agc004  (Alomone Labs)


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    Structured Review

    Alomone Labs agc004
    Cholesterol depletion reduces synaptic localization of NMDARs. ( A , C ) Colocalization of surface GluN2A (A, green) or GluN2B (C, green) and the postsynaptic marker Shank (red) in control and cholesterol-depleted neurons (10 mM MβCD pretreatment, 5 min). Scale bar 2 µm. ( B , D ) Bar graphs showing Pearson's coefficient for the colocalization indicate the reduction of synaptic localization of GluN2A and GluN2B after cholesterol depletion. ( E ) Colocalization of surface <t>GluA1</t> (green) and the postsynaptic marker Shank (red) in control and cholesterol-depleted neurons (MβCD). Scale bar 2 µm. ( F ) Bar graph showing Pearson's coefficient for the colocalization. ( G ) Examples of typical dual AMPAR-NMDAR mEPSCs in control autaptic neurons having various AMPAR to NMDAR ratio. ( H ) Examples of typical dual AMPAR-NMDAR mEPSCs in 10 mM MβCD-pretreated autaptic neurons. ( I ) Examples of NMDAR mEPSCs obtained from average dual mEPSCs after AMPAR mEPSC subtraction. A control neuron (top trace) and a cholesterol-depleted neuron (bottom trace). The arrows indicate mEPSC onsets. ( J ) The comparison of average amplitude of NMDAR mEPSCs in control neurons and in cholesterol-depleted neurons. (* p
    Agc004, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Cholesterol modulates presynaptic and postsynaptic properties of excitatory synaptic transmission"

    Article Title: Cholesterol modulates presynaptic and postsynaptic properties of excitatory synaptic transmission

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-69454-5

    Cholesterol depletion reduces synaptic localization of NMDARs. ( A , C ) Colocalization of surface GluN2A (A, green) or GluN2B (C, green) and the postsynaptic marker Shank (red) in control and cholesterol-depleted neurons (10 mM MβCD pretreatment, 5 min). Scale bar 2 µm. ( B , D ) Bar graphs showing Pearson's coefficient for the colocalization indicate the reduction of synaptic localization of GluN2A and GluN2B after cholesterol depletion. ( E ) Colocalization of surface GluA1 (green) and the postsynaptic marker Shank (red) in control and cholesterol-depleted neurons (MβCD). Scale bar 2 µm. ( F ) Bar graph showing Pearson's coefficient for the colocalization. ( G ) Examples of typical dual AMPAR-NMDAR mEPSCs in control autaptic neurons having various AMPAR to NMDAR ratio. ( H ) Examples of typical dual AMPAR-NMDAR mEPSCs in 10 mM MβCD-pretreated autaptic neurons. ( I ) Examples of NMDAR mEPSCs obtained from average dual mEPSCs after AMPAR mEPSC subtraction. A control neuron (top trace) and a cholesterol-depleted neuron (bottom trace). The arrows indicate mEPSC onsets. ( J ) The comparison of average amplitude of NMDAR mEPSCs in control neurons and in cholesterol-depleted neurons. (* p
    Figure Legend Snippet: Cholesterol depletion reduces synaptic localization of NMDARs. ( A , C ) Colocalization of surface GluN2A (A, green) or GluN2B (C, green) and the postsynaptic marker Shank (red) in control and cholesterol-depleted neurons (10 mM MβCD pretreatment, 5 min). Scale bar 2 µm. ( B , D ) Bar graphs showing Pearson's coefficient for the colocalization indicate the reduction of synaptic localization of GluN2A and GluN2B after cholesterol depletion. ( E ) Colocalization of surface GluA1 (green) and the postsynaptic marker Shank (red) in control and cholesterol-depleted neurons (MβCD). Scale bar 2 µm. ( F ) Bar graph showing Pearson's coefficient for the colocalization. ( G ) Examples of typical dual AMPAR-NMDAR mEPSCs in control autaptic neurons having various AMPAR to NMDAR ratio. ( H ) Examples of typical dual AMPAR-NMDAR mEPSCs in 10 mM MβCD-pretreated autaptic neurons. ( I ) Examples of NMDAR mEPSCs obtained from average dual mEPSCs after AMPAR mEPSC subtraction. A control neuron (top trace) and a cholesterol-depleted neuron (bottom trace). The arrows indicate mEPSC onsets. ( J ) The comparison of average amplitude of NMDAR mEPSCs in control neurons and in cholesterol-depleted neurons. (* p

