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The BRAF kinase plays a critical role in the MAPK signaling pathway and is mutated in ~8% of all human cancers including melanoma (~60%), thyroid (~60%), and lung adenocarcinoma (~10%). The most common mutation in BRAF is V600E (Class I), which is found in >70% in these cancers. Despite the clinical success of approved small molecule inhibitors of BRAF V600E (vemurafenib, dabrafenib and encorafenib), this remains an area of unmet medical need because nearly all patients progress, due to either primary or acquired resistance. A bifunctional degradation activating compound (or BiDACTM degrader) may address the liabilities of approved drugs by overcoming, or preventing the emergence of, resistance to BRAF inhibitors. Here we describe CFT1946, an orally bioavailable cereblon-based BiDAC degrader of BRAF V600 mutant proteins, and provide an overview of the medicinal chemistry path leading to its discovery. CFT1946 degrades BRAF V600 mutant proteins, while maintaining exquisite selectivity against the proteome, sparing wild type BRAF (BRAF-WT), ARAF, and CRAF. In A375 cells, CFT1946 potently degraded BRAF V600E and inhibited ERK phosphorylation and cell growth while having no effect in the mutant KRAS, BRAF-WT driven cell line HCT116. In the A375 xenograft model, oral delivery of CFT1946 at 10 mg/kg PO BID resulted in deeper and more durable tumor regression compared to a clinically relevant dose of encorafenib. Further evaluation of CFT1946 in an engineered, clinically relevant BRAFi-resistant A375 cell line (endogenous BRAF V600E + engineered expression of NRAS Q61K) demonstrated that CFT1946 both degraded BRAF V600E and caused a loss of viability in these cells, while treatment with encorafenib had no effect. In xenografts derived from this BRAFi-resistant cell line, oral dosing of CFT1946 as a single agent led to tumor growth inhibition, while treatment with a clinically relevant dose of encorafenib had no effect on tumor growth. Furthermore, dosing CFT1946 in combination with the MEK inhibitor, trametinib, resulted in significant tumor regression, whereas combining encorafenib with the same dose of trametinib had no effect. The medicinal chemistry campaign resulting in CFT1946 focused on the improvement of in vivo pharmacokinetics and rational linker design to achieve high oral bioavailability in a beyond Rule of 5 heterobifunctional degrader. The preclinical data presented herein support the planned Phase 1/2 clinical trial of CFT1946 for the treatment of BRAF-V600 mutant solid tumors. Citation Format: Yanke Liang, Mathew E. Sowa, Katrina L. Jackson, Jeffrey R. Simard, Bridget Kreger, Ping Li, Laura Poling, Joelle Baddour, Andrew Good, Hongwei Huang, Scott Eron, Christopher G. Nasveschuk, Robert Yu, Mark Fitzgerald, Victoria Garza, Morgan W. O’Shea, Gesine Veits, Jeremy Y. Yap, Moses Moustakim, Ashley Hart, Roman V. Agafonov, Grace Sarkissian, Joe S. Patel, Richard Deibler, Kyle S. Cole, David Cocozziello, Fazlur Rahman, Andrew J. Phillips, Elizabeth Norton, Adam S. Crystal, Roy M. Pollock, Stewart L. Fisher. The discovery and characterization of CFT1946: A potent, selective, and orally bioavailable degrader of mutant BRAF for the treatment of BRAF-driven cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3425.

Mid-life hypertension and cerebrovascular dysfunction are associated with increased risk of later life dementia, including Alzheimer’s disease (AD). The classical renin–angiotensin system (cRAS), a physiological regulator of blood pressure, functions independently within the brain and is overactive in AD. cRAS-targeting anti-hypertensive drugs are associated with reduced incidence of AD, delayed onset of cognitive decline, and reduced levels of Aβ and tau in both animal models and human pathological studies. cRAS activity is moderated by a downstream regulatory RAS pathway (rRAS), which is underactive in AD and is strongly associated with pathological hallmarks in human AD, and cognitive decline in animal models of CNS disease. We now show that enhancement of brain ACE2 activity, a major effector of rRAS, by intraperitoneal administration of diminazene aceturate (DIZE), an established activator of ACE2, lowered hippocampal Aβ and restored cognition in mid-aged (13–14-month-old) symptomatic Tg2576 mice. We confirmed that the protective effects of DIZE were directly mediated through ACE2 and were associated with reduced hippocampal soluble Aβ42 and IL1-β levels. DIZE restored hippocampal MasR levels in conjunction with increased NMDA NR2B and downstream ERK signalling expression in hippocampal synaptosomes from Tg2576 mice. Chronic (10 weeks) administration of DIZE to pre-symptomatic 9–10-month-old Tg2576 mice, and acute (10 days) treatment in cognitively impaired 12–13-month-old mice, prevented the development of cognitive impairment. Together these data demonstrate that ACE2 enhancement protects against and reverses amyloid-related hippocampal pathology and cognitive impairment in a preclinical model of AD. Electronic supplementary material The online version of this article (10.1007/s00401-019-02098-6) contains supplementary material, which is available to authorized users.

