What this is
- Corticosteroids disrupt circadian regulation of hippocampal function, leading to memory impairment.
- This study examines the effects of corticosteroid treatment on hippocampal gene regulation and synaptic plasticity in rats.
- Findings suggest that corticosteroid exposure misaligns circadian rhythms with natural light/dark cues, impacting cognitive function.
Essence
- Corticosteroid treatment disrupts circadian regulation of hippocampal function, leading to impaired memory. This disruption is linked to altered gene expression and synaptic plasticity.
Key takeaways
- Corticosteroid treatment for 5 days disrupts hippocampal rhythmic activity and impairs memory. This is mediated by in the hippocampus, which interfere with circadian gene regulation.
- The study demonstrates that synthetic corticosteroids do not affect the master circadian clock but significantly impact the hippocampus, resulting in misaligned circadian rhythms and cognitive deficits.
- Findings indicate that the disruption of hippocampal circadian function due to corticosteroids may underlie cognitive decline in patients receiving such treatments.
Caveats
- The study primarily uses male rats, limiting the understanding of corticosteroid effects in females. Future research is needed to explore gender differences in circadian and memory processes.
- No rescue strategy was established to mitigate the adverse effects of corticosteroid treatment, indicating a gap in potential therapeutic interventions.
Definitions
- Glucocorticoid Receptors (GRs): Proteins that bind glucocorticoids and mediate their effects on gene expression and cellular function.
- Long-Term Potentiation (LTP): A persistent strengthening of synapses based on recent patterns of activity, crucial for memory formation.
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Discussion
Corticosteroids are widely used in clinical medicine to relieve signs and symptoms of many inflammatory and autoimmune disorders (62, 63). However, in addition to this, their use is often reported with cognitive and psychiatric symptoms—inducing a range of psychiatric adverse effects including depression, mania, and cognitive and memory impairment. These relationships have been known for decades, yet the mechanisms underlying these disturbances and their clinical management have been poorly described.
In this reverse translational study, we demonstrate that 5 d of corticosteroid treatment disrupts the rhythmic activity of hippocampal function through GRs, induces NMDAR-dependent synaptic dysfunction, and subsequently impairs memory. This model is supported by several lines of evidence: 1) Corticosteroids do not influence the master clock (SCN), or light/dark entraining behaviors due to a lack of GR expression in the SCN; 2) the hippocampus is rich in GRs, which in the presence of corticosteroids, bind to GREs on the essential clock gene Per1 to mediate hippocampal activity in competition with central clock-mediated control; 3) synthetic corticosteroid treatment eliminates the circadian variation in circulating corticosteroids, and consequently the circadian variation in the activity of hippocampal GRs to influence clock gene expression and destabilize clock-entrained NMDAR/CamkII complexes; and 4) synthetic corticosteroid treatment blocks LTP, a cellular correlate of long-term memory, by disrupting NMDAR-dependent processes to impair long-term, but not short-term, hippocampal-dependent memory.
There are two regulators in normal chronophysiology. On the one hand, the SCN is the master regulator of circadian timing, entraining behaviors such as feeding, sleep, and memory processes to ~24 h (64). On the other hand, extra-SCN oscillators, such as those in the hippocampus and other forebrain regions, are entrained by SCN-dependent neural and humoral inputs (65). Pertinent to this study, one of the more powerful humoral signals is from adrenal corticosteroids (66), which exhibit a robust circadian secretory rhythm that peaks prior to wakening (2) in preparation for the neurocognitive activities that ensue. Synthetic long-acting corticosteroids, such as MPL, are therefore poised to mediate influence over these endogenous systems in the presence of the master clock.
Indeed, corticosteroids exert vast and variable effects on the body’s physiology, both peripherally and centrally (2, 67, 68). Consistent with previous studies, we found that MPL treatment suppressed appetite, and subsequently decreased body weight (SI Appendix, Fig. S1 D–F↗). However, interestingly, we identified distinct and specific actions of MPL on the brain. For instance, the SCN remained protected from MPL-mediated disruption, as previously reported with dexamethasone (69). However, the GR-rich hippocampus was sensitive to corticosteroid interference (70). The identification of GREs upstream of the Per1 gene, and its corticosteroid-induced transcription, underpins a mechanism whereby GR-expressing extra-SCN oscillators can be disturbed by corticosteroids, and may account for the loss of circadian rhythms often seen in hypercortisolemic states of Cushing’s disease and syndrome (71). Notably, our RNAseq dataset which encompassed whole hippocampus found that Per1 mRNA expression was up-regulated throughout the day with MPL treatment. Interestingly, Per1(−/−) mice also exhibit spatial memory deficits. These targeted manipulations of Per1 are opposing (i.e., overexpression vs. knockout) yet led to a similar outcome, suggesting that any disruption to the rhythmic expression of Per1 may be detrimental to spatial memory performance.
