Acute exercise enhances fear extinction through a mechanism involving central mTOR signaling

https://doi.org/10.1016/j.nlm.2020.107328Get rights and content

Highlights

  • A bout of wheel running after fear extinction enhances fear extinction retrieval.

  • Intra-cerebral ventricular rapamycin blocks exercise-augmentation of fear extinction.

  • A bout of wheel running increases mTOR signaling in the nucleus accumbens.

  • Rapamycin reduces mTOR signaling in brain regions involved in fear extinction.

  • Exercise-augmentation of fear extinction involves central mTOR signaling.

Abstract

Impaired fear extinction, combined with the likelihood of fear relapse after exposure therapy, contributes to the persistence of many trauma-related disorders such as anxiety and post-traumatic stress disorder. Identifying mechanisms to aid fear extinction and reduce relapse could provide novel strategies for augmentation of exposure therapy. Exercise can enhance learning and memory and augment fear extinction of traumatic memories in humans and rodents. One factor that could contribute to enhanced fear extinction following exercise is the mammalian target of rapamycin (mTOR). mTOR is a translation regulator involved in synaptic plasticity and is sensitive to many exercise signals such as monoamines, growth factors, and cellular metabolism. Further, mTOR signaling is increased after chronic exercise in brain regions involved in learning and emotional behavior. Therefore, mTOR is a compelling potential facilitator of the memory-enhancing and overall beneficial effects of exercise on mental health. The goal of the current study is to test the hypothesis that mTOR signaling is necessary for the enhancement of fear extinction produced by acute, voluntary exercise. We observed that intracerebral-ventricular administration of the mTOR inhibitor rapamycin reduced immunoreactivity of phosphorylated S6, a downstream target of mTOR, in brain regions involved in fear extinction and eliminated the enhancement of fear extinction memory produced by acute exercise, without reducing voluntary exercise behavior or altering fear extinction in sedentary rats. These results suggest that mTOR signaling contributes to exercise-augmentation of fear extinction.

Introduction

Exercise is well known to produce beneficial effects on cognition and mental health. These effects include improving learning and memory (Cassilhas et al., 2015, Hotting and Roder, 2013, Voss et al., 2011) and providing protection against stress-related psychiatric disorders, such as anxiety, depression and post-traumatic stress disorder (Chekroud et al., 2018, Harvey et al., 2018, Herring et al., 2010, Schuch et al., 2016, Schuch et al., 2016, Stubbs et al., 2017). In rats, voluntary wheel running can similarly enhance cognitive function (Hotting & Roder, 2013) and enable stress resilience (Greenwood and Fleshner, 2019, Mul, 2018). The majority of studies investigating the effects of exercise on cognitive and mental health outcomes have examined the effects of chronic exercise, and important duration-dependent effects have been observed (Burghardt et al., 2006, Greenwood et al., 2005). Recent studies, however, have revealed some benefits of fewer bouts of exercise. Single bouts of exercise can, for example, improve cognitive function (Keyan and Bryant, 2019a, Keyan and Bryant, 2019b, Loprinzi and Kane, 2015) and enhance long-term memory processes, such as sematic recall (Coles and Tomporowski, 2008, Winter et al., 2007, Won et al., 2019). Some of the beneficial effects of acute exercise bouts can be observed even in the absence of a history of prior exercise. The emerging benefits of single bouts of exercise are especially exciting, as adherence to long-term exercise programs is difficult (Van Roie, Bautmans, Coudyzer, Boen, & Delecluse, 2015), but single bouts of exercise could be relatively easy to implement in a clinical setting. One example of incorporating acute exercise bouts successfully in a clinical setting comes from recent studies on exercise and fear extinction.