Introduction: The chromatin factor BRD9 is a genetic dependency in some cancers, often referred to as SMARCB1-perturbed cancers. Two types of genetic alterations result in SMARCB1 perturbation: SS18-SSX gene fusion and SMARCB1 loss-of-function mutations. In synovial sarcoma, a rare and aggressive soft tissue malignancy comprising approximately 10% of all soft tissue sarcomas, the presence of the SS18-SSX fusion gene drives the disruption of SMARCB1 function and leads to a synthetic lethal dependence on BRD9. In SMARCB1-null solid tumors, for example malignant rhabdoid tumors (MRT), poorly differentiated chordomas, and epithelioid sarcomas, the absence of SMARCB1 protein results in a similar BRD9 dependence. Thus, in SMARCB1-perturbed cancers, including synovial sarcoma and SMARCB1-null cancers, degradation of BRD9 is hypothesized to result in an anticancer effect. CFT8634 is an orally bioavailable heterobifunctional degrader that induces ternary complex formation with BRD9 and an E3 ligase, leading to the ubiquitination of BRD9 and its subsequent degradation by the proteasome. Results: Here we describe the chemical structure of CFT8634 and an overview of the medicinal chemistry path leading to its discovery. In vitro, CFT8634 promotes rapid, potent, deep, and selective degradation of BRD9 with a half-maximal degradation concentration (DC50) of 2 nM in a synovial sarcoma cell line. In long-term growth assays, CFT8634 is effective at impairing cell growth in a concentration-dependent manner specifically in SMARCB1-perturbed contexts. In vivo, oral dosing of CFT8634 in xenograft models of SMARCB1-perturbed cancers leads to robust and dose-dependent degradation of BRD9, which translates to significant and dose-dependent inhibition of tumor growth in preclinical xenograft models. Conclusion: The preclinical data presented herein support the clinical development of CFT8634 for the treatment of synovial sarcoma and SMARCB1-null tumors. Citation Format: Katrina L. Jackson, Roman V. Agafonov, Mark W. Carlson, Prasoon Chaturvedi, David Cocozziello, Kyle Cole, Richard Deibler, Scott J. Eron, Andrew Good, Ashley A. Hart, Minsheng He, Christina S. Henderson, Hongwei Huang, Marta Isasa, R. Jason Kirby, Linda Lee, Michelle Mahler, Moses Moustakim, Christopher G. Nasveschuk, Michael Palmer, Laura L. Poling, Roy M. Pollock, Matthew Schnaderbeck, Stan Spence, Gesine K. Veits, Jeremy L. Yap, Ning Yin, Rhamy Zeid, Adam S. Crystal, Andrew J. Phillips, Stewart L. Fisher. The discovery and characterization of CFT8634: A potent and selective degrader of BRD9 for the treatment of SMARCB1-perturbed cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr ND09.

Introduction: Ikaros family zinc finger protein 1 and 3 (IKZF1/3) are essential transcription factors (TF) for terminal differentiation of B and T cells. Depletion of IKZF1/3 inhibits the growth of multiple myeloma (MM) cells, confirming their dependency on IKZF1/3. IMiDs (lenalidomide, pomalidomide) are effective therapies for treatment of MM and promote degradation of IKZF1/3 via their interaction with CRL4-CRBN E3 ligase. However, most patients treated with lenalidomide or pomalidomide eventually develop progressive disease due to acquired resistance, underscoring the unmet medical need. CFT7455 is a novel IKZF1/3 degrader optimized for high binding affinity to cereblon (CRBN), rapid and deep IKZF1/3 degradation, and potent dose-dependent efficacy in vivo. Results: A series of novel benzoimidazolone-based CRBN ligands with potent binding affinity were discovered and their binding modes were informed by CRBN co-crystal structures. Although the benzoimidazolone-based CRBN binders did not exhibit IKZF1/3 degradation activity, structural insights into their unique binding modes and knowledge of the IKZF1/3 degradation pharmacophore were combined to enable identification of a novel benzoisoindolone-based ligand that exhibited a 10-fold potency increase in biochemical CRBN binding and a 30-fold potency increase in H929 MM cell growth inhibition when compared to lenalidomide. Additional rounds of structure-based drug design, degradation and phenotypic profiling led to the discovery of CFT7455, a highly potent, selective and orally bioavailable degrader of IKZF1/3. CFT7455 demonstrated an 800 and 1600-fold improvement in CRBN binding compared to pomalidomide in biochemical and cellular NanoBRET assays, respectively. In H929 MM cells expressing HiBiT-tagged IKZF1, CFT7455 induced >75% degradation of IKZF1 within 1.5 hrs. The high binding affinity and degradation catalysis shown with CFT7455 enabled potent antiproliferative activity across a panel of MM cell lines, as well as H929 cells made resistant to IMiDs. In vivo, CFT7455 catalyzed deep and durable degradation of IKZF3, translating into potent antitumor activity in multiple myeloma xenograft models. CFT7455 also retained its activity in models resistant or insensitive to clinically approved IMiDs as single agent or in combination with standard of care agent dexamethasone. Conclusion: Overall, CFT7455 is a next generation IKZF1/3 degrader, with improved potency and anticancer efficacy in preclinical models compared to existing IMiDs. These features make CFT7455 an exciting drug candidate, as a single agent or for use in combination. CFT7455 is currently being studied in a Ph1 clinical trial. Citation Format: James A. Henderson, Scott J. Eron, Andrew Good, R Jason Kirby, Samantha Perino, Roman V. Agafonov, Prasoon Chaturvedi, Bradley Class, David Cocozziello, Ashley A. Hart, Christina S. Henderson, Marta Isasa, Brendon Ladd, Matt Schnaderbeck, Michelle Mahler, Adam S. Crystal, Roy M. Pollock, Christopher G. Nasveschuk, Andrew J. Phillips, Stewart L. Fisher, David A. Proia. The discovery and characterization of CFT7455: A potent and selective degrader of IKZF1/3 for the treatment of relapsed/refractory multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr ND13.

