Neurochemical, histological, and behavioral profiling of the acute, sub-acute, and chronic MPTP mouse model of Parkinson's disease.

Parkinson's disease (PD) is a heterogeneous multi-systemic disorder unique to humans characterized by motor and non-motor symptoms. Preclinical experimental models of PD present limitations and inconsistent neurochemical, histological, and behavioral readouts. The 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD is the most common in vivo screening platform for novel drug therapies; nonetheless, behavioral endpoints yielded amongst laboratories are often discordant and inconclusive. In this study, we characterized neurochemically, histologically, and behaviorally three different MPTP mouse models of PD to identify translational traits reminiscent of PD symptomatology. MPTP was intraperitoneally (i.p.) administered in three different regimens: (i) acute-four injections of 20 mg/kg of MPTP every 2 h; (ii) sub-acute-one daily injection of 30 mg/kg of MPTP for 5 consecutive days; and (iii) chronic-one daily injection of 4 mg/kg of MPTP for 28 consecutive days. A series of behavioral tests were conducted to assess motor and non-motor behavioral changes including anxiety, endurance, gait, motor deficits, cognitive impairment, circadian rhythm and food consumption. Impairments in balance and gait were confirmed in the chronic and acute models, respectively, with the latter showing significant correlation with lesion size. The sub-acute model, by contrast, presented with generalized hyperactivity. Both, motor and non-motor changes were identified in the acute and sub-acute regime where habituation to a novel environment was significantly reduced. Moreover, we report increased water and food intake across all three models. Overall, the acute model displayed the most severe lesion size, while across the three models striatal dopamine content (DA) did not correlate with the behavioral performance. The present study demonstrates that detection of behavioral changes following MPTP exposure is challenging and does not correlate with the dopaminergic lesion extent.

Cu, Fe, and Zn isotope ratios in murine Alzheimer's disease models suggest specific signatures of amyloidogenesis and tauopathy.

Alzheimer's disease (AD) is characterized by accumulation of tau and amyloid-beta in the brain, and recent evidence suggests a correlation between associated protein aggregates and trace elements, such as copper, iron, and zinc. In AD, a distorted brain redox homeostasis and complexation by amyloid-beta and hyperphosphorylated tau may alter the isotopic composition of essential mineral elements. Therefore, high-precision isotopic analysis may reveal changes in the homeostasis of these elements. We used inductively coupled plasma-mass spectrometry (ICP-MS)-based techniques to determine the total Cu, Fe, and Zn contents in the brain, as well as their isotopic compositions in both mouse brain and serum. Results for male transgenic tau (Line 66, L66) and amyloid/presenilin (5xFAD) mice were compared with those for the corresponding age- and sex-matched wild-type control mice (WT). Our data show that L66 brains showed significantly higher Fe levels than did those from the corresponding WT. Significantly less Cu, but more Zn was found in 5xFAD brains. We observed significantly lighter isotopic compositions of Fe (enrichment in the lighter isotopes) in the brain and serum of L66 mice compared with WT. For 5xFAD mice, Zn exhibited a trend toward a lighter isotopic composition in the brain and a heavier isotopic composition in serum compared with WT. Neither mouse model yielded differences in the isotopic composition of Cu. Our findings indicate significant pathology-specific alterations of Fe and Zn brain homeostasis in mouse models of AD. The associated changes in isotopic composition may serve as a marker for proteinopathies underlying AD and other types of dementia.

High-fat diet exacerbates cognitive and metabolic abnormalities in neuronal BACE1 knock-in mice - partial prevention by Fenretinide.

