Offline consolidation of spatial memory: Do the cerebellar output circuits play a role? A study utilizing a Morris water maze protocol in male Wistar rats.

To address whether the cerebellum takes part to spatial memory consolidation related to navigation, male Wistar rats were trained daily (4 days), in a Morris water maze to found a submerged escape platform by use of distal cues (place training test). Retention of the allocentric map was evaluated in the probe test (without platform), before the place test. Bilateral shutdown of deep cerebellar nuclei was carried by infusion of the GABA-A agonist muscimol (0.25 µl at 1 µg/µl) immediately after each place test. Histology revealed a dorsal dentate nucleus (DDN) group, with muscimol diffusion confined to dentate nuclei, and a ventromedial/dentate nuclei (VMDN) group, with muscimol additionally involving fastigial, interpositus and vestibular nuclei. In the place test, Vehicle, DDN and VMDN groups reduced latency and distance to the platform over the 4 days and within the single day, indicative of efficient acquisition and working memory; navigational trajectories however differed in that, while Vehicle and DDN groups evolved to use direct paths, VMDN group indulged to navigate in proximity to the platform, suggesting an impairment in refining the spatial map. In the probe test VMDN, unlike Vehicle and DDN animals, failed to develop a preference for the quadrant where the platform was previously located, indicating a consolidation deficit. In conclusion, ventromedial cerebellar related structures may contribute to the process of consolidation of an allocentric spatial memory: their inactivation may have impaired the offline integration of idiothetic information with allothetic signals within the navigational network, leading to a coarse resolution map.

Bisphenol-A differently affects estrogen receptors-alpha in estrous-cycling and lactating female rats.

The effect of long-term exposure to bisphenol-A (BPA) on estrogen receptor-alpha (ER) immunoreactivity was studied in the medial preoptic area, arcuate nucleus and the ventromedial nucleus of the hypothalamus of estrous cycling and lactating female rats. Pregnant/lactating or estrous cycling rats were exposed to BPA (40 mg/Kg/day) or peanut oil. Lactating females showed fewer ER-immunoreactive cells than non-lactating females in the medial preoptic area and ventromedial nucleus of the hypothalamus. BPA induced an increase in ER-immunoreactive cells in the medial preoptic nucleus irrespective of lactation. BPA only induced a decrease in ER-immunoreactive cells in the arcuate nucleus of the lactating group; oil induced an increase in ER-immunoreactive cells in the lactating with respect to non-lactating group. The results demonstrate that exposure of adult females to BPA modifies the number of ERs.

Perinatal exposure to the estrogenic pollutant bisphenol A affects behavior in male and female rats.

Bisphenol A (BPA) is an environmental estrogen with potentially aversive effects on public health. In rats, we studied the effects of perinatal exposure to BPA on nonsocial behaviors partly influenced by gonadal hormones. BPA was administered orally to one group of mother rats at a concentration within the range of human exposure from 10 days before mating until the weaning of the pups. In a second group, BPA was given at a higher dosage during a critical period for brain organization, i.e., from day 14 of gestation until day 6 after birth. The offspring of the treated mothers were tested in the holeboard and the elevated plus-maze at 85 days of age. Various aspects of nonsocial behavior were affected by BPA, differently in males and females, confirming that exposure to a weak environmental estrogen in the period of sexual differentiation of the brain can influence adult behavior. However, contrary to our expectation, a clear masculinization of females was not observed. In general, the factor analysis indicated that in treated males both the motivation to explore and anxiety are reduced, while in females, motor activity and motivation to explore are depressed. Because there were no substantial differences between the two modalities of BPA administration, we suggest that the prolonged treatment with the low dosage compensates for the higher dosage given during a shorter steroid-sensitive period. This may be a cause of concern for public health, given the greater incidence of prolonged exposure of humans to low concentrations released into the environment.

Neuroendocrine and behavioral effects of CRH blockade and stress in male rats.

Our previous data have shown that restraint (RT), a mild nonpainful stressor, acutely impairs nonsocial and social behavior in male rats. Corticotropin-releasing hormone (CRH) is a regulator of these behavioral responses. To evaluate whether CRH mediates the neuroendocrine and behavioral alterations present 24 h after restraint stress, we administered the CRH antagonist alpha-helical CRH(9-41) (alpha-hCRH) intracerebroventricularly to male rats and we compared its effects with those of saline. Twenty-four hours after treatment, nonsocial behaviors were significantly decreased by alpha-hCRH, this effect being independent of RT. Among social behaviors, only introductory activity showed significant differences as a result of both RT and alpha-hCRH. The concentrations of ACTH in the plasma and those of beta-endorphin in the anterior and neurointermediate lobes of the pituitary were affected by alpha-hCRH treatment. The effect on ACTH was simply related to the administration of the alpha-hCRH, while for beta-endorphin, significant interactions between alpha-hCRH and RT were found. On the whole, these results point to the role played by CRH in the control of neuronal mechanisms involved in the stress-induced effects.

