Social and sexual incentive properties of estrogen receptor alpha, estrogen receptor beta, or oxytocin knockout mice.

Social and sexual incentive motivation, defined as the intensity of approach to a social and a sexual incentive, respectively, were studied in female Swiss Webster mice. In the first experiment, the social incentive was a castrated mouse of the same strain as the females, whereas the sexual incentive was an intact male mouse of the same strain. Ovariectomized females were first tested after oil treatment and then after administration of estradiol benzoate + progesterone in doses sufficient to induce full receptivity. The hormones increased sexual incentive motivation while leaving social incentive motivation unaffected. This suggests that sexual incentive motivation in the female mouse is dependent on ovarian hormones. In the next experiment, ovariectomized females were tested with an intact, male estrogen receptor alpha knockout and its wild type as incentives, first without hormones and then when fully receptive. There were no differences in incentive properties between the wild type and the knockout. In a similar experiment, we used an intact male estrogen receptor beta knockout and its corresponding wild type as incentives. The wild type turned out to be a more attractive social incentive than the knockout, while they were equivalent as sexual incentives. Finally, an intact male oxytocin knockout and its wild type were used as incentives. The knockout turned out to be a superior incentive, particularly a superior sexual incentive. The fact that the estrogen receptor beta and oxytocin knockouts have incentive properties different from their wild types may be important to consider in studies of these knockouts' sociosexual behaviors.

Involvement of estrogen receptor alpha, beta and oxytocin in social discrimination: A detailed behavioral analysis with knockout female mice.

Social recognition, processing, and retaining information about conspecific individuals is crucial for the development of normal social relationships. The neuropeptide oxytocin (OT) is necessary for social recognition in male and female mice, with its effects being modulated by estrogens in females. In previous studies, mice whose genes for the estrogen receptor-alpha (alpha-ERKO) and estrogen receptor-beta (beta-ERKO) as well as OTKO were knocked out failed to habituate to a repeatedly presented conspecific and to dishabituate when the familiar mouse is replaced by a novel animal (Choleris et al. 2003, Proc Natl Acad Sci USA 100, 6192-6197). However, a binary social discrimination assay, where animals are given a simultaneous choice between a familiar and a previously unknown individual, offers a more direct test of social recognition. Here, we used alpha-ERKO, beta-ERKO, and OTKO female mice in the binary social discrimination paradigm. Differently from their wild-type controls, when given a choice, the KO mice showed either reduced (beta-ERKO) or completely impaired (OTKO and alpha-ERKO) social discrimination. Detailed behavioral analyses indicate that all of the KO mice have reduced anxiety-related stretched approaches to the social stimulus with no overall impairment in horizontal and vertical activity, non-social investigation, and various other behaviors such as, self-grooming, digging, and inactivity. Therefore, the OT, ER-alpha, and ER-beta genes are necessary, to different degrees, for social discrimination and, thus, for the modulation of social behavior (e.g. aggression, affiliation).

Oxytocin and estrogen receptor alpha and beta knockout mice provide discriminably different odor cues in behavioral assays.

Social behavior involves both the recognition and pro-duction of social cues. Mice with selective deletion(knockout) of either the gene for oxytocin (OT) or genes for the estrogen receptor (ER) -c or -B display impaired social recognition. In this study we demonstrate that these gene knockout mice also provide discriminably different social stimuli in behavioral assays. In an odor choice test, which is a measure of social interest and discrimination, outbred female Swiss-Webster mice discriminated the urine odors of male knock-outs IKO: OTKO, alphaERKO, betaERKO) from the odors of their wildtype littermates (WT: OTWT, alphaERWT, betaERWT). Females showed marked initial choices of the urine odors of OTWT and betaERWT males over those of OTKOand PERKO males, and alphaERKO males over alphaERWT males. The odors of OTKO and betaERKO males also induced aversive, analgesic responses, with the odors of WTs having no significant effects. Odors of both the alphaERWT andalphaERKO males induced aversive, analgesic responses,with the odors of the WT inducing significantly greater analgesia. The odors of restraint stressed WT and KO males also elicited analgesia with, again, females dis-playing significantly greater responses to the odors of stressed OTKO and betaERKO males than their WTs, and significantly lower analgesia to the odors of stressedalphaERKO than alphaERWT males. These findings show that the KO mice are discriminated from their WTs on the basis of odor and that the various KOs differ in the relative attractiveness/aversiveness of their odors. Therefore, in behavioral assays one causal route by which gene inactivation alters the social behavior of knockout mice may be mediated through the partners'modified responses to their odors.

