Orexinergic modulation of chronic jet lag-induced deficits in mouse cognitive flexibility

Orexinergic modulation of chronic jet lag-induced deficits in mouse cognitive flexibility | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Orexinergic modulation of chronic jet lag-induced deficits in mouse cognitive flexibility Markus Fendt, Julius Duske, Nicole D'Souza, Dana Mayer, Daniela Dieterich This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4713362/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 30 Oct, 2024 Read the published version in Neuropsychopharmacology → Version 1 posted You are reading this latest preprint version Abstract Cognitive flexibility and working memory are important executive functions mediated by the prefrontal cortex and can be impaired by circadian rhythm disturbances such as chronic jet lag (CJL) or shift work. In the present study, we used mice to investigate whether (1) simulated CJL impairs cognitive flexibility, (2) the orexin system is involved in such impairment, and (3) nasal administration of orexin A is able to reverse CJL-induced deficits in cognitive flexibility and working memory. Mice were exposed to either standard light-dark conditions or simulated CJL consisting of series of advance time shifts. Experiment (1) investigated the effects of a mild CJL protocol on cognitive flexibility using the attentional set shifting task. Experiment (2) used a stronger CJL protocol and examined CJL effects on the orexin system utilizing c-Fos and orexin immunohistochemistry. Experiment (3) tested whether nasal orexin application can rescue CJL-induced deficits in cognitive flexibility and working memory, the latter by measuring spontaneous alternation in the Y-maze. The present data show that CJL (1) impairs cognitive flexibility and (2) reduces activity of orexin neurons in the lateral hypothalamus. (3) Nasal administration of orexin A rescued CJL-induced deficits in working memory and cognitive flexibility. These findings suggest that executive functions impairments by circadian rhythm disturbances such as CJL are caused by dysregulation of orexinergic input to the prefrontal cortex. Compensation of decreased orexinergic input by nasal administration of orexin A could be a potential therapy for CJL- or shift work-induced human deficits in executive functions. Biological sciences/Neuroscience/Circadian rhythms and sleep/Hypocretin Biological sciences/Neuroscience/Learning and memory/Working memory Biological sciences/Neuroscience/Learning and memory/Cortex Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 INTRODUCTION Circadian rhythm sleep disorder (CRSD) is a disorder that affects the timing of sleep. It is caused by a mismatch of the endogenous clock and externally set stimuli such as light or dark outdoors, physical activity, or meal times. CRSD is either intrinsic such as advanced or delayed sleep-wake disorder or extrinsic CRSD including shift work disease or jet lag, which is more common. For both variants, the main clinical manifestations are insomnia and excessive daytime sleepiness [ 1 ]. Of interest, cognitive impairments and associated changes in the brain can be observed in CRSD. For example, chronic jet lag (CJL) in flight attendants has been associated with cognitive deficits, particularly in working memory, as well as atrophy of the temporal lobe [ 2 , 3 ]. In addition, impairments in attention and decision making due to chronic sleep deprivation have been demonstrated [ 4 ]. Although chronotherapy, photic treatment, and cognitive-behavioural therapy exist, pharmacological treatment for CRSD is mainly limited to melatonin [ 5 ]. Cognitive impairments have been also found in animal models of CRSD. In laboratory rodents, simulated CJL, especially when phase shortening simulates eastward travel, has been shown to reduce hippocampal neurogenesis, which was associated with impaired spatial learning [ 6 ]. However, there is poor knowledge about the effects of simulated CJL on executive functions in laboratory rodents. Executive functions include working memory, inhibitory control, attention, and cognitive flexibility [ 7 ] and can be measured in mice using specific behavioural paradigms [ 8 ]. A well-established paradigm to measure cognitive flexibility is the attentional set shifting task (ASST) [ 9 , 10 ]. The ASST is based on discrimination learning and includes different types of transfers such as reversals, intra- and extradimensional shifts. Working memory can be measured by spontaneous alternations in the Y-maze [ 11 , 12 ]. The aim of the present study is to investigate whether the orexin system in mice is involved in cognitive deficits induced by CRSD. The orexin system consists of neurons in the lateral hypothalamus that synthesize the neuropeptides orexin A and B and their brain-wide projections [ 13 – 15 ]. This system is involved in a variety of regulatory mechanisms including stress responses, emotions, motivation, eating behaviour and sleep-wake cycle, which is the major focus of this study [ 16 – 18 ]. The orexin system promotes wakefulness by stimulating the ascending reticular activating system [ 19 ]. Studies first in animal models [ 20 – 22 ] and later in humans [ 23 , 24 ] highlighted the importance of the orexin system in narcolepsy. This disease with symptoms such as excessive daytime sleepiness and sleep attacks is a consequence of loss of orexin neurons or disturbed orexin receptor signalling. This suggests that orexin or orexin receptor agonists could be a promising pharmacological treatment option for CRSD (as well as for narcolepsy). Such a treatment would not only address symptoms associated with disturbed sleep-wake cycle but potentially also cognitive symptoms because the orexin system also plays a role in cognition. In laboratory animals, orexin improves spatial learning and memory, rescues impaired discrimination learning, and improves executive functions such as cognitive flexibility or working memory. Thus, in the present study, we exposed male and female mice to simulated CJL, an animal model of CRSD. Then, we investigated whether (1) simulated CJL impairs cognitive flexibility, (2) the orexin system is involved in such an impairment, and if so, whether (3) nasal administration of orexin A is able to reverse CJL-induced deficits in cognitive flexibility and working memory. Our hypothesis is that (1) simulated CJL induces impairments in cognitive flexibility and working memory, (2) these impairments are associated with changes in the orexin system, and (3) nasal administration of orexin rescues CJL-induced cognitive deficits. MATERIALS AND METHODS Animals Female and male C57BL/6J mice were used in the experiments. Mice were housed in groups of up to 8 mice/cage under controlled conditions (humidity: 55 ± 10%, temperature: 22 ± 2°C) and – depending on the group – either in a standard 12 h light/12 h dark cycle (light on: 6:00 am) or in a CJL cycle (see below). Mice were between 12 and 15 weeks of age during testing. Food and water were provided ad libitum until 1 week before and during ASST, when food was restricted (approximately 2.5 g/mice/day) to maintain 90–95% of the mice’s basal body weight. All experiments were performed during the light period and were complied with International Guidelines for the Care and Use of Animals for Experimental Procedures (2010/63/EU) with confirmed approval from the local authorities (Landesverwaltungsamt Sachsen-Anhalt, Az. 42502-2-1618 UniMD). Simulation of Chronic Jet Lag (CJL) After an adaptation period of at least 1 week, CJL was simulated for the experimental groups by phase advancing of the light-dark cycle, while the control groups stayed in the standard light-dark cycle (ST). In experiment 1, the light-dark conditions were shifted by 6 hours every seventh day (mild CJL protocol; Fig. 1 A). Behavioural tests were performed after 4 or 8 time shifts, respectively (i.e., in week 4 or 8 after starting CJL). In experiments 2 and 3, the light-dark conditions were shifted by 8 hours every fifth day (strong CJL protocol; Fig. 1 B). Behavioural tests or immunohistochemistry, respectively, were performed after 6 time shifts, i.e., in week 4 after starting CJL. Behavioural experiments Attentional Set Shifting Task (ASST). The ASST was performed as previously described [ 25 – 27 ]. Briefly, custom-made boxes were used consisting of a waiting and a choice area that was divided into two compartments separated by a transparent wall, with sliding doors between the waiting area and the choice area compartments. Mice were provided with water and familiarized with bowls and rewards before testing. During habituation, mice were handled daily for a week, subjected to food restriction, and introduced to the setup and rewards. Training involved digging in bowls filled with bedding material to find rewards. For experiment 3, mice also underwent nasal saline application habituation. The testing phase began immediately after habituation, lasting 4 days. Bowls were presented with visual-tactile cues (digging media) or olfactory cues (odorants), with only one containing a reward. Mice had to associate the correct cue with the reward, with bowl positions randomized. Trials started with a 30-second wait period, then sliding doors were opened. Correct choices allowed mice to consume the reward, while incorrect choices were indicated by removing the empty bowl. After six consecutive correct decisions, a phase was considered to be completed and the next phase started. The ASST had several phases: Day 1; Simple Discrimination (SD): Mice chose between two exemplars of one stimulus dimension (odor or digging medium). Day 2; Compound Discrimination (CD): An additional, irrelevant stimulus dimension was introduced, followed by a reversal phase (Rev1) where contingencies changed. Day 3; Intradimensional Shift (IDS): New exemplars of both dimensions were presented, with the relevant dimension of the previous phases predicting the reward, followed by another reversal (Rev2). Day 4; Extradimensional Shift (EDS): New exemplars of both dimensions were used, with the previously irrelevant dimension now predicting the reward, followed by a final reversal phase (Rev3). Spontaneous alternation in the Y-maze. The custom-made Y-maze consisted