A retrieval-specific mechanism of adaptive forgetting in the mammalian brain.

Forgetting is a ubiquitous phenomenon that is actively promoted in many species. How and whether organisms' behavioral goals drive which memories are actively forgotten is unknown. Here we show that processes essential to controlling goal-directed behavior trigger active forgetting of distracting memories that interfere with behavioral goals. When rats need to retrieve particular memories to guide exploration, it reduces later retention of other memories encoded in that environment. As with humans, this retrieval-induced forgetting is competition-dependent, cue-independent and reliant on prefrontal control: Silencing the medial prefrontal cortex with muscimol abolishes the effect. cFos imaging reveals that prefrontal control demands decline over repeated retrievals as competing memories are forgotten successfully, revealing a key adaptive benefit of forgetting. Occurring in 88% of the rats studied, this finding establishes a robust model of how adaptive forgetting harmonizes memory with behavioral demands, permitting isolation of its circuit, cellular and molecular mechanisms.

Synaptic plasticity through activation of GluA3-containing AMPA-receptors.

Excitatory synaptic transmission is mediated by AMPA-type glutamate receptors (AMPARs). In CA1 pyramidal neurons of the hippocampus two types of AMPARs predominate: those that contain subunits GluA1 and GluA2 (GluA1/2), and those that contain GluA2 and GluA3 (GluA2/3). Whereas subunits GluA1 and GluA2 have been extensively studied, the contribution of GluA3 to synapse physiology has remained unclear. Here we show in mice that GluA2/3s are in a low-conductance state under basal conditions, and although present at synapses they contribute little to synaptic currents. When intracellular cyclic AMP (cAMP) levels rise, GluA2/3 channels shift to a high-conductance state, leading to synaptic potentiation. This cAMP-driven synaptic potentiation requires the activation of both protein kinase A (PKA) and the GTPase Ras, and is induced upon the activation of ß-adrenergic receptors. Together, these experiments reveal a novel type of plasticity at CA1 hippocampal synapses that is expressed by the activation of GluA3-containing AMPARs.

Motor Learning Requires Purkinje Cell Synaptic Potentiation through Activation of AMPA-Receptor Subunit GluA3.

Accumulating evidence indicates that cerebellar long-term potentiation (LTP) is necessary for procedural learning. However, little is known about its underlying molecular mechanisms. Whereas AMPA receptor (AMPAR) subunit rules for synaptic plasticity have been extensively studied in relation to declarative learning, it is unclear whether these rules apply to cerebellum-dependent motor learning. Here we show that LTP at the parallel-fiber-to-Purkinje-cell synapse and adaptation of the vestibulo-ocular reflex depend not on GluA1- but on GluA3-containing AMPARs. In contrast to the classic form of LTP implicated in declarative memory formation, this form of LTP does not require GluA1-AMPAR trafficking but rather requires changes in open-channel probability of GluA3-AMPARs mediated by cAMP signaling and activation of the protein directly activated by cAMP (Epac). We conclude that vestibulo-cerebellar motor learning is the first form of memory acquisition shown to depend on GluA3-dependent synaptic potentiation by increasing single-channel conductance.

Amyloid-ß effects on synapses and memory require AMPA receptor subunit GluA3.

Amyloid-ß (Aß) is a prime suspect for causing cognitive deficits during the early phases of Alzheimer's disease (AD). Experiments in AD mouse models have shown that soluble oligomeric clusters of Aß degrade synapses and impair memory formation. We show that all Aß-driven effects measured in these mice depend on AMPA receptor (AMPAR) subunit GluA3. Hippocampal neurons that lack GluA3 were resistant against Aß-mediated synaptic depression and spine loss. In addition, Aß oligomers blocked long-term synaptic potentiation only in neurons that expressed GluA3. Furthermore, although Aß-overproducing mice showed significant memory impairment, memories in GluA3-deficient congenics remained unaffected. These experiments indicate that the presence of GluA3-containing AMPARs is critical for Aß-mediated synaptic and cognitive deficits.

Visualization of Active Glucocerebrosidase in Rodent Brain with High Spatial Resolution following In Situ Labeling with Fluorescent Activity Based Probes.

