Single injection of rapamycin blocks post-food restriction hyperphagia and body-weight regain in rats.

Given the increasing prevalence of and severity of complications associated with obesity, there is great need for treatments resulting in prolonged weight loss. Long-term maintenance of weight loss requires sustained changes in food-intake and energy-expenditure strategies, which are unfortunately often taxing, resulting in a return to predieting weight. Therefore, drug therapies may facilitate greater adherence to a restricted diet and prolong weight loss. One such drug is rapamycin (RAP), a mechanistic target of rapamycin (mTOR) inhibitor. Here, we show that a single injection of RAP dampens the hyperphagic response in calorically restricted rats when they are returned to free feed immediately or 10 days after injection. Moreover, we demonstrate that a single injection of RAP given to calorically restricted rats prevents body-weight regain when animals are returned to free feed either immediately or 10 days after injection. Furthermore, we extend our previous findings that RAP does not produce malaise or illness and show that RAP does not produce any behavioral deficits that may inhibit an animal from eating. Thus, we suggest that mTOR may be a useful target in obesity research, given that its inhibition may decrease the hyperphagic response following caloric restriction. (PsycINFO Database Record (c) 2019 APA, all rights reserved).

Absence of neurogenic response following robust predator-induced stress response.

Traumatic events contribute to a variety of neuropsychiatric disorders including post-traumatic stress disorder (PTSD). Identifying the neural mechanisms that affect the stress response may improve treatment for stress-related disorders. Neurogenesis, the production of neurons, occurs within the adult brain and disturbances in neurogenesis in the subgranular zone (SGZ) of the hippocampus have been linked to mood and anxiety disorders. Chronic stress models have mainly suggested correlations with stress reducing adult SGZ neurogenesis, whereas acute stress models and those with a naturalistic component that are also associated with long-lasting behavioral changes have produced inconsistent results. Therefore, the goal of the current study was to examine the effects of acute predator stress on adult neurogenesis. Predator stress involved a single 10-min unprotected rat to cat exposure that has previously been shown to produce contextual fear, hyperarousal, and anxiety-like behavior lasting at least 3weeks. As expected, predator stress produced a stress response as detected by elevated corticosterone (CORT) levels immediately after stress. Despite this robust stress response, there was no significant difference between stressed and handled control rats in the number of proliferating or surviving cells as assessed by a 5-bromo-2'-deoxyuridine-immunoreactive (BrdU-IR) labeling 2h or 4weeks post-stress throughout the rostro-caudal axis of the SGZ, respectively. Additionally, 90% of 4-week-old BrdU-IR cells in both conditions expressed NeuN, suggesting no change in cell fate with stress exposure. Overall, these data give caution to the notion that acute predator stress can alter the production or survival of adult-generated cells.

Time-dependent effects of rapamycin on consolidation of predator stress-induced hyperarousal.

Previous studies have indicated that rapamycin, a potent inhibitor of the mammalian target of rapamycin (mTOR) pathway, blocks consolidation of shock-induced associative fear memories. Moreover, rapamycin's block of associative fear memories is time-dependent. It is unknown, however, if rapamycin blocks consolidation of predator stress-induced non-associative fear memories. Furthermore, the temporal pattern of mTOR activation following predator stress is unknown. Thus, the goal of the current studies was to determine if rapamycin blocks consolidation of predator stress-induced fear memories and if so, whether rapamycin's effect is time-dependent. Male rats were injected systemically with rapamycin at various time points following predator stress. Predator stress involves an acute, unprotected exposure of a rat to a cat, which causes long-lasting non-associative fear memories manifested as generalized hyperarousal and increased anxiety-like behaviour. We show that rapamycin injected immediately after predator stress blocked consolidation of stress-induced startle. However, rapamycin injected 9, 24 or 48h post predator stress potentiated stress-induced startle. Consistent with shock-induced associative fear memories, we show that mTOR signalling is essential for consolidation of predator stress-induced hyperarousal. However, unlike shock-induced fear memories, a second, persistent, late phase mTOR-dependent process following predator stress actually dampens startle. Consistent with previous findings, our data support the potential role for rapamycin in treatment of stress related disorders such as posttraumatic stress disorder. However, our data suggest timing of rapamycin administration is critical.

Single rapamycin administration induces prolonged downward shift in defended body weight in rats.

Manipulation of body weight set point may be an effective weight loss and maintenance strategy as the homeostatic mechanism governing energy balance remains intact even in obese conditions and counters the effort to lose weight. However, how the set point is determined is not well understood. We show that a single injection of rapamycin (RAP), an mTOR inhibitor, is sufficient to shift the set point in rats. Intraperitoneal RAP decreased food intake and daily weight gain for several days, but surprisingly, there was also a long-term reduction in body weight which lasted at least 10 weeks without additional RAP injection. These effects were not due to malaise or glucose intolerance. Two RAP administrations with a two-week interval had additive effects on body weight without desensitization and significantly reduced the white adipose tissue weight. When challenged with food deprivation, vehicle and RAP-treated rats responded with rebound hyperphagia, suggesting that RAP was not inhibiting compensatory responses to weight loss. Instead, RAP animals defended a lower body weight achieved after RAP treatment. Decreased food intake and body weight were also seen with intracerebroventricular injection of RAP, indicating that the RAP effect is at least partially mediated by the brain. In summary, we found a novel effect of RAP that maintains lower body weight by shifting the set point long-term. Thus, RAP and related compounds may be unique tools to investigate the mechanisms by which the defended level of body weight is determined; such compounds may also be used to complement weight loss strategy.