Objective: The ß-site APP-cleaving enzyme 1 (BACE1) is a rate-limiting step in ß-amyloid (Aß) production in Alzheimer's disease (AD) brains, but recent evidence suggests that BACE1 is also involved in metabolic regulation. Here, we aimed to assess the effects of highfat diet (HFD) on metabolic and cognitive phenotypes in the diabetic BACE1 knock-in mice (PLB4) and WT controls; we additionally examined whether these phenotypes can be normalized with a synthetic retinoid (Fenretinide, Fen) targeting weight loss.Methods: Five-month old male WT and PLB4 mice were fed either (1) control chow diet, (2) 45%-saturated fat diet (HFD), (3) HFD with 0.04% Fen (HFD + Fen) or (4) control chow diet with 0.04% Fen (Fen) for 10 weeks. We assessed basic metabolic parameters, circadian rhythmicity, spatial habituation (Phenotyper) and working memory (Y-maze). Hypothalami, forebrain and liver tissues were assessed using Western blots, qPCR and ELISAs.Results: HFD feeding drastically worsened metabolism and induced early mortality (-40%) in otherwise viable PLB4 mice. This was ameliorated by Fen, despite no effects on glucose intolerance. In HFD-fed WT mice, Fen reduced weight gain, glucose intolerance and hepatic steatosis. The physiological changes induced in WT and PLB4 mice by HFD (+/-Fen) were accompanied by enhanced cerebral astrogliosis, elevated PTP1B, phopsho-eIF2α and altered hypothalamic transcription of Bace1, Pomc and Mc4r. Behaviourally, HFD feeding exacerbated spatial memory deficits in PLB4 mice, which was prevented by Fen and linked with increased full-length APP, normalized brain Aß*56 oligomerization and astrogliosis.Conclusions: HFD induces early mortality and worsened cognition in the Alzheimer's-like BACE1 mice- partial prevention was achieved with Fenretinide, without improvements in glucose homeostasis.

Sequential habituation to space, object and stranger is differentially modulated by glutamatergic, cholinergic and dopaminergic transmission.

Novel object and social interaction tasks allow assessments of rodent cognition and social behavior. Here, we combined these tasks and defined unequivocal locations of interest. Our procedure, termed OF-NO-SI, comprised habituation to the open field (OF), novel object (NO) and social interaction (SI) stages. Habituation was measured within- and between-trials (10 minutes each, two per stage). Ambulation emerged as the appropriate proxy during the OF stage, but NO and SI trials were best quantified via direct exploration measures. We pharmacologically validated the paradigm using 5-month old C57BL/6J male mice, treated intraperitoneally with (1) 0.5 mg/kg scopolamine, (2) 0.05 mg/kg MK-801 and (3) 0.05 mg/kg SCH-23390 to block muscarinic (M1), NMDA, and D1 receptors, respectively, or (4) vehicle (distilled water). Activity and gross exploratory behavior were affected by all compounds cf. vehicle: scopolamine and MK-801 cohorts were hyperactive, while SCH-23390 caused hypo-locomotion throughout. Vehicle treated mice showed reliable habituation to all stages for time in interaction zone, directed exploration and number of visits. Exploration was severely impaired by scopolamine. MK-801 mostly affected within-session exploration but also increased exploration of the conspecific compared to the object. Interestingly, even though within-trial habituation was lacking in the SCH-23390 cohort, between-trial habituation was largely intact, despite reduced locomotion. Our data suggest that the OF-NO-SI task is a convenient and robust paradigm to measure habituation to different experimental settings and stimuli. It allows the dissociation of proxies related to activity and non-associative learning/memory, as revealed by distinct pharmacological treatment effects within- vs. between-trials.

Brain region-specific lipid alterations in the PLB4 hBACE1 knock-in mouse model of Alzheimer's disease.

BACKGROUND: Lipid dysregulation is associated with several key characteristics of Alzheimer's disease (AD), including amyloid-ß and tau neuropathology, neurodegeneration, glucose hypometabolism, as well as synaptic and mitochondrial dysfunction. The ß-site amyloid precursor protein cleavage enzyme 1 (BACE1) is associated with increased amyloidogenesis, and has been affiliated with diabetes via its role in metabolic regulation. METHODS: The research presented herein investigates the role of hBACE1 in lipid metabolism and whether specific brain regions show increased vulnerability to lipid dysregulation. By utilising advanced mass spectrometry techniques, a comprehensive, quantitative lipidomics analysis was performed to investigate the phospholipid, sterol, and fatty acid profiles of the brain from the well-known PLB4 hBACE1 knock-in mouse model of AD, which also shows a diabetic phenotype, to provide insight into regional alterations in lipid metabolism. RESULTS: Results show extensive region - specific lipid alterations in the PLB4 brain compared to the wild-type, with decreases in the phosphatidylethanolamine content of the cortex and triacylglycerol content of the hippocampus and hypothalamus, but increases in the phosphatidylcholine, phosphatidylinositol, and diacylglycerol content of the hippocampus. Several sterol and fatty acids were also specifically decreased in the PLB4 hippocampus. CONCLUSION: Collectively, the lipid alterations observed in the PLB4 hBACE1 knock-in AD mouse model highlights the regional vulnerability of the brain, in particular the hippocampus and hypothalamus, to lipid dysregulation, hence supports the premise that metabolic abnormalities have a central role in both AD and diabetes.

