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    Additional name(s) for this target protein: Secreted frizzled-related protein 2
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    86
    Thermo Fisher r34676
    R34676, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 86 stars, based on 1 article reviews
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    86
    Thermo Fisher rhod dextran
    Rhod Dextran, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rhod dextran/product/Thermo Fisher
    Average 86 stars, based on 1 article reviews
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    rhod dextran - by Bioz Stars, 2024-02
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    86
    Thermo Fisher ca 2 indicator rhod dextran
    Endogenous tagging of the Ca V 2 Ca 2+ channel Cacophony. A , Map of the cacophony locus indicating the genomic region targeted to incorporate sfGFP in all predicted isoforms. sfGFP is inserted between amino acids 107 and 108 of isoform cac-RN and immediately following the start codon in all other annotated isoforms. B , Schematic of the endogenously tagged Cac protein indicating the site of tag incorporation in the encoded channel (green dot). The longer N-terminus of the isoform encoded by cac-RN is represented by a dotted line. EC, Extracellular; IC, intracellular. C – E , Confocal Z -projections of a cac sfGFP-N larval NMJ ( C ), larval ventral ganglion ( D ), and adult brain ( E ) colabeled with antibodies against GFP and the active zone marker Brp.
    Ca 2 Indicator Rhod Dextran, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ca 2 indicator rhod dextran/product/Thermo Fisher
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ca 2 indicator rhod dextran - by Bioz Stars, 2024-02
    86/100 stars
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    86
    Thermo Fisher ca2 indicator 159 rhod dextran
    Endogenous tagging of the Ca V 2 Ca 2+ channel Cacophony. A , Map of the cacophony locus indicating the genomic region targeted to incorporate sfGFP in all predicted isoforms. sfGFP is inserted between amino acids 107 and 108 of isoform cac-RN and immediately following the start codon in all other annotated isoforms. B , Schematic of the endogenously tagged Cac protein indicating the site of tag incorporation in the encoded channel (green dot). The longer N-terminus of the isoform encoded by cac-RN is represented by a dotted line. EC, Extracellular; IC, intracellular. C – E , Confocal Z -projections of a cac sfGFP-N larval NMJ ( C ), larval ventral ganglion ( D ), and adult brain ( E ) colabeled with antibodies against GFP and the active zone marker Brp.
    Ca2 Indicator 159 Rhod Dextran, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ca2 indicator 159 rhod dextran/product/Thermo Fisher
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ca2 indicator 159 rhod dextran - by Bioz Stars, 2024-02
    86/100 stars
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    Image Search Results


    Endogenous tagging of the Ca V 2 Ca 2+ channel Cacophony. A , Map of the cacophony locus indicating the genomic region targeted to incorporate sfGFP in all predicted isoforms. sfGFP is inserted between amino acids 107 and 108 of isoform cac-RN and immediately following the start codon in all other annotated isoforms. B , Schematic of the endogenously tagged Cac protein indicating the site of tag incorporation in the encoded channel (green dot). The longer N-terminus of the isoform encoded by cac-RN is represented by a dotted line. EC, Extracellular; IC, intracellular. C – E , Confocal Z -projections of a cac sfGFP-N larval NMJ ( C ), larval ventral ganglion ( D ), and adult brain ( E ) colabeled with antibodies against GFP and the active zone marker Brp.

    Journal: The Journal of Neuroscience

    Article Title: Endogenous Tagging Reveals Differential Regulation of Ca 2+ Channels at Single Active Zones during Presynaptic Homeostatic Potentiation and Depression

    doi: 10.1523/JNEUROSCI.3068-18.2019

    Figure Lengend Snippet: Endogenous tagging of the Ca V 2 Ca 2+ channel Cacophony. A , Map of the cacophony locus indicating the genomic region targeted to incorporate sfGFP in all predicted isoforms. sfGFP is inserted between amino acids 107 and 108 of isoform cac-RN and immediately following the start codon in all other annotated isoforms. B , Schematic of the endogenously tagged Cac protein indicating the site of tag incorporation in the encoded channel (green dot). The longer N-terminus of the isoform encoded by cac-RN is represented by a dotted line. EC, Extracellular; IC, intracellular. C – E , Confocal Z -projections of a cac sfGFP-N larval NMJ ( C ), larval ventral ganglion ( D ), and adult brain ( E ) colabeled with antibodies against GFP and the active zone marker Brp.

