Misaligned Feeding Impairs Memories Dawn H Loh1,5*, Shekib a Jami2,5
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1 Misaligned feeding impairs memories 2 Dawn H Loh1,5*, Shekib A Jami2,5, Richard E Flores1, Danny Truong1, Cristina A Ghiani1,3, 3 Thomas J O’Dell4,5, Christopher S Colwell1,5*. 4 5 1Department of Psychiatry and Biobehavioral Sciences, David Geffen School of Medicine, 6 University of California Los Angeles, Los Angeles, CA 90095, USA. 7 2Molecular, Cellular, and Integrative Physiology Ph.D. Program, University of California Los 8 Angeles, Los Angeles, CA 90095, USA. 9 3Department of Pathology & Laboratory Medicine, David Geffen School of Medicine, 10 University of California Los Angeles, Los Angeles, CA 90095, USA. 11 4Department of Physiology, David Geffen School of Medicine, University of California Los 12 Angeles, Los Angeles, CA 90095, USA. 13 5UCLA Integrative Center for Learning and Memory, University of California Los Angeles, Los 14 Angeles, CA 90095, USA. 15 16 *Correspondence to: [email protected], [email protected]. 1 17 Abstract 18 Robust sleep/wake rhythms are important for health and cognitive function. Unfortunately, many 19 people are living in an environment where their circadian system is challenged by inappropriate 20 meal- or work-times. Here we scheduled food access to the sleep time and examined the impact 21 on learning and memory in mice. Under these conditions, we demonstrate that the molecular 22 clock in the master pacemaker, the suprachiasmatic nucleus (SCN), is unaltered while the 23 molecular clock in the hippocampus is synchronized by the timing of food availability. This 24 chronic circadian misalignment causes reduced hippocampal long term potentiation and total 25 CREB expression. Importantly this mis-timed feeding resulted in dramatic deficits in 26 hippocampal-dependent learning and memory. Our findings suggest that the timing of meals 27 have far-reaching effects on hippocampal physiology and learned behaviour. 2 28 Introduction 29 The circadian system is a finely tuned network of central and peripheral oscillators 30 headed by a master pacemaker, the suprachiasmatic nucleus (SCN), which governs daily rhythms 31 in physiology and behaviour, including cognition. This network regulates cognitive processes (1, 32 2), and the neural circuits involved in learning and memory also exhibit circadian rhythms in 33 gene expression and synaptic plasticity (3-6). Genetic disruption of these molecular oscillations 34 has severe consequences on cognition (7-9). Environmental perturbations also have the capacity 35 to disrupt synchrony and misalign this clock network (10-19) and are problematic as many 36 people in our modern society extend their work and recreation into the night hours. 37 There has been mounting evidence that the timing of when we eat is critical for our 38 metabolic health (20, 21). At this point, timing of food intake is well-established to have a major 39 impact on the phase of the molecular oscillations in peripheral organs such as the liver and 40 pancreas (22, 23). Mis-timed meals during the sleep phase accelerates weight gain compared 41 with animals fed during their wake phase (24, 25), whereas wake-phase feeding has a protective 42 effect against the cardiac and metabolic dysfunction caused by high fat diets (26, 27). Similar 43 disruptive effects are seen in humans, where misaligned mealtimes produce cardiac and 44 metabolic deficits, leading to a pre-diabetic state (28). We thus became interested in the 45 possibility that these ill consequences of eating at inappropriate phases of the daily cycle may 46 also be maladaptive for cognitive function. In this study, we sought to determine the effects of 47 chronic but stable misalignment of the circadian network by scheduling access to food at an 48 inappropriate phase of the daily cycle. We demonstrate that this simple manipulation has far- 49 reaching consequences for learning and memory. 3 50 Results 51 Misaligned feeding alters diurnal rhythms of activity and sleep. 52 Mice were allotted a six hour window in which food was made available either during the 53 middle of their active (aligned) or inactive (misaligned) phase (Figure 1). Mice adapted to the 54 feeding protocol within 6 days (P = 0.9, Figure 2A) and there were no significant differences in 55 body weight between the two groups at the time of testing (P = 0.5, Figure 2B). Daytime activity 56 was increased in mice subjected to misaligned feeding (P < 0.001; Figure 3A, B), and the 57 strength of daily rhythms of activity was reduced by misaligned feeding (P = 0.003; Figure 3C). 58 Similarly, the temporal pattern of sleep was altered by misaligned feeding (Figure 3D). 