It is well established that corticosteroids are secreted from the adrenal gland in a circadian pattern with underlying ultradian pulses, and common to several neuropsychiatric disorders are HPA axis hyperactivity or steroid therapy (72–74). We found that 5 d of MPL treatment induced a prolonged increase in activated nuclear GR levels throughout the full circadian cycle and disrupted the circadian expression of NMDAR and CAMKII subunits. Both NMDAR and CAMKII are known to be essential mediators of neural plasticity (75), a process that is causally linked with the successful encoding of mnemonic information (76). Further to this, CAMKII is essential for coupling time of day to behavioral rhythms (33) and improved memory performance (77).
Consistent with our findings, other synthetic corticosteroids, particularly dexamethasone, can impair memory (78). However, translational studies often assess memory performance during the early inactive period, when endogenous circulating corticosteroids are low, therefore not time relevant to biological rhythms (79) or peak cognitive performance of the participants. Similarly, many rodent studies assess both hippocampal synaptic plasticity processes and memory performance during the animals’ inactive period, leading to the somewhat incomplete conclusion that effective memory consolidation requires a strong stress association. These tests use aversive or fear conditioning-based tasks such as inescapable foot shock, which induces a robust stress response in the animal, resulting in similar elevated glucocorticoid levels to those circulating at the onset of the active phase. Consistent with our interpretation that elevated glucocorticoids during the inactive phase can influence the molecular physiology of the brain, acute administration of corticosteroids has been demonstrated to enhance memory consolidation during nonaversive memory tasks performed during rats’ inactive phase. (80, 81). The specificity of corticosteroid, duration of action, as well as the timing and type of experimental testing, may contribute to differences in corticosteroid-mediated memory formation in the literature. Importantly in this study, we have identified a mechanism involving dysregulation of the hippocampal circadian clock, in which neurocognitive decline is mediated by a commonly prescribed synthetic corticosteroid. A limitation of this work is that a rescue strategy is yet to be established, and this will require future investigation. Moreover, how various corticosteroids (endogenous and synthetic) contribute to such differential outcomes on hippocampal function warrants further investigation. This study also used male rats, and therefore does not consider the action of corticosteroids on circadian processes and memory performance in females. Indeed, it is well established that in hippocampal CA1, LTP is dependent on locally synthesized estrogen (82), and that blocking estradiol synthesis disrupts LTP in females only (82, 83). Therefore, understanding how endogenous and exogenous corticosteroids may interact with and disrupt these processes warrants investigation.
Nevertheless, our ex vivo physiology data suggest that time of day is a potent modulator of neuronal activity and plasticity processes in CA1 neurons. Crucially, we show that this circadian regulation is modified by long-acting corticosteroid treatment, disrupting the dynamic control of miniature postsynaptic currents (mPSCs) and preventing LTP induction. These data support the notion that circadian and ultradian corticosteroid fluctuations play an important role in maintaining the rhythmic activity of the hippocampus via dynamic transcription of target genes. This has major physiological consequences considering the importance of mPSCs in the maintenance of synaptic connections and dendritic spines (84), as long-term alterations to these dynamics can result in critical changes to hippocampal circuitry and function, as demonstrated by the prevention of hippocampal-dependent memory formation (Fig. 4).
In summary, a large proportion of patients prescribed with corticosteroids report cognitive decline and memory impairment (16). Our data reveal a GR-mediated pathway that underlies the circadian regulation of hippocampal-dependent memory formation which is vulnerable to long-acting corticosteroid treatment. Currently, there are no clinical guidelines for treating corticosteroid-induced adverse effects. It is, perhaps, quite surprising that the brain-specific effects of such treatment has had relatively little scientific investigation given the broad application, widespread clinical use, and adverse psychiatric outcomes (15, 85, 86). Advancing our knowledge of corticosteroid-mediated regulation of hippocampal function will take us a step closer to understanding the mechanisms underpinning several prevalent major mental illnesses and suggest tailoring treatment regimens for prevention and intervention of corticosteroid (endogenous and synthetic)-induced disorders.
