Systemic inhibition of mTOR kinase via rapamycin disrupts consolidation and reconsolidation of auditory fear memory.

The mammalian target of rapamycin (mTOR) kinase is a critical regulator of mRNA translation and is known to be involved in various long lasting forms of synaptic and behavioural plasticity. However, information concerning the temporal pattern of mTOR activation and susceptibility to pharmacological intervention during both consolidation and reconsolidation of long-term memory (LTM) remains scant. Male C57BL/6 mice were injected systemically with rapamycin at various time points following conditioning or retrieval in an auditory fear conditioning paradigm, and compared to vehicle (and/or anisomycin) controls for subsequent memory recall. Systemic blockade of mTOR with rapamycin immediately or 12h after training or reactivation impairs both consolidation and reconsolidation of an auditory fear memory. Further behavioural analysis revealed that the enduring effects of rapamycin on reconsolidation are dependent upon reactivation of the memory trace. Rapamycin, however, has no effect on short-term memory or the ability to retrieve an established fear memory. Collectively, our data suggest that biphasic mTOR signalling is essential for both consolidation and reconsolidation-like activities that contribute to the formation, re-stabilization, and persistence of long term auditory-fear memories, while not influencing other aspects of the memory trace. These findings also provide evidence for a cogent treatment model for reducing the emotional strength of established, traumatic memories analogous to those observed in acquired anxiety disorders such as posttraumatic stress disorder (PTSD) and specific phobias, through pharmacologic blockade of mTOR using systemic rapamycin following reactivation.

Glucocorticoids are required for extinction of predator stress-induced hyperarousal.

BACKGROUND: The role of glucocorticoids in extinction of traumatic memories has not been fully characterized despite its potential as a therapeutic target for acquired posttraumatic stress disorder (PTSD). The predator stress paradigm allows us to determine whether glucocorticoids mediate the extinction of both context-dependent and context-independent fear memories. METHODS: Male C57BL/6J mice were exposed to a predator (cat) then repeatedly exposed to the predator stress context in the absence of the cat. Context-dependent (associative) fear memory was assessed as suppression of activity during re-exposure to the predator stress context without the cat (extinction trials). Context-independent fear (non-associative) was assessed seven days after extinction trials using measures of hyperarousal and anxiety-like behaviours in environments unlike the predator stress context. To assess the role of glucocorticoids, mice were injected with metyrapone (50mg/kg) 90 min prior to extinction trials in predator stressed mice and context-dependent and context-independent fear memories were assessed. Finally, metyrapone-treated predator stressed mice were injected with corticosterone (5 or 10mg/kg) immediately following extinction trials and context-dependent and context-independent fear memories were assessed. RESULTS: Repeated re-exposure to the predator stress context without the cat present extinguished context-dependent fear memory, and also reduced hyperarousal, a generalized, chronic PTSD-like symptom. We show that extinction of context-independent predator stress-induced hyperarousal is dependent on endogenous glucocorticoids during the extinction trials. Furthermore, the inhibition of extinction by metyrapone on startle amplitude was reduced by exogenous administration of corticosterone following extinction trials. Overall, these data implicate glucocorticoids in the extinction of hyperarousal, a core symptom of PTSD.

Relationship of the predatory attack experience to neural plasticity, pCREB expression and neuroendocrine response.

Aggression takes at least two, an attacker and a target. This paper will address the lasting consequences of being a target of aggression. We review the lasting impact of predatory attack on brain and behavior in rodents. A single brief unprotected exposure of a rat to a cat lastingly alters affective responses of rats in a variety of contexts. Alterations of these behaviors resembles both generalized anxiety comorbid with post traumatic stress disorder (PTSD), and the hyper arousal expressed in enhanced startle in PTSD. Examination of neural transmission and neural plasticity in limbic circuits implicates changes in transmission in two connecting pathways in many but not all of the behavioral changes. Quantification of the predator encounter reveals that both the behavior of the predator and the reaction of the rat to attack are highly predictive of the effects of predatory attack on molecular biological (pCREB expression) and electrophysiological measures of limbic neuroplastic change. Moreover, a case will be made that the pattern of change of corticosteroid level over three hours after the predator encounter, in interaction with the predatory experience, plays an important part in initiation of lasting changes in brain and behavior.

Neural circuit changes mediating lasting brain and behavioral response to predator stress.