Gaucher disease is characterized by lysosomal accumulation of glucosylceramide due to deficient activity of lysosomal glucocerebrosidase (GBA). In cells, glucosylceramide is also degraded outside lysosomes by the enzyme glucosylceramidase 2 (GBA2) of which inherited deficiency is associated with ataxias. The interest in GBA and glucosylceramide metabolism in the brain has grown following the notion that mutations in the GBA gene impose a risk factor for motor disorders such as α-synucleinopathies. We earlier developed a ß-glucopyranosyl-configured cyclophellitol-epoxide type activity based probe (ABP) allowing in vivo and in vitro visualization of active molecules of GBA with high spatial resolution. Labeling occurs through covalent linkage of the ABP to the catalytic nucleophile residue in the enzyme pocket. Here, we describe a method to visualize active GBA molecules in rat brain slices using in vivo labeling. Brain areas related to motor control, like the basal ganglia and motor related structures in the brainstem, show a high content of active GBA. We also developed a ß-glucopyranosyl cyclophellitol-aziridine ABP allowing in situ labeling of GBA2. Labeled GBA2 in brain areas can be identified and quantified upon gel electrophoresis. The distribution of active GBA2 markedly differs from that of GBA, being highest in the cerebellar cortex. The histological findings with ABP labeling were confirmed by biochemical analysis of isolated brain areas. In conclusion, ABPs offer sensitive tools to visualize active GBA and to study the distribution of GBA2 in the brain and thus may find application to establish the role of these enzymes in neurodegenerative disease conditions such as α-synucleinopathies and cerebellar ataxia.

Tripeptidyl Peptidase II Mediates Levels of Nuclear Phosphorylated ERK1 and ERK2.

Tripeptidyl peptidase II (TPP2) is a serine peptidase involved in various biological processes, including antigen processing, cell growth, DNA repair, and neuropeptide mediated signaling. The underlying mechanisms of how a peptidase can influence this multitude of processes still remain unknown. We identified rapid proteomic changes in neuroblastoma cells following selective TPP2 inhibition using the known reversible inhibitor butabindide, as well as a new, more potent, and irreversible peptide phosphonate inhibitor. Our data show that TPP2 inhibition indirectly but rapidly decreases the levels of active, di-phosphorylated extracellular signal-regulated kinase 1 (ERK1) and ERK2 in the nucleus, thereby down-regulating signal transduction downstream of growth factors and mitogenic stimuli. We conclude that TPP2 mediates many important cellular functions by controlling ERK1 and ERK2 phosphorylation. For instance, we show that TPP2 inhibition of neurons in the hippocampus leads to an excessive strengthening of synapses, indicating that TPP2 activity is crucial for normal brain function.

Development and validation of a risk score for predicting death after pneumonectomy.

Pneumonectomy is associated with significant postoperative mortality. This study was undertaken to develop and validate a risk model of mortality following pneumonectomy. We reviewed our prospective database and identified 774 pneumonectomies from a total of 7792 consecutive anatomical lung resections in the years 2003 to 2010 (rate of pneumonectomy: 9.9%). Based on data from 542 pneumonectomies between 2003 and 2007 (i.e., the "discovery set"), a penalized multivariable logistic regression analysis was performed to identify preoperative risk factors. A risk model was developed and validated in an independent data set of 232 pneumonectomies that were performed between 2008 and 2010 (i.e., the "validation set"). Of the 542 patients in the discovery set (DS), 35 patients (6.5%) died after pneumonectomy during the same admission. We developed a risk prediction model for in-hospital mortality following pneumonectomy; that model included age, current alcohol use, coronary artery disease, preoperative leukocyte count and palliative indication as possible risk factors. The risk model was subsequently successfully validated in an independent data set (n = 232) in which 18 patients (7.8%) died following pneumonectomy. For the validation set, the sensitivity of the model was 53.3% (DS: 54.3%), the specificity was 88.0% (DS: 87.4%), the positive predictive value was 26.7% (DS: 22.9%) and the negative predictive value was 95.8% (DS: 96.5%). The Brier score was 0.062 (DS: 0.054). The prediction model is statistically valid and clinically relevant.

Quantitative in vitro to in vivo extrapolation of tissues toxicity.

Predicting repeated-dosing in vivo drug toxicity from in vitro testing and omics data gathering requires significant support in bioinformatics, mathematical modeling and statistics. We present here the major aspects of the work devoted within the framework of the European integrated Predict-IV to pharmacokinetic modeling of in vitro experiments, physiologically based pharmacokinetic (PBPK) modeling, mechanistic models of toxicity for the kidney and brain, large scale dose-response analyses methods and biomarker discovery tools. All of those methods have been applied to various extent to the drug datasets developed by the project's partners. Our approach is rather generic and could be adapted to other drugs or drug candidates. It marks a successful integration of the work of the different teams toward a common goal of predictive quantitative in vitro to in vivo extrapolation.

Role of medial prefrontal cortex serotonin 2A receptors in the control of retrieval of recognition memory in rats.