    Article Snippet: Severed segmental nerves were drawn into a filling pipette, and a 16:1 mixture of the Ca 2+ indicator rhod dextran (R34676; ThermoFisher Scientific) and a Ca 2+ -insensitive AlexaFluor 647 dextran (AF647 dextran; D22914; ThermoFisher Scientific) was applied to the cut nerve end for 15–45 min. Preparations were incubated in the dark for at least 3 h and rinsed every 30 min with fresh Schneider's insect medium.

    Techniques: Marker

    Endogenous tagging of Cacophony does not perturb presynaptic Ca 2+ influx or synaptic function. A , Live fluorescence images of type-Ib boutons from a cac sfGFP-N motor terminal showing Cac sfGFP-N (top) and AF647-dextran (center), which was co-loaded with rhod-dextran (image not shown). B , Ratiometric fluorescence changes (rhod-dextran relative to AF647-dextran) in presynaptic motorneuron terminals during stimulation in wild type and cac sfGFP-N . An action potential was initiated every second, for a period of 10 s, followed by a 1 s, 20 Hz train of action potentials. C , Plot of average Ca 2+ levels ( R ) in terminals before nerve stimulation for type-Ib and -Is terminals in wild-type and cac sfGFP-N boutons [wild-type Ib, 2.74 ± 0.44, one NMJ from each of 5 animals ( N = 5); cac sfGFP-N Ib, 2.47 ± 0.49, N = 8, p = 0.69; wild-type Is, 2.16 ± 0.47, N = 5; cac sfGFP-N Is, 2.94 ± 0.32, N = 6, p = 0.21; Student's t test). D , Plot of the average amplitude of single action potential-mediated Ca 2+ transients [Δ R ; wild-type Ib, 4.50 ± 0.81, one NMJ from each of 5 animals ( N = 5); cac sfGFP-N Ib, 3.90 ± 1.07, N = 8, p = 0.67; wild-type Is, 6.28 ± 1.79, N = 5; cac sfGFP-N Is, 7.33 ± 2.20, N = 6, p = 0.72; Student's t test]. E , Plot of the average amplitude of 1 s, 20 Hz action potential train-mediated Ca 2+ transients [wild-type Ib, 9.71 ± 1.53, one NMJ from each of 5 animals ( N = 5); cac sfGFP-N Ib, 8.13 ± 2.21, N = 8, p = 0.57; wild-type Is, 12.26 ± 3.36, N = 5; cac sfGFP-N Is, 12.94 ± 3.45, N = 6, p = 0.89; Student's t test]. F – H , Representative traces of EJPs and mEJPs recorded in 0.4 m m Ca 2+ at wild-type ( F ), cac sfGFP-N ( G ), and cac TagRFP-N NMJs ( H ). I , mEJP amplitude is unaffected in cac sfGFP-N and cac TagRFP-N (wild type, 0.91 ± 0.05, n = 9 NMJs from 4 larvae; cac sfGFP-N , 0.99 ± 0.04, n = 13 NMJs from 4 larvae, p = 0.17, Student's t test; cac TagRFP-N , 0.99 ± 0.04, n = 12 NMJs from 4 larvae, p = 0.20, Mann–Whitney U test). J , EJP amplitude is unchanged between wild-type, cac sfGFP-N and cac TagRFP-N NMJs (wild type, 30.87 ± 1.41, n = 9 NMJs from 4 larvae; cac sfGFP-N , 29.64 ± 0.52, n = 13 NMJs from 4 larvae, p = 0.84, Mann–Whitney U test; cac TagRFP-N , 26.70 ± 1.39, n = 12 NMJs from 4 larvae, p = 0.05, Student's t test). K , Quantal content is similar between wild-type, cac sfGFP-N and cac TagRFP-N NMJs (wild type, 34.8 ± 2.3, n = 9 NMJs from 4 larvae; cac sfGFP-N , 30.6 ± 1.4, n = 13 NMJs from 4 larvae, p = 0.12, Student's t test; cac TagRFP-N , 27.5 ± 1.7, n = 12 NMJs from 4 larvae, p = 0.03, Mann–Whitney U test). Not significant (ns) and * p < 0.05.