This paper reviews recent work which points to critical neural circuitry involved in lasting changes in anxiety like behavior following unprotected exposure of rats to cats (predator stress). Predator stress may increase anxiety like behavior in a variety of behavioral tests including: elevated plus maze, light dark box, acoustic startle, and social interaction. Studies of neural transmission in two limbic pathways, combined with path and covariance analysis relating physiology to behavior, suggest long term potentiation like changes in one or both of these pathways in the right hemisphere accounts for stress induced changes in all behaviors changed by predator stress except light dark box and social interaction. Findings will be discussed within the context of what is known about neural substrates activated by predator odor.

Expression of egr-1 (zif268) mRNA in select fear-related brain regions following exposure to a predator.

Research has demonstrated that immediate-early genes/inducible transcriptional factors (e.g., c-fos, egr-1) are increased in amygdala nuclei (lateral, basal and central nuclei) known to be involved in fear conditioning, footshock stress and novelty. Although these data suggest that expression of inducible transcriptional factors are involved in fear, other non-shock ethologically based paradigms (predator or predator odor exposure) do not appear to increase c-fos in the lateral and basal nuclei. While the lack of c-fos expression may indicate that predator stress does not engage the lateral and basal amygdala nuclei, it may be that c-fos in the amygdala is not responsive to predator exposure. Therefore, egr-1, which increases in the lateral nucleus following fear conditioning, footshock and novelty, was assessed to determine if its expression is induced in rats exposed to a cat. Five minutes of cat exposure did not increase expression of egr-1 mRNA in the lateral nucleus of the amygdala. egr-1 was increased in the paraventricular nucleus of the hypothalamus, indicating cat-induced stress, and visual cortex compared to rats that were either confined for 5 min or handled. In the lateral periaqueductal gray, handled rats displayed a left hemisphere dominance, which disappeared in both the cat-exposed and confined group, suggesting that immobility, induced by either cat-induced stress or unstressed confinement, increased right hemisphere egr-1 expression. The results are discussed in a context of differences and similarities in neural circuitry for conditioned and unconditioned fear.

Phosphorylated cyclic AMP response element binding protein expression induced in the periaqueductal gray by predator stress: its relationship to the stress experience, behavior and limbic neural plasticity.

Electrophysiological studies in cats and recently in rats implicate neuroplasticity in the periaqueductal gray (PAG) and its afferents in stressor-induced increases in fearful behavior and anxiety-like behavior (ALB). Such increases may model aspects of affective changes following traumatic stress in humans. The present study explored the role of neuroplasticity in PAG and its connection with the central nucleus of the amygdala (ACE) in male rodent anxiety-like response to predator stress. In the first of two studies, the effects of predator stress on the induction of phosphorylated cyclic AMP response element binding protein (pCREB) were investigated. pCREB expression in the PAG and ventromedial hypothalamus (VMH) was examined immunohistochemically. Predator stress increased the degree of pCREB expression in PAG cells (measured densitometrically) but did not increase the number of cells expressing pCREB (measured stereologically). Moreover, predator stress-specific increase in pCREB-like immunoreactivity (lir) was restricted to the right lateral column of the PAG. In addition, pCREB lir in the right lateral column likely reflects aspects of the stress experience because the stressor (cat behavior) and the response to the stressor (rat defensive behavior) are highly predictive of degree of pCREB expression. There was no effect of predator stress on pCREB lir in the VMH. Because pCREB expression has been associated with long-lasting potentiation (LLP) of neural transmission, we examined the effects of predator stress on transmission in the ACE-PAG pathway in a second study. Predator stress elevated evoked potential measures of ACE-PAG transmission in the right hemisphere but not in the left hemisphere 11-12 days after predator stress. This finding is consistent with the longer-lived effects of pharmacological stress on amygdalo-PAG transmission in the right hemisphere but not in the left hemisphere in cats. Of interest is the fact that the same aspects of the stressor experience and reaction to it, which are predictive of the degree of pCREB expression, are also highly predictive of the degree of potentiation of measures of ACE-PAG transmission. Behavioral analyses revealed that the most consistent effects of predator stress are on behavior in the plus maze (open arm exploration and risk assessment) and on startle. In addition, covariance analysis suggests that ACE-PAG potentiation mediates some but not all of the changes in ALB produced by predator stress. Because pCREB expression may be a precursor to neuroplastic changes in certain forms of memory and LLP, the present findings complement studies in the cat, showing that neuroplastic changes in the PAG underlie changes in affect following stress. Furthermore, these findings suggest that neuroplastic changes in PAG may be important mediators of predator stress-induced changes in affective behavior in rodents. Finally, consistent with cat and human studies, the right hemisphere appears particularly important in long-term response to stress.