Effects of gonadectomy and exogenous gonadal steroids on sex differences in open field behaviour of adult rats

Two experiments were carried out to investigate the contribution of gonadal hormones to the expression of sex differences in open field behaviour of adult female and male rats.In the first experiment rats were gonadectomized or sham-operated in adulthood and tested in the open field 1, 2, 3, 4, 5 or 6 weeks later (3 min/rat on 3 consecutive days during the light period of the day). It was found that following gonadectomy the well known sex difference in ambulation and rearing behaviour (females more than males), as well as in defaecation (females less than males) remained. Ovariectomy caused a decrease in ambulation and rearing, with an increase in defaecation, whereas castration had no significant effect on ambulation and rearing, but also increased defaecation. Also an overall increase in ambulation and rearing was found: animals tested 5 and 6 weeks following the operation were significantly more active than animals tested at 1–4 weeks after the operation.In the second experiment female and male rats were gonadectomized and 7 weeks later they received a silastic implant containing testosterone (T), dihydrotestosterone (DHT), oestradiol (E₂), DHT plus E₂, or nothing (control condition). Four weeks after the implantation the animals were tested in the open field (3 min/rat on 3 consecutive days). An overall sex difference was found for ambulation (females more than males) and for defaecation (females less than males). The only effect of hormone administration was found in DHT-treated animals: lowest ambulation and highest defaecation compared to other hormone and control animals. No differences were found between the T, E₂, DHT plus E₂, and control animals.From these data it was concluded that endogenous gonadal steroids seem to play a minor role in the expression of sex differences in adult open field behaviour in the rat.     

Sex and estrus cycle differences in the modulatory effects of morphine on seizure susceptibility in mice

PURPOSE: To evaluate the effects of sex and estrus cycle on biphasic anticonvulsant and proconvulsant modulation of seizure threshold by morphine. METHODS: The threshold for the clonic seizures (CST) induced by acute intravenous administration of gamma-aminobutyric acid (GABA)-antagonist pentylenetetrazole (PTZ) was assessed in male and female mice. Estrus cycle was assessed by vaginal smears. The effect of removing circulating sex hormones was assessed by gonadectomy. RESULTS: At baseline, diestrus females had a higher CST compared with males and estrus females. Morphine at lower doses (0.5-3 mg/kg) had a significant anticonvulsant effect in males and estrus females compared with that in vehicle-treated controls, whereas female mice in diestrus phase showed a relative subsensitivity to this effect. Morphine at higher doses (30 and 60 mg/kg) significantly decreased CST in males and diestrus females, with less relative effect in estrus mice. In both phases, morphine exerted stronger effects in males compared with females. Ovariectomy brought the baseline CST to the male level and resulted in significant expression of both phases of morphine effect but did not abolish the sex difference in responsiveness to morphine. CONCLUSIONS: The biphasic modulation of seizure threshold is subject to both constitutive sex differences in sensitivity to morphine and hormonal fluctuations during the estrus cycle.     

Sex Differences in the Expression of Sex Steroid Receptor mRNA in the Quail Brain

In Japanese quail, males will readily exhibit the full sequence of male‐typical sexual behaviors but females never show this response, even after ovariectomy and treatment with male‐typical concentrations of exogenous testosterone. Testosterone aromatisation plays a key‐limiting role in the activation of this behavior but the higher aromatase activity in the brain of males compared to females is not sufficient to explain the behavioural sex difference. The cellular and molecular bases of this prominent sex difference in the functional consequences of testosterone have not been identified so far. We hypothesised that the differential expression of sex steroid receptors in specific brain areas could mediate this behavioural sex difference. Therefore, using radioactive in situ hybridisation histochemistry, we quantified the expression of the mRNA coding for the androgen receptor (AR) and the oestrogen receptors (ER) of the α and β subtypes. All three receptors were expressed in an anatomically discrete manner in various nuclei of the hypothalamus and limbic system and, at usually lower densities, in a few other brain areas. In both sexes, the intensity of the hybridisation signal for all steroid receptors was highest in the medial preoptic nucleus (POM), a major site of testosterone action that is related to the activation of male sexual behaviour. Although no sex difference in the optical density of the AR hybridisation signal could be found in POM, the area covered by AR mRNA was significantly larger in males than in females, indicating a higher overall degree of AR expression in this region in males. By contrast, females tended to have significantly higher levels of AR expression than males in the lateral septum. ERα was more densely expressed in females than males throughout the medial preoptic and hypothalamic areas (including the POM and the medio‐basal hypothalamus), an area implicated in the control of female receptivity) and in the mesencephalic nucleus intercollicularis. ERβ was more densely expressed in the medio‐basal hypothalamus of females but a difference in the reverse direction (males > females) was observed in the nucleus taeniae of the amygdala. These data suggest that a differential expression of steroid receptors in specific brain areas could mediate at least certain aspects of the sex differences in behavioural responses to testosterone, although they do not appear to be sufficient to explain the complete lack of activation by testosterone of male‐typical copulatory behaviour in females.     