    Journal: The Journal of Neuroscience

    Article Title: Endogenous Tagging Reveals Differential Regulation of Ca 2+ Channels at Single Active Zones during Presynaptic Homeostatic Potentiation and Depression

    doi: 10.1523/JNEUROSCI.3068-18.2019

    Figure Lengend Snippet: Endogenous tagging of Cacophony does not perturb presynaptic Ca 2+ influx or synaptic function. A , Live fluorescence images of type-Ib boutons from a cac sfGFP-N motor terminal showing Cac sfGFP-N (top) and AF647-dextran (center), which was co-loaded with rhod-dextran (image not shown). B , Ratiometric fluorescence changes (rhod-dextran relative to AF647-dextran) in presynaptic motorneuron terminals during stimulation in wild type and cac sfGFP-N . An action potential was initiated every second, for a period of 10 s, followed by a 1 s, 20 Hz train of action potentials. C , Plot of average Ca 2+ levels ( R ) in terminals before nerve stimulation for type-Ib and -Is terminals in wild-type and cac sfGFP-N boutons [wild-type Ib, 2.74 ± 0.44, one NMJ from each of 5 animals ( N = 5); cac sfGFP-N Ib, 2.47 ± 0.49, N = 8, p = 0.69; wild-type Is, 2.16 ± 0.47, N = 5; cac sfGFP-N Is, 2.94 ± 0.32, N = 6, p = 0.21; Student's t test). D , Plot of the average amplitude of single action potential-mediated Ca 2+ transients [Δ R ; wild-type Ib, 4.50 ± 0.81, one NMJ from each of 5 animals ( N = 5); cac sfGFP-N Ib, 3.90 ± 1.07, N = 8, p = 0.67; wild-type Is, 6.28 ± 1.79, N = 5; cac sfGFP-N Is, 7.33 ± 2.20, N = 6, p = 0.72; Student's t test]. E , Plot of the average amplitude of 1 s, 20 Hz action potential train-mediated Ca 2+ transients [wild-type Ib, 9.71 ± 1.53, one NMJ from each of 5 animals ( N = 5); cac sfGFP-N Ib, 8.13 ± 2.21, N = 8, p = 0.57; wild-type Is, 12.26 ± 3.36, N = 5; cac sfGFP-N Is, 12.94 ± 3.45, N = 6, p = 0.89; Student's t test]. F – H , Representative traces of EJPs and mEJPs recorded in 0.4 m m Ca 2+ at wild-type ( F ), cac sfGFP-N ( G ), and cac TagRFP-N NMJs ( H ). I , mEJP amplitude is unaffected in cac sfGFP-N and cac TagRFP-N (wild type, 0.91 ± 0.05, n = 9 NMJs from 4 larvae; cac sfGFP-N , 0.99 ± 0.04, n = 13 NMJs from 4 larvae, p = 0.17, Student's t test; cac TagRFP-N , 0.99 ± 0.04, n = 12 NMJs from 4 larvae, p = 0.20, Mann–Whitney U test). J , EJP amplitude is unchanged between wild-type, cac sfGFP-N and cac TagRFP-N NMJs (wild type, 30.87 ± 1.41, n = 9 NMJs from 4 larvae; cac sfGFP-N , 29.64 ± 0.52, n = 13 NMJs from 4 larvae, p = 0.84, Mann–Whitney U test; cac TagRFP-N , 26.70 ± 1.39, n = 12 NMJs from 4 larvae, p = 0.05, Student's t test). K , Quantal content is similar between wild-type, cac sfGFP-N and cac TagRFP-N NMJs (wild type, 34.8 ± 2.3, n = 9 NMJs from 4 larvae; cac sfGFP-N , 30.6 ± 1.4, n = 13 NMJs from 4 larvae, p = 0.12, Student's t test; cac TagRFP-N , 27.5 ± 1.7, n = 12 NMJs from 4 larvae, p = 0.03, Mann–Whitney U test). Not significant (ns) and * p < 0.05.