Often, retrieval cues are not uniquely related to one specific memory, which could lead to memory interference. Controlling interference is particularly important during episodic memory retrieval or when remembering specific events in a spatiotemporal context. Despite a clear involvement of prefrontal cortex (PFC) in episodic memory in human studies, information regarding the mechanisms and neurotransmitter systems in PFC involved in memory is scarce. Although the serotoninergic system has been linked to PFC functionality and modulation, its role in memory processing is poorly understood. We hypothesized that the serotoninergic system in PFC, in particular the 5-HT2A receptor (5-HT2AR) could have a role in the control of memory retrieval. In this work we used different versions of the object recognition task in rats to study the role of the serotoninergic modulation in the medial PFC (mPFC) in memory retrieval. We found that blockade of 5-HT2AR in mPFC affects retrieval of an object in context memory in a spontaneous novelty preference task, while sparing single-item recognition memory. We also determined that 5-HT2ARs in mPFC are required for hippocampal-mPFC interaction during retrieval of this type of memory, suggesting that the mPFC controls the expression of memory traces stored in the hippocampus biasing retrieval to the most relevant one.

Stochastic time-concentration activity models for cytotoxicity in 3D brain cell cultures.

BACKGROUND: In vitro aggregating brain cell cultures containing all types of brain cells have been shown to be useful for neurotoxicological investigations. The cultures are used for the detection of nervous system-specific effects of compounds by measuring multiple endpoints, including changes in enzyme activities. Concentration-dependent neurotoxicity is determined at several time points. METHODS: A Markov model was set up to describe the dynamics of brain cell populations exposed to potentially neurotoxic compounds. Brain cells were assumed to be either in a healthy or stressed state, with only stressed cells being susceptible to cell death. Cells may have switched between these states or died with concentration-dependent transition rates. Since cell numbers were not directly measurable, intracellular lactate dehydrogenase (LDH) activity was used as a surrogate. Assuming that changes in cell numbers are proportional to changes in intracellular LDH activity, stochastic enzyme activity models were derived. Maximum likelihood and least squares regression techniques were applied for estimation of the transition rates. Likelihood ratio tests were performed to test hypotheses about the transition rates. Simulation studies were used to investigate the performance of the transition rate estimators and to analyze the error rates of the likelihood ratio tests. The stochastic time-concentration activity model was applied to intracellular LDH activity measurements after 7 and 14 days of continuous exposure to propofol. The model describes transitions from healthy to stressed cells and from stressed cells to death. RESULTS: The model predicted that propofol would affect stressed cells more than healthy cells. Increasing propofol concentration from 10 to 100 µM reduced the mean waiting time for transition to the stressed state by 50%, from 14 to 7 days, whereas the mean duration to cellular death reduced more dramatically from 2.7 days to 6.5 hours. CONCLUSION: The proposed stochastic modeling approach can be used to discriminate between different biological hypotheses regarding the effect of a compound on the transition rates. The effects of different compounds on the transition rate estimates can be quantitatively compared. Data can be extrapolated at late measurement time points to investigate whether costs and time-consuming long-term experiments could possibly be eliminated.

Role for staphylococci in misguided thrombus resolution of chronic thromboembolic pulmonary hypertension.

OBJECTIVE: Acute pulmonary emboli usually resolve within 6 months. However, in 0.1% to 3.8% of cases thrombus transforms into fibrous masses. If vascular obstruction is severe, the resulting condition is chronic thromboembolic pulmonary hypertension (CTEPH). Patients who carry ventriculo-atrial (VA-) shunts for the treatment of hydrocephalus and report a history of shunt infection are at an increased risk for CTEPH. Because CTEPH lacks traditional plasmatic risk factors for venous thromboembolism, we hypothesized that delayed thrombus resolution rather than abnormal coagulation is important, and that bacterial infection would be important for this misguidance. METHODS AND RESULTS: Human CTEPH thromboemboli were harvested during pulmonary endarterectomy. The effects of Staphylococcal infection on thrombus organization were examined in a murine model of stagnant-flow venous thrombosis. Staphylococcal DNA, but not RNA, was detected in 6 of 7 thrombi from VA shunt carriers. In the mouse model, staphylococcal infection delayed thrombus resolution in parallel with upregulation of transforming growth factor (TGF) beta and connective tissue growth factor. CONCLUSIONS: In the present work, we propose a mechanism of disease demonstrating that infection with Staphylococci enhances fibrotic vascular remodeling after thrombosis, resulting in misguided thrombus resolution. Thrombus infection appears to be a trigger in the evolution of CTEPH.