Neuronal dendritic and synaptic pruning are early features of neurodegenerative diseases, including Alzheimer’s disease. In addition to brain pathology, amyloid plaque deposition, microglial activation, and cell loss occur in the retinas of human patients and animal models of Alzheimer’s disease. Retinal ganglion cells, the output neurons of the retina, are vulnerable to damage in neurodegenerative diseases and are a potential opportunity for non-invasive clinical diagnosis and monitoring of Alzheimer’s progression. However, the extent of retinal involvement in Alzheimer’s models and how well this reflects brain pathology is unclear. Here we have quantified changes in retinal ganglion cells dendritic structure and hippocampal dendritic spines in three well-studied Alzheimer’s mouse models, Tg2576, 3xTg-AD and APPNL-G-F. Dendritic complexity of DiOlistically labelled retinal ganglion cells from retinal explants was reduced in all three models in an age-, gender-, and receptive field-dependent manner. DiOlistically labelled hippocampal slices showed spine loss in CA1 apical dendrites in all three Alzheimer’s models, mirroring the early stages of neurodegeneration as seen in the retina. Morphological classification showed that loss of thin spines predominated in all. The demonstration that retinal ganglion cells dendritic field reduction occurs in parallel with hippocampal dendritic spine loss in all three Alzheimer’s models provide compelling support for the use of retinal neurodegeneration. As retinal dendritic changes are within the optical range of current clinical imaging systems (for example optical coherence tomography), our study makes a case for imaging the retina as a non-invasive way to diagnose disease and monitor progression in Alzheimer’s disease.

Assessment of outcome in preclinical studies of vascular cognitive impairment (VCI) is heterogenous. Through an ARUK Scottish Network supported questionnaire and workshop (mostly UK-based researchers), we aimed to determine underlying variability and what could be implemented to overcome identified challenges. Twelve UK VCI research centres were identified and invited to complete a questionnaire and attend a one-day workshop. Questionnaire responses demonstrated agreement that outcome assessments in VCI preclinical research vary by group and even those common across groups, may be performed differently. From the workshop, six themes were discussed: issues with preclinical models, reasons for choosing functional assessments, issues in interpretation of functional assessments, describing and reporting functional outcome assessments, sharing resources and expertise, and standardization of outcomes. Eight consensus points emerged demonstrating broadly that the chosen assessment should reflect the deficit being measured, and therefore that one assessment does not suit all models; guidance/standardisation on recording VCI outcome reporting is needed and that uniformity would be aided by a platform to share expertise, material, protocols and procedures thus reducing heterogeneity and so increasing potential for collaboration, comparison and replication. As a result of the workshop, UK wide consensus statements were agreed and future priorities for preclinical research identified.

A healthy mitochondrial network is essential for the maintenance of neuronal synaptic integrity. Mitochondrial and metabolic dysfunction contributes to the pathogenesis of many neurodegenerative diseases including dementia. OPA1 is the master regulator of mitochondrial fusion and fission and is likely to play an important role during neurodegenerative events. To explore this, we quantified hippocampal dendritic and synaptic integrity and the learning and memory performance of aged Opa1 haploinsufficient mice carrying the Opa1Q285X mutation (B6; C3-Opa1Q285STOP; Opa1+/−). We demonstrate that heterozygous loss of Opa1 results in premature age-related loss of spines in hippocampal pyramidal CA1 neurons and a reduction in synaptic density in the hippocampus. This loss is associated with subtle memory deficits in both spatial novelty and object recognition. We hypothesize that metabolic failure to maintain normal neuronal activity at the level of a single spine leads to premature age-related memory deficits. These results highlight the importance of mitochondrial homeostasis for maintenance of neuronal function during ageing., Mitochondria generate energy required for memory function. Aged mice having genetically reduced amount of OPA1 protein necessary for energy production have subtle but persistent deficits in behavioural memory. Anatomical examination of the relevant brain region (hippocampal CA1) revealed changes affecting communication between cells and leading to premature age-related memory deficits., Graphical Abstract Graphical Abstract

Numerical modelling of the flow interactions between tidal turbines in arrays is a prerequisite to assess the energy production potential and to optimise the layout of tidal stream energy farms. It requires (i) a refined representation of the tide propagation and (ii) a reliable estimate of the flow characteristics around the turbines. The Actuator Disk (AD) theory is recognised as a reliable parameterisation to approach the far wake of fixed horizontal-axis turbines and was therefore implemented to provide recommendations as regards to the design of arrays, especially the optimal spacing between devices. However, analysis of the arrangement of tidal stream turbines are typically restricted to schematic test cases with idealised bathymetry and inflow conditions. The present study aims to investigate the flow interactions between turbines in a stream energy site with spatially- and time-varying tidal conditions. The site of application is the Alderney Race (English Channel), an area with strong potential for the exploitation of tidal energy in European shelf seas. The three-dimensional numerical model Telemac3D is used to capture the flow at both the regional and the array scales. Predictions are first assessed against ADCP measurements acquired around the island of Alderney. A series of simulations, representing turbines with the AD theory, are then performed to investigate the influence of the wake interactions on the energy production of individual devices. In those simulations, the turbines are represented with the AD theory. The zone occupied by the turbines is 20D-long and 15D-wide (D is the turbine diameter). Different layouts (isolated, aligned and staggered turbines) and different longitudinal and lateral spacings are considered to investigate the effect of the turbines’ arrangement and density on the array energy production. The results show that, for a given turbines density within the array, the staggered layout produces more than the aligned layout (+16%). When the turbines are staggered, a minimal lateral spacing of 5D is required to avoid wake overlapping. However, when the turbines are aligned, the lateral spacing has a small influence on the turbines production. It is therefore possible to pack the turbines close to each other in the lateral direction. The simulations also outline that the turbulence develops much faster within the array when the turbines are aligned.