of 3 arms, 120 degrees apart, 37 cm long, 10 cm wide, and 13 cm high. The walls were transparent, the floor of grey PVC. The experiment started with placing the mice in one arm of the maze, facing to the centre. Then, the mouse could freely explore the Y-maze for 5 min. The experimenter manually scored arm entries of the mice, defined by entering an arm with all four paws. Immunohistochemistry Mice were deeply anesthetized with i.p. administration of ketamine hydrochloride (100 mg/kg), xylazine hydrochloride (20 mg/kg), and acepromazine maleate (3 mg/kg) and transcardially perfused, first with saline (2 min), then with Zamboni solution (4% paraformaldehyde, 0.2% picric acid in PBS; 10 min). The brain was removed, post-fixed overnight, and then stored in 30% sucrose. Using a cryostat, coronal 40 µm sections were cut. Double-immunofluorescence staining was performed on every third section of the regions of interest (lateral hypothalamus, prefrontal cortex). For c-Fos staining, a rabbit anti-c-Fos antibody (1:3000, Synaptic Systems GmbH, Göttingen, Germany), a biotinylated goat anti-rabbit IgG antibody (1:1000, Vector Laboratories, Newark, USA), and an ABC kit (Vector Laboratories, Newark, USA) were used. For orexin A staining, a mouse anti-orexin A antibody (1:3000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), Cy 3-conjugated goat anti-mouse IgG antibody (1:400, Jackson ImmunoResearch, Laboratories, Inc., Ely, Cambridgeshire, United Kingdom), and Streptavidin Cy-2 (1:500, Life Technologies Corporation, Eugene, USA) were used. The stained slides were mounted on microscope slides and coverslips were added. Photomicrographs were taken using a Zeiss microscope (Laserscanningmicroscope 710, Carl Zeiss AG, Oberkochen, Germany) with respective software. For quantitative analysis, pre-defined frames were bilaterally placed in the regions of interest. In the lateral hypothalamus, c-Fos-positive neurons, orexin A-positive neurons and double-labelled neurons were manually counted by the blinded experimenter. c-Fos-positive neurons of the different subregions of the PFC were counted with a custom-made software. Drugs In experiment 3, mice were treated with either 10 µl vehicle (saline) or 10 µl orexin A solution (0.1 mM; Tocris Bioscience, Bristol, UK). Concentration and volume was based on published data [ 26 , 28 ]. Experimental procedure Experiment 1 : Mice were kept in the standard light/dark cycle (ST) or in a mild CJL (see Fig. 1 A). After 4 or 8 time shifts (ie., in cycle 4 or 8), respectively, mice were submitted to the ASST. Group sizes: ST: 5 females, 6 males; 1 month CJL: 5 females, 6 males; 2 months CJL: 5 females, 6 males. Experiment 2 : Mice were kept in ST or strong CJL for 6 cycles (see Fig. 1 B). Then, at the time when behavioural tests in experiment 1 and 3 began (9:00–10:00 am), the mice were transcardially perfused. The brain was then removed and processed for immunohistochemistry as described. Group sizes: ST: 7 females, 7 males; CJL: 7 females, 6 males. Experiment 3 : Mice were kept in ST or strong CJL (see Fig. 1 B). After 6 time shifts (ie., in cycle 6), mice were submitted to the Y-maze test and 5 min later to ASST. Thirty minutes before the start of the behavioural experiments, they were treated with nasal administrations of saline (SL) or orexin A (OxA). Of note, some mice were tested only in the Y-maze but not in the ASST (since throughput is limited in the latter but not in the former). However, all mice subjected to ASST were previously tested in the Y-maze. Group sizes (Y-maze/ASST): ST/Veh: 9/6 females, 9/6 males; CJL/Veh: 7/6 females, 9/6 males; ST/OxA: 9/6 females, 8/6 males; CJL/OxA: 7/6 females, 10/6 males. Statistical analysis Data analysis was performed using SYSTAT 13 (SPSS Inc.) and Prism 7.0 (GraphPad Software Inc., La Jolla, USA). Normal distribution of the data was verified using the Shapiro-Wilk normality test. Analysis of variance (ANOVA) was performed, followed by respective post-hoc multiple comparisons (two-linear step-up procedure of Benjamini, Krieger and Yekutieli). The significance level was set at p < 0.05. Results Experiment 1 A multi-factorial ANOVA using sex (female/male) and cycle condition (ST, 1 month CJL, 2 months CJL) as between-subject factors and ASST phase (SD, CD, Rev1, IDS, Rev2, EDS, Rev3) as within-subject factor was used to analyse ASST performance of the mice. Since this multifactorial ANOVA showed neither main effects of sex ( F s 0.21) nor interactions of sex with other factors ( F s 0.13) for trials to criterion (Fig. 2 A, B), errors to criterion (Fig. 2 C, D), and the errors types (Fig. 2 E, F), the data from both sexes were pooled for further analysis. Regarding trials and error to criterion, the analysis of the pooled data revealed main effects of cycle conditions ( F s > 6.84, p s 5.49, p s < 0.004) but no interaction between these two factors (Fs 0.45). Post-hoc comparisons showed that 1 month CJL did not induce a performance deficit ( q s 0.14) while 2 months CJL significantly impaired ASST performance ( q s > 3.68, p s 2.16, p s < 0.05; Fig. 2 B, D). Analysis of the error types in the reversal phases revealed a trend for an interaction for ASST phase and CJL on perseverative errors ( F (4,60) = 2.45, p = 0.055; post-hoc comparison ST vs. 1 month CJL in Rev1: t = 2.69, p = 0.02), as well as a main effect of phase ( F (4,60) = 3.64, p = 0.04). There were no significant effects on regressive errors ( F s 0.08). Separate analyses of female and male mice further revealed that effects of CJL on ASST performance were more pronounced in female than in male mice ( Fig. S1 , S2 ). Experiment 2 The number of c-Fos- and OxA-positive neurons, as well as the percentage of double-positive neurons (normalized to the number of OxA-positive neurons) in the lateral hypothalamus was analysed with a multi-factorial ANOVA using sex (female/male) and cycle condition (ST, CJL) as between-subject factors. Figure 3 A shows examples of the immunohistochemical staining. Of note, a strong CJL protocol was used in this experiment. Both, the number of OxA- and c-Fos-positive neurons were not affected by sex or cycle condition ( F s 0.57; Fig. 3 B). While these two factors did not interact regarding the number of OxA-positive neurons ( F (1,23) = 0.62, p = 0.44), there was a trend for such an interaction regarding the c-Fos-positive neurons ( F (1,23) = 2.95, p = 0.099). Of note, this interaction between sex and cycle condition was significant in the analysis of the percentage of double-positive neurons ( F (1,23) = 7.40, p = 0.01), whereas there were no main effects of sex and cycle condition ( F s 0.26). Post-hoc comparison revealed a significant decrease of double-positive neurons after CJL in female mice (t = 2.79, p = 0.01) but not in male mice ( t = 1.09, p = 0.29). In addition, c-Fos expression (Fig. 3 C, D) was analysed in the regions of the PFC associated with cognitive flexibility (ventromedial PFC, cingulate cortex, lateral/ventral orbitofrontal cortex (OFC) and medial OFC). There were no main effects of sex and cycle condition in the different PFC regions ( F s 0.12), expect a main effect of cycle condition in the medial OFC ( F (1,23) = 4.63, p = 0.04). Furthermore, there was again an interaction of sex and cycle condition when the number of c-Fos-positive neurons in all these PFC regions was averaged ( F (1,24) = 4.24, p = 0.05; Fig. 3 D). Post-hoc comparisons showed a decreased number of c-Fos-positive neurons in female mice after CJL exposure in both the medial OFC and the mean of all PFC regions ( t s > 2.49, p s < 0.04), while there were no effects on male mice ( t s 0.80). Of note, orexin-positive fibres were detected in all PFC regions (Fig. 3 C). Experiment 3 Spontaneous alternation in the Y-maze Figure 4 depicts percent alternation and total arm visits of the mice in the Y-maze. Data were analysed with a multi-factorial ANOVA using sex (female/male), cycle condition (ST/CJL), and treatment (Veh, OxA) as between-subject factors. Since no main effect of sex or interactions of sex with the other factors were found (F 0.24), the data of female and male mice were pooled. The ANOVA of the pooled data revealed a main effect of cycle condition ( F (1,64) = 11.00; p = 0.002) and an interaction of cycle condition with treatment ( F (1,64) = 9.41; p = 0.003; Fig. 4 A). Post hoc comparisons showed that CJL impaired the percentage of alternations in vehicle-treated mice ( t = 4.51, p < 0.0001) but not in OxA-treated mice ( t = 0.18, p = 0.86). CJL-exposed OxA-treated mice had significantly more alternations than CJL-exposed vehicle-treated mice ( t = 3.37, p = 0.001). Neither CJL nor OxA treatment had any effects on the total number of arm entries in the Y-maze ( F s 0.28; Fig. 4 B). Separate analyses of female and male mice further are shown in the Supplements ( Fig. S3, S4 ). The CJL effect was very similar in female and male mice, while the OxA effect was more robust in females. ASST Performance The performance of the mice in the ASST were analysed with multi-factorial ANOVAs using sex (female/male), cycle condition (ST/CJL), and treatment (Veh/OxA) as between-subject factors and ASST phase (SD/CD/Rev1/IDS/Rev2/EDS/Rev3) as within-subject factor. Six mice failed to complete all ASST phases and were excluded from the final analyses (CJL/OxA: n = 2 males; ST/SL: n = 2 females; ST/OxA: n = 1 female, 1 male). A multi-factorial ANOVA revealed no main effects of sex (F 0.21) or interactions of sex with other factors (F 0.38) for trials to criterion ( Fig. 6A, B ), errors to criterion ( Fig. 6C, D ), and the errors types ( Fig. 6E, F ). Therefore, data from both sexes were pooled for further analysis, but data for female and male mice are also separately shown in the figures. Regarding the trials and errors to criterion, no main effects of cycle conditions (Fs 0.05) and treatment (Fs 0.16) were found. However, there was an interaction between cycle condition and treatment (Fs > 4.25, ps = 0.045). Post-hoc comparisons showed that CJL increased the number of trials and errors in vehicle-treated mice ( t s > 2.71, p s < 0.001) but not in OxA-treated mice ( t s 0.66). In addition, there was a significant reduction of the number of trials in CJL-exposed mice after nasal OxA administration ( t s > 2.34, p s 3.00, p s 2.84, p s < 0.01), as well as of orexin A on the CJL-induced increase of perseverative errors ( t = 2.69, p = 0.02). Separate analyses of female and male mice are further shown in the supplementary material ( Fig. S5, S6 ). The described effects were very similar in female and male mice. DISCUSSION The aim of the present study was to investigate both the role and the potential of the orexin system in the presumably impairing effects of simulated chronic jet lag on executive functions in laboratory mice. The present data demonstrate that simulated chronic jet lag robustly impairs cognitive flexibility. In addition, simulated chronic jet lag decreased the activity of the orexin system and different subregions of the prefrontal cortex, a brain area critical for cognitive flexibility. Based on these findings, an interventional experiment was performed which demonstrated that nasal administration of orexin rescued impaired cognitive flexibility and working memory after simulated chronic jet lag. Of note, the observed changes in the orexin system and the prefrontal cortex were only observed in female mice while there were no sex differences in the behavioural experiments. However, the behavioural changes were usually more pronounced in female mice. In the present study, CJL was simulated to model CRSD. Specifically, CRSD caused by jet lag and/or shift work was modelled [ 29 ]. Although occasional jet lag after long distance travel is usually problem that resolves itself, chronic jet lag in humans who travel frequently can cause a range of symptoms, including cognitive impairments [ 2 , 3 ]. Additionally, between 10 and 15% of all workers are affected by shift work [ 29 , 30 ]. Particularly, around one third of shift workers suffer from shift work disorder [ 31 ], which is associated with cognitive impairments [ 32 – 34 ] and an increased risk of dementia [ 35 ]. Many of the described symptoms of CRSD are also observed in laboratory mice after simulated chronic jet lag. Decreased motivation, increased anxiety, depression-like behaviours, reduced recognition memory and impaired spatial learning [ 36 – 39 ], but also molecular changes in different brain regions [ 40 , 41 ] and even increased mortality [ 42 ] have been observed. While the detrimental effects of chronic jet lag on executive functions such as cognitive flexibility or working memory have been demonstrated in humans [ 32 , 34 ], to our knowledge this has never been investigated in laboratory mice. The present study has shown that both cognitive flexibility and working memory are impaired in mice after simulated chronic jet lag. Our study demonstrates this in two independent experiments with different protocols of simulated chronic jet lag conducted by two different experimenters. A week protocol of chronic jet lag (6 hours shift every seventh day) had no effects on cognitive flexibility after 1 months (4 shifts) but induced a significant impairment after 2 months (8 shifts). Therefore, a more severe protocol of chronic jet lag (8 hours shift every fifth day) was used for the next experiments. With this protocol, impaired cognitive flexibility and working memory was observed after 4 weeks (6 shifts). Together, these results strongly support the findings in humans mentioned above and indicate that simulated chronic jet lag in mice can be used to study potential causes and treatments of CRSD. The orexin system is crucial for the regulation of the sleep/wake cycle [ 43 , 44 ]; hence, it is reasonable to assume that this system is involved in CRSD [ 45 , 46 ]. However, to the best of our knowledge, no published studies have shown that CRSD is associated with changes in the orexin system. The present study now shows decreased activity of the orexin system after simulated CJL, a mouse model of CRSD. Surprisingly, the reduced activity was only observed in female mice, although the behavioural effects of CRSD were very similar in both sexes. The same is true for neural activity in the prefrontal cortex, a crucial brain region for executive functions that is innervated by the orexin system [ 47 ]. The absence of changes in male mice is unclear; however, it is possible that changes, if any, can simply not be detected with the c-Fos approach. An attempt was made to measure orexin levels in the PFC in the present study, however, the ELISA used also measured orexin levels in orexin-deficient mice, so we discontinued these measurements. Therefore, future experiments should include more sensitive methods to measure the activity of the orexin system and the orexin levels the PFC. The observed decrease in neuronal activity in the PFC might be caused by the impaired activity of the orexin system and could further be the cause for the behavioural deficits in the cognitive flexibility and working memory tests. If this is the case, a pharmacological intervention that replaces the missing orexin in the PFC should rescue the observed behavioural deficits. A relatively simple approach to administer orexin to the brain is via the nasal route [ 48 ], which has been shown to increase orexin levels in the brain [ 49 , 50 ] and to activate the PFC [ 51 ]. Indeed, after nasal administration of orexin, we observed a complete rescue of impaired cognitive flexibility and working memory after simulated CJL. This rescue effect of nasal orexin was also observed in male mice, although we could not detect impaired activity in the orexin system and the PFC in them. Overall, the present findings show that the orexin system is involved in the cognitive impairments observed in a mouse model of CRSD and that nasal orexin administration rescues these impairments. Although there were some sex differences – as frequently observed in orexin research [ 17 , 26 , 52 ] – in the effect of simulated CJL on neural activity, nasal orexin administration had similar effects in female and male mice. These data suggest that nasal orexin administration is a potential treatment option for CRSD. Intranasal orexin administration has been tested in narcoleptic patients who have very low or absent orexin levels in the brain [ 24 ] and could alleviate several symptoms in these patients such as olfactory dysfunction, attentional deficits and sleep abnormalities without significant side effects [ 53 – 55 ]. Therefore, nasal orexin administration should be tested in CRSD patients. Declarations ACKNOWLEDGEMENTS We thank Uwe Disterheft for the construction of the ASST setups, Evelyn Kahl for technical assistance and Kathrin Freke for animal care. AUTHOR CONTRIBUTIONS JD, ND, DCD and MF conceived and designed the study, JD, ND and DM conducted the experiments, JD, ND, DM and MF analysed the data, DCD and MF gathered the resources for the study, and JD and MF wrote the manuscript with contributions from all co-authors. FUNDING This study was supported by a grant (MF, DCD) and a scholarship (JD) of the Deutsche Forschungsgemeinschaft (SFB1436/A01; project ID 42589994). COMPETING INTERESTS The authors declare no competing interests. ADDITIONAL INFORMATION The online version contains supplementary material available at … References Pavlova M. Circadian Rhythm Sleep-Wake Disorders. Continuum (Minneap Minn). 2017;23:1051–63. Cho K. Chronic 'jet lag' produces temporal lobe atrophy and spatial cognitive deficits. Nat Neurosci. 2001;4:567–8. Cho K, Ennaceur A, Cole JC, Suh CK. Chronic jet lag produces cognitive deficits. J Neurosci. 2000;20:RC66. Alhola P, Polo-Kantola P. Sleep deprivation: Impact on cognitive performance. Neuropsychiatr Dis Treat. 2007;3:553–67. Sun S-Y, Chen G-H. Treatment of Circadian Rhythm Sleep–Wake Disorders. Curr Neuropharmacol. 2022;20:1022–34. Kott J, Leach G, Yan L. Direction-dependent effects of chronic "jet-lag" on hippocampal neurogenesis. Neurosci Lett. 2012;515:177–80. 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Genes Brain Behav. 2021;20:e12707. Durairaja A, Pandey S, Kahl E, Fendt M. Nasal administration of orexin A partially rescues dizocilpine-induced cognitive impairments in female C57BL/6 J mice. Behav Brain Res. 2023;450:114491. Durairaja A, Steinecke C-S, Fendt M. Intracerebroventricular infusion of the selective orexin 1 receptor antagonist SB-334867 impairs cognitive flexibility in a sex-dependent manner. Behav Brain Res. 2022;424:113791. Calva CB, Fayyaz H, Fadel JR. Effects of Intranasal Orexin-A (Hypocretin-1) Administration on Neuronal Activation, Neurochemistry, and Attention in Aged Rats. Front Aging Neurosci. 2019;11:362. Kim MJ, Lee JH, Duffy JF. Circadian Rhythm Sleep Disorders. J Clin Outcomes Manag. 2013;20:513–28. He M, Zhou W, Liu K, Wang X, Liu C, Shi F, et al. The prevalence of male rotating shift work correlates with reduced total fertility rate: an ecological study of 54,734 reproductive-aged males in 35 European countries between 2000 and 2015. Chronobiol Int. 2021;38:1072–82. Pallesen S, Bjorvatn B, Waage S, Harris A, Sagoe D. Prevalence of Shift Work Disorder: A Systematic Review and Meta-Analysis. Front Psychol. 2021;12:638252. Cheng P, Tallent G, Bender TJ, Tran KM, Drake CL. Shift Work and Cognitive Flexibility: Decomposing Task Performance. J Biol Rhythms. 2017;32:143–53. Marquié J-C, Tucker P, Folkard S, Gentil C, Ansiau D. Chronic effects of shift work on cognition: findings from the VISAT longitudinal study. Occup Environ Med. 2015;72:258–64. Özdemir PG, Selvi Y, Özkol H, Aydın A, Tülüce Y, Boysan M, et al. The influence of shift work on cognitive functions and oxidative stress. Psychiatry Res. 2013;210:1219–25. Lee K-W, Yang C-C, Chen C-H, Hung C-H, Chuang H-Y. Shift work is significantly and positively associated with dementia: A meta-analysis study. Front Public Health. 2023;11:998464. Acosta J, Crespo MT, Plano SA, Golombek DA, Chiesa JJ, Agostino PV. Chronic jet lag reduces motivation and affects other mood-related behaviors in male mice. Front Physiol. 2023;14:1225134. Horsey EA, Maletta T, Turner H, Cole C, Lehmann H, Fournier NM. Chronic Jet Lag Simulation Decreases Hippocampal Neurogenesis and Enhances Depressive Behaviors and Cognitive Deficits in Adult Male Rats. Front Behav Neurosci. 