    Techniques Used: Marker

    2) Product Images from "SAP97 Binding Partner CRIPT Promotes Dendrite Growth In Vitro and In Vivo"

    Article Title: SAP97 Binding Partner CRIPT Promotes Dendrite Growth In Vitro and In Vivo

    Journal: eNeuro

    doi: 10.1523/ENEURO.0175-17.2017

    CRIPT partially colocalizes GluA1-positive and SAP97-positive puncta on dendrites. Mixed spinal cord cultures at DIV21 were immunocytochemically analyzed. The upper set of panels display images of HA-CRIPT and EGFP-SAP97, and the lower set of panels display images of HA-CRIPT and GluA1. For the HA-CRIPT/EGFP-SAP97 panels, the first row of panels shows immunocytochemical staining for HA-CRIPT (red), EGFP-SAP97 (green), and merge. As in Figure 3 , HA-CRIPT immunoreactivity is seen within the soma and throughout the dendritic tree. EGFP-SAP97 is similarly distributed but appears more punctate. In the merge image, extensive colocalization is seen (yellow) both in the soma and the dendritic tree. Scale bar: 30 μm. Beneath each of the lower power images are higher magnification images of the area outlined in a white box. In the high-power HA-CRIPT panel, immunocytochemically positive material is again seen in various morphologies within the dendritic tree, including small or large round puncta and elongated dendritic shaft entities. HA-CRIPT appears inhomogenously within dendritic outgrowths that may represent spines or filopodia. EGFP-SAP97 is clearly more punctate than HA-CRIPT and in the merge image areas of colocalization are seen (yellow, denoted with > ). EGFP-SAP97 appears enriched at along the edges of HA-CRIPT immunoreactivity. Scale bar: 10 μm. For the HA-CRIPT/GluA1 panels, the first row of panels shows immunocytochemical staining for HA-CRIPT (red), GluA1 (green), and merge. Again, HA-CRIPT immunoreactivity is seen within the soma and throughout the dendritic tree. GluA1 is seen exclusively as puncta. In the merge image, colocalization is seen (yellow) in the dendritic tree. Scale bar: 20 μm. Beneath each of the lower power images are higher magnification images of the area outlined in a white box. In the high-power HA-CRIPT panel, immunocytochemically positive material is again seen in various morphologies within the dendritic tree including small or large round puncta and elongated dendritic shaft entities. HA-CRIPT appears inhomogenously within dendritic outgrowths that may represent spines or filopodia. GluA1 is exclusively punctate and in the merge image areas of colocalization are seen (yellow, denoted with > ). GluA1, like EGFP-SAP97, appears enriched at along the edges of HA-CRIPT immunoreactivity. Scale bar: 4.0 μm.
    Figure Legend Snippet: CRIPT partially colocalizes GluA1-positive and SAP97-positive puncta on dendrites. Mixed spinal cord cultures at DIV21 were immunocytochemically analyzed. The upper set of panels display images of HA-CRIPT and EGFP-SAP97, and the lower set of panels display images of HA-CRIPT and GluA1. For the HA-CRIPT/EGFP-SAP97 panels, the first row of panels shows immunocytochemical staining for HA-CRIPT (red), EGFP-SAP97 (green), and merge. As in Figure 3 , HA-CRIPT immunoreactivity is seen within the soma and throughout the dendritic tree. EGFP-SAP97 is similarly distributed but appears more punctate. In the merge image, extensive colocalization is seen (yellow) both in the soma and the dendritic tree. Scale bar: 30 μm. Beneath each of the lower power images are higher magnification images of the area outlined in a white box. In the high-power HA-CRIPT panel, immunocytochemically positive material is again seen in various morphologies within the dendritic tree, including small or large round puncta and elongated dendritic shaft entities. HA-CRIPT appears inhomogenously within dendritic outgrowths that may represent spines or filopodia. EGFP-SAP97 is clearly more punctate than HA-CRIPT and in the merge image areas of colocalization are seen (yellow, denoted with > ). EGFP-SAP97 appears enriched at along the edges of HA-CRIPT immunoreactivity. Scale bar: 10 μm. For the HA-CRIPT/GluA1 panels, the first row of panels shows immunocytochemical staining for HA-CRIPT (red), GluA1 (green), and merge. Again, HA-CRIPT immunoreactivity is seen within the soma and throughout the dendritic tree. GluA1 is seen exclusively as puncta. In the merge image, colocalization is seen (yellow) in the dendritic tree. Scale bar: 20 μm. Beneath each of the lower power images are higher magnification images of the area outlined in a white box. In the high-power HA-CRIPT panel, immunocytochemically positive material is again seen in various morphologies within the dendritic tree including small or large round puncta and elongated dendritic shaft entities. HA-CRIPT appears inhomogenously within dendritic outgrowths that may represent spines or filopodia. GluA1 is exclusively punctate and in the merge image areas of colocalization are seen (yellow, denoted with > ). GluA1, like EGFP-SAP97, appears enriched at along the edges of HA-CRIPT immunoreactivity. Scale bar: 4.0 μm.

