Normal aging is associated with specific changes in brain structure and connectivity, and frequently with declines in cognitive function that significantly affect quality of life. Signaling pathways that regulate the aging process have in common a role in the control of cellular metabolism in response to availability of nutrients or growth factors (Kapahi et al.). mTOR controls cellular metabolism and organismal lifespan in invertebrates and mammals (Kapahi and Zid, 2004
). It was recently shown that inhibition of mTOR activity in mice by chronic rapamycin treatment extends lifespan, possibly by delaying aging (Harrison et al., 2009
). To explore the effect of rapamycin on normal brain aging we determined cognitive and non-cognitive components of behavior throughout lifespan in C57BL/6 animals that were fed control- or rapamycin-supplemented chow. Our studies show that feeding encapsulated rapamycin in chow at 14 ppm for at least 16 weeks is sufficient to improve cognitive outcomes in both adult (8 month-old) and in old (25 month-old) C57BL/6 mice compared to control-fed littermates (). Even though the acute inhibition of TOR has generally been associated with defects in long-term plasticity (Tang et al., 2002
), inhibition of TOR can also block LTD (Huber et al., 2001
). Moreover, high TOR activity in neuronal progenitors or in neuronal/glial precursors during development results in significant plasticity and memory deficits (Hoeffer and Klann, 2009
) and aberrant excitability, suggesting that adequate plasticity requires that TOR activity be within a range that allows for appropriate regulation of protein synthesis at synaptic sites. Our results suggest that an approximate 30% reduction in TOR activity in brain [, (Spilman et al., 2010
)] for 16 weeks or longer improves performance of C57BL/6 mice in tasks that involve long-term plasticity and are dependent on hippocampus or on hippocampus and prefrontal cortex. Considered together with prior knowledge on mTOR function (Huber et al., 2001
, Tang et al., 2002
, Sarbassov et al., 2005
, Hoeffer and Klann, 2009
), our results suggest that, while acute and complete inhibition of TOR abolishes plasticity required for long-term memory (Tang et al., 2002
, Hoeffer and Klann, 2009
), chronically decreased TOR activity (in our studies, approximately 30%) can improve learning and retention of a spatial task in young mice and improve memory of an aversive event in old animals (). Consistent with these observations, caloric restriction, which decreases mTOR activity (Dogan et al.) and extends lifespan, improves cognitive function in some, but not all, rodent (Mattson et al., 2002
, Martin et al., 2007
, Adams et al., 2008
, Komatsu et al., 2008
), primate (Witte et al., 2009
, Dal-Pan et al.) or invertebrate (Burger et al.) experimental models. Thus, it is conceivable that long-term, partial reduction of mTOR activity by rapamycin administered in the chow mimics the effect of calorie restriction, enhancing cognitive outcomes in young mice and maintaining intact cognitive performance in older animals. Of note, cognitive effects of rapamycin feeding were observed when mice were fed for 16 weeks or as long as 40 weeks, suggesting that the effect of chronic rapamycin treatment is sustained for protracted lengths of time.
It has been suggested that there was little selective pressure to evolve effective mechanisms of neuroprotection in old age (Finch and Austad, 2012
). It is therefore conceivable that brain mTOR activity levels that are adequate during the reproductive years may become detrimental as mammals age (Blagosklonny, 2010
). In agreement with this hypothesis, we (Spilman et al., 2010
) and others (Caccamo et al 2010
) previously showed that chronic (> 16 weeks) inhibition of mTOR activity by rapamycin in brain preserves cognitive function in mice modeling AD, possibly by increasing autophagy.
It had been previously reported that treatment with a rapamycin analog everolimus may decrease anxiety and depressive-like behaviour (Lang et al., 2009
) although other studies did not confirm this effect on transplant patients treated with sirolimus or tacrolimus (Martinez-Sanchis et al.). The studies presented here show that C57BL/6 mice chronically fed encapsulated rapamycin exhibit reduced anxiety and decreased depressive-like behavior at all ages tested and in animals of both genders (). These results suggest that a partial decrease in TOR activity may impinge on the activity of limbic structures like the amygdala, which mediate emotion output such as fear. It is conceivable that at least one mechanism mediating the effect of long-term oral rapamycin on memory involves the downregulation of synaptic plasticity in limbic networks. This potential mechanism, however, cannot explain the observed effects of chronic rapamycin treatment on spatial learning and memory ().
In agreement with the observed changes in behavioral outcomes, however, rapamycin-treated animals showed significantly increased levels of three major monoamines (NE, DA and 5-HT) and their metabolites (DOPAC, HVA, and 5-HIAA) specifically in midbrain (). Even though monoamine levels were not increased in hippocampal tissue (), the observed increase of monoamine content in midbrain is consistent with effects on key target areas such as the septo-hippocampal complex through the mesolimbic pathway. The mesocorticolimbic dopamine system is widely implicated in reward and in reinforcement processes (Morgane et al., 2005
). The midbrain contains major dopaminergic projecting neurons in the substantia nigra pars compacta (PC) and ventral tegmental area (VTA). Thus, increases in monoamine content in midbrain is consistent with effects on key target areas such as the striatum (involved in reward/motivation) through the mesostriatal pathway, as well as on the limbic cortices, the septo-hippocampal complex, the amygdala and the nucleus accumbens (involved in emotion, long-term memory and olfaction) through the mesolimbic pathway. Pathways arising from dopaminergic midbrain nuclei also innervate the prefrontal and insular cortices, several thalamic and hypothalamic nuclei and the monoaminergic nuclei of the brain stem, superior colliculus, reticular formation and periaqueductal grey, all having important roles in regulating arousal as well as affective and cognitive processes. In addition, the PC is important in spatial learning and some studies have suggested its involvement in a response-based memory system that utilizes a dorsostriatal pathway and may function independently of the hippocampus (Da Cunha et al., 2003
). In addition, the VTA is strongly involved in the reward circuitry of the brain, with important roles in cognition and motivation (Margolis et al., 2006
). Dopaminergic input from VTA contributes to processing of emotion output from the amygdala, thus playing a role in avoidance and fear conditioning (). An increase in dopamine levels in midbrain, therefore, is expected to have a broad impact on both affect and cognitive domains. Thus, our results suggest that increased levels of DA in midbrain may underlie the memory-enhancing, anxiolytic and antidepressant-like effects observed in rapamycin-fed C57BL/6J mice.
