A functional role for an opiate system in snail thermal behavior.

The terrestrial snail Cepaea nemoralis, when placed on a 40 degrees C hot plate, lifts the anterior portion of its foot. The latency of this response is influenced by morphine and by naloxone in a dose-dependent and time-dependent manner. Morphine increases the time taken to respond, whereas naloxone reduces it. Furthermore, naloxone abolishes the effect of morphine. These results indicate that an opiate system may have a role in this behavior, which resembles that reported in vertebrates.

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).

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.

Parasites and behavior: an ethopharmacological analysis and biomedical implications.

Parasites and disease are increasingly recognized as agents of behavioral, ecological and evolutionary importance having a variety of influences on their hosts other than the more obvious pathological and immunological changes. Parasites can have significant behavioral effects even when parasitism is sub-clinical with these effects proposed to either benefit the parasite (parasite 'manipulation'), benefit the host, or to simply arise as side-effects of the infection (parasitic 'constraints'). However, until relatively recently little attention has been paid to the neuromodulatory substrates that mediate these behavioral changes. Ethopharmacology incorporates an evolutionary approach to the study of behavior with pharmacological analysis of neuromodulatory mechanisms. As such, this approach is appropriate for, and has been applied to, the analysis of the effects of ectoparasites (e.g. biting and blood-feeding flies) and endoparasites (e.g. protozoa, nematodes) on a number of behaviors (e.g. pain inhibition, learning and memory, responses to predators and anxiety, mate selection) in selected host-parasite systems. Ethopharmacology suggests a promising direction by which neuromodulatory mechanisms that underlie the effects of parasites on behavior, including that of humans, can be addressed.

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.

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.

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.

Aging and day-night rhythms in feeding in mice: effects of the putative sigma opiate agonist, N-allylnormetazocine (SKF-10,047).

Day-night rhythms in feeding behavior and response to the putative sigma opiate agonist, N-allylnormetazocine (+/- SKF-10,047, 0.10-10 mg/kg), were measured in young (1-2 months), mature (8-12 months) and old (24-30 months) male CF-1 mice. The mice consumed more food at night than in the day-time, though this nocturnal peak was markedly reduced in the mature and old animals. The young mice also displayed a significant nocturnal enhancement in SKF-10,047 (0.10-1.0 mg/kg) stimulated feeding, that could, in part, be suppressed by the opiate antagonist naloxone (1.0 mg/kg). The day-night rhythm in ingestive responses to SKF-10,047 (0.10-1.0 mg/kg) was reduced in the mature animals and absent in the old animals. The old mice failed to show any significant increase in ingestive response following opiate administration. A higher dose of SKF-10,047 (10 mg/kg) had no significant ingestive effects in any of the age groups of mice; the excitatory, psychotomimetic-related effects, being also reduced in the old animals.

Aging and daily rhythms of analgesia in mice: effects of natural illumination and twilight.

Day-night rhythms in aversive thresholds and morphine-induced analgesia were assessed in young (1-3 months) and old (22-30 months) male mice exposed to natural summer (43 degrees N lat.) lighting using a hot-plate technique. In both age groups peak aversive thresholds and morphine-induced analgesia were present at night, with significantly reduced response times in the day. Response times increased during the course of the day with maximum increases in aversive thresholds and analgesia occurring during the decreasing light levels of dusk. Maximum decreases in the nocturnal response times occurred during the increasing light levels of dawn. The old mice displayed significantly lower nocturnal aversive thresholds and morphine-induced analgesia, as well as less pronounced patterns of change in response times during the dawn and dusk twilight lighting transitions. No significant effects of age were evident during the day-time.

Pinealectomy modifies the thermoregulatory effects of bombesin in goldfish.

Intraventricular administration of the neuropeptide, bombesin, to intact and sham-pinealectomized fish significantly decreased their behaviorally selected internal body temperatures in horizontal thermal gradients. A behavioral hypothermia of 4-7 degrees C was evident regardless of the previous acclimatization temperature (10 or 25 degrees C) and preferred body temperatures selected by individual fish. Pinealectomy increased the body temperatures of all control fish by 2-4 degrees C while markedly reducing the hypothermic effects of administration of bombesin. The hypothermic effects of bombesin were most evident in the pinealectomized fish acclimatized to 10 degrees C. The possible relations between neuropeptides and the pineal gland in the regulation of body temperature are considered.

