probdnf  (Alomone Labs)


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    Structured Review

    Alomone Labs probdnf
    proBNDF-mediated p75NTR activation in cortical PV cells reduces their perisomatic boutons. A , Experimental approach. B , The intensity of perisomatic PV immunostaining (green) is reduced in the binocular visual cortex ipsilateral to the minipump-releasing <t>mut-proBDNF</t> (Ipsi) compared with the contralateral cortex (Contra) in the same animal. On the other hand, perisomatic PV intensity in the ipsilateral cortex of PV_Cre;p75 flx/flx mice is similar to that observed in the contralateral, untreated cortex. C , Low ( C1 ) and high ( C2 ) magnification of PNN (red, WFA staining) enwrapping PV cells (green) shows a dramatic reduction in both PNN density and intensity in the visual cortex infused with mut-proBFNF. This effect is abolished in PV_Cre;p75 flx/flx mice. Scale bars: C1 , 100 μm; B , C2 , 10 μm. D , Quantification of the mean intensity of perisomatic PV-positive puncta in ipsilateral compared with contralateral cortex. I/C ratio is obtained for each animal and then averaged between different animals. Mean I/C ratio is significantly reduced in Mut-proBDNF-infused p75 Ctrl mice compared with Mut-proBDNF-infused PV_Cre;p75 flx/flx mice (unpaired t test, df = 8, t = 6.077, p = 0.0003). E , The ratio of mean PNN intensity around PV cells in ipsilateral versus contralateral cortex is significantly lower in p75 Ctrl than PV_Cre;p75 flx/flx mice infused with mut-proBDNF (unpaired t test, df = 8, t = 15.33, p
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    Images

    1) Product Images from "p75 Neurotrophin Receptor Activation Regulates the Timing of the Maturation of Cortical Parvalbumin Interneuron Connectivity and Promotes Juvenile-like Plasticity in Adult Visual Cortex"

    Article Title: p75 Neurotrophin Receptor Activation Regulates the Timing of the Maturation of Cortical Parvalbumin Interneuron Connectivity and Promotes Juvenile-like Plasticity in Adult Visual Cortex

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.2881-18.2019

    proBNDF-mediated p75NTR activation in cortical PV cells reduces their perisomatic boutons. A , Experimental approach. B , The intensity of perisomatic PV immunostaining (green) is reduced in the binocular visual cortex ipsilateral to the minipump-releasing mut-proBDNF (Ipsi) compared with the contralateral cortex (Contra) in the same animal. On the other hand, perisomatic PV intensity in the ipsilateral cortex of PV_Cre;p75 flx/flx mice is similar to that observed in the contralateral, untreated cortex. C , Low ( C1 ) and high ( C2 ) magnification of PNN (red, WFA staining) enwrapping PV cells (green) shows a dramatic reduction in both PNN density and intensity in the visual cortex infused with mut-proBFNF. This effect is abolished in PV_Cre;p75 flx/flx mice. Scale bars: C1 , 100 μm; B , C2 , 10 μm. D , Quantification of the mean intensity of perisomatic PV-positive puncta in ipsilateral compared with contralateral cortex. I/C ratio is obtained for each animal and then averaged between different animals. Mean I/C ratio is significantly reduced in Mut-proBDNF-infused p75 Ctrl mice compared with Mut-proBDNF-infused PV_Cre;p75 flx/flx mice (unpaired t test, df = 8, t = 6.077, p = 0.0003). E , The ratio of mean PNN intensity around PV cells in ipsilateral versus contralateral cortex is significantly lower in p75 Ctrl than PV_Cre;p75 flx/flx mice infused with mut-proBDNF (unpaired t test, df = 8, t = 15.33, p
    Figure Legend Snippet: proBNDF-mediated p75NTR activation in cortical PV cells reduces their perisomatic boutons. A , Experimental approach. B , The intensity of perisomatic PV immunostaining (green) is reduced in the binocular visual cortex ipsilateral to the minipump-releasing mut-proBDNF (Ipsi) compared with the contralateral cortex (Contra) in the same animal. On the other hand, perisomatic PV intensity in the ipsilateral cortex of PV_Cre;p75 flx/flx mice is similar to that observed in the contralateral, untreated cortex. C , Low ( C1 ) and high ( C2 ) magnification of PNN (red, WFA staining) enwrapping PV cells (green) shows a dramatic reduction in both PNN density and intensity in the visual cortex infused with mut-proBFNF. This effect is abolished in PV_Cre;p75 flx/flx mice. Scale bars: C1 , 100 μm; B , C2 , 10 μm. D , Quantification of the mean intensity of perisomatic PV-positive puncta in ipsilateral compared with contralateral cortex. I/C ratio is obtained for each animal and then averaged between different animals. Mean I/C ratio is significantly reduced in Mut-proBDNF-infused p75 Ctrl mice compared with Mut-proBDNF-infused PV_Cre;p75 flx/flx mice (unpaired t test, df = 8, t = 6.077, p = 0.0003). E , The ratio of mean PNN intensity around PV cells in ipsilateral versus contralateral cortex is significantly lower in p75 Ctrl than PV_Cre;p75 flx/flx mice infused with mut-proBDNF (unpaired t test, df = 8, t = 15.33, p

    Techniques Used: Activation Assay, Immunostaining, Mouse Assay, Staining

    Modulation of tPA activity affects the formation of PV cell innervations during early postnatal development. A , Control EP18 PV cell ( A1 , green represents Ctrl). B , PV cell treated with the tPA inhibitor PPACK from EP10–EP18 shows simpler axonal arborization, contacting less potential targets ( B2 , blue represents NeuN-positive somata). C , PV cell treated with tPA in the same time window shows a very complex axonal arbor ( C2 ) and an increase in both terminal branching and perisomatic boutons ( C3 , arrowheads) compared with control cells ( A2 , A3 ). D , PV cell treated simultaneously with tPA and mut-proBDNF shows axonal branching and perisomatic innervation more similar to those formed by PV cell treated with mut-proBDNF alone, suggesting that the effects of tPA application may be mediated by a decrease in endogenous proBDNF/mBDNF ratio. Stars indicate NeuN-positive somata that are not innervated. Scale bars: A1–D1 , 50 μm; A2–D2 , 10 μm; A3–D3 , 5 μm. E , Perisomatic boutons density (one-way ANOVA, F (3,20) = 121.2, p
    Figure Legend Snippet: Modulation of tPA activity affects the formation of PV cell innervations during early postnatal development. A , Control EP18 PV cell ( A1 , green represents Ctrl). B , PV cell treated with the tPA inhibitor PPACK from EP10–EP18 shows simpler axonal arborization, contacting less potential targets ( B2 , blue represents NeuN-positive somata). C , PV cell treated with tPA in the same time window shows a very complex axonal arbor ( C2 ) and an increase in both terminal branching and perisomatic boutons ( C3 , arrowheads) compared with control cells ( A2 , A3 ). D , PV cell treated simultaneously with tPA and mut-proBDNF shows axonal branching and perisomatic innervation more similar to those formed by PV cell treated with mut-proBDNF alone, suggesting that the effects of tPA application may be mediated by a decrease in endogenous proBDNF/mBDNF ratio. Stars indicate NeuN-positive somata that are not innervated. Scale bars: A1–D1 , 50 μm; A2–D2 , 10 μm; A3–D3 , 5 μm. E , Perisomatic boutons density (one-way ANOVA, F (3,20) = 121.2, p

    Techniques Used: Activity Assay

    proBNDF-mediated p75NTR activation in cortical PV cells restores ocular dominance plasticity in adult visual cortex in vivo . A , Typical VEP responses to the stimulation of either contralateral (blue) or ipsilateral (red) eye to the cortex in which the recording is performed in p75NTR Ctrl mice infused with either vehicle or mut-proBDNF, and PV_Cre;p75NTR flx/flx mice infused with mut-proBDNF. Calibration bars: 50 μV, 100 ms. B , C/I VEP ratio mean values. Three days of monocular deprivation do not affect the C/I VEP ratio in adult mice, although it leads to a significant decrease in the C/I VEP ratio in animals treated with mut-proBDNF. Mut-proBDNF effects are, however, abolished in PV_Cre;p75 flx/flx mice (one-way ANOVA, F (2,18) = 8.903, p = 0.0021). p75NTR Ctrl + vehicle: n = 9 mice; p75NTR Ctrl + mut-proBDNF: n = 5 mice; PV_Cre;p75 flx/flx +mut-proBDNF: n = 7 mice. C , ODI of p75NTR Ctrl mice infused with vehicle solution and PV_Cre;p75 flx/flx mice infused with mut-proBDNF are not significantly different from those of undeprived animals, whereas ODIs in p75 Ctrl mice treated with mut-proBDNF are significantly shifted toward the open eye (one-way ANOVA, F (2,443) = 5.203, p = 0.0058). D , Mean spontaneous discharge is significantly increased only in p75 Ctrl mice treated with mut-proBDNF (one-way ANOVA, F (2,443) = 4.580, p = 0.0107). p75NTR Ctrl + vehicle: n = 9 mice, 174 cells; p75NTR Ctrl + mut-proBDNF: n = 5 mice, 147 cells; PV_Cre;p75 flx/flx +mut-proBDNF: n = 6 mice, 125 cells. Gray area represents the C/I VEP ratio ( B ) or the ODI range ( C ) (mean ± SEM) in adult nondeprived animals ( n = 5 mice, 99 cells). * indicate p
    Figure Legend Snippet: proBNDF-mediated p75NTR activation in cortical PV cells restores ocular dominance plasticity in adult visual cortex in vivo . A , Typical VEP responses to the stimulation of either contralateral (blue) or ipsilateral (red) eye to the cortex in which the recording is performed in p75NTR Ctrl mice infused with either vehicle or mut-proBDNF, and PV_Cre;p75NTR flx/flx mice infused with mut-proBDNF. Calibration bars: 50 μV, 100 ms. B , C/I VEP ratio mean values. Three days of monocular deprivation do not affect the C/I VEP ratio in adult mice, although it leads to a significant decrease in the C/I VEP ratio in animals treated with mut-proBDNF. Mut-proBDNF effects are, however, abolished in PV_Cre;p75 flx/flx mice (one-way ANOVA, F (2,18) = 8.903, p = 0.0021). p75NTR Ctrl + vehicle: n = 9 mice; p75NTR Ctrl + mut-proBDNF: n = 5 mice; PV_Cre;p75 flx/flx +mut-proBDNF: n = 7 mice. C , ODI of p75NTR Ctrl mice infused with vehicle solution and PV_Cre;p75 flx/flx mice infused with mut-proBDNF are not significantly different from those of undeprived animals, whereas ODIs in p75 Ctrl mice treated with mut-proBDNF are significantly shifted toward the open eye (one-way ANOVA, F (2,443) = 5.203, p = 0.0058). D , Mean spontaneous discharge is significantly increased only in p75 Ctrl mice treated with mut-proBDNF (one-way ANOVA, F (2,443) = 4.580, p = 0.0107). p75NTR Ctrl + vehicle: n = 9 mice, 174 cells; p75NTR Ctrl + mut-proBDNF: n = 5 mice, 147 cells; PV_Cre;p75 flx/flx +mut-proBDNF: n = 6 mice, 125 cells. Gray area represents the C/I VEP ratio ( B ) or the ODI range ( C ) (mean ± SEM) in adult nondeprived animals ( n = 5 mice, 99 cells). * indicate p

    Techniques Used: Activation Assay, In Vivo, Mouse Assay

    mut-proBDNF destabilizes PV cell innervation, even after it has reached maturity. A , Control PV cell ( A1 , Ctrl, green) at EP32 with exuberant innervation field characterized by extensive branching contacting the majority of potential targets, dense boutons along axons ( A2 ), and terminal branches with prominent and clustered boutons ( A3 ; arrowheads) around NeuN-positive somata (blue). B , PV cell treated with wt-proBDNF from EP26-EP32 shows overall similar axon size ( B1 ), percentage of potentially targeted neurons ( B2 ), and perisomatic innervations ( B3 ) as control, untreated PV cells. C , PV cell treated with mut-proBDNF from EP26-EP32 shows a drastic reduction both in percentage of innervated cells ( C2 ) and perisomatic innervation ( C3 ). Stars indicate NeuN-positive somata that are not innervated. Scale bars: A1–C1 , 50 μm; A2–C2 , 10 μm; A3–C3 , 5 μm. D , Perisomatic bouton density (one-way ANOVA, F (2,18) = 93.34, p
    Figure Legend Snippet: mut-proBDNF destabilizes PV cell innervation, even after it has reached maturity. A , Control PV cell ( A1 , Ctrl, green) at EP32 with exuberant innervation field characterized by extensive branching contacting the majority of potential targets, dense boutons along axons ( A2 ), and terminal branches with prominent and clustered boutons ( A3 ; arrowheads) around NeuN-positive somata (blue). B , PV cell treated with wt-proBDNF from EP26-EP32 shows overall similar axon size ( B1 ), percentage of potentially targeted neurons ( B2 ), and perisomatic innervations ( B3 ) as control, untreated PV cells. C , PV cell treated with mut-proBDNF from EP26-EP32 shows a drastic reduction both in percentage of innervated cells ( C2 ) and perisomatic innervation ( C3 ). Stars indicate NeuN-positive somata that are not innervated. Scale bars: A1–C1 , 50 μm; A2–C2 , 10 μm; A3–C3 , 5 μm. D , Perisomatic bouton density (one-way ANOVA, F (2,18) = 93.34, p

    Techniques Used:

    2) Product Images from "Peripheral Brain Derived Neurotrophic Factor Precursor Regulates Pain as an Inflammatory Mediator"

    Article Title: Peripheral Brain Derived Neurotrophic Factor Precursor Regulates Pain as an Inflammatory Mediator

