We describe an engineered family of highly antigenic molecules based on GFP-like fluorescent proteins. These molecules contain numerous copies of peptide epitopes and simultaneously bind IgG antibodies at each location. These “spaghetti monster” fluorescent proteins (smFPs) distribute well in neurons, notably into small dendrites, spines and axons. smFP immunolabeling localizes weakly expressed proteins not well resolved with traditional epitope tags. By varying epitope and scaffold, we generated a diverse family of mutually orthogonal antigens. In cultured neurons and mouse and fly brains, smFP probes allow robust, orthogonal multi-color visualization of proteins, cell populations and neuropil. smFP variants complement existing tracers, greatly increase the number of simultaneous imaging channels, and perform well in advanced preparations such as array tomography, super-resolution fluorescence imaging and electron microscopy. In living cells, the probes improve single-molecule image tracking and increase yield for RNA-Seq. These probes facilitate new experiments in connectomics, transcriptomics and protein localization.

SUMMARY Our understanding of mechanisms that regulate the differentiation of specific classes of synapses is limited. Here, we investigate the formation of synapses between hippocampal dentate gyrus (DG) neurons and their target CA3 neurons and find that DG neurons preferentially form synapses with CA3 rather than DG or CA1 neurons in culture, suggesting that specific interactions between DG and CA3 neurons drive synapse formation. Cadherin-9 is expressed selectively in DG and CA3 neurons, and downregulation of cadherin-9 in CA3 neurons leads to a selective decrease in the number and size of DG synapses onto CA3 neurons. In addition, loss of cadherin-9 from DG or CA3 neurons in vivo leads to striking defects in the formation and differentiation of the DG-CA3 mossy fiber synapse. These observations indicate that cadherin-9 bidirectionally regulates DG-CA3 synapse development and highlight the critical role of differentially expressed molecular cues in establishing specific connections in the mammalian brain.

The use of Sep-Pak C18 reverse-phase cartridges to adsorb gangliosides from aqueous solutions was studied. When upper phases formed from chloroform-methanol tissue extracts or aqueous salt solutions containing gangliosides are rapidly passed through the cartridges, the lipids are retained and the non-lipid components can be washed through. Gangliosides and other retained lipids can subsequently be eluted with methanol or chloroform-methanol

The function of the brain depends on highly specific patterns of connections between populations of neurons. The establishment of these connections requires the targeting of axons and dendrites to defined zones or laminae, the recognition of individual target cells, the formation of synapses on particular regions of the dendritic tree, and the differentiation of pre- and postsynaptic specializations. Recent studies provide compelling evidence that transmembrane adhesion proteins of the immunoglobulin, cadherin, and leucine-rich repeat protein families, as well as secreted proteins such as semaphorins and FGFs, regulate distinct aspects of neuronal connectivity. These observations suggest that the coordinated actions of a number of molecular signals contribute to the specification and differentiation of synaptic connections in the developing brain.

The UNC5Hs are axon guidance receptors that mediate netrin-1-dependent chemorepulsion, and dependence receptors that mediate netrin-1-independent apoptosis. Here, we report an interaction between UNC5H1 and NRAGE. Our experiments show that this interaction is responsible for apoptosis induced by UNC5H1, and this level of apoptosis is greater than the amount induced by either UNC5H2 or UNC5H3. We mapped the NRAGE binding domain of UNC5H1 to its ZU-5 domain and show that this region, in addition to an adjacent PEST sequence, is required for UNC5H1-mediated apoptosis. Chimeric UNC5H2 and UNC5H3 receptors, containing the NRAGE binding domain and PEST sequence of UNC5H1, bind NRAGE and cause increased levels of apoptosis. UNC5H1 expression does not induce apoptosis in differentiated PC12 cells, which down-regulate NRAGE, but induces apoptosis in native PC12 cells that endogenously express high levels of NRAGE and in differentiated PC12 cells when NRAGE is overexpressed. Together, these results demonstrate a mechanism for UNC5H1-mediated apoptosis that requires an interaction with the MAGE protein NRAGE.Apoptosis plays a critical role in determining the size and shape of the vertebrate nervous system (1). The execution phase of the apoptotic program in neurons is well characterized and, as with most cell types, depends on the activation of intracellular proteases, primarily caspases (2). In contrast, it is not well understood how cues from the environment regulate this process during development of the nervous system. UNC-5 was originally characterized in Caenorhabditis elegans as a gene required for axonal repulsion in netrin/UNC-6 responsive neurons (3, 4). The vertebrate members of this family (UNC5H1, 2, and 3) (5, 6), together with C. elegans UNC-5 (4) and Drosophila Unc5 (7), comprise a subgroup of the Ig superfamily of receptors. The UNC5s contain two Ig and two thrombospondin type-I repeats in the extracellular domain. In addition, their cytoplasmic domains contain regions of homology with other proteins: 1) a ZU-5 domain homologous to Zona Occludens-1, a protein implicated in tight-junction formation (8); and 2) a C-terminal death domain, a domain first identified as the pro-apoptotic region of tumor necrosis factor receptor-1 (9, 10). In a netrin-1 gradient, a complex of UNC5H1 and DCC mediates repulsion (11), although there is evidence suggesting that short range repulsion by netrin-1 may be mediated by UNC5 alone (3, 7). NRAGE (Dlxin-1, MAGE-D1) is a recently identified molecule belonging to the MAGE (melanoma antigen) protein family. There are currently over 25 MAGE proteins in humans, characterized by the presence of a MAGE homology domain. The expression of many MAGE proteins is restricted to cancer cells (12); however, recent studies have revealed a role for two MAGE proteins in the nervous system. One MAGE family member, necdin, is thought to maintain the differentiated state of post-mitotic neurons by preventing entry into the cell cycle (13,14). Another MAGE family member, NRAGE, is expressed in the...