Functional genomics of social recognition.

Although various types of group living are widespread in mammals, including humans, the study of the hormonal and genetic underpinnings of nonsexual social behaviour, is in its infancy compared to the analysis of sexual behaviour mechanisms. Oxytocin, vasopressin and gonadal hormones certainly play an important role. Social recognition, where animals identify and recognize other individual conspecifics, is a crucial prerequisite for the occurrence of a wide range of social behaviours. Social recognition is also important for coping with one major cost of life in a group: the increased risk of exposure to parasites and infection. We review recent functional genomic studies on the involvement of oxytocin and oestrogen-receptor genes in the regulation of social recognition in mice and in the ecologically relevant context of parasite recognition and avoidance. Based on quantitative studies of social recognition with gene-knockout mice and with antisense DNA, we propose a four-gene micronet contributing to social recognition. This micronet involves the genes coding for oestrogen receptors alpha (ER-alpha), beta (ER-beta), oxytocin and the oxytocin receptor. In this model, circulating oestrogens promote transcription of (i) oxytocin in the paraventricular nucleus of the hypothalamus through ER-beta and (ii) oxytocin receptor in the amygdala through ER-alpha. This model forms the core around which increasingly complex genetic, hormonal and neural interactions associated with social behaviours and recognition can be organized.

Impaired discrimination of and aversion to parasitized male odors by female oxytocin knockout mice.

A major cost of social behavior is the increased risk of exposure to parasites, with animals utilizing social information to recognize and avoid infected conspecifics. In mice, females can discriminate between infected and uninfected males on the basis of social cues, displaying aversive responses to the odors of infected males. In the present study, using female mice whose gene for oxytocin (OT) has been selectively deleted (OT knockout mice (OTKO)), we show that at least one normal allele for OT is required for the mediation of the recognition and avoidance of parasitized males. Female wild type (OTWT) and heterozygous (OTHZ) mice distinguished between the odors of individual males infected with the louse, Polyplax serrata, and uninfected males while the KO mice did not. Exposure to the odors of infected males induced analgesia in OTWT and OTHZ females, with OTKO females displaying attenuated analgesia. OTWT and OTHZ females, but not the OTKO females, also distinguished between the odors of novel and familiar infected males and modulated their analgesic responses on the basis of prior familiarity. In an odor choice test, OTWT and OTHZ females displayed a marked initial choice for the odors of uninfected males, whereas the OTKO females showed no consistent choice. This impairment was specific to the odors of infected males. OTKO females displayed normal analgesic responses to another aversive social odor, that of a stressed male, and an aversive non-social odor, that of a cat. The OTKOs had normal non-social olfactory memory, but were impaired in their social odor memory. These findings indicate that a normal OT gene comprises an essential part of the central recognition mechanism whereby females can both reduce the transmission of parasites to themselves and select for parasite-free males.

Learning to fear and cope with a natural stressor: individually and socially acquired corticosterone and avoidance responses to biting flies.

Animals learn to recognize and respond to a variety of dangerous factors, with biting and blood-feeding flies being among the most prevalent of natural stressors. Here we describe the behavioral avoidance and hormonal (corticosterone) stress responses to biting fly exposure and the roles of individual and social learning in the acquisition of these fear-associated responses. Male mice exposed to a single 30-min session of attack by intact biting flies (stable fly, Stomoxys calcitrans L.) exhibited increased plasma corticosterone levels and active self-burying responses to avoid the flies. When exposed 24 h later to altered flies whose biting mouth parts were removed and were incapable of biting, the mice displayed conditioned increases in corticosterone and avoidance responses. This conditioned increase in corticosterone and self-burying was also acquired through social learning without direct individual experience with the intact biting flies. Fly naive "observer" mice that witnessed other "demonstrator" mice being attacked by biting flies, but were not exposed to intact flies themselves, displayed increases in corticosterone levels and self-burying to avoid flies when exposed 24 h later to altered flies. The social learning was not due to social facilitation or sensitization. Observers had to witness the self-burying avoidance responses of the demonstrator to the biting flies in order to subsequently recognize a potential threat to themselves and display the appropriate responses. These individually and socially acquired conditioned fear responses are likely part of the mechanisms that allow animals to defend themselves from biting and blood-feeding arthropods.