Alzheimer's disease pathology and the unfolded protein response: prospective pathways and therapeutic targets.

Many vital interdependent cellular functions including proteostasis, lipogenesis and Ca homeostasis are executed by the endoplasmic reticulum (ER). Exogenous insults can impair ER performance: this must be rapidly corrected or cell death will ensue. Protective adaptations can boost the functional capacity of the ER and form the basis of the unfolded protein response (UPR). Activated in response to the accumulation of misfolded proteins, the UPR can halt protein translation while increasing protein-handling chaperones and the degradation of erroneous proteins through a conserved three-tier molecular cascade. However, prolonged activation of the UPR can result in the maladaptation of the system, resulting in the activation of inflammatory and apoptotic effectors. Recently, UPR and its involvement in neurodegenerative disease has attracted much interest and numerous potentially 'drugable' points of crosstalk are now emerging. Here, we summarize the functions of the ER and UPR, and highlight evidence for its potential role in the pathogenesis of Alzheimer's disease, before discussing several key targets with therapeutic potential.

Neuronal human BACE1 knockin induces systemic diabetes in mice.

AIMS: ß-Secretase 1 (BACE1) is a key enzyme in Alzheimer's disease pathogenesis that catalyses the amyloidogenic cleavage of amyloid precursor protein (APP). Recently, global Bace1 deletion was shown to protect against diet-induced obesity and diabetes, suggesting that BACE1 is a potential regulator of glucose homeostasis. Here, we investigated whether increased neuronal BACE1 is sufficient to alter systemic glucose metabolism, using a neuron-specific human BACE1 knockin mouse model (PLB4). METHODS: Glucose homeostasis and adiposity were determined by glucose tolerance tests and EchoMRI, lipid species were measured by quantitative lipidomics, and biochemical and molecular alterations were assessed by western blotting, quantitative PCR and ELISAs. Glucose uptake in the brain and upper body was measured via (18)FDG-PET imaging. RESULTS: Physiological and molecular analyses demonstrated that centrally expressed human BACE1 induced systemic glucose intolerance in mice from 4 months of age onward, alongside a fatty liver phenotype and impaired hepatic glycogen storage. This diabetic phenotype was associated with hypothalamic pathology, i.e. deregulation of the melanocortin system, and advanced endoplasmic reticulum (ER) stress indicated by elevated central C/EBP homologous protein (CHOP) signalling and hyperphosphorylation of its regulator eukaryotic translation initiation factor 2α (eIF2α). In vivo (18)FDG-PET imaging further confirmed brain glucose hypometabolism in these mice; this corresponded with altered neuronal insulin-related signalling, enhanced protein tyrosine phosphatase 1B (PTP1B) and retinol-binding protein 4 (RBP4) levels, along with upregulation of the ribosomal protein and lipid translation machinery. Increased forebrain and plasma lipid accumulation (i.e. ceramides, triacylglycerols, phospholipids) was identified via lipidomics analysis. CONCLUSIONS/INTERPRETATION: Our data reveal that neuronal BACE1 is a key regulator of metabolic homeostasis and provide a potential mechanism for the high prevalence of metabolic disturbance in Alzheimer's disease.

Mutant Tau knock-in mice display frontotemporal dementia relevant behaviour and histopathology.