The presence of an opioid system mediating behavioral thermoregulation in the terrestrial snail, Cepaea nemoralis.

Administration of the opiate agonist, morphine sulfate (0.1-10 micrograms per snail) resulted in a significant dose- and time-dependent increase in the temperatures selected by the terrestrial snail, Cepaea nemoralis, in a thermal gradient. These thermoregulatory effects could be blocked and reversed by naloxone hydrochloride, with the opiate antagonist by itself (1 and 10 micrograms) causing a significant decrease in preferred temperatures. After 6-10 days of daily administration of morphine, Cepaea displayed tolerance, the effects of morphine decreasing and thermal preferences becoming similar to those of saline-treated individuals. These results suggest that an opioid system is involved in the control of thermoregulation in snails, in a manner that is similar to that shown by vertebrates.

Tolerance to the morphine-influenced thermal response in the terrestrial snail, Cepea nemoralis.

The terrestrial snail, Cepea nemoralis, displayed stereotyped thermal-avoidance behavior, with morphine increasing and naloxone decreasing the latency of this response. After 5-7 days of daily administration of morphine sulfate (1 microliter of 0.10-10.0 mg/ml) the thermal responses of snails displayed tolerance to morphine. The response latency was reduced to that of saline-treated individuals. Pretreatment with the protein synthesis inhibitor, cycloheximide, blocked the development of tolerance. After termination of chronic treatment with morphine, tolerant snails exhibited withdrawal signs that included increased responsiveness to naloxone, decreased responsiveness to morphine and saline and behavioral alterations suggesting irritation and hyperactivity. These results suggest that the thermally-induced behavior of Cepea can serve as a useful model for the investigation of the development of opioid tolerance and physical dependence in mammals.

Levorphanol but not dextrorphan suppresses the foot-lifting response to an aversive thermal stimulus in the terrestrial snail, Cepaea nemoralis.

The terrestrial snail, Cepaea nemoralis, when placed on a surface heated at 40 degrees C lifts the anterior portion of its foot from the source of heat. This stereotyped response, which suggests aversion, can be inhibited by injections of small doses of morphine and levorphanol, but is not affected by similar or larger doses of dextrorphan. It is suggested that the "analgesic" effect of the opiates in these animals involves interaction with stereochemically specific opiate receptors that may be fundamentally similar to those occurring in mammals.

Exposure to rotating magnetic fields alters morphine-induced behavioral responses in two strains of mice.

An exposure for 60 min to a 0.5 Hz rotating magnetic field (1.5-90 G) significantly reduced the day-time analgesic and locomotory effects of morphine (10 mg/kg) in CF-1 and C-57BL strains of mice, respectively. Exposure to lower intensity 60 Hz magnetic fields (0.-1.0 G) had no effect on analgesia induced by morphine. The reduction in responsiveness to morphine after exposure to the greater intensity rotating field was not evident 24 hr later. No changes were seen in the latencies of basal thermal responses or levels of activity of saline-treated mice exposed to the magnetic stimuli.

The effects of opioid and FMRF-amide peptides on thermal behavior in the snail.

Administration of methionine-enkephalin, beta-endorphin or, as previously shown, the opiate agonist, morphine sulfate (0.10-10.0 micrograms per snail), resulted in significant dose-dependent increases in the latency of thermal (40 degrees C hot plate) avoidance behavior of the terrestrial snail, Cepaea nemoralis. The analgesic effects could be blocked by the opiate antagonist, naloxone, as well as by the non-opioid peptides, FMRF-amide and YGG-FMRF-amide. When administered by themselves the FMRF-amide peptides had significant bimodal effects either decreasing (0.10 and 10.0 micrograms) or increasing (1.0 micrograms) the latency of the response to the thermal stimulus. These results indicate that opioid and FMRE-amide peptides may be involved in the determination of thermal behavior in the snail. They also suggest that FMRF-amide peptides may function as endogenous modulators of opioid activity.