    Journal: Scientific Reports

    doi: 10.1038/srep27171

    Exogenous proBDNF induces pain hypersensitivity and spinal cord activation in mice. ( A ) Dosage effect of exogenous proBDNF protein on PWT by injection of proBDNF protein into the plantar (*P
    Figure Legend Snippet: Exogenous proBDNF induces pain hypersensitivity and spinal cord activation in mice. ( A ) Dosage effect of exogenous proBDNF protein on PWT by injection of proBDNF protein into the plantar (*P

    Techniques Used: Activation Assay, Mouse Assay, Injection

    Polyclonal Ab-proBDNF pretreatment attenuates inflammatory pain in mice. ( A,B ) proBDNF polyclonal antibody (5 ml/Kg) i.p pretreatment attenuated both phases of nociceptive responses induced by 5% formalin intra-plantar injection in Kunming mice. ( A ) Time course of biphasic nociceptive response (*P
    Figure Legend Snippet: Polyclonal Ab-proBDNF pretreatment attenuates inflammatory pain in mice. ( A,B ) proBDNF polyclonal antibody (5 ml/Kg) i.p pretreatment attenuated both phases of nociceptive responses induced by 5% formalin intra-plantar injection in Kunming mice. ( A ) Time course of biphasic nociceptive response (*P

    Techniques Used: Mouse Assay, Injection

    Characterization of proBDNF monoclonal antibody 2B11. ( A ) ELISA assay for the immunoreactivity of 2B11 against human proBDNF prodomain, and human, rat and mice proBDNF proteins, and human mature BDNF (mBDNF). 2B11 has strong immunoreactivity against proBDNF and prodomain, but not mBDNF; ( B ) Representative Western blot of human proBDNF and mBDNF detected by 2B11 (dilution 1:2000), note that 2B11 specifically recognizes proBDNF, but not mBDNF. ( C ) Representative images of neurosphere radiant migration treated by proBDNF, mBDNF, sheep polyclonal anti-proBDNF antibody, mouse monoclonal anti-proBDNF antibody 2B11 and co-treatment. ( D ) Statistical analysis of neurosphere migration radiance assay (***P
    Figure Legend Snippet: Characterization of proBDNF monoclonal antibody 2B11. ( A ) ELISA assay for the immunoreactivity of 2B11 against human proBDNF prodomain, and human, rat and mice proBDNF proteins, and human mature BDNF (mBDNF). 2B11 has strong immunoreactivity against proBDNF and prodomain, but not mBDNF; ( B ) Representative Western blot of human proBDNF and mBDNF detected by 2B11 (dilution 1:2000), note that 2B11 specifically recognizes proBDNF, but not mBDNF. ( C ) Representative images of neurosphere radiant migration treated by proBDNF, mBDNF, sheep polyclonal anti-proBDNF antibody, mouse monoclonal anti-proBDNF antibody 2B11 and co-treatment. ( D ) Statistical analysis of neurosphere migration radiance assay (***P

    Techniques Used: Enzyme-linked Immunosorbent Assay, Mouse Assay, Western Blot, Migration

    Upregulation of p75NTR and effect of local proBDNF injection on inflammatory reaction in mice. ( A ) Representative Western blot of p75NTR and sortilin ( a ) and the semi-quantitative analysis of their expression (b and c) after formalin intra-plantar injection (***p
    Figure Legend Snippet: Upregulation of p75NTR and effect of local proBDNF injection on inflammatory reaction in mice. ( A ) Representative Western blot of p75NTR and sortilin ( a ) and the semi-quantitative analysis of their expression (b and c) after formalin intra-plantar injection (***p

    Techniques Used: Injection, Mouse Assay, Western Blot, Expressing

    Upregulation of proBDNF in the local tissue in acute and persistent inflammatory pain in mice. ( A ) Representative Western blot (a) and their semi-quantitative analyses of mature BDNF (b), proBDNF (c) and their ratio (d) in the local tissue after 10 μL 5% formalin intra-plantar injection into Kunming mice (*p
    Figure Legend Snippet: Upregulation of proBDNF in the local tissue in acute and persistent inflammatory pain in mice. ( A ) Representative Western blot (a) and their semi-quantitative analyses of mature BDNF (b), proBDNF (c) and their ratio (d) in the local tissue after 10 μL 5% formalin intra-plantar injection into Kunming mice (*p

    Techniques Used: Mouse Assay, Western Blot, Injection

    3) Product Images from "BDNF–TrkB signaling in the nucleus accumbens shell of mice has key role in methamphetamine withdrawal symptoms"

    Article Title: BDNF–TrkB signaling in the nucleus accumbens shell of mice has key role in methamphetamine withdrawal symptoms

    Journal: Translational Psychiatry

    doi: 10.1038/tp.2015.157

    Depression-like behavior and levels of proBDNF and BDNF in the brain regions after withdrawal from repeated METH exposure. ( a ) Schedule of treatment and behavioral tests. Saline (10 ml kg −1 per day for 5 days) or METH (3 mg kg −1 per day for 5 days) was injected into mice. Behavioral tests were performed at days 7, 11 and 18 (SPT), and days 8, 12 and 19 (LMT, TST, FST). ( b ) SPT: sucrose preference of METH-treated mice was significantly lower than that of control (saline-treated) mice. LMT: there were no differences between control and METH-treated mice. TST and FST: the immobility time of METH-treated mice was significantly higher than that of control mice. * P
    Figure Legend Snippet: Depression-like behavior and levels of proBDNF and BDNF in the brain regions after withdrawal from repeated METH exposure. ( a ) Schedule of treatment and behavioral tests. Saline (10 ml kg −1 per day for 5 days) or METH (3 mg kg −1 per day for 5 days) was injected into mice. Behavioral tests were performed at days 7, 11 and 18 (SPT), and days 8, 12 and 19 (LMT, TST, FST). ( b ) SPT: sucrose preference of METH-treated mice was significantly lower than that of control (saline-treated) mice. LMT: there were no differences between control and METH-treated mice. TST and FST: the immobility time of METH-treated mice was significantly higher than that of control mice. * P

    Techniques Used: Injection, Mouse Assay, Single-particle Tracking

    4) Product Images from "The decline in synaptic GluN2B and rise in inhibitory neurotransmission determine the end of a critical period"

    Article Title: The decline in synaptic GluN2B and rise in inhibitory neurotransmission determine the end of a critical period

    Journal: Scientific Reports

    doi: 10.1038/srep34196

    CS synapses in the ventral spinal cord were eliminated upon increasing synaptic 2B. ( A ) Reduction of CS synaptic activity in the ventral side after the critical period in 2a −/− mice. Spatial distribution of CS synapses determined using optical CS-EPSPs in WT, proBDNF-treated or 2a −/− mice at 11–13 or 14–16 DIV. Scale bar = 250 μm. ( B ) Ventrodorsal ratios of optical CS-EPSPs in the indicated culture pairs from 11–13 DIV (0.40 ± 0.03 in WT mice, n = 12, Ns = 12, Nm = 6; 0.42 ± 0.03 in 2a −/− mice, n = 6, Ns = 6, Nm = 6) or from 14–16 DIV (0.46 ± 0.02 in WT mice, n = 17, Ns = 17, Nm = 8; 0.39 ± 0.02 in proBDNF-treated mice, n = 4, Ns = 4, Nm = 4; 0.31 ± 0.03 in 2a −/− mice, n = 7, Ns = 7, Nm = 6).
    Figure Legend Snippet: CS synapses in the ventral spinal cord were eliminated upon increasing synaptic 2B. ( A ) Reduction of CS synaptic activity in the ventral side after the critical period in 2a −/− mice. Spatial distribution of CS synapses determined using optical CS-EPSPs in WT, proBDNF-treated or 2a −/− mice at 11–13 or 14–16 DIV. Scale bar = 250 μm. ( B ) Ventrodorsal ratios of optical CS-EPSPs in the indicated culture pairs from 11–13 DIV (0.40 ± 0.03 in WT mice, n = 12, Ns = 12, Nm = 6; 0.42 ± 0.03 in 2a −/− mice, n = 6, Ns = 6, Nm = 6) or from 14–16 DIV (0.46 ± 0.02 in WT mice, n = 17, Ns = 17, Nm = 8; 0.39 ± 0.02 in proBDNF-treated mice, n = 4, Ns = 4, Nm = 4; 0.31 ± 0.03 in 2a −/− mice, n = 7, Ns = 7, Nm = 6).

    Techniques Used: Activity Assay, Mouse Assay

    Decline in 2B-containing NMDAR across the end of the critical period. ( A ) Developmental alterations of the total NMDA component of CS-EPSCs in the spinal cord. Averaged CS-EPSC traces recorded after application of NBQX to WT ( upper ) or proBDNF-treated ( middle ) and 2a −/− ( lower ) mice before ( black ) or after ( red ) Ro25-6981 treatment on 12 DIV. Calibration: 50 pA, 100ms. ( B ) Averaged 2B-CS-EPSCs in WT (55.0 ± 14.6 pA, 6–8 DIV, n = 21, Ns = 16, Nm = 32; 18.0 ± 1.99 pA, 12–15 DIV, n = 18, Ns = 10, Nm = 20), proBDNF-treated (44.1 ± 6.45 pA, 12–15 DIV, n = 13, Ns = 8, Nm = 16) and 2a −/− mice (54.2 ± 10.8 pA, 6–8 DIV, n = 7, Ns = 7, Nm = 14; 74.4 ± 30.1 pA, 12–15 DIV, n = 7, Ns = 6, Nm = 12). ( C ) Developmental alterations of synaptic 2B in WT or 2a −/− mice. Immunoblot analysis of synaptic expression of 2B (2B in SPMs), PSD-95 (in SPMs) and MAGUKs (in SPMs) in WT and 2a −/− mice. Full-length blots are presented in Supplementary Fig. 7S . (D) Ratio of 2B to PSD-95 or MAGUKs in SPMs: To quantitate synaptic 2B, the intensity of 2B in SPMs was normalized to that of PSD-95 or MAGUK in SPMs in WT or 2a −/− spinal cords (Ratio of 2B to PSD-95: 1.00, 6 DIV in WT mice; 0.44, 13 DIV in WT mice; 0.62, 6 DIV in 2a −/− mice; 0.66, 13 DIV in 2a −/− mice. Ratio of 2B to MAGUKs: 1.00, 6 DIV in WT mice; 0.48, 13 DIV in WT mice; 0.75, 6 DIV in 2a −/− mice; 0.66, 13 DIV in 2a −/− mice. Each sample represents an SPM extract prepared from 20 co-cultures from 4 mice). Data are represented as the mean ± standard error of the mean. Asterisks indicate statistical significance (Student t test). * p
    Figure Legend Snippet: Decline in 2B-containing NMDAR across the end of the critical period. ( A ) Developmental alterations of the total NMDA component of CS-EPSCs in the spinal cord. Averaged CS-EPSC traces recorded after application of NBQX to WT ( upper ) or proBDNF-treated ( middle ) and 2a −/− ( lower ) mice before ( black ) or after ( red ) Ro25-6981 treatment on 12 DIV. Calibration: 50 pA, 100ms. ( B ) Averaged 2B-CS-EPSCs in WT (55.0 ± 14.6 pA, 6–8 DIV, n = 21, Ns = 16, Nm = 32; 18.0 ± 1.99 pA, 12–15 DIV, n = 18, Ns = 10, Nm = 20), proBDNF-treated (44.1 ± 6.45 pA, 12–15 DIV, n = 13, Ns = 8, Nm = 16) and 2a −/− mice (54.2 ± 10.8 pA, 6–8 DIV, n = 7, Ns = 7, Nm = 14; 74.4 ± 30.1 pA, 12–15 DIV, n = 7, Ns = 6, Nm = 12). ( C ) Developmental alterations of synaptic 2B in WT or 2a −/− mice. Immunoblot analysis of synaptic expression of 2B (2B in SPMs), PSD-95 (in SPMs) and MAGUKs (in SPMs) in WT and 2a −/− mice. Full-length blots are presented in Supplementary Fig. 7S . (D) Ratio of 2B to PSD-95 or MAGUKs in SPMs: To quantitate synaptic 2B, the intensity of 2B in SPMs was normalized to that of PSD-95 or MAGUK in SPMs in WT or 2a −/− spinal cords (Ratio of 2B to PSD-95: 1.00, 6 DIV in WT mice; 0.44, 13 DIV in WT mice; 0.62, 6 DIV in 2a −/− mice; 0.66, 13 DIV in 2a −/− mice. Ratio of 2B to MAGUKs: 1.00, 6 DIV in WT mice; 0.48, 13 DIV in WT mice; 0.75, 6 DIV in 2a −/− mice; 0.66, 13 DIV in 2a −/− mice. Each sample represents an SPM extract prepared from 20 co-cultures from 4 mice). Data are represented as the mean ± standard error of the mean. Asterisks indicate statistical significance (Student t test). * p

    Techniques Used: Mouse Assay, Expressing

    CS axons in the ventral spinal cord regressed upon increasing synaptic 2B. ( A ) Images of CS axons labeled with ChR2-EYFP in WT ( left ), proBDNF-treated and 2a −/− spinal explants ( right ). The first image was taken at 12 DIV and the second at 15 or 16 DIV. Enumeration of CS axons on the ventral side that crossed the 70% line ( red bold line ) from the dorsal to the ventral edge of spinal gray matter. Scale bar = 250 µm. ( B ) Ratios of CS axons on the ventral side at 15 or 16 DIV to that at 12 DIV (15 DIV/12 DIV: 1.13 ± 0.06 in WT mice, n = 9, Ns = 9, Nm = 4; 16 DIV/12 DIV: 0.60 ± 0.05 in proBDNF-treated mice, n = 11, Ns = 11, Nm = 6; 0.81 ± 0.05 in 2a −/− mice, n = 11, Ns = 11, Nm = 8) ( right ).
    Figure Legend Snippet: CS axons in the ventral spinal cord regressed upon increasing synaptic 2B. ( A ) Images of CS axons labeled with ChR2-EYFP in WT ( left ), proBDNF-treated and 2a −/− spinal explants ( right ). The first image was taken at 12 DIV and the second at 15 or 16 DIV. Enumeration of CS axons on the ventral side that crossed the 70% line ( red bold line ) from the dorsal to the ventral edge of spinal gray matter. Scale bar = 250 µm. ( B ) Ratios of CS axons on the ventral side at 15 or 16 DIV to that at 12 DIV (15 DIV/12 DIV: 1.13 ± 0.06 in WT mice, n = 9, Ns = 9, Nm = 4; 16 DIV/12 DIV: 0.60 ± 0.05 in proBDNF-treated mice, n = 11, Ns = 11, Nm = 6; 0.81 ± 0.05 in 2a −/− mice, n = 11, Ns = 11, Nm = 8) ( right ).

