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Table of Contents
ORIGINAL ARTICLE
Year : 2019  |  Volume : 62  |  Issue : 6  |  Page : 245-255

Sex differences and the modulating effects of gonad intactness on behavioral conformity in a mouse model


1 Education Center of Humanities and Social Sciences, National Yang-Ming University, Taipei, Taiwan
2 Department of Physiology; Institute of Behavioral Medicine, National Cheng Kung University College of Medicine, Tainan, Taiwan

Date of Submission16-May-2019
Date of Acceptance05-Nov-2019
Date of Web Publication29-Nov-2019

Correspondence Address:
Prof. Chianfang G Cherng
Education Center of Humanities and Social Sciences, National Yang-Ming University, Taipei 11221
Taiwan
Prof. Lung Yu
Department of Physiology, National Cheng Kung University College of Medicine, Tainan 70101; Institute of Behavioral Medicine, National Cheng Kung University College of Medicine, Tainan 70101
Taiwan
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/CJP.CJP_42_19

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  Abstract 

Although gender differences in conformity are noticed in human studies, cultural norms and psychosocial factors inevitably affect such differences. Biological factors, especially the gonadal hormones and the brain regions involved, contributing to the sex differences in behavioral conformity remained scarcely explored. To prevent psychosocial and cultural norm confounds, intact and gonadectomized male and female mice were used to assess the modulating effects of gonadal hormones on behavioral conformity and such conformity-related brain regions using an approach of choice paradigm. Intact and gonadectomized mice' choices for the nonrewarded runway were assessed when these experimental mice were alone versus with a group, consisting of three same-sex noncagemates choosing the respective experimental mice' nonrewarded runway, in a double-J-shaped maze test. Although male and female mice exhibited comparable rewarded runway choices at the conclusion of the operant training procedures and in the test individually, male mice demonstrated greater conformity index as compared to female mice when group tested. Gonadectomy, done at their 4 or 9 weeks of age, decreased males' conformity index but did not affect females' when both sexes were group tested. Such gonadectomy did not affect the conditioning or conformity index when tested individually in either sex. Female mice had higher serum corticosterone (CORT) levels when group tested as compared to the female mice tested individually and male mice. Finally, the number of FOS-staining cells in high conformity-displaying mice was found less than it in the low conformity-performing mice in the nucleus accumbens. Taken together, we conclude that testis-derived hormones, at least, play a role in enhancing behavioral conformity in male mice. CORT and nucleus accumbal neuronal activity deserve further investigation for their involvement in behavioral conformity.

Keywords: Conformity, decision-making behavior, glucocorticoids, operant conditioning, orchidectomy, ovariectomy, sex difference


How to cite this article:
Cherng CG, Yu L. Sex differences and the modulating effects of gonad intactness on behavioral conformity in a mouse model. Chin J Physiol 2019;62:245-55

How to cite this URL:
Cherng CG, Yu L. Sex differences and the modulating effects of gonad intactness on behavioral conformity in a mouse model. Chin J Physiol [serial online] 2019 [cited 2019 Dec 8];62:245-55. Available from: http://www.cjphysiology.org/text.asp?2019/62/6/245/272024


  Introduction Top


The earliest investigations into conformity are carried out by social psychologists.[1],[2],[3] Ever since these pioneering studies on individual and a group's consensus in perceptual decisions, conformity has been regarded as an ostensibly generalized phenomenon that an individual is prone to act out in agreement with the decision and behavior of a group of others even if the group decision and behavior is against the individual's own experience/knowledge-based decision or behavioral habits.[4] Over the past three decades, behavioral conformity has been assessed under variant conditions, including the group size, task importance, personal mood, and motivation.[5],[6],[7],[8],[9] To understand the biological roots and neurohormonal underpinnings of behavioral conformity is inevitably hampered using humans as experimental subjects. First, it is nearly impossible to control for an individual's conformity-displaying experiences and in-born propensity inasmuch as behavioral conformity could stem from a personality trait (inclination) and/or behavioral choices adapting to the environments.[10] Second, sociopsychological and cultural norms very likely overshadow the assessment of biological basis of behavioral conformity, especially for the roles of the neurohormonal systems in priming and motivating behavioral conformity.[11] Third, it is not surprising to note that most, if not all, human experimental studies are devoid of providing precise threat to species' substantial loss and survival[12],[13] due to the limitations in ethical concerns using humans as participants.

