Chung S.,Sections on Lipid science |
Chung S.,University of Nebraska - Lincoln |
Chung S.,University of Florida |
Cuffe H.,Sections on Lipid science |
And 9 more authors.
Arteriosclerosis, Thrombosis, and Vascular Biology | Year: 2014
OBJECTIVE - Excessive caloric intake is associated with obesity and adipose tissue dysfunction. However, the role of dietary cholesterol in this process is unknown. The aim of this study was to determine whether increasing dietary cholesterol intake alters adipose tissue cholesterol content, adipocyte size, and endocrine function in nonhuman primates. APPROACH AND RESULTS - Age-matched, male African Green monkeys (n=5 per group) were assigned to 1 of 3 diets containing 0.002 (low [Lo]), 0.2 (medium [Med]), or 0.4 (high [Hi]) mg cholesterol/kcal. After 10 weeks of diet feeding, animals were euthanized for adipose tissue, liver, and plasma collection. With increasing dietary cholesterol, free cholesterol (FC) content and adipocyte size increased in a stepwise manner in visceral, but not in subcutaneous fat, with a significant association between visceral adipocyte size and FC content (r=0.298; n=15; P=0.035). In visceral fat, dietary cholesterol intake was associated with (1) increased proinflammatory gene expression and macrophage recruitment, (2) decreased expression of genes involved in cholesterol biosynthesis and lipoprotein uptake, and (3) increased expression of proteins involved in FC efflux. CONCLUSIONS - Increasing dietary cholesterol selectively increases visceral fat adipocyte size, FC and macrophage content, and proinflammatory gene expression in nonhuman primates. Visceral fat cells seem to compensate for increased dietary cholesterol by limiting cholesterol uptake/synthesis and increasing FC efflux pathways. © 2014 American Heart Association, Inc. Source
Ditewig A.C.,Cellular |
Bratcher N.A.,Office of Animal Welfare and Compliance |
Davila D.R.,Toxicology |
Dayton B.D.,630 East Golf Road |
And 6 more authors.
Journal of the American Association for Laboratory Animal Science | Year: 2014
Environmental enrichment in rodents may improve animal well-being but can affect neurologic development, immune system function, and aging. We tested the hypothesis that wood block enrichment affects the interpretation of traditional and transcriptomic endpoints in an exploratory toxicology testing model using a well-characterized reference compound, cyclophosphamide. ANOVA was performed to distinguish effects of wood block enrichment separate from effects of 40 mg/kg cyclophosphamide treatment. Biologically relevant and statistically significant effects of wood block enrichment occurred only for body weight gain. ANOVA demonstrated the expected effects of cyclophosphamide on food consumption, spleen weight, and hematology. According to transcriptomic endpoints, cyclophosphamide induced fewer changes in gene expression in liver than in spleen. Splenic transcriptomic pathways affected by cyclophosphamide included: iron hemostasis; vascular tissue angiotensin system; hepatic stellate cell activation and fibrosis; complement activation; TGFβ-induced hypertrophy and fibrosis; monocytes, macrophages, and atherosclerosis; and platelet activation. Changes in these pathways due to cyclophosphamide treatment were consistent with bone marrow toxicity regardless of enrichment. In a second study, neither enrichment nor type of cage flooring altered body weight or food consumption over a 28-d period after the first week. In conclusion, wood block enrichment did not interfere with a typical exploratory toxicology study; the effects of ingested wood on drug level kinetics may require further consideration. Copyright 2014 by the American Association for Laboratory Animal Science. Source