In an exploratory, block-randomised, parallel, double-blind, single-centre, placebo-controlled superiority study (ISRCTN12562026, funded by Cultech Ltd), 220 Bulgarian participants (30 to 65 years old) with BMI 25–34.9 kg/m2 received Lab4P probiotic (50 billion/day) or a matched placebo for 6 months. Participants maintained their normal diet and lifestyle. Primary outcomes were changes in body weight, BMI, waist circumference (WC), waist-to-height ratio (WtHR), blood pressure and plasma lipids. Secondary outcomes were changes in plasma C-reactive protein (CRP), the diversity of the faecal microbiota, quality of life (QoL) assessments and the incidence of upper respiratory tract infection (URTI). Significant between group decreases in body weight (1.3 kg, p 2, p p p p p = 0.0005). Greatest weight losses were among probiotic hypercholesterolaemic participants (−2.5%, p p = 0.0241). Improvements in QoL and the incidence rate ratio of URTI (0.60, p

Three experiments examined the ability of mice to forage efficiently for liquid rewards in pots located in an open field arena. Search behaviour was unconstrained other than by the walls of the arena. All mice acquired the task within 4 days of training, with one trial per day. Experiment 1 tested the hypothesis that hippocampal lesions would disrupt foraging behaviour using extramaze cues. Mice with hippocampal lesions showed normal latency to initiate foraging and to complete the task relative to sham-operated mice. However, lesioned mice showed increased perseverative responding (sensitization) to recently rewarded locations, increased total working memory errors and an increased propensity to search near previously rewarded locations. In Experiment 2, the extramaze cues were obscured and each pot was identified by a unique pattern. Under these conditions, mice with hippocampal lesions showed comparable working memory errors to control mice. However, lesioned mice continued to display increased perseverative responding and altered search strategies. Experiment 3 tested the hypothesis that age-related accumulation of amyloid would disrupt foraging behaviour in transgenic PDAPP mice expressing the V717F amyloid precursor protein (APP) mutation. Consistent with previous findings, PDAPP mice showed both age-dependent and age-independent behavioural changes. More specifically, 14-16 month-old PDAPP mice showed a deficit in perseverative responding and working memory errors. In contrast, changes in search behaviour, such as systematic circling, were present throughout development. The latter indicates that APP overexpression contributed to some features of the PDAPP behavioural phenotype, whereas working memory and flexible responding was sensitive to ageing and β-amyloid burden. In conclusion, the present study provided novel insight into the role of the hippocampus and the effects of APP overexpression on memory and search behaviour in an open-field foraging task.

A major risk factor for the development of Alzheimer's disease (AD) is increasing age, but the reason behind this association has not been identified. It is thought that the changes in endocytosis seen in AD patients are causal for this condition. Thus, we hypothesized that the increased risk of developing AD associated with ageing may be because of changes in endocytosis. We investigated using Western blotting whether the expression of endocytic proteins involved in clathrin-mediated and clathrin-independent endocytosis are altered by increasing age in a mouse model of amyloid pathology. We used mice transgenic for human amyloid precursor protein containing the V717I London mutation. We compared the London mutation mice with age-matched wild-type (WT) controls at three ages, 3, 9 and 18 months, representing different stages in the development of pathology in this model. Having verified that the London mutation mice overexpressed amyloid precursor protein and β-amyloid, we found that the expression of the smallest isoform of PICALM, a key protein involved in the regulation of clathrin-coated pit formation, was significantly increased in WT mice, but decreased in the London mutation mice with age. PICALM levels in WT 18-month mice and clathrin levels in WT 9-month mice were significantly higher than those in the London mutation mice of the same ages. The expression of caveolin-1, involved in clathrin-independent endocytosis, was significantly increased with age in all mice. Our results suggest that endocytic processes could be altered by the ageing process and such changes could partly explain the association between ageing and AD.

Summary Altered neural dynamics in the medial prefrontal cortex (mPFC) and hippocampus may contribute to cognitive impairments in the complex chromosomal disorder Down syndrome (DS). Here, we demonstrate non-overlapping behavioral differences associated with distinct abnormalities in hippocampal and mPFC electrophysiology during a canonical spatial working memory task in three partially trisomic mouse models of DS (Dp1Tyb, Dp10Yey, and Dp17Yey) that together cover all regions of homology with human chromosome 21 (Hsa21). Dp1Tyb mice show slower decision-making (unrelated to the gene dose of DYRK1A, which has been implicated in DS cognitive dysfunction) and altered theta dynamics (reduced frequency, increased hippocampal-mPFC coherence, and increased modulation of hippocampal high gamma); Dp10Yey mice show impaired alternation performance and reduced theta modulation of hippocampal low gamma; and Dp17Yey mice are not significantly different from the wild type. These results link specific hippocampal and mPFC circuit dysfunctions to cognitive deficits in DS models and, importantly, map them to discrete regions of Hsa21., Graphical Abstract, Highlights • Study of behavior and EEG in three partially trisomic mouse models of Down syndrome (DS) • These mutant mice show non-overlapping differences in spatial working memory function • Behavioral changes segregate with distinct EEG abnormalities in the hippocampus and mPFC • This links cognitive deficits to specific changes in hippocampal and mPFC circuit dynamics, Chang et al. examine three partially trisomic mouse models of Down syndrome that together cover all regions of homology with human chromosome 21. They identify non-overlapping differences in spatial working memory function associated with distinct abnormalities in hippocampal and medial prefrontal electrophysiology.

Neurodegeneration has been linked to changes in the gut microbiota and this study compares the neuroprotective capability of two bacterial consortia, known as Lab4 and Lab4b, using the established SH-SY5Y neuronal cell model. Firstly, varying total antioxidant capacities (TAC) were identified in the intact cells from each consortia and their secreted metabolites, referred to as conditioned media (CM). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Crystal Violet (CV) assays of cell viability revealed that Lab4 CM and Lab4b CM could induce similar levels of proliferation in SH-SY5Y cells and, despite divergent TAC, possessed a comparable ability to protect undifferentiated and retinoic acid-differentiated cells from the cytotoxic actions of rotenone and undifferentiated cells from the cytotoxic actions of 1-methyl-4-phenylpyridinium iodide (MPP+). Lab4 CM and Lab4b CM also had the ability to attenuate rotenone-induced apoptosis and necrosis with Lab4b inducing the greater effect. Both consortia showed an analogous ability to attenuate intracellular reactive oxygen species accumulation in SH-SY5Y cells although the differential upregulation of genes encoding glutathione reductase and superoxide dismutase by Lab4 CM and Lab4b CM, respectively, implicates the involvement of consortia-specific antioxidative mechanisms of action. This study implicates Lab4 and Lab4b as potential neuroprotective agents and justifies their inclusion in further in vivo studies.