Meta-analysis-tested formal models of potential mechanisms underlying females’ low autism-spectrum-disorder diagnosis rate compared to males’

BackgroundWhy is autism spectrum disorder (ASD) less prevalent among females than males? We constructed a statistical model for each of both existing classes of theories, and derived competing predictions for the essentialist expression hypothesis (females express less severe ASD traits so are diagnosed less) against the constructivist perception hypothesis (females’ expressions are socially perceived as less severe so are diagnosed less). Specifically, if the expression hypothesis is true, based on our models, diagnosed females should show less severe symptoms than their male counterparts, whereas the reverse should happen if the perception hypothesis is true.MethodWe conducted a meta-analysis (Data point N = 117,778 participant N = 16,209) on the differences in ASD symptom severity between females and males diagnosed with ASD, across age groups, IQ ranges, diagnostic criteria, and assessment tools.ResultsWe found strong new evidence that ASD-diagnosed females and males differ little in symptom severity, even in the face of a found publication bias in favor of reporting males’ symptoms relative to females’ and the common understanding of ASD as a so-called male disorder.ConclusionsWe argue the finding supports both classes of theories, implying that they are similar in size, though different in directions, in creating sex differences in symptom severity for diagnosed individuals. The sex disparity in ASD prevalence likely results from both the biological expression of, and the social perception toward individuals’ ASD symptoms.     

Estrogen receptor-alpha distribution in male rodents is associated with social organization

It has been hypothesized that site-specific reduction of estrogen receptor-alpha (ERalpha) is associated with the expression of male prosocial behaviors. Specifically, highly social males are predicted to express significantly lower levels of ERalpha than females and less social males in brain regions associated with prosocial behavior including the bed nucleus of the stria terminalis (BST) and the medial amygdala (MeA). This hypothesis was tested by comparing ERalpha immunoreactivity (IR) in three species of microtines, the polygynous montane (Microtus montanus) and meadow (M. pennsylvanicus) voles and the monogamous pine vole (M. pinetorum), and two species of cricetines that differ in the extent of social pair-bond formation, Siberian (Phodopus sungorus) and Djungarian (P. campbelli) hamsters. As predicted, ERalpha-IR was sexually dimorphic in the BST and MeA of the highly social species, with females expressing more ERalpha-IR cells than males. Male and female montane voles did not differ. Male and female meadow voles differed in the ventromedial hypothalamus, with females expressing more ERalpha-IR cells. Male pine voles expressed lower levels of ERalpha-IR in the MeA than male montane and meadow voles and in the BST relative to montane males. Male Djungarian hamsters, which show higher levels of parental care, had fewer ERalpha-IR cells in the BST than male Siberian hamsters. Results indicate that the distribution of ERalpha differs relative to the continuum of species-typical affiliative behavior and supports the hypothesis that ERalpha has a significant role in regulating species-specific social organization.     

Sexual dimorphism in brain weight of meadow voles: role of gonadal hormones

In most adult mammals, brain weights of males exceed those of females. The role of androgens in the genesis of this sex difference was assessed in meadow voles by acute neonatal or chronic postweaning manipulation of testosterone titers. Female voles given a single injection of testosterone propionate (TP) on the second day of postnatal life had brain weights in adulthood that were indistinguishable from those of male voles and significantly heavier than those of control females. Whole brain DNA content, a measure of cell number, was not increased by neonatal TP treatment. Females treated with TP from day 19 to 70 had lower brain weights than control females and males gonadectomized at 19 days of age had greater brain weights than did intact male voles at day 70. The sex dimorphism in brain weight reflects organizational effects of testosterone during perinatal development. Beginning at weaning, and continuing through postpubertal development, testosterone decreases brain weight in both sexes. We suggest that testosterone affects brain weight by altering cell size or non-cellular components rather than cell number.     