Humans often change their beliefs or behavior due to the behavior or opinions of others, suggesting the dual cognitive-behavioral nature of conformity. Interestingly, a phylogenetically controlled meta-analysis has revealed correlations of certain cognitive performances and behavioral characteristics with a significant amount of variation in effect size across the studies.[14] Among all the factors examined, sex is the most reliable factor in predicting the variation in the effect size across those empirical studies,[14] suggesting a critical role of sex in determining the cognitive-behavioral association. Although conflicting findings reside in literature, the relations between sex and conformity have been documented using meta-analysis methods.[15],[16],[17],[18] Greater conformity performances are found among females as compared to males, while having small size of the effect[16],[17] or no difference.[15],[18] Such minute or negligible effect sizes are very likely attributed to a ubiquitous confounding effect of an individual's conformity to his/her psychosocial and cultural norms. For example, a recent survey indicates that female college athletes exhibit greater concussion symptom reporting intention and behavior as compared to male college athletes, while both male and female athletes are likely to engage in the risk behavior of continued play throughout their postconcussion symptomatic period.[19] That is, greater conformity to the norm of risk-taking at all costs, characterizing one traditional masculinized norm of the contemporary sport ethos, has been found to associate with greater likelihood of continued play, regardless of the athletes' sex.[19] Moreover, sexes play a critical role in actually receiving a cosmetic surgery regardless of the attitudinal acceptance.[20] These results and many sex-related findings[21],[22],[23],[24],[25],[26],[27],[28] all reveal how psychosocial and subcultural norms may twist or overshadow the impact of biological sex differences in assessing human cognitive and behavioral performances such as behavioral conformity.

The aforementioned difficulties and concerns prompted us to use a mouse model to better control for subjects' conformity-displaying experiences and propensity and to parcel out the contributing motives stemming from personal beliefs in psychosocial and cultural norms. To this end, male and female C57BL/6 mice given birth by siblings were used as the experimental animals.[4] Food deprivation-induced hunger drive and likely loss in having access to sucrose pellets following experimental animals' behavioral choices were used to model a stress condition. Since an approach of choice paradigm was adopted, behavioral conformity was defined as the degree to which the behavioral choice of individual experimental mouse was similar to that of a group of three, same-sex mice while against the individual's previously trained choice. To assess sex differences and the modulating roles of gonadal hormones in priming such behavioral conformity, intact and gonadectomized male and female mice were used in this regard.

In primate species with high degrees of male competition, it is not surprising to note that male brain regions involving in aggressive behavior are emphasized in size, while females' mirroring (empathy) systems are stressed in constitutive neuronal activity.[20],[29] It is of importance to emphasize that the mirroring system is not an exclusively motivational system in defining the quality and quantity of all social interaction behaviors, and behavioral conformity is at best one form of social interaction behaviors. Nonetheless, the mirroring system, at least, consists of a monitoring system in gleaning the information, including any conflicting information; thus the monitoring system, in turn, may prepare animals for subsequent decision in behavioral adjustment or conformity.[30] In this regard, frontal cortex (Cx) seems to play a critical role in initiating and/or modulating such conflict detection and mismatching response adjustment.[30] Likewise, ventral striatum, nucleus accumbens, prefrontal Cx, and anterior cingulate gyrus seem to signal the updated estimates of rewards obtained from previously distinct actions and choices.[10],[31] These brain regions could play a role in mediating the processing and execution of the conformity behavior. To study the brain regions involved in behavioral conformity, FOS-staining methods were used and the number of FOS-positive neuron was assessed in orbitofrontal Cx, anterior cingulate gyrus, striatum, amygdala, prefrontal Cx, and nucleus accumbens in the high versus low conformity-displaying mice.


  Materials and Methods Top


Experimental mice

This study was performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23) revised in 1996. All procedures were approved (NCKUCM No. 99053) by the local Animal Care Committee at National Cheng Kung University College of Medicine. A pregnant C57BL/6 mouse was obtained from National Cheng Kung University College of Medicine Laboratory Animal Center. Pups delivered by the dam were used. These siblings were, then, used for further breeding purpose and their offspring served as the experimental mice. The number of experimental pups was culled to 6 per litter at day 1 postpartum (D1PP) and the largest, healthiest-looking pups were chosen. Six same-sex experimental mice were group housed in one plastic cage (29 cm × 18 cm × 15 cm) after weaning in a temperature- and humidity-controlled colony room on a 12-h light/dark cycle with lights on at 0700. Tap water was accessible ad libitum throughout the experiments. Three days before the operant trainings, the experimental mice underwent a food (Purina Mouse Chow, Richmond, IN, USA) deprivation procedure and their body weights were carefully monitored throughout the deprivation period as previously reported.[32] All experiments were conducted in a temperature (23 ± 1°C)- and humidity (70%)-controlled laboratory.