The new findings, based on wild house sparrows, and published today, show how changes in DNA that are linked to ageing and lifespan take place as body size gets bigger. Although larger types of animals tend to live longer than smaller ones – elephants live longer than mice – within many species the bigger individuals have shorter life spans than their smaller counterparts – a Jack Russell has a much longer life than a St Bernard. In humans, a recent study has shown that taller people are more prone to diseases including cancer. But biologists haven't been able to fully explain why. Research into telomeres, special DNA structures that all animals have at the ends of their chromosomes, described as functioning like "the protective plastic caps at the end of shoelaces" may provide the answer. The study, conducted jointly by the University of Glasgow's Institute of Biodiversity, Animal Health & Comparative Medicine and the Centre of Biodiversity Dynamics at the Norwegian University of Science and Technology, focused on a population of wild house sparrows on the isolated island of Leka in Norway. The research, published in the Proceedings of the Royal SocietyB: Biological Sciences, found that skeletally bigger house sparrows had shorter telomeres. This relationship was maintained during a period when a selective breeding programme on the island resulted in the sparrows becoming even larger. In tandem, their telomeres became even shorter. Everyone's telomeres erode over time, and telomere shortening has been linked to ageing and disease risk including cancer. Having naturally longer telomeres appears to give individuals an advantage when it comes to health and the biological aging process. The results shed light on a paradox that has puzzled biologists for a long time. If being bigger gives you a competitive advantage, why don't animals just get bigger and bigger? Part of the answer is that growing big can mean more telomere loss and faster ageing. Professor Pat Monaghan, Regius Chair of Zoology at the University of Glasgow, who supervised the telomere analysis, said: "Growing a bigger body means that cells have to divide more. As a result, telomeres become eroded faster and cells and tissues function less well as a result. "The reason why the bigger individuals have shorter telomeres might also be related to increased DNA damage due to growing faster. Being big can have advantages, of course, but this study shows that it can also have costs." Associate professor in population ecology Thor Harald Ringsby at Norwegian University of Science and Technology who was running the fieldwork together with his colleagues in Norway said: 'The results from this study are very exciting and broad reaching. It is especially interesting that we obtained these results in a natural population. The reduction in telomere size that followed the increase in body size suggests one important mechanism that limits body size evolution in wild animal populations" The study, entitled 'On being the right size: increased body size is associated with reduced telomere length under natural conditions' is published in the Proceedings of the Royal Society B: Biological Sciences journal. The research was funded by the European Research Council and the Research Council of Norway. Explore further: Researchers show telomere lengths predict life expectancy in the wild More information: On being the right size: increased body size is associated with reduced telomere length under natural conditons, Proceedings of the Royal Society B: Biological Sciences, rspb.royalsocietypublishing.org/lookup/doi/10.1098/rspb.2015.2331
While this event was first predicted almost twenty years ago, evidence for it has proved elusive. Now, researchers from the University of Glasgow have demonstrated the Meselson effect for the first time in any organism at a genome-wide level, studying a parasite called Trypanosoma brucei gambiense (T.b. gambiense). Their findings are to be published in the journal eLife. The research was conducted at the Wellcome Trust Centre for Molecular Parasitology in the University's Institute of Biodiversity Animal Health and Comparative Medicine. T.b. gambiense is responsible for causing African sleeping sickness in humans, leading to severe symptoms including fever, headaches, extreme fatigue, and aching muscles and joints, which do not occur until weeks or sometimes even months after infection. These symptoms extend to neurologic problems, such as progressive confusion and personality changes, when the infection invades the central nervous system. In order to demonstrate the Meselson effect in T.b. gambiense, the research team, led by Dr. Annette Macleod, sequenced the genomes of 85 isolates of the parasite, including multiple samples from disease focus points within Guinea, Cote d'Ivoire and Cameroon, collected over fifty years from 1952 to 2004. The similarity of the genomes studied from these different locations, together with