Brief exposure to female odors "emboldens" male mice by reducing predator-induced behavioral and hormonal responses.

In rodents, where chemical signals play a particularly important role in determining intersexual interactions, various studies have shown that male behavior and physiology is sensitive to female odor cues. Here we examined the effects of brief (1 min) and more prolonged (60 min) preexposure to the odors of a novel estrous female on the behavioral and hormonal responses of sexually experienced and inexperienced male mice, Mus musculus, to subsequent predator (cat and weasel) odor exposure and potential predator risk. Brief, but not prolonged, preexposure to the odors of an estrous female decreased the aversion and avoidance responses of male mice to cat odor in a Y-maze preference test, with the extent of responses being affected by a males prior sexual experience. Similarly, brief, but not prolonged, preexposure to female odors markedly attenuated the analgesic responses elicited in male mice by weasel odor. Brief exposure to a novel estrous female by itself had no significant immediate effects on either corticosterone or testosterone levels in the males. However, brief, but not prolonged, preexposure to the odors of an estrous female attenuated the marked increase in corticosterone and decrease in testosterone that were induced in males by exposure to weasel odor. The decreases in aversive responses to, and effects of, predator odor exposure that are induced by brief exposure to a novel estrous female may reflect a greater risk taking and boldness in males that could directly facilitate access to an immediately, and possibly transiently, available novel sexually receptive female.

Corticosterone rapidly reduces male odor preferences in female mice.

There is accumulating evidence for rapid, non-genomic behavioral effects of various steroids including that of the glucocorticoid, corticosterone. Using an odor preference test, the responses of which are indicative of mate preferences and sexual interest, we examined the effects of acute corticosterone on the responses of oestrous female mice to male odors. Control female mice displayed an overwhelming preference for the odors of male mice. Peripheral administration of corticosterone elicited a significant dose-related (1.0-5.0 mg/kg) decrease in female preference for male odors at 10 min, but not at 60 min, after administration. These inhibitory effects of corticosterone on odor preferences were significantly reduced by the competitive NMDA antagonist, NPC 12626, and enhanced by the GABA antagonist bicuculline. This indicates that corticosterone has rapid inhibitory effects on olfactory mediated female mate preferences and responses to male odor that in part involve interactions with NMDA and GABA receptor mechanisms.

Learning from others to cope with biting flies: social learning of fear-induced conditioned analgesia and active avoidance.

Although fear conditioning has received extensive attention, little is known about the roles of social learning whereby an individual may learn and acquire the fear responses of another. The authors examined individually and socially mediated acquisition of fear and analgesia to the natural aversive stimulus of biting flies. Exposure to biting flies elicited in individual naive mice analgesia and active self-burying to avoid the flies. When exposed 24 hr later to flies whose biting parts were removed, but not to nonbiting house flies, these mice displayed conditioned analgesia and self-burying. This "one-trial" conditioned analgesia and avoidance was also acquired through social learning without direct individual experience with biting flies. Naive "observer" mice that witnessed other "demonstrator" mice being attacked by biting flies exhibited analgesia and self-burying 24 hr later to altered flies.

A detailed ethological analysis of the mouse open field test: effects of diazepam, chlordiazepoxide and an extremely low frequency pulsed magnetic field.

