjects were placed for 5 consecutive days in a neutral cage for 15 min with an opponent of the Balb/c strain that had been previously isolated for the same amount of time. The first and the last test sessions were videotaped to evaluate the first approach and the establishment of the social role, respectively. A number of behavioural categories were later scored. When compared with wild-type controls, IL-6 -/- mice showed a higher degree of aggressive behaviour as indicated by a higher frequency of Offensive Upright Posture, an effect more pronounced on the fifth encounter. On the contrary, NSE-hIL-6 subjects showed a tendency to be more involved in affiliative-type social interactions, displaying a higher frequency and duration of behaviours such as Anogenital, Nose or Body Sniff. IL-6 -/- mice showed a clear tendency to exhibit less affiliative interactions compared with their controls while dopamine levels were found to be modified in a number of brain regions in these mice. Overall, these data suggest that IL-6 affects both aggressive and affiliative-type interactions, although the behaviour of the NSE-hIL-6 subjects was less affected than that of the IL-6 -/- group. The effects of the genetic background of the animal in screening the outcome of gene manipulations on behaviour are also discussed.

Postnatal NGF administration causes adult hyperalgesia and overreactivity to social stimuli but does not reverse capsaicin induced hypoalgesia.

The present longitudinal analysis was aimed at assessing (i) the effects of developmental capsaicin (CAPS) administration on nociceptive responsivity and on the response of adult mice to social stimuli; (ii) the action of NGF on the ontogeny of the same nociceptive response and social stimuli; (iii) whether capsaicin treatment could be reversed by subsequent treatment with NGF. CD-1 mouse pups were treated with either capsaicin (50 mg/kg, s.c.) or vehicle on postnatal days (PNDs) 5 and 8. Every other day from PND 9 to PND 21 the same pups received a daily injection of NGF (0.75 mg/kg, s.c.). During both the prepuberal stage (PNDs 14, 21, and 28) and adulthood, mice were repeatedly tested in a hot-plate apparatus (52 +/- 0.1 degrees C for 1 min). At adulthood they also underwent an aggressive behaviour test. NGF-treated mice showed a shorter latency to hindlimb licking response in the hot plate compared to both controls and NGF-CAPS groups. CAPS-treated subjects showed a long-lasting hypoalgesia at both prepuberal and adult stages that was not modified by subsequent NGF treatment. Finally, NGF-treated mice were more aggressive than both controls and CAPS-NGF animals.

NGF effects on hot plate behaviors in mice.

Adult CD-1 male mice were injected intravenously with 2.5 micrograms/g of highly purified murine NGF and then assessed for hot plate responding (52 degrees C) at 15, 30, 60, 180, and 360 min (repeated test) or at 30, 60, or 360 min (single test, i.e., exposure to hot plate only once). Control animals received cytochrome c (2.5 micrograms/g). In the repeated test, NGF produced hyperalgesia, increasing the number of jumps (particularly at 30 and 60 min postinjection), while in the single test the pain reaction of NGF-treated animals remained unaffected. Sensitization of C-fibers in the periphery or release of bioactive mediators from mast cells may account for NGF-induced changes in nociception.

Removal of the submaxillary salivary glands first increases and then abolishes the agonistic response of male mice in repeated social encounters.

Adult male mice of the CD-1 strain were sialectomized (bilateral removal of submaxillary salivary glands) under IP Nembutal anesthesia and then individually housed for 5 weeks. Control mice were sham operated. The behaviors of sialectomized and control mice towards untreated, intact, matched opponents were videotaped during the first 10 min of a 20-min social encounter repeated for 10 consecutive days (isolation days 36-46). On the first session, sialectomized mice exhibited significant increases in elements of aggression (attack, bite, offensive sideways, offensive upright, and tail rattling). These behavioral changes significantly decreased over the remaining encounters, while defensive behaviors (defensive upright, oblique, parry, and defensive sideways) and elements of arrested flight increased progressively. The results suggest that sialectomy, perhaps by removing salivary NGF, interferes with the coping response of mice towards repeated agonist challenge from a conspecific.

Substance P effects on intraspecific aggressive behaviour of isolated male mice: an ethopharmacological analysis.

Adult male mice of the Swiss CD1 strain were used to evaluate the effects on isolation-induced aggressive behaviour of a single intravenous administration of substance P (SP; 0.25, 1.0 or 2.5mg/kg dose). All mice were injected 15min before testing (10min videotaped dyadic encounters with an isolated male untreated opponent). Control mice were injected with vehicle. All animals were tested again 24h later in a drug-free state. SP treatment produced a decrease in offensive scores (Attacks and Rattling behaviour), a longer latency to the first Attack episode, and enhanced defensive displays. These effects were reversed 24h later. In no case did SP treatment affect locomotor activity levels or freezing behaviour. A role of SP in the regulation of murine aggressive response is strongly suggested through a direct action of the drug on the central nervous system and specifically on the hypothalamus.