    Article Snippet: Severed segmental nerves were drawn into a filling pipette, and a 16:1 mixture of the Ca 2+ indicator rhod dextran (R34676; ThermoFisher Scientific) and a Ca 2+ -insensitive AlexaFluor 647 dextran (AF647 dextran; D22914; ThermoFisher Scientific) was applied to the cut nerve end for 15–45 min. Preparations were incubated in the dark for at least 3 h and rinsed every 30 min with fresh Schneider's insect medium.

    Techniques: Fluorescence, MANN-WHITNEY

    Endogenous Cac is differentially regulated in chronic PHP and PHD. A – F , Chronic expression of PHP and PHD occurs normally in cac sfGFP-N animals lacking GluRIIA ( GluRIIA SP16 ) or overexpressing vGlut (vGlut-OE; OK371-Gal4 > UAS-vGlut ), respectively. A – C , Representative traces of EJPs and mEJPs recorded in 0.4 m m Ca 2+ at cac sfGFP-N ( A ), cac sfGFP-N ; GluRIIA NMJs ( B ), and cac sfGFP-N ; vGlut-OE ( C ). D , mEJP amplitude is decreased in cac sfGFP-N ; GluRIIA and increased in cac sfGFP-N ; vGlut-OE as expected ( cac sfGFP-N , 1.0 ± 0.08, n = 8 NMJs from 4 larvae; cac sfGFP-N ; GluRIIA , 0.48 ± 0.02, n = 7 NMJs from 4 larvae, p = 0.0001, t test with Welch's correction; cac sfGFP-N ; vGlut-OE, 1.5 ± 0.08, n = 10 NMJs from 4 larvae, p = 0.0017, Student's t test). E , EJP amplitude is unchanged between cac sfGFP-N , cac sfGFP-N ; GluRIIA and cac sfGFP-N ; vGlut-OE NMJs ( cac sfGFP-N , 30.3 ± 1.70, n = 8 NMJs from 4 larvae; cac sfGFP-N ; GluRIIA , 27.1 ± 0.60, n = 7 NMJs from 5 larvae, p = 0.1115, t test with Welch's correction; cac sfGFP-N ; vGlut-OE, 29.8 ± 0.81, n = 10 NMJs from 4 larvae, p = 0.80, Student's t test), as expected. F , Quantal content is increased in cac sfGFP-N ; GluRIIA and decreased in cac sfGFP-N ; vGlut-OE as expected ( cac sfGFP-N , 30.7 ± 3.0, n = 8 NMJs from 4 larvae; cac sfGFP-N ; GluRIIA , 56.6 ± 2.4, n = 7 NMJs from 4 larvae, p < 0.0001, Student's t test; cac sfGFP-N ; vGlut-OE, 21.0 ± 1.3, n = 10 NMJs from 4 larvae, p = 0.0145, t test with Welch's correction). G , Confocal Z -projections of Cac and Brp in cac sfGFP-N , in cac sfGFP-N ; GluRIIA and in cac sfGFP-N ; vGlut-OE motorneuron boutons colabeled with antibodies against GFP and Brp. H , Normalized intensity of Cac and Brp puncta averaged for each NMJ in control, GluRIIA and vGlut-OE animals (Cac levels: cac sfGFP-N , 1.00 ± 0.05, n = 22 NMJs from 9 larvae; cac sfGFP-N ; GluRIIA , 1.36 ± 0.06, n = 14 NMJs from 5 larvae, p < 0.0001, Student's t test; and cac sfGFP-N ; vGlut-OE, 1.07 ± 0.06, n = 11 NMJs from 5 larvae, p = 0.43, Student's t test. Brp levels: cac sfGFP-N , 1.00 ± 0.05, n = 22 NMJs from 4 larvae; cac sfGFP-N ; GluRIIA , 1.44 ± 0.04, n = 14 NMJs from 5 larvae, p < 0.0001, Student's t test; cac sfGFP-N ; vGlut-OE, 1.08 ± 0.06, n = 11 NMJs from 5 larvae, p = 0.19, Student's t test). I , Cumulative probability distributions of Cac and Brp puncta intensities demonstrate an increase in both Brp and Cac levels across AZs of GluRIIA animals [Cac levels: cac sfGFP-N , n = 1475 AZs; cac sfGFP-N ; GluRIIA , n = 1475; (3 data points are outside the x -axis limits shown); Brp levels: cac sfGFP-N , n = 1475 AZs; cac sfGFP-N ; GluRIIA , n = 1475 AZs]. J , Cumulative probability distributions of Cac and Brp puncta intensities show no decrease in Brp or Cac levels across AZs of vGlut-OE animals [Cac levels: cac sfGFP-N , n = 1475 AZs; cac sfGFP-N ; vGlut-OE, n = 1443 (7 data points are outside the x -axis limits shown); Brp levels: cac sfGFP-N , n = 1475 AZs; cac sfGFP-N ; vGlut-OE, n = 1443 AZs]. Not significant (ns), * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