Sex differences in benzodiazepine binding in the frontal cortex and amygdala of the rat 24 hours after restraint stress.

Marked sex differences have been reported in behavioural responses of rats 24 h after exposure to a brief period of restraint (RT) stress. In the present study, differences in benzodiazepine (BZ) binding between male and female rat litter-mates randomly allocated to control or RT groups were investigated 24 h after RT. Scatchard analysis, using [3H] flunitrazepam, was carried out on the the frontal cortex and amygdala. In the frontal cortex, females had a significantly lower affinity and a greater number of BZ receptors than males; males, but not females, showed increased affinity after RT. In the amygdala, there was a tendency towards a greater number of BZ receptors in females, with no effect of RT on receptor number or affinity. These results provide evidence of sex differences in BZ binding both under basal conditions and 24 h after RT, which could contribute to the behavioural sex differences already reported.

Involvement of the 5-HT1A subtype receptor in the neonatal organization of agonistic behaviour in the rat.

5-hydroxytryptamine (5-HT) interacts with testosterone (T) in the development of a number of neuronal systems controlling sexually dimorphic adult behaviours. In this report, we investigated this interaction on the organization of agonistic behaviour in males, females, androgenized females (250 micrograms/pup of T proprionate on the day of birth), and males castrated on the day of birth. We have shown previously that manipulating 5-HT2 activity over the 2nd week of life modulates adult agonistic behaviour, depending on genetic sex and the presence of T. In this report, we investigated the effects seen in adulthood of a 5-HT1A agonist [8-OH-DPAT, 0.25 mg/kg, intraperitoneally (i.p.)] and antagonist (WAY100135, 0.25 mg/kg, i.p.) given over days 8-16 postpartum. The test for agonistic behaviour was carried out in a neutral territory against a matched conspecific, and introductory, offensive and defensive activities were note. Results show that neonatal administration of the 5-HT1A antagonist WAY100135 increases introductory activity and defense in the presence of neonatal T, independent of genetic sex, because these effects were seen in sham-castrated males and androgenized females. Offence followed a similar pattern, in that it was increased by WAY100135, but only in males. In the case of defence, the effects of the antagonist were reinforced by the action of the agonist (8-OH-DPAT) in both males and females, indicating an inhibitory role of 5-HT1A perinatal activity on defence in the presence of malelike levels of circulating T and a facilitatory role when levels of T are low or negligible. These findings indicate that 5-HT1A activity is involved in the development of agonistic behaviour and the effects are influenced by T. The results also show that the offensive and defensive facets of agonistic activity are controlled differently.

Neonatal organizational effects of the 5-HT2 and 5-HT1A subsystems on adult behavior in the rat.

Males, females, neonatally androgenized females, and neonatally castrated males were treated over the second week of life with 0.25 mg/kg of either the 5-HT2 agonist 1-(2,5-dimethoxy-3-iodophenyl)-2-aminopropane HCl (DOI), the 5-HT2 antagonist ritanserin (Rit), the 5-HT1A agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), or the 5-HT1A antagonist WAY100135 (WAY). Exploration, anxiety, sociosexual preferences, and sexual behavior were measured in adulthood. Agents acting on 5-HT1A receptors do not appear to affect organization of any of the behavioral systems studied. DOI increased exploratory activity but in females only, which suggests that testosterone antagonizes the stimulatory effect of 5-HT2 activity on exploration. Neonatal ritanserin selectively reduced anxiety in females, and DOI had a similar effect in androgenized females. This indicates that neonatal 5-HT2 activity is anxiogenic in normal females, anxiolytic in androgenized females, and has no effect on anxiety in males. Males and androgenized females both showed a preference for the female teaser that was abolished by the 5-HT2 agonist, DOI. These results point out that 5-HT2 activity selectively suppresses heterosexual preference induced in the presence of neonatal testosterone. DOI also reduced both male sexual behavior in males and female sexual behavior in androgenized females. Thus, the 5-HT2 system antagonizes the action of testosterone in stimulating heterosexual orientation and sexual activity, and this is independent of genetic sex.

Differential effects of restraint and novelty on the social behaviour of female rats.

