Effects of suppressing gonadal hormones on response to novel objects in adolescent rats

Human adolescents exhibit higher levels of novelty-seeking behaviour than younger or older individuals, and novelty-seeking is higher in males than females from adolescence onwards. Gonadal hormones, such as testosterone and estradiol, have been suggested to underlie age and sex difference in response to novelty; however, empirical evidence in support of this hypothesis is limited. Here, we investigated whether suppressing gonadal hormone levels during adolescence affects response to novelty in laboratory rats. Previously, we have shown that male adolescent Lister-hooded rats (postnatal day, pnd, 40) exhibit a stronger preference than same-aged females for a novel object compared to a familiar object. In the current study, 24 male and 24 female Lister-hooded rats were administered with Antide (a gonadotrophin-releasing hormone antagonist), or with a control vehicle solution, at pnd 28. Antide provided long-term suppression of gonadal hormone production, as confirmed by ELISA assays and measurement of internal organs. Response to novel objects was tested at pnd 40 in Antide-treated and control subjects using a ‘novel object recognition’ task with a short (2-minute) inter-trial interval. In support of previous findings, control males exhibited a stronger preference than control females for novelty when presented with a choice of objects. Antide-treated males exhibited a significantly lower preference for novel objects compared to control males, whilst Antide-treated females did not differ significantly from control females in their preference for novelty. Antide treatment did not affect total time spent interacting with objects. We discuss how gonadal hormones might influence sex differences in preference for novelty during adolescence.     

Low luteinizing hormone enhances spatial memory and has protective effects on memory loss in rats

Though several studies have suggested that estradiol improves hippocampal-dependent spatial memory, the effects of other hormones in the hypothalamic–pituitary–gonadal axis on memory have largely been ignored. Estradiol and luteinizing hormone (LH) are generally inversely related and LH may significantly affect spatial memory. Ovariectomized (ovx) rats treated with Antide (a gonadotropin releasing hormone receptor antagonist) had low LH levels and showed enhanced spatial memory, comparable to treatment with estradiol. Antide-treated ovx females retained spatial memory longer than estradiol-treated ovx females. Deficits in spatial memory are a primary symptom of neurodegenerative disorders including Alzheimer's disease (AD). Treatment with Antide prevented spatial memory deficits in a neurotoxin-induced model typical of early AD. These data suggest that memory impairments seen in female rats after ovariectomy or women after menopause may be due to high LH levels and that a reduction in LH enhances memory. These results also implicate an LH lowering agent as a potential preventative therapy for AD.     

Low luteinizing hormone enhances spatial memory and has protective effects on memory loss in rats

Though several studies have suggested that estradiol improves hippocampal-dependent spatial memory, the effects of other hormones in the hypothalamic–pituitary–gonadal axis on memory have largely been ignored. Estradiol and luteinizing hormone (LH) are generally inversely related and LH may significantly affect spatial memory. Ovariectomized (ovx) rats treated with Antide (a gonadotropin releasing hormone receptor antagonist) had low LH levels and showed enhanced spatial memory, comparable to treatment with estradiol. Antide-treated ovx females retained spatial memory longer than estradiol-treated ovx females. Deficits in spatial memory are a primary symptom of neurodegenerative disorders including Alzheimer's disease (AD). Treatment with Antide prevented spatial memory deficits in a neurotoxin-induced model typical of early AD. These data suggest that memory impairments seen in female rats after ovariectomy or women after menopause may be due to high LH levels and that a reduction in LH enhances memory. These results also implicate an LH lowering agent as a potential preventative therapy for AD.     