Methods
Adult male Lister hooded rats (250 to 350 g, 9 to 11 wk) (Envigo, UK) were used in all experimental procedures. Male rats were used in this study to limit estrous cycle influence on corticosterone release (87). Animals were maintained in standard housing conditions under a 12:12 gradual light/dark cycle in sound-attenuating rooms. A red light was used during the night phase to allow researchers to manipulate the animals. During the night phase, no external light could enter the room when opening the door. Food and water were available ad libitum. Throughout all experimental procedures, the same researcher took care of the animals to limit stress-induced effects and was blinded to treatment groups. All procedures were carried out in accordance with the UK Animals (Scientific Procedures) Act 1986 under PPL 30/3114 and PIL I04092F5F.
Adrenalectomy and Jugular Vein Cannulation.
For studies identifying glucocorticoid-specific control of hippocampal gene transcription (SI Appendix, Fig. S4↗), rats received balanced anesthesia using veterinary isoflurane (Merial Animal Health, Woking, UK) prior to bilateral adrenalectomy and cannulation of the right jugular vein for infusion of corticosterone. Rats recovered for 5 d postsurgery on 15 µg/mL corticosterone in 0.9% saline drinking solution to maintain isotonic levels. This solution was replaced 12 h prior to experiments with 0.9% saline to ensure washout of circulating corticosterone.
Corticosterone Infusion.
Rats received either a 30-min infusion of corticosterone (0.75 mg/mL corticosterone-2-hydroxypropyl-β-cyclodextrin; Sigma-Aldrich, Gillingham, UK) dissolved in sterile 0.45% (w/v) NaCl, or sterile 0.45% (w/v) NaCl at ZT2. A New Era NE-1800 computer-driven infusion pump (World Precision Instruments, Aston, UK) delivered 1 mL/h for 30 min via the indwelling jugular cannula. Rats were killed 2 h post infusion start at ZT4 (SI Appendix, Fig. S4↗).
MPL Treatment.
For all studies assessing the role of MPL, rats (randomly assigned to treatment) were given MPL sodium succinate (Solu-Medrol; Pharmacia Ltd, Sandwich, UK) 1 mg/mL ad libitum in drinking water for 5 d (prolonged treatment protocol), prior to the start of all further procedures. Concentrations of MPL in drinking water (1 mg/mL) were optimized in previous experiments as the minimum dose required to reproducibly induce hippocampal GR activation and suppress endogenous corticosterone (20 mg/day). Body weight was monitored throughout all procedures. Ad libitum access to MPL in drinking water was designed to limit any external stimuli that may act as a zeitgeber (88) and also represents greater than 1% of the human population that are prescribed oral glucocorticoid treatment (15). At this dose, no difference in fluid consumption was observed across days, and circulating endogenous corticosteroids (measured at the end of 5-d treatment) were suppressed (SI Appendix, Fig. S3↗).
Radioimmunoassay.
Using an automated gamma counter (PerkinElmer, US), a corticosterone radioimmunoassay measured endogenous levels in blood samples collected immediately following rapid decapitation. An 11-point standard curve of known corticosterone concentrations was prepared in B-buffer (25 mM tri-sodium citrate, 50 mM sodium dihydrogen orthophosphate, 1 mg/mL bovine serum albumin: pH3). Plasma obtained was diluted in triplicate at a ratio of either 1:10 or 1:50 in B-buffer. A specific corticosterone antibody (kindly provided by G. Makara, Institute of Experimental Medicine, Budapest, Hungary) was diluted at a ratio of 1:50 in B-buffer and 50 μL added to 100 μL standards, unknown samples, and Quality Control (QC20 and QC100) tubes. Tracer (Izotop, Institute of Isotopes, Hungary) was diluted in B-buffer to give total counts of 3,750 cpm in 50 μL and added to all tubes (50 μL/tube). Tubes were incubated overnight at 4 °C. Charcoal suspension (5 g charcoal added to 0.5 g dextran T70 dissolved in 1L B-buffer) was prepared and 500 μL was added to all tubes and briefly vortexed. Blocks were centrifuged at 4,000 rpm at 4 °C and the resulting supernatant was aspirated off. Unknown samples were determined from interpolation of the standard curve.
In Situ Hybridization Histochemistry.