The open field test (OFT) is a widely used procedure for examining the behavioral effects of drugs and anxiety. Detailed ethological assessments of animal behavior are lacking. Here we present a detailed ethological assessment of the effects of acute treatment with the benzodiazepines, diazepam (DZ, 1.5mg/kg) and chlordiazepoxide (CDP, 5.0 and 10.0mg/kg), as well as exposure to a non-pharmacological agent, a specific pulsed extremely low frequency magnetic field (MAG) on open field behavior. We examined the duration, frequency and time course of various behaviors (i.e. exploration, walk, rear, stretch attend, return, groom, sit, spin turn, jump and sleep) exhibited by male mice in different regions of a novel open field. Both DZ and CDP consistently reduced the typical anxiety-like behaviors of stretch attend and wall-following (thigmotaxis), along with that of an additional new measure: 'returns', without producing any overall effects on total locomotion. The drugs also differed in their effects. CDP elicited a shift in the locomotor pattern from a 'high explore' to a 'high walk', while DZ mainly elicited alterations in sit and groom. The MAG treatment was repeated twice with both exposures reducing horizontal and vertical (rearing) activity and increasing grooming and spin turns. However, the anxiety-like behaviors of stretch attend and return were marginally reduced by only the first exposure. We conclude that a detailed ethological analysis of the OFT allows not only the detection of specific effects of drugs and non-pharmacological agents (i.e. pulsed magnetic field) on anxiety-like behaviors, but also permits the examination of non-specific effects, in particular those on general activity.

Locomotor activity changes following lipopolysaccharide treatment in mice: a multivariate assessment of behavioral tolerance.

To determine the effects of repeated, acute endotoxin exposure on locomotor behavior, male laboratory mice were injected intraperitoneally with lipopolysaccharide (LPS: 50, 100 or 200 microg/kg) or saline vehicle on experimental Days 1, 4 and 7. At 2 h after each treatment, locomotor activity was assessed in a nonnovel, automated open-field apparatus (Digiscan) for 30 min. On Day 1, all horizontal and vertical activity measures were significantly reduced to near zero values by each dose of LPS. Behavioral tolerance to LPS formed rapidly, as locomotor activity of the treated groups did not differ from the control group on Days 4 or 7. In a second study, mice were given LPS (50, 100 or 150 microg/kg ip) or saline vehicle on two test days, 28 days apart. Activity was assessed, 1 h after injection, in a novel open field on the first test day and in a nonnovel open field on the second test day. Significant locomotor activity decrements were readily apparent in LPS-treated mice only in the nonnovel open field. This latter finding indicates that environmental novelty mediates, at least partially, the locomotor-reducing effects of LPS in mice.

NMDA-mediated social learning of fear-induced conditioned analgesia to biting flies.

Although fear conditioning has received extensive neurobiological attention little is known about social learning whereby one individual may learn and acquire the fear responses of another. A 30 min exposure to intact biting flies (stable fly, Stomoxys colcitrans L.) elicits in individual fly-naive mice analgesia and active self burying responses to avoid the flies. Fly-naive observer mice that witnessed other demonstrator mice being attacked by biting flies exhibited analgesia and self-burying to avoid flies when exposed 24 h later to altered flies whose biting mouth parts were removed. The opiate antagonist naloxone, while reducing the analgesic responses elicited by exposure to a fly-stressed demonstrator, did not affect either the subsequent conditioned analgesia or self-burying. However, the specific NMDA receptor antagonist NPC 12626, given to observers prior to, but not after, presentation of fly attacked demonstrators blocked the socially determined conditioned analgesia and self burying avoidance. This supports NMDA involvement in the mediation of the social transmission and long-term (24h) retention of conditioned analgesia and fear.

Antipredator responses and defensive behavior: ecological and ethological approaches for the neurosciences.

In nature, animals are exposed to a wide range of threats and dangers with predators being amongst the more prominent and intensely studied of these. The responses of prey to predators and various predator avoidance and antipredator behaviors have been extensively evaluated from ecological and ethological perspectives and more recent ethopharmacological and neuroscience approaches. Unfortunately, there has been relatively little interchange between the ecological-ethological and neuroscience areas with the latter often using responses to predators just simply as another 'model' system. There is, however, now a growing realization that integrative approaches incorporating ecological, evolutionary and neurobiological explanations are required for the understanding of behavior and its functions. This necessitates an incorporation of ecological and ethological concepts and validity with neuroscience approaches to the analysis of antipredator responses and defensive behavior. A number of selected ecological approaches that are used for the investigation of predator avoidance mechanisms and antipredator defensive behavior patterns are briefly reviewed here. These include examinations of how predation risk and its variation affect decision making in animals and how learning affects these responses. The trade-offs that are involved, how the risk of predation affects decisions concerning foraging behavior, mating and reproduction, as well as how varying levels of risk affect decisions relative to the type of defensive mechanisms utilized are briefly outlined. The utility of these approaches and their relevance to the design and interpretation of various neuroscience studies is addressed here.

