During development, the formation of mature neural circuits requires the selective elimination of inappropriate synaptic connections. Here we show that C1q, the initiating protein in the classical complement cascade, is expressed by postnatal neurons in response to immature astrocytes and is localized to synapses throughout the postnatal CNS and retina. Mice deficient in complement protein C1q or the downstream complement protein C3 exhibit large sustained defects in CNS synapse elimination, as shown by the failure of anatomical refinement of retinogeniculate connections and the retention of excess retinal innervation by lateral geniculate neurons. Neuronal C1q is normally downregulated in the adult CNS; however, in a mouse model of glaucoma, C1q becomes upregulated and synaptically relocalized in the adult retina early in the disease. These findings support a model in which unwanted synapses are tagged by complement for elimination and suggest that complement-mediated synapse elimination may become aberrantly reactivated in neurodegenerative disease.

Synapse loss correlates with a cognitive decline in Alzheimer's disease (AD), but whether this is caused by fibrillar deposits known as senile plaques or soluble oligomeric forms of amyloid ␤ (A␤) is controversial. By using array tomography, a technique that combines ultrathin sectioning of tissue with immunofluorescence, allowing precise quantification of small structures, such as synapses, we have tested the hypothesis that oligomeric A␤ surrounding plaques contributes to synapse loss in a mouse model of AD. We find that senile plaques are surrounded by a halo of oligomeric A␤. Analysis of >14,000 synapses (represented by PSD95-stained excitatory synapses) shows that there is a 60% loss of excitatory synapses in the halo of oligomeric A␤ surrounding plaques and that the density increases to reach almost control levels in volumes further than 50 m from a plaque in an approximately linear fashion (linear regression, r 2 ‫؍‬ 0.9; P < 0.0001). Further, in transgenic cortex, microdeposits of oligomeric A␤ associate with a subset of excitatory synapses, which are significantly smaller than those not in contact with oligomeric A␤. The proportion of excitatory synapses associated with A␤ correlates with decreasing density (correlation, ؊0.588; P < 0.0001). These data show that senile plaques are a potential reservoir of oligomeric A␤, which colocalizes with the postsynaptic density and is associated with spine collapse, reconciling the apparently competing schools of thought of ''plaque'' vs. ''oligomeric A␤'' as the synaptotoxic species in the brain of AD patients.Alzheimer ͉ array tomography ͉ neurodegeneration ͉ synaptotoxicity L oss of connectivity caused by neuronal death and synapse loss is thought to underlie cognitive decline in neurodegenerative conditions, such as Alzheimer's disease (AD). Synapse loss appears to be particularly important in the pathogenesis of AD. Indeed, it is known that synapses are lost during AD and that in AD tissue, synapse loss correlates strongly with cognitive decline (1-3). There is a growing consensus, based primarily on cell-based assays, that amyloid ␤ (A␤), the main component of senile plaques, is toxic to synapses (4-6). In both AD patients and animal models of the disease, synapse loss is greatest near senile plaques, indicating a link between amyloid pathology and synaptotoxicity in vivo. Work by several groups has shown a decrease in dendritic spine density and synaptophysin-positive synapses radiating out from the surface of plaques in mouse models of AD (7-10). Whether this is caused by fibrillar plaques or soluble oligomeric A␤ is controversial. We used multiphoton imaging of the living brain to show that this spine loss is caused by impaired spine stability over time near plaques and postulated that a plaque-related diffusible bioactive molecule was responsible (11). Here, we test the hypothesis that oligomeric A␤ is directly synaptotoxic.We hypothesize that soluble oligomeric A␤ associates with the postsynaptic density and causes the loss of synapses and spines observ...

Many biological functions depend critically upon fine details of tissue molecular architecture that have resisted exploration by existing imaging techniques. This is particularly true for nervous system tissues, where information processing function depends on intricate circuit and synaptic architectures. Here, we describe a new imaging method, called array tomography, which combines and extends superlative features of modern optical fluorescence and electron microscopy methods. Based on methods for constructing and repeatedly staining and imaging ordered arrays of ultrathin (50-200 nm), resin-embedded serial sections on glass microscope slides, array tomography allows for quantitative, high-resolution, large-field volumetric imaging of large numbers of antigens, fluorescent proteins, and ultrastructure in individual tissue specimens. Compared to confocal microscopy, array tomography offers the advantage of better spatial resolution, in particular along the z axis, as well as depth-independent immunofluorescent staining. The application of array tomography can reveal important but previously unseen features of brain molecular architecture.

Summary A lack of methods for measuring the protein compositions of individual synapses in situ has so far hindered the exploration and exploitation of synapse molecular diversity. Here we describe the use of array tomography, a new high-resolution proteomic imaging method, to determine the composition of glutamate and GABA synapses in somatosensory cortex of Line-H-YFP Thy-1 transgenic mice. We find that virtually all synapses are recognized by antibodies to the presynaptic phosphoprotein synapsin I, while antibodies to 16 other synaptic proteins discriminate amongst 4 subtypes of glutamatergic synapses and GABAergic synapses. Cell-specific YFP expression in the YFP-H mouse line allows synapses to be assigned to specific presynaptic and postsynaptic partners and reveals that a subpopulation of spines on layer 5 pyramidal cells receives both VGluT1-subtype glutamatergic and GABAergic synaptic inputs. These results establish a means for the high-throughput acquisition of proteomic data from individual cortical synapses in situ.