Whole brains were cryosectioned into coronal 12 μm sections, and thaw mounted on gelatin/chrome alum-coated slides. The location of the SCN was determined according to coordinates in the rat brain atlas (89). 35S 3′- end-labeled deoxyoligonucleotide complementary to transcripts encoding Per1 (5′- CTC TTG TCA GGA GGA ATC CGG GGA GCT TCA TAA CCA GAG TGG ATG -3′), Per2 (5′- GTG GCC TTC CGG GAT GGG ATG TTG GCT GGG AAC TCG CAC TTT CTT -3′), and hnAVP (5′- GCA CTG TCA GCA GCC CTG AAC GGA CCA CAG TGG TAC -3′) was used. The in situ hybridization protocol has been previously described in detail (90). Briefly, sections were fixed in 4 % (w/v) formaldehyde for 5 min and incubated in saline containing 0.25 % (v/v) acetic anhydride and 0.1 M triethanolamine for 10 min. Sections were then dehydrated in ethanol, delipidated in chloroform, and partially rehydrated. Hybridization with a total count of 1 × 106 cpm was performed overnight at 37 °C in 45 μL hybridization buffer under Nescofilm (Bando Kagaku, Osaka, Japan). After hybridization, sections were washed 4 times with SSC (150 mM NaCl and 15 mM sodium citrate) for 1 h at 65 °C and for an additional hour with two changes of SSC at room temperature. Hybridized sections were exposed for autoradiography (Hyperfilm, Amersham, Bucks, UK) for 1 wk. The amount of bound probe was analyzed in comparison to 14C-labeled standards (Amersham, Bucks, UK) using image analysis software (NIH Image 1.6.2, W. Rasband, NIH, Bethesda, MD, USA). The obtained results were represented in arbitrary units setting the mean optical density (OD) obtained from sham-operated rats.
Chromatin Immunoprecipitation (ChIP).
Hippocampi were fixed for ChIP and processed to chromatin in sodium dodecyl sulfate (SDS) lysis buffer [2% SDS, 10 mM EDTA, 50 mM Tris-HCl (pH 8.1)] as previously described (91). Chromatin was sheared with a Sonifier 450 (Branson Ultrasonics, Danbury, CT) using 4× 10-s pulses at 10% output, and then cleared of cellular debris by centrifugation. For each IP, chromatin was diluted 1:10 in ChIP dilution buffer [167 mM NaCl, 16.7 mM Tris-HCl (pH 8.1), 1.1% Triton X-100, 1.2 mM EDTA, 0.01% SDS] supplemented with complete protease inhibitor (Sigma). Reactions were immunoprecipitated overnight at 4 °C with 2 µg anti-GR M-20X (Santa Cruz Biotechnology, US) or rabbit nonimmune serum (2µg sc-2027; Santa Cruz, USA) for the negative control. GR–DNA complexes were collected onto protein A–conjugated Dynabeads (Invitrogen, Paisley, UK) and washed to remove nonspecific binding. Purified DNA was resuspended in nuclease-free water (Ambion, Huntington, UK).
PCR primers (forward: 5′- CCAAGGCTGAGTGCATGTC -3′; reverse: 5′- GCGGCCAGCGCACTA -3′) were designed to amplify across a previously described GRE (20, 91) in the rat Period 1 gene promoter. Samples were amplified with Sybr Green master mix (Applied Biosystems) in accordance with the manufacturer’s instructions. GR binding for each sample was calculated relative to 1% input chromatin taken from each individual sample, using the %Input method, described in (ThermoFisher Scientific, http://bit.ly/ChIPAnalysisTFS↗).
RNA and qPCR.
Animals were anesthetized with isoflurane in the animal facility, and eight were killed every 4 h (six time points). The brain was quickly extracted, and the hippocampus was removed and rapidly frozen in liquid nitrogen. The time between decapitation and sample freezing was <1 min to limit RNA degradation. The collection of the hippocampal tissue per time point was <20 min (10 min either side of the hour mark).
Total RNA was extracted from frozen whole hippocampi using the miRNeasy total RNA extraction kit protocol (Qiagen, US) following the manufacturer’s guide and included a DNase digestion step. Samples were stored at −80 °C. All samples were assessed for RNA quality and quantity using a Nanodrop (ThermoScientific, US). Samples sent for RNAseq were further assessed for RNA integrity on the 2200 TapeStation system (Agilent, US). Sequenced samples had >8.0 RIN score.