a lack of recombination in the evolution of the parasite, suggests that this sub-species emerged from a single individual within the last 10,000 years. "It was around this time that livestock farming was developing in West Africa, allowing the parasite, which was originally an animal organism, to 'jump' from one species to the other via the Tsetse fly," says lead author Dr. Willie Weir. "Since then, mutations have built up and the lack of sexual recombination in T.b. gambiense means that the two chromosomes in each pair have evolved independently of each other, demonstrating the Meselson effect." Dr. Weir adds that the parasites' inability to recombine with each other prevents genes from being exchanged between strains. This could subsequently hamper the ability of the organism to develop resistance to multiple drugs. The team also uncovered evidence that the parasite uses gene conversion to compensate for its lack of sex. This mechanism essentially repairs the inferior, or mutated, copy of a gene on a chromosome by 'copying and pasting' the superior copy from the chromosome's partner. The future challenge will be to investigate the effectiveness of this mechanism in the long term, as evolutionary theory suggests that asexual organisms should eventually face extinction. If T.b. gambiense shares this fate, the major cause of African sleeping sickness will be eliminated - although it is impossible to predict when this might happen. Explore further: Sequence is scaffold to study sleeping sickness More information: William Weir et al. Population genomics reveals the origin and asexual evolution of human infective trypanosomes, eLife (2016). DOI: 10.7554/eLife.11473
The APP/PS1 (ref. 10) double-transgenic AD mice, originally described as Line 85, were obtained from Jackson Laboratory (stock number 004462). Under the control of mouse prion promoter elements, these mice express a chimaeric mouse/human APP transgene containing Swedish mutations (K595N/M596L) as well as a mutant human PS1 transgene (delta exon 9 variant). To label memory engram cells in APP/PS1 mice, we generated a triple-transgenic mouse line by mating c-Fos-tTA11, 28 transgenic mice with APP/PS1 double-transgenic mice. The PS1/APP/tau18 triple-transgenic AD mice were obtained from Jackson Laboratory (stock number 004807). These 3×Tg-AD mice express a mutant human PS1 transgene (M146V), a human APP transgene containing Swedish mutations (KM670/671NL) and a human MAPT transgene harbouring the P301L mutation. All mouse lines were maintained as hemizygotes. Mice had access to food and water ad libitum and were socially housed in numbers of two to five littermates until surgery. After surgery, mice were singly housed. For behavioural experiments, all mice were male and 7–9 months old. For optogenetic experiments, mice had been raised on food containing 40 mg kg−1 DOX for at least 1 week before surgery, and remained on DOX for the remainder of the experiments except for the target engram labelling days. For in vitro electrophysiology experiments, mice were 24–28 days old at the time of surgery. All experiments were conducted in accordance with US National Institutes of Health (NIH) guidelines and the Massachusetts Institute of Technology Department of Comparative Medicine and Committee of Animal Care. No statistical methods were used to predetermine sample size. Our previously established method11 for labelling memory engram cells combined c-Fos-tTA transgenic mice with a DOX-sensitive adeno-associated virus (AAV). However, in this study, we modified the method using a double-virus system to label memory engram cells in the early AD mice, which already carry two transgenes. The pAAV-c-Fos-tTA plasmid was constructed by cloning a 1 kb fragment from the c-Fos gene (550 bp upstream of c-Fos exon I to 35 bp into exon II) into an AAV backbone using the KpnI restriction site at the 5′ terminus and the SpeI restriction site at the 3′ terminus. The AAV backbone contained the tTA-Advanced29 sequence at the SpeI restriction site. The pAAV-TRE-ChR2-eYFP and pAAV-TRE-eYFP constructs were previously described11, 12. The pAAV-TRE-oChIEF-tdTomato20 plasmid was constructed by replacing the ChR2-eYFP fragment from the pAAV-TRE-ChR2-eYFP plasmid using NheI and MfeI restriction sites. The pAAV-CaMKII-oChIEF-tdTomato plasmid was constructed by replacing the