    Journal: The Journal of Neuroscience

    Article Title: Endogenous Tagging Reveals Differential Regulation of Ca 2+ Channels at Single Active Zones during Presynaptic Homeostatic Potentiation and Depression

    doi: 10.1523/JNEUROSCI.3068-18.2019

    Figure Lengend Snippet: Endogenous Cac is differentially regulated in chronic PHP and PHD. A – F , Chronic expression of PHP and PHD occurs normally in cac sfGFP-N animals lacking GluRIIA ( GluRIIA SP16 ) or overexpressing vGlut (vGlut-OE; OK371-Gal4 > UAS-vGlut ), respectively. A – C , Representative traces of EJPs and mEJPs recorded in 0.4 m m Ca 2+ at cac sfGFP-N ( A ), cac sfGFP-N ; GluRIIA NMJs ( B ), and cac sfGFP-N ; vGlut-OE ( C ). D , mEJP amplitude is decreased in cac sfGFP-N ; GluRIIA and increased in cac sfGFP-N ; vGlut-OE as expected ( cac sfGFP-N , 1.0 ± 0.08, n = 8 NMJs from 4 larvae; cac sfGFP-N ; GluRIIA , 0.48 ± 0.02, n = 7 NMJs from 4 larvae, p = 0.0001, t test with Welch's correction; cac sfGFP-N ; vGlut-OE, 1.5 ± 0.08, n = 10 NMJs from 4 larvae, p = 0.0017, Student's t test). E , EJP amplitude is unchanged between cac sfGFP-N , cac sfGFP-N ; GluRIIA and cac sfGFP-N ; vGlut-OE NMJs ( cac sfGFP-N , 30.3 ± 1.70, n = 8 NMJs from 4 larvae; cac sfGFP-N ; GluRIIA , 27.1 ± 0.60, n = 7 NMJs from 5 larvae, p = 0.1115, t test with Welch's correction; cac sfGFP-N ; vGlut-OE, 29.8 ± 0.81, n = 10 NMJs from 4 larvae, p = 0.80, Student's t test), as expected. F , Quantal content is increased in cac sfGFP-N ; GluRIIA and decreased in cac sfGFP-N ; vGlut-OE as expected ( cac sfGFP-N , 30.7 ± 3.0, n = 8 NMJs from 4 larvae; cac sfGFP-N ; GluRIIA , 56.6 ± 2.4, n = 7 NMJs from 4 larvae, p < 0.0001, Student's t test; cac sfGFP-N ; vGlut-OE, 21.0 ± 1.3, n = 10 NMJs from 4 larvae, p = 0.0145, t test with Welch's correction). G , Confocal Z -projections of Cac and Brp in cac sfGFP-N , in cac sfGFP-N ; GluRIIA and in cac sfGFP-N ; vGlut-OE motorneuron boutons colabeled with antibodies against GFP and Brp. H , Normalized intensity of Cac and Brp puncta averaged for each NMJ in control, GluRIIA and vGlut-OE animals (Cac levels: cac sfGFP-N , 1.00 ± 0.05, n = 22 NMJs from 9 larvae; cac sfGFP-N ; GluRIIA , 1.36 ± 0.06, n = 14 NMJs from 5 larvae, p < 0.0001, Student's t test; and cac sfGFP-N ; vGlut-OE, 1.07 ± 0.06, n = 11 NMJs from 5 larvae, p = 0.43, Student's t test. Brp levels: cac sfGFP-N , 1.00 ± 0.05, n = 22 NMJs from 4 larvae; cac sfGFP-N ; GluRIIA , 1.44 ± 0.04, n = 14 NMJs from 5 larvae, p < 0.0001, Student's t test; cac sfGFP-N ; vGlut-OE, 1.08 ± 0.06, n = 11 NMJs from 5 larvae, p = 0.19, Student's t test). I , Cumulative probability distributions of Cac and Brp puncta intensities demonstrate an increase in both Brp and Cac levels across AZs of GluRIIA animals [Cac levels: cac sfGFP-N , n = 1475 AZs; cac sfGFP-N ; GluRIIA , n = 1475; (3 data points are outside the x -axis limits shown); Brp levels: cac sfGFP-N , n = 1475 AZs; cac sfGFP-N ; GluRIIA , n = 1475 AZs]. J , Cumulative probability distributions of Cac and Brp puncta intensities show no decrease in Brp or Cac levels across AZs of vGlut-OE animals [Cac levels: cac sfGFP-N , n = 1475 AZs; cac sfGFP-N ; vGlut-OE, n = 1443 (7 data points are outside the x -axis limits shown); Brp levels: cac sfGFP-N , n = 1475 AZs; cac sfGFP-N ; vGlut-OE, n = 1443 AZs]. Not significant (ns), * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