In a previous work, we have described an increase of agonistic behaviour 24 h after a single 30-min restraint (RT) in female rats. The present work was aimed at assessing whether this effect was actually due to physical immobilization or was a mere consequence of the exposure to a novel environment (the restraining box in the experimental room). Dioestrous females were either left undisturbed in their home cages (Control), restrained (RT), or subjected to the same experimental schedule as the restrained ones, but placed in a living cage instead of in the restraining device (Novelty). Twenty-four hours after treatment, the social behaviour of the experimental females was recorded during a 20-min encounter, in a neutral arena, with an unfamiliar conspecific female in the same oestrous cycle phase. Novelty did affect behaviour, but in a way completely different from RT: while RT increased the frequencies of agonism and other social behaviours, novelty caused a selective decrease of agonism. The effects of RT on the social behaviour of female rats appear therefore to be specific and independent of those caused by novelty.

Effects of single restraint on the defensive behavior of male and female rats.

The effects of single aversive stimulation due to restraint (RT) on behavioral responses to unfamiliar conspecifics were studied in male and female rats. The Resident-intruder paradigm was adopted, RT animals and their controls playing the role of intruders. Introductory and agonistic behaviors of both intruders and residents were recorded. In males, RT increased both the number of subjects which showed freezing and freezing duration, and this was independent of the amount of aggression received by the residents. By contrast, no change was found in active defense. Increased passive defense was not paralleled by a complete inhibition of aggression. The latter was rare, but not absent, and occurred in RT males as often as in their controls. Females never showed freezing and, unlike males, resorted to a fully active defensive strategy. RT females were the preferential targets of residents' high-intensity aggression, but showed the same rate of defensive responding as control females. The crucial role played in studies of social behavior by testing conditions and mutual influences between the behavior of experimental subjects and residents are discussed.

Social stress by repeated defeat: effects on social behaviour and emotionality.

The consequences of repeated defeat stress on social and non-social behaviours were assessed in male rats 24 h after the last defeat. Aggressive, defensive, introductory and affiliative items of both experimental animals and their opponents were recorded in a social behaviour test, while emotionality, exploration and general motor activity were scored in the Emergency, Hole-Board, and Elevated Plus-Maze tests. In addition to a dramatic loss of body weight, a selective inhibition of aggression was observed in the stressed experimental subjects, paralleled by decreased defence in their opponents. In the stressed animals, no change was found in other social and non-social behaviours; in particular, defence and emotionality were unaffected. This shows that, under our experimental conditions, the inhibition of aggression, which has often been reported to parallel an increase in defence after social and non-social aversive stimulation, was not dependent on a concomitant activation of a prevailing defensive motivational system, sustained by increased emotionality and fear. As the same result, namely a selective inhibition of aggression with no effect on defence, was obtained after exposure to a non-social stressor (restraint), the hypothesis is advanced that the threshold for stress-induced behavioural changes is lower for aggression than for any other behavioural and motivational system, including that leading to defence. The inhibition of aggression would therefore be a direct response to stress and not a by-product of the activation of a fear-based defensive system.

Sex-dependent effects of restraint on nociception and pituitary-adrenal hormones in the rat.

The sex-dependent effects of acute restraint (RT) on nociceptive and pituitary-adrenal responses were investigated in the rat. In a first experiment, the effect of 30 min RT on pain sensitivity was evaluated through repeated use of the tail withdrawal test during and after treatment. RT induced an increase in the nociceptive threshold, i.e., analgesia, in males and females, but the duration and time-course of this effect varied between sexes. The latencies returned to approximately control values in females in the second half of RT, but in males they remained higher for the whole period of RT and immediately afterwards. Twenty-four hours later, males displayed longer latencies than controls in response to simple reexposure to the environment. In a second experiment, ACTH and corticosterone plasma levels were measured immediately after 15 or 30 min of RT. ACTH and corticosterone were higher in restrained animals than in controls after both periods of treatment, and in both sexes; however, females showed higher basal and stress corticosterone levels than males. The role played by corticosteroids in the nociceptive responses of the two sexes is discussed.

Interactions between 5-hydroxytryptamine (5-HT) and testosterone in the control of sexual and nonsexual behaviour in male and female rats.