2019;13:272. Liu JA, Bumgarner JR, Walker WH, Meléndez-Fernández OH, Walton JC, DeVries AC, et al. Chronic phase advances reduces recognition memory and increases vascular cognitive dementia-like impairments in aged mice. Sci Rep. 2024;14:7760. Iggena D, Winter Y, Steiner B. Melatonin restores hippocampal neural precursor cell proliferation and prevents cognitive deficits induced by jet lag simulation in adult mice. J Pineal Res. 2017;62. Gao Q, Khan S, Zhang L. Brain activity and transcriptional profiling in mice under chronic jet lag. Sci Data. 2020;7:361. Siddique R, Awan FM, Nabi G, Khan S, Xue M. Chronic jet lag-like conditions dysregulate molecular profiles of neurological disorders in nucleus accumbens and prefrontal cortex. Front Neuroinform. 2022;16:1031448. Davidson AJ, Sellix MT, Daniel J, Yamazaki S, Menaker M, Block GD. Chronic jet-lag increases mortality in aged mice. Curr Biol. 2006;16:R914-6. Sakurai T. The neural circuit of orexin (hypocretin): maintaining sleep and wakefulness. Nat Rev Neurosci. 2007;8:171–81. Pizza F, Barateau L, Dauvilliers Y, Plazzi G. The orexin story, sleep and sleep disturbances. J Sleep Res. 2022;31:e13665. Tsuneki H, Wada T, Sasaoka T. Chronopathophysiological implications of orexin in sleep disturbances and lifestyle-related disorders. Pharmacol Ther. 2018;186:25–44. Umetsu M. Circadian Rhythm Sleep Disorder in Alzheimer’s Disease-A consideration in relation with the Neuropathological and Neuroendocrinal alternation. Brain Disord Ther. 2014;03. Jin J, Chen Q, Qiao Q, Yang L, Xiong J, Xia J, et al. Orexin neurons in the lateral hypothalamus project to the medial prefrontal cortex with a rostro-caudal gradient. Neurosci Lett. 2016;621:9–14. Calva CB, Fadel JR. Intranasal administration of orexin peptides: Mechanisms and therapeutic potential for age-related cognitive dysfunction. Brain Res. 2020;1731:145921. Dhuria SV, Hanson LR, Frey WH. Intranasal drug targeting of hypocretin-1 (orexin-A) to the central nervous system. J Pharm Sci. 2009;98:2501–15. van de Bittner GC, van de Bittner KC, Wey H-Y, Rowe W, Dharanipragada R, Ying X, et al. Positron Emission Tomography Assessment of the Intranasal Delivery Route for Orexin A. ACS Chem Neurosci. 2018;9:358–68. Deadwyler SA, Porrino L, Siegel JM, Hampson RE. Systemic and nasal delivery of orexin-A (Hypocretin-1) reduces the effects of sleep deprivation on cognitive performance in nonhuman primates. J Neurosci. 2007;27:14239–47. Funabashi T, Hagiwara H, Mogi K, Mitsushima D, Shinohara K, Kimura F. Sex differences in the responses of orexin neurons in the lateral hypothalamic area and feeding behavior to fasting. Neurosci Lett. 2009;463:31–4. Weinhold SL, Seeck-Hirschner M, Nowak A, Hallschmid M, Göder R, Baier PC. The effect of intranasal orexin-A (hypocretin-1) on sleep, wakefulness and attention in narcolepsy with cataplexy. Behav Brain Res. 2014;262:8–13. Baier PC, Weinhold SL, Huth V, Gottwald B, Ferstl R, Hinze-Selch D. Olfactory dysfunction in patients with narcolepsy with cataplexy is restored by intranasal Orexin A (Hypocretin-1). Brain. 2008;131:2734–41. Baier PC, Hallschmid M, Seeck-Hirschner M, Burkert S, Diessner N, Göder R, et al. Intranasal orexin A (hypocretin-1) restores the key REM-sleep abnormalities in human narcolepsy with cataplexy. Pharmacopsychiatry. 2009;42. Additional Declarations There is NO conflict of interest to disclose. Supplementary Files Supplementaryinformation.pdf Supplementary information Cite Share Download PDF Status: Published Journal Publication published 30 Oct, 2024 Read the published version in Neuropsychopharmacology → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4713362","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":325391218,"identity":"dfa92956-c7a5-4d2c-a091-63880359624b","order_by":0,"name":"Markus Fendt","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBElEQVRIiWNgGAWjYHACZhDBD8QGDB8YGBgbkETxapEEqjRsnEGylmYeYrTotp99bMzzh0GCX7p5+2PbHBvZBvYe49c8DNZyuLSYnUk3TuZtY5CQnHOssDl3W5pxA88ZM2sehnRjnFoOpDEf5m1gqDO4kWMI1HI4sUEiLc2YhwHIwKXl/DPmwyCH2YO0WG77D9dSj1PLjTTmZB42BgkDCaAWxm0HgFqSDz8GaknA6bAbz5gN57ZJSEjcSCuc2bst2biN5/AxxjkG6Ya4HZbGLPHmj40E/4zkDR9+brOT7WdvbP7wpsJaHpctUCCBYLIBkQQwJZAGmD+QqGEUjIJRMAqGNwAAG3pSXz4NIu0AAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-3451-1226","institution":"Otto-von-Guericke University Magdeburg, Medical Faculty","correspondingAuthor":true,"prefix":"","firstName":"Markus","middleName":"","lastName":"Fendt","suffix":""},{"id":325391219,"identity":"95d1dfa6-d732-49ee-89b0-a3079dd6d405","order_by":1,"name":"Julius Duske","email":"","orcid":"","institution":"Otto-von-Guericke University Magdeburg, Medical Faculty","correspondingAuthor":false,"prefix":"","firstName":"Julius","middleName":"","lastName":"Duske","suffix":""},{"id":325391220,"identity":"72dfa211-e544-4dcd-a747-222289723fc5","order_by":2,"name":"Nicole D'Souza","email":"","orcid":"","institution":"Otto-von-Guericke University Magdeburg, Medical Faculty","correspondingAuthor":false,"prefix":"","firstName":"Nicole","middleName":"","lastName":"D'Souza","suffix":""},{"id":325391221,"identity":"eaf81423-3740-4619-9fa9-fc195989aaf9","order_by":3,"name":"Dana Mayer","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Dana","middleName":"","lastName":"Mayer","suffix":""},{"id":325391222,"identity":"c63212d9-a715-4105-b1cf-0afff6abf53f","order_by":4,"name":"Daniela Dieterich","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Daniela","middleName":"","lastName":"Dieterich","suffix":""}],"badges":[],"createdAt":"2024-07-09 16:01:46","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4713362/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4713362/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41386-024-02017-8","type":"published","date":"2024-10-30T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":60186516,"identity":"8346ddef-a065-4ded-a7f5-2ccf389bdc95","added_by":"auto","created_at":"2024-07-12 18:55:39","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":46187,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLight-dark cycles of the CJL protocols.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e The mild CJL protocol consisted of 6 h-shifts every 7 days. One experimental group was tested after 4 time shifts (i.e., in cycle 4), the other group after 8 time shifts (i.e., in cycle 8). \u003cstrong\u003eB\u003c/strong\u003e The strong CJL protocol consisted of 8 h-shifts every 5 days and the experiments were performed after 6 time shifts (i.e., in cycle 6). Abbreviations: CJL, chronic jet lag; ST, standard light-dark cycle.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4713362/v1/1d7b122ec6427f13a07717d4.png"},{"id":60186514,"identity":"8b322596-9e26-4fa9-896a-952a389d6ca7","added_by":"auto","created_at":"2024-07-12 18:55:39","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":30615,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eASST performance after 1 or 2 months of simulated CJL.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e Two but not one month of CJL increase the mean number of trials to criterion compared with standard light/dark cycle. \u003cstrong\u003eB\u003c/strong\u003e This effect was most pronounced in the CD, Rev1, IDS and Rev3 phases of ASST. \u003cstrong\u003eC\u003c/strong\u003eVery similar effects were observed on the mean number of errors to criterion. \u003cstrong\u003eD\u003c/strong\u003e These effects were most pronounced in the CD, Rev1 and IDS phases. \u003cstrong\u003eE\u003c/strong\u003ePerseverative and \u003cstrong\u003eF\u003c/strong\u003e regressive errors were not affected on a statistical level. *** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, post-hoc comparisons (as indicated) after significant effects in ANOVA. The dots represent the individual measures. Abbreviations: ASST, attentional set shifting task; CD, compound discrimination; CJL, chronic jet lag; EDS, extradimensional shift; IDS, intradimensional shift; Rev1-3, reversal 1-3; ST, standard light-dark cycle.\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4713362/v1/f06a685c2d76b9e00069ea45.png"},{"id":60186519,"identity":"8b3eb12a-821b-4706-b7d7-cfc8e335ccec","added_by":"auto","created_at":"2024-07-12 18:55:40","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1373742,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of cycle condition and sex on c-Fos expression in orexin-positive neurons in the lateral hypothalamus and neurons in ASST-associated PFC regions. A \u003c/strong\u003edepicts examples of orexin- and c-Fos-positive neurons in the lateral hypothalamus (LH) of mice exposed to the standard light-dark cycle (ST) or chronic jet lag (CJL). The arrows point to representative double-positive neurons.\u003cstrong\u003e B \u003c/strong\u003eExposure to CJL as well as sex did not affect the number of orexin- and c-Fos-positive neurons. However, in female mice, less orexin-positive neurons were also c-Fos-positive when they were exposed to CJL. This suggests a decreased activity of orexin neurons after CJL xposure. \u003cstrong\u003eC\u003c/strong\u003e shows c-Fos-positive neurons in the medial orbitofrontal cortex (mOFC) after exposure to the ST or CJL. Note the presence of orexin-positive fibres in the mOFC.\u003cstrong\u003e \u003c/strong\u003e\u0026nbsp;\u003cstrong\u003eD\u003c/strong\u003e Very similar sex-specific effects were observed in the regions of the PFC that are associated with cognitive flexibility. In female but not male mice, exposure to CJL reduced the number of c-Fos-positive neurons. This effect was most pronounced in the medial OFC. * p \u0026lt; 0.05, post-hoc comparison (as indicated) after significant effects in ANOVA. The dots represent the individual measures. Abbreviations: CJL, simulated chronic jet lag; LH, lateral hypothalamus; mOFC, medial orbitofrontal cortex; OFC, orbitofrontal cortex; PFC, prefrontal cortex; ST, standard light-dark cycle.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4713362/v1/77e7b30930bd9589dd5a6f53.jpg"},{"id":60186517,"identity":"5a579f40-ab83-4126-b5a0-c4b54cef6334","added_by":"auto","created_at":"2024-07-12 18:55:40","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":11123,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of CJL and OxA treatment on spontaneous alternations and arm entries in the Y-maze. A\u003c/strong\u003e CJL impaired the percentage of spontaneous alternations which was rescued by treatment with OxA. \u003cstrong\u003eB\u003c/strong\u003eBoth CJL and OxA did not have effects on the total arm entries. *** p \u0026lt; 0.001, ** p \u0026lt; 0.01, post-hoc comparison (as indicated) after significant effects in ANOVA. The dots represent the individual measures. Abbreviations: CJL, simulated chronic jet lag; OxA, orexin A; ST, standard light-dark cycle; Veh, vehicle.