www.nature.com/scientificreports OPEN Torpor enhances synaptic strength and restores memory performance in a mouse model of Alzheimer’s disease Christina F. de Veij Mestdagh1,2, Jaap A. Timmerman3, Frank Koopmans1, Iryna Paliukhovich1, Suzanne S. M. Miedema1, Maaike Goris2, Rolinka J. van der Loo1, Guido Krenning4,5, Ka Wan Li1, Huibert D. Mansvelder3, August B. Smit1, Robert H. Henning2 & Ronald E. van Kesteren1* Hibernation induces neurodegeneration-like changes in the brain, which are completely reversed upon arousal. Hibernation-induced plasticity may therefore be of great relevance for the treatment of neurodegenerative diseases, but remains largely unexplored. Here we show that a single torpor and arousal sequence in mice does not induce dendrite retraction and synapse loss as observed in seasonal hibernators. Instead, it increases hippocampal long-term potentiation and contextual fear memory. This is accompanied by increased levels of key postsynaptic proteins and mitochondrial complex I and IV proteins, indicating mitochondrial reactivation and enhanced synaptic plasticity upon arousal. Interestingly, a single torpor and arousal sequence was also sufcient to restore contextual fear memory in an APP/PS1 mouse model of Alzheimer’s disease. Our study demonstrates that torpor in mice evokes an exceptional state of hippocampal plasticity and that naturally occurring plasticity mechanisms during torpor provide an opportunity to identify unique druggable targets for the treatment of cognitive impairment. Hibernation is a state of inactivity during which animals undergo periods of extreme hypometabolism and hypothermia to escape energetically challenging environmental conditions1. During hibernation, bouts of hypo- metabolism (i.e., torpor) typically last several days to weeks, and are alternated with short periods of rapid restoration of metabolism to normal values (i.e., interbout arousals). Smaller species may use a diferent pattern of daily hibernation, during which they deploy 6–12 h of torpor on a daily basis, and efectively experience a full hibernation cycle of torpor and arousal within several hours2,3. Previous studies in seasonal hibernators such as Syrian hamster and ground squirrel have demonstrated an exceptionally high degree of structural plasticity in the brain during hibernation, including extensive hip- pocampal dendritic retraction and changes in spine morphology and spine numbers 4. Tese changes are paral- leled by widespread hyper-phosphorylation of the microtubule-associated protein Tau, reaching levels that are pathological in humans and trigger formation of intracellular Tau aggregates as observed in Alzheimer’s disease (AD). Remarkably, arousal fully restores dendritic and synaptic integrity and reverses Tau hyper-phosphorylation and aggregation without post-hibernation damage 5–7. Tis features hibernation-based plasticity as an interest- ing mechanism from which novel treatments can be derived for neurodegenerative diseases in which neuronal plasticity is impaired. To date, mechanisms underlying torpor-associated plasticity in the brain are still poorly understood. Previ- ous studies in seasonal hibernators failed to unambiguously identify efects of torpor on normal brain function. For instance, following hibernation, retention of pre-torpor memory has been reported to be either disrupted8,9, enhanced10,11, or unafected5,12, possibly depending on the species and memory paradigm used. Given the rapid 1Department of Molecular and Cellular Neurobiology, Center for Neurogenomics and Cognitive Research, VU University, Amsterdam, The Netherlands. 2Department of Clinical Pharmacy and Pharmacology, University Medical Center Groningen, Groningen, The Netherlands. 3Department of Integrative Neurophysiology, Center for Neurogenomics and Cognitive Research, VU University, Amsterdam, The Netherlands. 4Department of Pathology and Medical Biology, University Medical Center Groningen, Groningen, The Netherlands. 5Sulfateq B.V., Groningen, The Netherlands. *email: [email protected] Scientifc Reports | (2021) 11:15486 | https://doi.org/10.1038/s41598-021-94992-x 1 Vol.:(0123456789) www.nature.com/scientificreports/ Figure 1. Torpor induction in mice. (A) Steady torpor is induced in mice using ambient temperature reduction on day 1 (Ta 21 → 19 °C) and food restriction on day 2 (1.5 g from 9:00AM till 17:00PM), followed by a fasting period of maximally 40 h. During the second night of fasting, ~ 70% of the mice enter torpor. Torpor stages were defned as pre-torpor (PT; Tb > 36 °C / VO2 > 120 mL/h); torpor late (TL; Tb < 26 °C and/or VO2 < 40 mL/h for at least 6 h); arousal early (AE; Tb of ~ 30 °C and VO2 of ~ 80 mL/h); arousal late (AL; Tb 37 °C and VO2 > 120 mL/h for 2 h). Euthermic (EU) animals were held at normal housing temperature and fed ad lib. (B) Representative core body temperature (Tb) and metabolic rate (VO2) graphs of normal torpor sensitive mice (70%; n = 21/30 mice). An average torpor bout lasted 9 ± 0.65 h (n = 12) with Tb (blue line) dropping to ~ 21 °C and VO2 (red line) dropping to ~ 20 mL/h VO2. (C) Torpor