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.

Inhibition of mTOR kinase via rapamycin blocks persistent predator stress-induced hyperarousal.

Traumatic, stressful life events are thought to trigger acquired anxiety disorders such as post-traumatic stress disorder (PTSD). Recent data suggests that the mammalian target of rapamycin (mTOR) plays a key role in the formation of traumatic memories. The predator stress paradigm allows us to determine whether mTOR mediates the formation of both context-dependent (associative) and context-independent (non-associative) fear memories. Predator stress involves an acute, unprotected exposure of a rat to a cat which causes long-lasting non-associative fear memories manifested as generalized hyperarousal and increased anxiety-like behavior. Here, we show that rapamycin, an mTOR inhibitor, attenuates predator stress-induced hyperarousal, lasting at least three weeks. In addition, rapamycin blocks a subset of anxiety-like behaviors as measured in the elevated plus maze and hole board. Furthermore, when re-exposed to the predator stress context, rapamycin-treated stressed rats showed increased activity compared to vehicle controls suggesting that rapamycin blocks predator stress-induced associative fear memory. Taken together with past research, our results indicate that mTOR regulation of protein translation is required for the formation of both associative and non-associative fear memories. Overall, these data suggest that mTOR activation may contribute to the development of acquired anxiety disorders such as PTSD.

Dendritic morphology of amygdala and hippocampal neurons in more and less predator stress responsive rats and more and less spontaneously anxious handled controls.

We investigated the neurobiological bases of variation in response to predator stress (PS). Sixteen days after treatment (PS or handling), rats were grouped according to anxiety in the elevated plus maze (EPM). Acoustic startle was also measured. We examined the structure of dendritic trees of basolateral amygdala (BLA) output neurons (stellate and pyramidal cells) and of dorsal hippocampal (DHC) dentate granule cells of less anxious (LA) and more (extremely) anxious (MA) stressed animals (PSLA and PSMA). Handled controls (HC) which were less anxious (HCLA) and spontaneously more anxious (HCMA) equivalently to predator stressed subgroups were also studied. Golgi analysis revealed BLA output neurons of HCMA rats exhibited longer, more branched dendrites with higher spine density than the other groups of rats, which did not differ. Finally, spine density of DHC granule cells was equally depressed in HCMA and PSMA rats relative to HCLA and PSLA rats. Total dendritic length of BLA pyramidal and stellate cells (positive predictor) and DHC spine density (negative predictor) together accounted for 96% of the variance of anxiety of handled rats. DHC spine density was a negative predictor of PSMA and PSLA anxiety, accounting for 70% of the variance. Data are discussed in the context of morphological differences as phenotypic markers of a genetic predisposition to anxiety in handled controls, and a possible genetic vulnerability to predator stress expressed as reduced spine density in the DHC. Significance of findings for animal models of anxiety and hyperarousal comorbidities of PTSD are discussed.

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.

Long lasting effects of predator stress on pCREB expression in brain regions involved in fearful and anxious behavior.

Predator stress is one animal model of posttraumatic stress disorder (PTSD). Neural plasticity in amygdala afferent and efferent pathways underlies anxiogenic effects of predator stress. Predator stress increases pCREB expression in these pathways 20min after stress, implicating pCREB in stress-induced neural plasticity. Here we examined impact of predator stress on pCREB expression 6-24h and 7 days after stress in amygdala pathways and in the supramammillary nucleus (SuM). Patterns of change in pCREB expression were complex, time dependent, column dependent in the periaqueductal gray (PAG), and AP plane dependent in the amygdala. In contrast to past work at 20 min after stress, there were no stress-induced increases in pCREB in the amygdala in the anterior AP plane or in the lateral PAG at 6h onward after stress. However, dorsal PAG pCREB was increased bilaterally at 24h and 7 days after stress. In the mid AP plane of all amygdala nuclei there were bilateral stress-induced increases in pCREB at 6h followed by decreases at 24h post stress. A similar pattern was observed in the posterior AP plane. In addition, we found a persistent increase (6h to 7 days after stress) in pCREB expression in the SuM. Further study of this nucleus as a contributor to fear sensitization following predator stress is warranted. Overall, these data highlight persistent neuroplastic changes in key brain areas following traumatic stress. Identification of these changes may aid in understanding the neural mechanisms underlying acquired anxiety disorders such as PTSD.