Acute effects of corticosterone on LiCl-induced rapid gustatory conditioning in rats: a taste reactivity analysis.

Previous research has shown that acute corticosterone treatment can have rapid effects on learning and memory. Using the taste reactivity test (TRT), the present study examined the effect of acute administration of corticosterone on sucrose palatability and the development of LiCl-induced rapid gustatory conditioning. On each of two conditioning days rats were injected with either a low dose of lithium chloride (LiCl; 0.75 mEq, i.p.) or saline (NaCl; 0.9%, i.p.) and 10 min later, received a second injection of either corticosterone (5 mg/kg, i.p.) or cyclodextrin vehicle. Rats were then placed in the TRT chamber, where 1 min intraoral infusions of sucrose (0.3 M) were delivered every 10 min. Taste reactivity responses were videotaped and later analyzed for frequency of occurrence. Rats treated with both LiCl and corticosterone showed enhanced aversive responding and reduced ingestive responding relative to control rats treated with LiCl and vehicle. The implication that corticosterone may have a rapid enhancing effect on gustatory conditioning is discussed.

Parasites and behaviour: an ethopharmacological perspective.

Ethopharmacology combines an ethological approach to the understanding of the causes and functions of behaviour with pharmacological analysis of the underlying neuromodulatory mechanisms. Recently, this approach has been applied to the analysis of the responses of animals to parasitized individuals and to the effects of parasites on various host behaviours (eg. host defences, mate responses) and their neurobiological correlates. Martin Kavaliers, Douglas Colwell and Elena Choleris explain here how ethopharmacology can be used to address the mechanisms that underlie the often subtle effects of parasites on host behaviour.

Extremely low frequency magnetic fields can either increase or decrease analgaesia in the land snail depending on field and light conditions.

Results of prior investigations with opioid peptide mediated antinociception or analgaesia have suggested that these extremely low frequency (ELF) magnetic field effects are described by a resonance mechanism rather than mechanisms based on either induced currents or magnetite. Here we show that ELF magnetic fields (141-414 microT peak) can, in a manner consistent with the predictions of Lednev's parametric resonance model (PRM) for the calcium ion, either (i) reduce, (ii) have no effect on, or (iii) increase endogenous opioid mediated analgaesia in the land snail, Cepaea nemoralis. When the magnetic fields were set to parameters for the predictions of the PRM for the potassium ion, opioid-peptide mediated analgaesia increased and there was evidence of antagonism by the K(+) channel blocker, glibenclamide. Furthermore, these effects were dependent on the presence of light; the effects were absent in the absence of light. These observed increases and decreases in opioid analgaesia are largely consistent with the predictions of Lednev's PRM.

Examining the effects of lipopolysaccharide and cholecystokinin on water ingestion: comparing intake and palatability.

Lipopolysaccharide (LPS) and cholecystokinin (CCK) have been shown to have anorectic properties in a variety of species. The present study examined the effects of LPS and CCK, both alone and in combination, on two different aspects of water ingestion, water intake and palatability. On test days, animals were first injected intraperitoneally (i.p.) with either LPS (200 microg/kg) or NaCl vehicle, and 2 h later received a second injection of either CCK (8 microg/kg) or NaCl vehicle. In Experiment 1, water intake was monitored for 1 h on 3 separate test days 72 h apart; while in Experiment 2, water palatability was assessed using the taste reactivity test (TRT), on two separate test days 72 h apart. Both LPS and CCK significantly (p<0.05) reduced water intake, with the effects of combined LPS with CCK being more pronounced than either agent injected alone. Rats developed a rapid tolerance to the effects of LPS on water intake on subsequent exposures to LPS. Results from the TRT indicated that LPS enhanced water palatability (p<0.05), as evidenced by a high level of ingestive responding, whereas CCK produced a pattern of responding indicative of satiety. LPS plus CCK reduced ingestive responding on the first test day, but these responses were significantly increased on the second test day (p<0.05). These results demonstrate that although LPS reduces water intake, it enhances water palatability. The results further underscore the necessity for examining palatability changes in addition to intake measures when studying the regulation of feeding and drinking.