Peri-pubertal exposure to testicular hormones organizes response to novel environments and social behaviour in adult male rats

Previous research has shown that exposure to testicular hormones during the peri-pubertal period of life has long-term, organizational effects on adult sexual behaviour and underlying neural mechanisms in laboratory rodents. However, the organizational effects of peri-pubertal testicular hormones on other aspects of behaviour and brain function are less well understood. Here, we investigated the effects of manipulating peri-pubertal testicular hormone exposure on later behavioural responses to novel environments and on hormone receptors in various brain regions that are involved in response to novelty. Male rodents generally spend less time in the exposed areas of novel environments than females, and this sex difference emerges during the peri-pubertal period. Male Lister-hooded rats (Rattus norvegicus) were castrated either before puberty or after puberty, then tested in three novel environments (elevated plus-maze, light–dark box, open field) and in an object/social novelty task in adulthood. Androgen receptor (AR), oestrogen receptor (ER1) and corticotropin-releasing factor receptor (CRF-R2) mRNA expression were quantified in the hypothalamus, hippocampus and medial amygdala. The results showed that pre-pubertally castrated males spent more time in the exposed areas of the elevated-plus maze and light–dark box than post-pubertally castrated males, and also confirmed that peri-pubertal hormone exposure influences later response to an opposite-sex conspecific. Hormone receptor gene expression levels did not differ between pre-pubertally and post-pubertally castrated males in any of the brain regions examined. This study therefore demonstrates that testicular hormone exposure during the peri-pubertal period masculinizes later response to novel environments, although the neural mechanisms remain to be fully elucidated.     

Effects of gonadectomy and hormone replacement on a spontaneous novel object recognition task in adult male rats

Recent studies in adult male rats have shown that gonadal hormones influence performance on certain working memory and other types of cognitive tasks that are sensitive to lesions of the medial and/or orbital prefrontal cortices. This study asked whether gonadal hormone modulation of prefrontal cortical function in males also extends to the perirhinal division of the rat prefrontal cortex. Specifically, sham-operated control, gonadectomized, and gonadectomized rats supplemented with testosterone propionate or estradiol were tested on a spontaneous novel object recognition task, a paradigm where performance has been shown to be impaired by perirhinal cortical lesions. Using analyses of variance, regression analyses and post-hoc testing to evaluate group differences, it was found that during both the sample and test trials of the task all four groups spent similar absolute and proportional amounts of time ambulating, rearing, stationary, and exploring the two objects present. All groups also explored each of the two identical objects present during sample trials equally. However, during the test trials, only the control and gonadectomized rats given testosterone showed the expected increase in exploration of the novel objects presented, whereas the gonadectomized and gonadectomized, estradiol-supplemental groups continued to explore the novel and familiar objects equally. That regression analyses also identified significant correlations between low bulbospongiosus muscle weight and impaired novel vs. familiar object discrimination further indicates that gonadectomy in adult male rats adversely affects spontaneous novel object recognition in an androgen-sensitive, estrogen-insensitive manner.     

Influence of gonadal hormones on odours emitted by male meadow voles (Microtus pennsylvanicus).

Free-living male meadow voles (Microtus pennsylvanicus) emit odours that are attractive to females at the beginning, but not at the end, of the breeding season. The effect of gonadal hormones on female-attractant cues was examined in males born and reared in long (14 h light day-1) and short (10 h light day-1) photoperiods that simulate daylengths in the breeding and nonbreeding seasons, respectively. Gonadectomy affected the attractant properties of odours emitted by long photoperiod, but not short photoperiod, males. Long photoperiod females preferred odours of intact rather than those of gonadectomized long photoperiod males, and odours of gonadectomized long photoperiod males rather than those of intact short photoperiod males. Females did not show a preference between the odours of intact and castrated short photoperiod males. Gonadal hormone replacement in males affected female responses to the odours emitted by long photoperiod, but not short photoperiod, gonadectomized males. Long photoperiod females did not display a preference between odours of intact long photoperiod males and gonadectomized long photoperiod males treated with testosterone or oestradiol. We conclude that in spring and summer gonadal hormones increase attractiveness of male odours; this effect may require aromatization of testosterone to oestradiol. Substrates that control attractiveness of odour cues in male voles appear to be unresponsive to androgens during the nonbreeding season.     

