Research reportEffects of chronic stress in adolescence on learned fear, anxiety, and synaptic transmission in the rat prelimbic cortex
Introduction
The Austro-Hungarian researcher Hans Selye introduced the concept of stress, which he defined as a complex biological response common to all living organisms induced by environmental threats (i.e., stressors) [55]. Stress is oriented to restoring homeostasis and adaption to environment pressures [7], [32]. When the threat is too intense and persistent, stress responses may become maladaptive and affect the brain [32], [52]. In adulthood, chronic stress impairs limbic structures such as the hippocampus and amygdaloid complex, and the medial prefrontal cortex (mPFC) in both animal models and humans [27], [42], [62], [63], [64]. These brain areas regulate anxiety and fear [14], [22], [40].
Anxiety is a long-lasting state of apprehension elicited by threats that are not immediately present [12], while fear is an adaptive state activated by real threats, which begins rapidly and dissipates once the threat disappears [12]. The amygdala and Bed Nucleus of Stria Terminalis (BNST) are involved in anxiety-like behaviors, while the central nucleus of the amygdala (CeA) and the periaqueductal gray (PAG) nucleus neuronal pathway are associated with fear regulation [12], [13], [22] (Scheme 1). Anxiety levels are higher in rats subjected to chronic stress during adulthood [9], [62], which is associated with dendritic hypertrophy in both the basolateral amygdala (BLA) and BNST [61], [63], [64]. In addition, chronic stress induces dendritic atrophy in the pyramidal neurons of layers II/III and V of the mPFC in adult rats [5], [31], [42], [49], a brain area that regulates the recall of learned fear [22], [38], [47], [56].
Adolescence is the only transitional period between childhood and adulthood that is characterized in all mammals by several behavioral, hormonal, and neural changes [57], [58]. Adolescence is an extended period that includes puberty and culminates in reproductive maturation. Adolescence in male rats is considered to last from post-natal day (PND) 35 to 55 [39], with physical markers of sexual maturation observed from PND 45 to 48 in males [26].
The brain is particularly sensitive to stress in the adolescence [11]. It has been shown that adolescent rats display higher levels of stress responses than do adult animals [31], [45]. For example, adolescent rats subjected to chronic stress have higher plasma corticosterone levels than do adults under same condition. As well, the corticosterone levels of adolescent can take twice as long as those adults to return to the baseline after acute stress [16], [19]. The rat mPFC and amygdala, and the neuronal connectivity between them begin developing during adolescence and continue into adulthood [8], [24]. Chronic stress affects the dendritic morphology of these brain areas in adolescence and adulthood [10], [15], [63]. As well, it has been shown that after 21 days of stress-free recovery, adult rats continue showing enhanced anxiety-like behavior and dendritic hypertrophy in the amygdala, while mPFC neurons completely restore their dendritic structure and functions [18], [48], [64]. A comparable study with adolescent mice treated with corticosterone, shows that this hormone decreases spinal density in the infralimbic and orbitofrontal cortices of the mPFC, while in the amygdala this treatment increases spinal density [21]. After a corticosterone-free recovery period, dendritic changes in the prelimbic cortex and amygdala were reversed, while the dendritic changes in the orbitofrontal cortex remained unchanged after the recovery period [21]. It is not known whether the effects of stress on the mPFC and amygdala during adolescence are reversed in adulthood.
Fear conditioning is a three-phase behavioral paradigm used to study the neural circuit of fear [53]. The first phase is conditioning in which the rats are trained in a shuttle box to associate a conditioned stimulus (CS) (a tone) with an unconditioned stimulus (US) (e.g. a foot shock). Once learned, the CS will by itself elicit a conditioned response. For instance, in the fear conditioning paradigm freezing is a behavioral and physiologically conditioned response to fear [25]. It is supposed that fear memories acquired during conditioning are consolidated in the BLA area of the brain [25], [43], [54] (Scheme 1). The recall of learned fear is associated with increased neuronal activity in the BLA, which in turn activates the CeA. Direct projections are sent from this brain structure to the PAG and paraventricular nuclei (PVN) to elicit defensive fear-behaviors (Scheme 1) [25]. During the fear extinction phase, the CS is presented several times to the rats, which allows them to acquire a new memory that suppresses the retrieval of the previously learned fear. The mPFC regulates the expression of extinction memory via the amygdala [46]. In the recall phase, the CS is presented to the rats and they recall the conditioned fear extinction learned during the extinction phase, which in turn decreases the freezing behavior elicited in the animals [47].