Endogenous Sex Steroids Dampen Neuroinflammation and Improve Outcome of Traumatic Brain Injury in Mice

The role of biological sex in short-term and long-term outcome after traumatic brain injury (TBI) remains controversial. The observation that exogenous female sex steroids (progesterone and estrogen) reduce brain injury coupled with a small number of clinical studies showing smaller injury in women suggest that sex steroids may play a role in outcome from TBI. We used the controlled cortical impact (CCI) model of TBI in mice to test the hypothesis that after CCI, female mice would demonstrate less injury than male mice, related to the protective role of endogenous steroids. Indeed, adult females exhibit histological protection (3.7?±?0.5 mm³) compared to adult male mice (6.8?±?0.6 mm³), and females that lacked sex steroids (ovex) showed increased injury compared to intact females. Consistent with histology, sensorimotor deficits measured as reduced contralateral limb use were most pronounced in male mice (31.9?±?6.9% reduced limb use) compared to a 12.7?±?3.8% reduction in female mice. Ovex mice exhibited behavioral deficits similar to males (31.5?±?3.9% reduced limb use). Ovex females demonstrated increased microglial activation relative to intact females in both the peri-injury cortex and the reticular thalamic nucleus. Ovex females also demonstrated increased astrogliosis in comparison to both females and males in the peri-injury cortex. These data indicate that female sex steroids reduce brain sensitivity to TBI and that reduced acute neuroinflammation may contribute to the relative protection observed in females.     

The role of sex hormones on formalin-induced nociceptive responses

Many chronic pain conditions are more frequent in women than in men. This observation suggests that there is a potential role of sex hormones on pain perception. In the present study, we measured nociceptive responses to the formalin test in normal and gonadectomized male and female rats. The nociceptive responses to formalin injection were divided in four phases: acute (phase I), interphase and late phases (phases II and III). Four groups of rats were tested: (a) males (n = 15), (b) females (n = 16), (c) ovariectomized females (OVX) (n = 15) and (d) castrated males (CAST) (n = 15). Females presented significantly more nociceptive responses than males during phase I, interphase and phase II (P < 0.01). They also presented significantly more nociceptive responses than OVX females during the interphase (P < 0.05). CAST males presented significantly more nociceptive responses during the phases I (P < 0.01), II (P < 0.01) and III (P < 0.05) than the male rats. Finally, the responses of CAST males and OVX females were virtually identical, suggesting that the differences recorded between males and females in the formalin test were related to an activational effect of the sex hormones rather than an organizational effect. In conclusion, these results permit the support of the role of sex hormones on the modulation of pain perception. Interestingly, male and female sex hormones seem to act specifically on the different phases of the formalin test, suggesting some specific roles for sex hormones in different pain conditions.     

The definition of depression

Prevalence figures of depression usually show a sex ratio for males:females of 1:2. This unequal ratio has been examined by varying the time periods for the calculation of prevalance rates and the criteria for case definition. Based on results of a 20–23 yr-old cohort of 591 males and females studied in Switzerland, some evidence was found to support the hypothesis that an unequal sex ratio is partially due to unequal reporting of depressive symptoms and to differential forgetting, perhaps linked with the male role in society. It is not yet possible to extend the results of this study to older cohorts. Further research would be desirable.     

Sex differences in social investigation: effects of androgen receptors, hormones and test partner

We examined the role of the androgen receptor (AR) in the investigatory behaviour of conspecifics using mice carrying the testicular feminisation mutation (X(Tfm) Y). Responses to members of the same and opposite sex were evaluated in a habituation/dishabituation task. Adult mice were gonadectomised and treated with oestradiol (E(2) ) or testosterone. After E(2) treatment, regardless of the sex of the stimulus mouse, wild-type (WT) males engaged in significantly more investigation than WT females. X(Tfm) Y males treated with E(2) showed 'male-like' behaviour in response to a male but behaved 'female-like' when the stimulus was a female. Because WT and X(Tfm) Y males behaved the same in response to another male, we used two additional mouse models to ask whether sex chromosomes were responsible for this phenomenon. Regardless of sex chromosome complement, gonadal males displayed high levels of investigation. When mice were treated with testosterone, investigation by WT females was enhanced, which eliminated the sex differences. Most strikingly, X(Tfm) Y males receiving testosterone-treatment increased the investigation of females to levels equal to those shown by WT mice. Given that testosterone, but not its metabolite E(2) , caused X(Tfm) Y males to investigate female conspecifics at high levels, it is plausible that nonclassical actions of AR, and/or activation of a novel AR, may be involved in this behaviour. Taken together, our data show that AR activation during adulthood is not required for males to investigate mice of either sex. However, 'male-like' levels of investigation of a female stimulus may depend on neonatal activation of the classic nuclear AR.     