Spatial learning and hippocampal volume in male deer mice: relations to age, testosterone and adrenal gland weight.

Spatial learning and various physiological parameters were examined in old (57 month), middle aged (38 month), adult (18 month) and young (3-3.5 month) male deer mice (Peromyscus maniculatus). Performance during acquisition of a water maze task was not significantly reduced for middle aged and adult mice relative to young reproductively active (breeding) mice. Performance was deteriorated in old mice relative to young breeding mice on block 4 of training. Retention of this spatial task, however, was reduced in all three older groups relative to young breeding mice. Corrected hippocampal volume (corrected for brain weight) was reduced only in old mice relative to young breeding mice although absolute volumes of hippocampus were lower in all groups relative to young breeding mice. Old mice also were shown to have lower levels of plasma testosterone and lighter brains relative to young breeding mice. Spatial retention was not deficient in old, middle aged and adult mice relative to a group of young reproductively quiescent male (non-breeding) mice. Young breeding mice displayed better spatial performance and had significantly higher plasma testosterone levels, corrected hippocampal volume and brain weight relative to young non-breeding mice. These results indicate that retention of a spatial task is more sensitive to the age of male deer mice than acquisition of the task. Hippocampal volume, although a gross morphological feature, appears to be sensitive to the effects of ageing in male deer mice. Plasma testosterone levels do not appear to be a crucial factor underlying age-related deficits in retention of a spatial task.

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.

Discrimination by female mice between the odours of parasitized and non-parasitized males.

The detection and avoidance of parasitized males has been proposed to be a component of female mate choice. We investigated whether or not female laboratory mice, Mus musculus domesticus, could discriminate between parasitized and non-parasitized males on the basis of odour. Female mice were given a choice between the urine and other odorous secretions of either a male mouse sub-clinically infected for five days with the naturally occurring, enteric, single host, protozoan parasite, Eimeria vermiformis, or an uninfected male. Females showed a marked preference for the odours of non-parasitized male mice over those of the parasitized males; as measured by number of investigations, time spent per investigation, and total investigation time of the odours in a choice situation. Female mice also displayed an overwhelming initial, or first choice, preference for the odours of the non-parasitized male mice over those of the parasitized males. These observations show that female mice can distinguish between the odours of parasitized and non-parasitized males, and discriminate against parasitized males on the basis of odour. We suggest that the detection and avoidance of infected males by female mice through odour cues may function to reduce parasite transmission and potentially serve as a component of female mate selection or choice.

Parasitized female mice display reduced aversive responses to the odours of infected males.

The present study showed that parasites influence both the responses of uninfected females to males and the responses of female hosts to infected males. In female laboratory mice one of the consequences of exposure to the olfactory cues associated with an infected male was a reduction of the reactivity to a thermal surface, i.e. pain inhibition or analgaesia. Uninfected oestrous and non-oestrous female mice displayed marked analgaesic responses after exposure to the odours of males infected with either the enteric single-host nematode parasite, Heligmosomoides polygyrus, or the protozoan parasite, Eimeria vermiformis. The uninfected oestrous females distinguished between infected and physically stressed males, displaying a greater analgaesic response to the odours of infected males. These analgaesic responses and their anxiety/ fearfulness-associated behavioural correlates could elicit either a reduced interest in, or avoidance of, parasitized males by females. Oestrous female mice infected with H. polygyrus displayed a reduced analgaesic response to the odours of the infected males and differentially responded to the odours of males infected with either the same (H. polygyrus) or a different parasite (E. vermiformis). An exposure time of 1 min elicited minimal responses to the odours of males infected with the same parasite, H. polygyrus, and an attenuated, though significant, non-opioid peptide-mediated analgaesic response to males infected with E. vermiformis. An exposure time of 30 min elicited similar markedly reduced endogenous opioid peptide-mediated analgaesic responses to the odours of both of the categories of infected males. The responses to the odours of a stressed male were, however, unaffected by the parasitic infection. The reduced analgaesic responses of the parasitized females to the odours of infected males may involve either enhanced odour familiarity and responses to group odour templates and/or neuromodulatory shifts resulting in reduced fearfulness and potentially greater interest in the infected males.

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.