    Article Snippet: Severed segmental nerves were drawn into a filling pipette, and a 16:1 mixture of the Ca 2+ indicator rhod dextran (R34676; ThermoFisher Scientific) and a Ca 2+ -insensitive AlexaFluor 647 dextran (AF647 dextran; D22914; ThermoFisher Scientific) was applied to the cut nerve end for 15–45 min. Preparations were incubated in the dark for at least 3 h and rinsed every 30 min with fresh Schneider's insect medium.

    Techniques: Expressing

    Cacophony levels are rapidly upregulated following acute homeostatic challenge. A , B , Representative traces of EJPs and mEJPs recorded at cac sfGFP-N NMJs in 0.4 m m Ca 2+ with ( B ) or without ( A ) 10 min application of PhTx. C , PhTx exposure significantly reduces mEJP amplitude at cac sfGFP-N NMJs ( cac sfGFP-N , 100 ± 7.3%, n = 8 NMJs from 4 larvae; cac sfGFP-N + PhTx, 52 ± 2.2%, n = 10 NMJs from 4 larvae, p = 0.0002, t test with Welch's correction) as expected. In response to PhTx exposure, EJP amplitude is maintained ( cac sfGFP-N , 100 ± 5.6%, n = 8 NMJs from 4 larvae; cac sfGFP-N + PhTx, 95.5 ± 2.5%, n = 10 NMJs from 4 larvae, p = 0.4393, Student's t test) as quantal content is significantly increased at cac sfGFP-N NMJs ( cac sfGFP-N , 100 ± 9.7%, n = 8 NMJs from 4 larvae; cac sfGFP-N + PhTx, 179.1 ± 8.8%, n = 10 NMJs from 4 larvae, p < 0.0001, Student's t test). D , Confocal Z -projections of cac sfGFP-N motorneuron boutons colabeled with antibodies against GFP and Brp following vehicle (control) and PhTx treatment. E , Normalized intensity of Cac and Brp per AZ averaged for each NMJ following 10 min vehicle or PhTx treatment from three independent experiments (yellow, green, and purple symbols) reveal a significant increase in both Brp and Cac levels immediately following PhTx treatment (Cac levels: control, 1.0 ± 0.05, n = 65 NMJs from 18 larvae; PhTx, 1.54 ± 0.07, n = 68 NMJs from 18 larvae, p < 0.0001, Mann–Whitney U test. Brp levels: control, 1.0 ± 0.04, n = 65 NMJs from 18 larvae; PhTx, 1.58 ± 0.06, n = 68 NMJs from 18 larvae, p < 0.0001, t test with Welch's correction). F , Cumulative probability distributions of Cac and Brp intensities in control and PhTx-treated cac sfGFP-N animals [control, n = 13,908 AZs; PhTx, n = 15,925 AZs (80 data points are outside the x -axis limits shown)]. G , H , Frequency distributions of Cac and Brp intensities at individual AZs of control and PhTx-treated cac sfGFP-N animals reveal a rightward shift in intensities. Not significant (ns), *** p < 0.001, and **** p < 0.0001.