Models of Tau pathology related to frontotemporal dementia (FTD) are essential to determine underlying neurodegenerative pathologies and resulting tauopathy relevant behavioural changes. However, existing models are often limited in their translational value due to Tau overexpression, and the frequent occurrence of motor deficits which prevent comprehensive behavioural assessments. In order to address these limitations, a forebrain-specific (CaMKIIα promoter), human mutated Tau (hTauP301L+R406W) knock-in mouse was generated out of the previously characterised PLB1Triple mouse, and named PLB2Tau. After confirmation of an additional hTau species (~60kDa) in forebrain samples, we identified age-dependent progressive Tau phosphorylation which coincided with the emergence of FTD relevant behavioural traits. In line with the non-cognitive symptomatology of FTD, PLB2Tau mice demonstrated early emerging (~6months) phenotypes of heightened anxiety in the elevated plus maze, depressive/apathetic behaviour in a sucrose preference test and generally reduced exploratory activity in the absence of motor impairments. Investigations of cognitive performance indicated prominent dysfunctions in semantic memory, as assessed by social transmission of food preference, and in behavioural flexibility during spatial reversal learning in a home cage corner-learning task. Spatial learning was only mildly affected and task-specific, with impairments at 12months of age in the corner learning but not in the water maze task. Electroencephalographic (EEG) investigations indicated a vigilance-stage specific loss of alpha power during wakefulness at both parietal and prefrontal recording sites, and site-specific EEG changes during non-rapid eye movement sleep (prefrontal) and rapid eye movement sleep (parietal). Further investigation of hippocampal electrophysiology conducted in slice preparations indicated a modest reduction in efficacy of synaptic transmission in the absence of altered synaptic plasticity. Together, our data demonstrate that the transgenic PLB2Tau mouse model presents with a striking behavioural and physiological face validity relevant for FTD, driven by the low level expression of mutant FTD hTau.

Soluble pre-fibrillar tau and ß-amyloid species emerge in early human Alzheimer's disease and track disease progression and cognitive decline.

Post-mortem investigations of human Alzheimer's disease (AD) have largely failed to provide unequivocal evidence in support of the original amyloid cascade hypothesis, which postulated deposition of ß-amyloid (Aß) aggregates to be the cause of a demented state as well as inductive to tau neurofibrillary tangles (NFTs). Conflicting evidence suggests, however, that Aß plaques and NFTs, albeit to a lesser extent, are present in a substantial subset of non-demented individuals. Hence, a range of soluble tau and Aß species has more recently been implicated as the disease-relevant toxic entities. Despite the incorporation of soluble proteins into a revised amyloid cascade hypothesis, a detailed characterization of these species in the context of human AD onset, progression and cognitive decline has been lacking. Here, lateral temporal lobe samples (Brodmann area 21) of 46 human cases were profiled via tau and Aß Western blot and native state dot blot protocols. Elevations in phospho-tau (antibodies: CP13, AT8 and PHF-1), pathological tau conformations (MC-1) and oligomeric tau (TOC1) agreed with medical diagnosis (non-AD cf. AD) and Braak stage classification (low, intermediate and high), alongside elevations in soluble Aß species (MOAB-2 and pyro-glu Aß) and a decline in levels of the amyloid precursor protein. Strong correlations were observed between individual Braak stages and multiple cognitive measures with all tau markers as well as total soluble Aß. In contrast to previous reports, SDS-stable Aß oligomers (*56) were not found to be reliable for all classifications and appeared likely to be a technical artefact. Critically, the robust predictive value of total soluble Aß was dependent on native state quantification. Elevations in tau and Aß within soluble fractions (Braak stage 2-3 cf. 0) were evident earlier than previously established in fibril-focused disease progression scales. Together, these data provide strong evidence that soluble forms of tau and Aß co-localise early in AD and are closely linked to disease progression and cognitive decline.

Polymeric alkylpyridinium salts permit intracellular delivery of human Tau in rat hippocampal neurons: requirement of Tau phosphorylation for functional deficits.