Recognition memory in tree shrew (Tupaia belangeri) after repeated familiarization sessions

Recognition memories are formed during perceptual experience and allow subsequent recognition of previously encountered objects as well as their distinction from novel objects. As a consequence, novel objects are generally explored longer than familiar objects by many species. This novelty preference has been documented in rodents using the novel object recognition (NOR) test, as well is in primates including humans using preferential looking time paradigms. Here, we examine novelty preference using the NOR task in tree shrew, a small animal species that is considered to be an intermediary between rodents and primates. Our paradigm consisted of three phases: arena familiarization, object familiarization sessions with two identical objects in the arena and finally a test session following a 24-h retention period with a familiar and a novel object in the arena. We employed two different object familiarization durations: one and three sessions on consecutive days. After three object familiarization sessions, tree shrews exhibited robust preference for novel objects on the test day. This was accompanied by significant reduction in familiar object exploration time, occurring largely between the first and second day of object familiarization. By contrast, tree shrews did not show a significant preference for the novel object after a one-session object familiarization. Nonetheless, they spent significantly less time exploring the familiar object on the test day compared to the object familiarization day, indicating that they did maintain a memory trace for the familiar object. Our study revealed different time courses for familiar object habituation and emergence of novelty preference, suggesting that novelty preference is dependent on well-consolidated memory of the competing familiar object. Taken together, our results demonstrate robust novelty preference of tree shrews, in general similarity to previous findings in rodents and primates.     

Behavioral responses to physical vs. social novelty in male and female laboratory rats

Most behavioral tests used with laboratory rodents involve measuring behavioral responses to physical novelty. However, laboratory rodents are often derived from highly social species for which novel social stimuli may induce different levels of fear or curiosity compared to novel physical objects. We hypothesized that behavioral responses will differ in response to novel physical vs. social cues, and that females may show more exploration of social novelty, based on prior studies indicating that females more actively seek social support during duress compared to males. We compared young (55-day-old) Sprague-Dawley rats’ responses to an arena filled with novel objects (“physical”) or a novel same-sex caged conspecific (“social”). Rats were more active and spent twice as much time in contact with the novel social stimulus compared to novel physical stimuli. Although females were more active than males, females were not particularly more exploratory in the social arena compared to males. The results indicate that a novel social partner (even a caged one with limited ability to interact) elicits more exploration than novel objects for both male and female rats.     

Hormonal status and test condition, but not sexual experience, modulate partner preference in female rats

A series of experiments was conducted to determine the contributions of hormonal status, test condition, and sexual experience to the display of partner preference by female rats. Preference for a sexually active male rat over a sexually receptive female rat was assessed in independent groups of female rats tested in a condition limiting physical contact (No Contact) and a condition allowing for sexual interaction (Contact). Although hormonal status and test condition influenced the preference for a sexually active male, repeated testing and sexual experience had no effect. Experiment 1 demonstrated that independent of test condition, preference for the male is stronger in estrogen- and progesterone-primed rats than in rats receiving the vehicle. Moreover, independent of hormone condition, rats tested in the No Contact condition exhibit a stronger preference for the male than rats tested in the Contact condition, reflecting in part the active pacing of mating stimulation by sexually receptive rats tested in the Contact condition. Experiment 2 showed that the overall pattern of partner preference in proestrous and diestrous rats was similar to that observed in ovariectomized, estrogen- and progesterone-primed, and oil-treated rats, respectively. In Experiment 3, rats primed with estrogen alone did not exhibit a preference for the male even though fully receptive. Experiments 4 and 5 demonstrated that sexual experience does not affect the expression of preference for the male in estrogen- and progesterone-primed rats. The present findings demonstrate that the female rat's preference for the male is stable across repeated tests and is not affected by sexual experience. Our results also confirm that gonadal hormones influence the expression of a preference for a sexually active male versus a sexually receptive female and demonstrate that the magnitude of preference is modulated by test conditions.     

Spaced initial stimulus familiarization enhances novelty preference in Long-Evans rats

Berlyne [Berlyne, D.E., 1950. Novelty and curiosity as determinants of exploratory behaviour. Brit. J. Psychol. 41, 68–80] first illustrated that rats prefer to explore novel objects over ones with which they have had previous experience. Recently, variants on this novel object recognition (NOR) task have become widely popular and have been employed in numerous neuroscience and behavioral pharmacological studies investigating memory processes. Given this popularity, a thorough understanding of the various behavioral processes involved in novelty reaction and preference is essential. The current study compared the effects of spaced and massed initial stimulus exposures upon later object exploration and novel stimulus preference in Long-Evans rats. Results illustrated that a distributed initial stimulus familiarization procedure promoted greater novel object preference than did a massed procedure, and suggest that the novel object recognition task is sensitive to spacing effects in a similar fashion to more traditional learning paradigms. The mechanisms underlying such spacing effects are briefly discussed.     