    Techniques Used: Staining

    CRIPT knockdown leads to a selective reduction in the abundance of GluA1 and SAP97. Mixed spinal cord cultures were infected with HSV engineered to express a miRNA targeting CRIPT or a scrambled control. Two days later, lysates were prepared and subjected to Western blottings. No more than six independent experiments were performed for the quantitative image analysis. CRIPT knockdown leads to a reduction in GluA1 and SAP97 abundance and no effect on the abundance of GluA2, GluA4, NR1, NR2A, NR2B, or PSD95. Representative images of Western blottings with actin loading controls are shown and quantification of band intensity in the bar graphs below; *significant difference between groups, p
    Figure Legend Snippet: CRIPT knockdown leads to a selective reduction in the abundance of GluA1 and SAP97. Mixed spinal cord cultures were infected with HSV engineered to express a miRNA targeting CRIPT or a scrambled control. Two days later, lysates were prepared and subjected to Western blottings. No more than six independent experiments were performed for the quantitative image analysis. CRIPT knockdown leads to a reduction in GluA1 and SAP97 abundance and no effect on the abundance of GluA2, GluA4, NR1, NR2A, NR2B, or PSD95. Representative images of Western blottings with actin loading controls are shown and quantification of band intensity in the bar graphs below; *significant difference between groups, p

    Techniques Used: Infection, Western Blot

    3) Product Images from "A CDC42EP4/septin-based perisynaptic glial scaffold facilitates glutamate clearance"

    Article Title: A CDC42EP4/septin-based perisynaptic glial scaffold facilitates glutamate clearance

