Overnight neuronal plasticity and adaptation to emotional distress.

Expressions such as 'sleep on it' refer to the resolution of distressing experiences across a night of sound sleep. Sleep is an active state during which the brain reorganizes the synaptic connections that form memories. This Perspective proposes a model of how sleep modifies emotional memory traces. Sleep-dependent reorganization occurs through neurophysiological events in neurochemical contexts that determine the fates of synapses to grow, to survive or to be pruned. We discuss how low levels of acetylcholine during non-rapid eye movement sleep and low levels of noradrenaline during rapid eye movement sleep provide a unique window of opportunity for plasticity in neuronal representations of emotional memories that resolves the associated distress. We integrate sleep-facilitated adaptation over three levels: experience and behaviour, neuronal circuits, and synaptic events. The model generates testable hypotheses for how failed sleep-dependent adaptation to emotional distress is key to mental disorders, notably disorders of anxiety, depression and post-traumatic stress with the common aetiology of insomnia.

Seizure-mediated iron accumulation and dysregulated iron metabolism after status epilepticus and in temporal lobe epilepsy.

Neuronal dysfunction due to iron accumulation in conjunction with reactive oxygen species (ROS) could represent an important, yet underappreciated, component of the epileptogenic process. However, to date, alterations in iron metabolism in the epileptogenic brain have not been addressed in detail. Iron-related neuropathology and antioxidant metabolic processes were investigated in resected brain tissue from patients with temporal lobe epilepsy and hippocampal sclerosis (TLE-HS), post-mortem brain tissue from patients who died after status epilepticus (SE) as well as brain tissue from the electrically induced SE rat model of TLE. Magnetic susceptibility of the presumed seizure-onset zone from three patients with focal epilepsy was compared during and after seizure activity. Finally, the cellular effects of iron overload were studied in vitro using an acute mouse hippocampal slice preparation and cultured human fetal astrocytes. While iron-accumulating neurons had a pyknotic morphology, astrocytes appeared to acquire iron-sequestrating capacity as indicated by prominent ferritin expression and iron retention in the hippocampus of patients with SE or TLE. Interictal to postictal comparison revealed increased magnetic susceptibility in the seizure-onset zone of epilepsy patients. Post-SE rats had consistently higher hippocampal iron levels during the acute and chronic phase (when spontaneous recurrent seizures are evident). In vitro, in acute slices that were exposed to iron, neurons readily took up iron, which was exacerbated by induced epileptiform activity. Human astrocyte cultures challenged with iron and ROS increased their antioxidant and iron-binding capacity, but simultaneously developed a pro-inflammatory phenotype upon chronic exposure. These data suggest that seizure-mediated, chronic neuronal iron uptake might play a role in neuronal dysfunction/loss in TLE-HS. On the other hand, astrocytes sequester iron, specifically in chronic epilepsy. This function might transform astrocytes into a highly resistant, pro-inflammatory phenotype potentially contributing to pro-epileptogenic inflammatory processes.

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.

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.

The prion protein as a receptor for amyloid-beta.

Increased levels of brain amyloid-beta, a secreted peptide cleavage product of amyloid precursor protein (APP), is believed to be critical in the aetiology of Alzheimer's disease. Increased amyloid-beta can cause synaptic depression, reduce the number of spine protrusions (that is, sites of synaptic contacts) and block long-term synaptic potentiation (LTP), a form of synaptic plasticity; however, the receptor through which amyloid-beta produces these synaptic perturbations has remained elusive. Laurén et al. suggested that binding between oligomeric amyloid-beta (a form of amyloid-beta thought to be most active) and the cellular prion protein (PrP(C)) is necessary for synaptic perturbations. Here we show that PrP(C) is not required for amyloid-beta-induced synaptic depression, reduction in spine density, or blockade of LTP; our results indicate that amyloid-beta-mediated synaptic defects do not require PrP(c).

Metabotropic NMDA receptor function is required for ß-amyloid-induced synaptic depression.

The mechanisms by which ß-amyloid (Aß), a peptide fragment believed to contribute to Alzheimer's disease, leads to synaptic deficits are not known. Here we find that elevated oligomeric Aß requires ion flux-independent function of NMDA receptors (NMDARs) to produce synaptic depression. Aß activates this metabotropic NMDAR function on GluN2B-containing NMDARs but not on those containing GluN2A. Furthermore, oligomeric Aß leads to a selective loss of synaptic GluN2B responses, effecting a switch in subunit composition from GluN2B to GluN2A, a process normally observed during development. Our results suggest that conformational changes of the NMDAR, and not ion flow through its channel, are required for Aß to produce synaptic depression and a switch in NMDAR composition. This Aß-induced signaling mediated by alterations in GluN2B conformation may be a target for therapeutic intervention of Alzheimer's disease.