TRE fragment from the pAAV-TRE-oChIEF-tdTomato plasmid using BamHI and EcoRI restriction sites. The pAAV-TRE-DTR-eYFP25 plasmid was constructed by replacing the ChR2 fragment from the pAAV-TRE-ChR2-eYFP plasmid using EcoRI and AgeI restriction sites. AAV vectors were serotyped with AAV coat proteins and packaged at the University of Massachusetts Medical School Gene Therapy Center and Vector Core. Viral titres were 1.5 × 1013 genome copy (GC) ml−1 for AAV -c-Fos-tTA, AAV -TRE-ChR2-eYFP and AAV -TRE-eYFP, 1 × 1013 GC ml−1 for AAV -TRE-oChIEF-tdTomato, 4 × 1013 GC ml−1 for AAV -CaMKII-oChIEF-tdTomato and 2 × 1013 GC ml−1 for AAV -TRE-DTR-eYFP. Mice were anaesthetized with isoflurane or 500 mg kg−1 avertin for stereotaxic injections14. Injections were targeted bilaterally to the DG (−2.0 mm anteroposterior (AP), ±1.3 mm mediolateral (ML), −1.9 mm dorsoventral (DV)), MEC (−4.7 mm AP, ±3.35 mm ML, −3.3 mm DV) and LEC (−3.4 mm AP, ±4.3 mm ML, −4.0 mm DV). Injection volumes were 300 nl for DG and 400 nl for MEC and LEC. Viruses were injected at 70 nl min−1 using a glass micropipette attached to a 10 ml Hamilton microsyringe. The needle was lowered to the target site and remained for 5 min before beginning the injection. After the injection, the needle stayed for 10 min before it was withdrawn. A custom DG implant containing two optic fibres (200 mm core diameter; Doric Lenses) was lowered above the injection site (−2.0 mm AP, ±1.3 mm ML, −1.7 mm DV). The implant was secured to the skull with two jewellery screws, adhesive cement (C&B Metabond) and dental cement. An opaque cap derived from the top part of an Eppendorf tube protected the implant. Mice were given 1.5 mg kg−1 metacam as analgesic and allowed to recover for 2 weeks before behavioural experiments. All injection sites were verified histologically. As criteria, we only included mice with virus expression limited to the targeted regions. For seizure experiments11, mice were taken off DOX for 1 day and injected intraperitoneally with 15 mg kg−1 kainic acid (KA). Mice were returned to DOX food 6 h after KA treatment and perfused the next day for immunohistochemistry procedures. Mice were dispatched using 750–1,000 mg kg−1 avertin and perfused transcardially with PBS, followed by 4% paraformaldehyde (PFA). Brains were extracted and incubated in 4% PFA at room temperature overnight. Brains were transferred to PBS and 50-μm coronal slices were prepared using a vibratome. For immunostaining14, each slice was placed in PBS + 0.2% Triton X-100 (PBS-T), with 5% normal goat serum for 1 h and then incubated with primary antibody at 4 °C for 24 h. Slices then underwent three wash steps for 10 min each in PBS-T, followed by 1 h incubation with secondary antibody. After three more wash steps of 10 min each in PBS-T, slices were mounted on microscope slides. All analyses were performed blind to the experimental conditions. Antibodies used for staining were as follows: to stain for ChR2–eYFP, DTR–eYFP or eYFP alone, slices were incubated with primary chicken anti-GFP (1:1,000, Life Technologies) and visualized using anti-chicken Alexa-488 (1:200). For plaques, slices were stained using primary mouse anti-β-amyloid (1:1,000; Sigma-Aldrich) and secondary anti-mouse Alexa-488 (1:500). c-Fos was stained with rabbit anti-c-Fos (1:500, Calbiochem) and anti-rabbit Alexa-568 (1:300). Adult newborn neurons were stained with guinea pig anti-DCX (1:1,000; Millipore) and anti-guinea-pig Alexa-555 (1:500). Neuronal nuclei were stained with mouse anti-NeuN (1:200; Millipore) and Alexa-488 (1:200). DG mossy cell axons were stained with mouse anti-CR (1:1,000; Swant) and Alexa-555 (1:300). To characterize the expression pattern of ChR2–eYFP, DTR–eYFP, eYFP alone and oChIEF-tdTomato in control and AD mice, the number of eYFP+/tdTomato+ neurons were counted from 4–5 coronal slices per mouse (n = 3–5 mice per group). Coronal slices centred on coordinates covered by optic fibre implants were taken for DG quantification and sagittal slices centred on injection coordinates were taken for MEC and LEC. Fluorescence images were acquired using a Zeiss AxioImager.Z1/ApoTome microscope (×20). Automated cell counting analysis was performed using ImageJ software. The cell body layers of DG granule cells (upper blade), MEC or LEC cells were outlined as a region of interest (ROI) according