Protocol for Studying Extinction of Conditioned Fear in Naturally Cycling Female Rats

Extinction of conditioned fear has been extensively studied in male rodents. Recently, there have been an increasing number of studies indicating that ...

Massachusetts General Hospital

Harvard Medical School

Acute stress exposure enhances classical eyeblink conditioning in male rats, whereas exposure to the same event dramatically impairs performance in females (Wood and Shors, 1998; Wood et al., 2001). We hypothesized that stress affects learning differently in males and females because different brain regions and circuits are being activated. In the first experiment, we determined that neuronal activity within the medial prefrontal cortex (mPFC) during the stressful event is necessary to disrupt learning in females. In both males and females, the mPFC was bilaterally inactivated with GABA agonist muscimol before the stressor. Inactivation prevented only the impaired performance in females; it had no consequence for performance in males. However, in the second experiment, excitation of the mPFC alone with GABA antagonist picrotoxin was insufficient to elicit the stress effect that was prevented through the inactivation of this region in females. Therefore, we hypothesized that the mPFC communicates with the basolateral amygdala to disrupt learning in females after the stressor. To test this hypothesis, these structures were disconnected from each other with unilateral excitotoxic (NMDA) lesions on either the same or opposite sides of the brain. Females with contralateral lesions, which disrupt the connections on both sides of the brain, were able to learn after the stressful event, whereas those with ipsilateral lesions, which disrupt only one connection, did not learn after the stressor. Together, these data indicate that the mPFC is critically involved in females during stress to impair subsequent learning and does so via communication with the amygdala.

The prefrontal cortex communicates with the amygdala to impair learning after acute stress in females but not in males.

Women experience profound hormonal fluctuations throughout their reproductive lives. They are especially susceptible to disturbances in mood and cognition during the transition from pregnancy into postpartum and motherhood (Brummelte & Galea, 2010). Their behavioral and hormonal responses to stressful stimuli are also altered during this time. These changes are not limited to humans but occur in many mammalian species. Virgin female rats express a severe learning deficit in associative eyeblink conditioning after a stressful life event (Wood, Beylin, & Shors, 2001; Wood & Shors, 1998), but lactating females or those that are caring for young learn well even after the stressor (Leuner & Shors, 2006). However, we do not know whether maternal experience persistently alters learning after a stressful event. Here we hypothesized that females that had been maternal at some time in their lives would learn well even after exposure to a stressful event. To test this hypothesis, females that had at least one brood of young and expressed a normal estrous cycle were exposed to an acute stressful event that reliably impairs learning in virgin females. Animals were trained 24 hr later with classical eyeblink conditioning. Exposure to the stressor suppressed learning in virgins but not in females that had been mothers at some time in their lives. These data suggest that maternal experience induces a protective mechanism in mothers, which promotes associative learning long after the offspring have left their care.

Once a mother, always a mother: maternal experience protects females from the negative effects of stress on learning.

The dentate gyrus is a major site of plasticity in the adult brain, giving rise to thousands of new neurons every day, through the process of adult neurogenesis. Although the majority of these cells die within two weeks of their birth, they can be rescued from death by various forms of learning. Successful acquisition of select types of associative and spatial memories increases the number of these cells that survive. Here, we investigated the possibility that an entirely different form of learning, physical skill learning, could rescue new hippocampal cells from death. To test this possibility, rats were trained with a physically-demanding and technically-difficult version of a rotarod procedure. Acquisition of the physical skill greatly increased the number of new hippocampal cells that survived. The number of surviving cells positively correlated with performance on the task. Only animals that successfully mastered the task retained the cells that would have otherwise died. Animals that failed to learn, and those that did not learn well did not retain any more cells than those that were untrained. Importantly, acute voluntary exercise in activity wheels did not increase the number of surviving cells. These data suggest that acquisition of a physical skill can increase the number of surviving hippocampal cells. Moreover, learning an easier version of the task did not increase cell survival. These results are consistent with previous reports revealing that learning only rescues new neurons from death when acquisition is sufficiently difficult to achieve. Finally, complete hippocampal lesions did not disrupt acquisition of this physical skill. Therefore, physical skill training that does not depend on the hippocampus can effectively increase the number of surviving cells in the adult hippocampus, the vast majority of which become mature neurons.

Physical skill training increases the number of surviving new cells in the adult hippocampus.

Women are nearly twice as likely as men to suffer from anxiety and post-traumatic stress disorder (PTSD), indicating that many females are especially vulnerable to stressful life experience. A profound sex difference in the response to stress is also observed in laboratory animals. Acute exposure to an uncontrollable stressful event disrupts associative learning during classical eyeblink conditioning in female rats but enhances this same type of learning process in males. These sex differences in response to stress are dependent on neuronal activity in similar but also different brain regions. Neuronal activity in the basolateral nucleus of the amygdala (BLA) is necessary in both males and females. However, neuronal activity in the medial prefrontal cortex (mPFC) during the stressor is necessary to modify learning in females but not in males. The mPFC is often divided into its prelimbic (PL) and infralimbic (IL) subregions, which differ both in structure and function. Through its connections to the BLA, we hypothesized that neuronal activity within the PL, but not IL, during the stressor is necessary to suppress learning in females. To test this hypothesis, either the PL or IL of adult female rats was bilaterally inactivated with GABAA agonist muscimol during acute inescapable swim stress. About 24 h later, all subjects were trained with classical eyeblink conditioning. Though stressed, females without neuronal activity in the PL learned well. In contrast, females with IL inactivation during the stressor did not learn well, behaving similarly to stressed vehicle-treated females. These data suggest that exposure to a stressful event critically engages the PL, but not IL, to disrupt associative learning in females. Together with previous studies, these data indicate that the PL communicates with the BLA to suppress learning after a stressful experience in females. This circuit may be similarly engaged in women who become cognitively impaired after stressful life events.

The stressed female brain: neuronal activity in the prelimbic but not infralimbic region of the medial prefrontal cortex suppresses learning after acute stress.

Lisa Y. Maeng

Department of Psychiatry

Massachusetts General Hospital

Protocol for Studying Extinction of Conditioned Fear in Naturally Cycling Female Rats

Lisa Y. Maeng

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Psychiatry

Massachusetts General Hospital

Protocol for Studying Extinction of Conditioned Fear in Naturally Cycling Female Rats

Department of Psychiatry