59 Immobility-defined video monitoring of sleep behaviour of misaligned mice showed decreased 60 time spent asleep during the day (P < 0.001) and a corresponding increase in sleep during the 61 night (P < 0.001; Figure 3E). Misaligned mice no longer exhibit a day vs night difference in 62 sleep, sleeping equally during the day and night (P = 0.5). Crucially, the total time spent asleep 63 over a 24 hr day was not reduced by misaligned feeding (P = 0.2). The change in temporal 64 pattern of sleep quantity is also reflected in sleep fragmentation, where misaligned mice exhibit a 65 greater number of sleep bouts (P < 0.05) with a corresponding decrease in average sleep bout 66 duration (P < 0.001) during the day compared to aligned mice (Figure3-figure supplement 1). 67 This increased day-time sleep fragmentation is compensated by fewer (P < 0.05) and longer (P < 68 0.001) night sleep bouts in the misaligned animals, resulting in no significant change in the total 69 number (P = 0.9) and average duration (P = 0.6) of sleep bouts over a 24 hr period. 70 Misaligned feeding alters phase of hippocampus without shifting the SCN. 4 71 Using ex vivo organotypic cultures of explants from PER2::LUC reporter mice subjected 72 to aligned or misaligned feeding, we confirmed that daytime feeding shifts the phase of the liver 73 oscillator (P < 0.001) without altering the phase of the SCN oscillator (P = 0.07; Figure 4A,C,D 74 ). Importantly, we demonstrate that daytime feeding significantly misaligns the hippocampal 75 oscillator by 14.1 hr (P < 0.001; Figure 4B, D), with small but significant effects on the intrinsic 76 properties of the oscillator, including period (P = 0.05; Figure 4E) and damping rate (P = 0.02; 77 Figure4F). 78 Misaligned feeding blunts total CREB expression and reduces hippocampal long term 79 potentiation. 80 We measured expression of phosphorylated CREB (pCREB) and total CREB (tCREB) in 81 the hippocampus of aligned and misaligned mice at 6 hr intervals through a 24 hr day. pCREB 82 protein levels were reduced in the misaligned animals at each time point, although significant 83 differences were not detected (Figure 5A, B). Strikingly, expression of tCREB was significantly 84 reduced by misaligned feeding (P < 0.01; Figure 5A, C, D), with the strongest effects in the day 85 (post hoc P < 0.05 for ZT 2, 8, and 20). This decrease in tCREB levels was uniformly observed 86 throughout the hippocampus (Figure 5D). 87 Long term potentiation (LTP) responses of the dorsal hippocampus were measured 88 during the day from mice subjected to aligned or misaligned feeding. LTP was significantly 89 impaired in the misaligned group (P < 0.05; Figure 5E), indicating significant deficits in 90 synaptic plasticity in the misaligned hippocampus. This decrease in LTP is specific to the 91 misaligned feeding treatment and not due to an LTP-boosting effect by aligned feeding (Figure 92 5-figure supplement 2). To determine the impact of misaligned feeding on presynaptic 93 neurotransmitter release probability, paired pulse facilitation (PPF) experiments were performed 5 94 at 25, 50, 100 and 250 msec intervals. There were no significant differences between the aligned 95 and misaligned PPF ratios (P = 0.5; Figure 5F), and EPSP profiles were similar. 96 Misaligned feeding affects hippocampal-dependent learning and memory. 97 To test our hypothesis that misaligning the hippocampal oscillator from the SCN 98 oscillator is detrimental to learning and memory, we subjected aligned and misaligned mice to 99 hippocampal-dependent contextual fear conditioning. Mice were trained to associate a specific 100 novel context to a fearful stimulus in the form of a mild shock. Both aligned and misaligned mice 101 acquired freezing behaviour during the training trial (Figure 6-figure supplement 1). When 102 tested 24 hr later by replacing the mice in the same context, the misaligned mice exhibited a 103 significant reduction in fear-conditioned behaviour (ZT 2, P < 0.0001; Figure 6A, left), 104 indicating that circadian misalignment affects long term memory. Performance on the fear 105 conditioning test is dependent on time of day and performance peaks in the early day (2, 3, 14). 106 To test for the possibility that misaligned mice have an inverted peak performance time, we 107 trained and tested a separate cohort of mice at the opposite time of day (night, ZT 14). Both 108 aligned and misaligned groups acquired freezing behaviour (Figure 6- figure supplement 1). 109 Two way comparisons revealed a time of day effect on recall (P < 0.001) and an interaction 110 between time of day and treatment (P < 0.001). Recall was significantly reduced in the aligned 111 mice compared to the daytime tests (post hoc P < 0.001), but did not significantly change with 112 time of day in misaligned mice (post hoc P = 0.3). Night-tested aligned and misaligned mice 113 exhibited equally poor recall (post hoc P = 0.4; Figure 6A, right), ruling out the possibility that 114 the misaligned mice have an altered peak phase of cognition. 115 We applied the novel object recognition test to examine cognition that peaks during the 116 night in nocturnal rodents (13).