Each PCR contained 1 µL cDNA, with a total volume of 10 µL. qPCR runs consisted of an initial 95 °C holding stage for 20 s, followed by 40 cycles of 95 °C (1 s) and 60 °C (20 s), followed by a melt curve step, consisting of 40 cycles of 95 °C (15 s) and 60 °C (1 min), with a final denaturing step of 95 °C (15 s) using a StepOnePlus PCR machine (Applied Biosystems, Life Technologies, UK).
Whole-Genome RNAseq and Analyses.
RNAseq of hippocampal tissue was carried out using TruSeq Stranded Total RNA kit and protocols (Illumina, US). A 1 μg aliquot total RNA from each hippocampus was prepared for RNAseq with 48 samples in total. First, bioanalyzer traces were carried out on all samples for RNA quantity and quality check. RIN scores above 8.0 were deemed high enough quality to take through to RNAseq. Following library preparation, the samples were run on a HiSeq 2500 machine (Illumina, US). Eight samples were run in each lane, in a paired-end sequencing run, generating approximately 300 million reads across the eight samples.
High-throughput RNAseq raw data were uploaded to Galaxy (92), an open-access portal for next-generation sequencing analysis. For the circadian sequencing experiments, an N of 4 was used in each group for a total of 48 whole hippocampi samples, and three lanes of data were collected for each sample. For the acute corticosterone administration experiments, an N of 4 was used in each group for a total of 8 whole hippocampi samples. Sequencing files were aligned to the Rattus norvegicus (Rn6) genome-generating individual Binary Alignment Map (BAM) files. These BAM files from each of the three lanes were merged. The merged BAM files were analyzed for gene expression differences using Tophat2, Cufflinks, and CuffDiff analyses aligning to the Rn6 genome (93). The parameters included geometric library organization and pooled dispersion estimation, and the false discovery rate was set at 0.05. Minimum alignment count—10, multi-read correct, bias correction and Cufflinks effective length correction were also included. Differential expression was assessed across time using CuffDiff version 15 (2020-06-16), with all time points compared to ZT 2. Any gene that failed DE analysis because of low gene expression [based on minimum alignment count (94, 95)] at any time point was removed from further analyses. Therefore, in the circadian RNAseq analysis–vehicle-treated rats, DE analysis was carried out on 13,939 genes. In MPL-treated rats, DE analysis was carried out on 16,269 genes. In the acute corticosterone administration to ADX rats, DE analysis was carried out on 16,266 genes. Differential gene expression was calculated from fragments per kilobase per million mapped reads values. Multiple hypothesis correction was carried out using the Benjamini–Hochberg test. Data deemed statistically significant, FDR < 0.05.
The Database for Annotation, Visualization and Integrated Discovery (96) was used to determine GO and pathways. GO categories tested included biological processes and pathways from the Kyoto Encyclopedia of Genes and Genomes (KEGG).
RNAseq Rhythmicity Analysis.
For CTL and MPL-treated groups, we focused on 24 h periodicity using harmonic regression on the log2 transformed signals, as previously described (52), from gene expression data sampled every 4 h. Each hour on the clock face refers to the total number of genes that peak at that time of day.
Brain Slice Preparation.
The brain was rapidly removed at either ZT0030 (30 to 60 min following light change–inactive phase recording) or ZT1230 (30 to 60 min following light change–active phase recording) (SI Appendix, Fig. S5↗). These times were chosen based upon our RNAseq data. Peak Per1 mRNA expression in controls was between ZT 10 and ZT 14. Nadir Per1 mRNA expression in controls was between ZT 22 and ZT 2. A minimum of 2 h after peak and trough expression was chosen to allow for functional protein availability. Slices placed in ice-cold slicing solution contained (in mM): 52.5 NaCl, 2.5 KCl, 25 NaHCO3, 1.25 NaH2PO4, 5 MgCl2, 25 D-Glucose, 100 sucrose, 2 CaCl2, 0.1 kynurenic acid, bubbled with 95% O2/5% CO2. Parasagittal slices (400 μm) were cut using a vibrating blade microtome (Leica VT1000 S) while maintained in slicing solution. The hippocampus was then isolated and placed in a holding chamber containing artificial cerebrospinal fluid (aCSF) (containing (in mM) 124 NaCl, 3 KCl, 26 NaHCO3, 1.4 NaH2PO4, 1 MgSO4, 10 D-Glucose, 2 CaCl2) where it was incubated at 32 °C for 30 min, followed by RT for at least 30 min (for whole-cell recordings), or kept at room temperature for at least 1 h (for field electrophysiology) before being transferred to the recording chamber.
Extracellular Electrophysiology.