Patients suffering from tauopathies including frontotemporal dementia (FTD) and Alzheimer's disease (AD) present with intra-neuronal aggregation of microtubule-associated protein Tau. During the disease process, Tau undergoes excessive phosphorylation, dissociates from microtubules and aggregates into insoluble neurofibrillary tangles (NFTs), accumulating in the soma. While many aspects of the disease pathology have been replicated in transgenic mouse models, a region-specific non-transgenic expression model is missing. Complementing existing models, we here report a novel region-specific approach to modelling Tau pathology. Local co-administration of the pore-former polymeric 1,3-alkylpyridinium salts (Poly-APS) extracted from marine sponges, and synthetic full-length 4R recombinant human Tau (hTau) was performed in vitro and in vivo. At low doses, Poly-APS was non-toxic and cultured cells exposed to Poly-APS (0.5 µg/ml) and hTau (1 µg/ml; ~22 µM) had normal input resistance, resting-state membrane potentials and Ca(2+) transients induced either by glutamate or KCl, as did cells exposed to a low concentration of the phosphatase inhibitor Okadaic acid (OA; 1 nM, 24 h). Combined hTau loading and phosphatase inhibition resulted in a collapse of the membrane potential, suppressed excitation and diminished glutamate and KCl-stimulated Ca(2+) transients. Stereotaxic infusions of Poly-APS (0.005 µg/ml) and hTau (1 µg/ml) bilaterally into the dorsal hippocampus at multiple sites resulted in hTau loading of neurons in rats. A separate cohort received an additional 7-day minipump infusion of OA (1.2 nM) intrahippocampally. When tested 2 weeks after surgery, rats treated with Poly-APS+hTau+OA presented with subtle learning deficits, but were also impaired in cognitive flexibility and recall. Hippocampal plasticity recorded from slices ex vivo was diminished in Poly-APS+hTau+OA subjects, but not in other treatment groups. Histological sections confirmed the intracellular accumulation of hTau in CA1 pyramidal cells and along their processes; phosphorylated Tau was present only within somata. This study demonstrates that cognitive, physiological and pathological symptoms reminiscent of tauopathies can be induced following non-mutant hTau delivery into CA1 in rats, but functional consequences hinge on increased Tau phosphorylation. Collectively, these data validate a novel model of locally infused recombinant hTau protein as an inducer of Tau pathology in the hippocampus of normal rats; future studies will provide insights into the pathological spread and maturation of Tau pathology.

Knock-in of human BACE1 cleaves murine APP and reiterates Alzheimer-like phenotypes.

Key neuropathological hallmarks of Alzheimer's disease (AD) are elevated levels of amyloid ß-peptide (Aß) species generated via amyloid precursor protein (APP) endoproteolysis and cleavage by the rate-limiting ß-site enzyme 1 (BACE1). Because rodents do not develop amyloid pathologies, we here investigated whether AD-like endophenotypes can be created in mice by expression of human bace1. To avoid pitfalls of existing models, we introduced hbace1 via knock-in under the control of the CaMKII α promoter into the safe HPRT locus. We report amyloidogenic processing of murine APP in the hBACE1 mice (termed PLB4), resulting in the formation of toxic APP metabolites that accumulate intra- and extraneuronally in hippocampus and cortex. Pronounced accumulation of Aß*56 and Aß hexamers in the absence of plaque deposition was detected in brain tissue from symptomatic PLB4 mice. Heightened levels of inflammation (gliosis) also appeared in several AD-related brain regions (dentate gyrus, hippocampal area CA1, piriform and parietal cortices) at 6 and 12 months of age. Behaviorally, deficits in habituation to a novel environment and semantic-like memory (social transmission of food preference) were detected from 3 to 4 months of age. Impairments in spatial learning strategies in long-term reference (water maze) and working memory (Y-maze) tasks presented at 6 months, and were distinct from reductions in locomotor activity and anxiety. Overall, our data indicate for the first time that targeted, subtle forebrain-specific expression through single gene knock-in of hBACE1 is sufficient to generate AD-relevant cognitive impairments amid corresponding histopathologies, confirming human BACE as the key parameter in amyloid pathogenesis.

Modulation of food consumption and sleep-wake cycle in mice by the neutral CB1 antagonist ABD459.