    Techniques Used: Labeling, Mouse Assay

    5) Product Images from "HBpF-proBDNF: A New Tool for the Analysis of Pro-Brain Derived Neurotrophic Factor Receptor Signaling and Cell Biology"

    Article Title: HBpF-proBDNF: A New Tool for the Analysis of Pro-Brain Derived Neurotrophic Factor Receptor Signaling and Cell Biology

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0150601

    Endocytosis of HBpF-proBDNF in Hippocampal neurons. Primary hippocampal neurons cells were exposed to HBpF-proBDNF (250 ng/ml) conjugated to Streptavidin-Cy3 for 1h to 6h. After fixation and mounting, cells were analyzed by fluorescent microscopy. Hoechst staining was used as a nuclear marker. Quantification of fluorescence intensity was performed using ImageJ software on three independent experiments. 100 cells were measured for each experiment. (unpaired two-tailed t -test, * indicates a p-value
    Figure Legend Snippet: Endocytosis of HBpF-proBDNF in Hippocampal neurons. Primary hippocampal neurons cells were exposed to HBpF-proBDNF (250 ng/ml) conjugated to Streptavidin-Cy3 for 1h to 6h. After fixation and mounting, cells were analyzed by fluorescent microscopy. Hoechst staining was used as a nuclear marker. Quantification of fluorescence intensity was performed using ImageJ software on three independent experiments. 100 cells were measured for each experiment. (unpaired two-tailed t -test, * indicates a p-value

    Techniques Used: Microscopy, Staining, Marker, Fluorescence, Software, Two Tailed Test

    HBpF-proBDNF inhibits carbachol (CCh)-induced persistent firing in cortical pyramidal neurons. A. Representative trace of current-clamp recording from pyramidal neuron in layer V of the entorhinal cortex. Slices were perfused with 10μM CCh and the persistent activity was produced by a short depolarization (1s, 100pA). HBpF-proBDNF at 2ng/ml was next added in presence of 10μM CCh during 10 minutes (first cut in the trace) and cells were stimulated. HBpF-proBDNF was removed by perfusing a solution containing only 10μM CCh for 10 minutes (second cut in the trace) and before the stimulation of the cells. B. Quantification of the plateau amplitude and frequency of the persistent activity (unpaired two-tailed t -test, * indicates a p-value
    Figure Legend Snippet: HBpF-proBDNF inhibits carbachol (CCh)-induced persistent firing in cortical pyramidal neurons. A. Representative trace of current-clamp recording from pyramidal neuron in layer V of the entorhinal cortex. Slices were perfused with 10μM CCh and the persistent activity was produced by a short depolarization (1s, 100pA). HBpF-proBDNF at 2ng/ml was next added in presence of 10μM CCh during 10 minutes (first cut in the trace) and cells were stimulated. HBpF-proBDNF was removed by perfusing a solution containing only 10μM CCh for 10 minutes (second cut in the trace) and before the stimulation of the cells. B. Quantification of the plateau amplitude and frequency of the persistent activity (unpaired two-tailed t -test, * indicates a p-value

    Techniques Used: Activity Assay, Produced, Two Tailed Test

    HBpF-proBDNF can be isolated by a modified tandem affinity purification protocol. A. HEK293T cells were transfected with p75NTR, Sortilin, HBpF-proBDNF, and BirA expression plasmid as indicated. 48h after transfection, HEK293T cells were lysed (input) and pulled-down on Ni-NTA beads. The Ni-NTA eluate was then pulled-down on SA beads and then cleaved by PP overnight (PP eluate). Samples were analyzed by immunoblotting for p75NTR, sortilin, biotin and the Flag tag.
    Figure Legend Snippet: HBpF-proBDNF can be isolated by a modified tandem affinity purification protocol. A. HEK293T cells were transfected with p75NTR, Sortilin, HBpF-proBDNF, and BirA expression plasmid as indicated. 48h after transfection, HEK293T cells were lysed (input) and pulled-down on Ni-NTA beads. The Ni-NTA eluate was then pulled-down on SA beads and then cleaved by PP overnight (PP eluate). Samples were analyzed by immunoblotting for p75NTR, sortilin, biotin and the Flag tag.

    Techniques Used: Isolation, Modification, Affinity Purification, Transfection, Expressing, Plasmid Preparation, FLAG-tag

    HBpF-proBDNF induces growth cone collapse. Following 2 days in culture, hippocampal neuronal culture were stimulated with different concentrations of HBpF-proBDNF or proBDNF (25ng/ml and 100ng/ml) for 1h. Ni-NTA eluate from cells expressing only BirA was used as a negative control. Cells were then fixed and immunostained against beta-III-tubulin (Tuj-1) and phalloidin (scale bar = 10μm). Quantification of growth cone-collapse was done on three independent experiments and 50 growth-cones were counted for each experiment (unpaired two-tailed t -test, * indicates a p-value
    Figure Legend Snippet: HBpF-proBDNF induces growth cone collapse. Following 2 days in culture, hippocampal neuronal culture were stimulated with different concentrations of HBpF-proBDNF or proBDNF (25ng/ml and 100ng/ml) for 1h. Ni-NTA eluate from cells expressing only BirA was used as a negative control. Cells were then fixed and immunostained against beta-III-tubulin (Tuj-1) and phalloidin (scale bar = 10μm). Quantification of growth cone-collapse was done on three independent experiments and 50 growth-cones were counted for each experiment (unpaired two-tailed t -test, * indicates a p-value

    Techniques Used: Expressing, Negative Control, Two Tailed Test

    HBpF-proBDNF design and production. A. Schematic representation of recombinant HBpF-proBDNF protein. HBpF-proBDNF contains a signal peptide, an amino-terminal 6xHis-tag, followed by a Biotin-Acceptor-Peptide (BAP) sequence, a linker (L), a PreScission ™ Protease (PP) cleavage site and a Flag-tag (Flag). The ProBDNF sequence has been mutated with a KR to AA mutation at the furin dibasic cleavage site between the pro-domain and the mature part of BDNF. B. HEK293T cells were transfected with HBpF-proBDNF and BirA plasmids. After Ni-NTA pulldown and cleavage with the PreScission ™ Protease, the eluates were analyzed by Western blot and blotted with anti-biotin, anti-Flag and anti-BDNF. C. Purified HBpF-proBDNF was incubated with PC12 cells lysates, without a protease inhibitors, for 1h at room temperature. Incubation of HBpF-proBDNF with lysis buffer for 1h at room temperature was used as a control. Immunoblots against BDNF were performed to visualize the degradation of HBpF-proBDNF.
    Figure Legend Snippet: HBpF-proBDNF design and production. A. Schematic representation of recombinant HBpF-proBDNF protein. HBpF-proBDNF contains a signal peptide, an amino-terminal 6xHis-tag, followed by a Biotin-Acceptor-Peptide (BAP) sequence, a linker (L), a PreScission ™ Protease (PP) cleavage site and a Flag-tag (Flag). The ProBDNF sequence has been mutated with a KR to AA mutation at the furin dibasic cleavage site between the pro-domain and the mature part of BDNF. B. HEK293T cells were transfected with HBpF-proBDNF and BirA plasmids. After Ni-NTA pulldown and cleavage with the PreScission ™ Protease, the eluates were analyzed by Western blot and blotted with anti-biotin, anti-Flag and anti-BDNF. C. Purified HBpF-proBDNF was incubated with PC12 cells lysates, without a protease inhibitors, for 1h at room temperature. Incubation of HBpF-proBDNF with lysis buffer for 1h at room temperature was used as a control. Immunoblots against BDNF were performed to visualize the degradation of HBpF-proBDNF.

    Techniques Used: Recombinant, Sequencing, FLAG-tag, Mutagenesis, Transfection, Western Blot, Purification, Incubation, Lysis

    HBpF-proBDNF interacts with endogenous p75NTR and SorCS2 in PC12 cells. PC12 cells were stimulated with HBpF-proBDNF (100μg/ml) with or without 9μM GM6001 for 3h. BirA Ni-NTA eluate was used as control treatment. After stimulation, cells were lysed and HBpF-proBDNF and associated protein were recovered on SA beads. Cleavage with PP was performed for 16 hours and the resulting PP eluate was collected. Cell lysates (Input) and PP eluate samples were then analyzed by immunoblotting for p75NTR, SorCS2 and FLAG.
    Figure Legend Snippet: HBpF-proBDNF interacts with endogenous p75NTR and SorCS2 in PC12 cells. PC12 cells were stimulated with HBpF-proBDNF (100μg/ml) with or without 9μM GM6001 for 3h. BirA Ni-NTA eluate was used as control treatment. After stimulation, cells were lysed and HBpF-proBDNF and associated protein were recovered on SA beads. Cleavage with PP was performed for 16 hours and the resulting PP eluate was collected. Cell lysates (Input) and PP eluate samples were then analyzed by immunoblotting for p75NTR, SorCS2 and FLAG.

    Techniques Used:

    HBpF-proBDNF does not activate TrkB receptors. A. Following 2 days in culture, cerebellar granule neurons were stimulated with different concentrations of HBpF-proBDNF (2ng/ml; 25ng/ml and 100ng/ml) or proBDNF (2ng/ml and 25ng/ml) for 30min. After the incubation time, cells were lysed immediately in sample buffer and analyzed by Western blot against phospho-Trk and TrkB. For positive and negative controls, CGN were treated with BDNF (25ng/ml) or with BirA Ni-NTA eluate (BirA), as indicated.
    Figure Legend Snippet: HBpF-proBDNF does not activate TrkB receptors. A. Following 2 days in culture, cerebellar granule neurons were stimulated with different concentrations of HBpF-proBDNF (2ng/ml; 25ng/ml and 100ng/ml) or proBDNF (2ng/ml and 25ng/ml) for 30min. After the incubation time, cells were lysed immediately in sample buffer and analyzed by Western blot against phospho-Trk and TrkB. For positive and negative controls, CGN were treated with BDNF (25ng/ml) or with BirA Ni-NTA eluate (BirA), as indicated.

    Techniques Used: Incubation, Western Blot

    6) Product Images from "Dexmedetomidine Attenuates Neurotoxicity in Developing Rats Induced by Sevoflurane through Upregulating BDNF-TrkB-CREB and Downregulating ProBDNF-P75NRT-RhoA Signaling Pathway"

    Article Title: Dexmedetomidine Attenuates Neurotoxicity in Developing Rats Induced by Sevoflurane through Upregulating BDNF-TrkB-CREB and Downregulating ProBDNF-P75NRT-RhoA Signaling Pathway

    Journal: Mediators of Inflammation

    doi: 10.1155/2020/5458061

    Dexmedetomidine could decrease the level of proBDNF and restore the ratio of proBDNF/mBDNF and alleviates activation of the proBDNF-P75NRT-RHOA pathway after sevoflurane. (a) Western blot band. (b) ProBDNF/mBDNF. (c–e) Bar graph of Western blot. (f) Immunofluorescence of proBDNF (scale bar = 50 μ m). ∗ Compare with the control group, P
    Figure Legend Snippet: Dexmedetomidine could decrease the level of proBDNF and restore the ratio of proBDNF/mBDNF and alleviates activation of the proBDNF-P75NRT-RHOA pathway after sevoflurane. (a) Western blot band. (b) ProBDNF/mBDNF. (c–e) Bar graph of Western blot. (f) Immunofluorescence of proBDNF (scale bar = 50 μ m). ∗ Compare with the control group, P

    Techniques Used: Activation Assay, Western Blot, Immunofluorescence

    7) Product Images from "Platelets Selectively Regulate the Release of BDNF, But Not That of Its Precursor Protein, proBDNF"

    Article Title: Platelets Selectively Regulate the Release of BDNF, But Not That of Its Precursor Protein, proBDNF

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2020.575607

    Molar concentrations of proBDNF are lower in platelets and higher in plasma than those of BDNF. ELISA quantification of proBDNF and BDNF levels in the intraplatelet (A, B) and plasma (C, D) compartments. Concentrations are normalized for 250 x 10 6 platelets. Horizontal bar represents median, *p
    Figure Legend Snippet: Molar concentrations of proBDNF are lower in platelets and higher in plasma than those of BDNF. ELISA quantification of proBDNF and BDNF levels in the intraplatelet (A, B) and plasma (C, D) compartments. Concentrations are normalized for 250 x 10 6 platelets. Horizontal bar represents median, *p