Testicular hormone exposure during adolescence organizes flank-marking behavior and vasopressin receptor binding in the lateral septum

Adolescence is a period during which many social behaviors emerge. One such behavior, flank marking, is a testosterone-modulated scent marking behavior that communicates dominance status between adult male Syrian hamsters. Testosterone modulates flank-marking behavior by altering neural transmission of vasopressin within a forebrain circuit. This study tested whether testicular hormones secreted during adolescence play purely a transient activational role in the display of flank-marking behavior, or whether adolescent steroid hormone secretions also cause long-term organizational changes in vasopressin binding within brain regions underlying flank-marking behavior. We tested this hypothesis by manipulating whether testicular secretions were present during adolescent development and then tested for flank-marking behavior and vasopressin receptor binding within the flank-marking neural circuit in young adulthood. Specifically, males were gonadectomized immediately before or after adolescence, replaced with testosterone 6 weeks following gonadectomy in young adulthood, and behavior tested 1 week later. Adult testosterone treatment activated flank-marking behavior only in males that were exposed to testicular hormones during adolescence. In addition, males exposed to testicular hormones during adolescence exhibited significantly less vasopressin receptor binding within the lateral septum than males deprived of adolescent hormones, suggesting that hormone-dependent remodeling of synapses normally occurs in the lateral septum during adolescence. These data highlight the importance of gonadal steroid hormone exposure during adolescence for the organization of neural circuits and social behavior.     

Lack of gonadal control of the dorsal gland and sandbathing in male and female bannertail kangaroo rats (Dipodomys spectabilis)

Previous studies have demonstrated gonadal control of mammalian scent glands; castration leads to reduced scent-marking rates and smaller gland sizes. I investigated whether gonadal hormones control the size of the dorsal gland (a specialized sebaceous gland) and sandbathing as a scent-marking behavior in adult male and female bannertail kangaroo rats (Dipodomys spectabilis). Gland sizes of males and females were similar in all age classes, except adult males with larger body weights had proportionately larger glands than females. Male gland sizes declined 18% following castration but were not significantly smaller than those of intact males; females showed no change in dorsal gland size either as a result of ovariectomy or after estradiol benzoate implants. Sandbathing rates also did not decline as a result of gonadectomy. Rather, castrated males and overiectomized females sandbathed at higher frequencies than intact males and ovariectomized females with estradiol benzoate implants, respectively. Gonadal hormones apparently have little influence on the function of a specialized scent gland and may inhibit sandbathing as a scent-marking behavior in adult D. spectabilis.     

Endocrine influences on the actions of morphine: IV. Effects of sex and strain

The antinociceptive and temperature responses to morphine were compared in male and female rats from two different strains. Males of both the Sprague-Dawley and Wistar-Furth strains were slightly more responsive to the acute actions of morphine than were females of the same strain. However, Wistar-Furth animals required approximately twice the dose of morphine to display equivalent antinociceptive responses and four times the dose of display equivalent hypothermic responses when compared with Sprague-Dawley animals. During chronic morphine treatment, the development of tolerance was slightly more rapid in males than in females and in Sprague-Dawley animals than in Wistar-Furth animals. Gonadal hormones also influenced morphine responses. Ovariectomized rats were significantly more responsive acutely to morphine and developed tolerance less rapidly than estradiol-treated females. However, alterations of gonadal hormones in males did not affect morphine responses. These results indicate that morphine responses vary considerably between strains of animals and are influenced by gonadal hormones of females, but not of males.     

Measurement of gonadal hormones in dried blood spots versus serum: verification of menstrual cycle phase

In the present study, we show that blood spot assays for estradiol, progesterone, and testosterone are a reliable, accurate, and sensitive means for measuring circulating gonadal hormones. The lower limit of sensitivity of each blood spot assay is sufficient to determine gonadal hormone levels in adult females. Correspondence of serum to blood spot measures is high, with blood spot hormone levels explaining an average of 88.60% of the variance in serum gonadal hormones in females, but only 46.20% in males. We provide formulas for converting hormone levels in blood to hormone levels in serum (which traditional endocrinology studies report). Finally, we show that careful attempts to estimate hormone status by day-count methods are unreliable when compared to hormone assay in blood spots.     

