Assessment of conditioned fear extinction in male and female adolescent rats

https://doi.org/10.1016/j.psyneuen.2020.104670Get rights and content

Highlights

  • Adolescent female rats in proestrus or met/diestrus at extinction show impaired extinction compared to males, immature females and females in estrus on extinction day.

  • Adolescent female rats in met/diestrus at extinction day showed impaired extinction recall compared to males when tested 24 hours later.

  • Gonadectomy improves extinction in adolescent female rats, whereas male rats show impaired extinction when compared to rats that received sham surgery.

  • These effects are opposite to those seen in adult female rats, which suggests that the influence of estrous cycle on extinction of conditioned fear is age-dependent.

Abstract

Pavlovian fear conditioning and extinction have been widely studied across many species to understand emotional learning and memory. Importantly, it is becoming clear that these processes are affected by sex and age. In adult rodents and humans, sex differences are evident in extinction, with estradiol playing a significant role. In adolescence, an extinction deficit has been reported in rodents and humans. However, the influence of sex on extinction during adolescence is unknown. This is surprising, since adolescence coincides with the onset of hormone cycling, and therefore it might be expected that hormones fluctuations exert a more profound effect at this time. Therefore, we examined Pavlovian fear conditioning and extinction in adolescent male and female rats. In experiment 1, 35-day-old male and female rats were exposed to 6 pairings of a conditioned stimulus (CS, a tone) with an aversive unconditioned stimulus (US, a footshock). The next day they were extinguished in a contextually distinct chamber, via 60 presentations of the CS without the US. Extinction recall was tested 24 hours later in the extinction context. Estrous phase was monitored by cytology on vaginal smears taken 1 hour after each behavioral session. In experiment 2, male and female rats were given sham surgery or gonadectomy at 21 days of age. They were then trained and tested as for experiment 1. We observed that females in proestrus or met/diestrus during extinction showed delayed extinction and impaired extinction recall the next day compared to males. Ovariectomy enhanced extinction for female rats, but orchidectomy delayed extinction for males. Plasma analyses showed that met/di/proestrus phases were associated with high estradiol levels. These findings suggest that high plasma estradiol levels impair extinction for adolescent females. These results contradict what is reported in adult animals, suggesting that hormonal influences on extinction are dependent on age. Given that impaired extinction is widely used as a model to understand resistance to exposure-based therapies, our findings have important implications for understanding mental health treatments in adolescents.

Introduction

Pavlovian fear conditioning and extinction are widely used paradigms to understand the psychological and neural mechanisms of associative learning (Flores et al., 2018; Myers and Davis, 2007). Fear conditioning involves subjects being exposed to repeated pairings of a neutral conditioned stimulus (CS) with the unconditioned stimulus (US – often a mild electric shock). As a result, the CS alone can elicit defensive responses such as freezing in rodents. Extinction is the reduction in this response by repeated presentation of the CS without the US. It has been widely demonstrated that typically, extinction is a new inhibitory association that competes with the conditioned fear memory (Bouton, 2002).

Fear conditioning and extinction represent how a stimulus can acquire an emotional meaning, and then how the responding to that meaning can be inhibited. Due to such importance, molecular, circuit, and psychological processes of conditioning and extinction are extensively studied across species (Flores et al., 2018; Haaker et al., 2019; Kandel et al., 2014; Myers and Davis, 2007). However, most studies have only assessed adult male subjects, until recently. Through increasing the diversity of subjects, it is becoming clear that there are both age and sex differences, particularly in extinction (Ganella and Kim, 2014; Velasco et al., 2019). For example, juvenile male rats show more enduring extinction compared to adults (Kim et al., 2009; Kim & Richardson, 2008; Li et al., 2012). Such findings are not observed in juvenile females (Park et al., 2017). On the other hand, adolescents show impaired extinction, demonstrated in male rodents (Kim et al., 2011; Pattwell et al., 2012; Zbukvic et al., 2017), and male and female humans (Ganella et al., 2018; Pattwell et al., 2012). Sex differences have also been reported in adult rodent studies, although the findings are mixed (Day and Stevenson, 2019). Some studies showed facilitated conditioning in males compared to females (Gupta et al., 2001; Wiltgen et al., 2001; Baran et al., 2010; Kosten et al., 2005), while others observed no sex differences (Wiltgen et al., 2005), or a conditioning deficit in males (Chen et al., 2014). Regarding extinction, some studies showed that females show impaired extinction compared to males (Baran et al., 2009; Clark et al., 2019), while others showed equivalent performance (Milad et al., 2009). While sex differences in behavioral measures of fear during extinction are not always reported in humans, differences in neural activity within fear circuitry during extinction have been observed (Lebron-Milad et al., 2012). To date there have been no studies that investigate sex difference in fear conditioning or extinction in adolescents.

