Fluorescence microscopy remains one of the single most widely applied experimental approaches in neuroscience and beyond and is continuously evolving to make it easier and more versatile. The success of the approach is based on synergistic developments in imaging technologies and fluorophore labeling strategies that have allowed it to greatly diversify and be used across preparations for addressing structure as well as function. Yet, while targeted labeling strategies are a key strength of fluorescence microscopy, they reciprocally impose general limitations on the possible types of experiments and analyses. One recent development that overcomes some of these limitations is fluorescence microscopy shadow imaging, where membrane-bound cellular structures remain unlabeled while the surrounding extracellular space is made to fluoresce to provide a negative contrast shadow image. When based on super-resolution STED microscopy, the technique in effect provides a positive image of the extracellular space geometry and entire neuropil in the field of view. Other noteworthy advantages include the near elimination of the adverse effects of photobleaching and toxicity in live imaging, exhaustive and homogeneous labeling across the preparation, and the ability to apply and adjust the label intensity on the fly. Shadow imaging is gaining popularity and has been applied on its own or combined with conventional positive labeling to visualize cells and synaptic proteins in their parenchymal context. Here, we highlight the inherent limitations of fluorescence microscopy and conventional labeling and contrast these against the pros and cons of recent shadow imaging approaches. Our aim is to describe the brief history and current trajectory of the shadow imaging technique in the neuroscience field, and to draw attention to its ease of application and versatility.

Glycogen synthase kinase-3 (GSK3) is an important signalling protein in the brain and modulates different forms of synaptic plasticity. Neuronal functions of GSK3 are typically attributed to one of its two isoforms, GSK3β, simply because of its prevalent expression in the brain. Consequently, the importance of isoform-specific functions of GSK3 in synaptic plasticity has not been fully explored. We now directly address this question for NMDA receptor-dependent long-term depression (LTD) in the hippocampus. Here, we specifically target the GSK3 isoforms with shRNA knock-down in mouse hippocampus and with novel isoform-selective drugs to dissect their roles in LTD. Using electrophysiological and live imaging approaches, we find that GSK3α, but not GSK3β, is required for LTD. The specific engagement of GSK3α occurs via its transient anchoring in dendritic spines during LTD induction. We find that the major GSK3 substrate, the microtubule-binding protein tau, is required for this spine anchoring of GSK3α and mediates GSK3α-induced LTD. These results link GSK3α and tau in a common mechanism for synaptic depression and rule out a major role for GSK3β in this process.

In humans, a considerable number of the autopsy samples of cognitively normal individuals aged between 57 and 102 years have revealed the presence of amyloid plaques, one of the typical signs of AD, indicating that many of us use mechanisms that defend ourselves from the toxic consequences of Aß. The human APP NL/F (hAPP NL/F) knockin mouse appears as the ideal mouse model to identify these mechanisms, since they have high Aß42 levels at an early age and moderate signs of disease when old. Here we show that in these mice, the brain levels of the hemoprotein Neuroglobin (Ngb) increase with age, in parallel with the increase in Aß42. In vitro , in wild type neurons, exogenous Aß increases the expression of Ngb and Ngb over-expression prevents Aß toxicity. In vivo , in old hAPP NL/F mice, Ngb knockdown leads to dendritic tree simplification, an early sign of Alzheimer’s disease. These results could indicate that Alzheimer’s symptoms may start developing at the time when defense mechanisms start wearing out. In agreement, analysis of plasma Ngb levels in aged individuals revealed decreased levels in those whose cognitive abilities worsened during a 5-year longitudinal follow-up period.