Influence of a natural stressor (predator odor) on locomotor activity in the meadow vole (Microtus pennsylvanicus): modulation by sex, reproductive condition and gonadal hormones.

Sex differences in a variety of non-reproductive behaviors have been indicated to occur in seasonally breeding polygynous promiscuous rodents such as the meadow vole, Microtus pennsylvanicus. The present study was designed to assess the effects of reproductive and hormonal status on the locomotor responses of meadow voles following brief exposure to the odors of a natural predator, the Red fox (Vulpes vulpes). Adult male and female meadow voles, which are seasonal photoperiodically-induced breeders, were housed in either mixed sex pairs under a long, reproductively stimulatory photoperiod (simulating breeding: long light cycle, paired: LLC + P) or in same-sex pairs under a short, reproductively inhibitory photoperiod (simulated non-breeding: short light cycle, non-paired: SLC-NP). On 2 consecutive days following 1 day of baseline activity monitoring, voles were exposed individually for 3 min to fox odor and a novel pungent control odor (extract of almond). The levels of various measures of activity that were displayed by the voles were assessed by an automated Digiscan activity monitoring system. LLC + P (simulated breeding) voles displayed higher basal levels of activity relative to SLC + NP (simulated non-breeding) voles, with males displaying greater activity than females. LLC + P (simulated breeding) males displayed a significant reduction in activity levels following exposure to fox odor relative to control odor. The reductions in activity following fox odor exposure were related to plasma testosterone levels such that a larger behavioral response (i.e. greater reduction) was associated with higher levels of testosterone. Furthermore, dividing males into high and low testosterone groups based on the median levels of testosterone revealed that high but not low testosterone males displayed reductions in activity following exposure to fox odor relative to control odor. No changes in activity levels following exposure to fox odor were noted in SLC-NP males, and either SLC-NP or LLC + P females. These results show that this sexually dimorphic non-reproductive behavior is significantly influenced by reproductive condition and gonadal hormone levels.

Antinociceptive effects of the neuroactive steroid, 3alpha-hydroxy-5alpha-pregnan-20-one and progesterone in the land snail, Cepaea nemoralis.

Results of investigations with vertebrates have implicated neuroactive steroids and in particular 5alpha-reduced metabolites of progesterone such as 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha,5alpha-THP/3A5P and originally allopregnanolone) in the rapid modulation of diverse functions including that of nociceptive sensitivity. These effects have been indicated to involve modulation of GABA receptors. Results of recent phylogenetic studies have revealed the presence of GABA receptors in invertebrates that may also be subject to modulation by steroids and neuroactive steroids. The present study examined the effects of the neuroactive steroid, 3alpha-hydroxy-5alpha-pregnan-20-one, as well as progesterone on aversive thermal (nociceptive) responses in a mollusc, the land snail, Cepaea nemoralis. 3alpha-Hydroxy-5alpha-pregnan-20-one had significant dose-related (0.01-1.0 microg) antinociceptive effects in Cepaea increasing the latency of response to a 40 degrees C surface, with maximum effects being evident 15-30 min after administration. These effects of 3alpha-hydroxy-5alpha-pregnan-20-one were stereospecific, with the stereoisomer 3beta-hydroxy-5alpha-pregnan-20-one (3B5P) failing to affect nociceptive responses. Progesterone also had significant dose-related (0.10-10 microg) antinociceptive effects that, however, were delayed in onset and relatively prolonged (60-120 min), suggestive of the formation of active metabolites. The presence of endogenous progesterone (12.36+/-0.17 ng/g tissue) was ascertained by a radioimmunoassay further supporting a functional role for steroids in Cepaea. The antinociceptive effects of 3alpha-hydroxy-5alpha-pregnan-20-one and progesterone were blocked by the GABA antagonists, bicuculline and picrotoxin, while being relatively insensitive to opioid and N-methyl-D-aspartate antagonists. These results suggest an early evolutionary development and phylogenetic continuity of neuroactive steroid and GABA involvement in the mediation of nociception.