Metabotropic NMDA receptor function is required for NMDA receptor-dependent long-term depression.

NMDA receptor (NMDAR) activation controls long-term potentiation (LTP) as well as long-term depression (LTD) of synaptic transmission, cellular models of learning and memory. A long-standing view proposes that a high level of Ca(2+) entry through NMDARs triggers LTP; lower Ca(2+) entry triggers LTD. Here we show that ligand binding to NMDARs is sufficient to induce LTD; neither ion flow through NMDARs nor Ca(2+) rise is required. However, basal levels of Ca(2+) are permissively required. Lowering, but not maintaining, basal Ca(2+) levels with Ca(2+) chelators blocks LTD and drives strong synaptic potentiation, indicating that basal Ca(2+) levels control NMDAR-dependent LTD and basal synaptic transmission. Our findings indicate that metabotropic actions of NMDARs can weaken active synapses without raising postsynaptic calcium, thereby revising and expanding the mechanisms controlling synaptic plasticity.

The developmental stages of synaptic plasticity.

The brain is programmed to drive behaviour by precisely wiring the appropriate neuronal circuits. Wiring and rewiring of neuronal circuits largely depends on the orchestrated changes in the strengths of synaptic contacts. Here, we review how the rules of synaptic plasticity change during development of the brain, from birth to independence. We focus on the changes that occur at the postsynaptic side of excitatory glutamatergic synapses in the rodent hippocampus and neocortex. First we summarize the current data on the structure of synapses and the developmental expression patterns of the key molecular players of synaptic plasticity, N-methyl-d-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, as well as pivotal kinases (Ca(2+)/calmodulin-dependent protein kinase II, protein kinase A, protein kinase C) and phosphatases (PP1, PP2A, PP2B). In the second part we relate these findings to important characteristics of the emerging network. We argue that the concerted and gradual shifts in the usage of plasticity molecules comply with the changing need for (re)wiring neuronal circuits.

Synapto-depressive effects of amyloid beta require PICK1.

Amyloid beta (Aß), a key component in the pathophysiology of Alzheimer's disease, is thought to target excitatory synapses early in the disease. However, the mechanism by which Aß weakens synapses is not well understood. Here we showed that the PDZ domain protein, protein interacting with C kinase 1 (PICK1), was required for Aß to weaken synapses. In mice lacking PICK1, elevations of Aß failed to depress synaptic transmission in cultured brain slices. In dissociated cultured neurons, Aß failed to reduce surface α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subunit 2, a subunit of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors that binds with PICK1 through a PDZ ligand-domain interaction. Lastly, a novel small molecule (BIO922) discovered through structure-based drug design that targets the specific interactions between GluA2 and PICK1 blocked the effects of Aß on synapses and surface receptors. We concluded that GluA2-PICK1 interactions are a key component of the effects of Aß on synapses.

Contextual learning requires synaptic AMPA receptor delivery in the hippocampus.

The hippocampus plays a central role in learning and memory. Although synaptic delivery of AMPA-type glutamate receptors (AMPARs) contributes to experience-dependent synaptic strengthening, its role in hippocampus-dependent learning remains elusive. By combining viral-mediated in vivo gene delivery with in vitro patch-clamp recordings, we found that the inhibitory avoidance task, a hippocampus-dependent contextual fear-learning paradigm, delivered GluR1-containing AMPARs into CA3-CA1 synapses of the dorsal hippocampus. To block the synaptic delivery of endogenous AMPARs, we expressed a fragment of the GluR1-cytoplasmic tail (the 14-aa GluR1 membrane-proximal region with two serines mutated to phospho-mimicking aspartates: MPR-DD). MPR-DD prevented learning-driven synaptic AMPAR delivery in CA1 neurons. Bilateral expression of MPR-DD in the CA1 region of the rat impaired inhibitory avoidance learning, indicating that synaptic GluR1 trafficking in the CA1 region of the hippocampus is required for encoding contextual fear memories. The fraction of CA1 neurons that underwent synaptic strengthening positively correlated with the performance in the inhibitory avoidance fear memory task. These data suggest that the robustness of a contextual memory depends on the number of hippocampal neurons that participate in the encoding of a memory trace.