to the DAPI signal in each slice. The number of eYFP+/tdTomato+ cells per section was calculated by applying a threshold above background fluorescence. Data were analysed using Microsoft Excel with the Statplus plug-in. A similar approach was applied for quantifying amyloid-β plaques, c-Fos+ neurons and adult newborn (DCX+) neurons. Total engram cell reactivation was calculated as ((c-Fos+ eYFP+)/(total DAPI+)) × 100. Chance overlap was calculated as ((c-Fos+/total DAPI+) × (eYFP+/total DAPI+)) × 100. Percentage of adult newborn neurons expressing neuronal markers was calculated as ((NeuN+ DCX+)/(total DCX+) × 100. DAPI+ counts were approximated from five coronal/sagittal slices using ImageJ. All counting experiments were conducted blind to experimental group. Researcher 1 trained the animals, prepared slices and randomized images, while researcher 2 performed semi-automated cell counting. Statistical comparisons were performed using unpaired t-tests: *P < 0.05, **P < 0.01, ***P < 0.001. Engram cells were labelled using c-Fos-tTA-driven synthesis of ChR2–eYFP or eYFP alone. The eYFP signal was amplified using immunohistochemistry procedures, after which fluorescence z-stacks were taken by confocal microscopy (Zeiss LSM700) using a ×40 objective. Maximum intensity projections were generated using ZEN Black software (Zeiss). Four mice per experimental group were analysed for dendritic spines. For each mouse, 30–40 dendritic fragments of 10-μm length were quantified (n = 120–160 fragments per group). To measure spine density of DG engram cells with a focus on entorhinal cortical inputs, distal dendritic fragments in the middle-to-outer molecular layer (ML) were selected. For CA3 and CA1 engram cells, apical and basal dendritic fragments were selected. To compute spine density, the number of spines counted on each fragment was normalized by the cylindrical approximation of the surface of the specific fragment. Experiments were conducted blind to experimental group. Researcher 1 imaged dendritic fragments and randomized images, while researcher 2 performed manual spine counting. After isoflurane anaesthesia, brains were quickly removed and used to prepare sagittal slices (300 μm) in an oxygenated cutting solution at 4 °C with a vibratome14. Slices were incubated at room temperature in oxygenated artificial cerebrospinal fluid (ACSF) until the recordings. The cutting solution contained (in mM): 3 KCl, 0.5 CaCl , 10 MgCl , 25 NaHCO , 1.2 NaH PO , 10 d-glucose, 230 sucrose, saturated with 95% O –5% CO (pH 7.3, osmolarity of 340 mOsm). The ACSF contained (in mM): 124 NaCl, 3 KCl, 2 CaCl , 1.3 MgSO , 25 NaHCO , 1.2 NaH PO , 10 d-glucose, saturated with 95% O –5% CO (pH 7.3, 300 mOsm). Individual slices were transferred to a submerged experimental chamber and perfused with oxygenated ACSF warmed at 35 °C (±0.5 °C) at a rate of 3 ml min−1 during recordings. Current or voltage clamp recordings were performed under an IR-DIC microscope (Olympus) with a ×40 water immersion objective (0.8 NA), equipped with four automatic manipulators (Luigs & Neumann) and a CCD camera (Hamamatsu). Borosilicate glass pipettes (Sutter Instruments) were fabricated with resistances of 8–10 MΩ. The intracellular solution (in mM) for current clamp recordings was: 110 K-gluconate, 10 KCl, 10 HEPES, 4 ATP, 0.3 GTP, 10 phosphocreatine, 0.5% biocytin (pH 7.25, 290 mOsm). Recordings used two dual channel amplifiers (Molecular Devices), a 2 kHz filter, 20 kHz digitization and an ADC/DAC data acquisition unit (Instrutech) running on custom software in Igor Pro (Wavemetrics). Data acquisition was suspended whenever the resting membrane potential was depolarized above −50 mV or the access resistance (RA) exceeded 20 MΩ. Optogenetic stimulation was achieved using a 460 nm LED light source (Lumen Dynamics) driven by TTL input with a delay onset of 25 μs (subtracted offline for latency estimation). Light power on the sample was 33 mW mm−2. To test oChIEF expression, EC cells were stimulated with a single light pulse of 1 s, repeated 10 times every 5 s. DG granule cells were held at −70 mV. Optical LTP protocol: 5 min baseline (10 blue light pulses of 2 ms each, repeated every 30 s) was acquired before the onset of the LTP protocol (100 blue light pulses of 2 ms each at a frequency of 100 Hz, repeated 5 times every 3 min) and the effect on