    Journal: Nature Communications

    doi: 10.1038/ncomms10090

    Morphological analysis of the neuronal and glial components in Cdc42ep4 fl/fl and Cdc42ep4 −/− cerebellar cortices. ( a ) Double-label IF of WT and KO cerebellar cortices for a Purkinje cell marker Car8 (red) and a parallel fibre (that is, granule cell) marker VGluT1 (top, green) or a climbing fibre marker VGluT2 (bottom, green). No obvious morphological anomaly, including aberrant CF–PC innervation, was found in the major neuronal components of KO-derived samples. Scale bar, 20 μm. ( b ) Transmission electron microscopy images of WT and KO molecular layers. No obvious ultrastructural difference was found between the genotypes. PF, parallel fibre terminal or bouton. PC, dendritic spine of Purkinje cell. Bergmann glial processes are tinted. Scale bar, 200 nm. ( c ) Cumulative histogram of PSD length of the PF–PC synapses, showing no significant difference between the genotypes ( n =92 synapses from two littermates for each genotype, NS, P > 0.05 by Kolmogorov–Smirnov test). ( d ) Quantitative immunoblot of WT and KO cerebellar PSD fractions for GluA1, GluA2 and GluA4 (the major subunits of the AMPARs), each normalized with PSD-95. There was no significant quantitative difference by genotype ( n =3, NS, P > 0.05 by t -test).
    Figure Legend Snippet: Morphological analysis of the neuronal and glial components in Cdc42ep4 fl/fl and Cdc42ep4 −/− cerebellar cortices. ( a ) Double-label IF of WT and KO cerebellar cortices for a Purkinje cell marker Car8 (red) and a parallel fibre (that is, granule cell) marker VGluT1 (top, green) or a climbing fibre marker VGluT2 (bottom, green). No obvious morphological anomaly, including aberrant CF–PC innervation, was found in the major neuronal components of KO-derived samples. Scale bar, 20 μm. ( b ) Transmission electron microscopy images of WT and KO molecular layers. No obvious ultrastructural difference was found between the genotypes. PF, parallel fibre terminal or bouton. PC, dendritic spine of Purkinje cell. Bergmann glial processes are tinted. Scale bar, 200 nm. ( c ) Cumulative histogram of PSD length of the PF–PC synapses, showing no significant difference between the genotypes ( n =92 synapses from two littermates for each genotype, NS, P > 0.05 by Kolmogorov–Smirnov test). ( d ) Quantitative immunoblot of WT and KO cerebellar PSD fractions for GluA1, GluA2 and GluA4 (the major subunits of the AMPARs), each normalized with PSD-95. There was no significant quantitative difference by genotype ( n =3, NS, P > 0.05 by t -test).

    Techniques Used: Marker, Derivative Assay, Transmission Assay, Electron Microscopy

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    Alomone Labs guinea pig anti glur1 glua1 extracellular antibody
    Working model of increased surface AMPAR expression in D620N heterozygous mutants. In wild-type cells, postsynaptic VPS35 traffics <t>GluA1-containing</t> AMPARs. At the drosophila neuromuscular junction, VPS35 localizes to large endocytic structures resembling bulk-endocytosed membrane, raising the possibility that it participates in SV regeneration [ 25 ]; however, in mammalian neurons it localizes to only a subset of terminals [ 22 ] and retromer deficiency has no effect on SV endo- or exocytosis [ 22 ] or neurotransmitter release [ 16 , 21 ]. Thus, we propose that presynaptic VPS35 may participate in recycling or retrograde transport of presynaptic receptors, channels, and/or SV proteins. In heterozygous neurons, there is increased abundance of endosomal structures positive for VPS35 and FAM21, accumulation of Rab11 + ve recycling endosomes, and increased surface expression of GluA1, thus we propose that the D620N mutation causes increased surface recycling of GluA1. Given the proposed presynaptic functions of retromer, we hypothesize that the observed increase in the probability of glutamate release is the result of either increased SV regeneration by retromer, or complex alterations to the recycling and axonal trafficking of presynaptic proteins by retromer
    Guinea Pig Anti Glur1 Glua1 Extracellular Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Alomone Labs ampa receptor 1 glur1a
    Summary of results. (A) Cartoon depicting basal (no silencing) striatal projection neuron dendrite (SPN; green) with three postsynaptic spines and two filopodia, with presynaptic Synapsin-1 signal shown in red. There were no changes detected following 3 h TTX silencing or 3 h TTX + AP5+CNQX. Silencing with TTX for 24 h increased Synapsin-1 signal intensity specifically on dendritic spines (darker red), and began the process of spine elimination, without changes to filopodia density. The additional block of spontaneous glutamate receptor activation (AP5 + CNQX) prevented reductions in spine density but not increased Synapsin-1 signal intensity. Chronic silencing with 2-week exposure to TTX dramatically reduced spine density and decreased Synapsin-1 signal intensity; specifically, that associated with dendritic spines and dendrites. (B) Cartoon of control (no glycine) SPN with three spines and two filopodia with postsynaptic <t>GluA1</t> AMPA receptor signal shown in purple. A chemical LTP protocol (3 min glycine) increased spine density, GluA1 signal intensity, and the percentage of spines with clearly detected GluA1 clusters, without changing presynaptic cluster density.
    Ampa Receptor 1 Glur1a, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Working model of increased surface AMPAR expression in D620N heterozygous mutants. In wild-type cells, postsynaptic VPS35 traffics GluA1-containing AMPARs. At the drosophila neuromuscular junction, VPS35 localizes to large endocytic structures resembling bulk-endocytosed membrane, raising the possibility that it participates in SV regeneration [ 25 ]; however, in mammalian neurons it localizes to only a subset of terminals [ 22 ] and retromer deficiency has no effect on SV endo- or exocytosis [ 22 ] or neurotransmitter release [ 16 , 21 ]. Thus, we propose that presynaptic VPS35 may participate in recycling or retrograde transport of presynaptic receptors, channels, and/or SV proteins. In heterozygous neurons, there is increased abundance of endosomal structures positive for VPS35 and FAM21, accumulation of Rab11 + ve recycling endosomes, and increased surface expression of GluA1, thus we propose that the D620N mutation causes increased surface recycling of GluA1. Given the proposed presynaptic functions of retromer, we hypothesize that the observed increase in the probability of glutamate release is the result of either increased SV regeneration by retromer, or complex alterations to the recycling and axonal trafficking of presynaptic proteins by retromer