The brain endocannabinoid system is a potential target for the treatment of psychiatric and metabolic conditions. Here, a novel CB1 receptor antagonist (ABD459) was synthesized and assayed for pharmacological efficacy in vitro and for modulation of food consumption, vigilance staging and cortical electroencephalography in the mouse. ABD459 completely displaced the CB1 agonist CP99540 at a Ki of 8.6 nmol/l, and did not affect basal, but antagonized CP55940-induced GTPγS binding with a KB of 7.7 nmol/l. Acute ABD459 (3-20 mg/kg) reliably inhibited food consumption in nonfasted mice, without affecting motor activity. Active food seeking was reduced for 5-6 h postdrug, with no rebound after washout. Epidural recording of electroencephalogram confirmed that ABD459 (3 mg/kg) robustly reduced rapid eye movement (REM) sleep, with no alterations of wakefulness or non-REM sleep. Effects were strongest during 3 h postdrug, followed by a progressive washout period. The CB1 antagonist AM251 (3 mg/kg) and agonist WIN-55,212-2 (WIN-2: 3 mg/kg) also reduced REM, but variously affected other vigilance stages. WIN-2 caused a global suppression of normalized spectral power. AM251 and ABD459 lowered delta power and increased power in the theta band in the hippocampus, but not the prefrontal cortex. The neutral antagonist ABD459 thus showed a specific role of endocannabinoid release in attention and arousal, possibly through modulation of cholinergic activity.

Age-dependent changes in hippocampal synaptic transmission and plasticity in the PLB1Triple Alzheimer mouse.

Several genetically engineered models exist that mimic aspects of the pathological and cognitive hallmarks of Alzheimer's disease (AD). Here we report on a novel mouse model generated by targeted knock-in of transgenes containing mutated human amyloid precursor protein (APP) and microtubule-associated protein tau genes, inserted into the HPRT locus and controlled by the CaMKIIα regulatory element. These mice were crossed with an asymptomatic presenilin1A246E overexpressing line to generate PLB1Triple mice. Gene expression analysis and in situ hybridization confirmed stable, forebrain-specific, and gene-dose-dependent transgene expression. Brain tissue harvested from homozygous, heterozygous, and wild-type cohorts aged between 3 and 24 months was analyzed immunohistochemically and electrophysiologically. Homozygous PLB1Triple offspring presented with mostly intracellular cortical and hippocampal human APP/amyloid, first detected reliably at 6 months. Human tau was already uncovered at 3 months (phospho-tau at 6 months) and labeling intensifying progressively with age. Gene-dose dependence was confirmed in age-matched heterozygous females that accumulated less tau and amyloid protein. General excitability of hippocampal neurones was not altered in slices from PLB1Triple mice up to 12 months, but 2-year-old homozygous PLB1Triple mice had smaller synaptically evoked postsynaptic potentials compared with wild types. Synaptic plasticity (paired-pulse depression/facilitation and long-term potentiation) of synaptic CA1 pyramidal cell responses was deficient from 6 months of age. Long-term depression was not affected at any age or in any genotype. Therefore, despite comparatively subtle gene expression and protein build-up, PLB1Triple mice develop age-dependent progressive phenotypes, suggesting that aggressive protein accumulation is not necessary to reconstruct endophenotypes of AD.

Advanced qEEG analyses discriminate between dementia subtypes.

BACKGROUND: Dementia is caused by neurodegenerative conditions and characterized by cognitive decline. Diagnostic accuracy for dementia subtypes, such as Alzheimer's Disease (AD), Dementia with Lewy Bodies (DLB) and Parkinson's Disease with dementia (PDD), remains challenging. METHODS: Here, different methods of quantitative electroencephalography (qEEG) analyses were employed to assess their effectiveness in distinguishing dementia subtypes from healthy controls under eyes closed (EC) and eyes open (EO) conditions. RESULTS: Classic Fast-Fourier Transform (FFT) and autoregressive (AR) power analyses differentiated between all conditions for the 4-8 Hz theta range. Only individuals with dementia with Lewy Bodies (DLB) differed from healthy subjects for the wider 4-15 Hz frequency range, encompassing the actual dominant frequency of all individuals. FFT results for this range yielded wider significant discriminators vs AR, also detecting differences between AD and DLB. Analyses of the inclusive dominant / peak frequency range (4-15 Hz) indicated slowing and reduced variability, also discriminating between synucleinopathies vs controls and AD. Dissociation of periodic oscillations and aperiodic components of AR spectra using Fitting-Oscillations-&-One-Over-F (FOOOF) modelling delivered a reliable and subtype-specific slowing of brain oscillatory peaks during EC and EO for all groups. Distinct and robust differences were particularly strong for aperiodic parameters, suggesting their potential diagnostic power in detecting specific changes resulting from age and cognitive status. CONCLUSION: Our findings indicate that qEEG methods can reliably detect dementia subtypes. Spectral analyses comprising an integrated, multi-parameter EEG approach discriminating between periodic and aperiodic components under EC and EO conditions may enhance diagnostic accuracy in the future.