Hippocampal slices were placed in a submersion style recording chamber maintained at 30 °C and continuously perfused with oxygenated aCSF at a flow rate of ~2 mL/min. Field excitatory postsynaptic potentials were evoked at 0.033 Hz by placing bipolar stimulating electrodes on the Schaffer collateral fibers with the recording electrode positioned in CA1 stratum radiatum. Recording electrodes were prepared by pulling borosilicate capillary tubes with a P-97 Flaming/Brown micropipette puller (Sutter Instrument Co) to a tip resistance of 2 to 6 MΩ and were then back-filled with aCSF. Signals were amplified using an AxoClamp 2B amplifier (Molecular Devices), digitized using a BNC-2110 (National Instruments) board, and 50/60 Hz noise eliminated by a Hum Bug (Quest Scientific). Data were acquired and analyzed using WinLTP software (97). Signals were averaged over a period of 2 min and a stable baseline recording of 30 min was acquired before LTP induction by delivery of 10 Hz stimulation for 90 s.
Whole-Cell Electrophysiology.
Whole-cell recordings were taken from pyramidal neurons in the CA1 cell layer. Borosilicate pipettes (2 to 6 MΩ) were filled with an internal solution containing (in mM): 8 NaCl, 130 CsMeSO4, 10 HEPES, 0.5 EGTA, 4 MgATP, 0.3 NaGTP, and 5 QX-314. Recordings were accepted for analysis with an uncompensated series resistance of <2.5 times the pipette resistance. Recordings were not corrected for series resistance due to the small current amplitudes. During mEPSC and mIPSC recordings, 100 µM D-AP5 was added to the perfused aCSF to block NMDA receptor-mediated currents. mEPSCs were recorded at a membrane potential of −70 mV and for mIPSCs, membrane potential was held at 0 mV. Recordings were amplified using an AxoClamp 700B (Molecular Devices) for whole-cell voltage-clamp recordings. Data were acquired using WinLTP software at a sampling rate of 10 KHz, and filtered at 6 KHz, before being analyzed offline using ClampFit 9.2. mEPSCs and mIPSCs were identified when the rise time was faster than the decay time and had a peak amplitude >6 mV.
Object Location Memory (OLM) Task.
For all memory testing, different rats were used at all time points and treatment. Rats were transferred to a sound-attenuating behavior room in low light (40 to 50 lx on arena floor) at ZT11.
Rats were left to habituate to the room for at least an hour, prior to starting behavioral experiments. Similarly to our electrophysiology studies, this time was chosen based upon our RNAseq data. Peak Per1 mRNA expression in controls was between ZT 10 and ZT 14. A minimum of 2 h after peak expression was chosen to allow for functional protein availability. Rats were handled for 1 wk in the behavior room prior to experimental start, followed by habituation to the arena without stimuli for 10 min daily for 5 d. Training and testing occurred in an open-top arena (50 × 90 × 100 cm) made of wood. The walls inside the arena were black and floor covered in sawdust. An overhead camera recorded behavior for analysis. Exploration was scored when the rat head orientated toward the object and came within 1 cm of the object. The objects were constructed from Duplo blocks, which were too heavy for the animals to displace. During training, rats were exposed to two identical objects and allowed to explore for 4 min. These objects were placed in the far side of the arena, 10 cm away from the walls, to allow full access around the objects. During the retention test (1 h for short-term memory, 6 h for intermediate memory, or 24 h for long-term memory), rats were allowed to explore for 3 min. During testing, one of the objects was moved to a new location. The position of the object was counterbalanced between rats. Total exploration time was recorded, and preference for the novel item was expressed as a discrimination index.
Activity and Temperature Recording.
All rats were individually housed for telemetry recordings for technical reasons. Rats were implanted intraperitoneally with telemeters (PTD 4000 E-mitter, Starr Life Sciences Corp, US). Following recovery (>3 d), cages were placed upon receivers (ER-4000 receiver, Starr Life Sciences Corp, US). Locomotor activity and core body temperature data were collected every 10 min for five consecutive days. Data collected were analyzed with R CRAN package cosinor to measure bathyphase, acrophase, and circadian period under a 24-h period.
Statistical Analyses.
Results are presented as mean ± SEM. All statistical analyses were performed using GraphPad Prism (v 9.1, GraphPad software Inc., US), with parametric and nonparametric tests used where appropriate. Details of specific tests are provided in the figure legends. Statistical significance was set at P < 0.05.