Two 5-hydroxytryptamine2 (5-HT2) agents, ritanserin and 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane HCl (DOI) (both at 0.25 mg/kg IP), were administered to castrated males bearing graded testosterone implants (empty, 2.5-, 5-, and 10-mm length) and to normal and neonatally androgenized ovariectomized females bearing 10-mm testosterone implants. The results indicate that testosterone stimulates male sexual behaviour and appears to have a dose-related anxiolytic effect, but no effect on other nonsexual activities. 5-HT and testosterone had opposite effects on male sexual behavior, with ritanserin (5-HT antagonist) enhancing activity in both sexes and DOI (5-HT agonist) inhibiting behaviour in males, the latter being testosterone-dependent. Independent of testosterone, ritanserin reduced locomotion and exploration and increased anxiety in males, while DOI increased locomotion and exploration in both sexes. Ritanserin had a gender-specific effect on anxiety which was independent of testosterone, since in castrated males it was anxiogenic whether they bore testosterone implants or not, while in females it was anxiolytic whether the female were neonatally androgenized (250 micrograms/pup testosterone proprionate [TP] on day 1) or not. These results show that 5-HT and testosterone have opposite effects on male sexual behaviour and these may be interrelated. In adulthood, their effects on nonsexual activities are not inversely related and are independent of each other in contrast to the relationship seen in the neonatal period.

Effects of single and repeated restraint on the social behavior of male rats.

The effects of a mild, nonpainful stressor (restraint, RT) on the social behavior of male rats were explored. Twenty-four hours after single or repeated RT, experimental subjects were confronted in a neutral cage with unfamiliar and naive opponents of the same sex and strain, matched for age and body weight. Introductory, affiliative, defensive, and offensive behaviors of the experimental animals were recorded for 20 min. Single RT caused a selective and dramatic inhibition of aggressive behavior, with no effects on defense. Allo-grooming, considered to be an appeasing and affiliative behavior, increased. Repeated RT caused basically the same effects, with an additional increase in retreat, a low-intensity defensive behavior, and attend, possibly aimed at monitoring the unfamiliar partner. Habituation to repeated stress, therefore, did not occur, and the behavioral consequences of repeated RT were even greater than those of single stress. Additional tests of sexual behavior failed to reveal any deficit in sexual performance after single or repeated RT.

Immune and neuroendocrine response to restraint in male and female rats.

A parallel study of the modification in the opioid and immunological systems induced by acute restraint (RT) was carried out in male and female rats 24 hr after the treatment. beta-Endorphin-like immunoreactivity (beta-ELI) was measured in two brain areas (ventral hypothalamus [HYP] and periaqueductal gray matter [PAG]) and in the pituitary (anterior and neurointermediate lobes), together with plasma corticosterone (C) and ACTH. Immune function was measured as mitogen-induced Interferon-gamma (IFN-gamma) production by splenocytes. RT reduced beta-ELI levels in the PAG in males and females. Plasma levels of C and ACTH did not differ from the basal levels in restrained animals. RT reduced IFN-gamma production in both sexes, but this effect was more marked in females than in males. The possible relationship between the immune and opioid system is discussed.

Behavioural effects in adulthood of neonatal manipulation of brain serotonin levels in normal and androgenized females.

5HT concentrations in the hypothalamus are higher in females than males over the second week of life and this differentiation is testosterone-dependent. We have investigated the possible influence of 5HT over this period on the development of systems that control adult behaviour, in particular those influenced by neonatal testosterone. Neonatal androgenization (250 micrograms/pup testosterone propionate; TP; on day 1 postpartum) induced a masculine pattern of behaviour in females ovariectomised in adulthood and bearing a TP implant. The neonatal treatment reduced exploration, motor activity and female sexual behaviour and increased anxiety, orientation toward the incentive female and male sexual behaviour. Depletion of 5HT by pCPA (100 mg/kg days 8-16 postpartum) enhanced the TP-induced increment in locomotion and female sexual behaviour and increased sexual orientation toward the incentive female, while 5HTP (20 mg/kg days 8-16 postpartum) antagonised the reduction in exploration by TP. Thus 5HT may normally exert an inhibitory control on the action of neonatal testosterone on exploration, motor activity and sexual behaviour. Neonatal PCPA treatment also had a marked anxiolytic effect which was independent of the presence of T as it was noted in normal and androgenized females and previously has been observed in intact males. This might indicate a primary control by a serotonergic system on the development of the systems controlling anxiety.

Behavioural responses to single and repeated restraint in male and female rats.