The concept of resilience to Alzheimer's Disease: current definitions and cellular and molecular mechanisms.

Some individuals are able to maintain their cognitive abilities despite the presence of significant Alzheimer's Disease (AD) neuropathological changes. This discrepancy between cognition and pathology has been labeled as resilience and has evolved into a widely debated concept. External factors such as cognitive stimulation are associated with resilience to AD, but the exact cellular and molecular underpinnings are not completely understood. In this review, we discuss the current definitions used in the field, highlight the translational approaches used to investigate resilience to AD and summarize the underlying cellular and molecular substrates of resilience that have been derived from human and animal studies, which have received more and more attention in the last few years. From these studies the picture emerges that resilient individuals are different from AD patients in terms of specific pathological species and their cellular reaction to AD pathology, which possibly helps to maintain cognition up to a certain tipping point. Studying these rare resilient individuals can be of great importance as it could pave the way to novel therapeutic avenues for AD.

M13 phage grafted with peptide motifs as a tool to detect amyloid-ß oligomers in brain tissue.

Oligomeric clusters of amyloid-ß (Aß) are one of the major biomarkers for Alzheimer's disease (AD). However, proficient methods to detect Aß-oligomers in brain tissue are lacking. Here we show that synthetic M13 bacteriophages displaying Aß-derived peptides on their surface preferentially interact with Aß-oligomers. When exposed to brain tissue isolated from APP/PS1-transgenic mice, these bacteriophages detect small-sized Aß-aggregates in hippocampus at an early age, prior to the occurrence of Aß-plaques. Similarly, the bacteriophages reveal the presence of such small Aß-aggregates in post-mortem hippocampus tissue of AD-patients. These results advocate bacteriophages displaying Aß-peptides as a convenient and low-cost tool to identify Aß-oligomers in post-mortem brain tissue of AD-model mice and AD-patients.

Early life stress lastingly alters the function and AMPA-receptor composition of glutamatergic synapses in the hippocampus of male mice.

Early postnatal life is a sensitive period of development that shapes brain structure and function later in life. Exposure to stress during this critical time window can alter brain development and may enhance the susceptibility to psychopathology and neurodegenerative disorders later in life. The developmental effects of early life stress (ELS) on synaptic function are not fully understood, but could provide mechanistic insights into how ELS modifies later brain function and disease risk. We here assessed the effects of ELS on synaptic function and composition in the hippocampus of male mice. Mice were subjected to ELS by housing dams and pups with limited bedding and nesting material from postnatal days (P) 2-9. Synaptic strength was measured in terms of miniature excitatory postsynaptic currents (mEPSCs) in the hippocampal dentate gyrus at three different developmental stages: the early postnatal phase (P9), preadolescence (P21, at weaning) and adulthood at 3 months of age (3MO). Hippocampal synaptosome fractions were isolated from P9 and 3MO tissue and analyzed for protein content to assess postsynaptic composition. Finally, dendritic spine density was assessed in the DG at 3MO. At P9, ELS increased mEPSC frequency and amplitude. In parallel, synaptic composition was altered as PSD-95, GluA3 and GluN2B content were significantly decreased. The increased mEPSC frequency was sustained up to 3MO, at which age, GluA3 content was significantly increased. No differences were found in dendritic spine density. These findings highlight how ELS affects the development of hippocampal synapses, which could provide valuable insight into mechanisms how ELS alters brain function later in life.

Variance analysis as a method to predict the locus of plasticity at populations of non-uniform synapses.

Our knowledge on synaptic transmission in the central nervous system has often been obtained by evoking synaptic responses to populations of synapses. Analysis of the variance in synaptic responses can be applied as a method to predict whether a change in synaptic responses is a consequence of altered presynaptic neurotransmitter release or postsynaptic receptors. However, variance analysis is based on binomial statistics, which assumes that synapses are uniform. In reality, synapses are far from uniform, which questions the reliability of variance analysis when applying this method to populations of synapses. To address this, we used an in silico model for evoked synaptic responses and compared variance analysis outcomes between populations of uniform versus non-uniform synapses. This simulation revealed that variance analysis produces similar results irrespectively of the grade of uniformity of synapses. We put this variance analysis to the test with an electrophysiology experiment using a model system for which the loci of plasticity are well established: the effect of amyloid-ß on synapses. Variance analysis correctly predicted that postsynaptically produced amyloid-ß triggered predominantly a loss of synapses and a minor reduction of postsynaptic currents in remaining synapses with little effect on presynaptic release probability. We propose that variance analysis can be reliably used to predict the locus of synaptic changes for populations of non-uniform synapses.