    Journal: Molecular Brain

    Article Title: Endosomal traffic and glutamate synapse activity are increased in VPS35 D620N mutant knock-in mouse neurons, and resistant to LRRK2 kinase inhibition

    doi: 10.1186/s13041-021-00848-w

    Figure Lengend Snippet: Working model of increased surface AMPAR expression in D620N heterozygous mutants. In wild-type cells, postsynaptic VPS35 traffics GluA1-containing AMPARs. At the drosophila neuromuscular junction, VPS35 localizes to large endocytic structures resembling bulk-endocytosed membrane, raising the possibility that it participates in SV regeneration [ 25 ]; however, in mammalian neurons it localizes to only a subset of terminals [ 22 ] and retromer deficiency has no effect on SV endo- or exocytosis [ 22 ] or neurotransmitter release [ 16 , 21 ]. Thus, we propose that presynaptic VPS35 may participate in recycling or retrograde transport of presynaptic receptors, channels, and/or SV proteins. In heterozygous neurons, there is increased abundance of endosomal structures positive for VPS35 and FAM21, accumulation of Rab11 + ve recycling endosomes, and increased surface expression of GluA1, thus we propose that the D620N mutation causes increased surface recycling of GluA1. Given the proposed presynaptic functions of retromer, we hypothesize that the observed increase in the probability of glutamate release is the result of either increased SV regeneration by retromer, or complex alterations to the recycling and axonal trafficking of presynaptic proteins by retromer

    Article Snippet: We used the following primary antibodies: GFP (Abcam ab1218); VPS35 (Abnova H00055737); VPS26 (a kind gift from J. Bonifacino, NICHD); FAM21C (Millipore ABT79); NEEP21/NSG1 (Genscript A01442); Rab11 (Abcam ab95375); MAP2 (Abcam ab5392); GluA1 (Millipore 05-855R); PSD95 (Thermo Scientific MA1-045); VGluT1 (Millipore AB5905); GluA1 extracellular (Millipore ABN241); Rab10 (Abcam 237703); and GluA1 (Alomone AGP-009).

    Techniques: Expressing, Mutagenesis

    Surface GluA1 is increased in VKI and altered by acute LRRK2 kinase inhibition. A GFP-filled (cyan) cultured cortical cells immunostained for MAP2 (blue; to ensure no permeabilization) and surface GluA1 (magenta) (i, left panel); in silico neurite outlines with only GluA1 staining displayed (i, right panel). There was a significant genotype effect on GluA1 cluster density, due to opposing effects on heterozygous and homozygous cells (ii, Kruskal–Wallis p

    Journal: Molecular Brain

    Article Title: Endosomal traffic and glutamate synapse activity are increased in VPS35 D620N mutant knock-in mouse neurons, and resistant to LRRK2 kinase inhibition

    doi: 10.1186/s13041-021-00848-w

    Figure Lengend Snippet: Surface GluA1 is increased in VKI and altered by acute LRRK2 kinase inhibition. A GFP-filled (cyan) cultured cortical cells immunostained for MAP2 (blue; to ensure no permeabilization) and surface GluA1 (magenta) (i, left panel); in silico neurite outlines with only GluA1 staining displayed (i, right panel). There was a significant genotype effect on GluA1 cluster density, due to opposing effects on heterozygous and homozygous cells (ii, Kruskal–Wallis p