Sex differences and ovarian hormones in animal models of drug dependence

Increasing evidence indicates the presence of sex differences in many aspects of drug abuse. Most studies reveal that females exceed males during the initiation, escalation, extinction, and reinstatement (relapse) of drug-seeking behavior, but males are more sensitive than females to the aversive effects of drugs such as drug withdrawal. Findings from human and animal research indicate that circulating levels of ovarian steroid hormones account for these sex differences. Estrogen (E) facilitates drug-seeking behavior, while progesterone (P) and its metabolite, allopregnanalone (ALLO), counteract the effects of E and reduce drug seeking. Estrogen and P influence other behaviors that are affiliated with drug abuse such as drug-induced locomotor sensitization and conditioned place preference. The enhanced vulnerability to drug seeking in females vs. males is also additive with the other risk factors for drug abuse (e.g., adolescence, sweet preference, novelty reactivity, and impulsivity). Finally, treatment studies using behavioral or pharmacological interventions, including P and ALLO, also indicate that females show greater treatment effectiveness during several phases of the addiction process. The neurobiological basis of sex differences in drug abuse appears to be genetic and involves the influence of ovarian hormones and their metabolites, the hypothalamic pituitary adrenal (HPA) axis, dopamine (DA), and gamma-hydroxy-butyric acid (GABA). Overall, sex and hormonal status along with other biological risk factors account for a continuum of addiction-prone and -resistant animal models that are valuable for studying drug abuse prevention and treatment strategies.     

Evidence for extended action of gonadal hormones on the organization of sexually dimorphic behavior and morphology in gray short-tailed opossums (Monodelphis domestica)

Male and female gray short-tailed opossums were gonadectomized (GDX), or treated with the estrogen receptor antagonist tamoxifen citrate (TX), or corn oil (OIL) (control) during the 5th postnatal week, a time period equivalent to the 3rd postnatal week in rats and associated with high levels of circulating gonadal hormones and neural aromatase activity in this marsupial species. In adulthood following gonadectomy (for animals not previously gonadectomized) and replacement therapy with estradiol or testosterone, GDX males showed less male-typical scent marking and had shorter phalluses than OIL and TX males. Following replacement therapy with estradiol, GDX females were more likely to fight with and less likely to mate with stimulus males than TX females; OIL females were intermediate in these measures. Along with previous findings, these results suggest that gonadal hormones act over an extended postnatal period to organize sexually dimorphic behavior and morphology in male gray opossums and may have some effect on the organization of aggressive behavior in females of this species.     

Platelet serotonin levels and gonadal hormones in rats

The role of gonadal hormones in the control of platelet serotonin levels was studied by evaluating the effect of sexual maturation in rats of both sexes and the time-course of changes following gonadectomy performed either prepubertally or on sexually mature animals. In males, platelet serotonin levels remained fairly stable during sexual maturation as well as during the whole postgonadectomy period monitored (four months). In females, somewhat higher values of platelet serotonin levels in adult than in sexually immature animals were found (9%, p less than 0.001, N = 34). A slight decrease of platelet serotonin (10-18%, p less than 0.05) was observed following ovariectomy of sexually mature females, but it was of transient nature. When females were ovariectomized prepubertally a tendency towards permanently lower platelet serotonin levels was noticed. These results suggest that gonadal hormones have no major role in the control of platelet serotonin levels in rats, although a subtle hormonal modulation of this platelet variable in females may exist.     