\u003c/p\u003e","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4713362/v1/ad796d99600cf46555b5997c.png"},{"id":60186812,"identity":"9cb908b2-d2c6-4b0f-9e7f-72360a7cfa49","added_by":"auto","created_at":"2024-07-12 19:03:39","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":42358,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of oral administration of orexin A on impaired ASST performance after simulated CJL.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e CJL increases the mean number of trials to criterion in vehicle-treated mice but not in orexin A-treated mice. \u003cstrong\u003eB\u003c/strong\u003e These effects were most pronounced in the Rev1 phase of ASST. \u003cstrong\u003eC\u003c/strong\u003e Very similar effects were observed on the mean number of errors to criterion. \u003cstrong\u003eD\u003c/strong\u003e These effects were most pronounced in the Rev1 and Rev3 phase. \u003cstrong\u003eE\u003c/strong\u003ePerseverative and \u003cstrong\u003eF\u003c/strong\u003e regressive errors were both increased in the Rev1 phase. ** p \u0026lt; 0.01, * p \u0026lt; 0.05, post-hoc comparison (as indicated) after significant effects in ANOVA. The dots represent the individual measures. Abbreviations: ASST, attentional set shifting task; CD, compound discrimination; CJL, chronic jet lag; EDS, extradimensional shift; IDS, intradimensional shift; Rev1-3, reversal 1-3; ST, standard light-dark cycle; Veh, vehicle.\u003c/p\u003e","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4713362/v1/79c9f7d39a3ad79259d024ee.png"},{"id":67918802,"identity":"22513e1d-803f-4cbb-a73f-ac5da957505e","added_by":"auto","created_at":"2024-10-31 07:10:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2108802,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4713362/v1/1541e0cf-8e04-48d2-a0ac-bc2fc5a863c9.pdf"},{"id":60186518,"identity":"a54d609f-77ec-4ca0-8f56-1cfb90245a30","added_by":"auto","created_at":"2024-07-12 18:55:40","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1532886,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary information\u003c/p\u003e","description":"","filename":"Supplementaryinformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4713362/v1/b32236d2815d9f899d27d37e.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose.","formattedTitle":"Orexinergic modulation of chronic jet lag-induced deficits in mouse cognitive flexibility","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eCircadian rhythm sleep disorder (CRSD) is a disorder that affects the timing of sleep. It is caused by a mismatch of the endogenous clock and externally set stimuli such as light or dark outdoors, physical activity, or meal times. CRSD is either intrinsic such as advanced or delayed sleep-wake disorder or extrinsic CRSD including shift work disease or jet lag, which is more common. For both variants, the main clinical manifestations are insomnia and excessive daytime sleepiness [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Of interest, cognitive impairments and associated changes in the brain can be observed in CRSD. For example, chronic jet lag (CJL) in flight attendants has been associated with cognitive deficits, particularly in working memory, as well as atrophy of the temporal lobe [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. In addition, impairments in attention and decision making due to chronic sleep deprivation have been demonstrated [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Although chronotherapy, photic treatment, and cognitive-behavioural therapy exist, pharmacological treatment for CRSD is mainly limited to melatonin [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCognitive impairments have been also found in animal models of CRSD. In laboratory rodents, simulated CJL, especially when phase shortening simulates eastward travel, has been shown to reduce hippocampal neurogenesis, which was associated with impaired spatial learning [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. However, there is poor knowledge about the effects of simulated CJL on executive functions in laboratory rodents. Executive functions include working memory, inhibitory control, attention, and cognitive flexibility [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] and can be measured in mice using specific behavioural paradigms [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. A well-established paradigm to measure cognitive flexibility is the attentional set shifting task (ASST) [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The ASST is based on discrimination learning and includes different types of transfers such as reversals, intra- and extradimensional shifts. Working memory can be measured by spontaneous alternations in the Y-maze [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe aim of the present study is to investigate whether the orexin system in mice is involved in cognitive deficits induced by CRSD. The orexin system consists of neurons in the lateral hypothalamus that synthesize the neuropeptides orexin A and B and their brain-wide projections [\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. This system is involved in a variety of regulatory mechanisms including stress responses, emotions, motivation, eating behaviour and sleep-wake cycle, which is the major focus of this study [\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The orexin system promotes wakefulness by stimulating the ascending reticular activating system [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Studies first in animal models [\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] and later in humans [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] highlighted the importance of the orexin system in narcolepsy. This disease with symptoms such as excessive daytime sleepiness and sleep attacks is a consequence of loss of orexin neurons or disturbed orexin receptor signalling. This suggests that orexin or orexin receptor agonists could be a promising pharmacological treatment option for CRSD (as well as for narcolepsy). Such a treatment would not only address symptoms associated with disturbed sleep-wake cycle but potentially also cognitive symptoms because the orexin system also plays a role in cognition. In laboratory animals, orexin improves spatial learning and memory, rescues impaired discrimination learning, and improves executive functions such as cognitive flexibility or working memory.\u003c/p\u003e \u003cp\u003eThus, in the present study, we exposed male and female mice to simulated CJL, an animal model of CRSD. Then, we investigated whether (1) simulated CJL impairs cognitive flexibility, (2) the orexin system is involved in such an impairment, and if so, whether (3) nasal administration of orexin A is able to reverse CJL-induced deficits in cognitive flexibility and working memory. Our hypothesis is that (1) simulated CJL induces impairments in cognitive flexibility and working memory, (2) these impairments are associated with changes in the orexin system, and (3) nasal administration of orexin rescues CJL-induced cognitive deficits.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eFemale and male C57BL/6J mice were used in the experiments. Mice were housed in groups of up to 8 mice/cage under controlled conditions (humidity: 55\u0026thinsp;\u0026plusmn;\u0026thinsp;10%, temperature: 22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C) and \u0026ndash; depending on the group \u0026ndash; either in a standard 12 h light/12 h dark cycle (light on: 6:00 am) or in a CJL cycle (see below). Mice were between 12 and 15 weeks of age during testing. Food and water were provided \u003cem\u003ead libitum\u003c/em\u003e until 1 week before and during ASST, when food was restricted (approximately 2.5 g/mice/day) to maintain 90\u0026ndash;95% of the mice\u0026rsquo;s basal body weight. All experiments were performed during the light period and were complied with International Guidelines for the Care and Use of Animals for Experimental Procedures (2010/63/EU) with confirmed approval from the local authorities (Landesverwaltungsamt Sachsen-Anhalt, Az. 42502-2-1618 UniMD).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eSimulation of Chronic Jet Lag (CJL)\u003c/h2\u003e \u003cp\u003eAfter an adaptation period of at least 1 week, CJL was simulated for the experimental groups by phase advancing of the light-dark cycle, while the control groups stayed in the standard light-dark cycle (ST). In experiment 1, the light-dark conditions were shifted by 6 hours every seventh day (mild CJL protocol; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Behavioural tests were performed after 4 or 8 time shifts, respectively (i.e., in week 4 or 8 after starting CJL). In experiments 2 and 3, the light-dark conditions were shifted by 8 hours every fifth day (strong CJL protocol; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Behavioural tests or immunohistochemistry, respectively, were performed after 6 time shifts, i.e., in week 4 after starting CJL.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eBehavioural experiments\u003c/h2\u003e \u003cp\u003e \u003cem\u003eAttentional Set Shifting Task (ASST).\u003c/em\u003e The ASST was performed as previously described [\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Briefly, custom-made boxes were used consisting of a waiting and a choice area that was divided into two compartments separated by a transparent wall, with sliding doors between the waiting area and the choice area compartments. Mice were provided with water and familiarized with bowls and rewards before testing.\u003c/p\u003e \u003cp\u003eDuring habituation, mice were handled daily for a week, subjected to food restriction, and introduced to the setup and rewards. Training involved digging in bowls filled with bedding material to find rewards. For experiment 3, mice also underwent nasal saline application habituation.