Ageing is associated with changes in the gut microbiome that may contribute to age-related changes in cognition. Previous work has shown that dietary supplements with multi-species live microorganisms can influence brain function, including induction of hippocampal synaptic plasticity and production of brain derived neurotrophic factor, in both young and aged rodents. However, the effect of such dietary supplements on memory processes has been less well documented, particularly in the context of aging. The main aim of the present study was to examine the impact of a long-term dietary supplement with a multi-species live Lactobacillus and Bifidobacteria mixture (Lactobacillus acidophilus CUL60, L. acidophilus CUL21, Bifidobacterium bifidum CUL20 and B. lactis CUL34) on tests of memory and behavioural flexibility in 15-17-month-old male rats. Following behavioural testing, the hippocampus and prefrontal cortex was extracted and analysed ex vivo using 1H nuclear magnetic resonance (1H NMR) spectroscopy to examine brain metabolites. The results showed a small beneficial effect of the dietary supplement on watermaze spatial navigation and robust improvements in long-term object recognition memory and short-term memory for object-in-place associations. Short-term object novelty and object temporal order memory was not influenced by the dietary supplement in aging rats. 1H NMR analysis revealed diet-related regional-specific changes in brain metabolites; which indicated changes in several pathways contributing to modulation of neural signaling. These data suggest that chronic dietary supplement with multi-species live microorganisms can alter brain metabolites in aging rats and have beneficial effects on memory.

The conditions under which the hippocampus contributes to learning about spatio-temporal configural patterns are not fully established. The aim of Experiments 1–4 was to investigate the impact of hippocampal lesions on learning about where or when a reinforcer would be delivered. In each experiment, the rats received exposure to an identical set of patterns (i.e., spotted + morning, checked + morning, spotted + afternoon and checked + afternoon); and the contexts (Experiment 1), times of day (Experiment 2), or their configuration (Experiments 3 and 4) signalled whether or not a reinforcer would be delivered. The fact that hippocampal damage did not disrupt the formation of simple or configural associations involving spatio-temporal patterns is surprising, and suggests that the contribution of the hippocampus is restricted to mediated learning (or updating) involving spatio-temporal configurations.

Novel behavioral assays were used to assess the role of the entorhinal cortex in modulating attention to components of stimulus compounds. In Stage 1, rats received discrimination training with compounds constructed from 3 dimensions (auditory, visual, and tactile); in each compound the combination of components from 2 dimensions (e.g., auditory and visual) were relevant to the solution of the discrimination, and the remaining dimension (e.g., tactile) was irrelevant. In Stage 2, rats received a different discrimination in which the relevant dimensions were either congruent (auditory and visual) or incongruent (auditory and tactile) with those that were relevant in Stage 1. Sham-operated rats acquired the congruent discrimination more rapidly than the incongruent discrimination--a finding indicative of a process of attentional modulation--whereas rats with excitotoxic lesions of the entorhinal cortex acquired both discriminations equally readily.

Many of the discriminations made by animals involve some stimuli that are relevant to the solution, and some that are irrelevant. If the same stimuli are used for a new discrimination, then learning often progresses more readily with the previously relevant than irrelevant stimuli, (e.g. Lawrence, 1949; 1950). Furthermore, if relevant and irrelevant stimuli are from different dimensions, and the new discrimination involves novel stimuli from these dimensions, learning often progresses more readily with the novel stimuli that belong to the previously relevant rather than the previously irrelevant dimensions (e.g. Mackintosh & Little, 1969). These differences in conditionability, or associability, have been attributed to animals paying more attention to relevant than irrelevant stimuli during discrimination learning (e.g. Sutherland & Mackintosh, 1971; Mackintosh, 1975). The purpose of the present paper is to explore the generality of the circumstances that promote these changes in attention. One source of support for the claim that animals pay more attention to relevant than to irrelevant stimuli is the intradimensional shift - extradimensional shift (IDS-EDS) effect. Typically, subjects receive a number of discriminations of the form AX+ BX−, AY+ BY−, where A and B represent values from one dimension, say colour, which is relevant to the solution of the discriminations and X and Y represent values from another, irrelevant dimension, say orientation. Once the initial discriminations have been mastered, subjects are transferred to a set of new discriminations involving novel values from the two dimensions. It is generally found that animals receiving an IDS, where the significance of the two dimensions remains the same for both stages, acquire the second set of discriminations more readily than those receiving an EDS, where the significance of the dimensions is reversed for the second stage. Examples of this effect have been provided by Mackintosh and Little (1969) and George and Pearce (1999), using pigeons; Shepp and Eimas (1964) and Trobalon, Miguelez, McLaren, and Mackintosh (2003), using rats; and Roberts, Robbins, and Everitt (1988), using marmosets. Demonstrations of an IDS-EDS effect normally take place in conditioning chambers, where visual stimuli presented on a display signal the presence or absence of reward. A series of experiments by Trobalon et al. (2003) has indicated, however, that this effect can be found in rather different circumstances. Rats were trained to find food at the end of one of two arms that were connected to a central platform. During the test phase the spatial position of the arms indicated where food could be found. For an IDS group, the position of the arms also indicated where food was situated during the previous training phase, but they occupied different locations to those used for testing. In addition, the texture of the floor of each arm was different and varied randomly with respect to food during the training stage. For an EDS group, the texture of the floor was relevant to the occurrence of food during the training stage, whereas the location of the arms was irrelevant. The former group, which received an IDS for the test phase, solved the test discrimination more rapidly than the latter group, which received an EDS. The experiment by Trobalon et al. (2003) indicates that changes in attention to relevant and irrelevant cues are not confined to the conditioning chamber but, even in their study, the movement of the animals was constrained by the maze on which they were trained. It remains to be determined, therefore, whether similar changes can be seen when the animal must explore the entire environment in order to discover where reward is located. In order to address this issue, the experiments examined whether rats trained to escape from a pool of water, by swimming to a submerged platform, pay more attention to cues that are relevant rather than irrelevant for finding the platform. Tests for an IDS-EDS effect are not always successful (see Hall & Channel, 1985; Couvillon, Tennant, & Bitterman, 1967; Sutherland & Andelman, 1969). Dopson, Esber, and Pearce (2010) have argued that this effect may be elusive because it depends upon a change in attention to one set of stimuli generalising to others from the same dimension. If the stimuli selected for the test phase differ too much from those used initially, then the difference in the degree of attention paid to the previously relevant and irrelevant stimuli might generalize only slightly to the new stimuli, and render the test for an IDS-EDS effect insensitive. To circumvent this obstacle, Pearce, Esber, George, and Haselgrove (2008; see also Dopson, Esber et al., 2010; Haselgrove, Esber, Pearce & Jones, 2010) devised an alternative method for revealing changes in attention to relevant and irrelevant stimuli. This method involved training pigeons on a number of discriminations, including: AW+ CW−, AX+ CX−, BW+ DW−. Following this training, subjects were given an AW+ AX− BW− test discrimination. All three test compounds were followed by food during the initial training, and it was therefore expected that responding during each of them would be similar at the outset of testing. Of interest was the rate at which the two sub-discriminations, AW+ AX− and AW+ BW−, were solved. It was expected that by the end of the initial training, A and B would be paid considerable attention, as these stimuli were relevant by virtue of consistently signalling food. W and X on the other hand should be paid little attention, as these stimuli were irrelevant to the delivery of food. Thus the AW+ BW− component of the test discrimination, which is based on cues A and B, should be acquired readily. In contrast, the AW+ AX− discrimination, which is based on cues W and X, should be acquired with difficulty. The results of Pearce et al. (2008) confirmed these predictions. A variation of the method used by Pearce et al. (2008) was adopted for Experiment 1. The results were consistent with the claim that changes in attention to relevant and irrelevant stimuli can be found in a free exploration task. In order to extend further our understanding of the factors that promote changes in attention, Experiment 2 capitalised on the methodology of Experiment 1 to explore whether any enhancement of attention to cues that indicate the location of a goal, also occurs with cues that reliably indicate where the goal is not located. Thus far there is only a single report of such a change in attention, and this was obtained using autoshaping with pigeons (Dopson, Williams, Esber & Pearce, 2010).