    Article Snippet: We used the following primary antibodies: GFP (Abcam ab1218); VPS35 (Abnova H00055737); VPS26 (a kind gift from J. Bonifacino, NICHD); FAM21C (Millipore ABT79); NEEP21/NSG1 (Genscript A01442); Rab11 (Abcam ab95375); MAP2 (Abcam ab5392); GluA1 (Millipore 05-855R); PSD95 (Thermo Scientific MA1-045); VGluT1 (Millipore AB5905); GluA1 extracellular (Millipore ABN241); Rab10 (Abcam 237703); and GluA1 (Alomone AGP-009).

    Techniques: Inhibition, Cell Culture, In Silico, Staining

    Nogo-A regulates the synaptic insertion of calcium permeable-AMPARs. ( A , B ) Live-cell immunolabeling of surface AMPAR subunit (magenta) GluA1 ( A ) or GluA2 ( B ) followed by immunofluorescence for presynaptic marker synapsin (Syn1/2; green) and their merged images (bottom) in primary hippocampal neurons treated for 10 min either with the control (left) or the Nogo-A function-blocking (right) antibody. For illustration, all images underwent deconvolution and were equally increased in brightness and contrast by the same absolute values for visibility. Scale bar 2 μm. ( C , D ) Normalized data for density ( C ) and fluorescence intensity ( D ) of GluA1 immuno-positive puncta in hippocampal neurons treated with either control antibody (black, n = 40) or Nogo-A function-blocking antibody (red, n = 39) for 10 min. ( E ) Normalized values for the density of GluA1 clusters colocalized with Syn 1/2 immuno-positive puncta. ( F , G ) Normalized GluA2 cluster density ( F ) and fluorescence intensity ( G ) in hippocampal neurons upon 10 min application with control antibody (black, n = 36) or Nogo-A function-blocking antibody (red, n = 35). ( H ) Normalized density of GluA2 immuno-positive puncta colocalized with Syn 1/2. Data are presented as mean ± SEM. * p

    Journal: Cells

    Article Title: Nogo-A Modulates the Synaptic Excitation of Hippocampal Neurons in a Ca2+-Dependent Manner

    doi: 10.3390/cells10092299

    Figure Lengend Snippet: Nogo-A regulates the synaptic insertion of calcium permeable-AMPARs. ( A , B ) Live-cell immunolabeling of surface AMPAR subunit (magenta) GluA1 ( A ) or GluA2 ( B ) followed by immunofluorescence for presynaptic marker synapsin (Syn1/2; green) and their merged images (bottom) in primary hippocampal neurons treated for 10 min either with the control (left) or the Nogo-A function-blocking (right) antibody. For illustration, all images underwent deconvolution and were equally increased in brightness and contrast by the same absolute values for visibility. Scale bar 2 μm. ( C , D ) Normalized data for density ( C ) and fluorescence intensity ( D ) of GluA1 immuno-positive puncta in hippocampal neurons treated with either control antibody (black, n = 40) or Nogo-A function-blocking antibody (red, n = 39) for 10 min. ( E ) Normalized values for the density of GluA1 clusters colocalized with Syn 1/2 immuno-positive puncta. ( F , G ) Normalized GluA2 cluster density ( F ) and fluorescence intensity ( G ) in hippocampal neurons upon 10 min application with control antibody (black, n = 36) or Nogo-A function-blocking antibody (red, n = 35). ( H ) Normalized density of GluA2 immuno-positive puncta colocalized with Syn 1/2. Data are presented as mean ± SEM. * p

    Article Snippet: In the case of the AMPA receptors, the anti-AMPAR 1 GluA1 (Alomone Labs, Jerusalem, Israel, Cat# AGP-009, 1:50) and anti-AMPAR 2 GluA2 (Alomone Labs, Cat# AGC-005, 1:50) were co-applied with the Nogo-A or control antibodies for 10 min. After completion of the treatment, the coverslips were rinsed with pre-warmed NB- medium and fixed with 4% paraformaldehyde (PFA) in phosphate buffer (PB containing in mM 50 NaH2 PO4 *2H2 O, 85 Na2 HPO4 *2H2 O) for 10 min at room temperature (RT).