Fear extinction is the decline of a fear response after repeated exposure to a fear-eliciting conditioned stimulus (CS) previously associated with an aversive unconditioned stimulus (US; (Pavlov, 1927)). Fear extinction is the basis of exposure therapy, a common behavioral therapy used to treat fear and anxiety-related disorders in humans. Experiments in rats indicate that a single bout of voluntary wheel running either during (Mika, Bouchet, Bunker, Hellwinkel, & Spence, 2015) or immediately after (Bouchet et al., 2017, Siette et al., 2014, Tanner et al., 2018) fear extinction training can enhance fear extinction retrieval the following day, and reduce the subsequent renewal and spontaneous recovery of conditioned fear (for a review, see (Tanner et al., 2018)). Acute bouts of exercise can also enhance fear extinction in humans ((Keyan and Bryant, 2019a, Keyan and Bryant, 2019b), but see (Jacquart, Roquet, Papini, Powers, & Rosenfield, 2017)), potentially leading to improved outcomes in patients with trauma-related psychiatric disorders (Powers, Medina, Burns, Kauffman, & Monfils, 2015). However, the neurobiological mechanisms by which acute exercise augments fear extinction remain unknown. Identification of these mechanisms could help support the use of acute exercise bouts in a clinical setting and could reveal novel therapeutic targets for the treatment of anxiety and trauma-related disorders.

Fear extinction learning includes the critical phases of acquisition and consolidation. The association between the CS and the lack of the predicted aversive US is initially encoded during the acquisition phase. During consolidation, molecular processes such as protein synthesis take place in the neural circuits responsible for the retention of fear extinction (Santini, Huynh, & Klann, 2014). The observations that a bout of voluntary exercise either during (Mika et al., 2015) or after (Bouchet et al., 2017, Tanner et al., 2018) extinction training, but not before extinction training (Jacquart et al., 2017, Tanner et al., 2018), enhances later fear extinction retrieval, suggest that transient neurochemical effects of exercise during either the acquisition or consolidation phases could influence the molecular processes underlying the long-term memory of fear extinction. One factor that could underlie the long-term memory of fear extinction is the mammalian target of rapamycin (mTOR).

mTOR is a translation regulator and serine/threonine protein kinase involved in cell motility, proliferation, and synaptic plasticity (Hall, 2008). mTOR is a part of the phosphatidylinositol kinase-related kinases protein family and regulates protein synthesis by translational modification through the phosphorylation of several intracellular targets. These targets include the ribosomal S6 kinase and eIF4E-binding protein 1, which have been recognized as key initiators of translation (Raught et al., 2001, Saxton and Sabatini, 2017). Translation initiation and control subsequently initiates plastic changes required for learning and memory (Graber et al., 2013, Parsons et al., 2006). Considering that mTOR regulates translation initiators such as 4E-BP1 and S6 kinases, which are involved in neuronal plasticity, it is likely involved in memory consolidation. Indeed, previous studies reveal that inhibition of the mTOR pathway with rapamycin decreases p70s6 kinase expression in the amygdala (Parsons et al., 2006), and impairs consolidation of an auditory fear memory (Mac Callum, Hebert, Adamec, & Blundell, 2014), as well as the memory-enhancing effects of corticosterone (Xiong, Casse, Zhou, Zhou, & Xiong, 2015). mTOR has also been implicated in fear extinction learning. For example, one downstream target of mTOR is calcium/calmodulin-dependent protein kinase IIα (CaMKIIα) (Gong, Park, Abbassi, & Tang, 2006), blockade of which in the hippocampus prevents the enhancement of fear extinction caused by exposure to a novel environment (de Carvalho Myskiw et al., 2014). Additionally, ketamine increases mTOR signaling and, like exercise, has recently been reported to enhance fear extinction (Girgenti, Ghosal, LoPresto, Taylor, & Duman, 2017). Inhibition of mTOR in the medial prefrontal cortex blocks the ketamine-induced enhancement of fear extinction memory (Girgenti et al., 2017).

In addition to its role in learning and memory, mTOR is an attractive molecular candidate mediating the effects of exercise on learning and memory, given mTOR’s sensitivity to several factors known to be modulated by exercise. These factors include nutrient availability, metabolic signals like amino acids (Nave, Ouwens, Withers, Alessi, & Shepherd, 1999) and growth factors like brain-derived neurotrophic factor (BDNF; (Slipczuk et al., 2009)). Moreover, mTOR signaling is increased after 6 weeks of wheel running in brain areas involved in learning, fear regulation, and emotional behavior (Lloyd, Hake, Ishiwata, Farmer, & Loetz, 2017).