    Journal: The Journal of Neuroscience

    Article Title: Endogenous Tagging Reveals Differential Regulation of Ca 2+ Channels at Single Active Zones during Presynaptic Homeostatic Potentiation and Depression

    doi: 10.1523/JNEUROSCI.3068-18.2019

    Figure Lengend Snippet: Cacophony levels are rapidly upregulated following acute homeostatic challenge. A , B , Representative traces of EJPs and mEJPs recorded at cac sfGFP-N NMJs in 0.4 m m Ca 2+ with ( B ) or without ( A ) 10 min application of PhTx. C , PhTx exposure significantly reduces mEJP amplitude at cac sfGFP-N NMJs ( cac sfGFP-N , 100 ± 7.3%, n = 8 NMJs from 4 larvae; cac sfGFP-N + PhTx, 52 ± 2.2%, n = 10 NMJs from 4 larvae, p = 0.0002, t test with Welch's correction) as expected. In response to PhTx exposure, EJP amplitude is maintained ( cac sfGFP-N , 100 ± 5.6%, n = 8 NMJs from 4 larvae; cac sfGFP-N + PhTx, 95.5 ± 2.5%, n = 10 NMJs from 4 larvae, p = 0.4393, Student's t test) as quantal content is significantly increased at cac sfGFP-N NMJs ( cac sfGFP-N , 100 ± 9.7%, n = 8 NMJs from 4 larvae; cac sfGFP-N + PhTx, 179.1 ± 8.8%, n = 10 NMJs from 4 larvae, p < 0.0001, Student's t test). D , Confocal Z -projections of cac sfGFP-N motorneuron boutons colabeled with antibodies against GFP and Brp following vehicle (control) and PhTx treatment. E , Normalized intensity of Cac and Brp per AZ averaged for each NMJ following 10 min vehicle or PhTx treatment from three independent experiments (yellow, green, and purple symbols) reveal a significant increase in both Brp and Cac levels immediately following PhTx treatment (Cac levels: control, 1.0 ± 0.05, n = 65 NMJs from 18 larvae; PhTx, 1.54 ± 0.07, n = 68 NMJs from 18 larvae, p < 0.0001, Mann–Whitney U test. Brp levels: control, 1.0 ± 0.04, n = 65 NMJs from 18 larvae; PhTx, 1.58 ± 0.06, n = 68 NMJs from 18 larvae, p < 0.0001, t test with Welch's correction). F , Cumulative probability distributions of Cac and Brp intensities in control and PhTx-treated cac sfGFP-N animals [control, n = 13,908 AZs; PhTx, n = 15,925 AZs (80 data points are outside the x -axis limits shown)]. G , H , Frequency distributions of Cac and Brp intensities at individual AZs of control and PhTx-treated cac sfGFP-N animals reveal a rightward shift in intensities. Not significant (ns), *** p < 0.001, and **** p < 0.0001.

    Article Snippet: Severed segmental nerves were drawn into a filling pipette, and a 16:1 mixture of the Ca 2+ indicator rhod dextran (R34676; ThermoFisher Scientific) and a Ca 2+ -insensitive AlexaFluor 647 dextran (AF647 dextran; D22914; ThermoFisher Scientific) was applied to the cut nerve end for 15–45 min. Preparations were incubated in the dark for at least 3 h and rinsed every 30 min with fresh Schneider's insect medium.