    Techniques Used: Enzyme-linked Immunosorbent Assay

    Human platelets contain proBDNF. (A) ProBDNF immunoblotting of human washed platelet lysates (15 µg) from six different healthy volunteers. Recombinant proBDNF (3 ng) and human cortex lysate (3 µg) were used as positive controls. Molecular weight is indicated on the left (kDa) and primary antibody on the right. Experiments representative of n=9 for R-176 and n=10 for mab31751 antibody. IB, immunoblotting. (B) Immunoblotting of proBDNF and BDNF in different fractions of washed human platelets. P-Selectin was used as control protein in the membrane fraction, p65 NF-ĸB was used as control protein in the cytosol, and α-tubulin was used as control protein in the cytoskeleton. The equivalent of the protein content of 3 x 10 7 platelets was loaded for each fraction on the gel. Representative experiment of n=4 different volunteers. (C) ProBDNF treatment with PNGase F in washed human platelet lysates. U87-MG glioblastoma cells were used as a control. rProBDNF, recombinant proBDNF (3 ng); cortex, human cortex lysate (3 µg); platelets, whole human platelet lysate (representative experiment of n=4 different volunteers, 7.5 x 10 8 platelets per well); −PNGF, platelets treated with GlycoBuffer, and incubated at 37°C for 60 min without PNGase F; +PNGF, platelets treated with GlycoBuffer and incubated at 37°C for 60 min with PNGase F; PNGF alone, PNGase F incubated at 37°C for 60 min without platelet lysate. CD42b and sortilin were used as controls of protein deglycosylation in platelets and in U87-MG cells, respectively. n=3 different volunteers for PNGase treatments in human platelets and n=4 independent experiments for U87-MG cells. (D) Representative flow cytometry experiment showing surface and intracellular proBDNF in human washed platelets and in U87-MG and U251-MG glioblastoma cell lines. Mouse IgG 2b was used as isotype control. Percentage of expression are indicated on the figure. n=10 different healthy volunteers for human platelets; n=3 independent experiments for each glioblastoma cell line. (E) Confocal microscopy imaging of proBDNF in human permeabilized washed platelets (top) and in permeabilized U-251 MG cells (bottom). Mouse IgG 2b was used as isotype control. ProBDNF was labelled using Alexa488 fluorochrome (in green). Nuclei were stained with DAPI (in blue). Scale bar = 2 µm and 200 µm for washed platelets and U-251 MG cells images, respectively. (F) Immunoblotting of α 2 -macroglobulin at increasing quantities of loaded proteins (1–20 µg) obtained from a washed platelet lysate or platelet-poor plasma (PPP) from the same individual (#23). α-tubulin was used as loading control. Molecular weight is indicated on the left (kDa) and primary antibody on the right. PPP, platelet poor plasma; PLTs, platelets; IB, immunoblotting.
    Figure Legend Snippet: Human platelets contain proBDNF. (A) ProBDNF immunoblotting of human washed platelet lysates (15 µg) from six different healthy volunteers. Recombinant proBDNF (3 ng) and human cortex lysate (3 µg) were used as positive controls. Molecular weight is indicated on the left (kDa) and primary antibody on the right. Experiments representative of n=9 for R-176 and n=10 for mab31751 antibody. IB, immunoblotting. (B) Immunoblotting of proBDNF and BDNF in different fractions of washed human platelets. P-Selectin was used as control protein in the membrane fraction, p65 NF-ĸB was used as control protein in the cytosol, and α-tubulin was used as control protein in the cytoskeleton. The equivalent of the protein content of 3 x 10 7 platelets was loaded for each fraction on the gel. Representative experiment of n=4 different volunteers. (C) ProBDNF treatment with PNGase F in washed human platelet lysates. U87-MG glioblastoma cells were used as a control. rProBDNF, recombinant proBDNF (3 ng); cortex, human cortex lysate (3 µg); platelets, whole human platelet lysate (representative experiment of n=4 different volunteers, 7.5 x 10 8 platelets per well); −PNGF, platelets treated with GlycoBuffer, and incubated at 37°C for 60 min without PNGase F; +PNGF, platelets treated with GlycoBuffer and incubated at 37°C for 60 min with PNGase F; PNGF alone, PNGase F incubated at 37°C for 60 min without platelet lysate. CD42b and sortilin were used as controls of protein deglycosylation in platelets and in U87-MG cells, respectively. n=3 different volunteers for PNGase treatments in human platelets and n=4 independent experiments for U87-MG cells. (D) Representative flow cytometry experiment showing surface and intracellular proBDNF in human washed platelets and in U87-MG and U251-MG glioblastoma cell lines. Mouse IgG 2b was used as isotype control. Percentage of expression are indicated on the figure. n=10 different healthy volunteers for human platelets; n=3 independent experiments for each glioblastoma cell line. (E) Confocal microscopy imaging of proBDNF in human permeabilized washed platelets (top) and in permeabilized U-251 MG cells (bottom). Mouse IgG 2b was used as isotype control. ProBDNF was labelled using Alexa488 fluorochrome (in green). Nuclei were stained with DAPI (in blue). Scale bar = 2 µm and 200 µm for washed platelets and U-251 MG cells images, respectively. (F) Immunoblotting of α 2 -macroglobulin at increasing quantities of loaded proteins (1–20 µg) obtained from a washed platelet lysate or platelet-poor plasma (PPP) from the same individual (#23). α-tubulin was used as loading control. Molecular weight is indicated on the left (kDa) and primary antibody on the right. PPP, platelet poor plasma; PLTs, platelets; IB, immunoblotting.

    Techniques Used: Recombinant, Molecular Weight, Incubation, Flow Cytometry, Expressing, Confocal Microscopy, Imaging, Staining

    Unlike BDNF, intraplatelet proBDNF is not released during platelet activation. Intraplatelet (A, D) and plasma (B, E) concentrations of BDNF and proBDNF following platelet activation by different agonists. Intraplatelet concentrations are normalized for 250 x 10 6 platelets. Proportion of BDNF (C) and proBDNF (F) in plasma vs . in platelets are expressed in percentage. Error bar represents IQR, *p
    Figure Legend Snippet: Unlike BDNF, intraplatelet proBDNF is not released during platelet activation. Intraplatelet (A, D) and plasma (B, E) concentrations of BDNF and proBDNF following platelet activation by different agonists. Intraplatelet concentrations are normalized for 250 x 10 6 platelets. Proportion of BDNF (C) and proBDNF (F) in plasma vs . in platelets are expressed in percentage. Error bar represents IQR, *p

    Techniques Used: Activation Assay

    8) Product Images from "HBpF-proBDNF: A New Tool for the Analysis of Pro-Brain Derived Neurotrophic Factor Receptor Signaling and Cell Biology"

    Article Title: HBpF-proBDNF: A New Tool for the Analysis of Pro-Brain Derived Neurotrophic Factor Receptor Signaling and Cell Biology

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0150601

    Endocytosis of HBpF-proBDNF in Hippocampal neurons. Primary hippocampal neurons cells were exposed to HBpF-proBDNF (250 ng/ml) conjugated to Streptavidin-Cy3 for 1h to 6h. After fixation and mounting, cells were analyzed by fluorescent microscopy. Hoechst staining was used as a nuclear marker. Quantification of fluorescence intensity was performed using ImageJ software on three independent experiments. 100 cells were measured for each experiment. (unpaired two-tailed t -test, * indicates a p-value
    Figure Legend Snippet: Endocytosis of HBpF-proBDNF in Hippocampal neurons. Primary hippocampal neurons cells were exposed to HBpF-proBDNF (250 ng/ml) conjugated to Streptavidin-Cy3 for 1h to 6h. After fixation and mounting, cells were analyzed by fluorescent microscopy. Hoechst staining was used as a nuclear marker. Quantification of fluorescence intensity was performed using ImageJ software on three independent experiments. 100 cells were measured for each experiment. (unpaired two-tailed t -test, * indicates a p-value

    Techniques Used: Microscopy, Staining, Marker, Fluorescence, Software, Two Tailed Test

    HBpF-proBDNF inhibits carbachol (CCh)-induced persistent firing in cortical pyramidal neurons. A. Representative trace of current-clamp recording from pyramidal neuron in layer V of the entorhinal cortex. Slices were perfused with 10μM CCh and the persistent activity was produced by a short depolarization (1s, 100pA). HBpF-proBDNF at 2ng/ml was next added in presence of 10μM CCh during 10 minutes (first cut in the trace) and cells were stimulated. HBpF-proBDNF was removed by perfusing a solution containing only 10μM CCh for 10 minutes (second cut in the trace) and before the stimulation of the cells. B. Quantification of the plateau amplitude and frequency of the persistent activity (unpaired two-tailed t -test, * indicates a p-value
    Figure Legend Snippet: HBpF-proBDNF inhibits carbachol (CCh)-induced persistent firing in cortical pyramidal neurons. A. Representative trace of current-clamp recording from pyramidal neuron in layer V of the entorhinal cortex. Slices were perfused with 10μM CCh and the persistent activity was produced by a short depolarization (1s, 100pA). HBpF-proBDNF at 2ng/ml was next added in presence of 10μM CCh during 10 minutes (first cut in the trace) and cells were stimulated. HBpF-proBDNF was removed by perfusing a solution containing only 10μM CCh for 10 minutes (second cut in the trace) and before the stimulation of the cells. B. Quantification of the plateau amplitude and frequency of the persistent activity (unpaired two-tailed t -test, * indicates a p-value

    Techniques Used: Activity Assay, Produced, Two Tailed Test

    HBpF-proBDNF can be isolated by a modified tandem affinity purification protocol. A. HEK293T cells were transfected with p75NTR, Sortilin, HBpF-proBDNF, and BirA expression plasmid as indicated. 48h after transfection, HEK293T cells were lysed (input) and pulled-down on Ni-NTA beads. The Ni-NTA eluate was then pulled-down on SA beads and then cleaved by PP overnight (PP eluate). Samples were analyzed by immunoblotting for p75NTR, sortilin, biotin and the Flag tag.
    Figure Legend Snippet: HBpF-proBDNF can be isolated by a modified tandem affinity purification protocol. A. HEK293T cells were transfected with p75NTR, Sortilin, HBpF-proBDNF, and BirA expression plasmid as indicated. 48h after transfection, HEK293T cells were lysed (input) and pulled-down on Ni-NTA beads. The Ni-NTA eluate was then pulled-down on SA beads and then cleaved by PP overnight (PP eluate). Samples were analyzed by immunoblotting for p75NTR, sortilin, biotin and the Flag tag.

    Techniques Used: Isolation, Modification, Affinity Purification, Transfection, Expressing, Plasmid Preparation, FLAG-tag

    HBpF-proBDNF induces growth cone collapse. Following 2 days in culture, hippocampal neuronal culture were stimulated with different concentrations of HBpF-proBDNF or proBDNF (25ng/ml and 100ng/ml) for 1h. Ni-NTA eluate from cells expressing only BirA was used as a negative control. Cells were then fixed and immunostained against beta-III-tubulin (Tuj-1) and phalloidin (scale bar = 10μm). Quantification of growth cone-collapse was done on three independent experiments and 50 growth-cones were counted for each experiment (unpaired two-tailed t -test, * indicates a p-value
    Figure Legend Snippet: HBpF-proBDNF induces growth cone collapse. Following 2 days in culture, hippocampal neuronal culture were stimulated with different concentrations of HBpF-proBDNF or proBDNF (25ng/ml and 100ng/ml) for 1h. Ni-NTA eluate from cells expressing only BirA was used as a negative control. Cells were then fixed and immunostained against beta-III-tubulin (Tuj-1) and phalloidin (scale bar = 10μm). Quantification of growth cone-collapse was done on three independent experiments and 50 growth-cones were counted for each experiment (unpaired two-tailed t -test, * indicates a p-value

    Techniques Used: Expressing, Negative Control, Two Tailed Test

    HBpF-proBDNF design and production. A. Schematic representation of recombinant HBpF-proBDNF protein. HBpF-proBDNF contains a signal peptide, an amino-terminal 6xHis-tag, followed by a Biotin-Acceptor-Peptide (BAP) sequence, a linker (L), a PreScission ™ Protease (PP) cleavage site and a Flag-tag (Flag). The ProBDNF sequence has been mutated with a KR to AA mutation at the furin dibasic cleavage site between the pro-domain and the mature part of BDNF. B. HEK293T cells were transfected with HBpF-proBDNF and BirA plasmids. After Ni-NTA pulldown and cleavage with the PreScission ™ Protease, the eluates were analyzed by Western blot and blotted with anti-biotin, anti-Flag and anti-BDNF. C. Purified HBpF-proBDNF was incubated with PC12 cells lysates, without a protease inhibitors, for 1h at room temperature. Incubation of HBpF-proBDNF with lysis buffer for 1h at room temperature was used as a control. Immunoblots against BDNF were performed to visualize the degradation of HBpF-proBDNF.
    Figure Legend Snippet: HBpF-proBDNF design and production. A. Schematic representation of recombinant HBpF-proBDNF protein. HBpF-proBDNF contains a signal peptide, an amino-terminal 6xHis-tag, followed by a Biotin-Acceptor-Peptide (BAP) sequence, a linker (L), a PreScission ™ Protease (PP) cleavage site and a Flag-tag (Flag). The ProBDNF sequence has been mutated with a KR to AA mutation at the furin dibasic cleavage site between the pro-domain and the mature part of BDNF. B. HEK293T cells were transfected with HBpF-proBDNF and BirA plasmids. After Ni-NTA pulldown and cleavage with the PreScission ™ Protease, the eluates were analyzed by Western blot and blotted with anti-biotin, anti-Flag and anti-BDNF. C. Purified HBpF-proBDNF was incubated with PC12 cells lysates, without a protease inhibitors, for 1h at room temperature. Incubation of HBpF-proBDNF with lysis buffer for 1h at room temperature was used as a control. Immunoblots against BDNF were performed to visualize the degradation of HBpF-proBDNF.

    Techniques Used: Recombinant, Sequencing, FLAG-tag, Mutagenesis, Transfection, Western Blot, Purification, Incubation, Lysis

    HBpF-proBDNF interacts with endogenous p75NTR and SorCS2 in PC12 cells. PC12 cells were stimulated with HBpF-proBDNF (100μg/ml) with or without 9μM GM6001 for 3h. BirA Ni-NTA eluate was used as control treatment. After stimulation, cells were lysed and HBpF-proBDNF and associated protein were recovered on SA beads. Cleavage with PP was performed for 16 hours and the resulting PP eluate was collected. Cell lysates (Input) and PP eluate samples were then analyzed by immunoblotting for p75NTR, SorCS2 and FLAG.
    Figure Legend Snippet: HBpF-proBDNF interacts with endogenous p75NTR and SorCS2 in PC12 cells. PC12 cells were stimulated with HBpF-proBDNF (100μg/ml) with or without 9μM GM6001 for 3h. BirA Ni-NTA eluate was used as control treatment. After stimulation, cells were lysed and HBpF-proBDNF and associated protein were recovered on SA beads. Cleavage with PP was performed for 16 hours and the resulting PP eluate was collected. Cell lysates (Input) and PP eluate samples were then analyzed by immunoblotting for p75NTR, SorCS2 and FLAG.