Sex differences in the glutamate signaling pathway in juvenile rats

Females have been found to be at lower risk for the development of neurodevelopmental disorders than males. The greater neuroprotection in females is mostly due to female sex hormones. Estrogen is hypothesized to provide neuroprotection by suppressing the neuro‐excitotoxicity induced by glutamate (Glu). This study was conducted to understand the effect of sex in modulating Glu signaling in juvenile rats. Brain tissue homogenate of 15 Wistar albino rats (9 males, 6 females) weighing 60 to 80 g and aged approximately 28 days was used. Biochemical parameters related to Glu signaling, such as the absolute and relative concentrations of Glu, gamma aminobutyric acid (GABA), and glutamine, as well as glutamate transporter 1 (GLT1), glutamine synthetase (GS), glutaminase (GLN), and glutamate decarboxylase‐67 (GAD‐67), were measured by ELISA. The data obtained demonstrated that compared with the levels in males, female rats exhibited significantly lower levels of Glu (p = .001) and GLN/GS (p = .021). The Glu/GABA and Glu/GLT1 ratios as well as the levels of GAD‐67 were also lower in female rats, although the difference was not significant. The GLN/GAD‐67 ratio (p = .027) and levels of GS (p = .019) were significantly higher in female rats than in males. Multiple regression analysis confirmed the role of GLN/GS, together with the much higher affinity of GLT1 to Glu, in avoiding excitotoxicity in females. In conclusion, there was a significant difference in Glu signaling between female and male rats. The females exhibited a lower susceptibility to develop Glu‐induced excitotoxicity, an etiological mechanism for multiple neurodevelopmental disorders.     

Male and female differences in enzyme activities of energy metabolism in vastus lateralis muscle

To investigate sex differences in the organization of enzyme activities of energy supplying metabolism in skeletal muscle, samples of the vastus lateralis were extracted from active but untrained males (n = 16) and females (n = 17), ranging in age from 18 to 22 years. Muscle tissue from 2 different biopsy samples from each subject were analyzed for enzymes representative of the citric acid cycle (succinic dehydrogenase, SDH), beta-oxidation of fatty acids (3-hydroxyacyl CoA dehydrogenase, HAD), glycogenolysis (phosphorylase, PHOSPH), glycolysis (pyruvate kinase, PK; phosphofructokinase, PFK and lactate dehydrogenase, LDH) and glucose phosphorylation (hexokinase, HK). The results indicated that the maximal activities of PFK, PK, LDH and PHOSPH, HK and SDH averaged between 15 and 32% higher in the males than in the females. No significant differences between the sexes were found for HAD. When enzyme activity ratios were calculated, sex differences were only evident for the HAD/SDH ratio (mean +/- SD; females = 0.56 +/- 0.20; males = 0.41 +/- 0.11 and for the PFK/HAD ratio (females = 7.40 +/- 1.6; males = 9.58 +/- 1.9). The findings suggest that (1) the females have a significantly lower overall capacity for aerobic oxidation and for anaerobic glycolysis than the males; (2) the females have a greater capacity for beta-oxidation relative to the capacity of the citric acid cycle; and (3) the glycolytic potential relative to the potential for beta-oxidation is lower in the females.     

Oestrogen and androgen receptor activation contribute to the masculinisation of oxytocin receptors in the bed nucleus of the stria terminalis of rats

Oxytocin (OT) often regulates social behaviours in sex‐specific ways, and this may be a result of sex differences in the brain OT system. Adult male rats show higher OT receptor (OTR) binding in the posterior bed nucleus of the stria terminalis (pBNST) than adult female rats. In the present study, we investigated the mechanisms that lead to this sex difference. First, we found that male rats have higher OTR mRNA expression in the pBNST than females at postnatal day (P) 35 and P60, which demonstrates the presence of the sex difference in OTR binding density at message level. Second, the sex difference in OTR binding density in the pBNST was absent at P0 and P3, but was present by P5. Third, systemic administration of the oestrogen receptor (ER) antagonist fulvestrant at P0 and P1 dose‐dependently reduced OTR binding density in the pBNST of 5‐week‐old male rats, but did not eliminate the sex difference in OTR binding density. Fourth, pBNST‐OTR binding density was lower in androgen receptor (AR) deficient genetic male rats compared to wild‐type males, but higher compared to wild‐type females. Finally, systemic administration of the histone deacetylase inhibitor valproic acid at P0 and P1 did not alter pBNST‐OTR binding density in 5‐week‐old male and female rats. Interestingly, neonatal ER antagonism, AR deficiency, and neonatal valproic acid treatment each eliminated the sex difference in pBNST size. Overall, we demonstrate a role for neonatal ER and AR activation in setting up the sex difference in OTR binding density in the pBNST, which may underlie sexual differentiation of the pBNST and social behaviour.     