    Techniques: Immunolabeling, Immunofluorescence, Marker, Blocking Assay, Fluorescence

    Summary of results. (A) Cartoon depicting basal (no silencing) striatal projection neuron dendrite (SPN; green) with three postsynaptic spines and two filopodia, with presynaptic Synapsin-1 signal shown in red. There were no changes detected following 3 h TTX silencing or 3 h TTX + AP5+CNQX. Silencing with TTX for 24 h increased Synapsin-1 signal intensity specifically on dendritic spines (darker red), and began the process of spine elimination, without changes to filopodia density. The additional block of spontaneous glutamate receptor activation (AP5 + CNQX) prevented reductions in spine density but not increased Synapsin-1 signal intensity. Chronic silencing with 2-week exposure to TTX dramatically reduced spine density and decreased Synapsin-1 signal intensity; specifically, that associated with dendritic spines and dendrites. (B) Cartoon of control (no glycine) SPN with three spines and two filopodia with postsynaptic GluA1 AMPA receptor signal shown in purple. A chemical LTP protocol (3 min glycine) increased spine density, GluA1 signal intensity, and the percentage of spines with clearly detected GluA1 clusters, without changing presynaptic cluster density.

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Chronic and Acute Manipulation of Cortical Glutamate Transmission Induces Structural and Synaptic Changes in Co-cultured Striatal Neurons

    doi: 10.3389/fncel.2021.569031

    Figure Lengend Snippet: Summary of results. (A) Cartoon depicting basal (no silencing) striatal projection neuron dendrite (SPN; green) with three postsynaptic spines and two filopodia, with presynaptic Synapsin-1 signal shown in red. There were no changes detected following 3 h TTX silencing or 3 h TTX + AP5+CNQX. Silencing with TTX for 24 h increased Synapsin-1 signal intensity specifically on dendritic spines (darker red), and began the process of spine elimination, without changes to filopodia density. The additional block of spontaneous glutamate receptor activation (AP5 + CNQX) prevented reductions in spine density but not increased Synapsin-1 signal intensity. Chronic silencing with 2-week exposure to TTX dramatically reduced spine density and decreased Synapsin-1 signal intensity; specifically, that associated with dendritic spines and dendrites. (B) Cartoon of control (no glycine) SPN with three spines and two filopodia with postsynaptic GluA1 AMPA receptor signal shown in purple. A chemical LTP protocol (3 min glycine) increased spine density, GluA1 signal intensity, and the percentage of spines with clearly detected GluA1 clusters, without changing presynaptic cluster density.

    Article Snippet: The primary antibodies used were anti-GFP (Green Fluorescent Protein, mouse, Abcam Cat# ab1218 RRID: AB_298911 , 1:1,000), anti-synapsin1 (Synapsin-1, rabbit, Millipore Cat# AB1543P RRID: AB_90757 , 1:500), anti-GluA1 (AMPA Receptor, rabbit, Alomone Labs Cat# AGC-004 RRID: AB_2039878 , 1:500), anti-tRFP (tagRFP, rabbit, Axxora Cat# EVN-AB233, 1:500).