    Techniques Used:

    HBpF-proBDNF does not activate TrkB receptors. A. Following 2 days in culture, cerebellar granule neurons were stimulated with different concentrations of HBpF-proBDNF (2ng/ml; 25ng/ml and 100ng/ml) or proBDNF (2ng/ml and 25ng/ml) for 30min. After the incubation time, cells were lysed immediately in sample buffer and analyzed by Western blot against phospho-Trk and TrkB. For positive and negative controls, CGN were treated with BDNF (25ng/ml) or with BirA Ni-NTA eluate (BirA), as indicated.
    Figure Legend Snippet: HBpF-proBDNF does not activate TrkB receptors. A. Following 2 days in culture, cerebellar granule neurons were stimulated with different concentrations of HBpF-proBDNF (2ng/ml; 25ng/ml and 100ng/ml) or proBDNF (2ng/ml and 25ng/ml) for 30min. After the incubation time, cells were lysed immediately in sample buffer and analyzed by Western blot against phospho-Trk and TrkB. For positive and negative controls, CGN were treated with BDNF (25ng/ml) or with BirA Ni-NTA eluate (BirA), as indicated.

    Techniques Used: Incubation, Western Blot

    9) Product Images from "Human Immunodeficiency Virus-1 Alters Brain-derived Neurotrophic Factor Processing in neurons"

    Article Title: Human Immunodeficiency Virus-1 Alters Brain-derived Neurotrophic Factor Processing in neurons

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    doi: 10.1523/JNEUROSCI.0865-12.2012

    Neuronal localization of proBDNF in human brains
    Figure Legend Snippet: Neuronal localization of proBDNF in human brains

    Techniques Used:

    pBDNF and proBDNF levels in HIV subjects
    Figure Legend Snippet: pBDNF and proBDNF levels in HIV subjects

    Techniques Used:

    gp120 alters the release of mBDNF/proBDNF in cortical neurons
    Figure Legend Snippet: gp120 alters the release of mBDNF/proBDNF in cortical neurons

    Techniques Used:

    gp120 increases proBDNF
    Figure Legend Snippet: gp120 increases proBDNF

    Techniques Used:

    10) Product Images from "p75 Neurotrophin Receptor Activation Regulates the Timing of the Maturation of Cortical Parvalbumin Interneuron Connectivity and Promotes Juvenile-like Plasticity in Adult Visual Cortex"

    Article Title: p75 Neurotrophin Receptor Activation Regulates the Timing of the Maturation of Cortical Parvalbumin Interneuron Connectivity and Promotes Juvenile-like Plasticity in Adult Visual Cortex

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.2881-18.2019

    proBNDF-mediated p75NTR activation in cortical PV cells reduces their perisomatic boutons. A , Experimental approach. B , The intensity of perisomatic PV immunostaining (green) is reduced in the binocular visual cortex ipsilateral to the minipump-releasing mut-proBDNF (Ipsi) compared with the contralateral cortex (Contra) in the same animal. On the other hand, perisomatic PV intensity in the ipsilateral cortex of PV_Cre;p75 flx/flx mice is similar to that observed in the contralateral, untreated cortex. C , Low ( C1 ) and high ( C2 ) magnification of PNN (red, WFA staining) enwrapping PV cells (green) shows a dramatic reduction in both PNN density and intensity in the visual cortex infused with mut-proBFNF. This effect is abolished in PV_Cre;p75 flx/flx mice. Scale bars: C1 , 100 μm; B , C2 , 10 μm. D , Quantification of the mean intensity of perisomatic PV-positive puncta in ipsilateral compared with contralateral cortex. I/C ratio is obtained for each animal and then averaged between different animals. Mean I/C ratio is significantly reduced in Mut-proBDNF-infused p75 Ctrl mice compared with Mut-proBDNF-infused PV_Cre;p75 flx/flx mice (unpaired t test, df = 8, t = 6.077, p = 0.0003). E , The ratio of mean PNN intensity around PV cells in ipsilateral versus contralateral cortex is significantly lower in p75 Ctrl than PV_Cre;p75 flx/flx mice infused with mut-proBDNF (unpaired t test, df = 8, t = 15.33, p
    Figure Legend Snippet: proBNDF-mediated p75NTR activation in cortical PV cells reduces their perisomatic boutons. A , Experimental approach. B , The intensity of perisomatic PV immunostaining (green) is reduced in the binocular visual cortex ipsilateral to the minipump-releasing mut-proBDNF (Ipsi) compared with the contralateral cortex (Contra) in the same animal. On the other hand, perisomatic PV intensity in the ipsilateral cortex of PV_Cre;p75 flx/flx mice is similar to that observed in the contralateral, untreated cortex. C , Low ( C1 ) and high ( C2 ) magnification of PNN (red, WFA staining) enwrapping PV cells (green) shows a dramatic reduction in both PNN density and intensity in the visual cortex infused with mut-proBFNF. This effect is abolished in PV_Cre;p75 flx/flx mice. Scale bars: C1 , 100 μm; B , C2 , 10 μm. D , Quantification of the mean intensity of perisomatic PV-positive puncta in ipsilateral compared with contralateral cortex. I/C ratio is obtained for each animal and then averaged between different animals. Mean I/C ratio is significantly reduced in Mut-proBDNF-infused p75 Ctrl mice compared with Mut-proBDNF-infused PV_Cre;p75 flx/flx mice (unpaired t test, df = 8, t = 6.077, p = 0.0003). E , The ratio of mean PNN intensity around PV cells in ipsilateral versus contralateral cortex is significantly lower in p75 Ctrl than PV_Cre;p75 flx/flx mice infused with mut-proBDNF (unpaired t test, df = 8, t = 15.33, p

    Techniques Used: Activation Assay, Immunostaining, Mouse Assay, Staining

    Modulation of tPA activity affects the formation of PV cell innervations during early postnatal development. A , Control EP18 PV cell ( A1 , green represents Ctrl). B , PV cell treated with the tPA inhibitor PPACK from EP10–EP18 shows simpler axonal arborization, contacting less potential targets ( B2 , blue represents NeuN-positive somata). C , PV cell treated with tPA in the same time window shows a very complex axonal arbor ( C2 ) and an increase in both terminal branching and perisomatic boutons ( C3 , arrowheads) compared with control cells ( A2 , A3 ). D , PV cell treated simultaneously with tPA and mut-proBDNF shows axonal branching and perisomatic innervation more similar to those formed by PV cell treated with mut-proBDNF alone, suggesting that the effects of tPA application may be mediated by a decrease in endogenous proBDNF/mBDNF ratio. Stars indicate NeuN-positive somata that are not innervated. Scale bars: A1–D1 , 50 μm; A2–D2 , 10 μm; A3–D3 , 5 μm. E , Perisomatic boutons density (one-way ANOVA, F (3,20) = 121.2, p
    Figure Legend Snippet: Modulation of tPA activity affects the formation of PV cell innervations during early postnatal development. A , Control EP18 PV cell ( A1 , green represents Ctrl). B , PV cell treated with the tPA inhibitor PPACK from EP10–EP18 shows simpler axonal arborization, contacting less potential targets ( B2 , blue represents NeuN-positive somata). C , PV cell treated with tPA in the same time window shows a very complex axonal arbor ( C2 ) and an increase in both terminal branching and perisomatic boutons ( C3 , arrowheads) compared with control cells ( A2 , A3 ). D , PV cell treated simultaneously with tPA and mut-proBDNF shows axonal branching and perisomatic innervation more similar to those formed by PV cell treated with mut-proBDNF alone, suggesting that the effects of tPA application may be mediated by a decrease in endogenous proBDNF/mBDNF ratio. Stars indicate NeuN-positive somata that are not innervated. Scale bars: A1–D1 , 50 μm; A2–D2 , 10 μm; A3–D3 , 5 μm. E , Perisomatic boutons density (one-way ANOVA, F (3,20) = 121.2, p

    Techniques Used: Activity Assay

    proBNDF-mediated p75NTR activation in cortical PV cells restores ocular dominance plasticity in adult visual cortex in vivo . A , Typical VEP responses to the stimulation of either contralateral (blue) or ipsilateral (red) eye to the cortex in which the recording is performed in p75NTR Ctrl mice infused with either vehicle or mut-proBDNF, and PV_Cre;p75NTR flx/flx mice infused with mut-proBDNF. Calibration bars: 50 μV, 100 ms. B , C/I VEP ratio mean values. Three days of monocular deprivation do not affect the C/I VEP ratio in adult mice, although it leads to a significant decrease in the C/I VEP ratio in animals treated with mut-proBDNF. Mut-proBDNF effects are, however, abolished in PV_Cre;p75 flx/flx mice (one-way ANOVA, F (2,18) = 8.903, p = 0.0021). p75NTR Ctrl + vehicle: n = 9 mice; p75NTR Ctrl + mut-proBDNF: n = 5 mice; PV_Cre;p75 flx/flx +mut-proBDNF: n = 7 mice. C , ODI of p75NTR Ctrl mice infused with vehicle solution and PV_Cre;p75 flx/flx mice infused with mut-proBDNF are not significantly different from those of undeprived animals, whereas ODIs in p75 Ctrl mice treated with mut-proBDNF are significantly shifted toward the open eye (one-way ANOVA, F (2,443) = 5.203, p = 0.0058). D , Mean spontaneous discharge is significantly increased only in p75 Ctrl mice treated with mut-proBDNF (one-way ANOVA, F (2,443) = 4.580, p = 0.0107). p75NTR Ctrl + vehicle: n = 9 mice, 174 cells; p75NTR Ctrl + mut-proBDNF: n = 5 mice, 147 cells; PV_Cre;p75 flx/flx +mut-proBDNF: n = 6 mice, 125 cells. Gray area represents the C/I VEP ratio ( B ) or the ODI range ( C ) (mean ± SEM) in adult nondeprived animals ( n = 5 mice, 99 cells). * indicate p
    Figure Legend Snippet: proBNDF-mediated p75NTR activation in cortical PV cells restores ocular dominance plasticity in adult visual cortex in vivo . A , Typical VEP responses to the stimulation of either contralateral (blue) or ipsilateral (red) eye to the cortex in which the recording is performed in p75NTR Ctrl mice infused with either vehicle or mut-proBDNF, and PV_Cre;p75NTR flx/flx mice infused with mut-proBDNF. Calibration bars: 50 μV, 100 ms. B , C/I VEP ratio mean values. Three days of monocular deprivation do not affect the C/I VEP ratio in adult mice, although it leads to a significant decrease in the C/I VEP ratio in animals treated with mut-proBDNF. Mut-proBDNF effects are, however, abolished in PV_Cre;p75 flx/flx mice (one-way ANOVA, F (2,18) = 8.903, p = 0.0021). p75NTR Ctrl + vehicle: n = 9 mice; p75NTR Ctrl + mut-proBDNF: n = 5 mice; PV_Cre;p75 flx/flx +mut-proBDNF: n = 7 mice. C , ODI of p75NTR Ctrl mice infused with vehicle solution and PV_Cre;p75 flx/flx mice infused with mut-proBDNF are not significantly different from those of undeprived animals, whereas ODIs in p75 Ctrl mice treated with mut-proBDNF are significantly shifted toward the open eye (one-way ANOVA, F (2,443) = 5.203, p = 0.0058). D , Mean spontaneous discharge is significantly increased only in p75 Ctrl mice treated with mut-proBDNF (one-way ANOVA, F (2,443) = 4.580, p = 0.0107). p75NTR Ctrl + vehicle: n = 9 mice, 174 cells; p75NTR Ctrl + mut-proBDNF: n = 5 mice, 147 cells; PV_Cre;p75 flx/flx +mut-proBDNF: n = 6 mice, 125 cells. Gray area represents the C/I VEP ratio ( B ) or the ODI range ( C ) (mean ± SEM) in adult nondeprived animals ( n = 5 mice, 99 cells). * indicate p

    Techniques Used: Activation Assay, In Vivo, Mouse Assay, Mass Spectrometry

    mut-proBDNF destabilizes PV cell innervation, even after it has reached maturity. A , Control PV cell ( A1 , Ctrl, green) at EP32 with exuberant innervation field characterized by extensive branching contacting the majority of potential targets, dense boutons along axons ( A2 ), and terminal branches with prominent and clustered boutons ( A3 ; arrowheads) around NeuN-positive somata (blue). B , PV cell treated with wt-proBDNF from EP26-EP32 shows overall similar axon size ( B1 ), percentage of potentially targeted neurons ( B2 ), and perisomatic innervations ( B3 ) as control, untreated PV cells. C , PV cell treated with mut-proBDNF from EP26-EP32 shows a drastic reduction both in percentage of innervated cells ( C2 ) and perisomatic innervation ( C3 ). Stars indicate NeuN-positive somata that are not innervated. Scale bars: A1–C1 , 50 μm; A2–C2 , 10 μm; A3–C3 , 5 μm. D , Perisomatic bouton density (one-way ANOVA, F (2,18) = 93.34, p
    Figure Legend Snippet: mut-proBDNF destabilizes PV cell innervation, even after it has reached maturity. A , Control PV cell ( A1 , Ctrl, green) at EP32 with exuberant innervation field characterized by extensive branching contacting the majority of potential targets, dense boutons along axons ( A2 ), and terminal branches with prominent and clustered boutons ( A3 ; arrowheads) around NeuN-positive somata (blue). B , PV cell treated with wt-proBDNF from EP26-EP32 shows overall similar axon size ( B1 ), percentage of potentially targeted neurons ( B2 ), and perisomatic innervations ( B3 ) as control, untreated PV cells. C , PV cell treated with mut-proBDNF from EP26-EP32 shows a drastic reduction both in percentage of innervated cells ( C2 ) and perisomatic innervation ( C3 ). Stars indicate NeuN-positive somata that are not innervated. Scale bars: A1–C1 , 50 μm; A2–C2 , 10 μm; A3–C3 , 5 μm. D , Perisomatic bouton density (one-way ANOVA, F (2,18) = 93.34, p