Synaptic target specificity, whereby neurons make distinct types of synapses with different target cells, is critical for brain function, yet the mechanisms driving it are poorly understood. In this study, we demonstrate Kirrel3 regulates target-specific synapse formation at hippocampal mossy fiber (MF) synapses, which connect dentate granule (DG) neurons to both CA3 and GABAergic neurons. Here, we show Kirrel3 is required for formation of MF filopodia; the structures that give rise to DG-GABA synapses and that regulate feed-forward inhibition of CA3 neurons. Consequently, loss of Kirrel3 robustly increases CA3 neuron activity in developing mice. Alterations in the Kirrel3 gene are repeatedly associated with intellectual disabilities, but the role of Kirrel3 at synapses remained largely unknown. Our findings demonstrate that subtle synaptic changes during development impact circuit function and provide the first insight toward understanding the cellular basis of Kirrel3-dependent neurodevelopmental disorders.DOI: http://dx.doi.org/10.7554/eLife.09395.001

Netrin-1 is a bifunctional guidance cue that directs migrating neurons and axons based on specific receptors expressed on the cell surface. Attraction occurs through the receptor Deleted in Colorectal Cancer (DCC) and repulsion occurs through a receptor complex of DCC and UNC5H, the vertebrate homolog to Caenorhabditis elegans UNC-5, but how the specific surface expression of these receptors is achieved remains unknown. Here, we demonstrate that surface expression of UNC5H1 is regulated in neurons by protein interacting with C kinase-1 (PICK1) and protein kinase C (PKC), and show that one mechanism by which cells control their response to netrin-1 is by changing the surface availability of receptors. We identified PICK1 as a binding partner for UNC5H1 using the yeast two-hybrid system and found that the extreme three C-terminal amino acids of UNC5H1 interact with the PSD-95/Dlg/ZO-1 (PDZ) domain of PICK1. Coexpression of UNC5H1 and PICK1 in heterologous cells results in the recruitment of PICK1 to UNC5H1 clusters. Endogenous UNC5H1 and PICK1 coimmunoprecipitate from extracts of cultured hippocampal neurons and P4 cortices, and immunohistochemistry shows that UNC5H1, PICK1, and PKC are all present in growth cones. PKC activation induces the formation of UNC5H1/PICK1/PKC complexes and leads to the specific removal of UNC5H1, but not DCC, from the surface of neurons and growth cones via a PICK1/PKC-dependent mechanism. Lastly, we demonstrate that activating PKC, which decreases surface expression of UNC5H1, inhibits netrin-1-dependent collapse of hippocampal growth cones. Together, our results suggest that by regulating the surface expression of UNC5Hs, an axon can modulate its repellent response to netrin-1.

Summary Hippocampal CA3 neurons form synapses with CA1 neurons in two layers, stratum oriens (SO) and stratum radiatum (SR). Each layer develops unique synaptic properties but molecular mechanisms that mediate these differences are unknown. Here, we show SO synapses normally have significantly more mushroom spines and higher magnitude long-term potentiation (LTP) than SR synapses. Further, we discovered these differences require the Type II classic cadherins, cadherins-6, 9, and 10. Though cadherins typically function via trans-cellular homophilic interactions, our results suggest presynaptic cadherin-9 binds postsynaptic cadherins-6 and 10 to regulate mushroom spine density and high magnitude LTP in the SO layer. Loss of these cadherins has no effect on the lower magnitude LTP typically observed in the SR layer, demonstrating that cadherins-6, 9, and 10 are gatekeepers for high magnitude LTP. Thus, Type II cadherins may uniquely contribute to the specificity and strength of synaptic changes associated with learning and memory.