Neonatal capsaicin exposure affects isolation-induced aggressive behavior and hypothalamic substance P levels of adult male mice (Mus musculus).

Subcutaneous administration of capsaicin (50 mg/kg) at Postnatal Days 2 and 5 exerted long-term effects on isolation-induced aggressive behavior of adult mice (Mus musculus) of the CD-1 strain. Isolated capsaicin-treated mice (scored during a 10-min session) showed the highest frequency and the longest duration of total attacks, attacks, rattling, and offensive upright posture when compared with nonisolated capsaicin-treated subjects and both isolated and nonisolated vehicle control animals. Hypothalamic Substance P (SP) was assessed by radioimmunoassay. Capsaicin treatment significantly lowered hypothalamic SP content in both isolated and nonisolated mice. Moreover, individual scores of isolated capsaicin-treated subjects showed a significant correlation between SP depletion and expression of offensive upright posture. Isolation per se was revealed to play an important role in depleting SP from the hypothalamus.

Capsaicin affects aggressive behavior, but not hot plate responding, of adult male mice.

Adult male mice of albino Swiss-derived CD-1 strain were used to assess the effects of capsaicin (a powerful agent that produces a marked depletion of the undecapeptide substance P) on both intraspecific aggressive behavior (induced by 8 weeks of individual housing) and pain sensitivity. Capsaicin was given SC, 48 h before behavioral testing. Aggressive behavior, scored during a 5-min session under red light, was significantly enhanced by capsaicin treatment (50 or 100 microliters of a 7.5 mg/ml solution). In fact, Total Aggressive Episodes, Attacks, and Upright Offensive Posture were significantly higher in the two capsaicin-treated groups, while Latency to the first Attack was decreased, when compared to both vehicle or unhandled controls. A concomitant decrease in Submissive Postures and Flee was also evident in capsaicin mice. Hot plate testing (55 +/- 0.1 degrees C, cutoff time 30 s), carried out on nonisolated mice, did not reveal any difference among the two capsaicin groups (same doses) and vehicle or unhandled controls.

Prenatal oxazepam enhances mouse maternal aggression in the offspring, without modifying acute chlordiazepoxide effects.

In the rat, behavioral changes during lactation are in several ways similar to those produced by benzodiazepines (BDZ). Moreover, an increased activity at the GABA/BDZ receptor complex has been found in both conditions. We tested the hypothesis that early manipulation of this neurochemical system by prenatal BDZ exposure should affect typical responses of lactating dams, such as maternal aggression. Outbred CD-1 mice were treated with either oxazepam (15 mg/kg PO twice/day on days 12-16 of fetal life) or vehicle and fostered at birth to untreated dams. Female offspring were subsequently mated at the young-adult stage and used to assess maternal aggressive responses towards a male intruder. In a first 5-min test on postpartum day 6, the prenatal oxazepam animals showed a reduced Latency to the First Attack, a markedly enhanced frequency of several offensive scores (such as Fighting Episodes, Attacks, and Offensive Upright, On Top, and Kicking Postures), a decrease of Submissive Postures and a reduced duration of time spent lying still (Out of the Nest). The tests were repeated 48 h later after IP treatment by either chlordiazepoxide (10 mg/kg) or saline. The drug significantly enhanced locomotor activity as well as the frequency of Fighting Episodes and of Attacks, while decreasing the number of Submissive Postures and the time spent On Nest. These effects were not significantly modified by prenatal oxazepam exposure. This suggests that long-term and acute effects of benzodiazepines are produced either by changes in different regulatory systems or by different types of changes in the same system.

Amphetamine, conditioned stimulus, and nondebilitating preshock effects on activity and avoidance: further evidence for interactions between associative and nonassociative changes.

A literature survey and preliminary experiments with rats on the consequences of shock preexposure on subsequent activity and escape or avoidance showed the need for further work on the interactions between nondebilitating preshock and various test and treatment factors. The two main experiments used 16 preexposure conditions, namely, presence or absence of unavoidable punishment (36 shocks of 2.5 mA and 5 sec subdivided in three daily sessions), a light CS, a central partition in the shuttle-box, and dl-amphetamine sulfate (1 mg/kg ip 15 min before each session). In both experiments the four factors studied exerted more than additive effects on activity in preexposure sessions, leading to a very high frequency of crossing in the CS-shock-no-partition-drug condition. Upon retesting for activity (Experiment 1) suppression of locomotion by prior shock was less marked in animals preexposed to CS-US pairings in the absence of partition, while proactive amphetamine effects consisted mainly of a progressive increase of activity over successive retest sessions in the groups not preshocked. Upon retesting for light-cued, two-way avoidance acquisition (Experiment 2) the groups preexposed to US only were mostly retarded, while those preexposed to paired CS and US were mostly facilitated. Other changes, including drug pretreatment consequences, were negligible or unsystematic, but in general the data showed that the effects of various preexposure conditions on activity could not account for those on avoidance. Overall, it appears that the interactions between nondebilitating preshock and other test and treatment factors can be further exploited to clarify the respective roles of various associative and nonassociative mechanisms in modulation of activity and adaptive responding in aversive situations.