synaptic amplitude was recorded for 30 min (1 pulse of 2 ms every 30 s). Using the 5 min baseline recording data, EPSPs were normalized (Fig. 3j). Potentiation was observed in 6 out of 30 cells and results were statistically confirmed using a two-tailed paired t-test. Experiments were performed in the presence of 10 μM gabazine (Tocris) and 2 μM CGP55845 (Tocris). Recorded cells were recovered for morphological identification using streptavidin CF633 (Biotium). Multi-unit responses to optical stimulation were recorded in the DG of mice injected with a cocktail of AAV -c-Fos-tTA and AAV -TRE-oChIEF-tdTomato viruses into MEC/LEC. Mice were anaesthetized (10 ml kg−1) using a mixture of ketamine (100 mg ml−1)/xylazine (20 mg ml−1) and placed in the stereotactic system. Anaesthesia was maintained by booster doses of ketamine (100 mg kg−1). An optrode consisting of a tungsten electrode (0.5 MΩ) attached to an optic fibre (200-μm core diameter), with the tip of the electrode extending beyond the tip of the fibre by 300 μm, was used for simultaneous optical stimulation and extracellular recording. The power intensity of light emitted from the optrode was calibrated to about 10 mW, consistent with the power used in behavioural assays. oChIEF+ cells were identified by delivering 20-ms light pulses (1 Hz) to the recording site every 50–100 μm. After light-responsive cells were detected, multi-unit activity in response to trains of light pulses (200 ms) at 100 Hz was recorded. Data acquisition used an Axon CNS Digidata 1440A system. MATLAB analysis was performed, as previously described12. Experiments were conducted during the light cycle (7 a.m. to 7 p.m.). Mice were randomly assigned to experimental groups for specific behavioural assays immediately after surgery. Mice were habituated to investigator handling for 1–2 min on three consecutive days. Handling took place in the holding room where the mice were housed. Before each handling session, mice were transported by wheeled cart to and from the vicinity of the behaviour rooms to habituate them to the journey. For natural memory recall sessions, data were quantified using FreezeFrame software. Optogenetic stimulation interfered with the motion detection, and therefore all light-induced freezing behaviour was manually quantified. All behaviour experiments were analysed blind to experimental group. Unpaired Student’s t-tests were used for independent group comparisons, with Welch’s correction when group variances were significantly different. Given behavioural variability, initial assays were performed using a minimum of 10 mice per group to ensure adequate power for any observed differences. Experiments that resulted in significant behavioural effects were replicated three times in the laboratory. Following behavioural protocols, brain sections were prepared to confirm efficient viral labelling in target areas. Animals lacking adequate labelling were excluded before behaviour quantification. Two distinct contexts were employed14. Context A was 29 × 25 × 22 cm chambers with grid floors, opaque triangular ceilings, red lighting, and scented with 1% acetic acid. Four mice were run simultaneously in four identical context A chambers. Context B consisted of four 30 × 25 × 33 cm chambers with perspex floors, transparent square ceilings, bright white lighting, and scented with 0.25% benzaldehyde. All mice were conditioned in context A (two 0.60 mA shocks of 2 s duration in 5 min), and tested (3 min) in contexts A and B 1 day later. Experiments showed no generalization in the neutral context B. All experimental groups were counter-balanced for chamber within contexts. Floors of chambers were cleaned with quatricide before and between runs. Mice were transported to and from the experimental room in their home cages using a wheeled cart. The cart and cages remained in an anteroom to the experimental rooms during all behavioural experiments. For engram labelling, mice were kept on regular food without DOX for 24 h before training. When training was complete, mice were switched back to food containing 40 mg kg−1 DOX. Spontaneous motor activity was measured in an open field arena (52 × 26 cm) for 10 min. All mice were transferred to the testing room and acclimated for 30 min before the test session. During the testing period, lighting in the room was turned off. The