\u003c/p\u003e \u003cp\u003eThe testing phase began immediately after habituation, lasting 4 days. Bowls were presented with visual-tactile cues (digging media) or olfactory cues (odorants), with only one containing a reward. Mice had to associate the correct cue with the reward, with bowl positions randomized. Trials started with a 30-second wait period, then sliding doors were opened. Correct choices allowed mice to consume the reward, while incorrect choices were indicated by removing the empty bowl. After six consecutive correct decisions, a phase was considered to be completed and the next phase started.\u003c/p\u003e \u003cp\u003eThe ASST had several phases:\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eDay 1; Simple Discrimination (SD): Mice chose between two exemplars of one stimulus dimension (odor or digging medium).\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eDay 2; Compound Discrimination (CD): An additional, irrelevant stimulus dimension was introduced, followed by a reversal phase (Rev1) where contingencies changed.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eDay 3; Intradimensional Shift (IDS): New exemplars of both dimensions were presented, with the relevant dimension of the previous phases predicting the reward, followed by another reversal (Rev2).\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eDay 4; Extradimensional Shift (EDS): New exemplars of both dimensions were used, with the previously irrelevant dimension now predicting the reward, followed by a final reversal phase (Rev3).\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eSpontaneous alternation in the Y-maze.\u003c/em\u003e The custom-made Y-maze consisted of 3 arms, 120 degrees apart, 37 cm long, 10 cm wide, and 13 cm high. The walls were transparent, the floor of grey PVC. The experiment started with placing the mice in one arm of the maze, facing to the centre. Then, the mouse could freely explore the Y-maze for 5 min. The experimenter manually scored arm entries of the mice, defined by entering an arm with all four paws.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry\u003c/h2\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eMice were deeply anesthetized with i.p. administration of ketamine hydrochloride (100 mg/kg), xylazine hydrochloride (20 mg/kg), and acepromazine maleate (3 mg/kg) and transcardially perfused, first with saline (2 min), then with Zamboni solution (4% paraformaldehyde, 0.2% picric acid in PBS; 10 min). The brain was removed, post-fixed overnight, and then stored in 30% sucrose. Using a cryostat, coronal 40 \u0026micro;m sections were cut.\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eDouble-immunofluorescence staining was performed on every third section of the regions of interest (lateral hypothalamus, prefrontal cortex). For c-Fos staining, a rabbit anti-c-Fos antibody (1:3000, Synaptic Systems GmbH, G\u0026ouml;ttingen, Germany), a biotinylated goat anti-rabbit IgG antibody (1:1000, Vector Laboratories, Newark, USA), and an ABC kit (Vector Laboratories, Newark, USA) were used. For orexin A staining, a mouse anti-orexin A antibody (1:3000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), Cy 3-conjugated goat anti-mouse IgG antibody (1:400, Jackson ImmunoResearch, Laboratories, Inc., Ely, Cambridgeshire, United Kingdom), and Streptavidin Cy-2 (1:500, Life Technologies Corporation, Eugene, USA) were used. The stained slides were mounted on microscope slides and coverslips were added. Photomicrographs were taken using a Zeiss microscope (Laserscanningmicroscope 710, Carl Zeiss AG, Oberkochen, Germany) with respective software.\u003c/p\u003e \u003cp\u003eFor quantitative analysis, pre-defined frames were bilaterally placed in the regions of interest. In the lateral hypothalamus, c-Fos-positive neurons, orexin A-positive neurons and double-labelled neurons were manually counted by the blinded experimenter. c-Fos-positive neurons of the different subregions of the PFC were counted with a custom-made software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eDrugs\u003c/h2\u003e \u003cp\u003eIn experiment 3, mice were treated with either 10 \u0026micro;l vehicle (saline) or 10 \u0026micro;l orexin A solution (0.1 mM; Tocris Bioscience, Bristol, UK). Concentration and volume was based on published data [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eExperimental procedure\u003c/h2\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003e \u003cem\u003eExperiment 1\u003c/em\u003e: Mice were kept in the standard light/dark cycle (ST) or in a mild CJL (see Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). After 4 or 8 time shifts (ie., in cycle 4 or 8), respectively, mice were submitted to the ASST. Group sizes: ST: 5 females, 6 males; 1 month CJL: 5 females, 6 males; 2 months CJL: 5 females, 6 males.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cem\u003eExperiment 2\u003c/em\u003e: Mice were kept in ST or strong CJL for 6 cycles (see Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Then, at the time when behavioural tests in experiment 1 and 3 began (9:00\u0026ndash;10:00 am), the mice were transcardially perfused. The brain was then removed and processed for immunohistochemistry as described. Group sizes: ST: 7 females, 7 males; CJL: 7 females, 6 males.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cem\u003eExperiment 3\u003c/em\u003e: Mice were kept in ST or strong CJL (see Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). After 6 time shifts (ie., in cycle 6), mice were submitted to the Y-maze test and 5 min later to ASST. Thirty minutes before the start of the behavioural experiments, they were treated with nasal administrations of saline (SL) or orexin A (OxA). Of note, some mice were tested only in the Y-maze but not in the ASST (since throughput is limited in the latter but not in the former). However, all mice subjected to ASST were previously tested in the Y-maze. Group sizes (Y-maze/ASST): ST/Veh: 9/6 females, 9/6 males; CJL/Veh: 7/6 females, 9/6 males; ST/OxA: 9/6 females, 8/6 males; CJL/OxA: 7/6 females, 10/6 males.\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData analysis was performed using SYSTAT 13 (SPSS Inc.) and Prism 7.0 (GraphPad Software Inc., La Jolla, USA). Normal distribution of the data was verified using the Shapiro-Wilk normality test. Analysis of variance (ANOVA) was performed, followed by respective post-hoc multiple comparisons (two-linear step-up procedure of Benjamini, Krieger and Yekutieli). The significance level was set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eExperiment 1\u003c/h2\u003e \u003cp\u003eA multi-factorial ANOVA using sex (female/male) and cycle condition (ST, 1 month CJL, 2 months CJL) as between-subject factors and ASST phase (SD, CD, Rev1, IDS, Rev2, EDS, Rev3) as within-subject factor was used to analyse ASST performance of the mice.\u003c/p\u003e \u003cp\u003eSince this multifactorial ANOVA showed neither main effects of sex (\u003cem\u003eF\u003c/em\u003es\u0026thinsp;\u0026lt;\u0026thinsp;1.63; \u003cem\u003ep\u003c/em\u003es\u0026thinsp;\u0026gt;\u0026thinsp;0.21) nor interactions of sex with other factors (\u003cem\u003eF\u003c/em\u003es\u0026thinsp;\u0026lt;\u0026thinsp;2.17; \u003cem\u003ep\u003c/em\u003es\u0026thinsp;\u0026gt;\u0026thinsp;0.13) for trials to criterion (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B), errors to criterion (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, D), and the errors types (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, F), the data from both sexes were pooled for further analysis.\u003c/p\u003e \u003cp\u003eRegarding trials and error to criterion, the analysis of the pooled data revealed main effects of cycle conditions (\u003cem\u003eF\u003c/em\u003es\u0026thinsp;\u0026gt;\u0026thinsp;6.84, \u003cem\u003ep\u003c/em\u003es\u0026thinsp;\u0026lt;\u0026thinsp;0.004) and ASST phase (\u003cem\u003eF\u003c/em\u003es\u0026thinsp;\u0026gt;\u0026thinsp;5.49, \u003cem\u003ep\u003c/em\u003es\u0026thinsp;\u0026lt;\u0026thinsp;0.004) but no interaction between these two factors (Fs\u0026thinsp;\u0026lt;\u0026thinsp;1.00, \u003cem\u003ep\u003c/em\u003es\u0026thinsp;\u0026gt;\u0026thinsp;0.45). Post-hoc comparisons showed that 1 month CJL did not induce a performance deficit (\u003cem\u003eq\u003c/em\u003es\u0026thinsp;\u0026lt;\u0026thinsp;1.81, \u003cem\u003ep\u003c/em\u003es\u0026thinsp;\u0026gt;\u0026thinsp;0.14) while 2 months CJL significantly impaired ASST performance (\u003cem\u003eq\u003c/em\u003es\u0026thinsp;\u0026gt;\u0026thinsp;3.68, \u003cem\u003ep\u003c/em\u003es\u0026thinsp;\u0026lt;\u0026thinsp;0.002; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, C). This effect of 2 months CJL was most robust in the CD, Rev1, IDS and Rev3 phases (\u003cem\u003et\u003c/em\u003es\u0026thinsp;\u0026gt;\u0026thinsp;2.16, \u003cem\u003ep\u003c/em\u003es\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, D). Analysis of the error types in the reversal phases revealed a trend for an interaction for ASST phase and CJL on perseverative errors (\u003cem\u003eF\u003c/em\u003e(4,60)\u0026thinsp;=\u0026thinsp;2.45, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.055; post-hoc comparison ST vs. 1 month CJL in Rev1: \u003cem\u003et\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.69, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.02), as well as a main effect of phase (\u003cem\u003eF\u003c/em\u003e(4,60)\u0026thinsp;=\u0026thinsp;3.64, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.04). There were no significant effects on regressive errors (\u003cem\u003eF\u003c/em\u003es\u0026thinsp;\u0026lt;\u0026thinsp;2.74, \u003cem\u003ep\u003c/em\u003es\u0026thinsp;\u0026gt;\u0026thinsp;0.08).\u003c/p\u003e \u003cp\u003eSeparate analyses of female and male mice further revealed that effects of CJL on ASST performance were more pronounced in female than in male mice (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, S2\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eExperiment 2\u003c/h2\u003e \u003cp\u003eThe number of c-Fos- and OxA-positive neurons, as well as the percentage of double-positive neurons (normalized to the number of OxA-positive neurons) in the lateral hypothalamus was analysed with a multi-factorial ANOVA using sex (female/male) and cycle condition (ST, CJL) as between-subject factors. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA shows examples of the immunohistochemical staining. Of note, a strong CJL protocol was used in this experiment.\u003c/p\u003e \u003cp\u003eBoth, the number of OxA- and c-Fos-positive neurons were not affected by sex or cycle condition (\u003cem\u003eF\u003c/em\u003es\u0026thinsp;\u0026lt;\u0026thinsp;0.34, \u003cem\u003ep\u003c/em\u003es\u0026thinsp;\u0026gt;\u0026thinsp;0.57; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). While these two factors did not interact regarding the number of OxA-positive neurons (\u003cem\u003eF\u003c/em\u003e(1,23)\u0026thinsp;=\u0026thinsp;0.62, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.44), there was a trend for such an interaction regarding the c-Fos-positive neurons (\u003cem\u003eF\u003c/em\u003e(1,23)\u0026thinsp;=\u0026thinsp;2.95, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.099). Of note, this interaction between sex and cycle condition was significant in the analysis of the percentage of double-positive neurons (\u003cem\u003eF\u003c/em\u003e(1,23)\u0026thinsp;=\u0026thinsp;7.40, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.01), whereas there were no main effects of sex and cycle condition (\u003cem\u003eF\u003c/em\u003es\u0026thinsp;\u0026lt;\u0026thinsp;1.32, \u003cem\u003ep\u003c/em\u003es\u0026thinsp;\u0026gt;\u0026thinsp;0.26). Post-hoc comparison revealed a significant decrease of double-positive neurons after CJL in female mice (t\u0026thinsp;=\u0026thinsp;2.79, p\u0026thinsp;=\u0026thinsp;0.01) but not in male mice (\u003cem\u003et\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.09, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.29).\u003c/p\u003e \u003cp\u003eIn addition, c-Fos expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, D) was analysed in the regions of the PFC associated with cognitive flexibility (ventromedial PFC, cingulate cortex, lateral/ventral orbitofrontal cortex (OFC) and medial OFC). There were no main effects of sex and cycle condition in the different PFC regions (\u003cem\u003eF\u003c/em\u003es\u0026thinsp;\u0026lt;\u0026thinsp;2.51, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.12), expect a main effect of cycle condition in the medial OFC (\u003cem\u003eF\u003c/em\u003e(1,23)\u0026thinsp;=\u0026thinsp;4.63, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.04). Furthermore, there was again an interaction of sex and cycle condition when the number of c-Fos-positive neurons in all these PFC regions was averaged (\u003cem\u003eF\u003c/em\u003e(1,24)\u0026thinsp;=\u0026thinsp;4.24, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Post-hoc comparisons showed a decreased number of c-Fos-positive neurons in female mice after CJL exposure in both the medial OFC and the mean of all PFC regions (\u003cem\u003et\u003c/em\u003es\u0026thinsp;\u0026gt;\u0026thinsp;2.49, \u003cem\u003ep\u003c/em\u003es\u0026thinsp;\u0026lt;\u0026thinsp;0.04), while there were no effects on male mice (\u003cem\u003et\u003c/em\u003es\u0026thinsp;\u0026lt;\u0026thinsp;0.59, \u003cem\u003ep\u003c/em\u003es\u0026thinsp;\u0026gt;\u0026thinsp;0.80). Of note, orexin-positive fibres were detected in all PFC regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eExperiment 3\u003c/h2\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003eSpontaneous alternation in the Y-maze\u003c/h2\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e depicts percent alternation and total arm visits of the mice in the Y-maze. Data were analysed with a multi-factorial ANOVA using sex (female/male), cycle condition (ST/CJL), and treatment (Veh, OxA) as between-subject factors. Since no main effect of sex or interactions of sex with the other factors were found (F\u0026thinsp;\u0026lt;\u0026thinsp;1.40; p\u0026thinsp;\u0026gt;\u0026thinsp;0.24), the data of female and male mice were pooled. The ANOVA of the pooled data revealed a main effect of cycle condition (\u003cem\u003eF\u003c/em\u003e(1,64)\u0026thinsp;=\u0026thinsp;11.00; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002) and an interaction of cycle condition with treatment (\u003cem\u003eF\u003c/em\u003e(1,64)\u0026thinsp;=\u0026thinsp;9.41; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.003; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Post hoc comparisons showed that CJL impaired the percentage of alternations in vehicle-treated mice (\u003cem\u003et\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.51, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) but not in OxA-treated mice (\u003cem\u003et\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.18, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.86). CJL-exposed OxA-treated mice had significantly more alternations than CJL-exposed vehicle-treated mice (\u003cem\u003et\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3.37, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001). Neither CJL nor OxA treatment had any effects on the total number of arm entries in the Y-maze (\u003cem\u003eF\u003c/em\u003es\u0026thinsp;\u0026lt;\u0026thinsp;1.17, \u003cem\u003ep\u003c/em\u003es\u0026thinsp;\u0026gt;\u0026thinsp;0.28; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eSeparate analyses of female and male mice further are shown in the Supplements (\u003cb\u003eFig. S3, S4\u003c/b\u003e). The CJL effect was very similar in female and male mice, while the OxA effect was more robust in females.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eASST Performance\u003c/h2\u003e \u003cp\u003eThe performance of the mice in the ASST were analysed with multi-factorial ANOVAs using sex (female/male), cycle condition (ST/CJL), and treatment (Veh/OxA) as between-subject factors and ASST phase (SD/CD/Rev1/IDS/Rev2/EDS/Rev3) as within-subject factor. Six mice failed to complete all ASST phases and were excluded from the final analyses (CJL/OxA: n\u0026thinsp;=\u0026thinsp;2 males; ST/SL: n\u0026thinsp;=\u0026thinsp;2 females; ST/OxA: n\u0026thinsp;=\u0026thinsp;1 female, 1 male).\u003c/p\u003e \u003cp\u003eA multi-factorial ANOVA revealed no main effects of sex (F\u0026thinsp;\u0026lt;\u0026thinsp;1.64; p\u0026thinsp;\u0026gt;\u0026thinsp;0.21) or interactions of sex with other factors (F\u0026thinsp;\u0026lt;\u0026thinsp;0.81; p\u0026thinsp;\u0026gt;\u0026thinsp;0.38) for trials to criterion (\u003cb\u003eFig.\u0026nbsp;6A, B\u003c/b\u003e), errors to criterion (\u003cb\u003eFig.\u0026nbsp;6C, D\u003c/b\u003e), and the errors types (\u003cb\u003eFig.\u0026nbsp;6E, F\u003c/b\u003e). Therefore, data from both sexes were pooled for further analysis, but data for female and male mice are also separately shown in the figures.\u003c/p\u003e \u003cp\u003eRegarding the trials and errors to criterion, no main effects of cycle conditions (Fs\u0026thinsp;\u0026lt;\u0026thinsp;4.03, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) and treatment (Fs\u0026thinsp;\u0026lt;\u0026thinsp;1.94, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.16) were found. However, there was an interaction between cycle condition and treatment (Fs\u0026thinsp;\u0026gt;\u0026thinsp;4.25, \u003cem\u003eps\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.045). Post-hoc comparisons showed that CJL increased the number of trials and errors in vehicle-treated mice (\u003cem\u003et\u003c/em\u003es\u0026thinsp;\u0026gt;\u0026thinsp;2.71, \u003cem\u003ep\u003c/em\u003es\u0026thinsp;\u0026lt;\u0026thinsp;0.001) but not in OxA-treated mice (\u003cem\u003et\u003c/em\u003es\u0026thinsp;\u0026lt;\u0026thinsp;0.44, \u003cem\u003ep\u003c/em\u003es\u0026thinsp;\u0026gt;\u0026thinsp;0.66). In addition, there was a significant reduction of the number of trials in CJL-exposed mice after nasal OxA administration (\u003cem\u003et\u003c/em\u003es\u0026thinsp;\u0026gt;\u0026thinsp;2.34, \u003cem\u003ep\u003c/em\u003es\u0026thinsp;\u0026lt;\u0026thinsp;0.02). These effects were most pronounced in the Rev1 phase (\u003cem\u003et\u003c/em\u003es\u0026thinsp;\u0026gt;\u0026thinsp;3.00, \u003cem\u003ep\u003c/em\u003es\u0026thinsp;\u0026lt;\u0026thinsp;0.008). In this phase, the analyses also revealed effects of CJL on perseverative and regressive errors (\u003cem\u003et\u003c/em\u003es\u0026thinsp;\u0026gt;\u0026thinsp;2.84, \u003cem\u003ep\u003c/em\u003es\u0026thinsp;\u0026lt;\u0026thinsp;0.01), as well as of orexin A on the CJL-induced increase of perseverative errors (\u003cem\u003et\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.69, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.02).\u003c/p\u003e \u003cp\u003eSeparate analyses of female and male mice are further shown in the supplementary material (\u003cb\u003eFig. S5, S6\u003c/b\u003e). The described effects were very similar in female and male mice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe aim of the present study was to investigate both the role and the potential of the orexin system in the presumably impairing effects of simulated chronic jet lag on executive functions in laboratory mice. The present data demonstrate that simulated chronic jet lag robustly impairs cognitive flexibility. In addition, simulated chronic jet lag decreased the activity of the orexin system and different subregions of the prefrontal cortex, a brain area critical for cognitive flexibility. Based on these findings, an interventional experiment was performed which demonstrated that nasal administration of orexin rescued impaired cognitive flexibility and working memory after simulated chronic jet lag. Of note, the observed changes in the orexin system and the prefrontal cortex were only observed in female mice while there were no sex differences in the behavioural experiments. However, the behavioural changes were usually more pronounced in female mice.