Custom-made J-shaped maze

The custom-made, combined J-shaped maze consisted of a start box (22.5 cm × 15 cm × 20.5 cm), straight runway (45 cm × 8 cm), two curve runways, and goal boxes (17 cm × 8 cm)[4] [Figure 1]. The start box was separated from the straight runway by a manually controlled slide door.
Figure 1: A photo representation of the double J-shaped maze. The maze consists of a start box, straight runway, two curve runways, and trough-shaped metal goal boxes. Sucrose pellets are loaded in a 2 cm × 2 cm metal container at the bottom of the goal boxes in each trial of the operant conditioning.

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Sucrose pellet-supported operant conditioning protocols

Experimental mice first underwent a 30-min free navigation for acclimation in the J-shaped maze individually. These mice, then, underwent food deprivation procedures until an approximately 3%–5% reduction of the body weight was achieved before the beginning of the sucrose pellet conditionings. Sucrose pellet-rewarded runway choice/goal box-approaching behavior conditionings were done for 4 consecutive days. On the 1st day of the conditioning, the mice were individually placed into the start box and forced by a manually controlled wooden plate to reach one randomly assigned goal box via the respective curve runway to obtain one 14-mg sucrose pellet (Bio-Serv, Frenchtown, NJ, USA) as a reward for 10 trials (intertrial interval = 2 min). During each trial, the opposite curved runway was blocked by a shield door.[4] These mice were then individually trained to voluntarily reach the end of the assigned goal box to get the sucrose pellets at an FR-1 regimen for the following 3 days (2 sessions/day, intersession interval = 4–6 h) with both curve runways open. Each training session lasted for up to 20 sucrose pellets (i.e., accumulated 20 entries into the assigned goal box). Mice' rewarded and nonrewarded runway choices at the joint of two curved runways at the last round of training were recorded as an index, indicating these mice' operant learning and memory performances. Mice serving for the noncagemate “conformity” group started with the same training procedures, while underwent extra several days of training in a 3-mouse group to ensure their consistent and rapid rewarded runway/box choices. Such “conformity” groups were repeatedly used for the experiments; their rewarded runway/goal box choices were, thus, maintained by supply of sucrose pellet rewards throughout. An approach of choice paradigm was adopted in this study; thus, the rewarded runway/goal box of the “conformity” groups' was always opposite to the respective experimental mice' rewarded one. Because experimental mice' nonrewarded runway choice (miss) rates were reliably low with negligible variations at the last round of training following the operant conditioning procedures, the experimental mice were randomly subdivided into two groups. One group underwent the test individually, whereas the other group received the test with a respective conformity group.

Test procedure and the conformity index

The test consisted of twenty trials. Experimental mice underwent each trial individually or with a conformity group. Individual experimental mouse and the respective group were placed in the start box and started the trial after manual removal of the slide door between the start box and straight runway. In each trial, experimental mice' runway/goal box choices were defined as their hind paws stepping in the rewarded or nonrewarded runway for the first time. It was of importance to note that the experimental mice were removed from the maze immediately when their forepaws stepped into any goal box and the trial terminated. In contrast, the respective conformity group was allowed to get access to the sucrose pellets in each trial. The intertrial intervals were approximately 2 min. Since conformity was regarded as individual's behavioral choice in agreement with the group choice whereas against its own one, the conformity index for each experimental mouse was obtained accordingly by dividing the number of its choice for the previously nonrewarded curved runway by 20 and being multiplied by 100/100.

Corticosterone assay

Stress-induced corticosterone (CORT) secretion has been implicated to play a role in memory performances when the learning context is distinct from the retrieval one.[33] In this study, some experimental mice acquired the operant conditioning individually, while being tested with a “conformity” group in the test. In addition, experimental mice' curved runway/goal box approaches in the test were manually interrupted after their arrivals to any goal box. The stress-induced CORT secretion was, thus, assessed in both sexes and a total of 32 mice (n = 16 for each sex) were used. Following the operant training procedures, each sex of mice was divided into two groups. One group (n = 8 for each sex) of mice received the test individually, while the remaining group (n = 8 for each sex) with a conformity group. Twenty minutes after the conclusion of the test, these mice were killed by rapid decapitation and the trunk blood was collected. Serum CORT concentration was analyzed using a CORT enzyme immunoassay kit (Cayman Chemical Co., Ann Arbor, MI, USA) according to the manufacturer's protocols. The intra-assay variability was 2.8%.