The prelimbic cortex (PL) integrates auditory, contextual, and stress-related signals from several brain areas during fear conditioning to regulate fear expression by the basal amygdala (Scheme 1) [6]. PL activity is key for the expression of fear and memory extinction. For example, in vivo stimulation of the PL increases freezing during the conditioning phase [60]. Chronic stress-induced dendritic atrophy in the PL and dendritic hypertrophy in the lateral amygdala, both in adulthood, have been correlated to failure to express fear extinction [36], [48], [63], [66].
In general, the acquisition of the conditioned fear is regulated by BLA [25], [43], [54], while the mPFC controls the recall of the extinction of conditioned fear [47], [56]. In adulthood, hypertrophy of the rat BLA neurons induced by chronic stress persists after 3 weeks of stress-free recovery, while the morphological alterations induced by stress on the PL were reversed [48], [64]. However, the effects of stress on the PL during adolescence remain unclear.
We hypothesize that seven days of restraint stress applied in adolescence decreases excitatory synaptic transmission in the PL and slows extinction of learned fear, while enhancing anxiety-like behavior levels. These electrophysiological and behavioral alterations display different recovery patterns during adolescence and adulthood. The objective of this study was to test whether adolescent chronic stress affects synaptic transmission in the PL and recall of conditioned fear extinction as well as anxiety-like behavior. The main results of this study were that rats subjected to restraint stress in adolescence had significantly higher anxiety levels during adulthood, whereas stress-induced decreases in excitatory transmission in the PL and slowing of extinction of learned fear were reversed in adulthood.
Section snippets
Animals
Male Sprague Dawley rats (80–100 g, 21 days old at the start of the experiment) were housed in groups of three animals per cage, under a 12/12 light/dark cycle (lights on at 8:00 am). They were maintained in a temperature and humidity controlled room (20 ± 1 °C, 55%) and weighed every day on a digital scale (Model WLC2/A1, Radwag, Poland). All procedures relating to animal experimentation were in strict accordance with animal care standards outlined in the National Institute of Health (USA)
The effects of restraint stress on the stress markers and locomotor activity
The two-ways repeated-measures ANOVA (group × days) showed that chronic restraint stress significantly reduced body weight gain [F(1,220) = 401.6; p < 0.0001] (Fig. 1B). There was a interaction between days and experimental groups [F(9,220) = 31.90; p = 0.0001].
Rats subjected to restraint stress had significantly higher adrenal weights than controls (Stress = 9.08 ± 0.69; Control = 7.14 ± 0.35; p < 0.05) (Fig. 1C).
Restraint stress did not affect the total distance (p = 0.6496), average speed (p = 0.6709), and maximum
Discussion
The present study shows that chronic stress applied in the course of adolescence decreases excitatory synaptic transmission in the rat PL and extinction of learned fear. These alterations were reversed in adulthood. Moreover, stressed rats that showed increased anxiety-like behavior levels in adolescence were still anxious in adulthood.
The first step of our investigation was to analyze whether our stress protocol was effective in triggering stress responses in adolescence. The rats subjected to
Conclusions
The data presented here demonstrate that chronic stress in adolescence decreased the amplitude of evoked fEPSPs in the PL and slows the extinction of learned fear, while anxiety levels were higher in stressed rats than controls. The anxiogenic effect of restraint stress was still present in adulthood, while alterations in excitatory synaptic transmission in the PL were reversed and the recall of extinction of learned fear was recovered. Interestingly, it has been shown that psychosocial stress
Acknowledgements
This work was supported by FONDECYT N° 1100413 and Anillo de Ciencia y Tecnología N° ADI-09 grants (AD-S) and the CONICYT and MECESUP graduate fellowships (IN-O).
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2022, AlcoholCitation Excerpt :Chronic restraint stress lasting at least 7 days has mixed effects on fear conditioning in both sexes. In male rodents, restraint stress increases freezing behavior during cued fear conditioning in some studies (Blume, Padival, Urban, & Rosenkranz, 2019; Zhang & Rosenkranz, 2013), but not others (Baran et al., 2009; Negrón-Oyarzo, Pérez, Terreros, Muñoz, & Dagnino-Subiabre, 2014; Sanders et al., 2010). Likewise, studies have shown that restraint stress impairs (Zhang & Rosenkranz, 2013), or has no effect on (Baran et al., 2009; Blume et al., 2019; Negrón-Oyarzo et al., 2014), cued fear extinction, and may impair cued fear extinction recall in males (Baran et al., 2009; Negrón-Oyarzo et al., 2014).