A novel association can form between two memories even when the events to which they correspond are not physically present. For example, once an integrated memory has formed that binds the (when, where, and what) components of an event together, this memory can be triggered by one of its components, and updated with coincident information in the environment. The neural basis of this form of retrieval-mediated learning is unknown. Here, we show, for the first time, that NMDA receptors in the rat hippocampus are required for retrieval-mediated learning involving episodes, but not for the expression of such learning or for retrieval-mediated learning involving simple associations between the components of episodes. These findings provide a novel insight into learning processes that serve the desirable function of integrating stored information with new information, but whose operation might also provide a substrate for some of the cognitive symptoms of schizophrenia and Alzheimer's disease.

A mouse model of amyloid pathology was used to first examine using a cross sectional design changes in retrosplenial cortex activity in transgenic mice aged 5, 11, 17, and 23 months. Attention focused on: (1) overt amyloid labeled with β-amyloid((1-42)) and Congo Red staining, (2) metabolic function assessed by the enzyme, cytochrome oxidase, and (3) neuronal activity as assessed indirectly by the immediate-early gene (IEG), c-Fos. Changes in cytochrome oxidase and c-Fos activity were observed in the retrosplenial cortex in Tg2576 mice as early as 5 months of age, long before evidence of plaque formation. Subsequent analyses concentrating on this early dysfunction revealed at 5 months pervasive, amyloid precursor protein (APP)-derived peptide accumulation in the retrosplenial cortex and selective afferents (anterior thalamus, hippocampus), which was associated with the observed c-Fos hyporeactivity. These findings indicate that retrosplenial cortex dysfunction occurs during early stages of amyloid production in Tg2576 mice and may contribute to cognitive dysfunction.

Two experiments investigated the capacity of rats to learn configural discriminations requiring integration of contextual (where) with temporal (when) information. In Experiment 1, during morning training sessions, food was delivered in context A and not in context B, whereas during afternoon sessions food was delivered in context B and not in context A. Rats acquired this discrimination over the course of 20 days. Experiment 2 employed a directly analogous aversive conditioning procedure in which footshock served in place of food. This procedure allowed the acquisition of the discrimination to be assessed through changes in activity to the contextual + temporal configurations (i.e., inactivity or freezing) and modulation of the immediate impact of footshock presentations (i.e., post-shock activity bursts). Both measures provided evidence of configural learning over the course of 12 days, with a final test showing that the presentation of footshock resulted in more post-shock activity in the nonreinforced than reinforced configurations. These behavioral effects reveal important parallels between (i) configural discrimination learning involving components allied to episodic memory and (ii) simple conditioning.

Recent studies have questioned the importance of the entorhinal cortex (ERC) for normal hippocampal function. For example, fibre-sparing ERC lesions have been found to have no effect on spatial learning in the watermaze. There is also doubt as to the importance of the ERC for contextual fear conditioning, with previous studies having yielded conflicting results. In an attempt to resolve this issue, the present study compared aspiration and cytotoxic ERC lesioned rats, along with fimbria-fornix (FFX) lesioned animals and sham operated controls, on an unsignalled contextual fear conditioning paradigm. The results of the present study show that whereas lesions of the FFX disrupted contextual freezing, neither aspiration nor cytotoxic ERC lesions had any effect on this behaviour. Aspiration ERC lesioned rats, however, like FFX lesioned animals, did display hyperactivity prior to the delivery of footshock. These results suggest that whereas projections between the hippocampus and subcortical structures are important for normal levels of contextual freezing, projections from the entorhinal cortex are not essential.