The effects of single and repeated restraint (RT) on non-social behaviour were investigated in male and female rats. The animals underwent either 15-min or 30-min single RT (Experiment 1), or daily 30-min RT for 7 days (Experiment 2). Behavioural parameters recorded included locomotion and exploration in the hole-board and anxiety in the elevated plus-maze. Effects of RT depended on sex, parameter and schedule of RT administration. After 15-min single RT, anxiety increased and exploration decreased in males; after 30-min single RT, motor activity decreased in both sexes, and anxiety increased in females. After repeated RT, exploration and anxiety were unmodified in males, indicating habituation. Residual emotionality was suggested by increased time spent self-grooming and preferential locomotion in the peripheral squares of the open field. In females, habituation was observed in locomotion and anxiety. Interestingly, parameters which did not habituate suggested decreased emotionality: exploration increased, and ambulation in central squares was preferred to peripheral locomotion. Caution is needed in taking these marginal modifications of female behaviour as facilitatory effects, possibly concurring to increase individual fitness. In summary, both single and repeated RT caused less behavioural deficits in females than in males. Effects on exploration were sex-dependent, and different after single and repeated stress. Locomotion in peripheral vs. central squares, and self-grooming, were also affected by RT sex-dependently, but with similar effects after single and repeated stress. In contrast with the above-mentioned items, modifications of anxiety appeared uninfluenced by sex.

Sex-dependent effects of aversive stimulation on holeboard and elevated plus-maze behavior.

The behavioral effects of exposure to inescapable shocks (IS) were studied in both the holeboard and the elevated plus-maze, 24 and 72 h after IS in male and female Wistar rats. The following effects were observed at the 24-h interval. In both sexes, head-dipping in the holeboard was reduced by IS, whereas general activity (ambulation and rearing) was reduced in males and not in females. Furthermore, the results of a correlation analysis indicate that previous exposure to IS disrupts the dissociation observed in control groups between exploratory activity directed at the holes (head-dipping) and general activity in the holeboard (ambulation and rearing). Effects of IS on plus-maze behavior could be observed in a clear suppression of rearing in males and not in females. IS did not affect time spent on the open arms. At the 72-h interval, IS affected head-dipping in the holeboard only in males and not in females. The present findings show that the effect of IS on specified behavioral elements is sex-dependent, with stronger and longer-lasting effects in males than in females.

Pituitary and brain beta-endorphin in male and female rats: effects of shock and cues associated with shock.

The present experiment was designed to study whether or not prior exposure to inescapable shock is accompanied by sex-dependent changes in pituitary and central levels of immunoreactive beta-endorphin, which is proposed to play an important role in opioid analgesia induced by aversive stimulation. Further, the effects of brief reexposure (5 min) to the chamber where inescapable shock was experienced earlier, were established in both sexes. Elevated levels of beta-endorphin were found 24 hours after inescapable shock, in the anterior pituitary of males and in the midbrain periaqueductal gray of both males and females. Reexposure to the experimental chamber only affected beta-endorphin levels if shock had been experienced in this chamber. Reexposure after inescapable shock reduced beta-endorphin content of the arcuate nucleus of males and beta-endorphin content of the periaqueductal gray of males and females. The present results are related to previous findings of sensitization and conditioning of analgesic reactions. The sex differences found in the present experiment are discussed with respect to sex-dependent behavioral consequences of inescapable shock.

Response to intruders in female rabbit colonies is related to sex of intruder and rank of residents.

Behavioural reactions to unfamiliar conspecifics of both sexes were studied in female domestic rabbits, living in stable unisexual groups. Intrusion caused an abrupt increase in the frequency of social investigation and agonistic behaviours directed to both intruders and group-mates. Reactions depended on the rank of resident females and the sex of the intruder, and were generally more marked in the presence of the male than the female intruder. Dominant and sub-dominant females investigated the male more than the female intruder; the opposite held for subordinate females. In the presence of the female intruder, only dominant and subdominant females were aggressive towards the intruder and group-mates. In the presence of the male intruder, aggression was directed to the intruder and group-mates by dominant females only. They tended to frequently attack sub-dominants, which in turn fled away from them more often than they did from other group-mates.

Time-course of opioid and pituitary-adrenal hormone modifications during the immobility reaction in rabbits.

A series of experiments in male rabbits examined the influence of tonic immobility (animal hypnosis) on the opioid and pituitary-adrenal hormones. The aims of the experiments were to follow the temporal modifications in hormone parameters and to discriminate the effects of immobility from those of the manipulative procedure to induce the reaction. Results show that immobility elicits increases in plasma ACTH and beta-EP, but no modifications in pituitary beta-EP. The effects of induction procedure were dissociable from those of immobility, the most marked effect being a prolonged, sustained increase of corticosterone plasma levels. Results support the hypothesis of the opioid involvement in the physiological control of immobility reaction and indicate different mechanisms underlying the immobility response and the procedure of induction.