Hormonal regulation of axonal sprouting in the hippocampus

Removal of septal fibers to the rat hippocampal formation is followed by an ingrowth of sympathetic axons into the deafferented regions. We have shown previously that the pattern of sprouting is more restricted in males than in females when the lesions are made in mature animals, but is almost the same if lesions are made during the early postnatal period. In the present study, the relationship of circulating sex hormones to the sprouting response was investigated by comparing the extent of sympathetic axon in-growth following fimbrial lesions in intact or gonadectomized adult male and female rats. The effects of manipulating sex steroids during development was examined by comparing sympathetic axonal sprouting after fimbrial lesions in rats which were castrated (male) or treated with testosterone (females and castrated males) on postnatal day 2.We find that (1) gonadectomy of either adult female or male rats does not affect the sprouting response, but (2) neonatal castration of male rats permits sprouting in a pattern similar to normal females, and neonatal testosterone treatment of females or castrated males results in the more limited sprouting response characteristic of normal males. These results indicate that the sex-related differential response to fimbrial lesions may be determined by developmental differences in endogenous steroid levels.     

Sex differences in androgen receptor mRNA levels and regulation in hamster facial motoneurons

We have previously shown that testosterone propionate augments hamster facial nerve regeneration to a greater extent in males than females. Further, sex differences in facial nerve regeneration have been observed. From those studies, we hypothesized that sex differences in nerve regeneration could be due to inherent differences in androgen receptor (AR) mRNA content within facial motor neurons (FMN) of male and female hamsters. In the present study, that hypothesis was tested using in situ hybridization, and computerized image analysis to quantify levels and regulation of AR mRNA in individual FMN of hamsters of both sexes. Intact and gonadectomized (gdx) male and female hamsters were used in the initial experiments in the study. In subsequent experiments, exogenous testosterone propionate (TP) was administered to the aforementioned groups of animals by subcutaneous implantation of one 10-mm Silastic capsule for 1, 2 or 7 days. FMN of intact females contained approximately 50% less AR mRNA than their male counterparts. Gonadectomy in males downregulated AR mRNA levels by approximately 50%, whereas no effects of gonadectomy were observed in females. Thus, in all paradigms where TP levels were low relative to the intact males, AR mRNA levels were approximately half of those in the intact male FMN. TP administration induced AR mRNA levels in gdx males within 1 day. Significant effects of TP were not detected in gdx females, and only after 7 days in the intact females. To our knowledge, the results of this study are the first quantitative demonstration of sex differences in steroid receptor mRNA content in a given neuronal population and substantiate the idea that sex differences in the effects of androgens on peripheral nerve regeneration are based on intrinsic sex differences in the levels and regulation of receptor mRNA in motor neurons.     

The behavior of male and female Wistar rats pressing a lever for food is not affected by sex differences in food motivation

Male rats both eat more and weigh more than females. Differences in food intake and body weight result, at least partly, from differences in gonadal hormone secretions. The present experiments were designed to investigate whether sex differences in food motivation might contribute to the behavioral differences observed when food-deprived male and female rats are exposed to appetitively motivated operant learning tasks. Male and female Wistar rats were exposed to different progressive ratio schedules of reinforcement which have been shown to generate reliable indices of 'motivational' conditions. In progressive ratio schedules, subjects are required to make a systematically increasing number of responses for each successive reinforcer, until the requirement becomes so large that the subjects stop responding. Expt. Ia was designed to investigate whether or not food-deprived males would be more motivated than food-deprived females to obtain food. Expt. Ib investigated whether gonadectomy might differentially affect food motivation of male and female rats exposed to a progressive ratio schedule of reinforcement. Motivational differences between males and females were not observed. Males and females obtained an equal number of reinforcers, while differences in the total number of responses and response rate were not observed. Gonadectomy did not affect the total number of responses in the final completed ratio. The results of the present experiments do therefore not support the hypothesis that sex differences in food motivation might underlie sex differences in behavior when food-deprived male and female rats are exposed to operant schedules of positive reinforcement.     