Tau is a microtubule-associated neuronal protein found mainly in axons. However, increasing evidence indicates that it is also present in dendrites, where it serves as an essential mediator of synaptic NMDA (N-methyl-D-aspartate) receptor-dependent excitotoxicity. Of note, NMDA receptors can also be found outside synapses in the plasma membrane, and activation of extrasynaptic NMDA receptors has been shown to be more linked to excitotoxicity than the activation of synaptic ones. Little is known about the role of Tau in the activity of extrasynaptic NMDA receptors. Thus, we have used a Tau knockout mouse model (Tau −/− mice) to analyze the consequences of Tau absence in extrasynaptic NMDA receptor activity. We demonstrate that absence of Tau leads to a decrease in functional extrasynaptic NMDA receptors in the hippocampus. We propose that this impairment in extrasynaptic NMDA receptor activity may contribute to the well-known neuroprotective effect associated with Tau deficiency under pathological conditions.

NMDA receptor-dependent long-term depression (LTD) in the hippocampus is a well-known form of synaptic plasticity that has been linked to different cognitive functions. The core mechanism for this form of plasticity is thought to be entirely neuronal. However, we now demonstrate that astrocytic activity drives LTD at CA3-CA1 synapses. We have found that LTD induction enhances astrocyte-to-neuron communication mediated by glutamate, and that Ca2+ signaling and SNARE-dependent vesicular release from the astrocyte are required for LTD expression. In addition, using optogenetic techniques, we show that low-frequency astrocytic activation, in the absence of presynaptic activity, is sufficient to induce postsynaptic AMPA receptor removal and LTD expression. Using cell-type-specific gene deletion, we show that astrocytic p38α MAPK is required for the increased astrocytic glutamate release and astrocyte-to-neuron communication during low-frequency stimulation. Accordingly, removal of astrocytic (but not neuronal) p38α abolishes LTD expression. Finally, this mechanism modulates long-term memory in vivo.

The deposition of aggregated amyloid-β peptides derived from the pro-amyloidogenic processing of the amyloid precurson protein (APP) into characteristic amyloid plaques (APs) is distinctive to Alzheimer's disease (AD). Alternative APP processing via the metalloprotease ADAM10 prevents amyloid-β formation. We tested whether downregulation of ADAM10 activity by its secreted endogenous inhibitor secreted-frizzled-related protein 1 (SFRP1) is a common trait of sporadic AD. We demonstrate that SFRP1 is significantly increased in the brain and cerebrospinal fluid of patients with AD, accumulates in APs and binds to amyloid-β, hindering amyloid-β protofibril formation. Sfrp1 overexpression in an AD-like mouse model anticipates the appearance of APs and dystrophic neurites, whereas its genetic inactivation or the infusion of α-SFRP1-neutralizing antibodies favors non-amyloidogenic APP processing. Decreased Sfrp1 function lowers AP accumulation, improves AD-related histopathological traits and prevents long-term potentiation loss and cognitive deficits. Our study unveils SFRP1 as a crucial player in AD pathogenesis and a promising AD therapeutic target.

The regulated transport of AMPA-type glutamate receptors (AMPARs) to the synaptic membrane is a key mechanism to determine the strength of excitatory synaptic transmission in the brain. In this work, we uncovered a new role for the microtubule-associated protein MAP1B in modulating access of AMPARs to the postsynaptic membrane. Using mice and rats of either sex, we show that MAP1B light chain (LC) accumulates in the somatodendritic compartment of hippocampal neurons, where it forms immobile complexes on microtubules that limit vesicular transport. These complexes restrict AMPAR dendritic mobility, leading to the intracellular trapping of receptors and impairing their access to the dendritic surface and spines. Accordingly, increasing MAP1B-LC expression depresses AMPAR-mediated synaptic transmission. This effect is specific for the GluA2 subunit of the AMPAR and requires glutamate receptor interacting protein 1 (GRIP1) interaction with MAP1B-LC. Therefore, MAP1B-LC represents an alternative link between GRIP1-AMPARs and microtubules that does not result in productive transport, but rather limits AMPAR availability for synaptic insertion, with a direct impact on synaptic transmission. SIGNIFICANCE STATEMENT The ability of neurons to modify their synaptic connections, known as synaptic plasticity, is accepted as the cellular basis for learning and memory. One mechanism for synaptic plasticity is the regulated addition and removal of AMPA-type glutamate receptors (AMPARs) at excitatory synapses. In this study, we found that a microtubule-associated protein, MAP1B light chain (MAP1B-LC), participates in this process. MAP1B-LC forms immobile complexes along dendrites. These complexes limit intracellular vesicular trafficking and trap AMPARs inside the dendritic shaft. In this manner, MAP1B restricts the access of AMPARs to dendritic spines and the postsynaptic membrane, contributing to downregulating synaptic transmission.