Objective: To integrate and evaluate the pharmacokinetic, endocrine, and clinical effects of micronized progesterone formulations. Design: Published articles concerning the pharmacokinetics of orally administered progesterone and the potential clinical uses of oral micronized progesterone were reviewed. Results concerning their use for secondary amenorrhea, premenopausal bleeding disorders, luteal phase dysfunction, termination of premature labor, hormone replacement therapy, and premenopausal syndrome are summarized. Critical issues to be resolved through ongoing preclinical and clinical research are highlighted. Result(s): Because of the enhanced bioavailability of oral micronized progesterone, the compound may be useful for a variety of therapeutic indications. Oral micronized progesterone is available in France, and a formulation recently has been approved in the United States for the treatment of secondary amenorrhea and postmenopausal hormone replacement therapy. A large body of evidence, including the Postmenopausal Estrogen/Progestin Interventions study, suggests that the use of a combination of estrogen and oral micronized progesterone is optimal for long-term hormone replacement therapy. There also are data indicating that oral micronized progesterone could be of potential use for the treatment of premenopausal bleeding disorders, luteal phase disorders, and premature labor. Conclusion(s): Oral micronized progesterone has widespread clinical potential, particularly for the treatment of secondary amenorrhea and dysfunctional premenopausal bleeding, and as a component of postmenopausal hormone replacement therapy.

Rats with complete cytotoxic hippocampal lesions exhibited spatial memory impairments in both the water maze and elevated T maze. They were hyperactive in photocell cages; swam faster in the water maze; and were less efficient on a nonspatial, differential reinforcement of low rates (DRL) task. Performance on both spatial tasks was also impaired by selective dorsal but not ventral lesions; swim speed was increased by ventral but not dorsal lesions. Both partial lesions caused a comparable reduction in DRL efficiency, although these effects were smaller than those of complete lesions. Neither partial lesion induced hyperactivity when rats were tested in photocell cages, although both complete and ventral lesion groups showed increased activity after footshock in other studies (Richmond et al., 1999). These results demonstrate possible functional dissociations along the septotemporal axis of the hippocampus.

Novel assays were used to assess inter alia whether the hippocampus\ud is involved in detecting novelty per se or in an\ud associative mismatch process. During training, rats received\ud two audiovisual sequences (tone–left constant light and click–\ud left flashing light). In both sham-operated control rats and those\ud with excitotoxic hippocampal lesions, novel visual targets provoked\ud an orienting response that habituated during training.\ud Moreover, like sham-operated rats, rats with hippocampal lesions\ud acquired associations between the elements of two audiovisual\ud sequences. However, subsequent test trials in which\ud the auditory stimuli preceding the visual targets were switched\ud (click–left constant light and tone–left flashing light) provoked\ud renewed orienting to the visual targets in sham-operated rats\ud but not in hippocampal rats. These results support the view that\ud hippocampal damage results in a failure to detect (or act on)\ud mismatches that are generated when an auditory stimulus associatively\ud evokes the memory of one visual stimulus and a\ud different (familiar) visual stimulus is present in the environment.

Current therapies for Alzheimer’s disease only treat the symptoms of the disease. We have previously developed a novel monoclonal antibody, 2B3, which binds to the [beta]-secretase cleavage site in amyloid precursor protein (APP) and reduces the production of amyloid-[beta] (A[beta]) in human cell lines. To determine whether the antibody was likely to be effective in mouse models of amyloid pathology in vivo, we investigated whether 2B3 could also bind to APP in mouse primary cortical neurones. Primary cortical neurones were produced from E15.5–17.5 C57Bl/6 wild-type and transgenic APP/V717I (London mutation) embryos. The percentage of the neuronal population was determined by immunocytochemistry. Cells were treated with 10 µg/ml 2B3 or an irrelevant IgG for 48 h and A[beta]40 levels determined by ELISA. The population of cells was found to contain over 75% neurones and 2B3 bound effectively to these cells. No differences in A[beta]40 were detected between wild-type and transgenic cells. Importantly, 2B3 significantly inhibited the production of A[beta]40 by 75.15±1.37% of the media control, whereas an irrelevant IgG only significantly reduced A[beta]40 levels by 23.35±5.55% of the media control. The reduction in A[beta]40 produced by 2B3 was significantly greater than that caused by the IgG. These data indicate that 2B3 binds to APP in mouse neurones and can inhibit A[beta]40, similar to our previous findings. The antibody is probably therefore acting by steric hindrance of [beta]-secretase and these data suggest that it will be effective in mice in vivo and could be an alternative potential therapy for Alzheimer’s disease.

Animal models are making an increasing contribution to our understanding of the psychology and brain mechanisms underlying behavioral inhibition and impulsivity. The aim here was to develop, for the first time, a mouse analogue of the stop-signal reaction time task with high translational validity in order to be able to exploit this species in genetic and molecular investigations of impulsive behaviours. Cohorts of mice were trained to nose-poke to presentations of visual stimuli. Control of responding was manipulated by altering the onset of an auditory ‘stop-signal’ during the go response. The anticipated systematic changes in action cancellation were observed as stopping was made more difficult by placing the stop-signal closer to the execution of the action. Excitotoxic lesions of medial prefrontal cortex resulted in impaired stopping, whilst the clinically effective drugs methylphenidate and atomoxetine enhanced stopping abilities. The specific 5-HT2C receptor antagonist SB242084 also led to enhanced response control in this task. We conclude that stop-signal reaction time task performance can be successfully modelled in mice and is sensitive to prefrontal cortex dysfunction and drug treatments in a qualitatively similar manner to humans and previous rat models. Additionally, using the model we show novel and highly discrete effects of 5-HT2C receptor antagonism that suggest manipulation of 5-HT2C receptor function may be of use in correcting maladaptive impulsive behaviors and provide further evidence for dissociable contributions of serotonergic transmission to response control.