The goal of the current study was to test the hypothesis that acute voluntary exercise augments fear extinction through a mechanism involving central mTOR signaling. We inhibited mTOR signaling with an intracerebral-ventricular (ICV) injection of rapamycin prior to fear extinction training, to ensure the drug reached various extinction-related brain areas. Effectiveness of the drug to inhibit mTOR signaling was determined by immunohistochemistry for phosphorylated (p)S6, a down-stream target of mTOR (Saxton & Sabatini, 2017). Results indicate that central mTOR signaling contributes to acute exercise-augmentation of fear extinction, and adds to growing evidence that the mTOR pathway is an important target in memory and the treatment of trauma-related psychiatric disorders.

Section snippets

Subjects

A total of 40 young adult (P49 on arrival), male Long-Evans rats were ordered from Charles River labs (Wilmington, MA). Rats were pair-housed in ventilated cages (45 W × 25.2 D × 14.7H cm) with ad libitum access to food (standard Rat Chow) and water. The housing room was kept on a 12–12 h light–dark cycle with the lights on from 0600 to 1800 and maintained at a temperature of 25 °C. All rats were allowed to habituate to their housing conditions for 7 days prior to the start of any experimental

Quantification of immunohistochemistry

Quantification of pS6 occurred according to our published protocols (Lloyd et al., 2017). Images of the prelimbic (PL) and infralimbic (IL) cortices of PFC, dorsal lateral (DLS) and dorsal medial (DMS) striatum, nucleus accumbens core (NAcC) and nucleus accumbens shell (NAcS), central (CeA) and basolateral (BLA) amygdala, superior and inferior dentate gyrus (DG), cornu ammonis (CA1, CA2, CA3), and the ventral tegmental area (VTA) were captured digitally at 20X on an Olympus BX51microscope.

Effects of rapamycin and exercise on fear extinction and fear renewal

A timeline for the experiment is shown in Fig. 1. After exclusion of rats with misplaced cannulae, final group sizes were as follows: Veh/Sed, n = 7; RAP/Sed, n = 10; Veh/Run, n = 7; and RAP/Run, n = 10. Rats ran equal amounts during the wheel familiarization period regardless of subsequent group assignments (Fig. 2A). One wheel did not record running distance during the first night of the wheel running familiarization period, so running data for this rat is omitted. Rats exhibited no freezing

Discussion

Here we report the novel finding that the enhanced fear extinction memory retrieval produced by an acute bout of post-fear extinction wheel running can be prevented by ICV administration of the mTOR inhibitor RAP. RAP administration successfully reduced mTOR signaling, as indicated by a reduction in phosphorylation of S6, a downstream target of mTOR, in several brain regions implicated in fear extinction. These results add to prior data (Bouchet et al., 2017, Mika et al., 2015, Siette et al.,

Funding sources

This work was funded by the National Institutes of Health, R15 MH114026 (BNG).

CRediT authorship contribution statement

Nicolette A. Moya: Conceptualization, Investigation, Formal analysis, Visualization. Margaret K. Tanner: Methodology, Validation, Investigation. Abigail M. Smith: Investigation. Aleezah Balolia: Investigation. Jazmyne K.P. Davis: Investigation. Kelsey Bonar: Investigation. Jennifer Jaime: Investigation. Troy Hubert: Investigation. Jorge Silva: Investigation. William Whitworth: Investigation. Esteban C. Loetz: Investigation. Sondra T. Bland: Resources, Supervision. Benjamin N. Greenwood:

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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      In addition to its suspected role in ketamine's antidepressant effects, mounting evidence suggests mTOR signaling pathways may also be implicated in suicidality (Rengasamy et al., 2019). Studies from both blood and brain tissue samples support mTOR pathways may be important for understanding and predicting suicide attempts and deaths (Flory et al., 2017; Niculescu et al., 2015) as well as being linked to broader constructs with implications for suicidality including stress- and trauma-related psychopathology/disorders, learning, memory and brain development (Graber et al., 2013; Hoeffer and Klann, 2010; Lee, 2015; Jernigan et al., 2011; Ni et al., 2020; Moya et al., 2020; Fifield et al., 2013). Given this potential role in suicidality and the surprising outcomes of our recent study, here we conducted follow-up analyses to examine the role of rapamycin pretreatment on ketamine's antisuicidal effects.

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