Gonadectomy

To assess the impact of the intact gonads and gonadal hormones on behavioral conformity at two developmental stages,[34] 4- (n = 24 for each sex) and 9-week-old (n = 36 for each sex) experimental mice were used for the gonadectomy experiments. Mouse gonads were bilaterally removed under chloral hydrate anesthesia (400 mg/kg, i. p.) and such anesthesia was maintained with isoflurane (<1%) in oxygen (Northern Vaporisers, Skipton, England) throughout the surgery. It was of importance to note that gonadectomy surgery was done at least 2 weeks before the operant training procedures. In brief, orchidectomy surgery was done by removing bilateral testes and a part of epididymis through a midline incision on the ventral side of the scrotum in male mice (n = 12 for the 4-week-old and n = 18 for the 9-week-old mice).[35] For the surgery of ovariectomy, bilateral ovaries and a proportion of oviducts were removed from the ovarian fat pad in female mouse lower back (n = 12 for 4-week-old and n = 18 for 9-week-old mice).[35] The remaining male (n = 12 for the 4-week-old and n = 18 for the 9-week-old mice) and female mice (n = 12 for the 4-week-old and n = 18 for the 9-week-old mice) undergoing similar anesthesia and surgical procedures except gonad removal served as sham surgical controls.

FOS-staining protocol and quantification

It was noticed that two sexes of mice had indistinctive conformity indices when tested individually, while female mice performed lower behavioral conformity with the group as compared to males in the test. Female mice, thus, were used in the conformity-induced FOS expression experiment to conservatively assess the brain regions involving in the conformity performance. The trained female mice performing in the test with the respective conformity group were used and high (n = 5) and low (n = 5) conformity-performing ones (high conformity-performing: conformity index ≥0.4; low conformity-performing: conformity index <0.3) were chosen for further FOS-staining experiment. These mice were deeply anesthetized with sodium pentobarbital and transcardially perfused with ice-cold 0.1 M phosphate-buffered saline (PBS, pH adjusted to 7.4), followed by 4% paraformaldehyde in ice-cold 0.1 M PBS. Their brains were removed and postfixed in a 4% paraformaldehyde solution overnight at 4°C and subsequently cryoprotected in 30% sucrose solution for 48 h at 4°C. Moreover, the brains were then fast frozen in optical coherence tomography and stored at −80°C until sectioning. Coronal sections at 30 μm in thickness were made using a microtome (CryoStar NX50, Thermo Fisher Scientific, Cleveland, OH, USA). FOS staining was quantified in the orbitofrontal Cx (including lateral, medial, and ventral orbital Cx) (bregma: 2.22 to 2.80 mm), anterior cingulate gyrus (i.e., the Cg1 and Cg2 in combination) (bregma: −0.22 to 2.34 mm), caudate-putamen complex (bregma: −0.10 to 1.70 mm), amygdala (the lateral and basolateral nuclei of amygdala) (bregma: −0.94 to −2.30 mm), prefrontal Cx (prelimbic and infralimbic Cx in combination) (bregma: 1.54 to 2.80 mm), and nucleus accumbens (bregma: 0.74 to 1.94 mm) [Figure 2].[4] One section of every 60 μm was sampled for the FOS staining in each brain region of interest; thus, a total of 12 (orbitofrontal Cx), 23 (prefrontal Cx), 45 (anterior cingulate gyrus), 21 (nucleus accumbens), 30 (caudate-putamen complex), and 24 (amygdala) sections were used for FOS staining and counting from each mouse. The sections were washed 3 min × 10 min in 0.1 M PBS-Tween 20 (PBST, 0.5% Tween 20, pH adjusted to 7.2), and endogenous peroxidase was deactivated using 1% H2O2 in PBS for 30 min. The sections were then rinsed in 0.1 M PBST for 2 min × 10 min and preincubated in a blocking solution consisting of 5% normal goat serum and 0.2% Triton X-100 in 0.1 M PBS for 1 h. The sections were incubated in primary antibody (Ab-5) at 1:2000 in the same blocking solution for 48 h at 4°C. After that, the sections were washed 3 times with 0.1 M PBST and then incubated with secondary antibody (1:200) for 2 h at room temperature in blocking solution and followed by 3 min × 10 min 0.1 M PBST washes. The sections were then incubated for 1 h at room temperature in avidin–biotin complex (Vectastain ABC Elite Kit). After two washes in 0.1 M PBST and two washes in 0.05 M Tris-HCl buffer (pH = 7.4), the sections were reacted with nickel-enhanced 3,3'-diaminobenzidine (DAB kit). After one rinse in Tris-HCl buffer and one rinse in PBST, the sections were mounted on the slides, dehydrated, and coverslipped with glycerol gelatin. Total counts of FOS-stained cells in each brain region of interest were determined for each mouse by manual counting the number of black immunoreactive spots. The rater was blind to grouping in counting the FOS-positive cells.
Figure 2: Representations of mouse brain atlas coronal section illustrating the boundaries of regions used for quantification of FOS-positive cells. The dotted lines circle the boundaries of the respective brain regions.