How stra(i)nge are your controls? A comparative analysis of metabolic phenotypes in commonly used C57BL/6 substrains.

In recent years, insufficiently characterised controls have been a contributing factor to irreproducibility in biomedical research including neuroscience and metabolism. There is now a growing awareness of phenotypic differences between the C57BL/6 substrains which are commonly used as control animals. We here investigated baseline metabolic characteristics such as glucose regulation, fasted serum insulin levels and hepatic insulin signalling in five different C57BL/6 substrains (N, J, JOla, JRcc) of both sexes, obtained from two commercial vendors, Charles River Laboratories (Crl) and Envigo (Env). Our results indicate systematic and tissue-specific differences between substrains, affected by both vendor and sex, in all parameters investigated, and not necessarily mediated by the presence of the NntC57BL/6J mutation. Not only were there differences between 6J and 6N as expected, all three 6J substrains exhibited different profiles, even from the same breeder. Two distinct metabolic profiles were identified, one in which low insulin levels resulted in impaired glucose clearance (6JCrl; both sexes) and the other, where sustained elevations in fasted basal insulin levels led to glucose intolerance (male 6JRccEnv). Further, 6JRccEnv displayed sex differences in both glucose clearance and hepatic insulin signalling markers. In comparison, the two 6N substrains of either sex, irrespective of vendor, did not exhibit considerable differences, with 6NCrl animals presenting a good choice as a healthy baseline 'control' for many types of experiments. Overall, our data emphasise the importance of selecting and characterising control subjects regarding background, sex, and supplier to ensure proper experimental outcomes in biomedical research.

Comparison of automated video tracking systems in the open field test: ANY-Maze versus EthoVision XT.

BACKGROUND: ANY-Maze and EthoVision XT are two commonly used automated animal tracking systems employed to produce reliable and consistent results in behavioural paradigms. Data obtained with both tracking systems have presented differences, particularly when varying laboratory lighting conditions and contrasts of mice coat colour against the arena background in both water maze and tunnel maze. METHOD: In this study, two fluorescent lighting conditions (58 and 295 lux), local to our laboratory, and different coat-coloured mouse lines (C57BL/6 J - black; CD1 - agouti; C3H/HeN - white) were used to compare reproducibility in measures of tracking systems (ANY-Maze versus EthoVision) in the open field test. RESULTS: Differences between systems were reliant on the contrasts between coat and background colours. Surprisingly, black animals presented the greatest differences in read-outs between tracking systems, regardless of lighting conditions. Data from both video observation tools differed mainly in exploration-related parameters (distance travelled), but less in more static proxies (time in thigmotaxis zone). Overall, EthoVision XT returned higher values for most parameters analysed relative to ANY-Maze. More inconsistencies in recording and analysis can be expected from other video recording systems. CONCLUSION: Data analysis software provides an additional source of variation in need of consideration when reproducibility in behavioural neuroscience is required.

Apolipoprotein E loss of function: Influence on murine brain markers of physiology and pathology.

The canonical role of Apolipoprotein E (ApoE) is related to lipid and cholesterol metabolism, however, additional functions of this protein have not been fully described. Given the association of ApoE with diseases such as Alzheimer's Disease (AD), it is clear that further characterisation of its roles, especially within the brain, is needed. Therefore, using protein and gene expression analyses of neonatal and 6-month old brain tissues from an ApoE knockout mouse model, we examined ApoE's contribution to several CNS pathways, with an emphasis on those linked to AD. Early neonatal changes associated with ApoE-/- were observed, with decreased soluble phosphorylated tau (p-tau, -40 %), increased synaptophysin (+36 %) and microglial Iba1 protein levels (+25 %) vs controls. Progression of the phenotype was evident upon analysis of 6-month-old tissue, where decreased p-tau was also confirmed in the insoluble fraction, alongside reduced synaptic and increased amyloid precursor protein (APP) protein levels. An age comparison further underlined deviations from WT animals and thus the impact of ApoE loss on neuronal maturation. Taken together, our data implicate ApoE modulation of multiple CNS roles. Loss of function is associated with alterations from birth, and include synaptic deficits, neuroinflammation, and changes to key AD pathologies, amyloid-ß and tau.