Relationships between sex and the size and number of forebrain gonadotropin-releasing hormone-immunoreactive neurones in the ballan wrasse (Labrus berggylta), a protogynous hermaphrodite.

This study is the first to examine the brain gonadotropin-releasing hormone (GnRH) cell population phenotype in a protogynous and monandric sequentially hermaphroditic fish. Male ballan wrasse (Labrus berggylta) had on average higher numbers of GnRH-immunoreactive (GnRH-ir) cells within the brain preoptic area (POA) than females, a difference not found in GnRH-ir cells in other brain regions. Furthermore, in males, but not females, the number of these POA GnRH-ir cells correlated with body size. Maturational state (prespawning or postspawning) had marked effects on mean profile sizes (but not numbers) of both GnRH-ir cell bodies and cell nuclei, even when existing differences in body size and allometric relationships had been taken into account. Postspawning males tended to have larger GnRH-ir profiles in all brain regions relative to both prespawning males and females. Moreover, the GnRH-ir cell number in POA, and the cell body profile size in both POA and at the level of the anterior commissure, correlated with gonad size in spermiated prespawning males, indicating a relationship between both size and number of GnRH cells and male gonadal development. These results suggest that temporary changes in the size of brain GnRH-ir neurones are coupled to the male spawning cycle, and that permanent POA GnRH-ir cell number changes are involved in the process of sex change in sequential hermaphrodites. However, smaller males had no more preoptic GnRH-ir cells than equally sized females, which may argue against a proximate inducing role of GnRH cell number changes in naturally occurring sex reversal.     

Sex differences in children with autism spectrum disorders compared with their unaffected siblings and typically developing children

This study examined the nature of cognitive and behavioral sex differences in children with autism spectrum disorders (ASDs) and two comparison groups: a group of typically developing (TD) children and a group of unaffected siblings of ASD children. Sex differences in core autistic symptoms, co-occurring behavioral symptoms, and cognitive styles were assessed in each group. Females with ASD were less severely affected than IQ- and age-matched males with ASD in the communication and repetitive stereotyped behavior domains, as measured by the ADI-R, but such sex differences were less significant than they were in the unaffected sibling group. Several behavioral/emotional symptom scores were significantly higher in male siblings than in female siblings. However, the ASD and TD groups did not show sex differences in any behavioral/emotional symptom scores. Males were superior in systemizing relative to empathizing, while the opposite was true for females in the unaffected sibling group and the TD children group; however, both males and females were superior in systemizing relative to empathizing in the ASD group. Our findings support the extreme male brain theory of autism, and further studies are needed to understand the mechanisms behind and developmental perspectives on the nature of sex differences in ASD.     

Biological sex influences learning strategy preference and muscarinic receptor binding in specific brain regions of prepubertal rats

According to the theory of multiple memory systems, specific brain regions interact to determine how the locations of goals are learned when rodents navigate a spatial environment. A number of factors influence the type of strategy used by rodents to remember the location of a given goal in space, including the biological sex of the learner. We recently found that prior to puberty male rats preferred a striatum‐dependent stimulus‐response strategy over a hippocampus‐dependent place strategy when solving a dual‐solution task, while age‐matched females showed no strategy preference. Because the cholinergic system has been implicated in learning strategy and is known to be sexually dimorphic prior to puberty, we explored the relationship between learning strategy and muscarinic receptor binding in specific brain regions of prepubertal males and female rats. We confirmed our previous finding that at 28 days of age a significantly higher proportion of prepubertal males preferred a stimulus‐response learning strategy than a place strategy to solve a dual‐solution visible platform water maze task. Equal proportions of prepubertal females preferred stimulus‐response or place strategies. Profiles of muscarinic receptor binding as assessed by autoradiography varied according to strategy preference. Regardless of biological sex, prepubertal rats that preferred stimulus‐response strategy exhibited lower ratios of muscarinic receptor binding in the hippocampus relative to the dorsolateral striatum compared to rats that preferred place strategy. Importantly, much of the variance in this ratio was related to differences in the ventral hippocampus to a greater extent than the dorsal hippocampus. The ratios of muscarinic receptors in the hippocampus relative to the basolateral amygdala also were lower in rats that preferred stimulus‐response strategy over place strategy. Results confirm that learning strategy preference varies with biological sex in prepubertal rats with males biased toward a stimulus‐response strategy, and that stimulus‐response strategy is associated with lower ratios of muscarinic binding in the hippocampus relative to either the striatum or amygdala. © 2012 Wiley Periodicals, Inc.