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Statistical analysis

To assess the miss ratio differences at the last round of the operant training among groups, one-way ANOVAs were employed. A two-way ANOVA was used to determine the differences in the conformity index in male and female mice receiving the test individually versus with a conformity group. Likewise, two-way ANOVAs were used to detect the differences in the conformity index in sham surgical and orchidectomized male mice receiving the test individually versus with a conformity group. Likewise, two-way ANOVAs were used to detect the differences in the conformity index in sham surgical and ovariectomized female mice receiving the test individually versus with a conformity group. A two-way ANOVA was used to unveil CORT differences in male and female experimental mice receiving the test individually and with a conformity group. Finally, unpaired t-tests were used to examine FOS-staining spot differences in variant brain regions in high- and low-conformity-performing female experimental mice.


  Results Top


Male exhibited higher behavioral conformity (conformity index) as compared with female mice when tested with a conformity group

Male and female mice demonstrated low miss ratios for the respective trained runway-goal choice at the last round of the training, suggesting comparable learning and memory using this sucrose pellet-supported operant conditioning procedure in two sexes of mice [Figure 3]a. Likewise, individual male and female mice exhibited comparable choice ratio for the untrained runway goal in the test [Figure 3]b. A two-way (male vs. female and individually vs. with a conformity group) ANOVA revealed that two main (male vs. female: F(1, 36) = 8.696, P = 0.0056; individually vs. with a conformity group: F(1, 36) = 41.1, P < 0.0001) and interactive effects (F [1, 36] = 4.208, P = 0.0476) were evident for the conformity index in the test. These results indicated that both male and female mice exhibited greater conformity index choosing the untrained runway as these mice were tested with the conformity group versus individually. Importantly, male mice had greater conformity index as compared to female mice. Notably, given that twenty trials were subdivided into four bins (5 trials in a bin) in the test, the number of trained arm-goal choice behavior progressively increased across the four bins, regardless of sex or individual versus group test [Figure 3]c, [Figure 3]d, [Figure 3]e, [Figure 3]f.
Figure 3: Performances in the sucrose pellet-supported runway choice conditioning and test in male and female mice. (a) Male and female mice exhibited low and comparable miss rates of trained runway choice at the last round of operant training. (b) Male exhibited higher conformity index as compared with female mice when they were tested with a conformity group.aHigher than the other three groups.bHigher than the groups receiving the test individually. When the twenty trials were subdivided into four bins (five trials in a bin) in the test, the number of trained runway choice behavior progressively increased across the four bins in (c) males tested individually, (d) males tested with a group, (e) females tested individually, and (f) females tested with a group.

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Ovariectomy at 9 weeks of age did not affect females' behavioral conformity

When ovariectomy was done at females' 9 weeks of age, sham surgical and ovariectomized female mice demonstrated low and comparable miss ratios for the respective trained runway choice at the last round of the training, suggesting that the ovariectomy did not affect the learning or performances in the operant conditioning [Figure 4]a. Moreover, ovariectomized and sham surgical mice exhibited comparable choice ratios for the untrained runway in the test as tested individually [Figure 4]b. Furthermore, a two-way (ovariectomized vs. sham surgical and individually vs. with a conformity group) ANOVA revealed a main effect of individually versus with a conformity group (F [1, 32] = 91.57, P < 0.0001) on the conformity index [Figure 4]b. The latter result indicated that both ovariectomized and sham surgical female mice displayed greater conformity index choosing the untrained runway regardless of their tests with the conformity group or individually.
Figure 4: Performances in the sucrose pellet-supported runway choice conditioning and test in sham surgical and ovariectomized female mice. (a) When the surgery was done at 9-week-old female mice, sham surgical and ovariectomized mice exhibited low and comparable miss rates of trained runway choice at the last round of operant training. (b) Regardless of ovariectomy and sham surgery, female mice receiving the test with a group exhibited higher conformity index as compared with female mice receiving the test individually.aHigher than the groups receiving the conformity individually.