The Expression of Epac2 and GluA3 in an Alzheimer's Disease Experimental Model and Postmortem Patient Samples.

Alzheimer's disease (AD) is one of the most prevalent neurodegenerative diseases, characterized by amyloid beta (Aß) and hyperphosphorylated tau accumulation in the brain. Recent studies indicated that memory retrieval, rather than memory formation, was impaired in the early stage of AD. Our previous study reported that pharmacological activation of hippocampal Epac2 promoted memory retrieval in C57BL/6J mice. A recent study suggested that pharmacological inhibition of Epac2 prevented synaptic potentiation mediated by GluA3-containing AMPARs. In this study, we aimed to investigate proteins associated with Epac2-mediated memory in hippocampal postmortem samples of AD patients and healthy controls compared with the experimental AD model J20 and wild-type mice. Epac2 and phospho-Akt were downregulated in AD patients and J20 mice, while Epac1 and phospho-ERK1/2 were not altered. GluA3 was reduced in J20 mice and tended to decrease in AD patients. PSD95 tended to decrease in AD patients and J20. Interestingly, AKAP5 was increased in AD patients but not in J20 mice, implicating its role in tau phosphorylation. Our study points to the downregulation of hippocampal expression of proteins associated with Epac2 in AD.

PSD-95 protects synapses from ß-amyloid.

Beta-amyloid (Aß) depresses excitatory synapses by a poorly understood mechanism requiring NMDA receptor (NMDAR) function. Here, we show that increased PSD-95, a major synaptic scaffolding molecule, blocks the effects of Aß on synapses. The protective effect persists in tissue lacking the AMPA receptor subunit GluA1, which prevents the confounding synaptic potentiation by increased PSD-95. Aß modifies the conformation of the NMDAR C-terminal domain (CTD) and its interaction with protein phosphatase 1 (PP1), producing synaptic weakening. Higher endogenous levels or overexpression of PSD-95 block Aß-induced effects on the NMDAR CTD conformation, its interaction with PP1, and synaptic weakening. Our results indicate that increased PSD-95 protects synapses from Aß toxicity, suggesting that low levels of synaptic PSD-95 may be a molecular sign indicating synapse vulnerability to Aß. Importantly, pharmacological inhibition of its depalmitoylation increases PSD-95 at synapses and rescues deficits caused by Aß, possibly opening a therapeutic avenue against Alzheimer's disease.

Measuring Behavior in the Home Cage: Study Design, Applications, Challenges, and Perspectives.

The reproducibility crisis (or replication crisis) in biomedical research is a particularly existential and under-addressed issue in the field of behavioral neuroscience, where, in spite of efforts to standardize testing and assay protocols, several known and unknown sources of confounding environmental factors add to variance. Human interference is a major contributor to variability both within and across laboratories, as well as novelty-induced anxiety. Attempts to reduce human interference and to measure more "natural" behaviors in subjects has led to the development of automated home-cage monitoring systems. These systems enable prolonged and longitudinal recordings, and provide large continuous measures of spontaneous behavior that can be analyzed across multiple time scales. In this review, a diverse team of neuroscientists and product developers share their experiences using such an automated monitoring system that combines Noldus PhenoTyper® home-cages and the video-based tracking software, EthoVision® XT, to extract digital biomarkers of motor, emotional, social and cognitive behavior. After presenting our working definition of a "home-cage", we compare home-cage testing with more conventional out-of-cage tests (e.g., the open field) and outline the various advantages of the former, including opportunities for within-subject analyses and assessments of circadian and ultradian activity. Next, we address technical issues pertaining to the acquisition of behavioral data, such as the fine-tuning of the tracking software and the potential for integration with biotelemetry and optogenetics. Finally, we provide guidance on which behavioral measures to emphasize, how to filter, segment, and analyze behavior, and how to use analysis scripts. We summarize how the PhenoTyper has applications to study neuropharmacology as well as animal models of neurodegenerative and neuropsychiatric illness. Looking forward, we examine current challenges and the impact of new developments. Examples include the automated recognition of specific behaviors, unambiguous tracking of individuals in a social context, the development of more animal-centered measures of behavior and ways of dealing with large datasets. Together, we advocate that by embracing standardized home-cage monitoring platforms like the PhenoTyper, we are poised to directly assess issues pertaining to reproducibility, and more importantly, measure features of rodent behavior under more ethologically relevant scenarios.