Coexistence of Anhedonia and anxiety-independent increased novelty-seeking behavior in the chronic mild stress model of depression

Previous research demonstrated excessive decreases in reward sensitivity and increases in harm avoidance in depressed individuals. These results straightly lead to a hypothesis that depressed patients should avoid novelty or express reduced novelty-seeking behavior. Nevertheless, literature in this regard is inconsistent. Furthermore, whether the potentially altered novelty-associated behavior is dependent on changed anxiety/fear or related to altered goal-directed approaching tendency is unclear. Here, we tested novel object-approaching behavior in a free-exploration paradigm in chronic mild stress (CMS)-induced anhedonic and stress-resistant rats respectively. Other CMS-induced, emotional behaviors were also examined in a battery of behavioral tests including novel cage, exploration, locomotor activity and elevated plus maze (EPM). We found that compared with controls, stress-resistant rats who consistently showed lower anxiety level in EPM (time in open arms) and, open-field (OF) test (time in central area) showed no sign of enhanced novel object approaching behavior. To the contrary, the anhedonic ones who did not express any sign of reduced anxiety showed paradoxically intensified novelty-approaching behavior. We concluded that reduced anxiety would not necessarily lead to enhanced novelty-seeking behavior; anhedonia coexists with anxiety-independent, increased novelty-seeking behavior. The salient paradox of coexistence of anhedonia and increased novelty-seeking behavior was critically discussed.     

Ghrelin Influences Novelty Seeking Behavior in Rodents and Men

Recent discoveries indicate an important role for ghrelin in drug and alcohol reward and an ability of ghrelin to regulate mesolimbic dopamine activity. The role of dopamine in novelty seeking, and the association between this trait and drug and alcohol abuse, led us to hypothesize that ghrelin may influence novelty seeking behavior. To test this possibility we applied several complementary rodent models of novelty seeking behavior, i.e. inescapable novelty-induced locomotor activity (NILA), novelty-induced place preference and novel object exploration, in rats subjected to acute ghrelin receptor (growth hormone secretagogue receptor; GHSR) stimulation or blockade. Furthermore we assessed the possible association between polymorphisms in the genes encoding ghrelin and GHSR and novelty seeking behavior in humans. The rodent studies indicate an important role for ghrelin in a wide range of novelty seeking behaviors. Ghrelin-injected rats exhibited a higher preference for a novel environment and increased novel object exploration. Conversely, those with GHSR blockade drastically reduced their preference for a novel environment and displayed decreased NILA. Importantly, the mesolimbic ventral tegmental area selective GHSR blockade was sufficient to reduce the NILA response indicating that the mesolimbic GHSRs might play an important role in the observed novelty responses. Moreover, in untreated animals, a striking positive correlation between NILA and sucrose reward behavior was detected. Two GHSR single nucleotide polymorphisms (SNPs), rs2948694 and rs495225, were significantly associated with the personality trait novelty seeking, as assessed using the Temperament and Character Inventory (TCI), in human subjects. This study provides the first evidence for a role of ghrelin in novelty seeking behavior in animals and humans, and also points to an association between food reward and novelty seeking in rodents.     

Gonadal hormones modulate sex differences in judgments of relative numerousness in meadow voles, Microtus pennsylvanicus

Animals in a variety of taxa discriminate between a greater quantity and a lesser quantity of the same object, an ability that is referred to as relative numerousness judgment. For example, meadow voles can distinguish between areas containing more over-marks by one opposite-sex scent donor and fewer over-marks by another opposite-sex scent donor. Females appear to be able to make better discriminations between more or less over-marks than do males. In that gonadal hormones have been implicated in modulating cognitive function associated with spatial tasks, we tested the hypothesis that high titers of testosterone and estradiol are necessary for male and female voles, respectively, to distinguish between the top- and bottom-scent donors in an area containing mixed over-marks. We gonadectomized voles, giving them either gonadal hormone replacement (testosterone for males and estradiol for females) or no hormone replacement, and tested their spontaneous judgments of distinguishing between the top- and bottom-scent donors in an area containing mixed over-marks; a task involving judgments of relative numerousness. Female voles given replacement estradiol performed better than did female voles not given replacement estradiol in determining the top-scent and bottom-scent males in areas containing mixed over-marks. In contrast, males not treated with replacement testosterone performed better than did males treated with testosterone in determining the top-scent and bottom-scent males in areas containing mixed over-marks. Thus, high titers of estradiol and low titers of testosterone are associated with better performance on tasks involving relative numerousness in female and male voles, respectively. The results of this task on relative numerousness judgments are discussed in relation to the effects of gonadal steroid hormone on spatial ability, a closely related cognitive domain, and the social biology of meadow voles.