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Orchidectomy at 9 weeks of age prevented males' high behavioral conformity

When orchidectomy was done at males' 9 weeks of age, sham surgical and orchidectomized mice demonstrated low and comparable miss ratios for the respective trained runway choice at the last round of the training, suggesting that the orchidectomy did not affect the learning or performances in the operant conditioning [Figure 5]a. Likewise, individual orchiectomized and sham surgical mouse exhibited comparable choice ratios for the untrained runway in the test [Figure 5]b. A two-way (sham surgical vs. orchidectomized and individually vs. with a conformity group) ANOVA revealed that main (individually vs. with a conformity group: F [1, 32] = 36.23, P < 0.0001) and interactive effects (F [1, 32] = 5.784, P = 0.0221) were evident on the conformity index. However, the main effect of orchidectomized vs. sham surgical on the conformity index was shy of significance (F [1, 32] = 3.989, P = 0.0544). These results indicated that sham surgical male mice exhibited greater conformity index choosing the untrained runway when these mice were tested with a conformity group versus individually. However, orchidectomized male mice had comparable conformity indices as these mice were tested with the conformity group versus individually.
Figure 5: Performances in the sucrose pellet-supported runway choice conditioning and test in sham surgical and orchidectomized male mice. (a) When the surgery was done at 9-week-old male mice, sham surgical and orchidectomized mice exhibited low and comparable miss rates of trained runway choice at the last round of operant training. (b) Sham surgical, not orchidectomized, male mice receiving the test with a group exhibited higher conformity index as compared with the male mice receiving the test individually.aHigher than the groups receiving the conformity individually.

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Gonadectomy at 4 weeks of age caused decreases in males' behavioral conformity

When bilateral gonads were removed at mice' 4 weeks of age and 7 weeks awaited after the surgery, sham surgical and orchidectomized male mice demonstrated comparable miss ratios for the respective trained runway choice at the last round of the training [Figure 6]a. Likewise, sham surgical and ovariectomized female mice demonstrated comparable miss ratios for the respective trained runway choice [Figure 6]c. These results indicated that mice' learning and performances in the operant conditioning were not altered by the lacking of gonads when the surgery of gonadectomy was conducted as early as their 4 weeks of age. Although both orchidectomized and sham surgical mice exhibited higher conformity indices when these mice were tested with the conformity group versus individually, sham surgical male mice displayed higher conformity indices as compared to the orchidectomized mice (F [1, 20] = 5.084, P = 0.0355 for the interactive effect of surgery and alone vs. with a group on the conformity index) [Figure 6]b. In contrast, both ovariectomized and sham surgical female mice exhibited comparable and greater conformity indices when tested with a conformity group versus individually (F [1, 20] = 28.48, P < 0.0001 for the main effect of alone versus with a group on the conformity index) [Figure 6]d. These results, taken together, indicated that removal of testes as early as 4 weeks of age rendered decreases in behavioral conformity in male mice, while removal of ovaries at the same ages did not alter behavioral conformity in female mice [Figure 6]b and d].
Figure 6: Performances in the sucrose pellet-supported runway choice conditioning and test in sham surgical and gonadectomized male and female mice. (a) When the surgery was done at 4-week-old male mice, sham surgical and orchidectomized mice exhibited low and comparable miss rates of trained runway choice at the last round of operant training. (b) When mice received the test with a group, orchidectomized male mice exhibited lower conformity index as compared with the sham surgical male mice.aHigher than the other three groups.bHigher than the groups receiving the test individually. (c) When the surgery was done at their 4 weeks of age, sham surgical and ovariectomized female mice exhibited low and comparable miss rates of trained runway choice at the last round of the operant training. (d) When mice received the test with a group, sham surgical and ovariectomized female mice exhibited comparable conformity indices and higher than the female mice receiving the test individually.aHigher than the groups receiving the test individually.

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A stress response was eminent in female mice performing conformity test with a group

Stress-induced CORT secretion was assessed in two sexes. To this end, mouse serum CORT levels were assayed following the test. A two-way ANOVA (sex x individually versus with a conformity group) revealed that main (sex: F [1, 28] = 20.58, P < 0.0001) and interactive effects (F [1, 28] = 5.165, P = 0.0309) were evident. Post hoc analyses further revealed that female mice tested with the conformity group exhibited the highest serum CORT level among all groups [Figure 7].
Figure 7: Serum corticosterone levels in male and female mice receiving the test individually or with a conformity group. Female mice receiving the test with a group demonstrated the highest corticosterone levels among all groups.aSignificantly higher than the other three groups.