Contributions of the T cell receptor-associated CD3gamma-ITAM to thymocyte selection.

The immunoreceptor tyrosine-based activation motifs (ITAMs) in the CD3 chains associated with the T cell receptor (TCR) are crucial for TCR signaling. To probe the role of the CD3gamma-ITAM in T cell development, we created knock-in mice in which the CD3gamma chain of the TCR complex is replaced by a mutant signaling-deficient CD3gamma chain, lacking the CD3gamma-ITAM. This mutation results in considerable impairment in positive selection in the polyclonal TCR repertoire. When CD3gamma-deltaITAM mice are crossed to mice expressing transgenic F5 TCRs, their thymocytes are completely unable to perform positive selection in vivo in response to intrathymic ligands. Also, the in vitro positive selection response of double-positive (DP) thymocytes with F5-CD3gamma-deltaITAM mutant receptors to their agonist ligand and many of its variants is severely impaired or abrogated. Yet, the binding and dissociation constants of agonist ligands for the F5 receptor are not affected by the CD3gamma-deltaITAM mutation. Furthermore, DP thymocytes with mutant receptors can respond to agonist ligand with normal antigen sensitivity and to normal levels, as shown by their ability to induce CD69 up-regulation, TCR down-regulation, negative selection, and ZAP70 and c-Jun NH2-terminal kinase activation. In sharp contrast, induction of extracellular signal-regulated kinase (ERK) activation and linker for activation of T cells (LAT) phosphorylation are severely impaired in these cells. Together, these findings underscore that intrinsic properties of the TCR-CD3 complex regulate selection at the DP checkpoint. More importantly, this analysis provides the first direct genetic evidence for a role of the CD3gamma-ITAM in TCR-driven thymocyte selection.

Variance analysis as a tool to predict the mechanism underlying synaptic plasticity.

BACKGROUND: The strength of synaptic transmission onto a neuron depends on the number of functional vesicle release sites (N), the probability of vesicle release (Pr), and the quantal size (Q). Statistical tools based on the quantal model of synaptic transmission can be used to acquire information on which of these parameters is the source of plasticity. However, quantal analysis depends on assumptions that may not be met at central synapses. NEW METHOD: We examined the merit of quantal analysis to extract the mechanisms underlying synaptic plasticity by applying binomial statistics on the variance in amplitude of postsynaptic currents evoked at Schaffer collateral-CA1 (Sc-CA1) synapses in mouse hippocampal slices. We extend this analysis by combining the conventional inverse square of the coefficient of variation (1/CV2) with the variance-to-mean ratio (VMR). RESULTS: This method can be used to assess the relative, but not absolute, contribution of N, Pr and Q to synaptic plasticity. The changes in 1/CV2 and VMR values correctly reflect experimental modifications of N, Pr and Q at Sc-CA1 synapses. COMPARISON WITH EXISTING METHODS: While the 1/CV2 depends on N and Pr, but is independent of Q, the VMR is dependent on Pr and Q, but not on N. Combining both allows for a rapid assessment of the mechanism underlying synaptic plasticity without the need for additional electrophysiological experiments. CONCLUSION: Combining the 1/CV2 with the VMR allows for a reliable prediction of the relative contribution of changes in N, Pr and Q to synaptic plasticity.

The Effects of Sindbis Viral Vectors on Neuronal Function.

Viral vectors are attractive tools to express genes in neurons. Transduction of neurons with a recombinant, replication-deficient Sindbis viral vector is a method of choice for studying the effects of short-term protein overexpression on neuronal function. However, to which extent Sindbis by itself may affect neurons is not fully understood. We assessed effects of neuronal transduction with a Sindbis viral vector on the transcriptome and proteome in organotypic hippocampal slice cultures, and analyzed the electrophysiological properties of individual CA1 neurons, at 24 h and 72 h after viral vector injection. Whereas Sindbis caused substantial gene expression alterations, changes at the protein level were less pronounced. Alterations in transcriptome and proteome were predominantly limited to proteins involved in mediating anti-viral innate immune responses. Sindbis transduction did not affect the intrinsic electrophysiological properties of individual neurons: the membrane potential and neuronal excitability were similar between transduced and non-transduced CA1 neurons up to 72 h after Sindbis injection. Synaptic currents also remained unchanged upon Sindbis transduction, unless slices were massively infected for 72 h. We conclude that Sindbis viral vectors at low transduction rates are suitable for studying short-term effects of a protein of interest on electrophysiological properties of neurons, but not for studies on the regulation of gene expression.