The discrepancies described above may be due to the effect of estrous phase and related cycling gonadal hormones on conditioned fear (Maeng et al., 2017; Wegerer et al., 2014; Zeidan et al., 2011). For adult female humans and rodents, extinction facilitation is observed during the phase of estrous cycle where natural levels of estrogen are high (Wegerer et al., 2014; Zeidan et al., 2011). The impact of estrous phase on fear conditioning and extinction has never been examined during adolescence, a period of maturation marked by puberty and the onset of menarche in which estrous cycling is irregular (Abreu and Kaiser, 2016; Laffan et al., 2018). It remains to be determined whether the onset of puberty and related irregular estrous cycling has effects on fear conditioning and extinction. Assessing such effects is critical, considering the importance of puberty in the emergence of sex differences in brain and behavior (Abreu and Kaiser, 2016; Gennatas et al. 2017), and the significance of fear conditioning and extinction in our understanding of cognition and emotion (Haaker et al., 2019;Myers and Davis, 2002; Singewald and Holmes, 2019).

The aim of this study, therefore, was to investigate sex differences in Pavlovian fear conditioning and extinction in adolescent rats, and whether estrous cycling mediates such difference. In experiment 1, male and female rats underwent Pavlovian fear conditioning beginning on postnatal day (P)35. We hypothesized that females would show delayed extinction learning and impaired extinction recall compared to males. According to studies with adult animals, extinction impairments in female adolescents may also be the most pronounced during estrus, when estradiol level is the lowest in rodents (Smith et al., 1975; Staley and Scharfman, 2005). In experiment 2, we tested the hypothesis that gonadal hormones generally promote extinction. We manipulated gonadal hormone release by performing ovariectomy (OVX) or orchidectomy (ORX) surgery in P21 male and female rats, and then trained rats in Pavlovian fear conditioning at P35. We also verified plasma estradiol, progesterone, and testosterone levels at each estrus phase in these rats. This was done the day after the final test day. The hypothesis would be supported if OVX or ORX cause impaired extinction and extinction recall.

Section snippets

Subjects and surgery

Male and female Sprague Dawley rats were bred in-house at the Florey Institute of Neuroscience and Mental Health, Melbourne, Australia. Rats were weaned at P21 and housed for the remainder of experimental procedures in groups of 5-6 with same-sex littermates in individually ventilated cages under a 12/12 h cycle (lights on at 07:00) with food and water available ad libitum.

For experiment 2, all rats received ORX or OVX surgery before weaning on the same day. Rats were anaesthetized with an

Experiment 1: The relationship between natural estrous cycling and fear conditioning/extinction

In experiment 1 estrous phase was determined on P35, P36, P37, and P38 with vaginal lavages then cytology under a light microscope (Fig. 1a), unless the vagina was unopened. As expected, the number of rats in each phase per day significantly differed (Fig. 1b), χ2 (9,62) = 35.74, p < 0.05. Post-hoc tests confirmed that the number of rats in each phase at P35 was significantly different from P37 and P38 (ps < 0.05), driven by the decreasing number of female rats with unopened vaginas (confirming

Discussion

The present study first showed that adolescent female Sprague-Dawley rats undergo puberty throughout P35-38, with 31% of P35 rats not having begun estrous cycling. This is consistent with previous research (Goldman et al., 2000; Laffan et al., 2018). The cycle was somewhat irregular (Fig. 1c). This is also consistent with previous research reporting that for many mammalian species, including rats, the first few menstrual cycles are typically irregular (reviewed in Goldman et al., 2000; Laffan

Funding and disclosure

This work was supported by National Health and Medical Research Council (NHMRC)/Australian Research Council Dementia Research Development Fellowships awarded to CJP and XD, Baker Foundation Fellowship awarded to DEG, NHMRC project grant awarded to TYP, NHMRC Career Development Fellowships awarded to JHK and SW, and the Victorian State Government Operational Infrastructure Scheme. The authors declare that they do not have any conflicts of interest (financial or otherwise) related to the data

Acknowledgments

We thank Ms Liubov Lee-Kardashyan for carrying out manual scoring where required for both experiments.

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