Sex Differences in Behavior and Molecular Pathology in the 5XFAD Model.

BACKGROUND: The prevalence of Alzheimer's disease (AD) is greater in women compared to men, but the reasons for this remain unknown. This sex difference has been widely neglected in experimental studies using transgenic mouse models of AD. OBJECTIVE: Here, we studied behavior and molecular pathology of 5-month-old 5XFAD mice, which express mutated human amyloid precursor protein and presenilin-1 on a C57BL/6J background, versus their wild-type littermate controls, to compare both sex- and genotype-dependent differences. METHODS: A novel behavioral paradigm was utilized (OF-NO-SI), comprising activity measures (Open Field, OF) arena, followed by Novel Object exploration (NO) and Social Interaction (SI) of a sex-matched conspecific. Each segment consisted of two repeated trials to assess between-trial habituation. Subsequently, brain pathology (amyloid load, stress response and inflammation markers, synaptic integrity, trophic support) was assessed using qPCR and western blotting. RESULTS: Female 5XFAD mice had higher levels of human APP and amyloid-ß and heightened inflammation versus males. These markers correlated with hyperactivity observed in both sexes, yet only female 5XFAD mice presented with subtle deficits in object and social exploration. Male animals had higher expression of stress markers and neurotrophic factors irrespective of genotype, this correlated with cognitive performance. CONCLUSION: The impact of sex on AD-relevant phenotypes is in line with human data and emphasizes the necessity of appropriate study design and reporting. Differential molecular profiles observed in male versus female mice offer insights into possible protective mechanisms, and hence treatment strategies.

The impact of tethered recording techniques on activity and sleep patterns in rats.

Electrophysiological recordings in animals constitute frequently applied techniques to study neuronal function. In this context, several authors described tethered recordings as a semi-restraint situation with negative implications for animal welfare and suggested radiotelemetric setups as a refinement measure. Thus, we here investigated the hypothesis that tethered recordings exert measurable effects on behavioral and sleep patterns in Sprague-Dawley rats. Animals were kept in monitoring glass cages either with or without a head connection to a recording cable. Saccharin preference, nest building, serum corticosterone and fecal corticosterone metabolite levels were in a comparable range in both groups. The proportion of vigilance states was not affected by the cable connection. Minor group differences were detected in bout lengths distributions, with a trend for longer NREM and WAKE stages in animals with a cable connection. However, a relevant effect was not further confirmed by an analysis of the number of sleep/wake and wake/sleep transitions. The analysis of activity levels did not reveal group differences. However, prolonged exposure to the tethered condition resulted in an intra-group increase of activity. In conclusion, the comparison between freely moving vs tethered rats did not reveal major group differences. Our findings indicate that telemetric recordings only offer small advantages vs cabled set ups, though this may differ in other experimental studies where for example anxiety- or drug-induced effects are analyzed.

FDG-PET imaging, EEG and sleep phenotypes as translational biomarkers for research in Alzheimer's disease.

The lack of reliable translational procedures applicable to both patients and experimental models are a major obstacle for the advancement of basic research as well as for the development of therapeutics. This is particularly relevant to neurodegenerative disorders such as AD (Alzheimer's disease), where the predictive validity of animal models and procedures applied preclinically have met with little success. Two approaches available for human diagnostics are currently experiencing major advancements in preclinical research: in vivo imaging using MRI (magnetic resonance imaging) or PET (positron-emission tomography) and recordings of brain electrical activity via surface EEG (electroencephalogram). The present paper reviews the results obtained so far in rodent AD models, and summarizes advantages and disadvantages of such procedures.