Recent increases in pathology costs per scheme member are a concern to medical schemes and pathologists alike. To better understand the observed increasing costs, the National Pathology Group commissioned Prognosys to analyse the trends affecting these increases. We found that these increases are driven by inflation, increases in utilisation, and redistribution of the burden of cost. The identification of utilisation as a cost driver for pathology services is noteworthy as almost all pathology services are by referral from another doctor.

Synaptic plasticity dependent on N-methyl-D-aspartate (NMDA) receptors is thought to underlie certain types of learning and memory. In support of this, both hippocampal long-term potentiation and spatial learning in a watermaze are impaired by blocking NMDA receptors with a selective antagonist D(-)-2-amino-5-phosphonovaleric acid (AP5) or by a mutation in one of the receptor subunits. Here we report, however, that the AP5-induced learning deficit can be almost completely prevented if rats are pretrained in a different watermaze before administration of the drug. This is not because of stimulus generalization, and occurs despite learning of the second task remaining hippocampus dependent. An AP5-induced learning deficit is, however, still seen if the animals are pretrained using a non-spatial task. Thus, despite its procedural simplicity, the watermaze may involve multiple cognitive processes with distinct pharmacological properties; although required for some component of spatial learning, NMDA receptors may not be required for encoding the spatial representation of a specific environment.

In Experiment 1, we used six procedures in a series of unsuccessful attempts to obtain relational learning using trial-unique pictorial stimuli in pigeons. The Experiment began by testing conventional (three-key) matching-to-sample (MTS) and nonmatching-to-sample (NMTS); in subsequent stages of the experiment we progressively incorporated features of techniques that do obtain relational learning in a single-key apparatus. In Experiment 2, we found that acquisition of NMTS using pictorial stimuli proceeded no more rapidly than acquisition of a conditional discrimination. Experiment 3 showed that acquisition of NMTS was more rapid than acquisition of MTS when plain colored stimuli were used, but not when pictorial stimuli were used. These three experiments suggest that pigeons do not recognize pictorial stimuli shown on different keys. In Experiment 4, between-key recognition was obtained with familiar but not with novel pictorial stimuli. It is argued that perceptual learning facilitates the detection of the between-key identity of complex stimuli, and that perceptual processes may underlie the difficulty in demonstrating relational learning in pigeons.

Amyloid-β (Aβ) is cleaved from amyloid precursor protein (APP) predominantly after APP has trafficked through the secretory pathway and then become re-internalised by endocytosis. Clathrin-mediated and, more recently, clathrin-independent endocytosis have both been implicated in this process. Furthermore, endocytic abnormalities have been identified in cases of Alzheimer’s disease (AD), however, the relevance of these changes to the aetiology of the disease remains unclear. We therefore examined the expression of proteins related to these endocytic processes in the cortex of Tg2576 mice that overexpress the Swedish mutation in APP, and consequently overexpress Aβ, to determine if there were any changes in their associated pathways. We identified significant increases in the levels of clathrin, dynamin and PICALM, all proteins intimately involved with the clathrin-mediated endocytic pathway, in the transgenic animals. However, levels of proteins associated with flotillin or caveolin-mediated endocytic pathways remained unchanged. These results emphasise the importance of clathrin-mediated endocytosis in the aetiology of AD and reinforce the results of the recent GWAS studies that identified genes for clathrin-mediated endocytosis as susceptibility genes for AD. Such studies in transgenic mice will allow us to learn more about the role of clathrin-mediated endocytosis in AD.

Genetically modified mice, lacking the GluA1 AMPA receptor subunit, are impaired on spatial working memory tasks, but display normal acquisition of spatial reference memory tasks. One explanation for this dissociation is that working memory, win-shift performance engages a GluA1-dependent, non-associative, short-term memory process through which animals choose relatively novel arms in preference to relatively familiar options. In contrast, spatial reference memory, as exemplified by the Morris water maze task, reflects a GluA1-independent, associative, long-term memory mechanism. These results can be accommodated by Wagner's dual-process model of memory in which short and long-term memory mechanisms exist in parallel and, under certain circumstances, compete with each other. According to our analysis, GluA1−/− mice lack short-term memory for recently experienced spatial stimuli. One consequence of this impairment is that these stimuli should remain surprising and thus be better able to form long-term associative representations. Consistent with this hypothesis, we have recently shown that long-term spatial memory for recently visited locations is enhanced in GluA1−/− mice, despite impairments in hippocampal synaptic plasticity. Taken together, these results support a role for GluA1-containing AMPA receptors in short-term habituation, and in modulating the intensity or perceived salience of stimuli.

The GluA1 AMPA receptor subunit is a key mediator of hippocampal synaptic plasticity and is especially important for a rapidly-induced, short-lasting form of potentiation. GluA1 gene deletion impairs hippocampus-dependent, spatial working memory, but spares hippocampus-dependent spatial reference memory. These findings may reflect the necessity of GluA1-dependent synaptic plasticity for short-term memory of recently visited places, but not for the ability to form long-term associations between a particular spatial location and an outcome. This hypothesis is in concordance with the theory that short-term and long-term memory depend on dissociable psychological processes. In this study we tested GluA1−/− mice on both short-term and long-term spatial memory using a simple novelty preference task. Mice were given a series of repeated exposures to a particular spatial location (the arm of a Y-maze) before their preference for a novel spatial location (the unvisited arm of the maze) over the familiar spatial location was assessed. GluA1−/− mice were impaired if the interval between the trials was short (1 min), but showed enhanced spatial memory if the interval between the trials was long (24 h). This enhancement was caused by the interval between the exposure trials rather than the interval prior to the test, thus demonstrating enhanced learning and not simply enhanced performance or expression of memory. This seemingly paradoxical enhancement of hippocampus-dependent spatial learning may be caused by GluA1 gene deletion reducing the detrimental effects of short-term memory on subsequent long-term learning. Thus, these results support a dual-process model of memory in which short-term and long-term memory are separate and sometimes competitive processes.