\u003c/p\u003e \u003cp\u003eIn the present study, CJL was simulated to model CRSD. Specifically, CRSD caused by jet lag and/or shift work was modelled [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Although occasional jet lag after long distance travel is usually problem that resolves itself, chronic jet lag in humans who travel frequently can cause a range of symptoms, including cognitive impairments [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Additionally, between 10 and 15% of all workers are affected by shift work [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Particularly, around one third of shift workers suffer from shift work disorder [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], which is associated with cognitive impairments [\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] and an increased risk of dementia [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Many of the described symptoms of CRSD are also observed in laboratory mice after simulated chronic jet lag. Decreased motivation, increased anxiety, depression-like behaviours, reduced recognition memory and impaired spatial learning [\u003cspan additionalcitationids=\"CR37 CR38\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], but also molecular changes in different brain regions [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] and even increased mortality [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] have been observed. While the detrimental effects of chronic jet lag on executive functions such as cognitive flexibility or working memory have been demonstrated in humans [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], to our knowledge this has never been investigated in laboratory mice.\u003c/p\u003e \u003cp\u003eThe present study has shown that both cognitive flexibility and working memory are impaired in mice after simulated chronic jet lag. Our study demonstrates this in two independent experiments with different protocols of simulated chronic jet lag conducted by two different experimenters. A week protocol of chronic jet lag (6 hours shift every seventh day) had no effects on cognitive flexibility after 1 months (4 shifts) but induced a significant impairment after 2 months (8 shifts). Therefore, a more severe protocol of chronic jet lag (8 hours shift every fifth day) was used for the next experiments. With this protocol, impaired cognitive flexibility and working memory was observed after 4 weeks (6 shifts). Together, these results strongly support the findings in humans mentioned above and indicate that simulated chronic jet lag in mice can be used to study potential causes and treatments of CRSD.\u003c/p\u003e \u003cp\u003eThe orexin system is crucial for the regulation of the sleep/wake cycle [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]; hence, it is reasonable to assume that this system is involved in CRSD [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. However, to the best of our knowledge, no published studies have shown that CRSD is associated with changes in the orexin system. The present study now shows decreased activity of the orexin system after simulated CJL, a mouse model of CRSD. Surprisingly, the reduced activity was only observed in female mice, although the behavioural effects of CRSD were very similar in both sexes. The same is true for neural activity in the prefrontal cortex, a crucial brain region for executive functions that is innervated by the orexin system [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. The absence of changes in male mice is unclear; however, it is possible that changes, if any, can simply not be detected with the c-Fos approach. An attempt was made to measure orexin levels in the PFC in the present study, however, the ELISA used also measured orexin levels in orexin-deficient mice, so we discontinued these measurements. Therefore, future experiments should include more sensitive methods to measure the activity of the orexin system and the orexin levels the PFC.\u003c/p\u003e \u003cp\u003eThe observed decrease in neuronal activity in the PFC might be caused by the impaired activity of the orexin system and could further be the cause for the behavioural deficits in the cognitive flexibility and working memory tests. If this is the case, a pharmacological intervention that replaces the missing orexin in the PFC should rescue the observed behavioural deficits. A relatively simple approach to administer orexin to the brain is via the nasal route [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e], which has been shown to increase orexin levels in the brain [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e] and to activate the PFC [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Indeed, after nasal administration of orexin, we observed a complete rescue of impaired cognitive flexibility and working memory after simulated CJL. This rescue effect of nasal orexin was also observed in male mice, although we could not detect impaired activity in the orexin system and the PFC in them.\u003c/p\u003e \u003cp\u003eOverall, the present findings show that the orexin system is involved in the cognitive impairments observed in a mouse model of CRSD and that nasal orexin administration rescues these impairments. Although there were some sex differences \u0026ndash; as frequently observed in orexin research [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e] \u0026ndash; in the effect of simulated CJL on neural activity, nasal orexin administration had similar effects in female and male mice. These data suggest that nasal orexin administration is a potential treatment option for CRSD. Intranasal orexin administration has been tested in narcoleptic patients who have very low or absent orexin levels in the brain [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] and could alleviate several symptoms in these patients such as olfactory dysfunction, attentional deficits and sleep abnormalities without significant side effects [\u003cspan additionalcitationids=\"CR54\" citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Therefore, nasal orexin administration should be tested in CRSD patients.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch1\u003eACKNOWLEDGEMENTS\u003c/h1\u003e\n\u003cp\u003eWe thank Uwe Disterheft for the construction of the ASST setups, Evelyn Kahl for technical assistance and Kathrin Freke for animal care.\u0026nbsp;\u003c/p\u003e\n\u003ch1\u003eAUTHOR CONTRIBUTIONS\u003c/h1\u003e\n\u003cp\u003eJD, ND, DCD and MF conceived and designed the study, JD, ND and DM conducted the experiments, JD, ND, DM and MF analysed the data, DCD and MF gathered the resources for the study, and JD and MF wrote the manuscript with contributions from all co-authors.\u003c/p\u003e\n\u003ch1\u003eFUNDING\u003c/h1\u003e\n\u003cp\u003eThis study was supported by a grant (MF, DCD) and a scholarship (JD) of the Deutsche Forschungsgemeinschaft (SFB1436/A01; project ID 42589994).\u003c/p\u003e\n\u003ch1\u003eCOMPETING INTERESTS\u003c/h1\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003ch1\u003eADDITIONAL INFORMATION\u003c/h1\u003e\n\u003cp\u003eThe online version contains supplementary material available at \u0026hellip;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePavlova M. 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Pharmacopsychiatry. 2009;42.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4713362/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4713362/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Cognitive flexibility and working memory are important executive functions mediated by the prefrontal cortex and can be impaired by circadian rhythm disturbances such as chronic jet lag (CJL) or shift work. In the present study, we used mice to investigate whether (1) simulated CJL impairs cognitive flexibility, (2) the orexin system is involved in such impairment, and (3) nasal administration of orexin A is able to reverse CJL-induced deficits in cognitive flexibility and working memory. Mice were exposed to either standard light-dark conditions or simulated CJL consisting of series of advance time shifts. Experiment (1) investigated the effects of a mild CJL protocol on cognitive flexibility using the attentional set shifting task. Experiment (2) used a stronger CJL protocol and examined CJL effects on the orexin system utilizing c-Fos and orexin immunohistochemistry. Experiment (3) tested whether nasal orexin application can rescue CJL-induced deficits in cognitive flexibility and working memory, the latter by measuring spontaneous alternation in the Y-maze. The present data show that CJL (1) impairs cognitive flexibility and (2) reduces activity of orexin neurons in the lateral hypothalamus. (3) Nasal administration of orexin A rescued CJL-induced deficits in working memory and cognitive flexibility. These findings suggest that executive functions impairments by circadian rhythm disturbances such as CJL are caused by dysregulation of orexinergic input to the prefrontal cortex. Compensation of decreased orexinergic input by nasal administration of orexin A could be a potential therapy for CJL- or shift work-induced human deficits in executive functions.","manuscriptTitle":"Orexinergic modulation of chronic jet lag-induced deficits in mouse cognitive flexibility","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-12 18:55:32","doi":"10.21203/rs.3.rs-4713362/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8f79b636-5b87-405d-a1c1-eefe760519dc","owner":[],"postedDate":"July 12th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":34420792,"name":"Biological sciences/Neuroscience/Circadian rhythms and sleep/Hypocretin"},{"id":34420793,"name":"Biological sciences/Neuroscience/Learning and memory/Working memory"},{"id":34420794,"name":"Biological sciences/Neuroscience/Learning and memory/Cortex"}],"tags":[],"updatedAt":"2024-10-31T07:10:10+00:00","versionOfRecord":{"articleIdentity":"rs-4713362","link":"https://doi.org/10.1038/s41386-024-02017-8","journal":{"identity":"neuropsychopharmacology","isVorOnly":false,"title":"Neuropsychopharmacology"},"publishedOn":"2024-10-30 04:00:00","publishedOnDateReadable":"October 30th, 2024"},"versionCreatedAt":"2024-07-12 18:55:32","video":"","vorDoi":"10.1038/s41386-024-02017-8","vorDoiUrl":"https://doi.org/10.1038/s41386-024-02017-8","workflowStages":[]},"version":"v1","identity":"rs-4713362","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4713362","identity":"rs-4713362","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}