semi-sensitive mice (20%; n = 6/30 mice) did not reach stable Tb < 26 °C, and (D) no-torpor mice (10%; n = 3/30 mice) do not enter torpor afer two nights of fasting. Normal torpor graphs are shown in grey for reference. (E) Tb (°C) and VO2 (mL/h) were highly correlated (n = 12 mice; R2 = 0.74, p < 0.001). restoration of brain morphology and the extent of biochemical changes, arousal may be viewed as a period of exceptional neuroplasticity, and further insight into its underlying mechanisms may both advance our under- standing of adult brain plasticity and aid the identifcation of treatment targets for neurodegenerative diseases13,14. Te notion that laboratory mice are capable of daily torpor15–18 not only ofers the opportunity to explore arousal- associated plasticity mechanisms in wildtype mice, but also allows to test torpor-derived interventions in mouse models of disease. Here, we used fasting-induced torpor in mice to study arousal-associated structural, functional and molecular adaptations in the hippocampus and its efects on memory acquisition. We show that torpor in mice mainly acts on synaptic plasticity and mitochondria, and is associated with increased long-term potentiation (LTP) and memory performance afer arousal in wildtype and in an APP/PS1 mouse model of AD. Tus, plasticity mechanisms during torpor may provide a unique opportunity to identify novel targets for the treatment of neurodegenerative diseases. Results Fasting-induced torpor in mice. We frst established a method to induce stable torpor in mice. Torpor was induced by a reduction of ambient temperature (Ta) from 21 to 19 °C for 96 h with a limitation of food availability to 1.5 g during the frst inactive phase, followed by a maximum of 40 h of fasting (two times over- night till 12:00AM on day 4). Torpor was successfully induced when an animal’s core body temperature (Tb) reached < 26 °C for at least 6 h. Diferent torpor phases were defned (Fig. 1A): pre-torpor (PT) is the phase just before hypothermia is instigated; torpor late (TL) is at the end of the hypothermic phase (Tb < 26 °C for at least 6 h); arousal early (AE) is half way through the arousal phase when Tb reaches ~ 30 °C; and arousal late (AL) is when mice are fully aroused and have reached a T b > 36 °C for ~ 2 h. Euthermic (EU) control mice were main- Scientifc Reports | (2021) 11:15486 | https://doi.org/10.1038/s41598-021-94992-x 2 Vol:.(1234567890) www.nature.com/scientificreports/ tained on food ad libitum at a Ta of ~ 21 °C and did not enter torpor. Metabolic measurements confrmed that lowered body temperature coincides with a lowered oxygen consumption (VO2; mL/h) (Fig. 1B–D). Te torpor paradigm induced steady and profound torpor in 70% of mice (normal torpor-sensitive mice; Fig. 1B), while 20% of the mice did not reach temperatures below 26 °C for more than 6 h, yet showed intermittent Tb drops and metabolic rate reduction (torpor semi-sensitive mice; Fig. 1C), and 10% did not enter torpor at all afer two nights of fasting (no-torpor mice, used as metabolic control mice in this study; Fig. 1D). Since VO2 and Tb were always strongly correlated (Fig. 1E), only Tb was used to monitor animals in all subsequent experiments. Dendrite morphology and spine numbers are preserved during torpor in mice. CA1 hippocam- pal pyramidal neuron structure during torpor were assessed using Golgi-Cox staining. In particular, we analyzed dendrite complexity (dendritic branching) and size (total dendritic length) as well as spine numbers and spine head diameter (Fig. 1A), as these are strongly afected in seasonal hibernators4,5,19–21. Scholl analysis of CA1 pyramidal neurons (Fig. 2A) showed no diferences in dendritic branching between euthermic (EU) controls and mice at any hibernation stage (PT, TL, AE and AL) (Fig. 2B). Furthermore, neither total dendrite length, nor the number of basal or apical spines difered between groups (Fig. 2C–E). Tese data demonstrate that major structural changes such as dendritic retraction or spine loss, as observed in seasonal hibernators, are absent during or afer a single torpor bout in mice. When basal and apical spine morphology were assessed, a minor reduction in spine head diameter was observed during torpor (Fig. 2F–I) which only reached signifcance for apical spines at AE compared to EU animals (Fig. 2I). Spine head diameters were restored again in AL animals. Long-term potentiation and contextual fear memory are transiently enhanced after tor- por. Te observed changes in hippocampal spine morphology during torpor and arousal, in particular the normalization of apical spine head diameter in AL, prompted us to test whether hippocampal synaptic transmis- sion is altered during arousal. Field-stimulated long-term potentiation (LTP) was measured in the hippocampal CA1 region of AL, EU, metabolic control and 24 h post-torpor mice (Fig. 3A,B). Post-tetanic potentiation at 1 min afer tetanus stimulation and up to 30 min afer tetanus stimulation were signifcantly increased in AL compared to EU, while LTP in EU mice did not difer signifcantly from metabolic control mice (Fig.