    Techniques: MANN-WHITNEY

    Live imaging of Cac sfGFP reveals Ca 2+ channel accumulation across heterogeneous AZs during rapid homeostatic adaptation to PhTx application. A , Confocal Z -projection of Cac at AZs of a motorneuron branch in a live cac sfGFP-N preparation immediately before and after 10-min PhTx treatment. The heat map indicates PhTx-induced Cac accumulation (color) relative to baseline Cac levels (size) at each AZ. B , Cac intensity at single AZs of the NMJ shown in A before and after PhTx reveals Cac accumulation at the majority of AZs (baseline: 75.58 ± 8.55; post-PhTx minus vehicle: 94.25 ± 10.12, n = 34 AZs from 1 NMJ branch, p < 0.0001, paired t test). C , Cac intensity at single AZs across multiple animals also reveals Ca 2+ channel accumulation at most AZs (Baseline, 89.56 ± 3.24; post-PhTx minus vehicle, 105.7 ± 3.96, n = 496 AZs from 7 NMJs and 4 animals, p < 0.0001, Wilcoxon signed rank test). D , Cac intensity at individual AZs at baseline versus post-PhTx minus vehicle. The blue line is a least-squares fit to the data (slope = 1.16, R 2 = 0.94), whereas the dotted line would describe the data if there were no effect of PhTx (unity line, slope = 1), the green line would describe the data if there were an additive effect, and the orange line would describe the data if there were a multiplicative effect. E , AZs that experience either no change or a decrease in Ca 2+ channel content after PhTx occur more frequently in lower Cac-content AZs (bottom-third, 48%; middle-third, 28%, top-third, 13%; bottom vs middle, p = 0.003, middle vs top, p = 0.02, bottom vs top, p < 0.0001, Fisher's exact test). F , AZ number is similar between control and PhTx-treated cac sfGFP-N NMJs in fixed tissue preparations (control: 10.56 ± 0.60, n = 22 NMJs from 6 animals; PhTx: 11.73 ± 0.45, n = 23 NMJs from 6 animals, p = 0.12, Student's t test). Not significant (ns), * p < 0.05, ** p < 0.01, and **** p < 0.0001.

    Journal: The Journal of Neuroscience

    Article Title: Endogenous Tagging Reveals Differential Regulation of Ca 2+ Channels at Single Active Zones during Presynaptic Homeostatic Potentiation and Depression

    doi: 10.1523/JNEUROSCI.3068-18.2019

    Figure Lengend Snippet: Live imaging of Cac sfGFP reveals Ca 2+ channel accumulation across heterogeneous AZs during rapid homeostatic adaptation to PhTx application. A , Confocal Z -projection of Cac at AZs of a motorneuron branch in a live cac sfGFP-N preparation immediately before and after 10-min PhTx treatment. The heat map indicates PhTx-induced Cac accumulation (color) relative to baseline Cac levels (size) at each AZ. B , Cac intensity at single AZs of the NMJ shown in A before and after PhTx reveals Cac accumulation at the majority of AZs (baseline: 75.58 ± 8.55; post-PhTx minus vehicle: 94.25 ± 10.12, n = 34 AZs from 1 NMJ branch, p < 0.0001, paired t test). C , Cac intensity at single AZs across multiple animals also reveals Ca 2+ channel accumulation at most AZs (Baseline, 89.56 ± 3.24; post-PhTx minus vehicle, 105.7 ± 3.96, n = 496 AZs from 7 NMJs and 4 animals, p < 0.0001, Wilcoxon signed rank test). D , Cac intensity at individual AZs at baseline versus post-PhTx minus vehicle. The blue line is a least-squares fit to the data (slope = 1.16, R 2 = 0.94), whereas the dotted line would describe the data if there were no effect of PhTx (unity line, slope = 1), the green line would describe the data if there were an additive effect, and the orange line would describe the data if there were a multiplicative effect. E , AZs that experience either no change or a decrease in Ca 2+ channel content after PhTx occur more frequently in lower Cac-content AZs (bottom-third, 48%; middle-third, 28%, top-third, 13%; bottom vs middle, p = 0.003, middle vs top, p = 0.02, bottom vs top, p < 0.0001, Fisher's exact test). F , AZ number is similar between control and PhTx-treated cac sfGFP-N NMJs in fixed tissue preparations (control: 10.56 ± 0.60, n = 22 NMJs from 6 animals; PhTx: 11.73 ± 0.45, n = 23 NMJs from 6 animals, p = 0.12, Student's t test). Not significant (ns), * p < 0.05, ** p < 0.01, and **** p < 0.0001.

    Article Snippet: Severed segmental nerves were drawn into a filling pipette, and a 16:1 mixture of the Ca 2+ indicator rhod dextran (R34676; ThermoFisher Scientific) and a Ca 2+ -insensitive AlexaFluor 647 dextran (AF647 dextran; D22914; ThermoFisher Scientific) was applied to the cut nerve end for 15–45 min. Preparations were incubated in the dark for at least 3 h and rinsed every 30 min with fresh Schneider's insect medium.

    Techniques: Imaging