    Techniques Used:

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    Alomone Labs unimpaired probdnf
    Memory impairment in aged mice positively correlates with increases in hippocampal <t>proBDNF</t> levels. (A) Average (±SEM) reference memory (RM) and working memory (WM) errors over four 4-session blocks of a WRAM task in 24-month old aged mice and 4-month old young mice. RM and WM errors positively correlate with proBDNF levels. (B) Increase in proBDNF, but not brain-derived neurotrophic factor (BDNF), in 24-month old aged mice relative to young 4-month old mice. (C) Relative to young 4-month old mice, aged 24-month old mice show increased p75 Neurotrophin Receptor (p75NTR) and decreased p-trk140 and trk140. (D) Relative to young 4-month old mice, aged 24-month old mice show decreased Tissue Plasminogen Activator (tPA), but not carboxypeptidase E (CPE). * p
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    Memory impairment in aged mice positively correlates with increases in hippocampal proBDNF levels. (A) Average (±SEM) reference memory (RM) and working memory (WM) errors over four 4-session blocks of a WRAM task in 24-month old aged mice and 4-month old young mice. RM and WM errors positively correlate with proBDNF levels. (B) Increase in proBDNF, but not brain-derived neurotrophic factor (BDNF), in 24-month old aged mice relative to young 4-month old mice. (C) Relative to young 4-month old mice, aged 24-month old mice show increased p75 Neurotrophin Receptor (p75NTR) and decreased p-trk140 and trk140. (D) Relative to young 4-month old mice, aged 24-month old mice show decreased Tissue Plasminogen Activator (tPA), but not carboxypeptidase E (CPE). * p

    Journal: Frontiers in Aging Neuroscience

    Article Title: Increased Hippocampal ProBDNF Contributes to Memory Impairments in Aged Mice

    doi: 10.3389/fnagi.2017.00284

    Figure Lengend Snippet: Memory impairment in aged mice positively correlates with increases in hippocampal proBDNF levels. (A) Average (±SEM) reference memory (RM) and working memory (WM) errors over four 4-session blocks of a WRAM task in 24-month old aged mice and 4-month old young mice. RM and WM errors positively correlate with proBDNF levels. (B) Increase in proBDNF, but not brain-derived neurotrophic factor (BDNF), in 24-month old aged mice relative to young 4-month old mice. (C) Relative to young 4-month old mice, aged 24-month old mice show increased p75 Neurotrophin Receptor (p75NTR) and decreased p-trk140 and trk140. (D) Relative to young 4-month old mice, aged 24-month old mice show decreased Tissue Plasminogen Activator (tPA), but not carboxypeptidase E (CPE). * p

    Article Snippet: On d20–22 unimpaired mice in group “unimpaired + proBDNF” were injected with “uncleavable ” mouse proBDNF (proBDNF mut-mouse, Alomone, Jerusalem, Israel) 40 pg/0.4 μL/side.

    Techniques: Mouse Assay, Derivative Assay

    Theoretical model of the role of proBDNF in learning in memory in aged individuals. In young individuals, maturation of proBDNF to BDNF is controlled by plasmin and tPA, and is essential for learning and memory. Aged individuals show decreased levels of tPA and plasmin, and increased levels of uncleaved proBDNF, associated with increased spine remodeling and memory deficits. Blockade of p75NTR (e.g., by TAT-Pep5, as in the current study) leads to spine growth, and rescues learning and memory.

    Journal: Frontiers in Aging Neuroscience

    Article Title: Increased Hippocampal ProBDNF Contributes to Memory Impairments in Aged Mice

    doi: 10.3389/fnagi.2017.00284

    Figure Lengend Snippet: Theoretical model of the role of proBDNF in learning in memory in aged individuals. In young individuals, maturation of proBDNF to BDNF is controlled by plasmin and tPA, and is essential for learning and memory. Aged individuals show decreased levels of tPA and plasmin, and increased levels of uncleaved proBDNF, associated with increased spine remodeling and memory deficits. Blockade of p75NTR (e.g., by TAT-Pep5, as in the current study) leads to spine growth, and rescues learning and memory.

    Article Snippet: On d20–22 unimpaired mice in group “unimpaired + proBDNF” were injected with “uncleavable ” mouse proBDNF (proBDNF mut-mouse, Alomone, Jerusalem, Israel) 40 pg/0.4 μL/side.

    Techniques:

    Timeline of Experiments 1 and 2. In Experiment 2, mice were split into three groups based on their performance (unimpaired/impaired) and henceforth infused with different drugs (saline, proBDNF, TAT-Pep5). d, day of study; WRAM, behavioral training/testing in the water radial arm maze task; SAL, saline.

    Journal: Frontiers in Aging Neuroscience

    Article Title: Increased Hippocampal ProBDNF Contributes to Memory Impairments in Aged Mice

    doi: 10.3389/fnagi.2017.00284

    Figure Lengend Snippet: Timeline of Experiments 1 and 2. In Experiment 2, mice were split into three groups based on their performance (unimpaired/impaired) and henceforth infused with different drugs (saline, proBDNF, TAT-Pep5). d, day of study; WRAM, behavioral training/testing in the water radial arm maze task; SAL, saline.

    Article Snippet: On d20–22 unimpaired mice in group “unimpaired + proBDNF” were injected with “uncleavable ” mouse proBDNF (proBDNF mut-mouse, Alomone, Jerusalem, Israel) 40 pg/0.4 μL/side.

    Techniques: Mouse Assay

    Intra-hippocampal infusion of proBDNF impairs memory in well-performing (unimpaired) mice, while TAT-Pep5 infusion improves memory in poorly-performing (impaired) mice. (A) Average (±SEM) RM and WM errors in memory impaired 18-month old mice receiving intra-hippocampal saline infusions (impaired + SAL, n = 8, open triangles) and well-performing 18-month old aged mice receiving uncleavable proBDNF intra-hippocampal infusions (unimpaired + proBDNF, n = 16, open circles) over four daily sessions of a WRAM task. (B) Average (± SEM) RM and WM errors in memory impaired 18-month old mice infused with saline (impaired + SAL, n = 8, open triangles) and memory impaired 18-month old mice receiving intra-hippocampal infusions of TAT-Pep5 (impaired + TAT-Pep5, n = 9, closed circles) over four daily sessions of a WRAM task. (C) Representative images indicating the locations of drug infusions at two levels of the hippocampus. ns not significant; * p

    Journal: Frontiers in Aging Neuroscience

    Article Title: Increased Hippocampal ProBDNF Contributes to Memory Impairments in Aged Mice

    doi: 10.3389/fnagi.2017.00284

    Figure Lengend Snippet: Intra-hippocampal infusion of proBDNF impairs memory in well-performing (unimpaired) mice, while TAT-Pep5 infusion improves memory in poorly-performing (impaired) mice. (A) Average (±SEM) RM and WM errors in memory impaired 18-month old mice receiving intra-hippocampal saline infusions (impaired + SAL, n = 8, open triangles) and well-performing 18-month old aged mice receiving uncleavable proBDNF intra-hippocampal infusions (unimpaired + proBDNF, n = 16, open circles) over four daily sessions of a WRAM task. (B) Average (± SEM) RM and WM errors in memory impaired 18-month old mice infused with saline (impaired + SAL, n = 8, open triangles) and memory impaired 18-month old mice receiving intra-hippocampal infusions of TAT-Pep5 (impaired + TAT-Pep5, n = 9, closed circles) over four daily sessions of a WRAM task. (C) Representative images indicating the locations of drug infusions at two levels of the hippocampus. ns not significant; * p

    Article Snippet: On d20–22 unimpaired mice in group “unimpaired + proBDNF” were injected with “uncleavable ” mouse proBDNF (proBDNF mut-mouse, Alomone, Jerusalem, Israel) 40 pg/0.4 μL/side.

    Techniques: Mouse Assay

    Intra-hippocampal infusion of proBDNF increases p-cofilin levels in memory-unimpaired mice to levels seen in memory-impaired mice. (A) p-Cofilin to total cofilin ratio in memory-unimpaired mice, memory-impaired mice, and memory-unimpaired mice infused with proBDNF. (B) Representative p-cofilin and cofilin blots. ** p

    Journal: Frontiers in Aging Neuroscience

    Article Title: Increased Hippocampal ProBDNF Contributes to Memory Impairments in Aged Mice

    doi: 10.3389/fnagi.2017.00284

    Figure Lengend Snippet: Intra-hippocampal infusion of proBDNF increases p-cofilin levels in memory-unimpaired mice to levels seen in memory-impaired mice. (A) p-Cofilin to total cofilin ratio in memory-unimpaired mice, memory-impaired mice, and memory-unimpaired mice infused with proBDNF. (B) Representative p-cofilin and cofilin blots. ** p

    Article Snippet: On d20–22 unimpaired mice in group “unimpaired + proBDNF” were injected with “uncleavable ” mouse proBDNF (proBDNF mut-mouse, Alomone, Jerusalem, Israel) 40 pg/0.4 μL/side.

    Techniques: Mouse Assay

    Schematic diagram showing how proBDNF dampens CD4 + T cell activity and contributes to the pathogenesis of SAE. In sepsis, proBDNF expression is increased in peripheral blood and meningeal immune cells, which then decreases the infiltration of CD4 + T cells in the meninges. As a result, meningeal pro-inflammatory cytokines such as IL-6 and IL-1β are upregulated, but anti-inflammatory cytokines including IL-4 and IL-13 are downregulated, finally leading to SAE. SAE, sepsis-associated encephalopathy

    Journal: Journal of Neuroinflammation

    Article Title: ProBDNF promotes sepsis-associated encephalopathy in mice by dampening the immune activity of meningeal CD4+ T cells

    doi: 10.1186/s12974-020-01850-0

    Figure Lengend Snippet: Schematic diagram showing how proBDNF dampens CD4 + T cell activity and contributes to the pathogenesis of SAE. In sepsis, proBDNF expression is increased in peripheral blood and meningeal immune cells, which then decreases the infiltration of CD4 + T cells in the meninges. As a result, meningeal pro-inflammatory cytokines such as IL-6 and IL-1β are upregulated, but anti-inflammatory cytokines including IL-4 and IL-13 are downregulated, finally leading to SAE. SAE, sepsis-associated encephalopathy

    Article Snippet: As shown, proBDNF protein did not affect T cell subgroups in normal mice (Fig. a–c).

    Techniques: Activity Assay, Expressing

    Exogenous proBDNF protein reduced CD4 + T cells but increased CD8 + T cells of septic mice in vitro. Mice injected with saline or LPS (5 mg kg −1 ) for 5 days and the splenocytes were isolated and cultured for 3 days in vitro. Exogenous proBDNF did not alter the percentage of a CD3 + T cells in CD45 + cells or the percentage of b CD4 + T cells or c CD8 + T cells in CD3 + T cells in splenocytes from mice treated with saline. d Exogenous proBDNF did not alter the percentage of CD3 + T cells in CD45 + cells in splenocytes in septic mice. e–f ProBDNF treatment significantly decreased the percentage of e CD4 + T cells but increased the percentage of f CD8 + T cells in CD3 + T cells in splenocytes in LPS-treated mice. n = 4 in each group. Data were analyzed by one-way ANOVA and followed by Tukey post hoc test, * P

    Journal: Journal of Neuroinflammation

    Article Title: ProBDNF promotes sepsis-associated encephalopathy in mice by dampening the immune activity of meningeal CD4+ T cells

    doi: 10.1186/s12974-020-01850-0

    Figure Lengend Snippet: Exogenous proBDNF protein reduced CD4 + T cells but increased CD8 + T cells of septic mice in vitro. Mice injected with saline or LPS (5 mg kg −1 ) for 5 days and the splenocytes were isolated and cultured for 3 days in vitro. Exogenous proBDNF did not alter the percentage of a CD3 + T cells in CD45 + cells or the percentage of b CD4 + T cells or c CD8 + T cells in CD3 + T cells in splenocytes from mice treated with saline. d Exogenous proBDNF did not alter the percentage of CD3 + T cells in CD45 + cells in splenocytes in septic mice. e–f ProBDNF treatment significantly decreased the percentage of e CD4 + T cells but increased the percentage of f CD8 + T cells in CD3 + T cells in splenocytes in LPS-treated mice. n = 4 in each group. Data were analyzed by one-way ANOVA and followed by Tukey post hoc test, * P

    Article Snippet: As shown, proBDNF protein did not affect T cell subgroups in normal mice (Fig. a–c).