Myelin is best known for its role in increasing the conduction velocity and metabolic efficiency of long-range excitatory axons. Accordingly, the myelin observed in neocortical gray matter is thought to mostly ensheath excitatory axons connecting to subcortical regions and distant cortical areas. Using independent analyses of light and electron microscopy data from mouse neocortex, we show that a surprisingly large fraction of cortical myelin (half the myelin in layer 2/3 and a quarter in layer 4) ensheathes axons of inhibitory neurons, specifically of parvalbumin-positive basket cells. This myelin differs significantly from that of excitatory axons in distribution and protein composition. Myelin on inhibitory axons is unlikely to meaningfully hasten the arrival of spikes at their pre-synaptic terminals, due to the patchy distribution and short path-lengths observed. Our results thus highlight the need for exploring alternative roles for myelin in neocortical circuits.DOI: http://dx.doi.org/10.7554/eLife.15784.001

Glutamate is the predominant excitatory neurotransmitter in the mammalian brain. Once released, its rapid removal from the synaptic cleft is critical for preventing excitotoxicity and spillover to neighboring synapses. Despite consensus on the role of glutamate in normal and disease physiology, technical issues limit our understanding of its metabolism in intact cells. To monitor glutamate levels inside and at the surface of living cells, genetically encoded nanosensors were developed. The fluorescent indicator protein for glutamate (FLIPE) consists of the glutamate͞aspartate binding protein ybeJ from Escherichia coli fused to two variants of the green fluorescent protein. Three sensors with lower affinities for glutamate were created by mutation of residues peristeric to the ybeJ binding pocket. In the presence of ligands, FLIPEs show a concentration-dependent decrease in FRET efficiency. When expressed on the surface of rat hippocampal neurons or PC12 cells, the sensors respond to extracellular glutamate with a reversible concentration-dependent decrease in FRET efficiency. Depolarization of neurons leads to a reduction in FRET efficiency corresponding to 300 nM glutamate at the cell surface. No change in FRET was observed when cells expressing sensors in the cytosol were superfused with up to 20 mM glutamate, consistent with a minimal contribution of glutamate uptake to cytosolic glutamate levels. The results demonstrate that FLIPE sensors can be used for real-time monitoring of glutamate metabolism in living cells, in tissues, or in intact organisms, providing tools for studying metabolism or for drug discovery.aspartate ͉ hippocampal neuron ͉ neurotransmitter ͉ secretion ͉ transport I n addition to being an intermediate of primary metabolism in all biological cells, glutamate serves as the major excitatory amino acid neurotransmitter in the vertebrate central nervous system (1). As such, glutamate influences essentially all forms of behavior, including consciousness, sensory perception, motor control, and mood. Changes in the strength of connectivity at glutamatergic synapses in the form of long-term potentiation and long-term depression are considered to be the cellular mechanisms underlying learning and memory (2). In addition to its role in normal nervous system physiology, glutamate is also thought to be directly involved in neurologic damage occurring in stroke and neurodegenerative disorders, including AIDS-dementia complex, motor neuron disease, and Alzheimer's and Parkinson's diseases, through receptormediated toxicity (3).Despite its prominent role in normal and disease physiology, accurate and precise measurements of glutamate in living tissue are lacking. The concentration of glutamate in the cytoplasm and synaptic vesicles in neurons has been estimated by measurements of the amino acid in extracts (4). Extracellular glutamate concentrations have been measured by in vivo microdialysis techniques (5, 6). However, these techniques are limited in spatial and temporal resolution and are not suitable ...

Major histocompatibility complex Class I (MHCI) genes were discovered unexpectedly in healthy CNS neurons in a screen for genes regulated by neural activity. In mice lacking just 2 of the 50+ MHCI genes H2-Kb and H2-Db, ocular dominance (OD) plasticity is enhanced. Mice lacking PirB, an MHCI receptor, have a similar phenotype. H2-Kb and H2-Db are expressed not only in visual cortex, but also in lateral geniculate nucleus (LGN) where protein localization correlates strongly with synaptic markers and complement protein C1q. In KbDb-/- mice developmental refinement of retinogeniculate projections is impaired, similar to C1q-/- mice. These phenotypes in KbDb-/- mice are strikingly similar to those in β2m-/-TAP1-/- mice, which lack cell surface expression of all MHCIs, implying that H2-Kb and H2-Db can account for observed changes in synapse plasticity. H2-Kb and H2-Db ligands, signaling via neuronal MHCI receptors, may enable activity-dependent remodeling of brain circuits during developmental critical periods.

Amphiphysin is a nerve terminal-enriched protein thought to function in synaptic vesicle endocytosis, in part through Src homology 3 (SH3) domain-mediated interactions with dynamin and synaptojanin. Here, we report the characterization of a novel amphiphysin isoform (termed amphiphysin II) that was identified through a homology search of the data base of expressed sequence tags. Antibodies specific to amphiphysin II recognize a 90-kDa protein on Western blot that is brainspecific and highly enriched in nerve terminals. Like amphiphysin (now referred to as amphiphysin I), amphiphysin II binds to dynamin and synaptojanin through its SH3 domain. Further, both proteins bind directly to clathrin in an SH3 domain-independent manner. Taken together, these data suggest that amphiphysin II may participate with amphiphysin I in the regulation of synaptic vesicle endocytosis.