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Activated accumbal FOS activity was evident in female mice performing low behavioral conformity

Female mice exhibiting high behavioral conformity had comparable FOS-staining counts in the orbitofrontal Cx, anterior cingulate gyrus, caudate-putamen complex, amygdala, and prefrontal Cx as compared to the female mice exhibiting low behavioral conformity. In contrast, an unpaired t-test revealed that female mice performing low behavioral conformity had significantly greater FOS-staining counts in nucleus accumbens as compared to the female mice performing high behavioral conformity (t8 =2.884, P = 0.0204) [Figure 8].
Figure 8: Total FOS-positive cells in the brain region of interest in high versus low conformity-performing female mice. Female mice exhibiting high conformity indices had comparable FOS-positive counts (mean ± standard deviation) in (a) the orbitofrontal cortex, (b) anterior cingulate gyrus, (c) prefrontal cortex, (d) caudate-putamen complex, and (e) amygdala as compared to the female mice exhibiting low behavioral conformity. Female mice performing low behavioral conformity had significantly greater FOS-positive counts in (f) the nucleus accumbens as compared to the female mice performing high behavioral conformity. aSignificantly higher than the high behavioral conformity group.

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  Discussion Top


In this study, sucrose pellet-supported conditioning protocols were first used to train all experimental mice to choose the rewarded runway and goal box rather to the nonrewarded runway and box. Male and female mice exhibited comparable and low miss ratios in the previously rewarded runway choices at the conclusion of the conditioning protocol. Likewise, gonadectomized male and female mice demonstrated low and comparable miss ratios in those choices. These findings are in parallel with a recent report that the magnitudes of eyeblink trace conditioning are comparable in male and female rats using another associative conditioning paradigm.[36] Moreover, a report has documented that female and male rats exhibit comparable retentions of the fear-potentiated freezing responses following an extinction protocol.[37] Furthermore, using a discriminative cue and classical conditioning paradigm, males and females show comparable fear-potentiated freezing responses.[38] Finally, two sexes of rats demonstrate comparable magnitudes in the establishment of a conditioned cue-induced food-cup approaching behavior.[39],[40] Our findings indicate that adult gonads and gonadal hormones, at best, play a minor role in affecting the acquisition of natural reward-supported operant conditioning and memory.

Male and female mice maintained such comparable and eminent choice preferences for the previously rewarded runway/goal box when these mice were tested individually in the test. However, male mice demonstrated greater conformity index as compared to female mice when they were tested with a group in the test. That is, male mice were more prone to switch their own runway choices to a group's than female mice when the groups' choices were against their previously rewarded choices. When the gonadectomy surgery was performed in 4- or 9-week-old male mice, their conformity indices significantly descended when these gonadectomized male mice were tested with their respective groups in the test. When the gonadectomy surgery was done at mice' 4 or 9 weeks of age, ovariectomized and sham surgical female mice displayed comparable and low conformity indices as they were tested with the respective group making runway choices opposite to these female mice. These results, taken together, suggest that males demonstrate greater conformity as compared to females under the conditions that individual conformity propensity, experiences, and psychosocial and cultural norms are the best parceled out. Moreover, hormones secreted by the testes, at least, play a modulating role in priming adult male mice' conformity-related choices and/or suppressing their trained choices when individual's choice preferences are distinct from an unfamiliar group's.

We previously report that an expected risk may enhance behavioral conformity in male mice.[4] When an escapable risk and the learned risk-predictive cues are both present, there is a sex difference in behavioral phenotypes with high mobility in male while reduced mobility in female mice in the avoidance tasks.[41] An implication of the latter findings is that stressed female mice are prone to perform the prey-specific freezing responses, while stressed male mice attempt exploratory avoidance behavior of choice even in a passive avoidance task. In this study, intact female mice demonstrated higher cumulative count of previously rewarded runway choice across four bins of twenty trials (with five trials for each bin) as compared to intact male mice when these mice were tested with a group of three noncagemates in the test. It was of importance to note that the training and retrieval contexts changed in these experimental mice and the runway/goal box choices for each trial were terminated without any reward supply in the test regardless of these mice' choices. Thus, it was reasonable to suspect that stressed females, not the males, were more prone to make the previously trained, rather than to make exploratory alternative, runway choices. In contrast, male mice' alternative runway choices seemed to be irrelevant to the impact of stress or stress-induced CORT secretion. After all, we found that female mice undergoing the test with a group of three noncagemates had significantly higher CORT levels than males receiving the test individually or with a group and females receiving the test individually.