DA is a precursor to NE and then to EPI in these neurotransmitters’ biosynthetic pathway (Mains and Patterson, 1973
). The observed increase in NE in midbrain of rapamycin-treated animals may therefore be a direct consequence of increased DA levels (). A simple direct precursor relationship, however, is unlikely since EPI was not increased. In midbrain, noradrenergic neurons originate in the lateral tegmental field and exert effects on large areas of the brain, increasing alertness and arousal as well as influencing the reward system through activation of adrenergic receptors in amygdala, cingulated gyrus, hippocampus, hypothalamus, neocortex, striatum and thalamus. Midbrain NE has been associated with the regulation of anxiety (Simon et al., 2009
, Watt et al., 2009
) and motivation (Heimovics et al.). Moreover, increasing NE and 5-HT levels by inhibition of NE-DA uptake is a common intervention used to treat anxiety in depressive disorders (Tollefson et al., 1991
, Papakostas et al., 2008
). Uptake of NE-DA in prefrontal cortex is mediated by NET, and it was recently demonstrated (Siuta et al.) that animals with impaired mTORC2 function show increased prefrontal NET, resulting in increased levels of prefrontal NE concomitant with decreased DA. Our results show that neither mTORC2 activity () nor NET levels () are affected by rapamycin treatment, suggesting that increased noradrenergic input to cortex is intact in rapamycin-treated mice. Thus, increased NE levels in midbrain may further contribute to the observed memory-enhancing, anxiolytic and antidepressant-like effects of rapamycin treatment in C57BL/6J mice. 5-HT (serotonin) regulates mood, appetite and sleep as well as memory and learning (Hariri and Holmes, 2006
, Elliott et al.). The modulation of 5-HT levels at synapses is a major site of action for antidepressants (Sharp and Cowen). In midbrain, the 5-HT in the raphe nuclei (RN) controls the activity of ascending serotonergic systems and the release of 5-HT in forebrain (Adell et al., 2002
). 5-HT-containing neurons in the dorsal RN provide part of the serotonergic innervation to widely distributed areas in the brain (Monti). Dysfunctions in forebrain serotonergic innervation from the RN are thought to underlie the neural mechanisms of depression (Stamford et al., 2000
). NOR, gamma-amino butyric acid and glutamate regulate the extracellular concentration of 5-HT in raphe nuclei. Thus, it is possible that high NOR stimulates the synthesis and release of 5-HT in midbrain. The pivotal role of 5-HT in the regulation of mood and motivation as well as its role in the regulation of learning and memory provides an additional explanation for the observed improvements in cognitive and non-cognitive components of behavior in rapamycin-treated mice.
The question as to how TOR function is mechanistically linked to the regulation of midbrain monoamine levels, however, remains unanswered. Rapamycin has been shown to inhibit evoked dopamine release in striatal slices through the activation of macroautophagy [Hernandez et al (2012) Neuron
74:277], demonstrating that macroautophagy is necessary for the maintenance of adequate numbers of synaptic vesicles available for release at synaptic sites. Of note, Hernandez et al. noted that rapamycin induced decreased numbers of synaptic vesicles also in non-dopaminergic terminals in striatum, suggesting that the effect of macroautophagy in the maintenance of an adequately sized vesicle pool at nerve terminals may be general and not specific to dopaminergic cells. Decreases in the size of readily releasable vesicle pools mobilizes a reserve pool of neurotransmitter-containing synaptic vesicles [Venton et al. (2006) J Neurosci
26:3206]. Thus, it is conceivable that the increases in monoamine content that we have observed in midbrain may reflect the activation of synthetic pathways to replenish a decreasing pool of synaptic vesicles as a result of a chronic increase in macroautophagy induced by rapamycin treatment. Consistent with this hypothesis, we previously demonstrated that chronic rapamycin treatment increases autophagy in hippocampus of mice modeling AD (Spilman et al., 2010
). Since the specific midbrain/brainstem nuclei contain the major dopamine (substantia nigra) and serotonin (raphe nuclei) releasing clusters in the brain, this effect may be more pronounced, and thus readily detectable, in midbrain and brainstem.
In summary, the results of the present study demonstrate that chronic feeding with encapsulated rapamycin enhances memory in young C57BL/6 mice and delays cognitive decline associated with aging. Moreover, our results show that long-term feeding with encapsulated rapamycin has concomitant anxiolytic and antidepressant-like effects. Our findings are consistent with prior studies showing that long-term rapamycin treatment rescued learning and memory in mice modelling AD (Caccamo et al., Spilman et al., 2010
). Our results suggest chronic, partial inhibition of mTOR may have widespread effects in the regulation of arousal and attention as well as of affective and cognitive processes, possibly by stimulating major monoamine pathways in brain. Rapamycin and rapamycin analogs, already used in the clinic, may have potential for therapeutic intervention in cognitive and affective dysfunctions associated with aging.