    Techniques: Blocking Assay, Activation Assay

    A 3-min glycine cLTP induction protocol induced spine density and GluA1 cluster increases within 30 min and altered the decay time of miniature events. (A) Experimental timeline. Following glycine or control treatment, cells were fixed and immunostained with anti-GFP (green) and the presynaptic terminal marker Synapsin-1 (not shown) or the postsynaptic AMPA receptor subunit GluA1 (red). Separate coverslips were used for whole-cell voltage-clamp recordings. (B) Top: representative images (Olympus FV-1000, 60×, 2× zoom) of control (cLTP ctrl) and +glycine (cLTP) cells. Middle and bottom: expanded images (digital zoom) depicting the dendritic segment outlined in the white rectangle above. Middle row images show GFP fill with visible spines and GluA1 clusters, and in the lower panel, the GFP fill is outlined to depict the masked area for puncta quantification. (C) Structural and synaptic marker changes following glycine treatment in Mg 2+ -free ECS. (i,ii) The analysis revealed a ~30% increase in spine density relative to control-treated SPNs. (i) Unpaired t -test, ** p = 0.006; whereas no change was observed in filopodia density. (ii) p = 0.2. (iii,iv) GluA1 cluster intensity was significantly increased in glycine-treated relative to control SPNs. (iii) Unpaired t -test, ** p = 0.009; as was the percentage of spines colocalized with GluA1 clusters in glycine treated SPNs. (iv) Mann–Whitney test, * p = 0.022. (v) No changes in Synapsin-1 cluster density were observed. (D) Whole-cell patch-clamp recordings from cLTP and control SPNs. (i) Representative traces showing miniature excitatory postsynaptic currents (mEPSCs) in the control (top) and glycine-treated (bottom) SPN. (ii,iii) Despite a trend, there was no significant difference in mEPSC frequency. (ii) p = 0.1 and no change in amplitudes. (iii) p = 0.5. (iv) The mEPSC decay time (tau) was significantly faster, following glycine treatment (Mann–Whitney test, * p = 0.015).

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Chronic and Acute Manipulation of Cortical Glutamate Transmission Induces Structural and Synaptic Changes in Co-cultured Striatal Neurons

    doi: 10.3389/fncel.2021.569031

    Figure Lengend Snippet: A 3-min glycine cLTP induction protocol induced spine density and GluA1 cluster increases within 30 min and altered the decay time of miniature events. (A) Experimental timeline. Following glycine or control treatment, cells were fixed and immunostained with anti-GFP (green) and the presynaptic terminal marker Synapsin-1 (not shown) or the postsynaptic AMPA receptor subunit GluA1 (red). Separate coverslips were used for whole-cell voltage-clamp recordings. (B) Top: representative images (Olympus FV-1000, 60×, 2× zoom) of control (cLTP ctrl) and +glycine (cLTP) cells. Middle and bottom: expanded images (digital zoom) depicting the dendritic segment outlined in the white rectangle above. Middle row images show GFP fill with visible spines and GluA1 clusters, and in the lower panel, the GFP fill is outlined to depict the masked area for puncta quantification. (C) Structural and synaptic marker changes following glycine treatment in Mg 2+ -free ECS. (i,ii) The analysis revealed a ~30% increase in spine density relative to control-treated SPNs. (i) Unpaired t -test, ** p = 0.006; whereas no change was observed in filopodia density. (ii) p = 0.2. (iii,iv) GluA1 cluster intensity was significantly increased in glycine-treated relative to control SPNs. (iii) Unpaired t -test, ** p = 0.009; as was the percentage of spines colocalized with GluA1 clusters in glycine treated SPNs. (iv) Mann–Whitney test, * p = 0.022. (v) No changes in Synapsin-1 cluster density were observed. (D) Whole-cell patch-clamp recordings from cLTP and control SPNs. (i) Representative traces showing miniature excitatory postsynaptic currents (mEPSCs) in the control (top) and glycine-treated (bottom) SPN. (ii,iii) Despite a trend, there was no significant difference in mEPSC frequency. (ii) p = 0.1 and no change in amplitudes. (iii) p = 0.5. (iv) The mEPSC decay time (tau) was significantly faster, following glycine treatment (Mann–Whitney test, * p = 0.015).

    Article Snippet: The primary antibodies used were anti-GFP (Green Fluorescent Protein, mouse, Abcam Cat# ab1218 RRID: AB_298911 , 1:1,000), anti-synapsin1 (Synapsin-1, rabbit, Millipore Cat# AB1543P RRID: AB_90757 , 1:500), anti-GluA1 (AMPA Receptor, rabbit, Alomone Labs Cat# AGC-004 RRID: AB_2039878 , 1:500), anti-tRFP (tagRFP, rabbit, Axxora Cat# EVN-AB233, 1:500).

    Techniques: Marker, MANN-WHITNEY, Patch Clamp