On the other hand, behavioral and brain plasticity is vulnerable to acute stress especially in females, while insensitive in males.[3],[42],[43],[44],[45] A report has documented that female mice may switch their behavioral strategies in an escape test relying on their CORT receptor activation.[46] Moreover, when rats are under a long-term stress, females are found to show no alterations in the acquired object recognition or placement memory.[42] Interestingly, a recent study has revealed that females are less sensitive than males to deprivation of a membrane receptor trafficking protein in their memory performance.[47] Irrespective of appetitive motivation or phenotypic characteristics of the stimulus mice, no obvious sex difference is noticed in social interaction, a specific type of affiliative behavior, and social reward in adolescent C57BL/6J mice.[48] As per these results, we intend to provide a scenario that when a group showing choices different from individuals is present, females are inclined to maintain frequent behavioral choices (nonconformity) under stress conditions as compared with males, provided that individual conformity traits, conformity-performing experiences, beliefs in psychosocial, and cultural norms are removed from the analysis.

While ovarian-derived hormones did not demonstrate obvious impact on modulating females' conformity choice behavior using the present model, increases in CORT secretion were eminent following female, not male, mice' test with a group of three noncagemates in this study. A meta-analysis study shows that stress evokes sex differences in many learning tasks, while those differences are mediated by complicated interactions between stress and sex hormones.[49] An empirical study reveals that stress-stimulated increases in glucocorticoid secretion may modulate the memory at different menstrual status depending on the gonadal hormone levels in females.[50] Although the estrous cycle or acute stressor have no effect on female mice' spatial performance, brain region-specific loss of CORT receptor may disturb such spatial task performances when their estrogen levels are high.[51] These results, taken together, suggest a strong interaction between stress and gonadal hormones in priming and/or motivating females' decision-making process and behavior. Although our results have demonstrated that gonadal hormones do not seem to affect females' conformity magnitudes using the present model, interactive impact of stress-induced CORT secretion and circulating ovarian hormone levels on behavioral conformity deserves further study.

Behavioral conformity recruits a complex cognitive process that an individual finally decides to perform the choice of a group of others even the individual has perceived that the group choice is a mismatching of the individual's own. Prefrontal Cx, anterior cingulate gyrus, ventral striatum, nucleus accumbens, and amygdala have been known to be involved in monitoring the conflicting cues, updating the estimates of rewards from the most recent actions and choices, adjusting conflicting responses, and processing the conflict-related emotions.[10],[30],[31] These brain regions could serve as critical cognitive processing components of driving individual behavior to finally conform with a group's one. Stimulus-provoked neuronal activation and consequent FOS protein synthesis, in this regard, can be used to evaluate these regions' involvement in processing such cognitive processing of conformity. In this study, FOS-staining methods were used in high versus low conformity-performing female mice to reveal the region-specific differences in neuronal activity in response to their distinct magnitudes in performing conformity. Surprisingly, the number of FOS-staining neuron in low conformity-performing female mice significantly outnumbered it in high conformity-performing female mice only in the nucleus accumbens. The implications of such paradoxical association of low accumbal activity and high conformity performance are three-fold. First, it is reasonable for the high conformity-performing individuals to give up the real-time updating of their present rewarding experiences. Second, it is indispensable for the high conformity-performing animals to overlook the reward valence from the previous conditionings. Third, high conformity-performing animals ought to neglect the past behavioral habit in choosing the runway. High conformity-performing animals may execute any or all of these cognitive processes via active suppression on the neuronal activity of the nucleus accumbens. Further study should be done to test these possibilities.


  Conclusions Top


Although male and female mice exhibited comparable performances at the conclusion of the operant training procedures, male mice demonstrated greater conformity index as compared with female mice in the test. Moreover, gonadectomy, done at the 4 or 9 weeks of age, prevented the high behavioral conformity in males but did not affect females' when tested with a conformity group, while gonadectomy at the respective timings did not affect the conditioning or conformity performances when tested alone in either sex. Furthermore, female, not male, mice had higher serum CORT levels when tested with a conformity group as compared to the female mice tested individually. Finally, the number of FOS-staining neuron in female mice displaying high conformity was less than it in those mice showing low conformity in the nucleus accumbens. Taken together, we conclude that hormones secreted by the testis, at least, play a role in enhancing behavioral conformity in male mice.

Financial support and sponsorship

This research is supported, in part, by ROC MOST (Ministry of Science and Technology) grants 101-2811-H-006-013, 103-2410-H-006-028-MY3 to L.Y.

Conflicts of interest

There are no conflicts of interest.



 
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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]



 

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