apparatus was cleaned with quatricide before and between runs. Total movements (distance travelled and velocity) in the arena were quantified using an automated infrared (IR) detection system (EthoVision XT, Noldus). The tracking software plotted heat maps for each mouse, which was averaged to create representative heat maps for each genotype. Raw data were extracted and analysed using Microsoft Excel. For light-induced freezing behaviour, a context distinct from the CFC training chamber (context A) was used. These were 30 × 25 × 33 cm chambers with perspex floors, square ceilings, white lighting, and scented with 0.25% benzaldehyde. Chamber ceilings were customized to hold a rotary joint (Doric Lenses) connected to two 0.32-m patch cords. All mice had patch cords fitted to the optic fibre implant before testing. Two mice were run simultaneously in two identical chambers. ChR2 was stimulated at 20 Hz (15 ms pulse width) using a 473 nm laser (10–15 mW), for the designated epochs. Testing sessions were 12 min in duration, consisting of four 3 min epochs, with the first and third as light-off epochs, and the second and fourth as light-on epochs. At the end of 12 min, the mouse was detached and returned to its home cage. Floors of chambers were cleaned with quatricide before and between runs. One day after CFC training and engram labelling (DG plus PP terminals) in control and early AD groups, mice were placed in an open field arena (52 × 26 cm) after patch cords were fitted to the fibre implants. After a 15 min acclimatization period, mice with oChIEF+ PP engram terminals in the DG received the optical LTP23 protocol (100 blue light pulses of 2 ms each at a frequency of 100 Hz, repeated 5 times every 3 min). This in vivo protocol was repeated 10 times over a 3 h duration. After induction, mice remained in the arena for an additional 15 min before returning to their home cage. To apply optical LTP to a large portion of excitatory MEC neurons, an AAV virus expressing oChIEF-tdTomato under the CaMKII promoter, rather than a c-Fos-tTA/TRE virus (that is, engram labelling), was used. For protein synthesis inhibition experiments, immediately after the in vivo LTP induction protocol mice received 75 mg kg−1 anisomycin (Aniso) or an equivalent volume of saline intraperitoneally. Mice were then returned to their home cages. An hour later, a second injection of Aniso or saline was delivered. A 30 × 28 × 34 cm unscented chamber with transparent square ceilings and intermediate lighting was used. The chamber consisted of two sections, one with grid flooring and the other with a white light platform. During the conditioning session (1 min), mice were placed on the light platform, which is the less preferred section of the chamber (relative to the grid section). Once mice entered the grid section of the chamber (all four feet), 0.80 mA shocks of 2 s duration were delivered. On average, each mouse received 2–3 shocks per training session. After 1 min, mice were returned to their home cage. The next day, latency to enter the grid section of the chamber as well as total time on the light platform was measured (3 min test). Spatial memory was measured in a white plastic chamber (28 × 28 cm) that had patterns (series of parallel lines or circles) on opposite walls. The apparatus was unscented and intermediate lighting was used. All mice were transferred to the behavioural room and acclimated for 30 min before the training session. On day 1, mice were allowed to explore the chamber with patterns for 15 min. On days 2 and 3, mice were introduced into the chamber that had an object (7-cm-tall glass flask filled with metal beads) placed adjacent to either patterned wall. The position of the object was counter-balanced within each genotype. On day 4, mice were placed into the chamber with the object either in the same position as the previous exposure (familiar) or at a novel location based on wall patterning. Frequency of visits to the familiar and novel object locations was quantified using an automated detection system (EthoVision XT, Noldus). Total time exploring the object was also measured (nose within 1.5 cm of object). The tracking software plotted heat maps based on exploration time, which was averaged to create representative heat maps for each genotype. Raw data were extracted and analysed using Microsoft Excel.