Scopolamine and acquisition of go-no go avoidance: a further analysis of the perseverative antimuscarinic deficit.

Rats treated with scopolamine (0.5 mg/kg SC daily) during the acquisition of a discrimination task with symmetrical negative reinforcement (light-go, noise/light-no go) showed a learning impairment, with both active and passive avoidance deficits. In the initial stage of such training, however, fewer passive avoidance errors and more active avoidance errors were made by treated animals if active avoidance pretraining had occurred in the no-drug state. A similar experiment using the same stimulus arrangement with asymmetrical reinforcement (no punishment of intertrial, and no go signal, responses) showed a scopolamine effect consisting mainly of increased responding to extinction signals and during intertrial intervals, with little or no active avoidance deficit. Furthermore, interactions due to changes in treatment conditions in successive stages of training were minimized in the latter task, suggesting that the effects of the shift-no shift factor on distribution of errors in the early stages of active-passive avoidance learning were unlikely to have been due to a genuine drug dissociation. Overall, these results and others obtained previously in the same and related tasks tend to rule out some unidimensional explanations of antimuscarinic effects, e.g., response disinhibition (an exclusively motor deficit) or impairment of stimulus sensitivity (an exclusively sensory deficit). The data rather confirm the notion of a sensorimotor drug bias leading to a shift in response prepotencies depending jointly on stimuli, responses, and response consequences. Prior learning history and behavioural compensation for adverse treatment consequences at the reinforcement level may interact with the sensorimotor bias so as to produce "set perseveration" (perseveration of response tendencies).

Behaviorally augmented tolerance during chronic cholinesterase reduction by paraoxon.

Repeated injection of paraoxon to pretrained rats 2 hr before avoidance sessions, at a dose causing considerable intoxication symptoms and reduction of brain acetylcholinesterase (0.125 mg/kg SC daily), induced marked performance depression followed by progressive development of tolerance. Additional groups treated either after each session (i.e., 23.5 hr before each subsequent session), or treated and not tested, showed a substantial depression when shifted to treatment 2 hr before sessions after achievement of tolerance by the animals tested from the beginning of the experiment at the time of maximal paraoxon effect. This indicates that chronic paraoxon tolerance cannot be ascribed entirely to metabolic and/or physiological changes occurring as a consequence of repeated treatment per se, but must be explained at least in part by postulating a behaviorally augmented (or "learned") component. In an additional experiment chronic paraoxon animals (0.1 mg/kg SC daily) were indistinguishable from control rats with respect to acquisition of light/go, noise-light/no go discrimination, i.e., of an active-passive avoidance task known to be highly sensitive to the disrupting (response-disinhibiting) effect of antimuscarinics. Therefore, the enhanced sensitivity to antimuscarinics in organophosphate tolerant rats, which is usually ascribed to cholinergic receptor changes, does not appear to be associated with a spontaneous "antimuscarinic-like" syndrome.

Test factors affecting the time course of avoidance depression after DFP and paraoxon.

The time course of avoidance depression induced by DFP and Paraoxon in rats was measured in four experiments using sublethal doses which induced approximately equivalent changes at the time for maximal behavioral depression (3 hr after 1.1 mg/kg DPF or 0.25 mg/kg Paraoxon SC). The trends obtained with pretrained animals intoxicated for the first time, and not tested during the period between treatment and testing at any given interval (3, 8, 13, 18, and 24 hr after injection), served as baselines to assess (1) proactive consequences of one or more avoidance sessions on subsequent measurements, and (2) sensitivity changes upon repetition of treatment with the same or the other agent after a 5-week resting period. The changes in the time course of avoidance depression due to these factors were generally unimpressive. Some of the interactions observe, however, provided direct or indirect evidence (1) for an enhanced residual depression at long post-treatment intervals upon repetition of organophosphate intoxication; (2) for a proactive impairing effect sometimes appearing after behavioral testing at the time of maximal depression (3 hr), when total or near-total avoidance failure causes extensive exposure to shock; and (3) for a proactive facilitating effect sometimes appearing after testing at a time of moderate avoidance impairment (8 hr), which may be ascribed to behaviorally augmented tolerance ("learned" tolerance).