    Techniques: Mouse Assay, In Vitro, Injection, Isolation, Cell Culture

    Increased proBDNF expression in meningeal and peripheral blood immune cells in septic mice. Mice were i.p. injected with LPS (5 mg kg −1 ) and meninges and peripheral blood were harvested for immunofluorescence staining or flow cytometry. a Representative whole mount meningeal immunofluorescence images showed markedly increased proBDNF-positive staining cells in the meninges in mice at 1 day after LPS injection compared to saline injected mice. The high magnification images around the arrows are displayed in a white square as insets. Bar = 100 μm. b – f Representative meningeal single cell flow cytometry images ( upper panel ) and its statistical analysis ( lower panel ) indicated that proBDNF MFI was increased in meningeal b CD3 + T cells, c CD4 + T cells, d CD8 + T cells, and f CD11b + monocytes/macrophages at 1 day after LPS injection. proBDNF in meningeal e CD19 + B cells upregulated until 5 days after LPS injection. n = 10 in the Con group, n = 6 in LPS groups. g – k Upregulation of proBDNF in g CD3 + T cells, h CD4 + T cells, i CD8 + T cells, j CD19 + B cells, and k CD11b + monocytes/macrophages in peripheral blood in LPS-injected mice were detected. n = 9 in Con group, n = 8 in the LPS1d group, n = 4 in the LPS 5d group. Data b – k were analyzed by one-way ANOVA and followed by Tukey post hoc test, * P

    Journal: Journal of Neuroinflammation

    Article Title: ProBDNF promotes sepsis-associated encephalopathy in mice by dampening the immune activity of meningeal CD4+ T cells

    doi: 10.1186/s12974-020-01850-0

    Figure Lengend Snippet: Increased proBDNF expression in meningeal and peripheral blood immune cells in septic mice. Mice were i.p. injected with LPS (5 mg kg −1 ) and meninges and peripheral blood were harvested for immunofluorescence staining or flow cytometry. a Representative whole mount meningeal immunofluorescence images showed markedly increased proBDNF-positive staining cells in the meninges in mice at 1 day after LPS injection compared to saline injected mice. The high magnification images around the arrows are displayed in a white square as insets. Bar = 100 μm. b – f Representative meningeal single cell flow cytometry images ( upper panel ) and its statistical analysis ( lower panel ) indicated that proBDNF MFI was increased in meningeal b CD3 + T cells, c CD4 + T cells, d CD8 + T cells, and f CD11b + monocytes/macrophages at 1 day after LPS injection. proBDNF in meningeal e CD19 + B cells upregulated until 5 days after LPS injection. n = 10 in the Con group, n = 6 in LPS groups. g – k Upregulation of proBDNF in g CD3 + T cells, h CD4 + T cells, i CD8 + T cells, j CD19 + B cells, and k CD11b + monocytes/macrophages in peripheral blood in LPS-injected mice were detected. n = 9 in Con group, n = 8 in the LPS1d group, n = 4 in the LPS 5d group. Data b – k were analyzed by one-way ANOVA and followed by Tukey post hoc test, * P

    Article Snippet: As shown, proBDNF protein did not affect T cell subgroups in normal mice (Fig. a–c).

    Techniques: Expressing, Mouse Assay, Injection, Immunofluorescence, Staining, Flow Cytometry

    Effect of i.c.v. injection of anti-proBDNF antibody on fear memory and meningeal immune activity in the septic mice. Mice were bilateral i.c.v. injected with 1 μg McAb-proB 3 days before LPS injection. Behavior tests were performed 1 day after LPS injection. a Representative images showed the broad and thorough diffusion of drugs in cerebroventricular lumen following i.c.v. injection with methylene blue. b McAb-proB i.c.v. injection did not influence weight of mice. c–e There was no difference of c fear conditioning acquiring performance in each group, nor McAb-proB increased the freezing time of d contextual or e cued fear conditioning test as compared to IgG control after LPS injection. n = 6 in each group. Data b , c , and e were analyzed by repeated measures ANOVA and followed by Bonferroni post hoc test and data d was analyzed by one-way ANOVA and followed by Tukey post hoc test, * P

    Journal: Journal of Neuroinflammation

    Article Title: ProBDNF promotes sepsis-associated encephalopathy in mice by dampening the immune activity of meningeal CD4+ T cells

    doi: 10.1186/s12974-020-01850-0

    Figure Lengend Snippet: Effect of i.c.v. injection of anti-proBDNF antibody on fear memory and meningeal immune activity in the septic mice. Mice were bilateral i.c.v. injected with 1 μg McAb-proB 3 days before LPS injection. Behavior tests were performed 1 day after LPS injection. a Representative images showed the broad and thorough diffusion of drugs in cerebroventricular lumen following i.c.v. injection with methylene blue. b McAb-proB i.c.v. injection did not influence weight of mice. c–e There was no difference of c fear conditioning acquiring performance in each group, nor McAb-proB increased the freezing time of d contextual or e cued fear conditioning test as compared to IgG control after LPS injection. n = 6 in each group. Data b , c , and e were analyzed by repeated measures ANOVA and followed by Bonferroni post hoc test and data d was analyzed by one-way ANOVA and followed by Tukey post hoc test, * P

    Article Snippet: As shown, proBDNF protein did not affect T cell subgroups in normal mice (Fig. a–c).

    Techniques: Injection, Activity Assay, Mouse Assay, Diffusion-based Assay

    Systemic blockade of proBDNF ameliorated cognitive dysfunction and restored meningeal and peripheral CD4 + T cell ratio in septic mice. Mice were i.p. injected with proBDNF 30 min before LPS (5 mg kg −1 ) injection. Fear conditioning testing was performed 1 day after LPS injection. Meninges and peripheral blood were harvested 5 days after LPS injection for flow cytometry. a McAb-proB did not influence the weight of mice or b fear conditioning acquiring. c , d McAb-proB greatly alleviated memory deficit induced by LPS injection in mice as indicated by the increased freezing time in ( c ) contextual and ( d ) cued fear conditioning tests in the McAb-proB group relative to the IgG control. n = 8 in each group. Data a , b , and d were analyzed by repeated measures ANOVA and followed by Bonferroni post hoc tests. Data c was analyzed by one-way ANOVA and followed by Tukey post hoc test, * P

    Journal: Journal of Neuroinflammation

    Article Title: ProBDNF promotes sepsis-associated encephalopathy in mice by dampening the immune activity of meningeal CD4+ T cells

    doi: 10.1186/s12974-020-01850-0

    Figure Lengend Snippet: Systemic blockade of proBDNF ameliorated cognitive dysfunction and restored meningeal and peripheral CD4 + T cell ratio in septic mice. Mice were i.p. injected with proBDNF 30 min before LPS (5 mg kg −1 ) injection. Fear conditioning testing was performed 1 day after LPS injection. Meninges and peripheral blood were harvested 5 days after LPS injection for flow cytometry. a McAb-proB did not influence the weight of mice or b fear conditioning acquiring. c , d McAb-proB greatly alleviated memory deficit induced by LPS injection in mice as indicated by the increased freezing time in ( c ) contextual and ( d ) cued fear conditioning tests in the McAb-proB group relative to the IgG control. n = 8 in each group. Data a , b , and d were analyzed by repeated measures ANOVA and followed by Bonferroni post hoc tests. Data c was analyzed by one-way ANOVA and followed by Tukey post hoc test, * P

    Article Snippet: As shown, proBDNF protein did not affect T cell subgroups in normal mice (Fig. a–c).

    Techniques: Mouse Assay, Injection, Flow Cytometry

    Systemic blockade of proBDNF restored meningeal pro-inflammatory microenvironment in septic mice. Mice were i.p. injected with proBDNF 30 min before LPS (5 mg kg −1 ) injection. Meninges were harvested 5 days after LPS injection for qPCR. a The level of CD4 gene expression was higher in the meninges of the McAb-proB group than in IgG controls in septic mice. b–f Gene levels were significantly lower in b IL-1β and c IL-6 but higher in d IL-4, e IFN-γ, and f IL-13 in the meninges after LPS injection in the McAb-proB group as compared to IgG control. n = 5 in each group. All experiments were performed at least in triplicate. Data were analyzed by unpaired T test, * P

    Journal: Journal of Neuroinflammation

    Article Title: ProBDNF promotes sepsis-associated encephalopathy in mice by dampening the immune activity of meningeal CD4+ T cells

    doi: 10.1186/s12974-020-01850-0

    Figure Lengend Snippet: Systemic blockade of proBDNF restored meningeal pro-inflammatory microenvironment in septic mice. Mice were i.p. injected with proBDNF 30 min before LPS (5 mg kg −1 ) injection. Meninges were harvested 5 days after LPS injection for qPCR. a The level of CD4 gene expression was higher in the meninges of the McAb-proB group than in IgG controls in septic mice. b–f Gene levels were significantly lower in b IL-1β and c IL-6 but higher in d IL-4, e IFN-γ, and f IL-13 in the meninges after LPS injection in the McAb-proB group as compared to IgG control. n = 5 in each group. All experiments were performed at least in triplicate. Data were analyzed by unpaired T test, * P

    Article Snippet: As shown, proBDNF protein did not affect T cell subgroups in normal mice (Fig. a–c).

    Techniques: Mouse Assay, Injection, Real-time Polymerase Chain Reaction, Expressing

    proBNDF-mediated p75NTR activation in cortical PV cells reduces their perisomatic boutons. A , Experimental approach. B , The intensity of perisomatic PV immunostaining (green) is reduced in the binocular visual cortex ipsilateral to the minipump-releasing mut-proBDNF (Ipsi) compared with the contralateral cortex (Contra) in the same animal. On the other hand, perisomatic PV intensity in the ipsilateral cortex of PV_Cre;p75 flx/flx mice is similar to that observed in the contralateral, untreated cortex. C , Low ( C1 ) and high ( C2 ) magnification of PNN (red, WFA staining) enwrapping PV cells (green) shows a dramatic reduction in both PNN density and intensity in the visual cortex infused with mut-proBFNF. This effect is abolished in PV_Cre;p75 flx/flx mice. Scale bars: C1 , 100 μm; B , C2 , 10 μm. D , Quantification of the mean intensity of perisomatic PV-positive puncta in ipsilateral compared with contralateral cortex. I/C ratio is obtained for each animal and then averaged between different animals. Mean I/C ratio is significantly reduced in Mut-proBDNF-infused p75 Ctrl mice compared with Mut-proBDNF-infused PV_Cre;p75 flx/flx mice (unpaired t test, df = 8, t = 6.077, p = 0.0003). E , The ratio of mean PNN intensity around PV cells in ipsilateral versus contralateral cortex is significantly lower in p75 Ctrl than PV_Cre;p75 flx/flx mice infused with mut-proBDNF (unpaired t test, df = 8, t = 15.33, p

    Journal: The Journal of Neuroscience

    Article Title: p75 Neurotrophin Receptor Activation Regulates the Timing of the Maturation of Cortical Parvalbumin Interneuron Connectivity and Promotes Juvenile-like Plasticity in Adult Visual Cortex

    doi: 10.1523/JNEUROSCI.2881-18.2019

    Figure Lengend Snippet: proBNDF-mediated p75NTR activation in cortical PV cells reduces their perisomatic boutons. A , Experimental approach. B , The intensity of perisomatic PV immunostaining (green) is reduced in the binocular visual cortex ipsilateral to the minipump-releasing mut-proBDNF (Ipsi) compared with the contralateral cortex (Contra) in the same animal. On the other hand, perisomatic PV intensity in the ipsilateral cortex of PV_Cre;p75 flx/flx mice is similar to that observed in the contralateral, untreated cortex. C , Low ( C1 ) and high ( C2 ) magnification of PNN (red, WFA staining) enwrapping PV cells (green) shows a dramatic reduction in both PNN density and intensity in the visual cortex infused with mut-proBFNF. This effect is abolished in PV_Cre;p75 flx/flx mice. Scale bars: C1 , 100 μm; B , C2 , 10 μm. D , Quantification of the mean intensity of perisomatic PV-positive puncta in ipsilateral compared with contralateral cortex. I/C ratio is obtained for each animal and then averaged between different animals. Mean I/C ratio is significantly reduced in Mut-proBDNF-infused p75 Ctrl mice compared with Mut-proBDNF-infused PV_Cre;p75 flx/flx mice (unpaired t test, df = 8, t = 6.077, p = 0.0003). E , The ratio of mean PNN intensity around PV cells in ipsilateral versus contralateral cortex is significantly lower in p75 Ctrl than PV_Cre;p75 flx/flx mice infused with mut-proBDNF (unpaired t test, df = 8, t = 15.33, p

    Article Snippet: Recombinant mouse proneurotrophin, proBDNF (wt-proBDNF, 10 ng/ml, Alomone Labs, catalog #B-240) and cleavage-resistant, recombinant mouse proBDNF (mut-proBDNF, 10 ng/ml, Alomone Labs, catalog #B-243) were, respectively, added with the culture medium during the specific time window indicated in Results.

    Techniques: Activation Assay, Immunostaining, Mouse Assay, Staining

    Modulation of tPA activity affects the formation of PV cell innervations during early postnatal development. A , Control EP18 PV cell ( A1 , green represents Ctrl). B , PV cell treated with the tPA inhibitor PPACK from EP10–EP18 shows simpler axonal arborization, contacting less potential targets ( B2 , blue represents NeuN-positive somata). C , PV cell treated with tPA in the same time window shows a very complex axonal arbor ( C2 ) and an increase in both terminal branching and perisomatic boutons ( C3 , arrowheads) compared with control cells ( A2 , A3 ). D , PV cell treated simultaneously with tPA and mut-proBDNF shows axonal branching and perisomatic innervation more similar to those formed by PV cell treated with mut-proBDNF alone, suggesting that the effects of tPA application may be mediated by a decrease in endogenous proBDNF/mBDNF ratio. Stars indicate NeuN-positive somata that are not innervated. Scale bars: A1–D1 , 50 μm; A2–D2 , 10 μm; A3–D3 , 5 μm. E , Perisomatic boutons density (one-way ANOVA, F (3,20) = 121.2, p

    Journal: The Journal of Neuroscience

    Article Title: p75 Neurotrophin Receptor Activation Regulates the Timing of the Maturation of Cortical Parvalbumin Interneuron Connectivity and Promotes Juvenile-like Plasticity in Adult Visual Cortex

    doi: 10.1523/JNEUROSCI.2881-18.2019

    Figure Lengend Snippet: Modulation of tPA activity affects the formation of PV cell innervations during early postnatal development. A , Control EP18 PV cell ( A1 , green represents Ctrl). B , PV cell treated with the tPA inhibitor PPACK from EP10–EP18 shows simpler axonal arborization, contacting less potential targets ( B2 , blue represents NeuN-positive somata). C , PV cell treated with tPA in the same time window shows a very complex axonal arbor ( C2 ) and an increase in both terminal branching and perisomatic boutons ( C3 , arrowheads) compared with control cells ( A2 , A3 ). D , PV cell treated simultaneously with tPA and mut-proBDNF shows axonal branching and perisomatic innervation more similar to those formed by PV cell treated with mut-proBDNF alone, suggesting that the effects of tPA application may be mediated by a decrease in endogenous proBDNF/mBDNF ratio. Stars indicate NeuN-positive somata that are not innervated. Scale bars: A1–D1 , 50 μm; A2–D2 , 10 μm; A3–D3 , 5 μm. E , Perisomatic boutons density (one-way ANOVA, F (3,20) = 121.2, p

    Article Snippet: Recombinant mouse proneurotrophin, proBDNF (wt-proBDNF, 10 ng/ml, Alomone Labs, catalog #B-240) and cleavage-resistant, recombinant mouse proBDNF (mut-proBDNF, 10 ng/ml, Alomone Labs, catalog #B-243) were, respectively, added with the culture medium during the specific time window indicated in Results.