In this study, the authors show that PTEN alters synaptic function after PDZ-dependent recruitment into spines induced by amyloid-β. This mechanism is crucial for pathogenesis, as preventing PTEN-PDZ interactions renders neurons resistant to amyloid-β and rescues cognitive function in Alzheimer's disease models. This suggests that PTEN is a critical effector of the synaptic pathology associated with Alzheimer's disease.

Alzheimer's disease (AD) is the leading neurodegenerative disease worldwide and the expected rise in patient numbers is a critical threat to our health care systems. Current treatment strategies are of limited efficacy and only transiently active. Thus, there is an urgent need for a better understanding of the pathomechanisms involved, the precise sequence of events, and potential drug targets. Current research strategies are based on studies on animal models of the disease and mostly focus on interference with the key pathological hallmarks, beta-amyloid peptide accumulation and deposition and the neurofibrillary tangle formation. Further efforts need to be invested to complement our knowledge about AD and to identify new times and sites of therapeutic intervention.

The glutamate transporters GLAST and GLT-1 are mainly expressed in glial cells and regulate glutamate levels in the synapses. GLAST and GLT-1 are the targets of several signaling pathways. In this study we explore the possible functional interaction between these transporters and GSK3 beta. This kinase is involved in multiple cellular processes including neuronal development and synaptic plasticity. To evaluate whether GLT-1 and GLAST were regulated by GSK3 beta, we coexpresed these proteins in heterologous expression systems. In both COS-7 cells and Xenopus laevis oocytes, GSK3 beta stimulated the activity of GLT-1 and reduced that of GLAST. These effects were associated with corresponding changes in the amounts of GLT-1 or GLAST in the plasma membrane. These effects were suppressed by inhibitors of GSK3 beta or a catalytically inactive form of the kinase. GSK3 beta also decreases the incorporation of (32)Pi into GLT-1 and increases GLAST phosphorylation. Pharmacological inhibition of endogenous GSK3 beta in primary cultures of rat brain cortex also leads to a differential modulation of GLT-1 and GLAST. Our results suggest that constitutively active GSK3 beta is important in controlling the expression of functional glutamate transporters on the plasma membrane. This regulation might be relevant in physiological and pathological conditions in which glutamate transporters and GSK3 beta signaling are involved. (C) 2014 Elsevier Ltd. All rights reserved.

Phosphoinositide 3-kinase (PI3K) and PTEN have been shown to participate in synaptic plasticity during long-term potentiation (LTP) and long-term depression (LTD), respectively. Nevertheless, the dynamics of phosphatidylinositol-(3,4,5)-trisphosphate (PIP3) and the regulation of its synthesis and degradation at synaptic compartments is far from clear. Here, we have used fluorescence resonance energy transfer (FRET) imaging to monitor changes in PIP3 levels in dendritic spines from CA1 hippocampal neurons under basal conditions and upon induction of NMDA receptor (NMDAR)-dependent LTD and LTP. We found that PIP3 undergoes constant turnover in dendritic spines. Contrary to expectations, both LTD and LTP induction trigger an increase in PIP3 synthesis, which requires NMDARs and PI3K activity. Using biochemical methods, the upregulation of PIP3 levels during LTP was estimated to be twofold. However, in the case of LTD, PTEN activity counteracts the increase in PIP3 synthesis, resulting in no net change in PIP3 levels. Therefore, both LTP and LTD signaling converge towards PIP3 upregulation, but PTEN acts as an LTD-selective switch that determines the outcome of PIP3 accumulation.