    Techniques: Activity Assay

    proBNDF-mediated p75NTR activation in cortical PV cells restores ocular dominance plasticity in adult visual cortex in vivo . A , Typical VEP responses to the stimulation of either contralateral (blue) or ipsilateral (red) eye to the cortex in which the recording is performed in p75NTR Ctrl mice infused with either vehicle or mut-proBDNF, and PV_Cre;p75NTR flx/flx mice infused with mut-proBDNF. Calibration bars: 50 μV, 100 ms. B , C/I VEP ratio mean values. Three days of monocular deprivation do not affect the C/I VEP ratio in adult mice, although it leads to a significant decrease in the C/I VEP ratio in animals treated with mut-proBDNF. Mut-proBDNF effects are, however, abolished in PV_Cre;p75 flx/flx mice (one-way ANOVA, F (2,18) = 8.903, p = 0.0021). p75NTR Ctrl + vehicle: n = 9 mice; p75NTR Ctrl + mut-proBDNF: n = 5 mice; PV_Cre;p75 flx/flx +mut-proBDNF: n = 7 mice. C , ODI of p75NTR Ctrl mice infused with vehicle solution and PV_Cre;p75 flx/flx mice infused with mut-proBDNF are not significantly different from those of undeprived animals, whereas ODIs in p75 Ctrl mice treated with mut-proBDNF are significantly shifted toward the open eye (one-way ANOVA, F (2,443) = 5.203, p = 0.0058). D , Mean spontaneous discharge is significantly increased only in p75 Ctrl mice treated with mut-proBDNF (one-way ANOVA, F (2,443) = 4.580, p = 0.0107). p75NTR Ctrl + vehicle: n = 9 mice, 174 cells; p75NTR Ctrl + mut-proBDNF: n = 5 mice, 147 cells; PV_Cre;p75 flx/flx +mut-proBDNF: n = 6 mice, 125 cells. Gray area represents the C/I VEP ratio ( B ) or the ODI range ( C ) (mean ± SEM) in adult nondeprived animals ( n = 5 mice, 99 cells). * indicate p

    Journal: The Journal of Neuroscience

    Article Title: p75 Neurotrophin Receptor Activation Regulates the Timing of the Maturation of Cortical Parvalbumin Interneuron Connectivity and Promotes Juvenile-like Plasticity in Adult Visual Cortex

    doi: 10.1523/JNEUROSCI.2881-18.2019

    Figure Lengend Snippet: proBNDF-mediated p75NTR activation in cortical PV cells restores ocular dominance plasticity in adult visual cortex in vivo . A , Typical VEP responses to the stimulation of either contralateral (blue) or ipsilateral (red) eye to the cortex in which the recording is performed in p75NTR Ctrl mice infused with either vehicle or mut-proBDNF, and PV_Cre;p75NTR flx/flx mice infused with mut-proBDNF. Calibration bars: 50 μV, 100 ms. B , C/I VEP ratio mean values. Three days of monocular deprivation do not affect the C/I VEP ratio in adult mice, although it leads to a significant decrease in the C/I VEP ratio in animals treated with mut-proBDNF. Mut-proBDNF effects are, however, abolished in PV_Cre;p75 flx/flx mice (one-way ANOVA, F (2,18) = 8.903, p = 0.0021). p75NTR Ctrl + vehicle: n = 9 mice; p75NTR Ctrl + mut-proBDNF: n = 5 mice; PV_Cre;p75 flx/flx +mut-proBDNF: n = 7 mice. C , ODI of p75NTR Ctrl mice infused with vehicle solution and PV_Cre;p75 flx/flx mice infused with mut-proBDNF are not significantly different from those of undeprived animals, whereas ODIs in p75 Ctrl mice treated with mut-proBDNF are significantly shifted toward the open eye (one-way ANOVA, F (2,443) = 5.203, p = 0.0058). D , Mean spontaneous discharge is significantly increased only in p75 Ctrl mice treated with mut-proBDNF (one-way ANOVA, F (2,443) = 4.580, p = 0.0107). p75NTR Ctrl + vehicle: n = 9 mice, 174 cells; p75NTR Ctrl + mut-proBDNF: n = 5 mice, 147 cells; PV_Cre;p75 flx/flx +mut-proBDNF: n = 6 mice, 125 cells. Gray area represents the C/I VEP ratio ( B ) or the ODI range ( C ) (mean ± SEM) in adult nondeprived animals ( n = 5 mice, 99 cells). * indicate p

    Article Snippet: Recombinant mouse proneurotrophin, proBDNF (wt-proBDNF, 10 ng/ml, Alomone Labs, catalog #B-240) and cleavage-resistant, recombinant mouse proBDNF (mut-proBDNF, 10 ng/ml, Alomone Labs, catalog #B-243) were, respectively, added with the culture medium during the specific time window indicated in Results.

    Techniques: Activation Assay, In Vivo, Mouse Assay

    mut-proBDNF destabilizes PV cell innervation, even after it has reached maturity. A , Control PV cell ( A1 , Ctrl, green) at EP32 with exuberant innervation field characterized by extensive branching contacting the majority of potential targets, dense boutons along axons ( A2 ), and terminal branches with prominent and clustered boutons ( A3 ; arrowheads) around NeuN-positive somata (blue). B , PV cell treated with wt-proBDNF from EP26-EP32 shows overall similar axon size ( B1 ), percentage of potentially targeted neurons ( B2 ), and perisomatic innervations ( B3 ) as control, untreated PV cells. C , PV cell treated with mut-proBDNF from EP26-EP32 shows a drastic reduction both in percentage of innervated cells ( C2 ) and perisomatic innervation ( C3 ). Stars indicate NeuN-positive somata that are not innervated. Scale bars: A1–C1 , 50 μm; A2–C2 , 10 μm; A3–C3 , 5 μm. D , Perisomatic bouton density (one-way ANOVA, F (2,18) = 93.34, p

    Journal: The Journal of Neuroscience

    Article Title: p75 Neurotrophin Receptor Activation Regulates the Timing of the Maturation of Cortical Parvalbumin Interneuron Connectivity and Promotes Juvenile-like Plasticity in Adult Visual Cortex

    doi: 10.1523/JNEUROSCI.2881-18.2019

    Figure Lengend Snippet: mut-proBDNF destabilizes PV cell innervation, even after it has reached maturity. A , Control PV cell ( A1 , Ctrl, green) at EP32 with exuberant innervation field characterized by extensive branching contacting the majority of potential targets, dense boutons along axons ( A2 ), and terminal branches with prominent and clustered boutons ( A3 ; arrowheads) around NeuN-positive somata (blue). B , PV cell treated with wt-proBDNF from EP26-EP32 shows overall similar axon size ( B1 ), percentage of potentially targeted neurons ( B2 ), and perisomatic innervations ( B3 ) as control, untreated PV cells. C , PV cell treated with mut-proBDNF from EP26-EP32 shows a drastic reduction both in percentage of innervated cells ( C2 ) and perisomatic innervation ( C3 ). Stars indicate NeuN-positive somata that are not innervated. Scale bars: A1–C1 , 50 μm; A2–C2 , 10 μm; A3–C3 , 5 μm. D , Perisomatic bouton density (one-way ANOVA, F (2,18) = 93.34, p

    Article Snippet: Recombinant mouse proneurotrophin, proBDNF (wt-proBDNF, 10 ng/ml, Alomone Labs, catalog #B-240) and cleavage-resistant, recombinant mouse proBDNF (mut-proBDNF, 10 ng/ml, Alomone Labs, catalog #B-243) were, respectively, added with the culture medium during the specific time window indicated in Results.

    Techniques:

    proBNDF-mediated p75NTR activation in cortical PV cells reduces their perisomatic boutons and restores ocular dominance plasticity in adult visual cortex in vivo . (a) Experimental approach. (b) The number of immunolabeled PV-positive puncta (green) surrounding NeuN-positive neuronal somata (red) is reduced in the binocular visual cortex ipsilateral to the minipump releasing mut-proBDNF (Ipsi) compared to the contralateral cortex (Contra) in the same animal. On the other hand, the number of PV-positive puncta per NeuN-positive profile in the ipsilateral cortex of PV-CRE; p75 flx/flx mice is similar to that observed in the contralateral, untreated cortex. (c) Low (c1) and high (c2) magnification of PNN (red, WFA staining) enwrapping PV cells (green) show a dramatic reduction in both PNN density and intensity in the visual cortex infused with mut-proBFNF. This effects is abolished in PV-CRE; p75 flx/flx mice. Scale bar, c1: 100µm; b, c2: 10µm. (d) Quantification of the mean number of PV-positive puncta per NeuN-positive profile in ipsilateral compared to contralateral cortex. Ipsi/Contra ratio is obtained for each animal, and then averaged between different animals. Mean Ipsi/Contra ratio is significantly reduced in Mut-proBDNF infused p75 Ctrl but not in PV-CRE; p75 flx/flx mice (t-test, p

    Journal: bioRxiv

    Article Title: p75 Neurotrophin Receptor Regulates the Timing of the Maturation of Cortical Parvalbumin Cell Connectivity and Promotes Ocular Dominance Plasticity in Adult Visual Cortex

    doi: 10.1101/392159

    Figure Lengend Snippet: proBNDF-mediated p75NTR activation in cortical PV cells reduces their perisomatic boutons and restores ocular dominance plasticity in adult visual cortex in vivo . (a) Experimental approach. (b) The number of immunolabeled PV-positive puncta (green) surrounding NeuN-positive neuronal somata (red) is reduced in the binocular visual cortex ipsilateral to the minipump releasing mut-proBDNF (Ipsi) compared to the contralateral cortex (Contra) in the same animal. On the other hand, the number of PV-positive puncta per NeuN-positive profile in the ipsilateral cortex of PV-CRE; p75 flx/flx mice is similar to that observed in the contralateral, untreated cortex. (c) Low (c1) and high (c2) magnification of PNN (red, WFA staining) enwrapping PV cells (green) show a dramatic reduction in both PNN density and intensity in the visual cortex infused with mut-proBFNF. This effects is abolished in PV-CRE; p75 flx/flx mice. Scale bar, c1: 100µm; b, c2: 10µm. (d) Quantification of the mean number of PV-positive puncta per NeuN-positive profile in ipsilateral compared to contralateral cortex. Ipsi/Contra ratio is obtained for each animal, and then averaged between different animals. Mean Ipsi/Contra ratio is significantly reduced in Mut-proBDNF infused p75 Ctrl but not in PV-CRE; p75 flx/flx mice (t-test, p

    Article Snippet: To label control PV cells, slices were transfected with PG67 _GFP bullets, while for the p75NTR-/- PV cells were transfected with both PG67 _GFP and PG67_Cre. wt-proBDNF and mut-proBDNF (10 ng/ml, Alomone Labs) were respectively added with the culture medium during the specific time window indicated in the results section.

    Techniques: Activation Assay, In Vivo, Immunolabeling, Mouse Assay, Staining, T-Test

    mut-proBDNF can destabilize PV cell innervation even after it has reached maturity. (a) Control PV cell (a1, Ctrl, green) at EP32 with exuberant innervation field characterized by extensive branching contacting the majority of potential targets, dense boutons along axons (a2), and terminal branches with prominent and clustered boutons (a3; arrowheads) around pyramidal cell somata (NeuN immunostaining, blue). (b) PV cell treated with wt-proBDNF from EP26-32 shows overall similar axon size (b1), percentage of potentially targeted neurons (B2) and perisomatic innervations (b3) as control, untreated PV cells. (c) PV cell treated with mut-proBDNF from EP26-32 shows a drastic reduction both in percentage of innervated cells (c2) and perisomatic innervation (c3). Stars indicate pyramidal cells somata that are not innervated. Scale bar, a1-c1: 50µm; a2-c2: 10µm; a3-c3: 5µm. (d) Perisomatic bouton density (e) terminal branching and (f) percentage of innervated cells of the three experimental groups. One-way Anova, post hoc Tukey test, p

    Journal: bioRxiv

    Article Title: p75 Neurotrophin Receptor Regulates the Timing of the Maturation of Cortical Parvalbumin Cell Connectivity and Promotes Ocular Dominance Plasticity in Adult Visual Cortex

    doi: 10.1101/392159

    Figure Lengend Snippet: mut-proBDNF can destabilize PV cell innervation even after it has reached maturity. (a) Control PV cell (a1, Ctrl, green) at EP32 with exuberant innervation field characterized by extensive branching contacting the majority of potential targets, dense boutons along axons (a2), and terminal branches with prominent and clustered boutons (a3; arrowheads) around pyramidal cell somata (NeuN immunostaining, blue). (b) PV cell treated with wt-proBDNF from EP26-32 shows overall similar axon size (b1), percentage of potentially targeted neurons (B2) and perisomatic innervations (b3) as control, untreated PV cells. (c) PV cell treated with mut-proBDNF from EP26-32 shows a drastic reduction both in percentage of innervated cells (c2) and perisomatic innervation (c3). Stars indicate pyramidal cells somata that are not innervated. Scale bar, a1-c1: 50µm; a2-c2: 10µm; a3-c3: 5µm. (d) Perisomatic bouton density (e) terminal branching and (f) percentage of innervated cells of the three experimental groups. One-way Anova, post hoc Tukey test, p

    Article Snippet: To label control PV cells, slices were transfected with PG67 _GFP bullets, while for the p75NTR-/- PV cells were transfected with both PG67 _GFP and PG67_Cre. wt-proBDNF and mut-proBDNF (10 ng/ml, Alomone Labs) were respectively added with the culture medium during the specific time window indicated in the results section.

    Techniques: Immunostaining